A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand In fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2003 / Dietary iron (Fe) overload, originally referred to as Bantu Visceral Siderosis, is an Reloading condition that is still prevalent in rural populations of sub-Saharan Africa. The better known Fe loading disease, hereditary haemochromatosis (HFI) is frequently complicated by hepatocellular carcinoma (HCC) and, in rare instances this occurs in the absence of cirrhosis. The latter, together with recent evidence that dietary Fe overload in the Black African carries an increased risk for HCC, suggests that excessive hepatic iron may itself be carcinogenic. The aim of the study was to determine if Fe alone could induce HCC in experimental rat models and, if so, to investigate possible mechanisms of hepatocarcinogenesis. 360 Wistar albino rats (Rattus norvegicus) were divided into 6 groups. The first group, the control animals, was designated C group. Groups 2-6 were Fe-fed alone or in combination with other chemicals: group 2 Fe alone (Fe group), group 3 (Fe + V) vitamins A & E supplementation [50 mg all trans-retinol (vitamin A) and 500 mg a-tocopherol (vitamin E) per kg diet], group 4 (Fe - V) received a diet totally devoid of vitamins A & E, group 5 (Fe + ASA) received 20 mg aspirin (ASA) per day, group 6 (Fe + Cu) received 300 mg/kg diet of copper sulphate (CuS04) supplementation for 12 months followed by 3% copper hydroxide carbonate [CuC03»Cu(0H)2] / IT2018

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand In fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2003 / Dietary iron (Fe) overload, originally referred to as Bantu Visceral Siderosis, is an Fe- loading condition that is still prevalent in rural populations of sub-Saharan Africa. The better known Fe loading disease, hereditary haemochromatosis (HH) is frequently complicated by hepatocellular carcinoma (HCC) and, in rare instances this occurs in the absence of cirrhosis. The latter, together with recent evidence that dietary Fe overload in the Black African carries an increased risk for HCC, suggests that excessive hepatic iron may itself be carcinogenic. The aim of the study was to determine if Fe alone could induce HCC in experimental rat models and, if so, to investigate possible mechanisms of hepatocarcinogenesis. 360 Wistar albino rats (Rattus norvegicus) were divided into 6 groups. The first group, the control animals, was designated C group. Groups 2 - 6 were Fe-fed alone or in combination with other chemicals: group 2 Fe alone (Fe group), group 3 (Fe + V) vitamins A & E supplementation [50 mg all trans-retinol (vitamin A) and 500 mg a-tocopherol (vitamin E) per kg diet], group 4 (Fe - V) received a diet totally devoid of vitamins A & E, group 5 (Fe + ASA) received 20 mg aspirin (ASA) per day, group 6 (Fe + Cu) received 300 mg/kg diet of copper sulphate (CuS04) supplementation for 12 months / IT2018

Thesis advisor: Clare O'Connor / Within cells, reactive oxygen species (ROS) are synthesized naturally and in response to environmental stimuli. However, ROS have deleterious effects on a wide range of cellular molecules. Oxidative stress, caused by the ROS generated by the partial reduction of oxygen, is a major cause of cell damage linked to the initiation and progression of numerous diseases. Thioredoxin peroxidase (Tpx1) plays important roles in cellular defense against ROS. Although homologous genes and their functions have been identified in other eukaryotes, the level of activity as well as the necessity of this protective enzyme in S. pombe exposed to oxidative stress has yet to be fully elucidated. To explore the role of the Tpx1 protein in oxidative stress resistance, novel strains were constructed in which the tpx1 gene was overexpressed. The polymerase chain reaction was used to amplify txp1, and the amplified sequence was cloned into the yeast overexpression plasmid, pNMT41, which allows overexpression under the control of the powerful promoter. DNA sequencing was used to determine that the sequences had been properly inserted into the vector. The plasmids were transformed into two leu- yeast strains: FWP6 and TP108-3C. Production of the Tpx1 protein was ensured using Western Blot techniques. Experimentation to test the responses of the tpx1 strain to oxidative stress will employ a variety of reactive oxygen generators, including hydrogen n peroxide, menadione, tert-butyl hydroperoxide, and paraquat. The results generally supported the proposed role of Tpx1 to confer additional resistance against the oxidative stress. In a complementary line of investigation, knockout strains are being constructed to reduce the levels of the Tpx1 in S. pombe. Gene deletion cassettes were constructed for tpx1. Currently, the strains are being analyzed for the successful replacement of the endogenous tpx1 gene by homologous recombination. If the absence of the protein results in decreased cell viability, the role of Tpx1 indicated by the overexpression experiments could be supported. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Thesis advisor: Daniel A. Kirschner / Reactive oxygen species (ROS) are implicated in a range of degenerative conditions, including aging, neurodegenerative diseases, and neurological disorders such as multiple sclerosis. Myelin is a lipid-rich multilamellar assembly that facilitates rapid nerve conduction in higher animals, and may be intrinsically vulnerable to oxidative damage given the high energetic demands and low antioxidant capacity of myelinating cells. To determine whether ROS can cause structural damage to internodal myelin, whole mouse sciatic and optic nerves were incubated ex vivo with a previously-characterized copper (Cu)/hydrogen peroxide (HP)/o-phenanthroline (OP)-based hydroxyl radical-generating system followed by quantitative determination of myelin packing by x-ray diffraction. Exposure to Cu/OP/HP-mediated ROS caused irreversible myelin decompaction in both sciatic and optic nerves. The addition of the hydroxyl radical scavenger, sodium formate, to the ROS-producing incubation solution significantly prevented sciatic nerve myelin decompaction, implicating hydroxyl radical species in causing the damage. Furthermore, Cu/OP/HP-mediated decompaction could be prevented by the addition of EDTA, which can compete with OP for Cu binding and sequester the metal within the bulk solution. These findings suggest that Cu/OP/HP-dependent myelin decompaction is caused by OP-mediated membrane-targeted hydroxyl radical production. Myelin membranes are particularly enriched in plasmalogen phospholipids, which have been linked to antioxidant activity; this enrichment may constitute an endogenous ROS-defense mechanism that protects ROS-vulnerable myelin tissue from damage. Intriguingly, it was found that sciatic nerve myelin from plasmalogen deficient (Pex7 KO) mice was significantly more susceptible to ROS-mediated decompaction than that from WT mice, supporting the role of plasmalogens as endogenous antioxidants. / Thesis (MS) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Nitric oxide (NO) as well as reactive oxygen species (ROS) play an important role in defence signalling in plants. After successful recognition of an invading pathogen, an increase in ROS occurs, the ’oxidative burst’; and a ’nitrosative burst’ is also observed. This leads to the induction of defence responses, including the ’hypersensitive response’ (HR), a form of programmed cell death. A balanced production of hydrogen peroxide and NO is crucial for HR induction. In a process called S-nitrosylation, NO can react with cysteine thiols to form S-nitrosothiols, or react with glutathione to form S-nitrosoglutathione (GSNO). The enzyme GNSO reductase (GSNOR) indirectly regulates SNO levels by turning over GNSO. The Arabidopsis thaliana T-DNA insertion mutant atgsnor1-3 shows a complete loss of GNSOR activity and has drastically increased SNO levels, resulting in stunted growth, loss of apical dominance, increased HR, loss of salicylic acid (SA) accumulation and increased susceptibility to avirulent, virulent and non-host pathogens. Two recessive and allelic EMS suppressor mutants in the atgsnor1-3 background were isolated, which showed mostly wild type growth. The mutations were identified by map-based cloning as two different point mutations in At1g20620 or CAT3, one of three catalase genes in Arabidopsis. Catalases break down hydrogen peroxide, with CAT2 being the major catalase in Arabidopsis. All three catalases are structurally very similar, but show temporal and spatial differences in their expression patterns. The suppressor mutants recovered apical dominance, and partially recovered disease resistance to avirulent pathogens, but were still susceptible to virulent pathogens and showed decreased SA levels. The suppressor mutants showed wild type HR in response to different avirulent bacteria. Interestingly, loss-of-function of the other catalase genes as well as loss-of-function of other redox-related genes did not restore apical dominance of atgnsor1-3 plants. This effect seems to be highly specific to CAT3, possibly because of its expression pattern or its expression levels. Further research is needed to fully understand the mechanisms at work here, but these results certainly seem to show a direct connection between redox signalling and S-nitrosylation.

