La biologie des réactions redox est particulièrement difficile à étudier de par la compartimentation spatiale mais aussi cinétique des différents systèmes redox cellulaires. Les biosenseurs codés génétiquement, incluant la «redox-sensitive Yellow Fluorescent Protein» (rxYFP) sont une manière de contourner les limitations des méthodes conventionnelles de mesure du couple glutathion/glutathion disulfure (GSH/GSSG). Cette étude présente l’utilisation des biosenseurs rxYFP pour analyser les états redox dans des différents compartiments cellulaires, et leur dynamique en réponse au stress dans les cellules humaines. La rxYFP exprimée soit dans le cytosol, le noyau ou la matrice mitochondriale de cellules HeLa s’est révélée sensible aux changements de l’état redox intracellulaire provoqué par des traitements aussi bien réducteurs qu’oxydants. La rxYFP est capable de détecter des différences de l’état redox, entre les compartiments, mais aussi entre différentes lignées cellulaires. Les senseurs exprimés dans des kératinocytes humain de l’épiderme HEK001 ont réagi au stress induit par les UVA, de façon dose-dépendante, mais pas au stress induit par les UVB. De plus, ces senseurs ont pu détecter les changements redox induits par des faibles doses (30 µM) ainsi que par des doses modérées (100 µM) de peroxyde d’hydrogène (H2O2), de façon dynamique et spécifique des compartiments cellulaires. La rxYFP exprimé dans la matrice mitochondriale a montré une vitesse d’oxydation plus élevée que les senseurs rxYFP exprimés dans le cytosol ou le noyau, ce qui est attribuable à un pH local plus basique. De plus, la déplétion en GSH provoquée par un traitement au buthionine sulphoximine (BSO) a affecté spécifiquement le potentiel redox mitochondrial mais pas cytosolique ni nucléaire. Ces observations soutiennent l’idée que l’état redox du GSH mitochondrial est maintenu et régulé de façon indépendante par rapport à celui du cytosol ou du noyau. Nous avons également montré que dans les cellules humaines, les sondes rxYFP réagissent de façon prédominante avec le GSH/GSSG, puisque la déplétion en GSH ralentit la vitesse d’oxydation de la rxYFP en réponse à un traitement par H2O2. De plus, grâce à l’utilisation des sondes rxYFP et à l’analyse de l’état redox des antioxydants cellulaires, nous démontrons que l’oxydation des thiols se produit après l’activation des caspases au cours de l’apoptose induite par TRAIL. L’ensemble de nos données montrent la robustesse des senseurs rxYFP pour la mesure des changements d’état redox dans les cellules humaines. En complément d’autres senseurs redox ainsi que des méthodes conventionnelles de mesure des états redox, les senseurs rxYFP ciblés aux différents compartiments cellulaires sont un nouvel outil pour étudier l’homéostasie redox dans les cellules de mammifères, et permettent l’étude de l’état redox du glutathion et de la dynamique des changements redox avec une grande précision. / The kinetic and spatial separation of redox systems renders redox biology studies a particularly challenging field. Genetically encoded biosensors including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP) may provide an alternative way to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. This study describes the use of rxYFP sensors for investigating compartment-specific steady redox states and their dynamics in response to stress in human cells. RxYFP expressed either in the cytosol, nucleus or mitochondrial matrix of HeLa cells was responsive to the intracellular redox state changes induced by reducing as well as oxidizing agents. Compartment-targeted rxYFP sensors were able to detect different steady state redox conditions between the cytosol, nucleus and mitochondrial matrix as well as between the cell lines. These sensors expressed in human epidermal keratinocytes HEK001 responded to stress induced by UVA radiation in a dose-dependent manner but not to UVB radiation. Furthermore, rxYFP sensors were able to sense dynamic and compartment-specific redox changes caused by low dose (30 µM) and moderate dose (100 M) hydrogen peroxide (H2O2). Mitochondrial matrix-targeted rxYFP displayed a greater dynamics of oxidation in response to a H2O2 challenge than the cytosol- and nucleus-targeted sensors, largely due to a more alkaline local pH environment. Similarly, the depletion of glutathione induced by buthionine sulphoximine (BSO) affected selectively mitochondrial redox potential without inducing changes in cytosol and nucleus. Furthermore, using rxYFP probes and cellular antioxidants redox state analysis, we show that oxidation of thiols occurs after activation of caspases during TRAIL-induced apoptosis. These observations support the view that mitochondrial glutathione redox state is maintained and regulated independently from that of the cytosol and nucleus. We also showed that in human cells the rxYFP probes react predominantly with glutathione since the glutathione depletion slows down the dynamics of rxYFP oxidation in response to H2O2. Taken together, our data show the robustness of the rxYFP sensors to measure compartmental redox changes in human cells. Complementary to existing redox sensors and conventional redox measurements, compartment-targeted rxYFP sensors provide a novel tool for examining mammalian cell redox homeostasis, permitting high resolution readout of steady glutathione state and dynamics of redox changes.

