In the natural environment plants are confronted to a multitude of biotic and abiotic stress factors that must be perceived, transduced, integrated and signaled in order to achieve a successful acclimation that will secure survival and reproduction. Plants have to deal with excess excitation energy (EEE) when the amount of absorbed light energy is exceeding that needed for photosynthetic CO2 assimilation. EEE results in ROS formation and can be enhanced in low light intensities by changes in other environmental factors. The lesions simulating disease resistance (lsd1) mutant of Arabidopsis spontaneously initiates spreading lesions paralleled by ROS production in long day photoperiod and after application of salicylic acid (SA) and SA-analogues that trigger systemic acquired resistance (SAR). Moreover, the mutant fails to limit the boundaries of hypersensitive cell death (HR) after avirulent pathogen infection giving rise to the runaway cell death (rcd) phenotype. This ROS-dependent phenotype pointed towards a putative involvement of the ROS produced during photosynthesis in the initiation and spreading of the lesions. We report here that the rcd has a ROS-concentration dependent phenotype and that the light-triggered rcd is depending on the redox-state of the PQ pool in the chloroplast. Moreover, the lower stomatal conductance and catalase activity in the mutant suggested LSD1 was required for optimal gas exchange and ROS scavenging during EEE. Through this regulation, LSD1 can influence the effectiveness of photorespiration in dissipating EEE. Moreover, low and high SA levels are strictly correlated to lower and higher foliar H2O2 content, respectively. This implies an essential role of SA in regulating the redox homeostasis of the cell and suggests that SA could trigger rcd in lsd1 by inducing H2O2 production. LSD1 has been postulated to be a negative regulator of cell death acting as a ROS rheostat. Above a certain threshold, the pro-death pathway would operate leading to PCD. Our data suggest that LSD1 may be subjected to a turnover, enhanced in an oxidizing milieu and slowed down in a reducing environment that could reflect this ROS rheostat property. Finally, the two protein disulphide isomerase boxes (CGHC) present in the protein and the down regulation of the NADPH thioredoxin reductase (NTR) in the mutant connect the rcd to a putative impairment in the reduction of the cytosolic thioredoxin system. We propose that LSD1 suppresses the cell death processes through its control of the oxidation-reduction state of the TRX pool. An integrated model considers the role of LSD1 in both light acclimatory processes and in restricting pathogen-induced cell death.

Invadolysin (inv) is a member of the M8 class of zinc-metalloproteases and is conserved throughout metazoans. It is essential for development and invadolysin homozygous Drosophila mutants are third instar larval lethal. These larvae exhibit a reduced larval brain size and an absence of imaginal discs. Detailed analysis showed that inv mutants exhibit pleiotropic effects, including defects with chromosome architecture, cell cycle progression, spindle assembly, nuclear envelope dynamics, protein turnover and problems with germ cell migration. These findings indicated that Invadolysin must have a critical role in Drosophila. In order to better understand these roles, I set out to identify genetic interactors of invadolysin. I performed a genetic screen scoring for enhancer/suppressor modification of a ‘rough eye’ phenotype induced by invadolysin overexpression. Screening against the Drosdel ‘deficiency kit’ identified numerous genetic interactors including genes linked to energy regulation, glucose and fatty acid pathways. Immunofluorescence experiments in cultured cells showed that H. sapiens Invadolysin localises to the surface of lipid droplets (LD), and subcellular fractionation confirmed its enrichment to these structures. Lipid droplets are highly dynamic organelles involved not only in energy storage but also in protein sequestration, protein and membrane trafficking, and cell signaling. Drosophila fat bodies are enriched in LDs and therefore important energy stores. In addition, they are nutritional sensors and regulators, which are proposed to be the ortholog of vertebrate liver and adipose tissue. Mutant inv fat bodies appeared smaller and thinner than wild type fat body, and accumulated lower levels of triacylgylcerides. This indicated that the loss of invadolysin might be affecting lipid metabolism and storage, confirming the genetic data. However, it was not clear whether these effects were due to the direct action of Invadolysin. Hence, transgenic fly lines expressing either HA, RFP or FLAG tagged forms of Invadolysin were generated to identify physical interactors of Invadolysin. Subsequent mass spectrometry analysis detected ATP synthase-α, -β and -d as interactors. This result suggested that Invadolysin might play a role in regulating mitochondrial function, which might then be manifest in the fat body as the defects previously observed. Energy levels are known to affect the cell cycle, cell growth, lipid metabolism and inevitably development. Further in vivo and in vitro experiments confirmed this hypothesis. Genetic crosses confirmed the interaction of invadolysin with ATP-synthase subunit-α, whilst staining of mitochondria in mutant third instar larval fat bodies suggested decreased mitochondrial activity. Mutants also showed lower ATP levels and an accumulation of reactive oxygen species, hence indicating the possibility of a dysfunctional electron transport chain. Lipid droplets are known to interact with mitochondria, whilst ATP synthase has been found on lipid droplets by proteomic studies in Drosophila. Therefore, based on these data, we propose that Invadolysin is found, with ATP synthase, on lipid droplets, where Invadolysin (likely acting as a protease) could be aiding the normal processing or assembly of ATP synthase. This interaction is vital for the proper functioning of ATP synthase, and hence mitochondria. In this scenario, cellular ATP needs are not met, energy levels drop which results in an inhibition of fatty acid synthesis, cell and organismal growth defects.