Vaskuläre Komplikationen wie Atherosklerose sind bei Diabetikern weit verbreitet. Eine erhöhte Produktion reaktiver Sauerstoffspezies trägt zu einer Dysfunktion des Endothels bei Diabetes und hohen Glukosespiegeln bei. Glutathion (GSH) ist das häufigste zelluläre Thiol und stellt ein bedeutendens Antioxidans des menschlichen Organismus dar. Das Multidrug Resistance Protein 1 (MRP 1) ist im Endothel der Haupttransporter von oxidiertem GSH. Blockiert man MRP 1, so wird unter oxidativem Stress der intrazelluläre GSH-Spiegel erhalten. In dieser Arbeit wird der Einfluss von MRP 1 auf die endotheliale Funktion und Produktion reaktiver Sauerstoffspezies bei Diabetes und erhöhten Glukosespiegeln anhand von MRP 1-/- -Mäusen und Wildtyp-FVB-Tieren untersucht. Acht Wochen nach Injektion von STZ wurde die endothelabhängige Vasorelaxation an den isolierten thorakalen Aorten bestimmt. Diabetische Wildtyp-Tiere wiesen eine signifikant verminderte endothelabhängige Vasorelaxation auf. In MRP 1-/- -Tieren hingegen kam es zu keiner Beeinträchtigung der Endothelfunktion. Die endothelunabhängige Vasorelaxation war nicht signifikant unterschiedlich. STZ-induzierter Diabetes führte zu einer signifikant erhöhten Produktion von Superoxidanionen sowie Wasserstoffperoxid in Wildtyp-Tieren. Diabetische MRP 1-/- -Mäuse hingegen zeigten keinen Anstieg der Produktion reaktiver Sauerstoffspezies. Erhöhte Glukosekonzentrationen führten in vitro in humanen aortalen Endothelzellen ebenso zur erhöhten Superoxidanion-Produktion. In Zellen, in denen MRP 1 mittels siRNA herunterreguliert war, zeigte sich keine Erhöhung von Superoxidanionen. In Wildtyp-Mäusen führte Diabetes zu einer Verminderung des vaskulären GSH-Spiegels, wohingegen bei MRP 1-/- -Tieren keine Veränderung auftrat. Diese Daten weisen auf die wichtige Rolle von MRP 1 bei der unter hohen Glukosekonzentrationen auftretenden endothelialen Dysfunktion hin. MRP 1 stellt somit einen neuen Ansatzpunkt in der Behandlung der durch Diabetes ausgelösten vaskulären Dysfunktion dar. / Vascular complications and atherosclerosis are common in patients with diabetes. An increased production of reactive oxygen species contributes to endothelial dysfunction in diabetes. A major cellular defense against reactive oxygen species is Glutathione. The multidrug resistance associated protein 1 is the main transporter of oxidized glutathione in endothelial cells. Blockade of MRP 1 prevents endothelial cell dysfunction induced by reactive oxygen species. Diabetes was induced in 12 week old male MRP 1-/- mice or corresponding FVB background wildtype mice by injection of streptozotocin. Eight weeks thereafter endothelium-dependent vasorelaxation was blunted in isolated thoracic aortae. In aortae from diabetic mice lacking MRP 1, endothelium-dependent vasorelaxation was only mildly impaired. STZ induced diabetes increased aortic superoxide and hydrogen peroxide production in wildtype animals, while in aortae from MRP 1-/- mice the reactive oxygen species production was nearly unchanged by diabetic conditions. Aortic levels of reduced glutathione were diminished in diabetic FVB. Glutathione levels did not change in diabetic MRP 1-/- mice. These data indicate that MRP 1 plays an important role for endothelial dysfunction and reactive oxygen species production in diabetes and under conditions of high glucose. MRP 1 therefore may represent a therapeutic target in treatment of diabetes induced vascular dysfunction.

