Cancer cells are believed to exist in a condition of metabolic oxidative stress compared to normal cells because of inherent mitochondrial dysfunction. Cancer cells up regulate antioxidant defense mechanisms to combat the toxic effect of reactive oxygen species (ROS). Many anticancer agents block ROS detoxification mechanisms and utilize oxidative stress to cause cytotoxicity to cancer cells. However, ROS also up-regulate many pro-survival signaling pathways that may mediate resistance to chemotherapy. I hypothesize that ROS induces both cytotoxicity and pro-survival mechanisms in cells treated with chemotherapeutic agents such as the EGFR inhibitor erlotinib. This thesis explores how oxidative stress may induce both pro-survival and pro-death mechanisms in HNSCC cells and how this can be exploited to increase the cytotoxicity of erlotinib. The combined use of buthionine-[S,R]-sulfoximine, an inhibitor of glutathione and auranofin, an inhibitor of thioredoxin metabolism enhanced human head and neck cancer cell killing by a mechanism involving oxidative stress both in vitro and in vivo and sensitized cells to erlotinib in vitro. However, in other studies erlotinib as a single agent induced oxidative stress and this was mediated by NADPH oxidase 4 (NOX4). NOX4 mediated oxidative stress activated a process called autophagy which protected cancer cells from cytotoxic effect of erlotinib and inhibition of autophagy sensitized cells to erlotinib in vitro. These studies show that oxidative stress may have a dual role in cancer chemotherapy. ROS generated from various drug treatments can cause oxidative damage of cells culminating in cell death. However, it may also activate autophagy protecting cells against the stress and leading to decreased efficacy of the treatment. Hence inhibiting autophagy and hydroperoxide metabolism can be effective treatment modalities to enhance the cytotoxicity of erlotinib and achieve maximum therapeutic efficacy.

Philosophiae Doctor - PhD / The blood-brain barrier (BBB) is a critical interface between the blood circulation and brain tissue which performs critical selection of circulating molecules that gain access to the brain tissue. Its unique ability to adjust to changes in the constituents of the blood circulation confer in the BBB a dynamic nature enabling changes in its properties to suit the homeostatic needs of the brain. Dysfunction of the BBB has been established to be pivotal to the initiation and/or maintenance of an array of neurological disorders, most of which involve the production of excess reactive oxygen species (ROS) and oxidative stress in their pathophysiology. Thus, clinical trials of exogenous antioxidant agents have been proposed and initiated, with most results being inconclusive. Extensive studies of the impact, capacity and plasticity of endogenous antioxidants in the cells that constitute the blood-brain barrier, especially the brain endothelial cells, therefore, became necessary for the rational choice, timing, and the mode of application of antioxidants in the management of oxidative stress-mediated neurological diseases.

Background: Neutrophil extracellular traps (NETs) constitute a network of chromatin fibres containing histone and antimicrobial peptides that are released by activated neutrophils. NETs protect the host against infection by trapping and facilitating phagocytosis of potentially harmful pathogens. Objectives: The aim of the current study was to investigate the effects cigarette smoke condensate (CSC) on phorbol-ester (PMA)-mediated NETosis in vitro, as well as the effects of cigarette. Methods: Isolated human blood neutrophils were exposed to PMA (6.25 ng/ml) in the presence or absence of CSC (40-80 μg/ml) for 90 min at 37oC. Alternatively neutrophils of non-smokers and smokers were activated with PMA (6.25 ng/ml) for 90 min at 37oC. NET formation was measured using a spectrofluorimetric procedure to detect extracellular DNA and fluorescence microscopy was used to visualize nets. Oxygen consumption by PMA-activated neutrophils was measured using an oxygen sensitive electrode. Cotinine levels were measured in smokers and non-smokers for objective confirmation of smoking status Results: Activation of neutrophils with PMA was associated with induction of NETosis that was significantly attenuated in the presence of CSC (40 and 80 μg/ml), with mean fluorescence intensities of 65% and 66% of that observed with untreated cells, respectively, and confirmed by fluorescence microscopy. The rate and magnitude of oxygen consumption by activated neutrophils pre-treated with CSC (80 μg/ml) was significantly less than that observed with untreated cells (73% of the control system), indicative of decreased production of reactive oxidant species in the presence of CSC. When comparing smokers and non- smokers, neutrophils from smokers showed a decrease in both oxygen consumption and the number of NET-forming cells consistent with attenuation of NET formation due to inhalation of cigarette smoke. Conclusion: The inhibition of NETosis observed in the presence of CSC and CS (in smokers) correlated with attenuation of oxygen consumption by PMA-activated neutrophils suggesting a mechanistic relationship between these events. Smoking-related attenuation of NETosis may impair host immune responses and increase the risk of respiratory infections, in vivo. / Dissertation (MSc)--University of Pretoria, 2016. / Immunology / MSc / Unrestricted