This literature review posits that the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an attractive candidate therapeutic target in the setting of acute myocardial infarction (AMI). This transcription factor binds to antioxidant response elements (ARE) in the promoter region of a battery of genes that collectively encode an array of antioxidant, phase II drug metabolism, metabolically stabilizing, and overall cytoprotective enzymes, facilitating their transcription at basal levels and increasing transcription in response to various cellular stressors. Following a brief background tutorial on normal cardiac myocyte cellular physiology, key events that occur early in ischemia and reperfusion are outlined and integrated. These include ionic and metabolic dysregulation, electron transport chain uncoupling, mitochondrial depolarization, and the generation of reactive oxygen species (ROS). Abrupt changes in response to ischemia prime opening of the mitochondrial permeability transition pore (MPTP) and cardiac myocytes to generate a burst of ROS upon reperfusion–two key events that contribute to the umbrella term ischemia-reperfusion injury (IRI). How ROS damage cells is then outlined, and through a ROS-centric viewpoint, a case will be made as to how exogenous upregulation of Nrf2 could protect and/or salvage at-risk tissue immediately subjected to infarction and neighboring tissue in the peri-infarct zone (PIZ). The history of how Nrf2 came to be known as the "master regulator of oxidative stress" is reviewed, as well as the discovery of the canonical mechanism of Nrf2 regulation via Kelch-like ECH-associated protein 1 (Keap1) and other alternative mechanisms of endogenous Nrf2 regulation. Finally, compiling interdisciplinary evidence from research publications around the world, the benefits of therapeutically targeting Nrf2 are considered given the timescale and context of acute MI. Drug delivery methods, potential challenges, and limitations are then considered. Cardiac tissue is a dynamic substrate that exhibits changes for up to 90 days after AMI and patient outcomes are directly related to the extent of tissue lost following infarction/reperfusion. Targeting Nrf2 addresses an unmet need, as current clinical therapies focus on precluding occlusions and prompt reperfusion of infarcted tissue, but do not explicitly target at-risk tissue following infarcts and/or present-day reperfusion methodologies.

Cyanine dyes that absorb light in the near infrared to far red region of the electromagnetic spectrum are desirable as photosensitizers for photodynamic cancer therapy. Light of wavelengths in this range is able to deeply penetrate tissue allowing for practical in vivo use of these dyes. A series of three structurally similar pentamethine cyanine dyes that absorb light ~800 nm to ~500 nm was tested to determine the effects of structural influences on the yields of supercoiled DNA photo-converted to nicked or linear forms. Possible mechanisms and optimal parameters for near- quantitative DNA photocleavage with a symmetrical quinoline pentamethine cyanine dye are discussed.

Hypoxia-induced pulmonary hypertension (HPH) is a progressive disease that is mainly caused by chronic exposure to high altitude, chronic obstructive lung disease, and obstructive sleep apnea. The increased pulmonary vascular resistance and increased pulmonary arterial pressure result in increased right ventricular afterload, leading to right heart failure and increased morbidity. There are several clinical reports suggesting a link between PH and diabetes, insulin resistance, or obesity; however, it is unclear whether HPH is associated with diabetes as a progressive complication in diabetes. The major goal of this study is to examine the effect of diabetic ''preconditioning'' or priming effect on the progression of HPH and define the molecular mechanisms that explain the link between diabetes and HPH. Our data show that HPH is significantly enhanced in diabetic mice, while endothelium-dependent relaxation in pulmonary arteries is significantly attenuated in chronically hypoxic diabetic mice (DH). In addition, we demonstrate that mouse pulmonary endothelial cells (MPECs) isolated from DH mice exhibit a significant increase in mitochondrial reactive oxygen species (ROS) concentration and decreased SOD2 protein expression. Finally, scavenging mitochondrial ROS by mitoTempol restores endothelium-dependent relaxation in pulmonary arteries that is attenuated in DH mice. These data suggest that excessive mitochondrial ROS production in diabetic MPECs leads to the development of severe HPH in diabetic mice exposed to hypoxia.

