Dissertations / Theses on the topic 'Reactive oxigen species (ROS)'

Les leucémies aiguë myéloïde (LAM) sont un groupe d’hémopathies malignes, dont le traitement est généralement composé de deux génotoxiques : la cytarabine (Ara-C) et la daunorubicine (DNR). Nous avons montré que l’Ara-C et la DNR induisent la déconjugaison rapide de SUMO (Small Ubiquitin-related Modifier) de ses protéines cibles. Cette deSUMOylation est dûe à l'inactivation des enzymes E1 et E2 de SUMOylation par les espèces réactives de l'oxygène (ROS) produites par l’Ara-C et la DNR et est impliquée dans l'activation de l'apoptose. En outre, cet axe ROS/SUMO est anergisé dans les LAM chimiorésistantes. Cependant, il peut être réactivé par des pro-oxydants ou par inhibition de la voie SUMO par l'acide anacardique. Pour identifier les protéines contrôlées par l’axe ROS/SUMO nous avons effectué une approche de spectrométrie de masse quantitative (SILAC). Parmi les 1000 protéines SUMOylées identifiées, la plupart des 114 protéines qui perdent leur SUMOylation lors du traitement sont impliquées dans la régulation de l'expression des gènes. De plus, un ChIP-Seq avec des anticorps anti SUMO-2 a permis de montrer que les génotoxiques, en particulier la DNR, induisent une diminution massive de la présence de protéines SUMOylées sur la chromatine. La recherche de motifs au sein des séquences fixant SUMO a permis d’identifier le motif de liaison de CTCF à l’ADN. De plus, CTCF a été trouvé dans la SILAC comme l’une des protéines déSUMOylées par les traitements. En utilisant des données publiques de Chip-Seq pour CTCF, nous avons identifié 55 gènes qui fixent à la fois CTCF et SUMO et dont l’expression est régulée par les traitements. Dans la dernière partie de ce travail, nous avons étudié le groupe de 19 protéines dont la SUMOylation augmente suite aux traitements génotoxiques. Parmi ces protéines, nous avons trouvé diverses protéines centromériques, y compris CENP-B et CENP-C. En utilisant le PLA (Proximity Ligation Assay) nous avons pu montrer que CENP-B et CENP-C colocalisent avec SUMO et yH2AX après traitement. Cela suggère que la SUMOylation des protéines centromériques se produit sur les sites de cassure et pourrait jouer un rôle dans la réparation des dommages de l'ADN
Acute Myeloid Leukemias (AML) are a group a severe hematological malignancies, which treatment is generally composed of two genotoxics: Cytarabine (Ara-C) and Daunorubicin (DNR). We have shown that these drugs induce the rapid deconjugation of the Small Ubiquitin-related Modifier (SUMO) from its target protein. This is due to the inactivation of SUMO E1 and E2 enzymes by Reactive oxygen species (ROS). This deSUMOylation participated in the activation of specific genes and is involved the induction of apoptosis. In addition, this ROS/SUMO axis is anergized in chemoresistant AMLs. However, it can be reactivated by pro-oxidants or inhibition of the SUMO pathway with anacardic acid, an inhibitor of the SUMO E1. To identify which proteins are regulated by this ROS/SUMO axis, we performed a quantitative mass spectrometry approach. Among the 1000 identified SUMO targets, most of the 114 proteins, which SUMOylation decrease upon treatment, are involved in the regulation of gene expression. In addition, we showed by ChIP-Seq with SUMO-2 antibodies that genotoxics, in particular DNR, induce a massive decrease of the presence of SUMOylated proteins on the chromatin. Motif search analysis of the SUMO binding sequences in these genes identified CTCF binding motif. Interestingly, CTCF was found in the SILAC as deSUMOylated by the drugs. Using publicly available ChIP-Seq data for CTCF, we found 55 genes which are occupied by both SUMO-2 and CTCF and which expression is regulated by the drugs. In the last part of this work, we got interested in the 19 proteins that get up-SUMOylated upon treatment. Among them, we found centromeric proteins, including CENP-B and CENP-C. Using Proximity Ligation Assay, we could show that CENP-B and CENP-C colocalize with both SUMO and yH2AX upon DNR treatment. Altogether, this suggests that centromeric protein up-SUMOylation occurs at sites of DNA damage and might play a role in DNA damage repair

Oxidative stress has been related in numerous cardiovascular and muscular pathologies, but the causal relationship between ROS accumulation and contractile impairment is not clear yet. We hypothesized that upon oxidative stress myofibrillar proteins (MPs) are oxidized providing a relevant contribution to the decrease of contractile performance. The evidence provided in the present work demonstrates the key role of mitochondrial dysfunction in muscle injury. We demonstrated that ROS produced in mitochondria alter MPs causing contractile dysfunction in cardiac and skeletal muscle. These results demonstrated that MP oxidation is an important target of ROS. In addition, this study provides evidence that the MAO-dependent ROS accumulation is responsible for both oxidative modifications of MPs and cell death. In fact, inhibition of MAO protects dystrophic skeletal muscle by reducing ROS production and myofiber degeneration. These results demonstrate the pivotal role of mitochondria and suggest a therapeutic potential for MAO inhibition.
Lo stress ossidativo è stato riconosciuto come uno dei meccanismi alla base di molte patologie cardiovascolari e muscolari, ma non è ancora stata chiarita quale possa essere la relazione tra l’elevato accumulo di ROS e la disfunzione contrattile. Noi abbiamo ipotizzato che in presenza di stress ossidativo le proteine miofibrillari (MPs) possano venire ossidate, contribuendo cosi al danno contrattile. I risultati ottenuti in questo lavoro dimostrano che la disfunzione mitocondriale svolge un ruolo chiave nel danno muscolare, sia nelle malattie cardiovascolari che nella distrofia muscolare. Abbiamo dimostrato che le ROS prodotte nel mitocondrio modificano le MPs causando una disfunzione contrattile. Questi risultati dimostrano che le ossidazioni alle MPs sono un importante bersaglio delle ROS. Inoltre questo studio mette in evidenza che le ROS prodotte dalla monoamino ossidasi (MAO) sono responsabili sia delle modifiche a carico delle MPs che della morte cellulare. Infatti l’inibizione farmaceutica della MAO protegge il fenotipo distrofico riducendo la produzione delle ROS e la degenerazione delle fibre muscolari. Questo dimostra il ruolo chiave dei mitocondri nella disfunzione contrattile, suggerendo un nuovo potenziale terapeutico per gli inibitori della MAO.

Cerium oxide nanoparticles have the unique ability to accept and donate electrons, making them powerful antioxidants. Their redox nature is due to oxygen defects in the lattice structure, which are more abundant at the nanoscale. Reactive oxygen species (ROS) are pro-oxidants whose presence is increased during periods of inflammation in the body. ROS damage tissues and cellular function by stripping electrons from proteins, lipids, and DNA. We investigated the ability of nanoceria to quench ROS in vitro and in vivo, and examined the biodistribution and biocompatibility of nanoceria in murine models. Nanoceria was internalized in vitro by macrophages, is non-toxic at the concentrations we investigated, and proteins, mRNA, and oxidative markers of ROS were abated with nanoceria pretreatment in immune stimulated cells as measured by western blot, real time RT PCR, and Greiss assay respectively. In vivo, nanoceria was deposited in the spleen and liver, with trace amounts in the lungs and kidneys as determined by ICP-MS. Using IVIS in vivo imaging, it appeared that nanoceria deposition occurred in lymph tissue. Histology grades show no overt pathology associated with nanoceria deposition, although white blood cell (WBC) counts were generally elevated with nanoceria treatment. Nanoceria suspect particles were seen in lysosomes from kidney samples of IV injected mice in HRTEM images. Lastly, IV nanoceria treatment appears to reduce markers of oxidative stress in mice treated with carbon tetrachloride (CCl4) to induce ROS production. Taken together, our data suggest that nanoceria treatment has the potential to reduce oxidative stress.
Master of Science

Side effects of radiation therapy (RT) remain the most challenging issue for pancreatic cancer treatment. In this report we determined whether and how cerium oxide nanoparticles (CONPs) sensitize pancreatic cancer cells to RT. CONP pretreatment enhanced radiation-induced reactive oxygen species (ROS) production preferentially in acidic cell-free solutions as well as acidic human pancreatic cancer cells. In acidic environments, CONPs favor the scavenging of superoxide radical over the hydroxyl peroxide resulting in accumulation of the latter whereas in neutral pH CONPs scavenge both. CONP treatment prior to RT markedly potentiated the cancer cell apoptosis both in culture and in tumors and the inhibition of the pancreatic tumor growth without harming the normal tissues or host mice. Mechanistically, CONPs were not able to significantly impact RT-induced DNA damage in cancer cells, thereby ruling out sensitization through increased mitotic catastrophe. However, JNK activation, which is known to be a key driver of RT-induced apoptosis, was significantly upregulated by co-treatment with CONPs and RT in pancreatic cancer cells in vitro and human pancreatic tumors in nude mice in vivo compared to CONPs or RT treatment alone. Further, CONP-driven increase in RT-induced JNK activation was associated with marked increases in Caspase 3/7 activation, indicative of apoptosis. We have shown CONPs increase ROS production in cancer cells; ROS has been shown to drive the oxidation of thioredoxin (TRX) 1 which results in the activation of Apoptosis Signaling Kinase (ASK) 1. The dramatic increase in ASK1 activation following the co-treatment of pancreatic cancer cells with CONPs followed by RT in vitro suggests that increased the c-Jun terminal kinase (JNK) activation is the result of increased TRX1 oxidation. The ability of CONPs to sensitize pancreatic cancer cells to RT was mitigated when the TRX1 oxidation was prevented by mutagenesis of a cysteine residue, or the JNK activation was blocked by an inhibitor,. Additionally, angiogenesis in pancreatic tumors treated with CONPs and RT was significantly reduced compared to other treatment options. Taken together, these data demonstrate an important role and mechanisms for CONPs in specifically killing cancer cells and provide novel insight into the utilization of CONPs as a radiosensitizer and therapeutic agent for pancreatic cancer.
Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences

Protein nitration is considered as a marker of reactive nitrogen species formation. Heme oxygenases (HOs) are important for the defence against oxidative stress. We evaluated the involvement of the neuronal (nNOS), the endothelial (eNOS), and the inducible (iNOS) in nitrotyrosine formation and localitzation, and both the expression and funcional significance (HO inhibition and contractility studies) of HOs in sepsis-induced muscle contractile dysfunction. Sepsis was elicited by injecting rats and transgenic mice deficient in either nNOS, eNOS, or iNOS isoforms with E.Coli lipolysaccharide (LPS). Nitrotyrosine formation and HO expressions were assessed by immunoblotting. Oxidative stress was assessed measuring protein oxidation, lipid peroxidation, and muscle glutathione. We conclude that protein tyrosine nitration occurs in normal muscles, and sepsis-mediated increase in nitrotyrosine formation is limited to the mitochondria and membrane muscle fractions. The iNOS isoform is mostly involved in nitrotyrosine formation. HOs protect normal and septic muscles from the deleterious effects of oxidants.
En un model de sepsi de disfunció diafragmàtica, s´ha avaluat el paper de les sintetases de l'òxid nítric (NOS) en la formació i localitzacio de 3-nitrotirosina, i l´expressió i significat biològic de les hemo oxigenases (HOs) (inhibidor de les HOs i estudis de contractilitat) davant l' estrès oxidatiu. La sepsi s'induí mitjançant injecció de 20 mg/kg del lipolisacàrid (LPS) d´Escherichia Coli a rates, i a ratolins deficients en les NOS induïble (iNOS), neuronal (nNOS) i endotelial (eNOS). Les proteïnes nitrificades i les HOs es van detectar amb anticossos específics. L' estrès oxidatiu s' avaluà mitjançant l' oxidació proteica, la peroxidació lipídica i el glutation muscular. Concloem que hi han proteïnes nitrificades en el múscul normal i aquestes s'incrementen durant la sepsi en les fraccions mitocondrial i membranar. L'isoforma iNOS és majorment responsable de la formació de nitrotirosina. Les HOs protegirien el múscul normal i sèptic dels efectes deleteris dels oxidants.

Mestrado em Biologia Molecular e Celular
Graphene oxide (GO) is a compound with application in several fields, especially biomedicine and environment, due to its unique properties which confer excellent characteristics. Despite the nowadays wide application of nanomaterials, the lack of information regarding the risks to human health and the environment is still remaining. Consequently, the investigation about the toxicity of nanoparticles must be a priority. The lung is one of the main routes of entry for nanoparticles into the body, therefore, the aim of this study is to evaluate the cytotoxic potential of graphene oxide in lung, using as model the human carcinoma epithelial cell line (A549). The morphology and viability of A549 cells were evaluated after 24h of exposure to GO at concentrations from 10μg/ml to 200μg/ml. The uptake of GO and the production of reactive oxygen species were also investigated by flow cytometry. The results suggest that GO has no obvious toxicity to A549 cells when assessed by WST-8 assay, though the cell cycle showed a slightly alteration in the S and G2 phase at 50μg/mL with arrest at G2 phase. Also, GO showed an increasing in the ROS production at the lowest doses (10ug/mL and 37μg/mL). The intracellular uptake increased for the highest concentration. Together these results suggest that this form of GO shows biocompatibility for lung cells.
O óxido de grafeno (GO) é um composto com aplicação em diversas áreas, especialmente biomedicina e ambiente, devido as suas propriedades únicas que lhe conferem excelentes características. Apesar da atual ampla utilização de nanomateriais, a falta de informação sobre os riscos para a saúde humana e para o ambiente ainda permanece. Consequentemente, a investigação sobre a toxicidade das nanopartículas deve ser uma prioridade. Sendo o pulmão uma das principais vias de entrada das nanopartículas no organismo, o objetivo deste trabalho é avaliar o potencial citotóxico de óxido de grafeno no pulmão, usando com modelo uma linha celular epitelial de carcinoma do pulmão humano (A549). A morfologia e a viabilidade das células A549 foram avaliados após 24 horas de exposição a concentrações de GO entre 10μg/mL a 200μg/mL. A captação de GO e a produção de espécies reativas de oxigênio foram avaliadas por citometria de fluxo. Os resultados sugerem que GO não apresenta óbvia toxicidade para as células A549, quando avaliada pelo ensaio WST-8. No entanto, no ciclo celular observou-se uma ligeira alteração na fase S e na fase G2 em 50μg/mL, com paragem na fase G2. GO induziu também um aumento na produção de ROS em doses mais baixas (10μg/mL e 37μg/mL). A captação intracelular de GO aumentou para a dose mais elevada. Em conjunto, estes resultados sugerem que esta forma de GO apresenta biocompatibilidade para as células de pulmão.

