Ischemia is characterized by reduced blood flow to an area of the body which can then cause cellular injury through the generation of reactive oxygen species (ROS), activation of inflammation, and induction of apoptosis. Although rapid reestablishment of flow is required to prevent organ death, the reperfusion phase of this injury can cause its own deleterious effects often exacerbating the initial insult. The combined action of the two injuries is termed ischemia/reperfusion (I/R) injury. Oxidative stress that results from ischemia/reperfusion injury is a common pathological condition that accompanies many human diseases including stroke, heart attack and traumatic injury. In addition, neurodegenerative diseases including Parkinson’s, Alzheimer’s, and Huntington’s disease appear to involve oxidative stress. Although actively investigated by the medical and pharmaceutical industry; limited progress has been made to ameliorate I/R injury and to date there is no drug approved for treatment for I/R injury. Therapeutic approaches to treat I/R injury have included the administration of compounds to scavenge ROS or induce protective pathways or genetic responses. It was previously reported that caffeic acid phenethyl ester (CAPE), a plant-derived polyphenol, displayed cytoprotective effects against menadione (MD)-induced oxidative stress in human umbilical vein endothelial cells (HUVEC), and the induction of heme oxygenase-1 (HMOX1), a phase II enzyme, played an important role for CAPE cytoprotection. In an effort to improve this cytoprotection, other phase II enzyme inducers were investigated and, 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (CDDO-Im) and 2-cyano-3,12-dioxooleana-1,9-dien-28-oyl methyl ester (CDDO-Me), were found to be potent inducers with a rapid onset of action. CDDO-Im and CDDO-Me, synthetic olenane triterpenoids, developed as anticancer agents were compared to CAPE revealing that CDDO-Im was a more potent inducer of Phase II enzymes including HMOX1 and provided better cytoprotection than CAPE. Gene expression profiling showed that CDDO-Im was more potent inducer of protective genes like HMOX1 than CAPE and additionally induced heat shock proteins. To better understand the mechanism of action of CDDO-IM, a gene expression time-course was undertaken to identify early initiators of the transcriptional response preceding cytoprotection. Application of systems pharmacology identified molecular networks of cell mediating processes.

Carbon/Carbon (C/C) composite is defined as a carbon fiber reinforced carbon matrix. Since 1958 research has been carried out on the C/C composites. The main reason for the development of new C/C composites is the number of advantages it has to offer when compared with the regular materials. The areas where C/C composites are being used extensively are aerospace, military, etc. These C/C composites have better physical, mechanical, thermal properties when compared to steel. That is the reason C/C brakes made a huge impact in the aerospace industry. The main drawback associated with the C/C brakes which are used in aerospace applications is the oxidation of the composite at higher temperatures. Also other problem linked with the C/C brake is the migration of the inhibitors on to the friction surface of the brake which can eventually decrease the friction coefficient of the brake material. So, characterizing the commercially available Anti-Oxidant(A/O) system, developing a new A/O system which can not only provide better oxidation protection, but also an improved anti-oxidant migration resistance will be our main goal of this project.

