Excess excitation energy, mechanical injury and defence against pathogens, each trigger rapid production of reactive oxygen species (ROS) in Arabidopsis thaliana leaves. ROS, such as hydrogen peroxide (H2O2), are required for the induction of systemic acquired acclimation and may lead to redox changes in photosynthetic electron transport (PET). On one hand, enhanced ROS production during stress can destroy cells, and on the other, ROS can also act as signals for the activation of stress responsive and defensive pathways. In this work, physiological and molecular analyses of Arabidopsis mutants and transgenic lines were applied to investigate the signalling network controlling biotic and abiotic stress responses. A key enzyme of the antioxidant network is encoded by ASCORBATE PEROXIDASE 2 (APX2). Wounded leaves showed low induction of APX2 expression and when exposed to excess light, APX2 expression was increased synergistically. Signalling pathways dependent upon jasmonic acid, chitosan and abscisic acid were not involved in the wound-induced expression of APX2, but PET was required, and APX2 induction was preceded by a depressed rate of CO2 fixation. Analysis of lsd1 (LESION SIMULATING DISEASE 1) strongly suggests that light acclimatory processes and pathogen defences are genetically and functionally linked. It is important to know that LSD1 type of mutants have mainly been studied with regard to pathogenesis. From this work, it reveals that association of LSD1 with hypersensitive response may only be supplementary. GLUTATHIONE PEROXIDASES (GPXs) are another major family of ROS scavenging enzymes. Analysis of the Arabidopsis genome database revealed a new open-reading frame, thus increasing the total number of AtGPX gene family to eight (AtGPX1-AtGPX8). Arabidopsis thaliana transgenic lines with reduced expression of both putative chloroplastic isoforms (AtGPX1 and AtGPX7) and AtGPX7 knock-out mutant (ko-GPX7) were more sensitive to photo-oxidative stress but had a reduced bacterial growth rate when inoculated with virulent strains Pseudomonas syringae pv. tomato DC3000 and P.s.t. maculicola strain ES4326, indicating increased resistance to pathogenesis. This, to our knowledge, is the first functional and genetic analysis of chloroplastic GPXs in plants, and confirms that light and chloroplastic ROS metabolism is important for basal resistance against virulent pathogens. The above results confirm that light sensing, light acclimatory processes and photo-produced ROS also govern pathogen defence pathways. This has a great ecological relevance for Darwinian fitness of plants growing in the natural environment, where simultaneous pathogen attack and fluctuations in light, temperature and other environmental factors make rapid acclimation a constant necessity. Molecular, biochemical and physiological analysis of pathogen responses in mutants impaired in light sensing, EEE-dissipatory mechanisms, and similar analysis of light acclimatory processes in mutants impaired in pathogen defences may prove to be seminal.

Oxidative stress is a natural consequence of photosynthetic oxygen evolution and redox enzyme processes. Trp oxidation to N-formylkynurenine (NFK) is a specific, reactive oxygen species (ROS)-mediated reaction. This thesis work describes the identification and functional characterization of NFK in oxygen evolving Photosystem II (PSII). Although proteomics studies have confirmed NFK modifications in many types of proteins, limited knowledge on the biochemical significance exists. In vitro studies in thylakoids and PSII membranes were used to establish a correlation between oxidative stress, NFK formation, and photoinhibition. The in vivo effect of preventing Trp oxidation to NFK was assessed by site-directed mutation in the cyanobacteria Synechocystis sp. PCC 6803. This work provides insight into the role of NFK in photosynthetic oxygen evolution and photoinhibition. Based on the current knowledge of NFK, ROS, and repair, a new model is described. In this modified model for photoinhibition and repair, NFK plays a role in signaling for turnover of damaged proteins. NFK may play a similar role in replacement of damaged proteins in other systems.

