O sulfito e o tiossulfato encontram-se acumulados na deficiência da sulfito oxidase (SO), ao passo que o tiossulfato também se acumula na deficiência da proteína da encefalopatia etilmalônica 1 (ETHE1). Os pacientes apresentam principalmente encefalopatia progressiva e convulsões neonatais graves, resultando geralmente em morte prematura. Neste estudo, investigamos os efeitos in vivo do sulfito em estruturas encefálicas de ratos com deficiência da SO, e da administração intraestriatal de sulfito e tiossulfato em ratos normais sobre a homeostase redox e mitocondrial. Também avaliamos alterações nesses parâmetros em fibroblastos de pacientes. Inicialmente, observamos que o sulfito diminuiu os níveis de GSH e as atividades da glutationa redutase (GR) e glutationa S-transferase (GST) no córtex cerebral, e da GST no cerebelo de animais deficientes para a SO. Além disso, o sulfito aumentou as atividades dos complexos II e II-III em estriado e do complexo II no hipocampo, mas diminuiu a atividade do complexo IV no estriado de animais com deficiência da SO. Nesses animais, o sulfito também reduziu o potencial de membrana mitocondrial no córtex cerebral e no estriado, além de diminuir as atividades da malato e glutamato desidrogenase. Já nos animais que receberam injeção intraestriatal de sulfito ou tiossulfato, ambos os compostos diminuíram as atividades da creatina cinase e da citrato sintase, enquanto que o sulfito reduziu a massa mitocondrial. O sulfito ainda diminuiu os níveis de GSH e as atividades da glutationa peroxidase (GPx), GR, GST e glicose-6-fosfato desidrogenase (G6PDH), enquanto que o sulfito e o tiossulfato aumentaram a atividade da catalase. O sulfito também diminui os níveis nucleares de PGC-1α e induziu reatividade glial e dano neuronal. As alterações causadas pelo sulfito foram prevenidas pelo tratamento com bezafibrato. Por fim, nos estudos realizados em fibroblastos, utilizamos células de quatro pacientes com deficiência da ETHE1 e de um paciente com deficiência da SO. Observamos diminuição da respiração mitocondrial em todos os tipos celulares, e diminuição de ATP em duas linhagens com deficiência da ETHE1 e na linhagem com deficiência da SO. Também verificamos alterações variáveis no conteúdo de proteínas de dinâmica mitocondrial, e uma diminuição do conteúdo de proteínas envolvidas na comunicação entre retículo endoplasmático (RE) e mitocôndria. Um aumento nos níveis de DDIT3, marcadora de estresse de RE, na produção de superóxido e apoptose também foram verificados em todos os tipos celulares. O tratamento com JP4-039, um antioxidante mitocondrial, diminuiu os níveis de superóxido em todas as linhagens celulares e aumentou a respiração mitocondrial em duas linhagens com deficiência da ETHE1 e na linhagem com deficiência da SO. Os achados deste trabalho evidenciam que alterações na homeostase energética e redox, na biogênese e dinâmica mitocondrial, bem como na comunicação entre mitocôndria e RE são mecanismos patológicos envolvidos nas deficiências da SO e da ETHE1. Além disso, visto que o bezafibrato e o JP4-039 exerceram efeitos protetores nos diferentes modelos, pode ser sugerido que esses compostos são promissores para o desenvolvimento de novas estratégias terapêuticas para as deficiências da SO e da ETHE1. / Sulfite and thiosulfate are accumulated in tissues of patients affected by sulfite oxidase (SO) deficiency, whereas thiosulfate also accumulates in the deficiency of ethylmalonic encephalopathy protein 1 (ETHE1). Patients present progressive encephalopathy and severe neonatal seizures, often resulting in early childhood death. In this study, we investigated the effects of sulfite in encephalic structures of SO-deficient rats, and of an intrastriatal injection of sulfite or thiosulfate in normal rats on redox and mitochondrial homeostasis. We also investigated possible alterations in these parameters in fibroblasts of patients. Initially, we observed that sulfite decreased reduced glutathione (GSH) levels and the activities of glutathione reductase (GR) and glutathione S-transferase (GST) in cerebral cortex, and of GST in cerebellum of SO deficient rats. Moreover, sulfite increased the activities of the respiratory chain complexes II and II-III in striatum and of complex II in hippocampus, whereas complex IV activity was decreased in striatum of SO deficient