Plants have been utilized as a source of medicines since ancient times. They contain a vast range of secondary metabolites which play important roles in different diseases. The Scope of this study is to define the function of secondary metabolites from Andrographis paniculata (Kalmegh) and Silybum marianum (Milk Thistle) against DNA damage, which initiates many diseases. Sequential extraction of both plant materials was performed with different polarity solvents. Qualitative analysis was performed with Gas (GC) and High Performance Liquid Chromatography (HPLC). Primary extracts screening studies were performed against cytotoxicity, antioxidant and soluble collagen assays (Sircol dye). Further bioactivity was confirmed using the single-cell gel electrophoresis (Comet assay) to estimate levels of DNA damage. Fourier Transform Infrared analysis of bioactive extracts was performed to identify the functional groups present in them . Subsequently, bioactive extracts were further separated into acid, base, phenol and neutral fractions. These fractions and bioactive extracts were screened with the free radical assays to identify the scavenging activity. Chemical mapping of the bioactive fraction was performed with High Performance Liquid Chromatography (HPLC). Preparative-HPLC was performed to separate the compounds which were present in the bioactive fractions. MTT assay, Hydroxyl radical and nitric oxide radical scavenging activity assays were performed to screen the fractions and DNA protective activity of the bioactive fractions was confirmed with single-cell gel electrophoresis. These bioactive fractions were de-replicated with hyphenated techniques like LCMS and LCMS/MS to identify the molecular weight of the compounds and Quadrupole time-of-flight mass spectrometry was performed to identify the accurate mass of the compounds. Sequential extraction separates the non-polar and polar compounds present in the plant material. Qualitative analysis confirmed the presence of fatty acids in the non-polar extracts of both plants using GC and the presence of standard constituents in the polar extracts of both plants using HPLC. It also helps in chemical mapping of the extracts. Acetone, Methanol1 and Methanol2 extracts from either plant are non-cytotoxic. The high antioxidant activity is observed in methanol extracts from Andrographis paniculata and in acetone/methanol2 extracts from Silybum marianum. Extracts that protect against UVA and UVB damage also increased soluble collagen production in Human Dermal Fibroblast (HDF) in culture. Primary Screening helped to select six extracts out of twelve extracts for further analysis. Comet assay confirmed DNA protective activity in Methanol1 extract of Milk thistle and Acetone, Methanol2 extracts from Kalmegh. These three extracts were further fractionated into 38 fractions out of which three fractions that are F1, F13 and F31 fractions confirm the DNA protection activity. De-replication of the bioactive fractions was performed with LC-ESI-MS/MS which confirm twenty one compounds and accurate mass of fifteen compounds was determined using Q-tof mass spectrometry.

Os linfomas constituem um grupo de neoplasias que têm origem nas células linforreticulares e acomete tecidos linfóides primários, secundários como o baço e linfonodos e demais tecidos onde há presença de linfócitos circulantes, comumente descritos em cães, gatos e humanos. As síndromes paraneoplásicas são definidas como alterações sistêmicas não relacionadas com lesões metastáticas de forma direta ou indireta no hospedeiro. Dentre as alterações descritas como síndrome paraneoplásica, as alterações hematológicas constituem as mais freqüentes, apresentando destaque para os processos anêmicos. A anemia presente pode ser decorrente de alterações do metabolismo do ferro, perda sanguínea, distúrbios hemolíticos, infiltrado medular e hiperesplenismo. Atualmente tem se associado a redução da vida média das hemácias frente a presença do estresse oxidativo, que gera um desequilíbrio entre a excessiva produção de espécies reativas ao oxigênio e/ou a diminuição dos mecanismos de defesa antioxidante das hemácias. Esse processo pode ocasionar a peroxidação de lipídios da membrana eritrocitária, gerando hemólise e assim resultando em anemia e/ou exacerbando a mesma. Com o objetivo de avaliar a existência do estresse oxidativo e a presença de anemia assim como, a sua correlação com o estado redox, foram avaliadas as concentrações eritrocitárias de glutationa reduzida, glutationa redutase, glutationa peroxidase e superóxido redutase em 23 gatos sadios e 17 felinos com linfoma. Também foram determinadas as concentrações plasmáticas de malonaldeído como indicador da presença de peroxidação lipídica em ambos os grupos. Para avaliar o estado redox foi mensurada a concentração plasmática do estado antioxidante total para o grupo experimental e grupo controle. Não foram observadas diferenças significantes para as enzimas eritrocitárias glutationa redutase, glutationa peroxidade e superóxido dismutase, assim como para a mensuração plasmática de malonaldeído. Entretanto, foram observados valores significativamente menores (p=0,018) de glutationa reduzida nos felinos com linfoma quando comparados ao grupo controle. O mesmo pôde ser observado em relação à mensuração do estado antioxidante total (p=0,003). Os resultados obtidos indicam a presença de estresse oxidativo em felinos com linfoma, porém, não houve evidencias da relação entre a presença de estresse oxidativo e a presença de anemia. / Lymphomas are a group of cancers that originate in cells and lymphoreticular affects lymphoid tissues in primary, secondary as the spleen and lymph nodes and other tissues where there is presence of circulating lymphocytes, commonly described in dogs, cats and humans. Paraneoplastic syndromes are defined as systemic changes unrelated metastatic lesions either directly or indirectly in the host. Among the changes described as a paraneoplastic syndrome, hematological changes are the most frequent, with emphasis on the processes anemic. This anemia may be due to changes in iron metabolism, blood loss, hemolytic disorders, bone marrow infiltration and hypersplenism. Today has been associated with reduced average life span of red cells before the presence of oxidative stress, which creates an imbalance between excessive production of reactive oxygen species and / or decreased antioxidant defense mechanisms of red blood cells. This process can lead to lipid peroxidation of the membrane, causing hemolysis and thus resulting in anemia and / or exacerbating it. Aiming to evaluate the existence of oxidative stress and anemia as well as its correlation with redox state, were evaluated erythrocyte concentrations of reduced glutathione, glutathione reductase, glutathione peroxidase and superoxide reductase in 23 healthy cats and 17 cats with lymphoma. We also determined plasma concentrations of malondialdehyde as an indicator of the presence of lipid peroxidation in both groups. To assess the redox state was measured plasma concentration of total antioxidant status for the experimental group and control group. No significant differences were observed for enzymes erythrocyte glutathione reductase, glutathione peroxidase and superoxide dismutase, as well as for measurement of plasma malondialdehyde. However, observed values were significantly lower (p = 0.018) reduced glutathione in cats with lymphoma when compared to the control group. The same could be observed in relation to the measurement of total antioxidant status (p=0,003). The results indicate the presence of oxidative stress in cats with lymphoma, however, no evidence of the relationship between oxidative stress and anemia.