Listeria monocytogenes is an intracellular bacterium that utilizes two phospholipases C (PLCs) and a pore-forming cytolysin (listeriolysin O, LLO) to escape the phagosome. However, prior to escape, the bacterium must overcome a number of phagosomal defenses, including autophagy and NOX2 NADPH oxidase production of reactive oxygen species (ROS). Autophagy, the cellular process of self-digestion, is a key component of innate immunity. Previously, it has been shown that L. monocytogenes is targeted by autophagy (LC3+) at 1 h post infection (p.i.) but the mechanism remains elusive. Here, I show that at 1 h p.i., diacylglycerol (DAG) and ROS production are required for autophagy targeting to the bacteria, which are predominantly in phagosomes. It has been shown that autophagy targeting of cytosolic L. monocytogenes is mediated via protein ubiquitination. However, protein ubiquitination is not associated with LC3+ bacteria at 1 h p.i.. Thus, my data suggest that distinct signals mediate autophagy targeting of L. monocytogenes depending on the location within host cells. Given that ROS mediate autophagy targeting to L. monocytogenes and that previous studies have demonstrated that ROS production limits bacterial escape, I investigated how L. monocytogenes overcomes ROS production prior to phagosomal escape. I found that LLO inhibits ROS production by preventing NOX2 NADPH oxidase localization to L. monocytogenes-containing phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit ROS production. While PLCs can activate ROS production, this effect is alleviated by LLO pore-formation. Therefore, the combined activities of PLCs and LLO allow L. monocytogenes to efficiently escape the phagosome while avoiding microbicidal ROS. Together, this thesis provides a clearer understanding of the balance between host defense versus bacterial evasion. Greater insight into host-bacterial interaction may lead to better therapeutics that can “tip the balance” in the host’s favour.

Uncoupling protein-2 (UCP2) may be linked to the attenuation of reactive oxygen species (ROS), but it is unclear whether this phenomenon pertains to the pancreatic beta cell. In this study, a UCP2-deficient mouse model was used to assess the importance of UCP2 to beta cell viability. We investigated the effect of UCP2 absence in response to a beta cell cytotoxic model of diabetes induction. In vivo treatment by the cytotoxic agent streptozotocin led to overall beta cell loss, but severity was not exacerbated by UCP2 deficiency. We also examined ROS production and cell viability in islet cells exposed to various stressors associated with oxidative stress. In vitro measurements of ROS and cell death in islet cells demonstrated that the response was not influenced by UCP2 expression. In contrast with UCP2 overexpression studies showing cytoprotection, this study reveals that beta cell survival is not compromised by the absence of UCP2.

α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs), where it has been shown to have potent anti-apoptotic properties. The mechanism by which cryAB prevents apoptosis has not been fully characterized. Therefore, I was interested in elucidating its protective mechanism in CMs. I identified its sub-cellular localization and its binding interactors following H2O2 exposure. I found that cryAB is found in the cytosol under control conditions and that following H2O2 exposure it becomes phosphorylated and translocates to the mitochondria. CryAB silencing resulted in increased apoptosis levels in CMs. Co-immunoprecipitation revealed an apparent increased interaction of cryAB and PcryAB with mitochondrial VDAC, caspase 12 and uncleaved caspase 3 in stressed hearts relative to controls. These results suggest that the cardio-protective effects of cryAB are mediated by its translocation to the mitochondria and its interaction with VDAC, caspase 12 and caspase 3 following exposure to H2O2.

α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs), where it has been shown to have potent anti-apoptotic properties. The mechanism by which cryAB prevents apoptosis has not been fully characterized. Therefore, I was interested in elucidating its protective mechanism in CMs. I identified its sub-cellular localization and its binding interactors following H2O2 exposure. I found that cryAB is found in the cytosol under control conditions and that following H2O2 exposure it becomes phosphorylated and translocates to the mitochondria. CryAB silencing resulted in increased apoptosis levels in CMs. Co-immunoprecipitation revealed an apparent increased interaction of cryAB and PcryAB with mitochondrial VDAC, caspase 12 and uncleaved caspase 3 in stressed hearts relative to controls. These results suggest that the cardio-protective effects of cryAB are mediated by its translocation to the mitochondria and its interaction with VDAC, caspase 12 and caspase 3 following exposure to H2O2.

