Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 129-137.

Thesis (M.S.)--University of Missouri-Columbia, 2005. / "December 2005" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (Ph.D.)--McMaster University, 2004. / Advisor: G. J. Schrobilgen. Includes bibliographical references (leaves 333-352). Also available online.

Orientador: Ione Salgado / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T11:16:49Z (GMT). No. of bitstreams: 1 Wulff_Alfredo_M.pdf: 2480773 bytes, checksum: f1cbea2506b80bbad1e739df2bd5c1c4 (MD5) Previous issue date: 2008 / Resumo: O objetivo deste trabalho foi estudar os mecanismo s de síntese e de degradação do radical óxido nítrico (NO) pela mitocôndria vegetal, com ênfase à influência das NAD(P)H desidrogenases e da oxidase alternativa (AOX) nestes processos. Os experimentos foram realizados com mitocôndrias isoladas de células em cultura da planta modelo Arabidopsis thaliana. Inicialmente estabeleceu-se um protocolo para o isolamento e purificação de mitocôndrias, cuja integridade funcional foi determinada pelo controle respiratório, potencial elétrico de membrana e transporte eletroforético de cálcio. Os processos de síntese e de degradação de NO foram analisados monitorando-se o consumo de oxigênio e as concentrações de NO no meio de reação, com eletrodos específicos. Os resultados mostraram que o nitrito inibiu a respiração das mitocôndrias de A. thaliana em competição com o oxigênio do meio. Esta inibição foi revertida por cPTIO, um seqüestrador de NO. Quando a AOX foi inibida por n-propilgalato, o efeito inibitório do nitrito na respiração mitocondrial foi aumentado, enquanto que, na presença de myxothiazol, que previne a redução da citocromo c oxidase (COX), o nitrito não teve efeito na respiração. Os efeitos observados do nitrito no consumo de oxigênio sugeriram que NO foi sintetizado pelas mitocôndrias de A. thaliana através de uma atividade redutora de nitrito. Entretanto, a síntese de NO a partir de nitrito foi principalmente detectada em anaerobiose. Em altas concentrações de nitrito (1-5 mM), a produção de NO se mostrou praticamente insensível aos inibidores da cadeia respiratória, enquanto que, em concentrações menores de nitrito (100 -200 ?M) a COX, mas não a AOX, contribuiu para a síntese de NO. A exposição das mitocôndrias ao NO ou ao nitrito causou uma inibição transiente e menor no consumo de oxigênio quando NAD(P)H f oi utilizado como substrato respiratório, em relação ao succinato. Estas inibições foram potencializadas por superóxido dismutase, sugerindo um mecanismo de degradação de NO dependente de NAD(P)He de oxigênio pelas mitocôndrias de A. thaliana. O conjunto dos resultados mostra que o NO pode ser produzido pela mitocôndria de A. thaliana através de uma atividade redutora de nitrito e que as NAD(P)H desidrogenases e a AOX, além da COX, podem interferir nesta produção. Em aerobiose, a quantidade de NO produzido pode ser controlada pelas atividades de degradação das NAD(P)H desidrogenases e pela COX. A AOX contribui de maneira oposta, ajudando a manter a concentração de NO. Em anaerobiose a atividade redutora de nitrito é favorecida sendo o NO produzido nestas condições pela COX e também devido ao estado mais reduzido da cadeia respiratória. Ainda, em anaerobiose, os mecanismos de degradação de NO, dependentes de oxigênio, estão inibidos. Em conclusão, os resultados deste trabalho mostram a importância das proteínas alternativas da cadeia respiratória da mitocôndria para a homeostase do NO na planta / Abstract: The objective of this work was to study the mechanisms of synthesis and degradation of NO by plant mitochondria, with emphasis on the influence of the NAD(P)H dehydrogenases and the alternative oxidase (AOX) in these processes. The experiments were carried out with mitochondria isolated from cells in culture of the model plant Arabidopsis thaliana. Initially, a protocol for the isolation and purification of mitochondria was established, and their functional integrity was determined by respiratory control, electric membrane potential and electrophoretic calcium transport. The processes of synthesis and degradation of NO were analyzed by monitoring oxygen consumption and NO concentration in the reaction medium with specific electrodes. The results showed that nitrite inhibited respiration of A. thaliana mitochondria, in competition with oxygen in the medium. This inhibition was reversed by cPTIO, an NO scavenger. When