Abstract Antioxidants function as blockers of radical processes and eliminate harmful reactive oxygen species (ROS) produced during normal cellular metabolism. A complex antioxidant defence system has evolved to protect the cellular homeostasis. This system includes antioxidant enzymes (AOEs), such as superoxide dismutases (SODs), which are intracellular MnSOD and CuZnSOD and extracellular ECSOD, H2O2 scavenging enzymes catalase and glutathione peroxidase, and hemeoxygenase-1 (HO-1), an important enzyme in heme metabolism, which has also been suggested to have antioxidant capacities. ROS play an important role in the pathogenesis of interstitial lung diseases. These diseases represent a group of disorders with different etiology, histopathology, treatment and prognosis. Sarcoidosis, extrinsic allergic alveolitis and two different forms of idiopathic pulmonary fibrosis, usual interstitial pneumonia (UIP) and desquamative interstitial pneumonia (DIP) were included in this study. The purpose of this research was to evaluate the expressions of inducible nitric oxide synthase (i-NOS), endothelial nitric oxide synthase (e-NOS) and xanthine oxidase (XAO), oxidant generating enzymes commonly associated with tissue injury, and, on the other hand, the expressions of AOEs suggested to be involved in the defence of lung tissue against oxidant stress. The methods included immunohistochemistry on lung biopsies (n=48) and Western blotting, Northern blotting or reverse polymerase chain reaction (RT-PCR) on human inflammatory cells and cells obtained from bronchoalveolar lavage. I-NOS was intensively expressed in inflammatory, but not in fibrotic lesions, similar e-NOS expression was found in control lung and in all interstitial lung diseases, while XAO was mainly negative. MnSOD and HO-1 were highly expressed in the granulomas of sarcoidosis. In contrast the expressions of MnSOD and HO-1 in late fibrotic lesions of UIP were low or undetectable by immunohistochemistry. CuZnSOD and catalase showed similar immunoreactivity in healthy and diseased lung. A cell specific expression and regulation of various enzymes may play an important role during acute inflammatory diseases and also in the progression of lung fibrogenesis.