Die Restenose stellt ein zentrales Problem der interventionellen Kardiologie dar und ist häufigste Komplikation nach perkutanen Angioplastieverfahren. Hauptursache dieser Wiederverengung des Gefäßes ist die Bildung einer Neointima durch die Proliferation transdifferenzierter vaskulärer glatter Muskelzellen und die Sekretion extrazellulärer Matrix. Die Entstehung reaktiver Sauerstoffspezies (ROS) und die Entzündungsreaktion nach der Gefäßverletzung werden als frühe, die Neointimabildung induzierende Prozesse diskutiert. Im Rahmen dieser Arbeit wurden mehrere Projekte bearbeitet, die Aufschluss über die während der Neointimabildung statt findenden Prozesse geben sollen. Mit Hilfe eines Verletzungsmodells der murinen Femoralarterie wurde der Einfluss der Entzündung und der ROS-Bildung auf die Neointimabildung in der Maus untersucht. Die Behandlung mit dem mitochondrialen Superoxiddismutase-Mimetikum MitoTEMPO verminderte die Bildung der Neointima besser, als die Behandlung mit dem globalen ROS-Fänger N-Acetylcystein. Die stärkste Hemmung der Neointimabildung wurde jedoch durch die Immunsuppression mit Rapamycin erreicht. Interferon-γ (INFγ) ist ein wichtiges Zytokin der Th1-Immunantwort, das in Folge der Gefäßverletzung freigesetzt wird und die proinflammatorischen Chemokine CXCL9 (MIG, Monokine Induced by INF), CXCL10 (IP-10, INF inducible Protein of 10 kDa) und CXCL11 (I-TAC, Interferon inducible T cell-Chemoattractant) induziert. CXCL9, CXCL10 und CXCL11 sind Liganden des CXC-Chemokinrezeptors 3 (CXCR3) und locken chemotaktisch CXCR3 positive Entzündungszellen zum Ort der Gefäßverletzung. Daher wurde die spezielle Bedeutung des Chemokins CXCL10 in der Restenose untersucht. Dazu wurden CXCL10-defiziente Mäuse dem Femoralisverletzungsmodell unterzogen und die Gefäße nach 14 Tagen morphometrisch und immunhistologisch untersucht. CXCL10-Defizienz führte in Mäusen zu einer verminderten Neointimabildung, die mit einer verringerten Inflammation, Apoptose und Proliferation im verletzten Gefäß korrelierte. Neben der Inflammation beeinflusst aber auch die Reendothelialisierung der verletzten Gefäßwand die Restenose. Interessanterweise war im Vergleich zu Wildtyp-Mäusen in den CXCL10-Knockout-Mäusen auch die Reendothelialisierung erheblich verbessert. Offensichtlich ist das CXCR3-Chemokinsystem also in völlig unterschiedliche biologische Prozesse involviert und beeinflusst nicht nur die Bildung der Neoimtima durch die Förderung der Entzündung, sondern auch die Unterdrückung der Reendothelialisierung der verletzten Gefäßwand. Tatsächlich wird der CXCR3 nicht nur auf Entzündungszellen, sondern auch auf Endothelzellen exprimiert. Zur separaten Untersuchung der Rolle des CXCR3 in der Inflammation und der Reendothelialisierung wurde im Rahmen dieser Arbeit damit begonnen konditionelle CXCR3-Knockout-Mäuse zu generieren, in denen der CXCR3 entweder in Entzündungszellen oder in Endothelzellen ausgeschaltet ist. Zum besseren Verständnis der molekularen Mechanismen, mit denen der CXCR3 seine Funktionen vermittelt, wurde zudem untersucht ob dieser mit anderen G-Protein-gekoppelten Rezeptoren (GPCR) interagiert. Die Analyse von Coimmunpräzipitaten deutet auf eine Homodimerisierung der beiden CXCR3 Splicevarianten CXCR3A und CXCR3B, sowie auf die Heterodimerbildung von CXCR3A und CXCR3B mit sich, sowie jeweils mit CCR2, CCR3, CCR5 und den Opioidrezeptoren MOR und KOR hin. Die getestete Methode des Fluoreszenz-Resonanz-Energietransfers (FRET) erwies sich jedoch als ungeeignet zur Untersuchung von CXCR3, da dieser in HEK293T-Zellen nicht korrekt transient exprimiert wurde. Insgesamt deuten die Ergebnisse dieser Arbeit darauf hin, dass das CXCR3-Chemokinsystem eine zentrale Rolle in unterschiedlichen, die Neointimabildung beeinflussenden Prozessen spielt. Damit könnten der CXCR3 und insbesondere das Chemokin CXCL10 interessante Zielmoleküle in der Entwicklung neuer verbesserter Therapien zur Verhinderung der Restenose darstellen. / Restenosis represents a central problem after coronary angioplasty procedures and is caused by intimal hyperplasia, also called neointima, as a result of transdifferentiation, proliferation of vascular smooth muscle cells and secretion of extracellular matrix. Formation of reactive oxygen species (ROS) and inflammation after vascular injury caused by angioplasty are discussed as early inducers of neointima formation. In several projects the processes causing the development of intimal hyperplasia were investigated. First of all, the impact of inflammation and ROS in neointima formation was investigated using the mouse femoral injury model. The mitochondrial superoxide dismutase mimetic mitoTEMPO could reduce neointima formation better than the global ROS scavenger N-acetylcystein. However, the strongest reduction of neointima formation was achieved by the treatment with the immunosuppressant rapamycin. Interferon-γ(INFγ) is a major cytokine of the Th1 immune response. It is released as a result of vessel injury and induces the proinflammatory chemokines CXCL9 (MIG, Monokine Induced by INF), CXCL10 (IP-10, INF inducible Protein of 10 kDa) and CXCL11 (I-TAC, Interferon inducible T-cell-Chemoattractant), which are ligands of the CXC chemokine receptor 3 (CXCR3) and by this chemotactically recruit CXCR3 positive cells to the site of vessel injury. In this work the special role of CXCL10 in restenosis was investigated. Therefore, CXCL10 decient mice underwent the mouse femoral injury model. The vessels were analysed morphometrically and immunohistologically 14 days after injury. CXCL10 deciency lead to decreased neointima formation that correlated with a reduced recruitment of inflammatory cells as well as diminished numbers of apoptotic and proliferating cells at the site of vessel injury. In addition to inflammation the reconstitution of the endothelium has also impact on the development of restenosis. Interestingly reendothelialisation was strongly improved in CXCL10 decient mice compared to wildtype mice. Obviously the CXCR3 chemokine system is involved in different biological prosesses and impairs neointima formation on one hand by the advancement of inflammation and on the other hand by the suppression of reendothelialisation. In fact the CXCR3 is not only expressed on inflammatory cells but also on endothelial cells. To investigate the role of CXCR3 in inflammation and reendothelialisation separatly the generation of conditional CXCR3 knockout mice with a CXCR3 knockout in T-cells or endothelial cells was started in an additional project. For a better understanding of the molecular mechanisms on which the CXCR3 mediates its biological functions the protein-protein interactions of the CXCR3 with other G-protein coupled recteptors (GPCR) was analysed. Coimmunoprecipitation showed homodimerization of the CXCR3 splice variants CXCR3A and CXCR3B, as well as heterodimerization of CXCR3A and CXCR3B with each other and with the chemokine receptors CXCR4, CCR2, CCR3, CCR5 and the opioid receptors MOR and KOR. The additional tested Fluorecence resonance energy transfer (FRET) method proved to be not suitable to measure interactions of CXCR3, since this receptor could not be expressed correctly on the cell surface after transient transfection. To summarise, the results indicate that the CXCR3 chemokine system plays a central role in different processes that mediate neointima formation. Thus, the CXCR3 and especially the chemokine CXCL10 could be interesting therapeutic targets in the development of new or improved treatments to reduce the risk of restenosis.