Ultraviolet radiation (UVR) is known to be highly mutagenic. What types of DNA lesions that are induced by different UVR wavelengths are still a matter of debate. UVR induces mutagenesis mostly by the formation of photoproducts and the induction of reactive oxygen species (ROS). ROS can give rise to mutations via oxidation of nucleotides in the DNA or the nucleotide pool. Oxidized nucleotides in the nucleotide pool can thereby be incorporated into the DNA during replication and ultimately give rise to mutations. MTH1 however, dephosphorylates oxidized nucleotides in the nucleotide pool, in particular 8-oxo-dGTP and 2-OH-dATP, and inhibits their incorporation into the DNA.The aim of the present study was to investigate the role of MTH1 in mutagenesis and cytogenetic damage induced by UVR in a human lymphoblastoid TK6 cell line. The clonogenic survival, mutant frequency and micronucleus frequency were measured following exposure to UVA, UVB and UVC in MTH1-knockdown and wild-type TK6 cells. As a biomarker for oxidative damage the level of intracellular and extracellular 8-oxo-dG was measured in TK6 cells exposed to UVA. The mutational spectra of UVA-induced mutations at the thymidine kinase gene in MTH1-knockdown and wild-type TK6 cells were investigated.The results show that MTH1 protects against UVA and UVB mutagenesis significantly. MTH1, however, has been shown to offer no protection against UVR-induced cytogenetic damage and is therefore suggested to mainly inhibit mutagenesis. The mutational spectra show that GC&gt;AT and AT&gt;GC transitions are the dominant mutation types in cells exposed to UVA.In conclusion, MTH1 protects TK6 cells against mutagenesis induced by longer wavelengths of UVR. This indicates that the nucleotide pool is a significant target in mutagenesis for longer wavelengths of UVR. The type of mutations induced by UVA, GC&gt;AT and AT&gt;GC, can be formed by the incorporation of 2-OH-dATP from nucleotide pool into the DNA. UVA is therefore suggested to induce mutations by induction of oxidized nucleotides such as 2-OH-dATP. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

<p>The ability of photocatalytic nanoparticles (NPs) to produce reactive oxygen species (ROS) has inspired research into several new applications and technologies, including water purification, contaminant remediation, and self-cleaning surface coatings. As a result, NPs continue to be incorporated into a wide variety of increasingly complex products. With the increased use of NPs and nano-enabled products and their subsequent disposal, NPs will make their way into the environment. Currently, many unanswered questions remain concerning how changes to the NP surface chemistry that occur in natural waters will impact reactivity. This work seeks to investigate potential influences on photoreactivity – specifically the impact of functionalization, the influence of anions, and interactions with biological objects - so that ROS generation in natural aquatic environments may be better understood.</p><p>To this aim, titanium dioxide nanoparticles (TiO2) and fullerene nanoparticles (FNPs) were studied in terms of their reactive endpoints: ROS generation measured through the use of fluorescent or spectroscopic probe compounds, virus and bacterial inactivation, and contaminant degradation. Physical characterization of NPs included light scattering, electron microscopy and electrophoretic mobility. These systematic investigations into the effect of functionalization, sorption, and aggregation on NP aggregate structure, size, and reactivity improve our understanding of trends that impact nanoparticle reactivity.</p><p>Engineered functionalization of FNPs was shown to impact NP aggregation, ROS generation, and viral affinity. Fullerene cage derivatization can lead to a greater affinity for the aqueous phase, smaller mean aggregate size, and a more open aggregate structure, favoring greater rates of ROS production. At the same time however, fullerene derivatization also decreases the 1O2 quantum yield and may either increase or decrease the affinity for a biological surface. These results suggest that the biological impact of fullerenes will be influenced by changes in the type of surface functionalization and extent of cage derivatization, potentially increasing the ROS generation rate and facilitating closer association with biological targets.</p><p>Investigations into anion sorption onto the surface of TiO2 indicate that reactivity will be strongly influenced by the waters they are introduced into. The type and concentration of anion impacted both aggregate state and reactivity to varying degrees. Specific interactions due to inner sphere ligand exchange with phosphate and carbonate have been shown to stabilize NPs. As a result, waters containing chloride or nitrate may have little impact on inherent reactivity but will reduce NP transport via aggregation, while waters containing even low levels of phosphate and carbonate may decrease “acute” reactivity but stabilize NPs such that their lifetime in the water column is increased.</p><p>Finally, ROS delivery in a multicomponent system was studied under the paradigm of pesticide degradation. The presence of bacteria or chlorpyrifos in solution significantly decreased bulk ROS measurements, with almost no OH detected when both were present. However, the presence of bacteria had no observable impact on the rate of chlorpyrifos degradation, nor chlorpyrifos on bacterial inactivation. These results imply that investigating reactivity in simplified systems may significantly over or underestimate photocatalytic efficiency in realistic environments, depending on the surface affinity of a given target.</p><p>This dissertation demonstrates that the reactivity of a system is largely determined by NP surface chemistry. Altering the NP surface, either intentionally or incidentally, produces significant changes in reactivity and aggregate characteristics. Additionally, the photocatalytic impact of the ROS generated by a NP depends on the characteristics of potential targets as well as on the characteristics of the NP itself. These are complicating factors, and the myriad potential exposure conditions, endpoints, and environmental systems to be considered for even a single NP highlight the need for functional assays that employ environmentally relevant conditions if risk assessments for engineered NPs are to be made in a timely fashion so as not to be outpaced by, or impede, technological advances.</p> / Dissertation