Abstract Manganese superoxide dismutase (MnSOD) is an important intracellular antioxidant enzyme, which has been suggested to play a role in tumour biology. In the present study, the expression and possible role of MnSOD in malignant pleural mesothelioma was investigated. Mesothelial cells in healthy visceral pleural tissue showed no MnSOD immunoreactivity in five out of six cases, whereas moderate or high immunoreactivity for MnSOD was detected in 30 out of 42 (71%) cases of mesothelioma. Only two of the 21 cases with metastatic adenocarcinoma of the pleura showed moderate MnSOD immunoreactivity, the remaining 19 (90.5%) showing negative or weak reactivity (p < 0.001, by Fisher's exact test compared to mesothelioma). The immunostaining of catalase, a hydrogen peroxide scavenging antioxidant enzyme, was detectable in 27 of the 35 (77%) mesothelioma cases studied, whereas all the five samples of healthy pleural mesothelium were negative. Reactive mesothelium showed positive immunoreactivity for MnSOD and catalase, suggesting that induction of these enzymes is not specific for mesothelioma. Two continuous human mesothelioma cell lines showed higher MnSOD activity, immunoreactive protein and mRNA levels than non-malignant mesothelial cells. In addition, mesothelioma cells expressing the highest MnSOD levels had the highest levels of catalase and copper-zinc superoxide dismutase. The mitochondria of these cells expressed higher MnSOD and lower superoxide levels than non-malignant mesothelial cells. The mesothelioma cells with the highest antioxidant enzyme levels were most resistant to oxidant- and drug-induced injury and to drug-induced apoptosis compared to non-malignant mesothelial cells and mesothelioma cells with lower MnSOD and catalase levels. The extent of cell proliferation and apoptosis of mesothelioma tissue were 14.1±13.2% and 1.1±1.2%, respectively. MnSOD expression was inversely associated with cell proliferation (p = 0.02 by t-test), and a tendency for a better prognosis among patients with moderate or strong MnSOD expression was demonstrated. Patients displaying a tumour with enhanced proliferation or apoptosis had a poorer prognosis (p < 0.001 by Log Rank test). In conclusion, the MnSOD level is usually high in pleural mesothelioma, which may affect the proliferation and drug-resistance of mesothelioma cells. MnSOD immunostaining can thus possibly be used to distinguish mesothelioma from metastatic adenocarcinoma but not from reactive mesothelium.

Abstract Glutaredoxins (Grx) are small thiol disulphide oxidoreductases with a conserved active site sequence -CXXC/S- and a glutathione (GSH) binding site. They catalyze the reduction of protein disulphides, preferring protein-GSH mixed disulphides as substrates. The accumulation of protein-GSH mixed disulphides has been observed during oxidative stress, where they may serve both a regulatory and an antioxidant function by protecting the enzymes from irreversible oxidation. Once oxidative stress has been removed the GSH-protein mixed disulphides are reduced by GSH or, more efficiently, by Grx. The present study showed for the first time that Grx1 and Grx2 can be detected in healthy human lung. Highly specific expression of Grx1 was observed in alveolar macrophages, but it could also be detected from sputum supernatant. Grx1 levels in alveolar macrophages were lower in selected inflammatory diseases than in control lung samples. Grx1 was also mainly negative in the fibrotic areas in usual interstitial pneumonia, an aggressive fibrotic lung disease. Overall, the present study suggests that Grx1 is a potential redox modulatory protein regulating the intracellular as well as extracellular homeostasis of glutathionylated proteins and GSH not only in healthy lung, but also in inflammatory and fibrotic lung diseases. In order to study the mechanism of action of glutaredoxins in vitro, a new real-time fluorescence-based method for measuring the deglutathionylation activity of glutaredoxins using a glutathionylated peptide as a substrate was developed. The first reaction intermediate in the deglutathionylation reaction was shown to be exclusively Grx-GSH mixed disulphide and this specificity was solely dependent on the unusual γ-linkage present in glutathione. The study also demonstrated the role of conserved residues in the proximity of proposed GSH binding site to the GSH binding specificity of E. coli Grx1. Opening the binding groove and removing charged residues enabled Grx to form more readily mixed disulfides with other molecules besides GSH. Different members of the PDI family showed considerably lower activity levels compared to glutaredoxins and, in contrast to the glutaredoxin-GSH mixed disulphide, the only intermediate in the PDI catalysed reaction was PDI-peptide mixed disulphide.