Redox reactions are an essential part of the cell’s metabolism, differentiation, and responses to the prevailing environmental conditions. In plants, dramatic changes in cellular redox status are observed upon exposure to environmental stresses, including pathogen attack. These changes affect the oxidative status of reactive cysteine thiols in regulatory proteins. To control oxidative protein modifications, plant cells employ the antioxidant enzymes S-nitrosoglutathione Reductase 1 (GSNOR1) and members of the Thioredoxin (TRX) superfamily. Immune signalling by the hormone salicylic acid (SA) is particularly dependent on the activity of these enzymes. SA is synthesized in response to challenge by plant pathogens for the establishment of local and systemic immunity. SA accumulation is regulated by cellular levels of S-nitrosoglutathione (GSNO), a redox molecule capable of S-nitrosylating proteins (i.e., covalent attachment of nitric oxide to cysteines). GSNOR1 is thought to regulate cellular GSNO and global S-nitrosylation levels, but it is unknown how GSNOR1 regulates SA biosynthesis. Furthermore, SA recruits the activities of selected TRX enzymes that act as ubiquitous thiol reductases to counteract cysteine oxidation of SA-responsive regulatory proteins, thereby modulating their activities. However, it is unclear how SA controls nuclear redox processes involved in SAresponsive gene activation. Here we show that GSNOR1 regulates SA accumulation by regulating the expression of SA biosynthetic genes and their transcriptional activators. Moreover, we describe Nucleoredoxins (NRX) that represent novel, potentially nuclear localized members of the TRX superfamily. Mutant nrx1 plants displayed enhanced disease resistance, which was associated with enhanced expression of genes involved in synthesis of salicylic acid. Unlike classical TRX, NRX enzymes contain multiple active sites, suggesting they may exhibit significant reductase or remodelling activities. Indeed, insulin turbidity assays indicated that NRX proteins show an unusual form of disulphide reduction activity. Taken together, the data presented in this thesis demonstrate that GSNOR1 and NRX enzymes play critical roles in regulating synthesis of and signalling by SA in plant immunity.

This thesis presents the synthesis, characterization and reactivity of a series of palladium complexes that contain redox-active verdazyl ligands. This work was motivated by the possibility of discovering new and interesting reactivity that may eventually lead to the development of new chemical reactions. A bidentate verdazyl radical ligand that contains an aryl phosphine was synthesized. Reaction of this ligand with (PhCN)2PdCl2 yielded a square planar (verdazyl)PdCl2 complex. Structural and spectroscopic data suggest that this compound consists of a ligand-centered radical coordinated to a Pd(II) center. The radical complex was chemically reduced by one-electron to generate a binuclear chloride-bridged [(verdazyl)PdCl]2 complex. In this reduced complex, both metals were still Pd(II) and the verdazyl ligand was determined to be in its singly reduced, monoanionic charge state. The original radical PdCl2 complex could be regenerated via one-electron oxidation of the reduced complex using PhICl2. The verdazyl ligands in the reduced complex could also be reversibly protonated to generate “leuco” verdazyl complex (verdazyl-H)PdCl2. Reaction of the radical (verdazyl)PdCl2 complex with water triggers a ligand-centered redox disproportionation reaction. A series of bis(verdazyl) palladium complexes were synthesized using a bidentate pyridine-substituted verdazyl ligand. Reaction of two equivalents of radical ligand with (CH3CN)4Pd2+ yielded a (verdazyl)2Pd(solvent)2+ complex (solvent = CH3CN or DMSO). In this complex, one verdazyl radical ligand chelates to palladium and the other binds as a monodentate ligand. Two-electron reduction of this complex generated a (verdazyl)2Pd complex in which two monoanionic verdazyl ligands are bound to a central Pd(II) ion. This reduced complex could also be made via reaction of 0.5 equivalents of Pd(0)2(dba)3 with two equivalents of radical ligand. In this reaction, the metal is oxidized by two electrons and each ligand is reduced by a single electron. Two-electron oxidation of the reduced complex in the presence of DMSO yielded the original bis(radical)complex, (verdazyl)2Pd(DMSO)2+. Chlorination of the reduced complex using one equivalent of PhICl2 (two-electron oxidation) resulted in dissociation of one verdazyl ligand to afford a 1:1 mixture of free verdazyl : (verdazyl)PdCl2, in which both of the verdazyls are neutral radicals. Reaction of the reduced complex with 0.5 equivalents of PhICl2 (one-electron oxidation) yielded a (verdazyl)2PdCl