AOX was inhibited by n-propylgalate, the inhibitory effect of nitrite in mitochondrial respiration was increased, whereas in the presence of myxothiazol, which prevents the reduction of cytochrome c oxidase (COX), nitrite had no effect on respirati on. The observed effects of nitrite in the oxygen consumption suggested that NO was synthes ized by A. Thaliana mitochondria through a nitrite reductase activity. Meanwhile, the synthesis of NO from nitrite was mainly detected under anaerobiosis. At high concentrations of nitrite (1-5 mM), the production of NO was virtually insensitive to inhibitors of the respiratory chain, while at lower concentrations of nitrite (100-200 ?M) COX, but not AOX, contributed for the synthesis of NO. The exposure of mitochondria to NO or nitrite caused a transient and lower inhibition in oxygen consumption when NAD(P)H was used as respiratory substrate, compared to succinate. These inhibitions were enhanced by superoxide dismutase, suggesting an NAD(P)H- and oxygendependent mechanism for NO degradation by mitochondria of A. thaliana. The overall results show that NO can be produced by A. thaliana mitochondria through a nitrite reductase activity and that NAD(P)H dehydrogenases and AOX, in addition to COX, can interfere in this production. In aerobiosis the amount of NO produced can be controlled by the degradation activities of NAD(P)H dehydrogenases and COX. The AOX has an opposite effect, keeping the concentration of NO. In anaerobiosis the nitrite reductase activity is favore d and NO is produced in these circumstances by COX, and also due the more reduced state of the respiratory chain. Additionally, in anaerobiosis, the oxygen -dependent mechanisms of NO degradation are inhibited. In conclusion, the results of this study show the importance of the alternative proteins of the mitochondrial respiratory chain for NO homeostasis in the plant / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Everardo Magalhães Carneiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T18:02:10Z (GMT). No. of bitstreams: 1 Cappelli_AnaPaulaGameiro_D.pdf: 5548263 bytes, checksum: a858b8eb10853e19811981be3356a0a4 (MD5) Previous issue date: 2013 / Resumo: Neste trabalho estudamos o efeito da desnutrição, obesidade e da suplementação com taurina na sensibilidade à insulina nos tecidos muscular e hepático, e sobre a secreção de insulina. Foram utilizados camundongos C57BL6 machos controle (C), controle + dieta hiperlipídica (CH) e controle + dieta hiperlipídica + taurina (CHT), restrito (R), restrito + hiperlipídica (RH) e restrito + hiperlipídica + taurina (RHT). Os animais receberam dieta entre os 21 e 105 dias de vida. Após esse período os animais receberam uma aplicação intraperitoneal de salina ou glicose (2g/Kg) e foram sacrificados. Medimos o conteúdo protéico da SOD1, GPx1, CAT, p-IR, p-AKT, p-PTP1B e p-PTEN no fígado e músculo. Verificamos a secreção de insulina em ilhotas expostas a alta concentração de glicose (16,7mM) + DPI (inibidor da NADPH oxidase). Nossos resultados mostraram que a taurina foi eficiente em melhorar a sinalização da insulina no fígado, na presença ou não de uma carga de glicose e mostrou, também, uma possível associação entre o efeito observado no conteúdo da p-PTEN sobre a atividade da AKT nos grupos CHT e RHT, porém sem alteração no teste de sensibilidade à insulina. Nossos resultados sugerem que a taurina atua na modulação redox da PTEN, favorecendo a sinalização da insulina no tecido hepático tanto no grupo CHT quanto no grupo RHT. Em adição, notamos que a suplementação com taurina aumenta o conteúdo protéico das enzimas antioxidantes no tecido muscular, no grupo RHT. A suplementação com taurina, apesar de não ter apresentado diferença estatística, parece atenuar o efeito causado pela dieta hiperlipídica no grupo CHT sobre a sinalização da insulina no músculo, porém sem efeito sobre o grupo RHT. Dessa maneira, concluímos que a taurina no grupo CH melhora a sinalização da insulina, no fígado e músculo, e a tolerância à glicose, sem alteração da secreção de insulina. No grupo RH, a taurina melhora a sinalização da insulina no fígado, mas não altera a sinalização da insulina no músculo e melhora parcialmente, sem diferença estatística, a tolerância à glicose, sem alteração da secreção de insulina. Assim, a suplementação com taurina poderia ser utilizada como terapia complementar no tratamento da resistência à insulina e diabetes tipo 2, no entanto, essa