Prior studies have demonstrated the effect of diazoxide in protecting against apoptosis via mitochondrial KATP channel opening in vitro. The current investigations are designed to determine if sildenafil, a phosphodiesterase-5 inhibitor and known mitochondrial KATP channel opener, would protect against chronic doxorubicin cardiomyopathy both in vivo and in vitro.Male ICR mice were randomized to 1 of 4 treatments: saline, sildenafil (0.7 mg/kg IP), doxorubicin (5 mg/kg IP), and sildenafil (0.7 mg/kg IP)+doxorubicin. Apoptosis was determined using the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling and in situ oligo ligation methods. Desmin distribution was determined via immunofluorescence. Bcl-2 was analyzed by Western blot. Left ventricular function was measured in Langendorff mode. Electrocardiographical analysis measured changes indicative of doxorubicin cardiotoxicity (ST-prolongation). In vitro studies using adult ventricular cardiomyocytes were exposed to doxorubicin (1 μM), sildenafil (1 μM) with or without NG-nitro-L-arginine methyl ester (L-NAME; 100 μM), or 5-hydroxydecanoate (5-HD; 100 μM) 1 hour before doxorubicin and incubated for 18 hours. Doxorubicin-treated mice demonstrated increased apoptosis and desmin disruption, which was attenuated in the sildenafil+doxorubicin group. Bcl-2 decreased in the doxorubicin group but was maintained at basal levels in the sildenafil+doxorubicin group. Left ventricular developed pressure and rate pressure product were significantly depressed in the doxorubicin group but attenuated in the sildenafil+doxorubicin group. ST-interval significantly increased in the doxorubicin group over 8 weeks. In the sildenafil+doxorubicin group, ST-interval remained unchanged from baseline. Doxorubicin significantly increased apoptosis, caspase-3 activation, and disruption of mitochondrial membrane potential in vitro,. In contrast, sildenafil significantly protected against doxorubicin cardiotoxicity; however, protection was abolished by both L-NAME and 5-HD. Cell viability studies using spectrophotometer and flow cytometric techniques demonstrated that sildenafil did not affect the antitumor efficacy of doxorubicin in PC-3 cells in vitro. In fact, flow cytometry data indicate that sildenafil, when combined with doxorubicin, was synergistic in the antineoplastic action of doxorubicin. Prophylactic treatment with sildenafil prevented apoptosis and left ventricular dysfunction in a chronic model of doxorubicin-induced cardiomyopathy. Moreover, these studies provide relevant clinical data on the safety and efficacy of sildenafil, leading the way for clinical trials in humans receiving doxorubicin chemotherapy.