Listeria monocytogenes is an intracellular bacterium that utilizes two phospholipases C (PLCs) and a pore-forming cytolysin (listeriolysin O, LLO) to escape the phagosome. However, prior to escape, the bacterium must overcome a number of phagosomal defenses, including autophagy and NOX2 NADPH oxidase production of reactive oxygen species (ROS). Autophagy, the cellular process of self-digestion, is a key component of innate immunity. Previously, it has been shown that L. monocytogenes is targeted by autophagy (LC3+) at 1 h post infection (p.i.) but the mechanism remains elusive. Here, I show that at 1 h p.i., diacylglycerol (DAG) and ROS production are required for autophagy targeting to the bacteria, which are predominantly in phagosomes. It has been shown that autophagy targeting of cytosolic L. monocytogenes is mediated via protein ubiquitination. However, protein ubiquitination is not associated with LC3+ bacteria at 1 h p.i.. Thus, my data suggest that distinct signals mediate autophagy targeting of L. monocytogenes depending on the location within host cells. Given that ROS mediate autophagy targeting to L. monocytogenes and that previous studies have demonstrated that ROS production limits bacterial escape, I investigated how L. monocytogenes overcomes ROS production prior to phagosomal escape. I found that LLO inhibits ROS production by preventing NOX2 NADPH oxidase localization to L. monocytogenes-containing phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit ROS production. While PLCs can activate ROS production, this effect is alleviated by LLO pore-formation. Therefore, the combined activities of PLCs and LLO allow L. monocytogenes to efficiently escape the phagosome while avoiding microbicidal ROS. Together, this thesis provides a clearer understanding of the balance between host defense versus bacterial evasion. Greater insight into host-bacterial interaction may lead to better therapeutics that can “tip the balance” in the host’s favour.