Turmeric polar extract (TPE) was obtained by dielectric-precipitation of turmeric slurry and found to contain three proteins with two in the 10-11 KDa range being dominant. Antioxidative activity and persistence (AP) of TPE (5%, w/v) respectively showed 87% and 85% greater generation of alkoxy- and peroxyl radicals compared the non-redox-active buffer alone showing significant (p<0.05) pro-oxidative behavior. Conversely, purified curcumin (CU) (0.1% w/v) was dramatically antioxidative with AA and AP values of 2,828 and 1,129%, respectively, compared to the blank. However, a combination of the two at the same concentration dropped these values to 590 and 389%, respectively, reflecting dramatic dampening of the efficacy of CU. Ultraviolet radiation significantly modulated the efficacy of CU where UVB (300 nm) exposure gave the highest enhancement when limited to five min. Data showed that turmeric contains highly pro-oxidant polar proteins that significantly dramatically diminishes the beneficial antioxidative efficacy of its principal phytochemical, CU.

Yes / This study was focused on developing a new catalyst using metal oxide (10 %Mn) over Nano- activated Carbon (Nano-AC) particles and designing a new reactor (digital basket reactor, DBR) for the sulfur removal from kerosene oil via oxidative desulfurization (ODS). The new homemade Nano-catalyst was prepared by utilizing impregnation process and was characterized by SEM, EDX, BET, and FTIR techniques. The performance of ODS process under moderate operating conditions was significantly enhanced by the application of the new catalyst and the new reactor. The results showed that 94 % of the sulfur could be achieved at oxidation temperature of 80 ºC, oxidation time of 35 min and agitation rate of 750 rpm. The reactivity of catalyst was examined after four consecutive ODS cycles under the optimal experimental parameters and the used catalyst showed excellent stability based on oxidation efficiency. The spent catalyst was treated by methanol, ethanol and iso-octane solvents for regenerated it, and the result proved that iso-octane carried out the maximum regeneration performance. An optimization method depending on minimizing the sum of the squared error among the experimental and model predicted data of ODS technology was employed to evaluate the optimal kinetic model parameters of the reaction system. The ODS process model was able to predict the results obtained experimentally for a wide range of conditions very well by absolute average errors<5 %.

A new class of fluorous materials was developed to create a hybrid solid-solution phase strategy for the expedient preparation of 125I-labelled compounds, without the need of HPLC purification. The system is referred to as a hybrid platform in that it combines solution phase labelling and fluorous solid-phase purification in one step as opposed to two separate individual processes. Initial success was achieved by treating fluorous stannanes, coated on fluorous silica, with [125I]NaI and chloramine-T (CAT) as the oxidant, where the desired nonfluorous radiolabelled products were isolated in minutes in biocompatible solutions in high purity (>98%) free from excess starting material and unreacted radioiodine. This platform was initially developed through a model system based on a fluorous benzoic acid derivative. The platform was then validated with simple aryl and heterocyclic derivatives, known radiopharmaceuticals including meta-iodobenzylguanidine (MIBG) and iododeoxyuridine (IUdR), and a new agent with high affinity for prostate-specific membrane antigen (PSMA). The limitation of the platform was the presence of non-radioactive UV impurities which came from the oxidants employed. To resolve this issue a new class of fluorous oxidants based on chloramine-T (CAT, F-CAT) were prepared. F-CAT, was prepared in 87% overall synthesis yield from commercially available starting materials and found to be effective in labelling arylstannanes and proteins with [125I]NaI. The utility of the oxidant was further demonstrated in successfully preparing a radioiodinated tetrazine (125I-Tz) through a concomitant oxidation-halodemetallation reaction. 125I-Tz can be used to label biomolecules through bioorthogonal coupling reactions with prosthetic groups containing strained alkenes including norbornene and trans-cyclooctene (TCO). The reported hybrid platform labelling approach is readily accessible and requires minimal radiochemistry expertise and should therefore find widespread use. It is also noteworthy that a second generation of the fluorous oxidant, F-CAT2, was also prepared with the aim of obtaining an oxidant which has a higher solubility in perfluorinated solvents. Application of F-CAT2 for oxidation of hydrogen sulfide to elemental sulphur in a fluorous-aqueous biphasic system was demonstrated. This approach offers a new metal-free approach to scrubbing sour gas wells and demonstrates that the fluorous oxidants developed here have utility beyond radiochemistry. / Thesis / Doctor of Philosophy (PhD)