animals. In these animals, sulfite also reduced mitochondrial membrane potential in the cerebral cortex and in the striatum, as well as inhibited the activities of malate and glutamate dehydrogenase in cerebral cortex. Regarding the rats that received sulfite or thiosulfate via intrastriatal injection, both compounds reduced creatine kinase and citrate synthase activities, while sulfite decreased mitochondrial mass. Sulfite also decreased GSH levels and the activities of glutathione peroxidase (GPx), GR, GST, glucose-6-phosphate dehydrogenase (G6PDH), whereas both sulfite and thiosulfate increased catalase activity. In addition, sulfite decreased PGC-1α nuclear levels and induced glial reactivity and neuronal damage. Bezafibrate prevented the alterations induced by sulfite in striatum. Finally, in the experiments with fibroblasts, we used four cell lines with ETHE1 deficiency and one cell line with SO deficiency. We observed a decrease in basal and maximal respiration in all cell lines, and ATP depletion in two ETHE1 deficient cell lines and in the SO deficient fibroblasts. We also verified variable alterations in the content of proteins involved in mitochondrial dynamics, and a decrease in the content of proteins involved in endoplasmic reticulum (ER)-mitochondria communication. Increased content of DDIT3, an ER stress marker, as well as high levels of superoxide and apoptosis induction were further seen in all cell lines. Treatment with the mitochondria-targeted free radical scavenger JP4-039 decreased superoxide levels in all cells lines and increased basal and maximal respiration in two ETHE1 deficient cell lines and in the SO deficient cells. Our findings provide evidence that alterations in energy and redox homeostasis, mitochondrial biogenesis and dynamics, as well as in the communication between mitochondria and ER are pathological mechanisms involved in the SO and ETHE1 deficiencies. Furthermore, since bezafibrate and JP4-039 exerted protective effects, it may be suggested that these compounds are attractive agents for the development of new therapeutic strategies aiming to improve the prognosis of patients affected by SO and ETHE1 deficiency.

O sulfito e o tiossulfato encontram-se acumulados na deficiência da sulfito oxidase (SO), ao passo que o tiossulfato também se acumula na deficiência da proteína da encefalopatia etilmalônica 1 (ETHE1). Os pacientes apresentam principalmente encefalopatia progressiva e convulsões neonatais graves, resultando geralmente em morte prematura. Neste estudo, investigamos os efeitos in vivo do sulfito em estruturas encefálicas de ratos com deficiência da SO, e da administração intraestriatal de sulfito e tiossulfato em ratos normais sobre a homeostase redox e mitocondrial. Também avaliamos alterações nesses parâmetros em fibroblastos de pacientes. Inicialmente, observamos que o sulfito diminuiu os níveis de GSH e as atividades da glutationa redutase (GR) e glutationa S-transferase (GST) no córtex cerebral, e da GST no cerebelo de animais deficientes para a SO. Além disso, o sulfito aumentou as atividades dos complexos II e II-III em estriado e do complexo II no hipocampo, mas diminuiu a atividade do complexo IV no estriado de animais com deficiência da SO. Nesses animais, o sulfito também reduziu o potencial de membrana mitocondrial no córtex cerebral e no estriado, além de diminuir as atividades da malato e glutamato desidrogenase. Já nos animais que receberam injeção intraestriatal de sulfito ou tiossulfato, ambos os compostos diminuíram as atividades da creatina cinase e da citrato sintase, enquanto que o sulfito reduziu a massa mitocondrial. O sulfito ainda diminuiu os níveis de GSH e as atividades da glutationa peroxidase (GPx), GR, GST e glicose-6-fosfato desidrogenase (G6PDH), enquanto que o sulfito e o tiossulfato aumentaram a atividade da catalase. O sulfito também diminui os níveis nucleares de PGC-1α e induziu reatividade glial e dano neuronal. As alterações causadas pelo sulfito foram prevenidas pelo tratamento com bezafibrato. Por fim, nos estudos realizados em fibroblastos, utilizamos células de quatro pacientes com deficiência da ETHE1 e de um paciente com deficiência da SO. Observamos diminuição da respiração mitocondrial em todos os tipos celulares, e diminuição de ATP em duas linhagens com deficiência da ETHE1 e na linhagem com deficiência