Une présence en excès d'espèces réactives oxygénées induit un déséquilibre redox cellulaire pouvant conduire à des pathologies liées au stress oxydatif, notamment les pathologies cardiovasculaires. Connue et étudiée pour ses propriétés antioxydantes, l’astaxanthine, molécule de la famille des caroténoïdes, présente un intérêt thérapeutique potentiel. Cependant, sa structure chimique lui confère un caractère hydrophobe ainsi qu’une sensibilité à l’air, à la lumière et à la chaleur. Dans cette thèse, tout d’abord, un système de complexation de l’astaxanthine avec l’hydroxypropyl-b-cyclodextrine a été élaboré (CD-A). Nous démontrons que cette complexation améliore la stabilité de l’astaxanthine en solution aqueuse tout en préservant ses activités antioxydantes, mesurées par des méthodes chimiques et biologiques. L’action du CD-A semble être médiée par les voies de signalisation PTEN/AKT, Nrf2/OH1/NQO1 dans des cellules endothéliales soumises au stress oxydatif. Puis, afin de libérer l’astaxanthine in situ sur le site du stress, nous avons élaboré deux systèmes de type matriciel en PVA/dextrane ou en pullulane/dextrane chargés en CD-A. Nous avons évalué, comme preuve de concept, la faisabilité de ces dispositifs pour le traitement local de la pathologie d’ischémie/reperfusion. Les patchs de PVA/dextrane/CD-A ont montré une bonne compatibilité in vitro, ainsi qu’une grande stabilité et tenue mécanique sans modification des propriétés antioxydantes. Leur biocompatibilité in vivo et suturabilité au muscle cardiaque ont aussi été étudiées. Le deuxième système à base de pullulane/dextrane/CD-A a été évalué in vitro et in vivo dans un modèle d’ischémie/reperfusion du membre inférieur à différentes périodes d'implantation. Les résultats ont montré l’activation d’un mécanisme de défense normal lié à la présence d’un matériel étranger et une diminution de la translocation du Nrf2 pouvant indiquer un effet protecteur dans les tissus traités par le CD-A. Ce manuscrit présente des arguments en faveur du potentiel thérapeutique de systèmes de libération d’astaxanthine agissant au niveau du stress oxydatif lié aux pathologies cardiovasculaires. / An over concentration of reactive oxygen species induces a redox imbalance within the cell inducing oxidative tissue damage and leading to oxidative stress related diseases, particularly cardiovascular pathologies. Astaxanthin, a well-known and studied antioxidant molecule, member of the xanthophyll carotenoid family, presents an important therapeutic potential. However, the chemical structure confers to astaxanthin a hydrophobic character and renders it susceptible to air, light and temperate degradation. During this thesis, a carrier system based on astaxanthin inclusion within hydroxypropyl-β-cyclodextrin(CD-A) was developed. We demonstrate that after astaxanthin inclusion, not only its stability was enhanced by also the antioxidant scavenging capabilities were preserved, confirmed by chemical and biological tests. The action of CD-A seems to be mediated by PTEN/AKT, Nrf2/OH1/NQO1 signaling pathways of endothelial cells submitted to oxidative stress. Then, two systems based on PVA/dextran and Pullulan/Dextran loaded within CD-A were evaluated for astaxanthin in situ delivery in the stressed environment. The feasibility of using these systems in the local treatment of ischemia/reperfusion injury was evaluated as a proof of concept. PVA/Dextran patches showed good in vitro compatibility, high mechanical and stability properties, while preserving CD-A antioxidant capabilities, also the path suturability to the cardiac muscle and the in vivo biocompatibility were studied. The second system based on pullulan/dextran scaffolds were evaluated in vitro and in vivo in an ischemic/reperfusion model at different implantation periods. Results showed an inner body defense mechanism to foreign materials. Additionally, the Nrf2 translocation could indicate a protective effect of CD-A in treated tissues. This manuscript provides a support evidence of the therapeutic potential of CD astaxanthin delivery system, to act against oxidative stress linked to cardiovascular conditions.