Dans la recherche de thérapie antitumorale de plus en plus innovante, nous avons évalué un traitement local basé sur l’utilisation de plasma froid. Le plasma froid (dans ce cas, <40°C) est un gaz ionisé par un apport d’énergie. Il contient des charges (électrons, ions), des radicaux libres et des molécules excitées. Il peut être généré à l’extrémité de cathéter permettant un traitement locorégional comme le traitement de dysplasie ou encore de tumeurs non résécables. Une activité antitumorale importante du plasma a été mise en évidence in vitro sur diverses lignées tumorales (colorectale, pulmonaire, pancréatique et cérébrale). Par ailleurs les cellules tumorales sont plus sensibles au plasma que les cellules normales. Les ROS générés sont à l’origine des principaux mécanismes d’action du plasma. Ils induisent de nombreux dommages à l’ADN, suivi d’un arrêt du cycle cellulaire conduisant à l’apoptose des cellules. Les études de tolérance ont mis en évidence l’innocuité de faibles doses de plasma sur le tissu traité permettant de définir les doses de plasmas utilisable dans le cadre de traitements antitumoraux. En utilisant des tumeurs xénogreffées en sous cutané et l’imagerie de bioluminescence, une activité antitumorale du plasma froid a été mise en évidence pour la première fois in vivo avec une augmentation de la survie des souris traitées d’environ 60%. Le traitement induit un arrêt de la prolifération tumoral avec une induction d’apoptose dans l’ensemble de la tumeur sans augmenter la surface de nécrose. L’effet antitumoral a également été démontré en utilisant le plasma gun sur un modèle de tumeurs colique et pancréatique en situation orthotopique chez la souris avec une augmentation de la survie (115%) accompagné d’une diminution de la métastasie. Ces résultats obtenues dans une démarche de recherche translationnelle montrent l’intérêt potentiel du plasma comme nouvel agent antitumoral. / In the context of new innovated antitumor treatment discovery, we evaluated the efficacy of a new local treatment based on non-thermal plasma (NTP). NTP is a cold (in this case, <40°C) ionized gas (a ir or noble gas) thanks to an electric discharge. It contains free charges (electrons, ions), free radicals and excited molecules. It can be generated at the end of a catheter allowing a local treatment that is compatible with usual endoscopes for dysplasia or non resecable tumors. We showed that NTP has a significant antitumor effect in vitro on various cell lines including colorectal, pancreatic, lung and brain tumor cells. The major action mechanisms of NTP was linked to a high rate generation of ROS in the vicinity of tumors cells and others plasma components have a minor implications. These ROS induce lethal DNA damages leading to a multiphase cell cycle arrest and finally to apoptosis. In vivo, a good tolerance of plasma treatment was highlighted and NTP treatment parameter was defined. Using subcutaneous xenografts and bioluminescence imaging, we showed a major antitumor effect of plasma in vivo with a 60% increase of mice life span. NTP treatments of tumor induce a tumor cell cycle arrest with a significant apoptosis induction in the whole tumor without increase of necrotic area. This in vivo antitumor effect was also observed with an in situ treatment using plasma gun of colorectal and pancreatic orthotopic tumor xenografts. A significant increase of mice lifespan (115%) was obtained together with a metastasis decrease. These results obtained in translational research showed the potential antitumor activity of NTP as a new type of treatment for cancer treatment.

Aconitase is an enzyme of the Krebs cycle that catalyzes the isomerization of citrate to isocitrate. In addition to its enzymatic activity, Aco1 has been reported to bind to mitochondrial DNA (mtDNA) and mediate its maintenance in the budding yeast S. cerevisiae. In the absence of Aco1, cells rapidly lose mtDNA and become “petite” mutants. The purpose of this study is to uncover the mechanism behind mtDNA loss due to an aco1 deletion mutation. We found that an aco1 mutation activates the mitochondria-to-nucleus retrograde (RTG) signaling pathway, resulting in increased expression of citrate synthases (CIT) through the activation of two transcription factors Rtg1 and Rtg3. Increased activity of CIT leads to increased iron accumulation in cells, which is known to raise reactive oxygen species (ROS). By deleting RTG1, RTG3, genes encoding citrate synthases, orMRS3 and MRS4, encoding two irontransporters in the mitochondrial inner membranes, mtDNA loss can be prevented in aco1 deletion mutant cells. We further show that the loss of SOD1, encoding the cytoplasmic isoform of superoxide dismutase, but not SOD2, encoding the mitochondrial isoform of superoxide dismutase, prevents mtDNA loss in aco1 mutant cells. Altogether, our data suggest that mtDNA loss in aco1 mutant cells is caused by the activation of the RTG pathway and subsequent iron accumulation and toxicity in the mitochondria.