terapia seria mais eficiente em indivíduos que receberam dieta normoproteica e hiperlipídica do que em indivíduos que receberam dieta hipoproteica e hiperlipídica / Abstract: We studied the effect of malnutrition, obesity and taurine supplementation on insulin sensitivity in liver and muscle, and on insulin secretion. Male mice C57BL6 control (C), control + fat diet (CH) and control + fat diet + taurine (CHT), restricted (R), + restricted diet (HR) and restricted + fat diet + taurine (RHT) were treated between 21 and 105 days old. After this period the animals received intraperitoneal saline or glucose (2g/Kg) and were sacrificed. We measured the protein content of SOD1, GPx1, CAT, p-IR, p-AKT, p-PTEN and p-PTP1B in liver and muscle. We also measured insulin secretion in islets exposed to high glucose concentration (16,7mM) + DPI (inhibitor of NADPH oxidase). Our results showed that taurine was effective in improving insulin signaling in the liver, when stimulated or not with a glucose load, and also showed a possible association between the content of p-PTEN on the activity of AKT in CHT and RHT groups, but no change in insulin sensitivity test. Our results suggest that taurine has a role on the redox PTEN modulation, favoring the signaling of insulin in the hepatic tissue in both CHT and RHT groups. In addition, we noted that taurine supplementation increases antioxidant enzyme protein content in muscle in the RHT group. The taurine supplementation, although not statistically different, seems to attenuate the effect caused by high fat diet in the CHT groups on the insulin signaling in muscle, but no effects on RHT group were reported. Thus, we concluded that taurine in CH group improves insulin signaling in liver and muscle, as well as glucose tolerance, without alteration in insulin secretion. In RH group, taurine improves insulin signaling in the liver, but does not alter insulin signaling in muscle and improves partly glucose tolerance, without alteration in insulin secretion. Thus, supplementation with taurine could be used as adjunctive therapy in the treatment of insulin resistance and type 2 diabetes, however, this therapy would be more efficient in subjects who received normal and hyperlipidic diets than in subjects who received high-fat and low-protein diets / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Orientador: Carlos Francisco Sampaio Bonafé / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T10:21:51Z (GMT). No. of bitstreams: 1 Landini_GustavoFraga_D.pdf: 2852152 bytes, checksum: a366fb6b71cf198fd01af8b55114c8a9 (MD5) Previous issue date: 2007 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

The new political dispensation in South Africa has seen a lot of changes taking place. The democratic wind, which has been blowing in all spheres of the political arena, could not leave out Education. This has led to the transformation in education and the revision of the curriculum guided by the Outcomes-Based Education philosophy (OBE). Thus, require education authorities as well as educators to look at education more comprehensively. The challenge posed to educators now is to develop tools and strategies that will make learning accessible to as many learners as possible and to teach for understanding and construction of knowledge. The principal objective of this study was to investigate the important role the learner's prior knowledge plays and the use of different tools and strategies in stimulating conceptual understanding and construction of knowledge of redox reactions. This was done using learners' own investigations, practical activities, teaching settings and a workshop. The findings show that the learners lacked organized and structured prior knowledge. Learners could not integrate prior experience with new experience. The main issue seems to be the failure of learners to relate classroom experience to everyday redox phenomena. Possible reasons are discussed with some implications for teaching redox. The study further postulates that to assist learners to develop conceptual understanding of redox reactions, different tools and strategies should be employed and teaching made relevant to real-life situations. In so doing, redox concepts would not be abstract to learners.

The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H2O2, which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H2O2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening.

