The aim of the thesis was to elucidate the role of High light inducible proteins (Hlips) in the protection/regulation of the biogenesis of photosynthesis machinery. During the project two Hlip proteins (HliC and HliD) were isolated from the cyanobacterium Synechocystis PCC 6803; either as a pure oligomer (HliC protein) or as a small complex with a putative Photosystem II assembly factor Ycf39 (HliD protein). Pigments bound to purified Hlips were analyzed by state-of-art spectroscopic techniques to elucidate the mechanism of thermal energy dissipation. In addition, this work explained the mechanism of how the HliC protein regulates the interaction between chlorophyll synthase enzyme and the Ycf39 protein. This conceptually new mechanism is based on the replacement of HliD dimers in chlorophyll synthase complexes by stress-induced HliD-HliC heterodimers, which changes the affinity of Ycf39 towards chlorophyll synthase.

Aging, a major risk factor in Alzheimer's disease (AD), is associated with an increased free radical (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, glutathione (GSH) is the major ROS detoxifying redox buffer within the cell and cysteines are the major reducing buffer in the extracellular matrix. Since the relationship of the prominent ROS damage to aging and AD is unclear, we wanted to know whether A) an oxidative redox shift precedes these markers and leads to macromolecular damage, B) age and AD-related changes can be reversed using redox interventions in neurons and C) modification of the extracellular Cys/CySS redox can change intracellular redox and increase neuron survival. Hippocampal/cortical neurons were isolated across the age-span from non-transgenic (non-Tg) and a triple transgenic Alzheimer's mouse model (3xTg-AD) and cultured in common nutrients to control for age-related hormonal and vascular differences. We found an increase of NAD(P)H levels and redox state in non-Tg neurons until middle age, followed by a decline in old age. The 3xTg-AD neurons maintained much lower resting NAD(P)H and redox state after 4 months, but the NADH regenerating capacity continuously declined with age beginning at 2 months. Compared to non-Tg neurons, 3xTg-AD neurons had lower glutathione (GSH) levels, which preceded age-related increases in ROS levels. The redox deficits in NAD(P)H and GSH were partially reversed using the NADH precursor, nicotinamide. To determine the relative importance of GSH to ROS and cell death, we simultaneously determined GSH depletion and ROS elevation in live neurons across the age-span by titrating with buthionine sulfoximine (BSO), an inhibitor of the rate-limiting enzyme for GSH synthesis, γ-glutamylcysteine synthetase, subunit c (GCLC). We observed that in old age, neuron loss was more dependent on GSH depletion than ROS elevation. Remarkably, the rate of neuron loss with ROS was the same for both genotypes, indicating that cognitive deficits in the AD-model were not caused by ROS. Therefore, we targeted activation of the redox sensitive transcription factor, Nrf2 (Nuclear erythroid-related factor 2) for neuroprotection by 18α-glycyrrhetinic acid to stimulate glutathione synthesis through GCL. . By combining the Nrf2 activator together with nicotinamide, we increased neuron survival against glutathione depletion and beta-amyloid stress in an additive manner. Further, we explored the relative importance of NAD(P)H and GSH to neuron loss in aging and AD. Neurons stressed by either depleting NAD(P)H or GSH indicated that NAD(P)H redox control is upstream of GSH levels and compared to GSH depletion, higher neuron loss was observed with declining NAD(P)H, especially in old age and in the 3xTg-AD neurons. We also observed an age-dependent loss of gene expression of key redox dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyl transferase) and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age-related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age-related declines in NAD(P)H. Lastly, since extracellular redox deficits are seen in aging and AD, and Cys/CySS is the major redox buffer in the extracellular microenvironment, we determined the effects of extracellular redox modification on intracellular redox state, neuron survival and signaling through pAkt/Akt. We found that a reductive shift in extracellular Cys/CySS improved neuron survival, maintained intracellular GSH and NAD(P)H as well as increased pAkt/Akt in aging. Overall, our results strongly support the EORS theory of aging that an oxidative redox shift precedes ROS-mediated damage. A therapeutic reductive redox shift might be used to minimize aging and treat AD