da SO. Também verificamos alterações variáveis no conteúdo de proteínas de dinâmica mitocondrial, e uma diminuição do conteúdo de proteínas envolvidas na comunicação entre retículo endoplasmático (RE) e mitocôndria. Um aumento nos níveis de DDIT3, marcadora de estresse de RE, na produção de superóxido e apoptose também foram verificados em todos os tipos celulares. O tratamento com JP4-039, um antioxidante mitocondrial, diminuiu os níveis de superóxido em todas as linhagens celulares e aumentou a respiração mitocondrial em duas linhagens com deficiência da ETHE1 e na linhagem com deficiência da SO. Os achados deste trabalho evidenciam que alterações na homeostase energética e redox, na biogênese e dinâmica mitocondrial, bem como na comunicação entre mitocôndria e RE são mecanismos patológicos envolvidos nas deficiências da SO e da ETHE1. Além disso, visto que o bezafibrato e o JP4-039 exerceram efeitos protetores nos diferentes modelos, pode ser sugerido que esses compostos são promissores para o desenvolvimento de novas estratégias terapêuticas para as deficiências da SO e da ETHE1. / Sulfite and thiosulfate are accumulated in tissues of patients affected by sulfite oxidase (SO) deficiency, whereas thiosulfate also accumulates in the deficiency of ethylmalonic encephalopathy protein 1 (ETHE1). Patients present progressive encephalopathy and severe neonatal seizures, often resulting in early childhood death. In this study, we investigated the effects of sulfite in encephalic structures of SO-deficient rats, and of an intrastriatal injection of sulfite or thiosulfate in normal rats on redox and mitochondrial homeostasis. We also investigated possible alterations in these parameters in fibroblasts of patients. Initially, we observed that sulfite decreased reduced glutathione (GSH) levels and the activities of glutathione reductase (GR) and glutathione S-transferase (GST) in cerebral cortex, and of GST in cerebellum of SO deficient rats. Moreover, sulfite increased the activities of the respiratory chain complexes II and II-III in striatum and of complex II in hippocampus, whereas complex IV activity was decreased in striatum of SO deficient animals. In these animals, sulfite also reduced mitochondrial membrane potential in the cerebral cortex and in the striatum, as well as inhibited the activities of malate and glutamate dehydrogenase in cerebral cortex. Regarding the rats that received sulfite or thiosulfate via intrastriatal injection, both compounds reduced creatine kinase and citrate synthase activities, while sulfite decreased mitochondrial mass. Sulfite also decreased GSH levels and the activities of glutathione peroxidase (GPx), GR, GST, glucose-6-phosphate dehydrogenase (G6PDH), whereas both sulfite and thiosulfate increased catalase activity. In addition, sulfite decreased PGC-1α nuclear levels and induced glial reactivity and neuronal damage. Bezafibrate prevented the alterations induced by sulfite in striatum. Finally, in the experiments with fibroblasts, we used four cell lines with ETHE1 deficiency and one cell line with SO deficiency. We observed a decrease in basal and maximal respiration in all cell lines, and ATP depletion in two ETHE1 deficient cell lines and in the SO deficient fibroblasts. We also verified variable alterations in the content of proteins involved in mitochondrial dynamics, and a decrease in the content of proteins involved in endoplasmic reticulum (ER)-mitochondria communication. Increased content of DDIT3, an ER stress marker, as well as high levels of superoxide and apoptosis induction were further seen in all cell lines. Treatment with the mitochondria-targeted free radical scavenger JP4-039 decreased superoxide levels in all cells lines and increased basal and maximal respiration in two ETHE1 deficient cell lines and in the SO deficient cells. Our findings provide evidence that alterations in energy and redox homeostasis, mitochondrial biogenesis and dynamics, as well as in the communication between mitochondria and ER are pathological mechanisms involved in the SO and ETHE1 deficiencies. Furthermore, since bezafibrate and JP4-039 exerted protective effects, it may be suggested that these compounds are attractive agents for the development of new therapeutic strategies aiming to improve the prognosis of patients affected by SO and ETHE1 deficiency.