complex that contained a bidentate reduced verdazyl ligand and a monodentate radical ligand. All three of the oxidation reactions described above adhere to ligand-centered redox chemistry. Reaction of the reduced (verdazyl)2Pd complex with excess HCl resulted in protonation of both the anionic verdazyl ring and the pyridyl group to generate a leuco/pyridinium tetrachloropalladate salt, (verdazyl-H2)2(PdCl4). The protonated salt could be converted back to the original (verdazyl)2Pd complex by deprotonation with water. Palladium complexes of a tridentate NNN-chelating verdazyl ligand were prepared and their redox chemistry was explored. Reaction of the radical ligand with (CH3CN)4Pd2+ yielded radical complex (verdazyl)Pd(NCCH3)2+. The tridentate ligand was also prepared in its reduced, leuco form (verdazyl-H). Reaction of the leuco verdazyl with (CH3CN)2PdCl2 generated HCl and a (verdazyl)PdCl complex in which the ligand is in its monoanionic charge state. The reduced (verdazyl)PdCl complex was reacted with AgBF4 to afford (verdazyl)Pd(NCCH3)+ via chloride abstraction; the verdazyl remained in its reduced charge state following the reaction. Both reduced complexes (chloro and acetonitrile) were oxidized by a single electron to afford the corresponding radical complexes. These radical complexes could be reduced by a single electron to regenerate the original reduced complexes. Like the previous two projects, all of the redox chemistry was ligand-centered. The reactivity of these complexes with primary amines was also explored. Reaction of radical complex (verdazyl)Pd(NCCH3)2+ with n-butylamine resulted in one-electron reduction of the verdazyl ligand. We were unable to determine the mechanism of the reaction, but the reactivity that was observed demonstrates the potential for verdazyl-palladium complexes to be used in the design of new radical reactions. / Graduate / 2018-07-17

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Intracellular redox potential is a delicately balanced property in cells. It plays an important role in the regulation of cellular processes and dysfunction of the redox state is believed to get involved in initiation of many kinds of diseases. However, lack of suitable techniques for quantitatively monitoring the redox potential in cells with a wide range is a significant challenge. My project aims to develop SERS nanosensors for measuring intracellular redox state quantitatively and applying them to quantify hypoxia, which is generally described by the cell having a reducing environment and defined as a form of “reductive stress”. Four redox active probe molecules have been synthesised and characterised. Their Raman spectra all change as a function of local redox potential. Since these probe molecules can be assembled on gold nanoshells which are able to enhance the Raman Effect significantly, we can calculate the redox potential from simple optical SERS measurements. Transmission Electron Microscopy was used to investigate the cellular delivery of nanosensors. TEM images confirmed that either single nanosensor or small aggregates can be taken up controllably by cells and translocated in the cytoplasm. The introduction of nanosensors was also found not to be toxic to the cells. The nanosensor has been used to monitor the redox potential in resting cells as well as the redox changes when cells responded to pharmacologically induced hypoxia and oxidative stress. These measurements demonstrated that the SERS based nanosensor developed is able to monitor the intracellular redox change in a reversible, noninvasive way and respond to cellular hypoxia quantitatively.

EVALUATION OF REDOX POTENTIAL AS A NOVEL BIOMARKER OF OXIDATIVE STRESS, INFLAMMATORY RESPONSE, AND SHOCK USING NANOPOROUS GOLD ELECTRODES Background: Redox potential is a chemical species’ affinity for electrons. Increased oxidant concentration is associated with disease1,2, yet there is not a way to measure systemic redox status.3 Redox potentiometry uses metal electrodes that do not work in blood because protein molecules adhere on the metal surface, blocking electron exchange. Methods: Nanoporous gold electrodes have large surface areas that allowed electron exchange to continue in blood.4 Redox potential was measured in blood with ascorbic acid, in cardiac bypass patients and pigs undergoing hemorrhagic shock and resuscitation. Results: Blood redox decreased with ascorbic acid addition, both in vitro and in vivo. It was more positive in patients undergoing cardiac surgery compared to healthy volunteers. Conclusions: Preliminary studies were limited, but appear to show correlation to disease processes and medical therapies. More work needs to be done to further examine the relation of redox to disease and treatment.