Uncoupling protein-2 (UCP2) may be linked to the attenuation of reactive oxygen species (ROS), but it is unclear whether this phenomenon pertains to the pancreatic beta cell. In this study, a UCP2-deficient mouse model was used to assess the importance of UCP2 to beta cell viability. We investigated the effect of UCP2 absence in response to a beta cell cytotoxic model of diabetes induction. In vivo treatment by the cytotoxic agent streptozotocin led to overall beta cell loss, but severity was not exacerbated by UCP2 deficiency. We also examined ROS production and cell viability in islet cells exposed to various stressors associated with oxidative stress. In vitro measurements of ROS and cell death in islet cells demonstrated that the response was not influenced by UCP2 expression. In contrast with UCP2 overexpression studies showing cytoprotection, this study reveals that beta cell survival is not compromised by the absence of UCP2.

Photodynamic therapy (PDT), which utilizes the combination of light and a photosensitizing drug to cause tissue damages, has emerged as a novel clinical approach for the treatment of numerous cancers, as well as some other non-malignant conditions. Although a few photosensitizers have been approved for clinical uses, the mechanism of drug action, especially the initial photochemical reactions that lead to the formation of the reactive oxygen species (ROS), is still not well understood. Moreover, the PDT efficiency of currently used drugs is limited due to the strong attenuation of light by tissues in the wavelength range of 630-690 nm, where these drugs are photo-activated. Photosensitizers which are sensitive to near infrared (NIR) light are believed to be able to overcome this limitation. In this thesis work, the molecular mechanism of action of indocyanine green (ICG), a potential NIR PDT drug, was investigated using our femtosecond time-resolved laser spectroscopy. Femtosecond time-resolved fluorescence decay profiles of ICG in water were obtained using the fluorescence up-conversion technique. The lifetime of ICG excited singlet state was determined to be about 150 ps, directly from the fluorescence decay kinetic traces. The excited triplet-state yield of ICG in water was found to be extremely low, according to the result of the ground-state bleaching recovery measurement. This observation is contrary to the conventional understanding that the ROS would be generated mainly from the excited triplet state of the photosensitizer and, therefore, suggests the existence of a new reaction pathway. Pump-probe transient absorption spectroscopy was applied to study the reaction between ICG and oxygen in more details. The results reveal that the formation of ICG and oxygen ground-state complexes ([ICG]_m:[O₂]_n) is a key step in the generation of the ROS. Electron transfer from the excited singlet state of ICG to oxygen has been proposed to be a possible pathway for the generation of ROS.