The current practice of red blood cell banking for transfusion medicine relies primarily on a six-week liquid storage. A growing demand for red blood cell (RBC) products has prompted the search for alternative preservation methods including dry storage. Being desiccation sensitive, attempts to recover RBCs from the dry state have failed. This dissertation offers a new mechanistic understanding of desiccation-induced cellular injury that is correlated with the oxidative state of the hemoglobin. The general hypothesis states that RBC desiccation is accompanied with non-physiological oxidation of hemoglobin and, consequently, the release of toxic products capable of compromising cellular recovery through oxidative injury. Data acquired for this dissertation demonstrates that water loss induces a drastic increase in the rate of hemoglobin oxidation, formation of intracellular reactive oxygen species (ROS), and hemolysis. Pharmacological treatments of the hemoglobins oxygen binding site reveal that hemoglobin-induced cellular injury is more prominent in RBC samples that are partially dehydrated (about 3.5 to 5.5 g H2O/g dry weight) than in samples that are relatively dry ( 2 g H2O/g dry weight). Furthermore, partially dehydrated RBC samples contain higher levels of oxidized lipids than more fully dried samples. This dissertation also examined the role that glucose and glutathione play in enhancing desiccation tolerance of RBCs. Glucose treatment (5 mmol/L) significantly reduced ROS formation and hemolysis levels in partially dehydrated RBC samples (5.8 0.3 g H2O/g dry weight), but not in samples that are relatively dry (2.8 0.5 g H2O/g dry weight). Treating RBCs with DL-buthionine-(S,R)-sulfoximine, a glutathione depleting agent, was correlated with reduced levels of desiccation-induced hemolysis. This study suggests that desiccation-induced oxidative injury in RBCs is water dependent corresponding to earlier stages of water loss, in which cells can retain metabolic activity. Pharmacological treatments at this stage can significantly affect cell recovery as demonstrated with modifying the hemoglobins oxygen binding site, glutathione depletion, and glucose supplementation. On the other hand, increased cytoplasmatic viscosity compromises biochemical reactions at lower residual moisture contents, and cellular injury is likely the result of physical and mechanical stress. These differences should be taken into consideration in the design of innovative approaches to RBC preservation.

Le facteur de transcription NF-κB joue un rôle majeur dans lorchestration de nombreux processus biologiques, tels que les réponses immunitaires innée et adaptative, la division cellulaire, lapoptose et le développement. Le NF-κB est activé en réponse à un grand nombre de stimuli, comme les cytokines pro-inflammatoires, les agents viraux et bactériens et la stimulation antigénique des cellules du système immunitaire. Il peut être aussi activé dans des conditions de stress oxydant, par exemple après une exposition à des concentrations physiologiques (de lordre du µM) de peroxyde dhydrogène (H2O2). Au début de ce travail, le mécanisme conduisant à lactivation du NF-κB par le stress oxydant était mal connu et sujet à maintes controverses, ce qui nous a poussé à investiguer cette voie plus avant. Dans une première partie, nous nous sommes attachés à étudier le mécanisme dactivation du NF-κB dans des lymphocytes T soumis à un stress oxydant. Les cellules du système immunitaire sont en effet très sensibles à lenvironnement redox, et sont fréquemment en contact avec des espèces réactives de loxygène libérées par les cellules phagocytaires (monocytes/macrophages et neutrophiles) lors dune réponse inflammatoire. Comme le NF-κB est une protéine cruciale pour le développement et lhoméostasie des lymphocytes T, létude de sa modulation lors dun stress oxydant savère particulièrement importante. Nous avons pu mettre en évidence une activation importante du facteur de transcription lors dun traitement oxydant, ainsi quune dégradation presque compète de linhibiteur IκBα. Létude approfondie du mécanisme menant à cette dégradation a mis au jour un mécanisme dactivation tout à fait inédit, impliquant lactivation du complexe IKK via lintervention de lInositol Phosphatase SHIP-1. Dans cette première partie, nous avons également mis en évidence le rôle crucial de la protéine SHIP-1 dans la protection des lymphocytes T contre lapoptose induite par le stress oxydant, ce qui en fait une protéine clé dans la lhoméostasie des lymphocytes T. Dans un second temps, nous nous sommes intéressés au mécanisme influençant la fixation du NF-κB à lADN. Nous avons pu démontrer le rôle important de la protéine IKKα comme déterminant la fixation du NF-κB aux promoteurs de certains gènes, mettant ainsi au jour un mode daction inconnu pour cette protéine. Ce rôle est dautant plus intéressant quil est spécifique, ce qui pourrait déboucher sur des applications thérapeutiques intéressantes.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.