Wolbachia is a vertically transmitted, obligate intracellular bacterium infecting ~40% of all known species of arthropods, as well as filarial nematodes. The nature of Wolbachia-host interactions ranges from reproductive parasitism to increased fecundity and pathogen protection. Wolbachia reduces the ability of mosquitoes to transmit human pathogens, which is being explored as a novel method for the control of vector-borne diseases like Dengue and Zika. The mechanisms of Wolbachia blocking the transmission of these diseases are not fully understood. There are studies indicating that Wolbachia-induced changes in the insect immunity could block the virus, however there is no consensus in the literature. A necessary step in the transmission of these diseases is the viral entry into the insect vector. This occurs trough the gut epithelium, highlighting the importance of understanding the interaction of this tissue with microorganisms. We have recently shown that Wolbachia colonizes the Drosophila gut epithelium and affects the gut microbiome composition. Wolbachia’s presence did not affect the gene expression of immune effector molecules from the main regulators of gut immunity, Imd and ROS pathways. Our understanding of the mechanisms of Wolbachia’s colonization of the gut epithelium and modulation of gut microbiome are still very limited. This work characterizes Wolbachia’s kinetics of colonization in Drosophila midguts. Imaging analysis revealed that Wolbachia colonizes adult and larval midguts in different patterns. We have also characterized a preferential colonization in specific adult midgut sub-regions. We observed that Wolbachia patches are confined to specific midgut subregions, in a pattern similar to the arrangement of intestinal stem cell (ISC) clones. These results led us to hypothesize that Wolbachia colonizes Drosophila midguts by infecting intestinal progenitor cells and spreading vertically to their progeny with limited lateral transmission between neighboring cells. We provide evidence to support this hypothesis by showing that Wolbachia is present in intestinal progenitor cells in all stages of the fly’s life cycle as well as by analyzing the infection status of ISC clones and differentiated cells surrounding ISCs. Finally, we found that ISC proliferation is reduced by the intracellular presence of Wolbachia, which also decreases ISC tumor incidence triggered by the downregulation of Notch signaling specifically in ISCs. These findings will aid in our understanding of Wolbachia tropisms and its phenotypic consequences. It has been shown that in the Wolbachia wMelPop strain excessive growth of intracellular bacteria leads to damage to the host cell, suggesting a mechanism of controlling intracellular growth in other strains. To better understand the molecular mechanisms behind Wolbachia-Drosophila interactions, we turned to the gonads, as Wolbachia colonization of these tissues has been well characterized. We chose to investigate the interplay between Reactive Oxygen Species (ROS) and Wolbachia, as intracellular ROS could regulate bacterial density but also be affected by Wolbachia and play a role in symbiont-related phenotypes. Using direct and indirect measurements of ROS, we show that the pathogenic strain wMelPop increases ROS in the germarium, while the symbiotic strains wMel and wMelCS reduce ROS in the terminal filaments. None of the Wolbachia strains tested affected ROS levels in the testes. In addition, genetically altering ROS levels in the germline or systemically in the fly did not affect Wolbachia levels in the ovaries. We conclude that ROS does not significantly affect Wolbachia in the fruit fly gonads.