Mitochondria are the major effectors of cardioprotection by procedures that open the mitochondrial ATP-sensitive potassium channel (mitoKATP), including ischemic and pharmacological preconditioning. MitoKATP opening leads to increased reactive oxygen species (ROS), which then activate a mitoKATP-associated PKCε, which phosphorylates mitoKATP and leaves it in a persistent open state (Costa, ADT and Garlid, KD. Am J Physiol 295, H874-82, 2008). Superoxide (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (HO•) have each been proposed as the signaling ROS but the identity of the ROS responsible for this feedback effect is not known. Superoxide was excluded in earlier work on the basis that it does not activate PKCε and does not induce mitoKATP opening.To further examine the identity of the signaling ROS, respiring rat heart mitochondria were preincubated with ATP and diazoxide to induce the phosphorylation-dependent open state, together with agents that may interrupt feedback activation of mitoKATP by ROS scavenging or by blocking ROS transformations. Swelling assays of the preincubated mitochondria revealed that dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, trolox, and bromoenol lactone (BEL) each blocked the ROS-dependent open state but catalase did not interfere with this step. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO• excludes H2O2 as the endogenous signaling ROS and focuses attention on HO•. In support of the hypothesis that HO• is required, we also found that HO•-scavenging by DMF blocked cardioprotection by both ischemic preconditioning and diazoxide in the Langendorff perfused rat heart. HO• itself cannot act as a signaling molecule, because its lifetime is too short and it reacts immediately with nearest neighbor phospholipids and proteins. Therefore, these findings point to a product of phospholipid peroxidation, such as hydroperoxy-fatty acids. Indeed, this hypothesis was supported by the finding that hydroperoxylinoleic acid (LAOOH) opens the ATP-inhibited mitoKATP in isolated mitochondria. This effect was blocked by the specific PKCε inhibitor peptide εV1-2, showing that LAOOH activates the mitoKATP-associated PKCε. During ischemia, catabolism of mitochondrial phospholipids is accelerated, causing accumulation of plasmalogens and free fatty acids (FA) in the heart by the action of calcium independent phospholipases A2 (iPLA2). We first assessed the role of FAs and hydroxy FAs on mitoKATP opening and cardioprotection. Swelling assays of isolated rat heart mitochondria showed that naturally formed free FAs inhibit mitoKATP opening and that they are more potent inhibitors of the pharmacological open state of mitoKATP than the phosphorylation-dependent open state. That is, sustained mitoKATP opening induced by the phosphorylation-dependent feedback loop is more resistant to FA inhibition than direct mitoKATP opening by a potassium channel opener. Moreover, rat hearts perfused with micromolar concentrations of FA were resistant to cardioprotection by diazoxide or ischemic preconditioning. Racemic bromoenol lactone (BEL), a selective inhibitor of iPLA2, confers protection to otherwise untreated Langendorff perfused hearts by preventing ischemic FA release. To bring this story full circle, BEL blocks protection afforded by preconditioning and postconditioning by preventing the iPLA2-mediated release of FAOOH generated in the conditioned heart. HO• resulting from mitoKATP opening oxidizes polyunsaturated fatty acid components of the membrane phospholipids, resulting in a peroxidized side chain. FAOOH must be released in order to act on the mitochondrial PKCε, and this is achieved by the action of iPLA2. iPLA2 is essential for most modes of cardioprotection because it catalyzes the release of FAOOH. This fully supports the hypothesis that the second messenger of cardioprotective ROS-mediated signaling is hydroperoxy fatty acid (FAOOH), a downstream oxidation product of HO•.

In the aerobic cell, oxygen can be converted into a series of reactive metabolites, together called as "reactive oxygen species" (ROS). This large group include both radical and non-radical species such as superoxide anion (02"), hydroxyl radical ("0H), H202, nitric oxide (N0') and lipid hydroperoxides (LOOH). ROS are generated in very small amounts at all stages of aerobic life, and probably have a role in cellular regulation. However, their formation in excess leads to toxicity and damage to tissues. This situation, called 'oxidative stress', is responsible, atleast in part, to the pathophysioiogy of a number of disease states such as inflammation, arthritis, cancer, ageing, ischemia-reperfusion and several neurodegenerative disorders. Compared to other organs in the animal body, brain tissue is more vulnerable to oxidative stress. This is due to three major reasons; (1) brain has a high oxygen consumption (2) high content of polyunsaturated fatty acids and iron, that can promote lipid peroxidation, and (3) low levels of antioxidant enzymes such as catalase and glutathione peroxidase. The inability of neurons to regenerate also contributes to exacerbate an oxidant damage in the brain. The main objective of this investigation was to identify biochemical systems in the brain that are susceptible to ROS, on the following two issues: 1. What are the targets for the action of H2O2 and NO in the glycolytic cycle, the major route for the oxidation of glucose in brain? 2. What are the targets for the action of polyunsaturated fatty acids and their oxidative metabolites among the enzymes of phosphoinositide cycle (PI cycle), the ubiquitous signal transduction event in the brain? Using sheep brain cytosol , it was found that among the various glycolytic enzymes, only glyceraldehyde 3-phosphate dehydrogenase (GAPD) was inhibited by H2O2. The enzyme was purified to homogeneity from sheep brain and its inactivation with H202 was studied in detail. Commercial preparations of rabbit skeletal muscle GAPD was also used in this study. An unusual requirement of glutathione for the complete inactivtion of the enzyme by H2O2 was observed. The H2O2-inactivated GAPD was partially reactivated by prolonged treatment with thiol compounds. Using CD-spectral analysis, a significant change was found in the secondary structure in H2O2-treated GAPD. GAPD was inactivated by NO only in presence of high concentrations of DTT and after prolonged incubation. The N0-inactivated GAPD was partially reactivated by treatment with thiol compounds. A new activity, namely ADP-ribosylation (ADPR) emerged in the NO-treated mammalian, but not in yeast. GAPD, ADPR activity could be generated in GAPD through NO-independent treatments such as incubation with NADPH and aerobic dialysis. During NADPH treatment no loss of dehydrogenase activity occurred. Thus, it was concluded that loss of dehydrogenase activity and emergence of ADPR in NO-treated GAPD were not correlated but coincidental, and that NO treatment yielded small amounts of modified-GAPD that had ADPR activity. In the brain, onset of ischemia is characterized by a significant elevation in free fatty acid (FFA) levels, predominantly, arachidonic acid (AA). It is suggested that AA can be oxidised to its metabolites like prostaglandins and 15-hydroperoxy arachidonic acid (15-HPETE) and some of these might exert toxic effects during reperfusion. Using whole membranes or tissue slices prepared from rat brain, effects of polyunsaturated fatty acids and their oxidative metabolites on five enzymes of PI cycle namely PI synthase, PI and PIP kinases, agonist-stimulated PLC and DG kinase was studied. Hydroperoxides of linoleic- and arachidonic acids inactivated PI synthase selectively among the PI cycle enzymes. Interestingly, AA selectively stimulated DG kinase in neural membranes. Docasahexaenoic acid (DHA) a highly unsaturated fatty acid found in the brain, also stimulated DG kinase activity while saturated, mono-and di-unsaturated fatty acids were ineffective. It was concluded that AA and DHA have a role in modulating neural DG kinase. The data presented in the thesis indicate that ROS have selective targets in cells and the consequent protein modifications can be used to modulate cellular functions under normal and oxidative stress conditions.

This thesis presents a series of scientific studies exploring the initiation of various chemical reactions with reactive oxygen species (ROS), mainly singlet oxygen. These studies have revealed new mechanistic insights in environmental, industrial and biological systems, have described the associated set of reactions, have illustrated the detection of new radicals i.e., environmentally persistent free radicals (EPFR), and have provided a new insight explaining the spontaneous fire in coal mines. Comprehensive experimental and quantum-mechanical calculations afforded the investigation of oxidation reactions of singlet oxygen with wastewater organic contaminants, for example, the photodegradation of Phenol and Aniline in water. Detailed experimental studies on modelled surrogates, i.e., Anisole, resolved the fundamentals of thermal interaction of coal with iron oxide Fe2O3 nanoparticles. Along the same line of interest, enhancing the combustion efficiency of fuel constitutes a mainstream strategy in the pursuit of meeting the ever-increasing energy demand. Therefore, this thesis also provides a comprehensive mechanistic and thermo-kinetic accounts underpinning the reaction of fuel surrogates, namely Toluene, with singlet oxygen in the internal combustion (IC) engines. Finally, this work extends insights into biological systems, mapping the Alloxan-Glutathione redox cycle to expose the formation of ROS, species that eventually cause necrosis of the pancreatic insulin-producing beta cells and prompt the insulin-dependent diabetes mellitus (IDDM). The methodology involve customised LED-photoreactors, thermal packed-bed reactor, and various reaction product-monitoring systems, e.g., Fourier transform infrared spectroscopy (FTIR) to quantitate the ignition temperatures of fuel surrogates, in-situ electron paramagnetic resonance (EPR) to elucidate the formation of environmentally-persistent free radicals (EPFR) as well as intermediate radical species, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) to monitor the chemisorption of organic substrates on the nanoparticles, X-ray diffraction for particles characterisation, as well as broad-scan UV-Vis spectroscopy and high-performance liquid chromatography (HPLC) to identify and quantify the intermediate and product species in solutions. Results obtained in this thesis elucidate, for the very first time, the formation of para-semibenzoquinone anion (PSBQ) supporting the reaction pathway leading to the formation of para-benzoquinone during the reaction of phenol (and aniline) with singlet oxygen. These results have practical application to quantify the degradation of organic pollutants in wastewater. Investigations regarding combustion applications shows that the presence of singlet oxygen considerably lowers the activation energy of the initiation channels of aromatic hydrocarbons (e.g., in IC engines), resulting in an energetically improved combustion process, the relative reactivity of singlet oxygen, based on the reaction rate constants, follows the order of OH > H > CH3 > 1O2 > HO2 > 3O2. Furthermore, the chemisorption of anisole on α-Fe2O3 surfaces has been elucidated to follow a direct dissociation of the O–CH3 (and OCH2–H), leading to the formation of surface-bound phenoxy radicals and gaseous species at temperatures as low as 25 °C. This insight applies to free-radical chain reactions that induce spontaneous fires of coal, as low-ranked coal comprises ferric oxide nanoparticles, and equally, to coexistence of aromatic fuels with thermodynamically reactive Fe2O3 surface, e.g., in fly ash, at the cooled-down tail of combustion stacks. Results from alloxan-glutathione redox cycle clarified, for the first time, the direct synchronised generation of dialuric acid radical (DA˙) and glutathione radical (GS˙), assigning the nature of the mysterious “compound 305” to the DA˙- GS˙ complex. These results explain the alloxan-induced diabetes on precise molecular bases. This thesis provides new perspectives on opportunities in understanding the influence of ROS, mainly singlet oxygen (1O2) and superoxide (O2−) in germane chemical reactions. Such attempts will advance the existing ROS-related technologies, and improve the fundamental theories in supports of environmental management and application decisions.

Psoriasis is a chronic inflammatory skin disorder that affects around two percent of the population. There are many treatments available for the management of psoriasis including topical therapy, systemic agents and phototherapy. Despite the number of treatments available, however, there are still problems in the management of psoriasis. It is suggested here that the thioredoxin enzyme system may play a role in the pathology of psoriasis. Using specific molecular modelling techniques, a lead compound, RDP00060, was identified as a potential inhibitor of thioredoxin reductase, a key enzyme in the thioredoxin system. In vitro RDP00060 showed moderate inhibitory activity against the thioredoxin enzyme system with an IC50 value of 1.4 mM. RDP00060 also showed powerful activity in an MTT assay using a human papilloma virus immortalized keratinocyte (HPV-16) cell line. To increase the inhibitory activity towards thioredoxin reductase, molecular modelling techniques were used to identify analogues of RDP00060 with a high binding affinity for thioredoxin reductase. Several novel compounds were then synthesized, characterized and evaluated for inhibitory activity towards the thioredoxin system. One of the compounds, N-(3,4-bis-(toluene-4- sulfonylamino)phenyl)-2-furamide (33f) showed good inhibitory activity against the thioredoxin enzyme with an IC50 value of 37 μM. It is anticipated that N-(3,4- bis-(toluene-4-sulfonylamino)phenyl)-2-furamide (33f) binds to thioredoxin reductase irreversibly through a 1,4-conjugate addition mechanism. This compound also showed powerful activity in the MTT assay using an HPV-16 immortalized keratinocyte cell line. Further testing revealed that N-(3,4-bis-(toluene-4-sulfonylamino)phenyl)-2- furamide (33f) also showed apoptotic and antiproliferative properties in human Tcells. As a result of this work, N-(3,4-bis-(toluene-4-sulfonylamino)phenyl)-2- furamide (33f) has been selected for further investigation as a potential antipsoriatic agent.

The tumor suppressor p53 has a close relation with reactive oxygen species (ROS). As an indispensable component of the cellular redox system, ROS not only have been established to be involved in p53-dependent apoptosis, but also regulate p53 activity. Recent studies revealed several novel actions of p53, such as transactivation of antioxidative proteins, mitochondria translocation and inhibition of glycolysis. The fate of cells where p53 signaling pathways are initiated is either survival or death. In this review, we examine the hypothesis that ROS regulate cell fate through p53, in a way that physiological ROS levels trigger the protective pathways, while p53 behaves more like a cell killer under cytotoxic oxidative stress.