Oncolytic Viruses (OVs) are often attenuated to increase their safety profile, however this can lead to reduced efficacy in heterogeneous malignancies and result in resistance to OV therapy. Our group utilizes small molecule enhancers of OV therapy termed viral sensitizers. These small molecules have been shown to enhance the replication and spread of oncolytic rhabdovirus VSVΔ51 in vitro and prolong survival in tumour-bearing mice. In this study, we evaluate the ef-fect of these viral sensitizers on the innate antiviral response in order to identify the mechanism of action responsible for their viral-sensitizing properties. Our previous data suggest that VSe1 and its structural analogues affect the type I IFN antiviral response and have the potential to af-fect cellular redox homeostasis. We hypothesized that VSe1 and its structural analogues potenti-ate VSV∆51 activity by inhibiting the type I IFN response via redox-mediated dysregulation. In this study, we demonstrate that the viral sensitizers inhibit the nuclear translocation and transcrip-tional activity of NFκB, which in turn dampens the expression of antiviral cytokines IFN-, TNFα and IL-6. We also provide evidence supporting the possibility that the NFκB inhibition may be a result of the formation of ROS intermediates by the viral sensitizers, which leads to re-duced nuclear translocation of NFκB subunits, thereby preventing NFκB-mediated cytokine production. Overall, this work contributes to the identification of the mechanism of action of our viral sensitizers and highlights the finding that oncolytic VSV infection can be enhanced through redox-mediated modulation of the innate antiviral response.

Parkinson’s disease (PD) is the most common movement neurodegenerative disease affecting approximately 1% of the population over 60. Though originally thought to be sporadic in nature, a genetic component is increasingly being linked to the disease. Of these genes, mutations in DJ-1 (PARK7) cause early onset autosomal recessive PD. Initial workup of the DJ-1 protein has suggested that it may act in the cell by combatting oxidative stress though the mechanism by which it does so is unclear. Thus, though much work has attempted to elucidate a function at the biochemical, cellular and organismal level, the overt physiological role of DJ-1 remains elusive. In this dissertation, we explore the mechanisms through which DJ-1 confers neuroprotection, particularly in the case of oxidative stress insult. We demonstrate that DJ-1 acts through the pro-survival protein AKT to accomplish its neuroprotective function. Moreover, we note that DJ-1 likely serves its role as an antioxidant through the NRF2 master antioxidant regulator pathway a pathway that is, itself, likely to be regulated by AKT. Together, our results demonstrate that neuroprotection by DJ-1 is done through a signaling pathway involving both AKT and NRF2 and that disruption of the former in PD likely results in abolishing this signaling pathway. Finally, to generate a better animal model of PD, we demonstrate that backcrossing DJ-1 null mice - which originally did not demonstrate any gross histopathological or behavioral phenotypes – display unilateral dopaminergic degeneration that progresses to bilateral degeneration with aging, a feature reminiscent of classical PD progression. Collectively, this thesis takes a two-sided approach to address the biochemical and physiological functions of DJ-1 within the cell and the mouse in hopes of elucidating mechanisms of neuronal death to devise better translational therapies.

The introduction of autonomous vehicles has the potential to reduce the number of accidents and save countless lives. These benefits can only be realized if autonomous vehicles can prove to be safer than human drivers. There is a large amount of active research around developing robust algorithms for all parts of the autonomous vehicle stack including sensing, localization, mapping, perception, prediction, planning, and control. Additionally, some of these research projects have involved the use of the Robot Operating System (ROS). However, another key aspect of realizing an autonomous vehicle is a fault-tolerant design that can ensure the safe operation of the vehicle under unfavorable conditions. The goal of this thesis is to evaluate the feasibility of adding a dedicated fault tolerance module into a ROS based architecture. The fault tolerance module is used to implement a safety controller that can take over safety-critical operations of the system when a fault is detected in the main computer. A Xilinx Zynq-7000 SoC with a dual-core ARM Cortex-A9 and an FPGA programmable logic region is chosen as the platform. The platform works in the Asymmetric Multiprocessing (AMP) configuration with a Linux based operating system on one core and a real-time operating system (RTOS) on the other. Results are gathered from an implementation done on a ROS based mobile robot platform.

Dexrazoxane (DEX) is clinically used to reduce cardiotoxic efects of anthracycline cytostatics. Its cardioprotective efect is caused by chelatation of free iron and defends myocard against dangerous hydroxyl radicals. This research finds out how dexrazoxane works in ischemic-reperfusion damages of rat's heart. Each rat was infused by DEX (50, 150, 450 mg/kg) or by control solution. Isolated perfused rat's hearts were exposed to local ischemia for 30 minutes than 10 minutes of reperfusion for studing ischemic arrhythmias followed by 15 minutes of local ischemia and 10 minutes of reperfusion to examine reperfusion arrhythmias. For evaluation of EKG (ventricular arrhythmias) was used software CAR and Lambeth convention. Global ischemias (15 min.) were induced in rat's hearts (DEX 150 mg/kg) and left ventricules were used for HPLC to determinate concentration of glutathion. In vivo experiments rats were infused by DEX 50, 150 mg/kg or control solution and were exposed for 20 minutes to local ischemia and for 3 hours to reperfusion. Infarct size was evaluated based on the cross section of heart (GIMP, Ellipse). Maximum total number of ischemic arrhytmias decreased by DEX 150 mg/kg (64% comparing to controls). Reperfusion score was reduced by DEX 150 to 48% and percents of ventricular fibrilation was...