Summary. Despite its being the most efficacious drug for symptom reversal in Parkinson's disease (PD), there is concern that chronic levodopa (L-DOPA) treatment may be detrimental. In this paper we review the potential for L-DOPA to 1) autoxidize from a catechol to a quinone, and 2) generate other reactive oxygen species (ROS). Overt toxicity and neuroprotective effects of L-DOPA, both in vivo and in vitro, are described in the context of whether L-DOPA may accelerate or delay progression of human Parkinson's disease.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12816号 / 論医博第2078号 / 新制||医||1004(附属図書館) / 31303 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 長田 重一, 教授 川口 義弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18851号 / 医博第3962号 / 新制||医||1007(附属図書館) / 31802 / 京都大学大学院医学研究科医学専攻 / (主査)教授 別所 和久, 教授 鈴木 茂彦, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Yes / Platinum complexes are the most widely used anticancer drugs; however, new generations of agents are needed. The organoiridium(III) complex [(η5-Cpxbiph)Ir(phpy)(Cl)] (1-Cl), which contains π-bonded biphenyltetramethylcyclopentadienyl (Cpxbiph) and C^N-chelated phenylpyridine (phpy) ligands, undergoes rapid hydrolysis of the chlorido ligand. In contrast, the pyridine complex [(η5-Cpxbiph)Ir(phpy)(py)]+ (1-py) aquates slowly, and is more potent (in nanomolar amounts) than both 1-Cl and cisplatin towards a wide range of cancer cells. The pyridine ligand protects 1-py from rapid reaction with intracellular glutathione. The high potency of 1-py correlates with its ability to increase substantially the level of reactive oxygen species (ROS) in cancer cells. The unprecedented ability of these iridium complexes to generate H2O2 by catalytic hydride transfer from the coenzyme NADH to oxygen is demonstrated. Such organoiridium complexes are promising as a new generation of anticancer drugs for effective oxidant therapy. / We thank the ERC (247450), SNSF (PA00P2_145308 for N.P.E.B.), IAS (for I.R.C.), BBSRC (for J.M.H.), Science City (AWM and ERDF), and the EPSRC for support, and Prof. Timothy Bugg and members of EC COST Action CM1105 for stimulating discussions. We also thank Professor Pat Unwin, Mike Snowden, and Rob Lazenby for their help with the electrochemical experiments and the National Cancer Institute for NCI-60 human tumor cell panel screening.

No / Accumulation of large-scale mitochondrial DNA (mtDNA) deletions and chronic, subclinical inflammation are concomitant during skin aging, thus raising the question of a causal link. To approach this, we generated mice expressing a mutant mitochondrial helicase (K320E-TWINKLE) in the epidermis to accelerate the accumulation of mtDNA deletions in this skin compartment. Mice displayed low amounts of large-scale deletions and a dramatic depletion of mtDNA in the epidermis and showed macroscopic signs of severe skin inflammation. The mtDNA alterations led to an imbalanced stoichiometry of mitochondrial respiratory chain complexes, inducing a unique combination of cytokine expression, causing a severe inflammatory phenotype, with massive immune cell infiltrates already before birth. Altogether, these data unraveled a previously unknown link between an imbalanced stoichiometry of the mitochondrial respiratory chain complexes and skin inflammation and suggest that severe respiratory chain dysfunction, as observed in few cells leading to a mosaic in aged tissues, might be involved in the development of chronic subclinical inflammation. / Deutsche Forschungsgemeinschaft (Wi 889/6-3 to RJW, SFB 829 A14 to RJW, Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases–CECAD to RJW, BR2304/9-1 to BB, and SFB 829 A1, A5, and Z2 to CMN) and the Center of Molecular Medicine Cologne of the Medical Faculty (CMMC, to RJW)