Mitochondria are key generators of cellular ATP, vital to complex life. Historically, mitochondrial generation of reactive oxygen species (ROS) was considered to be an unregulated process, produced by dysfunctional mitochondria. More recently, mitochondrial ROS generated by complex I, particularly by the process of reverse electron transfer (RET), has emerged as a potentially biologically relevant signal that is tightly-regulated and dependent on mitochondrial status. ROS production by RET is reported to play a role in the innate immune response and lifespan extension in fruit flies. One way in which mitochondrial ROS may behave as a signal is by altering the activity of AMP-activated protein kinase (AMPK), a key metabolic sensor and regulator of cell metabolism, which is activated when cellular ATP levels decrease during energy demand. Mitochondria can signal to AMPK via the magnitude of the cellular ATP/AMP and ATP/ADP ratios, which alter in response to mitochondrial function. Our view is mitochondria may also signal to AMPK via ROS. Important studies have helped to clarify the role of exogenous or cytosolic ROS in AMPK regulation. However, the effects of mitochondrial ROS on AMPK activity, specifically that generated by complex I, remain unclear and is the main focus of this thesis. I characterized the effects of exogenous H2O2 on cellular AMPK activity, ATP/ADP ratios and cellular redox state in a cell model. I then compounded this with selective mitochondria generated ROS by the mitochondria-targeted redox-cycler, MitoParaquat (MPQ). AMPK activity appeared to correlate with decreasing cell ATP/ADP ratios, indicating that both sources of ROS primarily activate AMPK in an AMP/ADP-dependent mechanism. In parallel, I developed an approach for analyzing the redox state of candidate proteins, an important step in determining if a protein is directly regulated by ROS. I also initiated development of a cell model for studying the downstream effects of mitochondrial ROS production by RET, by expressing alternative respiratory enzymes in a mammalian cell line.

The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment.

Traditionally, reactive oxygen species (ROS) have been regarded as toxic by-products of aerobic metabolism. However, in recent years it has become apparent that plants actively produce ROS as signalling molecules. ROS are able to mediate adaptive responses to various environmental stresses as well as processes such as stomatal closure and development. Downstream signalling events that are modulated by ROS include calcium mobilisation, protein phosphorylation and gene expression. This study investigated signalling proteins acting downstream of ROS, in order to understand how ROS are perceived and transduced to elicit such a wide range of responses. To establish a molecular profile provoked by ROS, a microarray experiment of Arabidopsis plants exposed to exogenous H(_2)O(_2) was analysed. Of the 895 differentially expressed transcripts, a substantial proportion had predicted functions in cell rescue and defence, including heat shock, disease resistance and antioxidant genes. Genes encoding candidate H(_2)O(_2) signalling components were identified from this microarray experiment and their H(_2)O(_2) - induced expression was verified by northern RNA-blot analysis. Two transcription factors of the ethylene response factor (ERF) family (AtERFS [At5g47230]) and AtERF6 [At4g17490])and an ankyrin protein kinase (APK [At4g18950]) were selected for further study. Northern blot analysis and comparison with publicly available transcriptome data sets demonstrated that the expression of these three genes was induced by various stress treatments, such as UV-B irradiation, cold and elicitor challenge. To unravel the potential in vivo function of these proteins, loss- and gain-of-function lines were generated and analysed. No abnormal plant phenotypes were observed during development or in response to the stress and hormone treatments tested. A high level of functional redundancy may exist between AtERFS and AtERF6. Microarray analyses were performed on the over-expression lines. Genes that were differentially regulated in APK over-expressor lines gave no indication of its function. However, the microarray analyses revealed that AtERFS and AtERF6 have roles in the plant pathogen defence response, since their over-expression induced defence gene expression. Analysis of cis elements in the promoters of the ERF-differentially regulated genes revealed that both transcription factors displayed GCC box binding activity. However, the GCC box was not over-represented in the promoters of H202-differentially regulated genes, which suggests that this element has a ROS independent regulation.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS), saccharide (i.e. monosaccharide, disaccharide and polysaccharide), are continuously generated, transformed and consumed in the living systems. As a consequence of their significant value towards human health in aerobic life, it is very important and has drawn much attention in the chemical and biological sensing of the species. It is our long-standing interest in the recognition of monosaccharide (e.g. glucose) through exploration of various boronate-based fluorescence probes, thus, based on the previous work, we started on the design, synthesis and evaluation of novel fluorescent chemosensors for breakthrough discoveries in the detection of saccharide and ROS selectively and specifically, which are made up of different receptors and diverse singaling fluorophores, e.g. anthracene, coumarin, fluorescein, naphthalimine. Firstly, “integrated” and “insulated” boronate-based fluorescent probes (2-naphthylboronic acid and N-Methyl-o-(aminomethyl)phenylboronic acid) have been evaluated for the detection of hydrogen peroxide in the presence of saccharides (i.e. D-fructose). In the presence of D-fructose the initial fluorescence intensity of the “insulated” system is much higher and produces a blue visible fluorescence. Based on the experimental observation above in the boronate-based systems (i.e. B-N bond protection), a new water-soluble boronate-based fluorescent probe was designed and evaluated for the detection of peroxynitrite (much stronger oxidant) in the presence of D-fructose. The enhanced fluorescence of probe when bound with D-fructose was switched off in the presence of peroxynitrite. While, other reactive oxygen/nitrogen species led to only slight fluorescence decreases due to protection by the internal N-B interaction. The interaction of probe with D-fructose not only strengthens the fluorescence signal, but also protects the boronic acid to oxidation by other ROS/RNS. Therefore, under conditions generating various ROS/RNS, the boronate-based sugar complex preferentially reacts with peroxynitrite (ONOO−). The sensor displays good “on-off” response towards peroxynitrite both in RAW 264.7 cells and HeLa cells. A new ICT (internal charge transfer) sensing system was developed for the detection of hydrogen peroxide and peroxynitrite. The probe displayed an enhanced fluorescence change when bound with D-fructose due to the prolonged N-B distance. The fluorescence intensity of the probe dropped down both in the detection of H2O2 and ONOO− which was attributed to the oxidation of arylboronic acid even though in the presence of D-fructose. Using the self-assembly of aromatic boronic acids with Alizarin Red S (ARS), we developed a new chemo/biosensor for the selective detection of peroxynitrite. Phenylboronic acid, benzoboroxole and 2-(N, N-dimethylaminomethyl) phenylboronic acid were employed to bind with ARS to form the complex probes. In particular the ARS-NBA system with a high binding affinity can preferably react with peroxynitrite over hydrogen peroxide and hypochlorite due to the protection of the boron via the solvent-insertion B-N interaction. Our simple system produces a visible naked-eye colorimetric change and on-off fluorescence response towards peroxynitrite. By coupling a chemical reaction that leads to an indicator displacement, we have developed a new sensing strategy, referred to herein as RIA (Reaction-based Indicator displacement Assay). Next, we developed a novel class of simple materials for sensing monosaccharides by the functionalization of graphene oxide (GO) with boronate-based fluorescence probes. The composite materials were characterized by atomic force microscopy, Raman spectroscopy, and UV-vis/fluorescence spectroscopy. The strong fluorescence of the fluorescence probes is quenched in the presence of GO through fluorescence resonance energy transfer (FRET). The BA@GO composite sensors formed provide a useful platform for fluorogenic detection of monosaccharides based on the strong affinity between the boronic acid receptor and monosaccharides. The BA@GO composite sensor displayed a “turn-on” fluorescence response with a good linear relationship towards fructose over a range of other saccharides. Next, new water-soluble copper (II) complex fluorescence probes were developed and evaluated for the detection of nitric oxide and nitroxyl in a physiological condition. A significant fluorescence “off-on” response displayed by using the copper (II) complex for the detection of NO and HNO (Na2N2O3 as a donor). Under pathological conditions generating various ROS/RNS, the copper (II) complex fluorescent probe preferentially reacts with NO/HNO over other reactive oxygen species. The dual-analyte recognitions of the simple, sensitive probe were further applied in living cell for the exogenous NO/HNO. In the following work, we synthesised a phosphorous-based compound for the detection of HNO which derived from Angeli’s salt in a biological condition. Significantly, it displayed a high sensitivity and selectivity toward HNO over other various ROS species, especially NO since they have a similar chemical property. The underlying mechanism was attributed to the cleavage of C-O bond induced by Staudinger Ligation.

Arsenical-induced carcinogenesis in human bladder has been established through epidemiological evidence, but unfortunately, no mode of action had been determined for this phenomenon. UROtsa cells, a normal, immortalized cell culture model of human urothelium does not form tumors when injected into immuno-compromised mice nor does it have anchorage-independent growth. UROtsa cells were shown to be malignantly transformed following low-level exposure to both arsenite [As(III)] and its more toxic metabolite, monomethylarsonous acid [MMA(III)] providing additional models for studying arsenical-induced carcinogenesis of the bladder. These transformed cell lines allow researchers the ability to investigate the process of urothelial tumorigenesis at multiple time points of arsenical exposure. In the studies discussed here in, environmentally relevant levels of As(III) and MMA(III) were chosen. UROtsa cells were exposed to As(III) and MMA(III) both acutely and chronically to begin investigations into signaling pathway alterations that can lead to carcinogenesis in the human bladder upon exposure to arsenicals. In acute studies, it was shown that As(III) and MMA(III) generate oxidative stress response in UROtsa at low, environmentally relevant levels. The ROS generated by MMA(III) led to an increased 8-oxo-dG formation after 30 min, supporting the importance of MMA(III) in damage caused in the bladder by arsenicals. Because ROS has been linked to MAPK signaling, it was shown that 50 nM MMA(III) and 1 µM As(III) induce MAPK signaling following acute exposures and this increase is dependent on the production of ROS.Next, it was necessary to begin to look at changes that occur during transformation of UROtsa with MMA(III). Chronic exposure to 50 nM MMA(III) constitutively increases the amounts of EGFR, activated Ras, and COX-2 protein in MSC cells. Chronic upregulation of COX-2 in MSC52 cells is due to increased levels of ROS. Phenotypic changes seen in MSC52 cells (hyperproliferation and anchorage independent growth) are dependent on the secondary generation of excess ROS in MSC52 cells. These data clearly present evidence supporting a role for ROS in both acute and chronic toxicities associated with low-level arsenical exposure, and gives evidence that ROS are important in cellular transformation following MMA(III) exposure.

Kenji Takemoto, Etsuro Hatano, Keiko Iwaisako, Masatoshi Takeiri, Naruto Noma, Saori Ohmae, Kan Toriguchi, Kazutaka Tanabe, Hirokazu Tanaka, Satoru Seo, Kojiro Taura, Keigo Machida, Norihiko Takeda, Shigehira Saji, Shinji Uemoto, Masataka Asagiri, Necrostatin-1 protects against reactive oxygen species (ROS)-induced hepatotoxicity in acetaminophen-induced acute liver failure, FEBS Open Bio, Volume 4, 2014, Pages 777-787, ISSN 2211-5463
Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第18678号
医博第3950号
新制||医||1007(附属図書館)
31611
京都大学大学院医学研究科医学専攻
(主査)教授 松原 和夫, 教授 渡邊 直樹, 教授 一山 智
学位規則第4条第1項該当

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.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

Nous avons utilisé la régénération de la nageoire caudale du poisson zèbre comme modèle physiologique pour comprendre le rôle des cellules apoptotiques dans le recrutement des cellules souches chez l'adulte. Peu de temps après l'amputation, les cellules formant le moignon répondent à la blessure par dédifférenciation, en acquérant une identité de cellule progénitrice. Nous avons récemment identifié la mort cellulaire et les espèces réactives de l'oxygène (ou ROS, pour Reactive Oxygen Species) comme étant des signaux précoces régulés après blessure et amputation. La cicatrisation induit une mort cellulaire rapide et locale, tandis que l'amputation induit une seconde vague d'apoptose, spécifique de la régénération. Nous avons ensuite posé la question de la voie de signalisation engagée par les cellules apoptotiques, et un criblage chimique nous a permis d'identifier un signal purinergique. Par ailleurs, l'inhibition de la régénération par inhibition de l'apoptose peut être restaurée par de l'adénosine exogène. De façon surprenante, l'adénosine seule est suffisante pour augmenter le nombre de cellules progénitrices ainsi que pour stimuler la régénération. Afin de mieux comprendre la diffusion du signal apoptotique in vivo, nous avons mis au point des outils optogénétiques. Ces systèmes nous permettent d'induire l'apoptose et suivre sa progression in vivo. Combiné à des senseurs physiologiques fluorescents, ces outils nous permettent d'étudier de façon dynamique les effets de l'apoptose sur les cellules avoisinantes
We used regeneration of the zebrafish caudal fin as a physiological model to understand the role of apoptotic cells in stem cell recruitment in adult. Shortly after amputation, cells from the stump respond to injury by dedifferentiating and acquiring a progenitor identity. We recently identified cell death and ROS signalling as early events regulated after healing and amputation. Wound healing induces fast local cell death, while amputation induces a second round of apoptosis, specific of the regeneration. We then ask for the signalling pathway engaged by apoptotic cells and chemical screening allows us to identify a purinergic signalling. Moreover, the inhibition regeneration by inhibition of apoptosis could be rescued by exogenous adenosine. Surprisingly, adenosine alone is sufficient to enhance the number of progenitor cells and to stimulate fin regeneration. To better understand the spreading of apoptosis signalling in vivo, we implemented optogenetic tools. This system allows us to induce apoptosis and follow its progression in vivo. Combined with physiological fluorescent sensors, this allows us to investigate the dynamical effects of apoptosis on neighbouring cells

Endothelial cells are a vital region in the pathophysiology of the vasculature because it is the interface between blood flow and the vessel. One way that the structure of the vessels wall can change is by the accumulation of reactive oxygen species (ROS), which has been correlated to aneurysm formation. Four main ROS sources in endothelial cells are: NADPH oxidase, mitochondria electron transport chain, eNOS uncoupling, and xanthine oxidase. Endothelial cells are an essential component of vasculature that has distinct functions and morphology. The aorta and brain arteries are highly populated by endothelial cells but the morphology and cellular signaling has been shown to be different. This study focuses on the difference between brain and aorta ROS production and how flow affects ROS. Joeseph Moran-Guiati and Jason Kushner provided the brain and aortic endothelial cultures for these studies. NADPH oxidase complex is the main contributor in both cell types but more in brain. Surprisingly, both cell types contain approximately the same number of NOX subunits, suggesting that the difference in ROS production is dependent on how activated these subunits are. Mitochondrial ROS was only significantly generated in brain cells and is verified because brain endothelium contains higher numbers of mitochondria. Both uncoupling of eNOS and xanthine oxidase did not contribute to ROS generation in static cultures. ROS production increased even further in both cell types when cells were exposed to flow and even higher in brain, suggesting that flow effects ROS generation. These results provide useful information in the difference between ROS generation and how it can be harmful in possibly causing intracranial aneurysm formation.