The role of mitochondria is multidimensional and ranges in vast areas, including apoptosis, cellular response towards stress, metabolism, which is regulated by a plethora of proteins, acting together to maintain cellular and organellar homeostasis. In spite of the presence of mitochondrial DNA, most of the mitochondrial proteins are nuclear encoded and translocated inside the organelle through dedicated translocases present on outer and inner membrane of mitochondria. To fulfil the cellular energy demand, mitochondria efficiently generate ATP by oxidative phosphorylation, and thus are considered as "power house of cell." There occurs a transfer of electrons from various oxidizable substrates to oxygen, which is achieved by a series of redox reactions with generation of water as a byproduct. This process is coupled with ATP synthesis, involves five protein-complexes present in the inner mitochondrial membrane. During this process, it generates extremely reactive intermediate species of oxygen as a byproduct collectively referred as Reactive Oxygen Species (ROS) through partial reduction of oxygen. These intermediate metabolites of oxygen include superoxide anion (O2-º), H2O2 and highly reactive hydroxyl radicals (OHº). Although ROS are produced by different cellular sources, such as widely expressed and evolutionary conserved NADPH Oxidases, xanthine oxidase, cyclooxygenases, lipoxygenases and cytochrome P450 enzymes but mitochondria are one of the major contributors of cellular ROS. Earlier, reactive oxygen species were considered as harmful but for past few decades, the role ROS has been appreciated as signalling molecules. Because of their high reactivity, these species can cause redox mediated modifications to cellular components and thus have an ability to participate in signalling process. The regulation of signalling pathway by ROS is governed by either alterations in cellular redox conditions or by oxidative modifications of certain residues in proteins, which are involved in signalling cascades. Reactive Oxygen Species can modify amino acid residues, interact with Fe-S clusters or other metal complexes and induce dimerization of proteins to alter protein structure and function. ROS causes modifications to critical amino acids, mainly by oxidation of cysteine residues, where oxidation of sulfhydryl group (-SH) of a single cysteine residue leads to formation of sulfenic (-SOH), sulfinic (-SO2H), sulfonic (-SO3H), or S-glutathionylated (-SSG) derivatives. Thus, by incorporating these modifications, ROS affects the function of proteins, thereby modulating the cellular signalling process. On the other hand, the accumulation of higher level of reactive oxygen species may damage cellular components causing oxidative stress. Therefore, it is necessary to maintain the ROS levels and regulation of intracellular redox homeostasis depends upon a complex network of antioxidant molecules. These antioxidants range from low molecular weight glutathione to large proteins like glutathione peroxidases. Cell has an array of antioxidants with different subcellular locations. Superoxide Dismutase which catalyzes dismutation of superoxides and converts them to H2O2, localizes in cytosol, mitochondrial intermembrane space and extracellular matrix. Different isoforms of Glutatione Peroxidases (GPx) and Peroxiredoxins (Prx) are located in cytosol as well as in mitochondria and scavenge H2O2 by using glutathione (GSH) and thioredoxin (Trx) respectively, as co-factors. During this peroxidase activity of GPx and Prx, GSH and Trx get oxidized and recycled back to the reduced form by Glutathione Reductase (GR) and Thioredoxin Reductase (TR) correspondingly, with the help of NADPH. Thus, GPx system (GPx, GR, GSH and NADPH) and Prx system (Prx, Trx, TR and NADPH) helps in maintenance of redox balance by scavenging H2O2. Catalase is present in peroxisomes for the catalytic degradation of H2O2. Along with Thioredoxin, glutaredoxin (Grx) also reduces protein disulphides and maintains the redox homeostasis. Although, reactive oxygen species are important for normal physiological process, oxidative stress caused by imbalanced ROS levels is thought to be involved in progression of many disorders. However, in most of the diseases, the role of ROS is not yet clear. Elevated oxidative stress is observed with insulin resistance and progression of type II diabetes mellitus, and the resultant high glucose levels alter mitochondrial physiology, leading to the fragmentation of organelle. However, on contrary it has also been observed that ROS improves insulin sensitivity. ROS is directly involved in progression of neurodegenerative disorders, which are characterized by oxidative stress mediated neuronal loss. Interestingly, in case of cancer ROS plays a differential role. At moderately higher levels, ROS helps cancer cells to detach from the matrix and thus assist in metastasis but the higher accumulation of ROS leads to oxidative stress mediated cell death. Thus, cancer cells have an enhanced expression level of antioxidants to maintain the optimum ROS concentration for their survival and proliferation. The role of ROS in cellular signalling and progression of diseases highlights the importance of redox regulation. Mitochondria being the major source of ROS, harbours various redox regulators such as a mitochondrial permeability transition pore (mPTP), inner membrane anion channel (IMAC), Ca++ ions, etc. In addition, certain proteins like Hsp31/DJ1 class also translocate into the organelle in a stress dependent manner to maintain redox homeostasis. These proteins are encoded by the nuclear genome and translocated in the organelle, suggesting the importance of mitochondrial import machinery in regulation of redox balance. Another such example is MIA pathway of protein import, where MIA40 regulates ROS indirectly by catalyzing folding of disulfide containing proteins such as SOD-1 in a redox coupled process. However, under most cases, the physiological disorders lead to uncontrolled production of reactive oxygen species, thereby overloading the cellular antioxidant defence machinery. The failure of