Mitochondria are the major effectors of cardioprotection by procedures that open the mitochondrial ATP-sensitive potassium channel (mitoKATP), including ischemic and pharmacological preconditioning. MitoKATP opening leads to increased reactive oxygen species (ROS), which then activate a mitoKATP-associated PKCε, which phosphorylates mitoKATP and leaves it in a persistent open state (Costa, ADT and Garlid, KD. Am J Physiol 295, H874-82, 2008). Superoxide (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (HO•) have each been proposed as the signaling ROS but the identity of the ROS responsible for this feedback effect is not known. Superoxide was excluded in earlier work on the basis that it does not activate PKCε and does not induce mitoKATP opening.To further examine the identity of the signaling ROS, respiring rat heart mitochondria were preincubated with ATP and diazoxide to induce the phosphorylation-dependent open state, together with agents that may interrupt feedback activation of mitoKATP by ROS scavenging or by blocking ROS transformations. Swelling assays of the preincubated mitochondria revealed that dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, trolox, and bromoenol lactone (BEL) each blocked the ROS-dependent open state but catalase did not interfere with this step. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO• excludes H2O2 as the endogenous signaling ROS and focuses attention on HO•. In support of the hypothesis that HO• is required, we also found that HO•-scavenging by DMF blocked cardioprotection by both ischemic preconditioning and diazoxide in the Langendorff perfused rat heart. HO• itself cannot act as a signaling molecule, because its lifetime is too short and it reacts immediately with nearest neighbor phospholipids and proteins. Therefore, these findings point to a product of phospholipid peroxidation, such as hydroperoxy-fatty acids. Indeed, this hypothesis was supported by the finding that hydroperoxylinoleic acid (LAOOH) opens the ATP-inhibited mitoKATP in isolated mitochondria. This effect was blocked by the specific PKCε inhibitor peptide εV1-2, showing that LAOOH activates the mitoKATP-associated PKCε. During ischemia, catabolism of mitochondrial phospholipids is accelerated, causing accumulation of plasmalogens and free fatty acids (FA) in the heart by the action of calcium independent phospholipases A2 (iPLA2). We first assessed the role of FAs and hydroxy FAs on mitoKATP opening and cardioprotection. Swelling assays of isolated rat heart mitochondria showed that naturally formed free FAs inhibit mitoKATP opening and that they are more potent inhibitors of the pharmacological open state of mitoKATP than the phosphorylation-dependent open state. That is, sustained mitoKATP opening induced by the phosphorylation-dependent feedback loop is more resistant to FA inhibition than direct mitoKATP opening by a potassium channel opener. Moreover, rat hearts perfused with micromolar concentrations of FA were resistant to cardioprotection by diazoxide or ischemic preconditioning. Racemic bromoenol lactone (BEL), a selective inhibitor of iPLA2, confers protection to otherwise untreated Langendorff perfused hearts by preventing ischemic FA release. To bring this story full circle, BEL blocks protection afforded by preconditioning and postconditioning by preventing the iPLA2-mediated release of FAOOH generated in the conditioned heart. HO• resulting from mitoKATP opening oxidizes polyunsaturated fatty acid components of the membrane phospholipids, resulting in a peroxidized side chain. FAOOH must be released in order to act on the mitochondrial PKCε, and this is achieved by the action of iPLA2. iPLA2 is essential for most modes of cardioprotection because it catalyzes the release of FAOOH. This fully supports the hypothesis that the second messenger of cardioprotective ROS-mediated signaling is hydroperoxy fatty acid (FAOOH), a downstream oxidation product of HO•.

INTRODUCTION: Inflammatory bowel disease (IBD) is a complex, chronic, autoimmune disease of the gastrointestinal tract. Reactive oxygen species (ROS), a product of active leukocytes, have been implicated in the pathogenesis of IBD. The ability to reliably measure ROS in blood, urine, and stool samples could represent a new approach to assessing disease activity and response to therapy in pediatric patients with IBD. OBJECTIVES: To assess the relationship between redox measurements and clinical disease activity in pediatric patients with IBD. METHODS: Biological specimens, including stool, urine, blood plasma, and intestinal aspirates, were collected from patients at Boston Children’s Hospital. Each sample’s oxidation-reduction potential was measured by two oxidation-reduction potential probes (an Arrowdox probe and a Mettler Toledo probe). Probes were directly immersed into the sample, returning a millivolt measurement of oxidation-reduction potential. Linear regression was performed to explore the relationship between patient-reported outcome measures (PROMs) and redox measurements of biological specimens. Patients were also stratified by disease severity, and ANOVA testing was performed to test for differences in oxidation-reduction potential observed in patients with remittent, mild, moderate, and severe disease activity. RESULTS: Redox values in stool, urine, plasma, and intestinal aspirate did not significantly correlate with PROMs or differ significantly among groups categorized by disease severity. CONCLUSIONS: Measurements of oxidation-reduction potential from stool, urine, plasma, and intestinal aspirate do not appear to be useful for assessing disease severity in pediatric patients with inflammatory bowel disease.