Valproic Acid Promotes Acetylation of Superoxide Dismutase-2 During Neurogenesis. Valproic acid (VPA) is a known developmental toxicant associated with a high prevalence of neural tube defects (NTD). The mechanism of VPA-induced NTD is unclear, but oxidative stress may be implicated. To understand how embryotoxic oxidative stress may occur, we measured superoxide dismutase (SOD) activity following VPA treatment in the embryonic pluripotent P19 mouse carcinoma cell line. In undifferentiated P19 cultures treated with VPA (5 mM), dichlorofluorescein fluorescence increased 15% compared to untreated controls over 20 min, indicating a modest, yet statistically significant increase in ROS generation. Undifferentiated P19 cells were treated with VPA for 6 h, after which total SOD and mitochondrial SOD (SOD2) activities were measured. VPA treatment decreased total SOD activity by approximately 20% but SOD2 activity was undetectable; but this was not a consequence of changes to SOD (SOD1 or SOD2) protein concentrations. Interestingly, glutathione redox state increased from -262 mV to -245 mV after a 6 h treatment with VPA, indicating significant oxidation of the cellular redox environment. Measurement of mitochondrial superoxide levels showed an increase following VPA treatments. While it is unlikely that VPA works directly as an oxidant, these data suggest that VPA may promote oxidative stress through an alternative means, such as via the inhibition of SOD activity and thus, allow for an increase in ROS. Importantly, VPA is a known deacetylase inhibitor, and SOD2 function is regulated by acetylation. As such, we evaluated the acetylation state of SOD2 to determine potential disruption via acetylation. Treated undifferentiated P19 cells showed a significant increase in SOD2 acetylation. However, in fully differentiated P19-derived neurons, cells showed no such SOD2 acetylation. Additionally, pretreatment with dithiole-3-thione (D3T), a Nrf2 activator of the antioxidant response, attenuated VPA-induced mitochondrial ROS production and SOD2 acetylation and improved SOD2 activity, suggesting Nrf2 as a potential means to reduce VPA-mediated oxidative stress. To evaluate the effects in the embryo proper, gestational day 8 mouse embryos were treated with VPA in culture for 6 h. Similar to P19 cells, VPA-treated neurulating embryos showed significant SOD2 acetylation and a concomitant decrease in total SOD activity. These data support a similar consequence of VPA-induced oxidative stress in embryos as is demonstrated in our cellular model. Since no SOD2 acetylation is observed in differentiated neurons and VPAinduced SOD2 acetylation occurs more prevalently in undifferentiated/differentiating cells, these data purport means by which VPA preferentially induces oxidative stress in developing systems.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2 • −) have been shown to serve as messengers in biological signal transduction, and many prokaryotic and eukaryotic proteins are now known to have their function controlled via ROS-mediated oxidation reactions occurring on critical cysteine residues. The tumour-suppressor protein p53 is involved in the regulation of a diverse range of cellular processes including apoptosis, differentiation, senescence, DNArepair, cell-cycle arrest, autophagy, glycolysis and oxidative stress. However, little is understood about the specific molecular mechanisms that allow p53 to discriminate between these various different functions. p53 is a multiple cysteine-containing protein and there is mounting evidence to suggest that redox-modification of p53 Cys residues participate in control of its biological activity. Furthermore, p53 activity has been linked to intracellular ROS levels. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers superior mass resolving power and mass measurement accuracy, which is beneficial for the study of intact proteins and the characterisation of their posttranslational modifications (PTMs). The primary goal of the work described in this thesis was to employ FT-ICR mass spectrometry to investigate the molecular details of p53 redox-regulation. The relative reactivity of each of the ten cysteine residues in the DNA-binding core domain of recombinant human p53 was characterised by treatment with the Cys-alkylating reagent N-ethylmaleimide (NEM) under various conditions. A combination of top-down and middle-down FT-ICR MS was used to unambiguously identify Cys182 and Cys277 as sites of preferential alkylation. These results were confirmed by site-directed mutagenesis. Interestingly, Cys182 and Cys277 have previously been implicated in p53 redox-regulation. Alkylation beyond these two residues was found to trigger rapid alkylation of the remaining Cys residues, presumably accompanied by protein unfolding. These observations have implications for the re-activation of mutant p53 with Cys-targeting compounds which result in the death of cancer-cells. Furthermore, the molecular interaction between p53 and the ROS hydrogen peroxide was investigated. p53 was found to form two disulfide bonds upon treatment with H2O2. An enrichment strategy was developed to purify oxidised p53 and top-down FT-ICR mass spectrometry revealed unambiguously that Cys176, 182, 238 and 242 were the oxidised residues. Interestingly, Cys176, 238 and 242 are Zn2+- binding residues suggesting that p53 contains a zinc-redox switch. The mechanism of H2O2 oxidation was investigated, and revealed that oxidation via an alternative pathway results in indiscriminate over-oxidation of p53. Moreover, Cys176, 238 or 242 was shown to act as a nucleophile, and the intracellular antioxidant glutathione (GSH) did not prevent oxidation of the Zn2+-binding Cys residues, providing further evidence for a role in p53 redox-regulation. This study has revealed hitherto unknown details regarding the chemistry of cysteine residues within the important tumour-suppressor protein p53. Furthermore, the analytical power of FT-ICR MS for the study of multiple Cys-containing proteins has been very clearly demonstrated.

Mestrado em Biologia Molecular e Celular
Microglial cells are the resident immune cells of central nervous system (CNS) and the major players in neuroinflammation. These cells are also responsible for surveilling the neuronal microenvironment, and upon injury to the CNS they change their morphology and molecular profile and become activated. Activated status is associated with microglia proliferation, migration to injury foci, increased phagocytic capacity, production and release of reactive oxygen species (ROS), cytokines (pro- or anti-inflammatory) and reactive nitrogen species. Microglia activation is crucial for tissue repair in the healthy brain. However, their chronic activation or deregulation might contribute for the pathophysiology of neurodegenerative diseases. A better understanding of the mechanisms underlying microglial cell activation is important for defining targets and develop appropriate therapeutic strategies to control the chronic activation of microglia. It has been observed an increase in profilin (Pfn) mRNA in microglial cells in the rat hippocampus after unilateral ablation of its major extrinsic input, the entorhinal cortex. This observation suggested that Pfn might be involved in microglia activation. Pfn1 is an actin binding protein that controls assembly and disassembly of actin filaments and is important for several cellular processes, including, motility, cell proliferation and survival. Here, we studied the role of Pfn1 in microglial cell function. For that, we used primary cortical microglial cell cultures and microglial cell lines in which we knocked down Pfn1 expression and assessed the activation status of microglia, based on classical activation markers, such as: phagocytosis, glutamate release, reactive oxygen species (ROS), pro- and anti-inflammatory cytokines. We demonstrated that Pfn1 (i) is more active in hypoxia-challenged microglia, (ii) modulates microglia pro- and anti-inflammatory signatures and (iii) plays a critical role in ROS generation in microglia. Altogether, we conclude that Pfn1 is a key protein for microglia homeostasis, playing an essential role in their activation, regardless the polarization into a pro or anti-inflammatory signature.
As células da microglia são células imunes residentes no sistema nervoso central (SNC) e desempenham um papel importante em processos neuroinflamatórios. Estas células são responsáveis por monitorizar o parênquima neuronal, sendo capazes de responder rapidamente a danos no SNC. Após ativação, a microglia altera a sua morfologia e o seu perfil de expressão de proteínas. O processo de ativação induz a proliferação, migração para a foco da lesão, aumento da capacidade fagocítica, bem como produção e libertação de espécies reativas de oxigénio (EROs), espécies reativas de azoto e citocinas (pro- e anti-inflamatórias). A ativação da microglia é essencial para a reparação de tecidos e a manutenção da homeostasia do SNC. No entanto, a ativação crónica ou a sua desregulação podem contribuir para a patofisiologia de doenças neurodegenerativas. Assim sendo, o estudo dos mecanismos subjacentes à ativação das células da microglia é importante para ajudar a definir e desenvolver estratégias terapêuticas apropriadas para prevenir a sua ativação crónica. Um estudo anterior reportou o aumento dos níveis de RNAm da profilina (Pfn) em células da microglia no hipocampus de ratos após lesão unilateral no córtex entorrinal, sugerindo que a Pfn poderá estar envolvida no processo de ativação da microglia. A Pfn1 é uma proteína de ligação à actina que regula a polimerização do citoesqueleto de actina, sendo importante em diversos processos celulares, incluindo motilidade, proliferação e sobrevivência. Neste trabalho, nós estudamos o papel da Pfn1 na função da microglia. Para tal, utilizamos linhas celulares e células primárias de microglias corticais de rato nas quais reduzimos a expressão da Pfn1 e avaliamos o seu estado de ativação com base em marcadores clássicos de ativação, tais como: fagocitose, libertação de glutamato, produção e libertação de EROs e citocinas pro- e anti-inflamatórias. Nós demonstramos que a Pfn1 (i) se encontra mais ativa após estímulo da microglia por hipoxia, (ii) modula as assinaturas pro- e anti-inflamatória da microglia e (iii) desempenha um papel importante na produção de EROs pela microglia. Nesse estudo concluímos que a Pfn1 é uma proteína importante para o funcionamento da microglia, desempenhando um papel essencial na ativação da microglia, independentemente da polarização pró ou anti-inflamatória.

Cardiomyopathies and arrhythmias are major causes of death in untreated hereditary haemochromatosis, acute iron poisoning and during secondary iron overload resulting from repeated blood transfusions in β-thalassaemia. Iron overload cardiomyopathies are associated with systolic and diastolic dysfunction, suggesting that Ca2+ homeostasis is impaired. However, the cellular mechanisms of these dysfunctions are unknown. The data presented in this thesis establishes for the first time iron effects on cardiomyocyte Ca2+ handling, as well as the potential cellular substrates responsible for this impairment during iron overload. Exposure of isolated rat ventricular cardiomyocytes to 200μM iron led to biphasic changes in systolic Ca2+ release. Phase 1: an initial reduction of systolic Ca2+ release followed by; Phase 2: increased Ca2+ release with arrhythmogenic spontaneous Ca2+ release, cell contracture and cell death. There is evidence that Fe2+ enters cardiomyocytes via L-type Ca2+ channels (LTCC) and reduces the Ca2+ trigger. The close apposition of LTCCs to cardiac ryanodine receptors (RyR2) suggests RyR2 may be a first target. Indeed RyR2 activity was drastically reduced on exposure to nanomolar [Fe2+] in single channel studies. Together with evidence that Fe2+ may reduce the Ca2+ trigger from LTCC, this is consistent with iron reducing sarcoplasmic reticulum (SR) Ca2+ release during Phase 1. In Phase 2, the presence of spontaneous Ca2+ release events is consistent with SR Ca2+ overload. Indeed, in single rat ventricular cardiomyocytes SR Ca2+ content was found to be increased by 27% during Phase 2. The cellular substrates responsible for this increased SR Ca2+ content were 2-fold: 1) through reduced extrusion via both the Na+ Ca2+ Exchanger (NCX) and Plasmalemmal Ca2+ ATPase (PMCA) and 2) through increased resequestration via the SR Ca2+ ATPase. Iron catalyses the production of reactive oxygen species (ROS) during the Fenton reaction. To investigate whether iron effects might be due to ROS, I used the cell permeant ROS scavenger Tempol. Tempol attenuated Phase 2 effects but Phase 1 effects were not affected. This is consistent with the hypothesis that Phase 1 effects were due to direct effects of Fe2+ affecting LTCC trigger and RyR2 function. The attenuation of Phase 2 effects suggests that ROS damage to key Ca2+ handling mechanisms, such as NCX and PMCA might account for a reduced Ca2+ extrusion and subsequent SR Ca2+ overload.

Overall, this doctoral study aimed to contribute to the existing knowledge and clinical practice regarding male infertility, especially in relation to the potential sources and clinical impact of sperm apoptosis and reactive oxygen species (ROS) in the context of assisted reproductive technology (ART). This thesis encompasses three independent studies, each of which addresses a single aim related to different aspects of male infertility and ART practices. Study 1 examined the relationship between sperm apoptosis and ROS levels and ART outcomes. On the day of oocyte retrieval, the levels of sperm apoptosis (measured using Annexin V staining) and intracellular ROS (measured using dihydroethidium staining) were measured in neat and processed semen samples from a total of 170 male patients whose partners were undergoing in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) treatment cycles. In general, higher levels of apoptosis and ROS were associated with lower sperm motility, sperm count and advanced paternal age. No correlation was observed between sperm apoptosis and fertilisation rate, blastulation rate, day 5 embryo quality (D5EQ) and clinical pregnancy. Similarly, there was no correlation between sperm ROS levels and fertilisation rate and clinical pregnancy. However, higher levels of ROS might be associated with poorer early stages of embryonic development, as indicated by blastulation rate and D5EQ. Together, the findings suggest that although sperm apoptosis and ROS had an adverse effect on sperm parameters, the effect on clinical outcomes was minimal. Study 2 compared the efficacy of three sperm preparation methods, namely density gradient centrifugation (DGC), the swim-up method and SeaforiaTM, in reducing the levels of sperm apoptosis and ROS. The measurement of sperm apoptosis and ROS levels were performed on a total of 17 pooled semen samples before and after sperm preparation. The main findings suggest that all three methods effectively reduced sperm apoptosis levels in the neat samples. However, only swim-up and SeaforiaTM were effective in reducing the ROS levels. Compared with DGC and swim-up, SeaforiaTM also produced samples with a higher total yield. Study 3 investigated the effects of different time intervals during ART sperm preparation on clinical outcomes. Three time intervals including pre-wash interval (time between ejaculation and sperm preparation), post-wash interval (time between sperm preparation and insemination/sperm injection) and total interval (time between ejaculation and insemination/sperm injection) were calculated and retrospectively analysed from a total of 8,079 IVF/ICSI cycles. The results showed that prolonged pre- and post-wash and total intervals had an adverse effect on sperm motility and clinical outcomes including fertilisation rate and pregnancy outcomes. Intracellular ROS production during prolonged incubation was considered to be the most likely facilitator of this observed effect. In summary, this thesis demonstrates that although their effect on ART outcomes were at low levels, higher levels of sperm apoptosis and ROS were associated with poorer sperm parameters, and suggestive of, a lower chance of natural conception. The cumulative findings of this thesis are indicative of the central role of ROS levels in sperm physiological functions and suggest that minimising intracellular ROS production through sperm preparation techniques and time optimisation may be beneficial to improving ART outcomes

L’activation électrique des cardiomyocytes, via le courant de dépolarisation qu’elle induit, est primordiale dans la contraction cardiaque qui requiert l’adéquation de la production d'énergie par les mitochondries et des besoins énergétiques de l’appareil contractile. Les espèces radicalaires de l'oxygène (ROS) ont été récemment impliquées dans la régulation de nombreux acteurs du couplage excitation-contraction cardiaque. L’objectif de ce travail est d’explorer l'implication des ROS d'origine mitochondriale dans la régulation du couplage excitation-contraction au niveau du cardiomyocyte, en conditions physiologiques et pathologiques. C’est à cette fin qu’un modèle de surproduction endogène de ROS par addition de succinate - substrat énergétique mitochondrial - dans des cardiomyocytes isolés de cœur de rat a été mis au point. Différents protocoles pharmacologiques utilisant différents antioxydants (Trolox, MitoTEMPO et EUK) nous ont permis d’établir l'origine mitochondriale des ROS produits en présence de succinate. Enfin une étude pharmacologique utilisant la molécule OP2113 (inhibiteur spécifique de la production mitochondriale de ROS au niveau du complexe I de la chaine respiratoire- Brevet Philippe Diolez) nous a permis d'établir qu'environ 80% de cette production provenait du complexe I de la chaîne respiratoire. Notre travail s’est alors attaché à étudier, sur des cardiomyocytes isolés battants, les effets des ROS mitochondriaux sur différents paramètres du couplage excitation-contraction. Le succinate, via une surproduction de ROS mitochondriaux, provoque une baisse de 50% de l'amplitude de la contraction. L’amplitude de la contraction initiale est rétablie en présence de Trolox, MitoTEMPO ou OP2113, ce qui indique l'implication des ROS mitochondriaux et plus spécifiquement ceux produits au niveau du site IQ. Cette surproduction de ROS induit également une diminution de l’amplitude du transitoire calcique impliquant une diminution de la concentration en calcium systolique au cours de la contraction. Ces effets sont annulés en présence de Trolox et OP2113. Nous nous sommes alors attachés à moduler la concentration mitochondriale de calcium grâce à la cyclosporine A (inhibiteur du pore de transition mitochondrial) et au Ru360 (inhibiteur de l'entrée de calcium dans la mitochondrie). Il s’est avéré que ces inhibiteurs induisent une production de ROS insensible au Trolox et à OP2113 dont l'origine reste à établir. En conclusion une augmentation de la production mitochondriale de ROS se traduit par une diminution de la contraction intracytosolique et du transitoire calcique. L'ensemble de nos résultats illustre donc l’importance majeure de la mitochondrie dans le couplage excitation-contraction. Nos résultats ouvrent de nouvelles perspectives thérapeutiques dans le contexte de l’insuffisance cardiaque aigue ou chronique
The electrical activation of the cardiomyocyte through a generated depolarisation current is essential in the cardiac contraction, which requires the adequacy of the mitochondrial energy production and the energy needs of the contractile system. Radical oxygen species (ROS) have recently been involved in the regulation of many actors of the excitation-contraction coupling. The aim of this study was to explore the involvement of mitochondrial ROS in the regulation of the excitation-contraction coupling in cardiomyocytes, under physiological and pathological conditions. A model of endogenous ROS overproduction with the use of succinate was developed in isolated rat cardiomyocytes. Different pharmacological protocols, using various antioxidants (Trolox, Mito-Tempo,EUK and OP2113) allowed us to establish the mitochondrial origin of ROS production. Finally, the use of OP2113, (a specific inhibitor of mitochondrial ROS production, Patent P.Diolez) enabled us to establish that approximately 80 % of ROS production came from complex I in the respiratory chain. To start with, isolated cardiomyocytes were used to study the effects of mitochondrial ROS on different excitation-contraction coupling parameters. Succinate induced an overproduction of mitochondrial ROS, which lead to a drop of 50% of the contraction amplitude. The initial amplitude of contraction wasrecovered with addition of Trolox, Mito Tempo or OP2113, which demonstrates the implication of mitochondrial ROS produced at the site of Iq. Secondly, the overproduction of ROS leadsto a decrease of the calcium transient amplitude, due to a decrease of systolic calcium concentration during contraction. These effects were inhibited by Trolox and OP2113. Finally, mitochondrial calcium concentration was modulated with the use of Cyclosporin A (mitochondrial transition pore inhibitor) and Ru360 (mitochondrial calcium entry inhibitor). The inhibitors induced a ROS production unresponsive to Trolox and OP2113. The origin of which remains to be established. To conclude, an increase of mitochondrial ROS production results in a decrease of contraction amplitude and calcium transients. Our overall results demonstrate the critical significance of the mitochondrion in the excitation-contraction coupling. Our results open new therapeutic perspectives in the context of acute or chronic heart failure

The goal of cancer immunotherapies is to boost the immune system's ability to detect tumor antigens and mount an effective anti-tumor immune response. Currently, adoptive T cell transfer therapy (ACT), the administration of ex vivo expanded autologous tumor-specific T cells, is one of the most promising immunotherapies under development; however, its efficacy has been limited so far with a mere 10% complete remission rate in the most successful clinical trials. The prolonged ex vivo culture process is a potential reason for this ineffectiveness because the transfused cells may reach replicative senescence and immunosenescence prior to patient transfer. The objective of this thesis is to offer two approaches towards an improvement of treatment efficacy. First, we generated a 'senescence metric' from the identification of biomarkers that can be used in the clinic towards predicting age and responsiveness of ex vivo expanded T cells. The second approach is to understand at the molecular level the changes that occur during ex vivo expansion to devise improved ACT protocols. In particular, we focused on the shift towards a pro-oxidizing environment and its potential effects on calcium signaling. The combined development and application of microfluidic technologies and computational models in this thesis facilitated our investigations of the phenotypic and signaling changes occurring in T cells during the progression towards immunosenescence. Our findings of altered T cell properties over long term culture provide insight for the design of future cancer immunotherapy protocols.

Scopo della ricerca sviluppata durante questi tre anni di Dottorato è quello di spiegare i meccanismi sottesi alla risposta allo stress ossidativo nell'uomo a livello integrativo e molecolare. Per questo fine, nel primo anno di Dottorato, si è innanzi tutto implementato un metodo quantitativo assoluto atto a valutare la produzione dei radicali liberi. Come è noto ciò che importa è l’equilibrio tra produzione di Specie Reattive dell’Ossigeno (ROS) e la disponibilità di difese antiossidanti. Quando questo equilibrio viene meno, si parla di Stress Ossidativo, con conseguenze a carico degli acidi nucleici, dei lipidi e delle proteine, compromettendo il metabolismo e la vitalità cellulare, fino a indurre apoptosi o necrosi. Questo è un fenomeno ricorrente in molte malattie acute e croniche oltre che nel fisiologico processo di invecchiamento. Allo scopo si è utilizzata la tecnica di Risonanza Paramagnetica Elettronica (EPR) accoppiata a specifiche sonde molecolari (spin trapping) per sviluppare un metodo quantitativo “in vivo” in grado di monitorare la produzione di ROS in campioni biologici quale il sangue e sue frazioni. Gli inizi del mio periodo di Dottorato sono fortunatamente coincisi con l’acquisizione da parte dell’Istituto di Bioimmagini e Fisiologia Molecolare (IBFM) del CNR (Lita di Segrate), di uno strumento EPR che risponde alle esigenze di maneggevolezza e portabilità (e-scan Bruker BioSpin, Germania), di recente immissione sul mercato, che opera in onda continua (CW), nella banda X delle µW, in grado di rilevare concentrazioni molto basse (nM) in piccoli campioni (50 µl), utilizzando una cavità risonante le cui proprietà ottimizzano il fattore di riempimento, conferendo un’elevata sensibilità allo strumento. L’attendibilità dell'efficacia del metodo sviluppato è stata verificata con correlazioni tra metodi classici enzimatici e misure EPR in vari stati: fisiologici e parafisiologici. Trattandosi della messa a punto di un metodo di misura completamente innovativo e mancando pertanto dati di letteratura cui fare riferimento, si sono rese necessarie molte prove sperimentali da campioni ematici, in un vasto gruppo di soggetti, con tantissime acquisizioni modificando e ottimizzando di volta in volta i parametri e le condizioni (T, pO2) Questo procedimento è stato lento e laborioso; una volta superate le difficoltà tecniche, si è avuto a disposizione un piccolo catalogo di misurazioni che è servito ad ottenere i primi dati sperimentali che saranno illustrati nei primi paragrafi di questa tesi. Si è inoltre cercata una correlazione statistica tra la produzione di ROS determinata con tecnica EPR ed i marcatori dello stress ossidativo ottenuti dai vari test convenzionali enzimatici riportati in letteratura quali il dosaggio da sostanze reattive all'acido tiobarbiturico (TBARS) e carbonili proteici (PC) in grado di quantizzare il danno “a posteriori” prodotto dai ROS su lipidi e proteine rispettivamente. Una correlazione sistematica tra i dati EPR e i dati enzimatici non era certa, in quanto questi ultimi rivelano un danno conclamato. Tuttavia, è stata trovata una significativa correlazione (ANOVA, p<0.05) in soggetti a riposo, tra la produzione di ROS/TBARS e PC (rispettivamente: r2=0.74 e r2=0.60). Per rispondere allo scopo della ricerca, abbiamo assunto come modello l’ipossia studiata 1) come situazione che induce uno sbilanciamento tra Stress Ossidativo e capacità antiossidante e 2) nel tentativo di identificare i possibili meccanismi di ripristino dell’equilibrio. Dall'insieme dei dati raccolti, al di là dei risultati specifici, possiamo evincere che anche se la misura quantitativa assoluta della produzione di ROS (umol . mim-1) non può essere un parametro generalizzato, perché funzione di variabili fisico-chimiche (pO2, Temperatura, concentrazione della sonda), acquisizione ed elaborazione dei parametri vanno comunque prese in considerazione. Dal calcolo dei limiti di detenzione (LOD): 30 · 10-3 mM, e quantificazione (LOQ): 100 · 10-3 mM, lo strumento si è rilevato sensibile e affidabile. Allo stesso tempo, va tenuto presente che il dato ottenuto rappresenta la produzione di ROS non tamponati dal sistema antiossidante. Per questo, si è ritenuto che la misura su campione ematico fosse rappresentativa della risposta sistemica integrativa. In particolare, il sangue capillare, oltre alla sua elevata sensibilità e riproducibilità (0,5% di differenza tra misure ripetute), ci ha permesso di disegnare un protocollo agile mini-invasivo. Da quanto fin qui esposto, possiamo ricavare che, al di là della ricerca di un dato assoluto da riportare in letteratura, è una valida procedura che ogni soggetto, monitorato nel tempo risulti controllo di se stesso, ovvero che a indice della risposta equilibrio/squilibrio vengano quantizzate le variazioni. Nel tentativo di identificare i possibili meccanismi di ripristino dell’equilibrio dello stress ossidativo, gli esperimenti sono stati condotti su: A) soggetti sani, che come tali sono caratterizzati da una condizione di equilibrio tra ROS e difese antiossidanti, B) soggetti affetti da patologie neurodegenerative, per i quali era logico attendersi, come confermato dalla realtà, che fossero caratterizzati da una condizione di conclamato squilibrio, rivelabile da un significativo incremento di ROS prodotti in condizioni di riposo. In soggetti sani, si sono disegnati, messi a punto e applicati differenti protocolli sperimentali, per testare l’effetto: a) dell’esercizio fisico (stato ipossico transitorio) di breve (hockeisti e nuotatori) e lunga durata (atleti di triathlon); b) dell’allenamento (nuotatori); c) dell’ipossia normobarica e ipobarica acuta e prolungata (giovani soggetti sedentari); d) dell’assunzione di molecole antiossidanti sulla produzione di ROS. In patologie neurodegenerative (pazienti affetti da: Neuropatia Diabetica (tipo II DN), decadimento cognitivo lieve (MCI) e Sclerosi Laterale Amiotrofica sporadica (sSLA)) dopo aver rilevato uno squilibrio in condizioni basali, è stato studiato l’effetto dell’esercizio/allenamento (SLA) o dell’assunzione di antiossidanti (tipo II DN) sulla possibilità di un potenziale ripristino a valori di equilibrio. Importanti risultati sono emersi dall’analisi dei dati registrati nelle varie condizioni sperimentali e sono illustrati in questa tesi. ll tema comune di tutti i test, oltre all’evidente studio sulla produzione dei ROS, è stato quello dell’ipossia. Infatti l’Ipossia, nelle sue varie espressioni (intese come deficienze parziali o totali di O2 nel nostro organismo), è stato elemento caratterizzante in tutti i protocolli che abbiamo testato. I protocolli di studio su pazienti sono tuttora in corso; pertanto i dati illustrati devono intendersi come preliminari. Nei soggetti patologici si è costatata una differente produzione di ROS in condizioni di riposo, mostrando un valore significativamente (p<0,05) più alto nei pazienti affetti da SLA (+20%) rispetto ai soggetti sani. L’allenamento, in questi pazienti, sembra giocare un ruolo favorevole nel diminuire la produzione di ROS: un decremento del 7% è stato calcolato nei valori a riposo pre e post allenamento. Un sicuro effetto positivo dell’allenamento controllato, dopo otto settimane, è stato osservato nei nuotatori (-25%) sulla produzione di ROS (2.24 ± 0.14 μmol · min-1). Tutti questi esperimenti sono stati condotti e i risultati ottenuti durante il secondo e terzo anno della mia Scuola di Dottorato. A parte gli specifici risultati ottenuti dal set di dati raccolti, osservazioni generali possono essere riassunte come segue: 1) Non vi è alcun modo per ottenere dati per quantificare il volume totale di ROS prodotta da un soggetto in qualsiasi condizione. Il dato EPR ricavato dalle misurazioni rappresenta l’eccesso di ROS che il sistema ha prodotto (μmol · min-1) in una determinata condizione, ed è quindi un tasso di produzione di ROS Inoltre si è scelto di effettuare la misura da sangue invece che da campioni di tessuto in modo da ottenere dati il più possibile sistematici. In particolare, il sangue capillare (rispetto al sangue venoso o plasma) ha dato la possibilità di sviluppare un metodo di misurazione affidabile, relativamente semplice e mini-invasivo. Tuttavia quando si passa da misurazioni di sangue capillare a venoso, bisogna prendere in considerazione la differente pO2. (la produzione di ROS in sangue venoso è stata trovata circa il 18% inferiore a quella nel sangue capillare: 1.79 ± 0.12 vs 1.48 ± 0.29 μmol·min-1 rispettivamente). 2) I dati non possono essere presi come parametri generali, poiché dipendono strettamente da variabili fisico-chimiche (T, pO2) Da tutti questi punti, si può evincere che, al di là della ricerca di un dato assoluto da riportare in letteratura, è una valida procedura che ogni soggetto, monitorato nel tempo risulti controllo di se stesso, ovvero che a indice della risposta equilibrio/squilibrio vengano quantizzate le variazioni. 3) Tuttavia si è consapevoli che il dato fornito da EPR deve essere integrato con dati sistemici quali: analisi ematochimiche di laboratorio (ematocrito, ferro, colesterolo, piastrine, neutrofili, linfociti, monociti), saturazione dell’ossigeno, cinetica degli scambi gassosi (VO2max). Si ritiene che il metodo possa trovare applicazione i) medicina dello sport offrendo un valido aiuto nei centri sportivi per monitorare l’efficacia dell’allenamento e gli eventuali effetti dello stress provocato ed eventualmente migliorare i protocolli; ii) alla medicina di montagna per lo studio delle patologie da alta quota, poiché l’ipossia è il primo e più delicato problema dell’altitudine sin dalla quota media (1800m); iii) ai centri clinici/diagnostici per monitorare gli effetti delle terapie/trattamenti sulla progressione di patologia. Seconda parte della mia attività di ricerca, è stato lo studio in vitro dell’ipossia da un punto di vista molecolare mediante misurazioni spettroscopiche EPR. Partendo da una visione molecolare, l’ipossia orchestra una moltitudine di processi e di pathway molecolari. Nel tradizionale meccanismo dell’ipossia si ha la presenza di una proteina eme in grado di rilevare il legame reversibile di O2 il quale causa un cambiamento allosterico nell’emo-proteina: da inattivo (forma ossigenata) ad attivo (forma de-ossigenata). Quindi l'attività di una proteina emo è determinata dalla presenza o assenza dell’ossigeno legato. Nella presente ricerca, la rilevanza di stati ipossici, dal punto di vista molecolare, atti ad essere rilevati sia in condizioni fisiologiche che patologiche è stato attribuito all’Ossido Nitrico (NO) e al ruolo della Mioglobina (Mb). E’ noto, infatti, che l’NO è un radicale libero particolarmente espresso in condizioni ipossiche in quanto è in grado di regolare la vasodilatazione, quindi aumenta l’afflusso di sangue e al tempo stesso può essere però dannoso andando ad inibire la catena respiratoria a livello del complesso IV. In letteratura sono riportate cinque isoforme di Mb umana (Mb I-V) presenti nel muscolo scheletrico in percentuali diverse (Mb I: 75%; Mb II: 20%; Mb III-V: 5%) le cui funzioni non sono ancora state chiarite. Prendendo a riferimento Mb II, queste proteine differiscono solo per un singolo residuo aminoacidico: E54K (Mb I), K133N(Mb III), R139Q (Mb IV) a R139W (Mb V). Inoltre popolazioni tibetane, native dell’alta quota, presentano come risposta all’ipossia alte capacità antiossidanti, alto flusso ematico sistemico, metabolismo ed efficienza meccanica migliore, alti livelli di metaboliti circolanti biologicamente attivi all’NO e sono caratterizzate da un significativo aumento della concentrazione di Mb a livello muscolare con particolare riferimento all’ isoforma 54E (Mb II). Il presente studio, grazie all’analisi combinata EPR e simulazioni di Dinamica Molecolare (MD), contribuisce ulteriormente a sottolineare il ruolo importante svolto dalla Mb in condizioni di ipossia; inoltre segna un primo passo verso l'identificazione dei diversi ruoli svolti dalle isoforme della Mb Importanti risultati funzionali si sono ottenuti dallo studio EPR: l’NO ha capacità di legame significativamente maggiore con l’isoforma 54E rispetto all’isoforma 54K; questa è più circoscritta alla struttura dello stato de-ossigenato e la capacità di legame diventa significativamente inferiore (p<0.01) e quasi identica per le due proteine in presenza di O2 anche a differenti livelli di pO2 (40 e 100 mmHg). Concludendo, osservazioni e prospettive future possono essere condensate nella convinzione, che confidiamo saranno condivise e verificate anche dal lettore, della validità del nuovo del metodo sviluppato e dei dati raccolti. Alcuni risultati, in particolar modo quelli concernenti gli stati patologici, sono da considerarsi preliminari; tuttavia la strada da percorrere è estremamente interessante e richiede ulteriori indagini.
The aim of the research developed in the time course of the three years of my Doctoral course in Molecular Medicine was to shed light into the mechanisms involved in Oxidative Stress responses in man at both integrative and molecular levels. To reach this general and high-level purpose we firstly focused our attention towards developing a method capable of returning an absolute quantitative estimation of Free Radicals production level. This particular aim was the subject of the first year of my researching activity. As a matter of fact, as is well known, in any physiological and/or pathological condition a subject could be involved, ‘oxidative stress’ results from the imbalance between Reactive Oxygen Species (ROS) production and his antioxidant capacity. Electron Paramagnetic Technique (EPR) resulted the method of choice since, as is well known, it is the only technique capable of returning, in a non-invasive way, the ‘intrinsic’ quantitative information (the signal is proportional to the number of excited electron spins). Indeed the beginning of my Doctoral period fortunately coincided with the acquisition by the Molecular Bioimaging and Physiology (IBFM) Institute of CNR (Lita Segrate) of an EPR instrument (e-scan Bruker BioSpin, Germany) of recent design, responding to the innovative characteristics of easy portability and handling. The spectrometer operates at the common X-Band microwave frequency and deals with very low concentration levels (nM) even in small sample volumes (50 μl). Aware of the novelty of the method, we first looked for a correlation between the attained EPR experimental data and those returned when adopting the classical enzymatic methods (e.g. ThioBarbituric Acid Reactive Substances (TBARS) and Protein Carbonyls (PC) data collected from the same subject). Indeed, a correlation could not, by principle, be expected, since, compared to the EPR data, enzymatic methods give a quantitative “a posteriori” evaluation of the damage produced by ROS, on lipids and cellular proteins. As a matter of fact, a positive correlation (ANOVA, p<0.05) was found between EPR ROS production data and TBARS (r2 ~ 0.7) as well as PC concentration (r2 ~ 0.6), which was found in resting subjects. Then, in order to achieve the overall objective of the research, we assumed ‘hypoxia’ as the ‘particular’ condition to be investigated, since it was viewed as a peculiar state capable of: 1) producing un imbalance between oxidative stress and anti-oxidant capacity; 2) offering the possibility of the identification of mechanisms involved in the perturbed balance restoring. To attain this ‘secondary’ aim, the experiments were carried out on: A) healthy subjects, that as such, were expected to start from a balance equilibrium condition; B) subjects affected by neurodegenerative pathologies, for whom, on the contrary, a sure imbalance condition was expected, that would be confirmed by a significant increment of ROS production, even at rest. However a fully innovative method had been developed, so that we lacked of referring literature data: a series of preliminary measurements on a homogeneous group of healthy subjects had to be firstly carried out, not only to optimize the acquisition and environmental conditions (T, pO2 that have to be carefully defined since they greatly affect EPR data), but also to reach reliable resting physiological values and range of variability. A great reproducibility of the data (μmol . min-1) was found on blood capillary samples taken from the same healthy subject six hours apart (r² = 0.99, ~ 0.5% discrepancy) and was calculated; limit of detection (LOD) and quantification (LOQ) of 30 ∙ 10-3 mM and 100 ∙ 10-3 mM were calculated respectively. In healthy subjects, the experimental protocols were designed in order to study the effects of: a) exercise (as inducing a transitory hypoxic state) of both short (in hockey players, swimmers) and long duration (in triathletes); b) training (as inducing a balance restore, in swimmers); c) hypoxia, both hypo- and normobaric (to study the effect of the assumed model condition itself, in young sedentary subjects); d) antioxidant molecules administration as an external way helping to restore the balance. In neurodegenerative pathologies (patients affected by: type II Diabetic Neuropathy (type II DN), Mild Cognitive Impairment (MCI) and sporadic Amyotrophic Lateral Sclerosis (sALS)) after checking the expected unbalance throughout a significant increase of ROS production (sALS versus healthy subjects: +20%, p<0.05) under resting conditions, the effect of training on the possibility of approaching a normal ROS production level was investigated. The collected data thus far, that have to be nevertheless considered as preliminary results, of the training effect in sALS patients showed a beneficial effects; an decrease ROS production resting value (about -7%) was calculated after twelve weeks of training by the patients. in swimmers a significantly lower (about -25%) basal ROS production value (2.24 ± 0.14 μmol · min-1) after eight weeks training period was calculated. All these experiments were carried out and results obtained during my second and third year of my Doctoral School. Besides and beyond the specific results obtained from the collected data set, pointed out throughout my thesis, general observations can be summarized as follows: 1) There is no way to obtain data quantifying the total amount of ROS produced by a subject under any condition. Rather, EPR data return amount of ROS there were not scavenged by the antioxidant system. In other words, under a given condition, they represent the excess of the ROS produced in a given time, that is ROS production rate (μmol · min-1). Moreover we chose to carry out the measurement on blood instead of tissue samples to obtain most possible integrative data. In particular capillary blood (with respect to venous blood or plasma) gave us the opportunity of developing a mini-invasive, reliable and easy measurement method. Nevertheless when going from capillary to venous data, the different blood pO2 must be taken into account. (ROS production in venous was found about 18% lower than in capillary blood: e.g. 1.79 ± 0.12 vs 1.48 ± 0.29 µmol · min-1 respectively). 2) The obtained data cannot be taken as general parameters, since they strictly depend on the physic-chemical variables (T, pO2) assumed for the measurement that must be taken into great account. From all these considerations, we can therefore conclude that beyond the possibility of obtaining absolute data to report for the first time in literature, this method was found as quite a sensitive and reliable mean to monitor the changes of each subject during time progression, therefore making each subject the control. 3) Despite the reliability and high reproducibility of the afore-developed method, we are anyway aware that EPR data have to be compared and integrated by collecting systemic data: hematochemical parameters (e.g. hematocrit, iron, cholesterol, platelets, neutrophils, lymphocytes, monocytes), oxygen saturation, maximal oxygen consumption (VO2max). Finally we are confident that our method will be in the near future considered suitable to be applied into different medical fields: i) sport medicine, as an effective available help in sports centers to set up and adjusts training protocols by monitoring training effects on oxidative stress. ii) mountain medicine, in studying pathologies induced by hypoxia following high altitude acute and chronic staying; iii) clinic and diagnostic centers, helping in monitoring progression of pathologies and/or effects of therapies. On the other side, second part of my researching activity, the study of hypoxia throughout EPR measurements, was carried out in vitro at a molecular level. In fact, hypoxia orchestrates a multitude of processes of molecular pathway responses. Nevertheless the traditional mechanism of sensing hypoxia involves the presence of a heme protein so that hypoxia can be detected by a reversible binding of O2 at the heme site, which causes an allosteric shift in the hemoprotein from inactive (oxyform) to active (deoxy) form. The activity of a hemoprotein is therefore determined by the presence or absence of bound oxygen. Among heme proteins, the relevance of hypoxic states, suitable to be encountered in both physiological and pathological conditions was, in the present research, made in conjunction with the supporting role recently attributed to deoxy-Myoglobin (Mb) as a ROS scavenger, with particular attention to a peculiar reactive species molecule, that is nitric oxide (NO). NO is a highly diffusive and reactive molecule, produced, in the cells, by the NO synthases enzymes (NOS), using L-arginine and O2 as substrates. Under normoxic conditions, if NO concentration becomes too high, oxy-Mb is able to scavenge and oxidize it into NO3-, maintaining mitochondrial O2 consumption, thus cellular respiration, at the optimum. On the contrary, under hypoxia, the low O2 availability leads to NOS inactivation (being substrate limited). The NO produced by Mb down-regulates mitochondria O2 consumption, contributing to elongate the intracellular oxygen gradient and limiting the formation of deleterious reactive oxygen species. In addition, Mb-produced NO might contribute to hypoxic vasodilatation. Thus, Mb’s nitrite reductase activity (very low pO2 level) might participate both in the modulation of tissue mitochondrial respiration and in the limitation of ischemia-reperfusion injury, under hypoxic. The relevance of hypoxic states, in conjunction with the supporting role recently attributed to deoxy-Myoglobin throughout its scavenging and vasodilatory properties was the rational basis for this study. As regard as Mb, in human’s skeletal muscle, up to five different Mb isoforms are reported in the literature (Mb I-V). Also different Mb percentages are present in the muscle: Mb I 75%, Mb II 29%, Mb III-V 5%. Taking Mb-II as a reference, these proteins differ only for one external amino acid and, namely, they can be seen as the E54K (Mb I), K133N (Mb III), R139Q (Mb IV) and R139W (Mb V) mutants, whose specific functions, if any, are completely unknown. In addition, these general observations were supported by specific findings on populations born and living in extreme hypoxic conditions possibly explaining their better metabolic and mechanic efficiency by the increase of Mb concentration, with particular reference to the 54E isoform (Mb II). Throughout a combined EPR and MD simulations analysis, the present study aimed to give experimental evidenced able to further underline the relevant role played by Mb under hypoxic conditions, then marking a first step towards the identification of different roles played by Mb isoforms, whose presence, among mammals, is a distinct feature of human Mb. Functional results obtained by the EPR study showed up not only a significantly greater NO binding capacity (more than double; p<0.01) of the 54E with respect to the 54K isoform was calculated from the EPR spectra integrated areas but even more circumscribed this property to the deoxygenate state, the binding capacity becoming significantly lower and almost the same for the two proteins in the presence of O2 at the two analyzed pO2 levels (40 and 100 mmHg). This novel and quite intriguing result was supported by structural considerations emerged by the MD simulation analysis and in turn mutually supported by structural EPR results. Concluding remarks and future perspectives can be summarized in our firm belief, which we are confident will be verified and accorded by the reader as well, of the novelty of the developed method and of the reported data. Indeed some of them, especially when concerning pathological subjects, have to considered ‘in fieri’, nevertheless the road to take is extremely interesting, and calls for further investigations.

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.

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.

Changes in intracellular [Ca2+] ([Ca2+]I) homeostasis, mitochondrial dysfunction and reactive oxygen species (ROS) are generally intertwined. Therefore, it is difficult to establish sequence of events and causal relationships. For instance, an increase in [Ca2+]I affects mitochondrial [Ca2+] and in most cases it is associated with an increased mitochondrial ROS formation, leading to the opening of the permeability transition pore (PTP), mitochondrial dysfunction and cell death. On the other hand, ROS modify [Ca2+]I homeostasis by acting at various sites involved in intracellular Ca2+ fluxes. The changes induced by [Ca2+]I on ROS generation and vice versa are very rapid, making it difficult to elucidate the primary event under both physiological and pathological conditions. ROS are produced at several intracellular sites, and in cardiac myocytes mitochondria are the most redox-active compartment. Mitochondria are very susceptible to oxidation, representing both a source and a target of ROS. Although the enzymes responsible for mitochondrial ROS formation under physiopathological conditions are still a matter of investigation. An additional matter of complexity is related to available techniques. While various methods are available to induce a primary and direct rise in [Ca2+]I, the existing protocols to trigger ROS production are far from being specific. An increase in ROS is generally obtained as a consequence of the exogenous administration of an oxidant (mostly H2O2) or by applying pathological stimuli (i.e., respiratory chain inhibition) that inevitably trigger several other effects, including alterations in [Ca2+]I homeostasis. Here we aimed at solving these issues by evaluating the effects of a primary dose-dependent increase in mitochondrial ROS levels on cell physiology, especially in neonatal rat ventricular myocytes (NRVMs). In order to induce a primary increase in mitochondrial ROS levels, we treated NRVMs with a paraquat analogue targeted to mitochondria (Mitochondrial Paraquat, MitoPQ). This compound accumulates selectively in the mitochondrial matrix and elicits ROS formation by means of redox cycling at the flavin site of Complex I of the electron transport chain (ETC). Initially, we established a dose-response curve with different doses of MitoPQ by evaluating ROS formation, mitochondrial function and cell function. We observed that cells exposed to high doses of MitoPQ displayed a dose-dependent increase in mitochondrial ROS levels. High doses led to decreased mitochondrial membrane potential (ΔΨm), PTP opening, impaired cytosolic [Ca2+] homeostasis and cell death. Subsequently, we wondered whether high doses of MitoPQ could trigger mitochondrial pathway involved in ROS formation. We focused our attention on the activity of monoamine oxidases (MAOs) that have been recently shown to contribute to oxidative stress in cardiac diseases. MAO inhibition prevented MitoPQ-induced ROS formation along with mitochondrial dysfunction and cell death. Besides this amplification pathway, these findings provide the direct evidence that ROS produced within mitochondria can affect cytosolic processes and especially [Ca2+]I. The cross-talk between Ca2+, ROS and mitochondrial function plays an essential role in diabetic cardiomyopathy (DCM). Therefore we extended the results obtained with MitoPQ, and especially MAO-induced amplification and alterations in [Ca2+]I homeostasis induced by mitochondrial ROS to a cellular model of DCM. NRVMs treated with high glucose (HG) and interleukin-1β (IL-1β) displayed a significant increase in mitochondrial H2O2 levels, both after prolonged (48 h) and short (1 h) treatment. MAO inhibitor pargyline prevented this observed increase in ROS levels. Hence, we tested the effect of MAO inhibition on cytosolic [Ca2+] homeostasis in cells exposed to the diabetes-like protocol. Surprisingly, HG and IL-1β did not perturb significantly Ca2+ oscillatory parameters (i.e. amplitude, frequency and area under curve), while pretreatment with pargyline or N-(2-Mercaptopropionyl)glycine (MPG) worsened contractility defects induced by HG and IL-1β, compromising the oscillatory pattern. These unexpected findings suggest that a basal oxidative tone is necessary for a proper Ca2+ cycling in cardiomyocytes exposed to HG and IL-1β. Noteworthy, diabetes is related to pancreatic β-cells dysfunction. In pancreatic β-cell, short bursts of ROS have been shown to contribute to [Ca2+]I homeostasis and Ca2+-dependent insulin secretion. Although in diabetes a sustained oxidative stress leads to β-cell dysfunction, a primary and moderate increase in ROS levels could shape Ca2+ transients promoting insulin secretion. We tested this hypothesis on Min6 β-cells, an insulinoma cell line that can be used as a model of diabetes applied to pancreatic β-cells. We evoked a primary increase in intracellular ROS levels by treating Min6 β-cells with aluminium phthalocyanine chloride (AlClPc), a photosensitizer commonly used in photodynamic therapy. Photoactivation of AlClPc induces chemical changes into neighboring molecules leading to ROS production in amount depending on the intensity of the LED illumination. Results obtained showed that modulation of ROS induced by photoactivation of AlClPc or Sarco/Endoplasmic Reticulum Ca2+ ATPases (SERCA) inhibition by thapsigargin (Tg) resulted in acceleration of Ca2+ to a similar degree, indicating that a primary and controlled increase in cytosolic ROS levels modifies but not abolishes Ca2+ oscillation via a modulation of SERCA. Overall, our data showed that low levels of mitochondrial ROS slightly modulate [Ca2+]I homeostasis without affecting mitochondrial and cell function. On the other hand, mitochondrial ROS can be elevated by increase in mitochondrial Ca2+ uptake. We used this notion to investigate whether mitochondrial Ca2+ uniporter (MCU) overexpression could modify mitochondrial ROS formation and consequently mitochondrial and cardiomyocyte viability. Cells overexpressing MCU displayed an increase in ROS levels that was causally related to an increased tolerance to anoxia/reoxygenation (A/R) injury in a process that involved AKT activation. These findings demonstrate that a slight increase in mitochondrial ROS formation exerts a protective effect. To test this hypothesis, we investigated whether mitochondrial ROS induced by low doses of MitoPQ could decrease the susceptibility to A/R injury. Not only this hypothesis was verified, but also in this case protection was due to AKT activation. The data from this work demonstrate that mitochondrial ROS formation can trigger a wide array of responses with a clear separation between ROS levels required to elicit beneficial or detrimental effects. Changes in [Ca2+]I homeostasis can be upstream but also downstream of mitochondrial ROS formation and the cardioprotective effect linked to a slight increase in mitochondrial ROS levels appears to depend on AKT activation.
Variazioni dell’omeostasi del [Ca2+] ([Ca2+]I) intracellulare, disfunzione mitocondriale e specie reattive dell’ossigeno (ROS) sono generalmente interconnessi. Di conseguenza, è difficile stabilire una sequenza di eventi e relazioni di causa ed effetto. Per esempio, un aumento di [Ca2+]I influenza il [Ca2+] mitocondriale e, in molti casi, è associato con un aumento di ROS mitocondriali che portano all’apertura del poro di transizione mitocondriale (PTP), disfunzione mitocondriale e morte cellulare. D’altro canto, i ROS modificano l’omeostasi del [Ca2+]I interagendo con vari siti coinvolti nei flussi di Ca2+ intracellulari. I cambiamenti indotti dal [Ca2+]I sulla formazione dei ROS e viceversa sono molto rapidi, rendendo difficile definire l’evento primario sia in seguito sia ad un evento fisiologico che ad un evento patologico. I ROS sono prodotti in vari siti intracellulari e nei cardiomiociti i mitocondri sono il compartimento a più alto potenziale ossidoriduttivo. I mitocondri sono molto sensibili all’ossidazione, rappresentando sia una fonte che un bersaglio dei ROS. Tuttavia gli enzimi responsabili per la formazione dei ROS mitocondriali in condizioni patofisiologiche sono ancora motivo di discussione. Un’ulteriore complicazione è legata alle tecniche disponibili. Mentre esistono vari metodi per indurre un aumento primario e diretto di [Ca2+]I, i protocolli esistenti per innescare una produzione di ROS sono tutt’altro che specifici. Un aumento di ROS è in genere ottenuto come conseguenza della somministrazione di un ossidante esogeno (soprattutto H2O2) o applicando stimoli patologici (i.e. inibizione della catena respiratoria) che innescano inevitabilmente diversi altri effetti, incluse alterazioni nell’omeostasi del [Ca2+]I. In questo lavoro ci poniamo come obiettivo la risoluzione di questi problemi valutando gli effetti di un aumento primario dose-dipendente dei livelli di ROS mitocondriali sulla fisiologia cellulare, specialmente in cardiomiociti neonatali di ratto (NRVMs). Per indurre un aumento primario dei livelli di ROS mitocondriali, abbiamo trattato i NRVMs con un analogo del paraquat diretto ai mitocondri (Mitochondrial Paraquat, MitoPQ). Questo composto si accumula selettivamente nella matrice mitocondriale e provoca la formazione di ROS per mezzo di un meccanismo ossidoriduttivo che coinvolge il complesso flavinico del complesso I della catena di trasporto degli elettroni (ETC). Inizialmente, abbiamo stabilito una curva dose-risposta con dosi diverse di MitoPQ valutando la formazione di ROS, la funzione mitocondriale e la funzione della cellula. Abbiamo osservato che cellule trattate con alte dosi di MitoPQ mostravano un aumento dose-dipendente dei livelli di ROS mitocondriali. Alti dosaggi hanno portato ad una diminuzione del potenziale di membrana mitocondriale (ΔΨm), apertura del PTP, omeostasi citosolica del [Ca2+] compromessa e morte cellulare. Successivamente, ci siamo chiesti se alte dosi di MitoPQ potessero innescare vie di segnalazione mitocondriali coinvolte nella formazione dei ROS. Ci siamo focalizzati sull’attività delle monoammino ossidasi (MAOs) che sono state recentemente identificate come contribuenti dello stress ossidativo nelle patologie cardiache. L’inibizione della MAO ha prevenuto la formazione di ROS indotta dal MitoPQ, così come la disfunzione mitocondriale e la morte cellulare. Oltre a questa via di amplificazione, questi risultati forniscono la prova diretta che i ROS prodotti nei mitocondri possono influenzare i processi citosolici, specialmente l’omeostasi del [Ca2+]I. L’interazione tra Ca2+, ROS e funzione mitocondriale ha un ruolo essenziale nella cardiomiopatia diabetica (DCM). Pertanto abbiamo esteso i risultati ottenuti con il MitoPQ, specialmente l’amplificazione indotta dalla MAO e l’alterazione dell’omeostasi del [Ca2+]I indotta dai ROS mitocondriali, a un modello cellulare di DCM. NRVMs trattati con alti livelli di glucosio (HG) e interleuchina-1β (IL-1β) hanno manifestato un aumento significativo di livelli di H2O2 mitocondriale, sia dopo un trattamento prolungato (48 h) che breve (1 h). L’inibitore della MAO pargilina ha prevenuto questo aumento dei livelli di ROS. Quindi, abbiamo testato l’effetto dell’inibizione della MAO sull’omeostasi del [Ca2+] citosolico in cellule trattate con il protocollo diabetico. Sorprendentemente, HG e IL-1β non hanno modificato significativamente i parametri oscillatori del Ca2+ (i.e. ampiezza, frequenza, area sotto la curva), mentre il pretrattamento con la pargilina o con la N-(2-Mercaptopropionil)glicina (MPG) ha peggiorato il difetto di contrattilità indotto da HG e IL-1β, compromettendo le oscillazioni. Questo risultato inaspettato suggerisce che un tono ossidativo di base sia necessario per un appropriato riciclo del Ca2+ in cardiomiociti trattati con HG e IL-1β. Bisogna sottolineare che il diabete è correlato alla disfunzione delle β cellule pancreatiche. Nelle β cellule pancreatiche è stato dimostrato che lievi scariche di ROS contribuiscono all’omeostasi del [Ca2+]I e la secrezione di insulina dipendente dal Ca2+. Nonostante nel diabete un marcato stress ossidativo conduca alla disfunzione delle β cellule, un aumento primario e moderato di ROS potrebbero modulare i transienti di Ca2+ promuovendo la secrezione di insulina. Abbiamo testato quest’ipotesi sulle β cellule Min6, una linea cellulare di insulinoma che può essere usata come modello di diabete applicato alle β cellule pancreatiche. Abbiamo innescato un aumento di livello di ROS intracellulari trattando le Min6 con alluminio ftalocianina cloruro (AlClPc), un fotosensitizzatore comunemente usato in terapia fotodinamica. La fotoattivazione dell’AlClPc induce cambiamenti chimici nelle molecole circostanti inducendo la produzione di ROS in quantità dipendente dall’intensità dell’illuminazione del LED. I risultati ottenuti hanno evidenziato che la modulazione dei ROS indotta dalla fotoattivazione dell’AlClPc o l’inibizione della pompa Calcio/ATPasi del reticolo endoplasmico (SERCA) per mezzo della tapsigargina (Tg) hanno indotto una simile accelerazione delle oscillazioni di Ca2+, suggerendo che un aumento primario e controllato dei livelli di ROS citosolici modificano ma non bloccano le oscillazioni di Ca2+ modulando la SERCA. I nostri dati mostrano che bassi livelli di ROS mitocondriali modulano leggermente l’omeostasi di [Ca2+]I senza interferire con la funzione mitocondriale e citosolica. Abbiamo usato quest’informazione per studiare se la sovraespressione del trasportatore di Ca2+ mitocondriale (MCU) potesse modificare la formazione di ROS mitocondriali e conseguentemente la sopravvivenza mitocondriale e cellulare dei cardiomiociti. Cellule sovraesprimenti MCU hanno mostrato un aumento nei livelli di ROS che era correlato causalmente ad un aumento della tolleranza al danno da anossia/riossigenazione (A/R) in un processo che ha coinvolto l’attivazione di AKT. Queste scoperte dimostrano che un leggero aumento della formazione di ROS mitocondriali produce un effetto protettivo. Per testare quest’ipotesi, abbiamo studiato se i ROS mitocondriali indotti da basse dosi di MitoPQ potessero diminuire la suscettibilità al danno da A/R. Non solo quest’ipotesi è stata verificata, ma anche in questo caso la protezione è stata attribuita all’attivazione di AKT. I dati di questo lavoro dimostrano che la formazione di ROS mitocondriali può innescare un ampio spettro di risposte con una chiara separazione tra livelli di ROS richiesti per evocare effetti benefici o dannosi. Cambiamenti nell’omeostasi del [Ca2+]I possono essere a monte ma anche a valle della formazione dei ROS mitocondriali e l’effetto cardioprotettivo collegato al leggero aumento dei livelli di ROS mitocondriali sembra dipendere dall’attivazione di AKT.

This study presents the first attempt to combine the fields of ultraviolet (UV) photobiology, plant cell wall biochemistry, aerobic methane production and reactive oxygen species (ROS) mechanisms to investigate the effect of UV radiation on vegetation foliage. Following reports of a 17% increase in decomposition rates in oak (Quercus robur) due to increased UV, which were later ascribed to changes in cell wall carbohydrate extractability, this study investigated the effects of decreased UV levels on ash (Fraxinus excelsior), a fast-growing deciduous tree species. A field experiment was set up in Surrey, UK, with ash seedlings growing under polytunnels made of plastics chosen for the selective transmission of either all UV wavelengths, UV-A only, or no UV. In a subsequent field decomposition experiment on end-of-season leaves, a significant increase of 10% in decomposition rate was found after one year due to removal of UV-B. However, no significant changes in cell wall composition were found, and a sequential extraction of carbohydrate with different extractants suggested no effects of the UV treatments on cell wall structure. Meanwhile, the first observations of aerobic production of methane from vegetation were reported. Pectin, a key cell wall polysaccharide, was identified as a putative source of methane, but no mechanism was suggested for this production. This study therefore tested the effect of UV irradiation on methane emissions from pectin. A linear response of methane emissions against UV irradiation was found. UV-irradiation of de-esterified pectin produced no methane, demonstrating esters (probably methyl esters) to be the source of the observed methane. Addition of ROS-scavengers significantly decreased emissions from pectin, while addition of ROS without UV produced large quantities of methane. Therefore, this study proposes that UV light is generating ROS which are then attacking methyl esters to create methane. The study also demonstrates that this mechanism has the potential to generate several types of methyl halides. These findings may have implications for the global methane budget. In an attempt to demonstrate ROS generation in vivo by UV irradiation, radio-labelling techniques were developed to detect the presence of oxo groups, a product of carbohydrate attack by ROS. Using NaB3H4, the polysaccharides of ash leaflets from the field experiment were radio-labelled, but did not show any significant decrease in oxo groups due to UV treatments. However, UV-irradiation of lettuce leaves showed a significant increase in radio-labelling, suggesting increased UV irradiation caused an increase in the production of ROS. The study shows that the use of this radio-labelling technique has the potential to detect changes in ROS production due to changes in UV levels and could be used to demonstrate a link between ROS levels and methane emissions.

Phosphorescent transition metal complexes make up an important group of compounds that continues to attract intense research owing to their intrinsic bioimaging applications that arise from bright emissions, relatively long excited state lifetimes, and large stokes shifts. Now for biomaging assay a model organism is required which must meet certain criteria for practical applications. The organism needs to be small, with a high turn-over of progeny (high fecundity), a short lifecycle, and low maintenance and assay costs. Our model organism C. elegans met all the criteria. The ideal phosphor has low toxicity in the model organism. In this work the strongly phosphorescent platinum (II) pyrophosphito-complex was tested for biological applications as a potential in vivo hypoxia sensor. The suitability of the phosphor was derived from its water solubility, bright phosphorescence at room temperature, and long excited state lifetime (~ 10 µs). The applications branched off to include testing of C. elegans survival when treated with the phosphor, which included lifespan and fecundity assays, toxicity assays including the determination of the LC50, and recovery after paraquat poisoning. Quenching experiments were performed using some well knows oxygen derivatives, and the quenching mechanisms were derived from Stern-Volmer plots. Reaction stoichiometries were derived from Job plots, while percent scavenging (or antioxidant) activities were determined graphically. The high photochemical reactivity of the complex was clearly manifested in these reactions.

No início da lactação as vacas podem apresentar várias doenças metabólicas e infecciosas, como a mastite, devido às abruptas mudanças fisiológicas, entre elas a supressão da reposta imune, concomitante com a queda da ingestão de matéria seca e da concentração sérica de vitamina E. Com o objetivo de estudar a influência da suplementação de vitamina E nas atividades funcionais dos neutrófilos do leite de bovinos, catorze novilhas da raça holandesa foram divididas aletoriamente em dois grupos, sendo um com sete animais não suplementados (controle) e outro com sete animais suplementados com 1000 UI/dia de vitamina E durante 30 dias antes do parto previsto e 10 dias após o mesmo. Na primeira semana após o parto foi colhido o leite dos quartos mamários CMT negativo, após estimulação da leucocitose com a infusão intramamária de solução de glicogênio de ostra a 0,1% e 0,5%, 36 e 12 horas respectivamente, antes da colheita do leite. As células foram isoladas, contadas e estimou-se a viabilidade das mesmas. Em seguida foram realizados os testes de fagocitose incubando-se zimosan opsonizado (2x108/ml) com neutrófilos (2x107/ml), contados no microscópio óptico. Suspensões na concentração de 2x108/ml de cepas de Staphylococcus aureus, Streptococcus agalactiae, Corynebacterium bovis, Escherichia coli e Prototheca zopfii procedendes de casos de mastite clínica bovina foram incubados com neutrófilos (2x107/ml). A solução foi plaqueada antes da incubação para fagocitose, após a fagocitose, após o tratamento com antibióticos e após a lise dos neutrófilos para a determinação dos patógenos destruídos. As análises estatíscas foram feitas através da ANOVA. A porcentagem de células que englobaram três ou mais partículas de zimosan opsonizado foi de 52% no grupo não suplementado e 66% no grupo suplementado (P<0,05). Não foram detectadas diferenças estatísticas para a porcentagem de fagocitose de microrganismos opsonizados, sendo 30,2% e 28,8% para o grupo controle e suplementado, respectivamente. A destruição intracelular de patógenos foi de 99%, não diferindo entre os grupos. Em seguida, determinou-se a produção de O2- e H2O2, sendo 1,6 e 3,0 nmol de O2- e 0,83 e 0,67 µM de H2O2 para o grupo controle e suplementado, respectivamente. A atividade enzimática no grupo controle e suplementado foi de 4,3 e 3,6 µmol/min/mg proteína para a catalase, 43 e 49 nmol/min/mg de proteína para a glutationa redutase, 16,2 e 30 nmol/min/mg proteína para a glutaiona peroxidase, 3,85 e 6,32 U/mg proteína para a superóxido dismutase e 0,67 e 0,29 U/mg proteína para a mieloperoxidase, sendo que não foram encontradas diferenças estatísticas entre os grupos. A suplementação com vitamina E aumentou a habilidade fagocitária dos neutrófilos somente no ensaio com zimosan e não alterou a atividade enzimática assim como a produção de espécies reativas de oxigênio. O equilíbrio entre as espécies reativas de oxigênio, importante para a destruição intracelular de patógenos, e os antioxidantes é extremamente complexo, não sendo afetado pela suplementação de um único agente antioxidante
At the early lactation, lactating cows are more exposed to metabolic and infectious diseases as mastitis, due to the physiological modifications accomplished with diminishing of dry matter ingestion, immune response depressed and the vitamin E plasmatic concentration level reduction. Therefore, to study the influence of vitamin E supplementation on the function of milk neutrophils, fourteen primigravid holstein heifers were casually allocated in two groups: one was oral supplemented with vitamin E (1000UI/day) 30 days pre parturition and 10 days post parturition, the other group did not receive vitamin E supplementation (control group). During the first week post-parturition, milk was collected from negative CMT quarter after induction of leucocytosis with a intramammary infusion of oyster glycogen infusion (0,1% e 0,5%, 36 e 12 hours respectively). The cells were isolated from milk, counted and the viability was estimated. After these trials the assays: phagocytosis with opsonized zymosan and phagocytosis with microorganisms that cause mastitis were performed. zymosan opzonized particles (2x108/mL) were incubated with neutrophils (2x107/mL) and counted on the optic microscope. The microorganisms (2x108/mL) Staphylococcus aureus, Streptococcus agalactiae, Corynebacterium bovis, Escherichia coli and Prototheca zopfii were incubated with neutrophils (2.107/mL) from milk. Solution were spread plated pre phagocytosis assay, after phagocytosis, post treatment with antibiotics and after break of neutrophils. The results were analysed from the statistical ANOVA. For the phagocytosis assays, 52% of neutrophils from control group phagocyted three or more zymosan particles, and in the supplemented group, 66% of the cells (P< 0,05). For the microorganisms, did not have statistical difference and the results were 30.7% and 28.2% for the control and supplemented groups. The average of the pathogens intracellular kill was 99% in the both groups. In sequence the tests for production O2- and H2O2, enzimatic activity of catalase, superoxide dismutase, glutathione redutase, glutathione peroxidase and myeloperoxidase were made. It were no detected any statiscal differences in respect to the production of O2- and H2O2. The production of O2- was 1,6 nmol and 3,0 nmol and the H2O2 production was 0,83 µM and 0,67 µM for the groups control and supplemented. The control and supplemented group enzymatic activity results obtained were: catalase 4.3 and 3.6 µmol/min/mg protein, glutathione redutase 43 and 49 nmol/min/mg protein, glutathione peroxidase 16.2 and 30 nmol/min/mg protein, superoxide dismuthase 3.85 and 6.32 U/mg protein and myeloperoxidase 0,67 and 0,29 U/mg protein. In relation to these results it was not detected any statistical difference. Vitamin E supplementation increased zymosan phagocytosis and did not affect enzyme activities involved in the mechanisms of protection against oxigen reactive species and microorganism phagocytosis in the neutrophils. The vitamin E supplementation didn´t change the complex balance among the reactions of antioxidants and oxigen reactive species. It can be concluded that due to the complexity of the mechanisms involved the supplementation with just one isolate antioxidant did not interfered with the defence neutrophil activities

Breast cancer is a disease associated with excess exposures to estrogens. While the mode of cancer causation is unknown, others have shown that oxidative stress induced by prolonged exposure to estrogens mediates renal, liver, endometrial and mammary tumorigenesis though the mechanism(s) underling this process is unknown. In this study, we show that 4-hydroxyl 17β-estradiol (4-OHE2), a catechol metabolite of estrogen, induces mammary tumorigenesis in a redox dependent manner. We found that the mechanism of tumorigenesis involves redox activations of nuclear respiratory factor-1 (NRF1); a transcriptions factor associated with regulation of mitochondria biogenesis and oxidative phosphorylation (OXPHOS), as well as mediation of cell survival and growth of cells during periods of oxidative stress. Key findings from our study are as follows: (i) Prolonged treatments of normal mammary epithelial cells with 4-OHE2, increased the formation of intracellular reactive oxygen species (ROS). (ii) Estrogen-induced ROS activates redox sensitive transcription factors NRF1. (iii) 4-OHE2 through activation of serine-threonine kinase and histone acetyl transferase, phosphorylates and acetylate NRF1 respectively. (iv) Redox mediated epigenetic modifications of NRF1 facilitates mammary tumorigenesis and invasive phenotypes of breast cancer cells via modulations of genes involved in proliferation, growth and metastasis of exposed cells. (v) Animal engraftment of transformed clones formed invasive tumors. (vi) Treatment of cells or tumors with biological or chemical antioxidants, as well as silencing of NRF1 expressions, prevented 4-OHE2 induced mammary tumorigenesis and invasive phenotypes of MCF-10A cells. Based on these observations, we hypothesize that 4-OHE2 induced ROS epigenetically activate NRF1 through its phosphorylation and acylation. This, in turn, through NRF1-mediated transcriptional activation of the cell cycle genes, controls 4-OHE2 induced cell transformation and tumorigenesis.

The significantly increased copper and oxidative stress levels are characteristic hallmarks of cancer cells. These differences provide a unique opportunity for selective targeting of cancer cells. D-penicillamine (D-pen) has been proposed to generate reactive oxygen species (ROS) in presence of copper. Therefore, these studies were aimed at investigating the potential application of a currently marketed copper chelator, D-pen, as a novel cytotoxic anti-cancer agent. D-pen was shown to produce ROS, specifically hydrogen peroxide (H2O2), in the presence of cupric sulfate through a copper catalyzed oxidation reaction. During this process D-pen was converted to D-pen disulfide. The experimental proof of the H2O2 generation was conclusively shown with the aid of a novel High Performance Liquid Chromatography (HPLC) assay. The in-vitro cytotoxicity of D-pen co-incubated with cupric sulfate was examined in human beast cancer (MCF-7 and BT474) and leukemia cells (HL-60, HL-60/VCR, and HL-60/ADR). D-pen was shown to cause concentration dependent cytotoxicity in both leukemia and breast cancer cells. A direct correlation between the detection of intracellular ROS and cytotoxicity was established. The treatment of D-pen plus cupric sulfate resulted in a significant reduction in the intracellular thiol content. D-pen is highly hydrophilic and is rapidly eliminated from the body; therefore to improve the intracellular uptake and to protect the thiol group of D-pen, we carried out the synthesis and the in-vitro characterization of a novel gelatin-D-pen conjugate. It was shown that D-pen alone does not enter cells. Confocal microscopy was employed to exhibit the uptake of the novel gelatin-D-pen conjugate by cancer cells. As the cancer cells in-vitro do not accumulate the same levels of copper as reported for cancer cells in-vivo, cancer cells were pre-treated with cupric sulfate to simulate the elevated copper levels. The cupric sulfate pretreatment resulted in reduced thiol level and significantly increased cellular copper content compared to untreated cells. Whereas both free D-pen and gelatin-D-pen conjugate lacked cytotoxicity in un-treated cells, both agents caused concentration dependent cytotoxicity in cupric sulfate pre-treated leukemia cells. Therefore, it was shown that the administration of D-pen as polymer conjugate would potentially provide cytotoxicity and specificity in the treatment of cancer.