the antioxidant machinery leads to enhanced disease progression. Under such disease conditions where the upheaval of redox homeostasis leads to the accumulation of ROS, artificial antioxidants can be used to protect cells against oxidative damage. Artificial systems such as Cyclodextrins, metal complexes, porphyrins, polymers, supramolecules and biomolecules such as nucleic acids, catalytic antibodies and proteins, have been created to mimic the structures and functions of natural enzymes through various approaches. In the present thesis, we have elucidated the role of two mitochondrial proteins, which are part of mitochondrial import motor, as redox regulators and the effect of artificial antioxidants in maintenance of redox homeostasis under stress. A detailed description on importance of ROS in cellular signalling and disease progression has been included in Chapter I, which gives a preface for the work mentioned in this thesis. Chapter II to chapter V elucidates the main objectives of the present thesis, which are: 1. Identification of novel human mitochondrial regulators of redox homeostasis • Role of NEF in redox sensing (Chapter II) • Evolved function of J-like protein in ROS regulation (Chapter III) 2. Characterization of potential artificial antioxidants as redox therapeutics • Organo-selenium compounds as potential artificial antioxidants (Chapter IV) • Use of nanoparticles as a natural antioxidant mimics (Chapter V) Chapter II: Mitochondrial Hsp70 (mtHsp70) plays a critical role for the import of the precursor proteins. The import activity of mtHsp70 is attributed by cyclic binding and release of precursor proteins which in turn is regulated by co-chaperones J-proteins and nucleotide exchange factor (NEF). The affinity for substrate is governed by the binding of ADP or ATP at the N-terminal nucleotide binding pocket of mtHsp70. The affinity for substrate is higher in ADP bound state as compared to ATP bound state. mtHsp70 by its ATPase activity hydrolyze ATP (low-affinity state) to ADP (high-affinity state), which is replaced back to ATP by NEF thus maintaining the mtHsp70 cycle for protein import. In the present study, we have biochemically and functionally characterized GrpEL1 and GrpEL2 as a nucleotide exchange factor for mtHsp70. We observed that like their yeast ortholog Mge1, both the mammalian NEFs interacts with mtHsp70 and exchange ADP from ATP to maintain the cycle of mtHsp70. Interestingly, we observed that both the NEFs are part of human mitochondrial import motor and are recruited at the import motor as hetero-subcomplex. The formation of GrpEL1-EL2 hetero-subcomplex is important to maintain the stability of both the NEFs. In this study, we have elucidated that the interplay between the two NEFs governs organellar response towards oxidative stress. Chapter III: Redox imbalance generates multiple cellular damages leading to oxidative stress mediated pathological conditions such as neurodegenerative diseases, diabetes, ageing and cancer progression. Therefore, maintenance of ROS homeostasis is most important, that involves well-defined antioxidant machinery. In the present chapter, we have identified for first time a component of mammalian protein translocation machinery, Magmas, to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues, cancer cells and tissues of developmental origin that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance towards oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of ETC-complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as an ROS sensor was found to be independent of its role in protein import, underlying its dual role in human mitochondria. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cellular tolerance to oxidative stress even in yeast model organism. The cyto-protective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress related diseases. Chapter IV: The dysregulation of antioxidant machinery in oxidative stress mediated disorders lead to accumulation of excess ROS, highlighting the importance of artificial antioxidants. For the therapeutics of oxidative stress related disorders, artificial antioxidants have been used as combination redox therapy. In order to realize potent biocompatible antioxidants with minimum toxicity, we have utilized two approaches – synthesis of organic compounds and nanoparticle based enzyme mimetics. We have synthesized novel isoselenazoles with high glutathione peroxidase (GPx) and peroxiredoxin (Prx) activities, which provide remarkable cytoprotection to human cells, mainly by exhibiting antioxidant activities in the presence of cellular thiols. The cytotoxicity of the isoselenazoles is found to be significantly lower than that of ebselen, which is being widely clinically evaluated by several research groups for the treatment of reperfusion injuries and stroke, hearing loss, and bipolar disorder. The compounds reported in this study has the potential to be used as therapeutic agents for disorders mediated by reactive oxygen species.. Chapter V: Nanomaterials with enzyme-like properties have attracted significant interest, although limited information is available on their biological activities in cells. Here, we show that V2O5 nanowires (Vn) functionally mimic the antioxidant enzyme, glutathione peroxidase by using cellular glutathione as a co-factor. Although a bulk V2O5 is known to be toxic to the cells, the property is altered when converted into a nanomaterial form. The Vn nanozymes readily internalize into mammalian cells of multiple origins (kidney, neuronal, prostate, cervical) and exhibit robust enzyme-like activity by scavenging the reactive oxygen species, when challenged against intrinsic and extrinsic oxidative stress. The Vn nanozymes fully restore the redox balance without perturbing the cellular antioxidant defense, thus providing an important cytoprotection for biomolecules against harmful oxidative damage. Based on our findings, we envision that biocompatible Vn nanowires can provide future therapeutic potential to prevent ageing, cardiac disorders and several neurological conditions, including Parkinson’s and Alzheimer’s disease.

Abstract The hepatic inducible Cytochrome P450s (CYPs) generally prime xenobiotics for elimination. Murine CYP2A5 and human CYP2A6 share similar xenobiotic substrates and some regulatory features. Recently, they were shown to oxidize bilirubin, a byproduct of heme catabolism and a dose-dependent anti- or pro-oxidant, to biliverdin. In this study, the putative role of the redox-sensitive, cytoprotective transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the regulation of hepatic Cyp2a5 expression and induction under diverse hepatotoxic conditions and altered heme homeostasis was characterized. The coordination of Cyp2a5 and the Nrf2 target gene Heme oxygenase-1 (Hmox1), which determines bilirubin formation from heme, responses to heavy metals and modulators of heme homeostasis, was studied in cultured wildtype and Nrf2(-/-) mouse primary hepatocytes. Nrf2 was essential for the basal hepatic expression of CYP2A5 in the endoplasmic reticulum (ER) and mitochondria, as well as for its induction by cadmium, lead, methyl mercury and phenethyl isothiocyanate. A functional Nrf2 binding antioxidant response element (ARE) about -2.4 kilobases upstream of the Cyp2a5 transcriptional start site was identified. In contrast to Hmox1, a target of BTB and CNC homology 1 (Bach)-mediated repression via AREs, the regulation of Cyp2a5 did not clearly involve Bach1. Excessive heme induced mainly ER-localized CYP2A5 via Nrf2, which was limited by the Nrf2-independent HMOX1 induction. In heme synthesis blockades, CYP2A5 was enhanced via Nrf2 and additional factors, such as the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). The typical CYP2A5 inducers phenobarbital, dibutyryl-cyclic adenosine monophosphate (db-cAMP) and PGC-1α enhance heme synthesis; CYP2A5 was induced via Nrf2 in acute but not chronic phenobarbital exposure without a clear connection to heme, while the responses to db-cAMP and PGC-1α were sensitized in the absence of Nrf2. This suggests novel crosstalk between Nrf2 and PGC-1α. In this study, Cyp2a5 was identified as a sensitive indicator of hepatic Nrf2 pathway activation that could be used, e.g. for in vitro screening of drug candidate hepatotoxicity. The similar subcellular localization and coordination of CYP2A5 and HMOX1 expression in altered heme metabolism support the postulated role for CYP2A5 in bilirubin homeostasis. / Tiivistelmä Vierasaineet stimuloivat maksan Sytokromi P450 (CYP)-entsyymejä, mikä yleensä lisää niiden eliminaatiota. Hiiren CYP2A5 ja ihmisen CYP2A6 ovat lähisukua katalyyttisten ja osin säätelyllisten yhteneväisyyksiensä puolesta. Vastikään niiden osoitettiin katalysoivan hemin hajoamistuotteen, bilirubiinin hapettumista biliverdiiniksi, mikä saattaisi säädellä sen annosriippuvaisia vaikutuksia antioksidanttina ja oksidanttina. Työssä tutkittiin solustressiä aistivan, suojaavan transkriptiotekijän Nrf2 osuutta Cyp2a5-geenin aktivaatiossa maksatoksisissa olosuhteissa ja hemimetabolian muutoksissa. Cyp2a5:n ja bilirubiinin tuotosta vastaavan, Nrf2-säädellyn Hemioksigenaasi-1 (Hmox1):n vasteita verrattiin viljellyissä villityypin ja poistogeenisen Nrf2(-/-) hiiren primaarimaksasoluissa. Tulokset osoittavat, että Nrf2 ylläpitää CYP2A5:n ilmentymistä endoplasmisella kalvostolla (ER) ja mitokondrioissa sekä välittää sen stimulaation altisteilla kadmium, lyijy, metyylielohopea ja fenetyyli-isotiosyanaatti. Toimiva Nrf2-vasteinen antioksidanttivaste-elementti (ARE) tunnistettiin n. -2,4 kiloemäsparia Cyp2a5-geenin luennan aloituskohdasta ylävirtaan. BTB ja CNC homologia 1 (Bach1)-tekijä, joka on tärkeä Hmox1-säätelijä ja ARE-välitteinen transkription estäjä, ei selkeästi osallistu Cyp2a5:n säätelyyn. Hemin ylimäärä stimuloi CYP2A5:n määrää ER-kalvostolla, Nrf2-riippumattomasti stimuloituvan HMOX1 rajoittaessa Nrf2-reitin aktivaatiota. Hemisynteesin estyessä Nrf2 aktivoi Cyp2a5-geeniä muiden mekanismien kuten peroksisomiproliferaattori-aktivoituva reseptori gamman koaktivaattori-1α (PGC-1α) kanssa. Fenobarbitaali (PB), dibutyryyli-syklinen adenosiinimonofosfaatti (db-cAMP) ja PGC-1α lisäävät tunnetusti hemisynteesiä. Nrf2 havaittiin Cyp2a5:n aktivaatiolle välttämättömäksi akuutissa mutta ei kroonisessa PB-altistuksessa ilman selkeästi havaittua hemin osuutta. Cyp2a5-geenin db-cAMP- ja PGC-1α-vasteinen stimulaatio voimistui merkittävästi toimivan Nrf2-reitin puuttuessa, mikä osoittaa vuoropuhelua Nrf2 ja PGC-1α välillä. Väitöskirjatyössä Cyp2a5 tunnistettiin herkäksi Nrf2-reitin aktivaation maksamarkkeriksi, jota voitaisiin hyödyntää esim. lääkeainekandidaattien maksatoksisuuden seulonnassa soluviljelyssä. CYP2A5:n ja HMOX1:n solunsisäinen kohdentuminen ja ekspressio koordinoituvat hemimetabolian muutoksissa, mikä tukee teoriaa CYP2A5:n roolista bilirubiinin metaboliassa maksassa.

O sulfito e o tiossulfato encontram-se acumulados na deficiência da sulfito oxidase (SO), ao passo que o tiossulfato também se acumula na deficiência da proteína da encefalopatia etilmalônica 1 (ETHE1). Os pacientes apresentam principalmente encefalopatia progressiva e convulsões neonatais graves, resultando geralmente em morte prematura. Neste estudo, investigamos os efeitos in vivo do sulfito em estruturas encefálicas de ratos com deficiência da SO, e da administração intraestriatal de sulfito e tiossulfato em ratos normais sobre a homeostase redox e mitocondrial. Também avaliamos alterações nesses parâmetros em fibroblastos de pacientes. Inicialmente, observamos que o sulfito diminuiu os níveis de GSH e as atividades da glutationa redutase (GR) e glutationa S-transferase (GST) no córtex cerebral, e da GST no cerebelo de animais deficientes para a SO. Além disso, o sulfito aumentou as atividades dos complexos II e II-III em estriado e do complexo II no hipocampo, mas diminuiu a atividade do complexo IV no estriado de animais com deficiência da SO. Nesses animais, o sulfito também reduziu o potencial de membrana mitocondrial no córtex cerebral e no estriado, além de diminuir as atividades da malato e glutamato desidrogenase. Já nos animais que receberam injeção intraestriatal de sulfito ou tiossulfato, ambos os compostos diminuíram as atividades da creatina cinase e da citrato sintase, enquanto que o sulfito reduziu a massa mitocondrial. O sulfito ainda diminuiu os níveis de GSH e as atividades da glutationa peroxidase (GPx), GR, GST e glicose-6-fosfato desidrogenase (G6PDH), enquanto que o sulfito e o tiossulfato aumentaram a atividade da catalase. O sulfito também diminui os níveis nucleares de PGC-1α e induziu reatividade glial e dano neuronal. As alterações causadas pelo sulfito foram prevenidas pelo tratamento com bezafibrato. Por fim, nos estudos realizados em fibroblastos, utilizamos células de quatro pacientes com deficiência da ETHE1 e de um paciente com deficiência da SO. Observamos diminuição da respiração mitocondrial em todos os tipos celulares, e diminuição de ATP em duas linhagens com deficiência da ETHE1 e na linhagem com deficiência da SO. Também verificamos alterações variáveis no conteúdo de proteínas de dinâmica mitocondrial, e uma diminuição do conteúdo de proteínas envolvidas na comunicação entre retículo endoplasmático (RE) e mitocôndria. Um aumento nos níveis de DDIT3, marcadora de estresse de RE, na produção de superóxido e apoptose também foram verificados em todos os tipos celulares. O tratamento com JP4-039, um antioxidante mitocondrial, diminuiu os níveis de superóxido em todas as linhagens celulares e aumentou a respiração mitocondrial em duas linhagens com deficiência da ETHE1 e na linhagem com deficiência da SO. Os achados deste trabalho evidenciam que alterações na homeostase energética e redox, na biogênese e dinâmica mitocondrial, bem como na comunicação entre mitocôndria e RE são mecanismos patológicos envolvidos nas deficiências da SO e da ETHE1. Além disso, visto que o bezafibrato e o JP4-039 exerceram efeitos protetores nos diferentes modelos, pode ser sugerido que esses compostos são promissores para o desenvolvimento de novas estratégias terapêuticas para as deficiências da SO e da ETHE1. / Sulfite and thiosulfate are accumulated in tissues of patients affected by sulfite oxidase (SO) deficiency, whereas thiosulfate also accumulates in the deficiency of ethylmalonic encephalopathy protein 1 (ETHE1). Patients present progressive encephalopathy and severe neonatal seizures, often resulting in early childhood death. In this study, we investigated the effects of sulfite in encephalic structures of SO-deficient rats, and of an intrastriatal injection of sulfite or thiosulfate in normal rats on redox and mitochondrial homeostasis. We also investigated possible alterations in these parameters in fibroblasts of patients. Initially, we observed that sulfite decreased reduced glutathione (GSH) levels and the activities of glutathione reductase (GR) and glutathione S-transferase (GST) in cerebral cortex, and of GST in cerebellum of SO deficient rats. Moreover, sulfite increased the activities of the respiratory chain complexes II and II-III in striatum and of complex II in hippocampus, whereas complex IV activity was decreased in striatum of SO deficient animals. In these animals, sulfite also reduced mitochondrial membrane potential in the cerebral cortex and in the striatum, as well as inhibited the activities of malate and glutamate dehydrogenase in cerebral cortex. Regarding the rats that received sulfite or thiosulfate via intrastriatal injection, both compounds reduced creatine kinase and citrate synthase activities, while sulfite decreased mitochondrial mass. Sulfite also decreased GSH levels and the activities of glutathione peroxidase (GPx), GR, GST, glucose-6-phosphate dehydrogenase (G6PDH), whereas both sulfite and thiosulfate increased catalase activity. In addition, sulfite decreased PGC-1α nuclear levels and induced glial reactivity and neuronal damage. Bezafibrate prevented the alterations induced by sulfite in striatum. Finally, in the experiments with fibroblasts, we used four cell lines with ETHE1 deficiency and one cell line with SO deficiency. We observed a decrease in basal and maximal respiration in all cell lines, and ATP depletion in two ETHE1 deficient cell lines and in the SO deficient fibroblasts. We also verified variable alterations in the content of proteins involved in mitochondrial dynamics, and a decrease in the content of proteins involved in endoplasmic reticulum (ER)-mitochondria communication. Increased content of DDIT3, an ER stress marker, as well as high levels of superoxide and apoptosis induction were further seen in all cell lines. Treatment with the mitochondria-targeted free radical scavenger JP4-039 decreased superoxide levels in all cells lines and increased basal and maximal respiration in two ETHE1 deficient cell lines and in the SO deficient cells. Our findings provide evidence that alterations in energy and redox homeostasis, mitochondrial biogenesis and dynamics, as well as in the communication between mitochondria and ER are pathological mechanisms involved in the SO and ETHE1 deficiencies. Furthermore, since bezafibrate and JP4-039 exerted protective effects, it may be suggested that these compounds are attractive agents for the development of new therapeutic strategies aiming to improve the prognosis of patients affected by SO and ETHE1 deficiency.

Mutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress, and several in vitro studies have demonstrated that the nucleoporin ALADIN is involved in both the cellular oxidative stress response and adrenal steroidogenesis. It is known that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome. The objective of this study was to determine whether the application of chronic oxidative stress by ingestion of paraquat would generate a triple A-like phenotype in ALADIN null mice. Adult male mice were fed either a paraquat (0.25 g/kg diet) or control diet for 11 days. After application of chronic oxidative stress, ALADIN knock-out mice presented with an unexpected compensated glutathione metabolism, but lacked a phenotype resembling human triple A syndrome. We did not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN. This study stresses the species-specific role of the nucleoporin ALADIN, which in mice involves a novel compensatory mechanism for regulating the cellular glutathione redox response.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23043号 / 理博第4720号 / 新制||理||1676(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 秋山 秋梅, 教授 曽田 貞滋, 教授 沼田 英治 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Sexual ornaments important for mating success in many species are often assumed to evolve as condition-dependent signals of individual quality. Ornament expression can be associated with age and survival, thereby signalling individual viability. Here, we have tested viability signalling function of tail streamers and their importance for within-pair and extra-pair fertilisation success in the European barn swallow (Hirundo rustica rustica). In contrast to previous studies on this subspecies, our data suggest that tail length is not associated with fertilisation success in our population. Instead, the most important predictors of within-pair and extra-pair fertilisation success were female and male age, respectively. Our data supported viability signalling function of male tail streamers, as documented by age-related within- individual increase in their length. There was no evidence for senescence in this trait. Contrary to some previous studies, the viability signalling function of tail streamers was further supported by observed selective disappearance of males with shorter tails. Several physiological mechanisms have been proposed as maintaining signalling honesty. Among them, oxidative stress from highly reactive species (RS), including free radicals, attracted a considerable attention. Given...