Cardiac stem cells (CSCs)-based therapy provides a promising avenue for the management of ischemic heart diseases. However, engrafted CSCs are subjected to acute cell apoptosis in the ischemic microenvironment. Here, stem cell antigen 1 positive (Sca-1+) CSCs proved to own therapy potential were cultured and treated with H2O2 to mimic the ischemia situation. As autophagy inhibitor, 3-methyladenine (3MA), inhibited H2O2-induced CSCs apoptosis, thus we demonstrated that H2O2 induced autophagy-dependent apoptosis in CSCs, and continued to find key proteins responsible for the crosstalk between autophagy and apoptosis. Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), increased upon cardiomyocyte injury with unknown functions in CSCs, was increased by H2O2. NR4A2 siRNA attenuated H2O2 induced autophagy and apoptosis in CSCs, which suggested an important role of NR4A2 in CSCs survival in ischemia conditions. Reactive oxygen species (ROS) and NF- κB (P65) subunit were both increased by H2O2. Either the ROS scavenger, N-acetyl-lcysteine (NAC) or NF-κB signaling inhibitor, bay11-7082 could attenuate H2O2-induced autophagy and apoptosis in CSCs, which suggested they were involved in this process. Furthermore, NAC inhibited NF-κB activities, while bay11-7082 inhibited NR4A2 expression, which revealed a ROS/NF-κB/NR4A2 pathway responsible for H2O2- induced autophagy and apoptosis in CSCs. Our study supports a new clue enhancing the survival rate of CSCs in the infarcted myocardium for cell therapy in ischemic cardiomyopathy.

Biotic stresses such as infection by bacteria negatively affect plant growth and pose a severe threat to human food production. Improving our understanding of the immune systems of plants should help ensure food supplies in the years ahead. Bacterial infections induce Pattern-Triggered Immunity (PTI), a process in which plants perceive bacterial molecules and trigger an immune response. Mitogen- Activated Protein Kinase (MAPK) cascades are key players in this immunity process. Since the MAP Kinases (MPKs) 3/4/6 are mainly responsible for flg22- dependent phosphorylation events, we sought to find out how oxidation of MPK4 affects its ability to respond to stresses. Previous studies have shown varying kinase activity of MPK4 upon oxidation. Therefore, this project aims to provide an insight into the oxidative defense signaling mechanism of A. thaliana by investigating the role of MPK4 Cysteine181 in vitro and in vivo. Analysis of oxidation-mimicking as well as oxidation-dead mutants gave first hints that Cysteine181, which is located in the MPK4 substrate binding pocket, is a highly important regulatory residue of oxidative stress signaling by affecting MPK4 kinase activity and the activation of MPK3 and MPK6. Binding studies revealed that those events are due to sterical hindrance within the binding pocket of MPK4 and the blockage of upstream activator binding. The second part of this study characterizes compositional and post-translational changes of plant ribosomes during pathogen infection. Ribosomal proteins selectively participate in the formation of polysomes under different environmental and developmental conditions. However, the function of these changes still remains elusive. The current research project attempts to understand the plant ribosomal changes that occur upon exposure to bacterial pathogens. To observe ribosomal changes, A. thaliana plants were treated with a pathogen associated molecular pattern (PAMP), flg22. Mass spectrometric analysis identified quantitative changes of PAMP-induced ribosomal proteins in polysomes as well as changes in post-translational modifications. Spatial simulations of ribosomes revealed specific regions within the ribosomes to be PTI specific. This study demonstrates that MPK6 contributes to modification of P-stalk composition and phosphorylation status. The MPK6 mediated modifications may affect translation and in combination indicate a mechanism of PTI-related translational control.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19764号 / 農博第2160号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4980(農学部図書室) / 32800 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 阪井 康能, 教授 植田 和光, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20310号 / 薬科博第79号 / 新制||薬科||9(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 金子 周司, 教授 竹島 浩, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM