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.

IMPACTS OF AIR POLLUTION ON OXIDATIVE DNA DAMAGE Vlasta Svecova Department of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR, v.v.i. Videnska 1083, 142 20 Prague 4 Tel.: +420 241 062 669, fax.: +420 241 062 785, e-mail: svecova@biomed.cas.cz This thesis deal with impacts of air pollution on human health. The biomarkers of biologically effective dose, biomarkers of oxidative damage to DNA, lipids and proteins, were studied. We aimed at importance of individual pollutants, measured the personal exposure to these pollutants and analyzed the biomarkers of oxidative damage to macromolecules. c-PAHs (carcinogenic polycyclic aromatic hydrocarbons) bound to airborne PM2.5 (particulate matter ≤ 2.5 µm) and volatile organic compounds (benzene, toluene, ethylbenzene and m,p,o-xylenes, BTEX) were studied as ones of the biologically most important pollutants. Personal and outdoor concentrations of c-PAHs together with personal exposure to BTEX were measured. The concentrations of pollutants were correlated with biomarker levels in different seasons and localities. Bus drivers in Prague, 6-10 years old children from Teplice and Prachatice and policemen with office workers from Ostrava region were the model populations. Oxidative damage to DNA were measured by 8-oxodeoxyguanosine (8-oxodG), 15-F2t-...

O fator de transcrição mitocondrial A (TFAM) pertence ao grupo das proteínas de alta mobilidade, apresentando um importante papel para a replicação, transcrição e estrutura/organização do DNA mitocondrial (DNAmt). O DNAmt está organizado em um complexo nucleoprotéico, chamado de nucleóide, do qual TFAM é o principal componente protéico, empacotando o DNAmt de forma análoga às histonas no DNA nuclear. Em analogia ao DNA nuclear, foi sugerido que esse empacotamento pode proteger o DNAmt do ataque de espécies oxidantes, enquanto que, por outro lado, poderia também impedir o acesso das enzimas de reparo. Este trabalho visou esclarecer qual o papel de TFAM na proteção do DNAmt e entender como TFAM influencia o reparo do DNAmt. Nossos resultados indicaram que o empacotamento do DNAmt por TFAM pode proteger o DNA da formação de lesões em condições de estresse oxidativo. Células com redução na expressão de TFAM apresentaram taxas alteradas de proliferação e uma menor viabilidade celular após o tratamento com o fotossensibilizador azul de metileno, indicando que TFAM pode contribuir para a manutenção da integridade funcional da mitocondria. A velocidade do reparo do DNAmt, em células Kd-TFAM, foi aparentemente maior, o que indicou a importância da modulação da interação de TFAM com o DNAmt para um reparo rápido e eficiente das lesões oxidadas. Portanto, TFAM desempenha um papel importante para a estabilidade genômica mitocondrial, protegendo o DNAmt dos efeitos deletérios das lesões oxidadas no estresse oxidativo, e também modulando a velocidade do reparo do DNAmt, provavelmente através de modificações/interações que permitam que as enzimas de reparo acessem as lesões no DNAmt. / The mitochondrial transcription factor A (TFAM) belongs to the high mobility group box proteins, and is essencial for replication, transcription and structure/organization of the mitochondrial DNA (mtDNA).The mtDNA is organized in a nucleoproteic complex called the nucleoid, where TFAMis the main protein component,packaging mtDNA in a manner similar to histones in the nuclear DNA. In analogy to the histone role in nuclear DNA, it was suggested that mtDNA packaging by TFAM could protect the mtDNA against oxidized lesions. On the other hand, it could also prevent the access of repair enzymes. This study aimed to understand whether TFAM plays a role in mtDNA stability through these opposing effects of protecting from damage and preventing repair. Our results indicated that TFAM protects the mtDNA against lesion formation upon oxidative stress. Cells with reduced expression of TFAM showed altered proliferation and lower cellular viability after treatment with the photoactivated dye methylene blue, indicating an important role for TFAM in maintaining mitochondrial function and cell survival. MtDNA repair rate was apparently higherin Kd-TFAM cells, which indicated the importance of modulating the interaction of TFAM with mtDNA for a quick and efficient repair of oxidized lesions. Therefore, TFAM plays an important role in maintaining mitochondrial genomic stability by protecting the mtDNA of the deleterious effects of oxidized lesions in oxidative stress, also modulating mtDNA repair, likely through modifications/interactions that modulate its DNA binding activity and access to lesions in mtDNA by DNA repair enzymes.

A deficiência de ferro é o resultado de um longo período de balanço negativo do ferro, culminando na exaustão do estoque de ferro do organismo. Isto é revelado quando a concentração de hemoglobina declina para valores abaixo dos limites normais e então aparece a anemia, que ocorre devido à deficiente síntese de hemoglobina, com diminuição da proliferação eritrocitária. A anemia ferropênica é considerada o maior problema de saúde no idoso e está associada com várias complicações, incluindo doença cardiovascular, disfunção cognitiva, aumento no risco de morte, e principalmente tem um significante efeito na qualidade de vida. Estudos revelam que a anemia por deficiência de ferro causa maior susceptibilidade aos agentes oxidantes. Utilizando técnica espectrofotométrica, foram determinadas as atividades das enzimas antioxidantes catalase (CAT), glutationa peroxidase (GPx) e superóxido dismutase (SOD) e quantificada a glutationa total (GSH) nos eritrócitos dos pacientes. Também determinou-se o dano oxidativo nas proteínas plasmáticas e no hemolisado celular pelo método do carbonil a 360 nm. Os níveis da peroxidação lipídica (MDA) e da vitamina C foram determinados por cromatografia líquida de alta performance (HPLC). Os participantes da pesquisa foram selecionados de Ambulatórios de Atenção Básica de Saúde da Região Norte do Rio Grande do Sul, sendo que 17 indivíduos apresentaram anemia ferropênica e hipertensão primária e 18 apenas hipertensão primária devido à dificuldade de selecionar pacientes sem nenhuma patologia associada. Todos os indivíduos assinaram o termo de consentimento livre e esclarecido. Os dados obtidos foram expressos como médias ± desvio padrão e analisados utilizando-se o Teste t-Student. Os resultados do trabalho mostram que os indivíduos com anemia ferropênica apresentam atividade significativamente elevada da SOD e da CAT em relação aos indivíduos controle, mas não apresentaram diferença na atividade da GPx nem da GSH. Observamos também, dano oxidativo em proteínas plasmáticas, mas não no hemolisado celular. Os pacientes com anemia ferropênica mostraram dano oxidativo em proteínas plasmáticas significativamente maiores, assim como um aumento significativo da produção de MDA no soro, como um indicativo do aumento da auto-oxidação dos lipídios sob condições de estresse oxidativo. Os níveis séricos da vitamina C não mostraram diferença significativa entre os grupos. Estes dados revelam o envolvimento de espécies reativas de oxigênio no agravamento da anemia carencial. Sendo assim, reforça-se a necessidade de diagnosticar e tratar esses pacientes precocemente, diminuindo a magnitude dos efeitos da anemia na saúde do idoso. / Iron deficiency is the outcome of a long period of negative iron balance, culminating in exhaustion of the body’s iron stores. This is revealed when hemoglobin concentration declines to values below normal levels and anaemia is detected, due to a deficient synthesis of hemoglobin, with decrease in eritrocitary proliferation. Iron deficiency anaemia is considered the most important health problem in elderly and it is associated with various complications, including cardiovascular disease, cognitive dysfunction, increased risk of death and, mostly, it has a meaningful effect in the quality of life. Studies reveal that iron deficiency anaemia cause higher susceptibility to oxidant agents. Using spectrophotometric techniques, the activities of antioxidant enzymes, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and total glutathione (GSH) were quantified in the erythrocytes of the patients. We also determined oxidative damage of plasma proteins and in hemolysate using the carbonyl assay at 360 nm. The levels of lipid peroxidation (MDA) and vitamin C were determined by highperformance liquid chromatography (HPLC). Participants in this study were selected in Ambulatories of Basic Health Attention in the North of the State of Rio Grande do Sul. 17 individuals presented iron deficiency anaemia and primary hypertension and 18 only primary hypertension, because it was difficult to select patients without any associated deficiency. All individuals signed the term of free and clarified consent to participate in this study. Data were analyzed using t- Student Test. Results show that individuals with iron deficiency anaemia show significantly higher activity of SOD and CAT when compared with those from the control group, but they do not show difference in the activity of GPx or GSH. Oxidative damage was also observed in plasma proteins, but not in the cellular hemolysate. Patients with iron deficiency anaemia showed oxidative damage in plasma proteins significantly higher when compared with the control group. And they also showed a significant increase in the production of MDA in the serum as an indicative of the increased auto-oxidation of lipids under oxidative stress. Serum vitamin C levels did not show significant difference between the groups. These data reveal the involvement of the reactive oxygen species in carential anaemia. Therefore, we reinforce the need to diagnose and treat these patients early to reduce the magnitude of the effects of anaemia on the heath of the elderly.

O fator de transcrição mitocondrial A (TFAM) pertence ao grupo das proteínas de alta mobilidade, apresentando um importante papel para a replicação, transcrição e estrutura/organização do DNA mitocondrial (DNAmt). O DNAmt está organizado em um complexo nucleoprotéico, chamado de nucleóide, do qual TFAM é o principal componente protéico, empacotando o DNAmt de forma análoga às histonas no DNA nuclear. Em analogia ao DNA nuclear, foi sugerido que esse empacotamento pode proteger o DNAmt do ataque de espécies oxidantes, enquanto que, por outro lado, poderia também impedir o acesso das enzimas de reparo. Este trabalho visou esclarecer qual o papel de TFAM na proteção do DNAmt e entender como TFAM influencia o reparo do DNAmt. Nossos resultados indicaram que o empacotamento do DNAmt por TFAM pode proteger o DNA da formação de lesões em condições de estresse oxidativo. Células com redução na expressão de TFAM apresentaram taxas alteradas de proliferação e uma menor viabilidade celular após o tratamento com o fotossensibilizador azul de metileno, indicando que TFAM pode contribuir para a manutenção da integridade funcional da mitocondria. A velocidade do reparo do DNAmt, em células Kd-TFAM, foi aparentemente maior, o que indicou a importância da modulação da interação de TFAM com o DNAmt para um reparo rápido e eficiente das lesões oxidadas. Portanto, TFAM desempenha um papel importante para a estabilidade genômica mitocondrial, protegendo o DNAmt dos efeitos deletérios das lesões oxidadas no estresse oxidativo, e também modulando a velocidade do reparo do DNAmt, provavelmente através de modificações/interações que permitam que as enzimas de reparo acessem as lesões no DNAmt. / The mitochondrial transcription factor A (TFAM) belongs to the high mobility group box proteins, and is essencial for replication, transcription and structure/organization of the mitochondrial DNA (mtDNA).The mtDNA is organized in a nucleoproteic complex called the nucleoid, where TFAMis the main protein component,packaging mtDNA in a manner similar to histones in the nuclear DNA. In analogy to the histone role in nuclear DNA, it was suggested that mtDNA packaging by TFAM could protect the mtDNA against oxidized lesions. On the other hand, it could also prevent the access of repair enzymes. This study aimed to understand whether TFAM plays a role in mtDNA stability through these opposing effects of protecting from damage and preventing repair. Our results indicated that TFAM protects the mtDNA against lesion formation upon oxidative stress. Cells with reduced expression of TFAM showed altered proliferation and lower cellular viability after treatment with the photoactivated dye methylene blue, indicating an important role for TFAM in maintaining mitochondrial function and cell survival. MtDNA repair rate was apparently higherin Kd-TFAM cells, which indicated the importance of modulating the interaction of TFAM with mtDNA for a quick and efficient repair of oxidized lesions. Therefore, TFAM plays an important role in maintaining mitochondrial genomic stability by protecting the mtDNA of the deleterious effects of oxidized lesions in oxidative stress, also modulating mtDNA repair, likely through modifications/interactions that modulate its DNA binding activity and access to lesions in mtDNA by DNA repair enzymes.

The lens of the eye needs to be transparent with a high refractive index to focus images on the retina. In cataracts the lens becomes opaque, eventually leading to blindness. There are many possible causes of cataract but a lot of evidence implicates oxidative damage as contributing to opacification. This includes epidemiological studies showing that diets rich in antioxidants lowered the prevalence of cataract. This research tested the hypothesis that if cataracts were at least partially caused by oxidative damage then their progression would be slowed by application of antioxidants. The antioxidants used were two plant compounds found in the diet, resveratrol and quercetin. The system used was sheep lenses cultured in Eagles Minimal Essential Medium (EMEM). Lenses remained transparent for up to 7 days in EMEM but became opaque within 24 h when exposed to 1 mM hydrogen peroxide (H2O2). The lens is exposed to H2O2 in vivo as it is found in the aqueous humor. Prior Lenses pre-treated with quercetin reduced but did not prevent opacification. Lens cell death, as determined by measurement of leakage of lactate dehydrogenase, was found to increase with H2O2 and the increase was prevented by pre-treatment with antioxidants. The role of the endogenous antioxidant glutathione was also investigated. It was found that H2O2 decreased the amount of reduced glutathione in the lens cortex and increased the levels of oxidised glutathione but only at levels of 2 mM and above. Thus the results of this research indicate that H2O2 at low concentration (1 mM) is able to damage lens cells and cause opacification without affecting the reduced glutathione levels and that the exogenous antioxidants have some ability to protect the lens.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-02-08T18:24:25Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) karine_beatriz_costa.pdf: 1519823 bytes, checksum: a9100527a90f32a6eb92e5f97960ea08 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-03-09T19:22:14Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) karine_beatriz_costa.pdf: 1519823 bytes, checksum: a9100527a90f32a6eb92e5f97960ea08 (MD5) / Made available in DSpace on 2018-03-09T19:22:14Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) karine_beatriz_costa.pdf: 1519823 bytes, checksum: a9100527a90f32a6eb92e5f97960ea08 (MD5) Previous issue date: 2017 / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O desbalan?o entre a produ??o de esp?cies reativas de oxig?nio e nitrog?nio e a a??o dos sistemas de defesa antioxidante ? uma condi??o conhecida como desequil?brio redox. O exerc?cio f?sico m?ximo ? associado ao aumento da produ??o de esp?cies reativas e pode ser utilizado como modelo fisiol?gico para o estudo do desequil?brio redox. Condi??es pr?-anal?ticas de manejo de amostras biol?gicas, como o tempo de congelamento, podem interferir na integridade de analitos. Assim, esse estudo avaliou o efeito do tempo de congelamento da amostra na quantifica??o de biomarcadores de estado redox no gastrocn?mio, cora??o e c?rebro de camundongos swiss submetidos a uma sess?o de exerc?cio m?ximo. Vinte e seis camundongos foram divididos em grupo controle, que n?o realizou exerc?cio, e grupo exerc?cio, submetido a uma sess?o de exerc?cio em piscina, com aumento progressivo de carga, at? a exaust?o. Os tecidos foram coletados imediatamente ap?s o protocolo experimental e seccionados para avalia??o a fresco e ap?s 1, 3 e 6 meses de congelamento a -80 ?C. O exerc?cio m?ximo modificou o estado redox de tecidos frescos, demonstrado pelo aumento da peroxida??o lip?dica, da atividade das enzimas antioxidantes catalase e super?xido dismutase e diminui??o da capacidade antioxidante total em todos os tecidos analisados e pelo aumento de derivados carbon?licos em prote?nas no gastrocn?mio e c?rebro. O efeito do exerc?cio sobre a peroxida??o lip?dica foi reduzido no gastrocn?mio congelado por 6 meses e no c?rebro e cora??o isso ocorreu j? com um m?s de congelamento da amostra. Por outro lado, o tempo de congelamento n?o alterou o efeito do exerc?cio sobre os derivados carbon?licos em prote?nas e a capacidade antioxidante total n?o enzim?tica. A resposta ao exerc?cio da catalase, em todos os tecidos, e da super?xido dismutase no gastrocn?mio foi reduzida ap?s um m?s de congelamento. J? no c?rebro e cora??o a resposta da super?xido dismutase ao exerc?cio foi reduzida apenas ap?s tr?s meses de congelamento. De maneira geral, os resultados desse estudo mostram que o tempo de congelamento afeta, de maneira dependente do tecido e do marcador em an?lise, a resposta de biomarcadores do estado redox a uma sess?o de exerc?cio m?ximo. / Disserta??o (Mestrado) ? Programa Multic?ntrico de P?s-gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / The imbalance between the production of oxygen and nitrogen species and the antioxidant system function is known as redox imbalance. Maximum physical exercise is associated with increased production of reactive species and can be used as a physiological model for the study of redox imbalance. Pre-analytical conditions, such as sample freezing duration, may interfere with analyte integrity. Thus, this study evaluated the effect of sample freezing duration on the quantification of redox biomarkers in the gastrocnemius, heart and brain of Swiss mice submitted to a maximal exercise session. Twenty-six mice were divided into control group, who did not exercise, and exercise group, who performed a maximal swim test. The tissues were collected immediately after the experimental protocol and sectioned for fresh evaluation and after 1, 3 and 6 months of freezing at -80 ? C. The maximal exercise modified the redox status of fresh tissues, demonstrated bythe increase on levels of lipid peroxidation, activity of the antioxidant enzymes catalase and superoxide dismutase and reduction of the total antioxidant capacity, in all the analyzed tissues, and by the increase of protein carbonyl content in the gastrocnemius and brain. The effect of exercise on lipid peroxidation was reduced in gastrocnemius frozen for 6 months and in the brain and heart this occurred in samples after one month of freezing. On the other hand, the freezing duration did not alter the exercise effect on proteins carbonyl content and the total non-enzymatic antioxidant capacity. The catalase response to exercise in all tissues and the superoxide dismutase response in the gastrocnemius were reduced after one month of freezing. However in the brain and heart the response of superoxide dismutase to exercise was reduced only after three months of freezing. This study shows that freezing duration, depending on the tissue and marker under analysis, affects the evaluation of the redox state response to a maximal exercise session.

Sabe-se que concentrações elevadas de homocisteína (Hcy) estão associadas com o aumento do risco de doenças cardiovasculares e com o dano celular causado pela formação das espécies ativas de oxigênio (EAO). Sabe-se também que o estrogênio atua como antioxidante não enzimático envolvido na proteção cardiovascular. Foram objetivos deste trabalho avaliar o efeito da homocistinúria sobre parâmetros de estresse oxidativo em tecido cardíaco; e avaliar o efeito da hiperhomocisteinemia (HHcy) sobre parâmetros de estresse oxidativo e hemodinâmicos em ratas com e sem estrogênio. Este trabalho foi divido em 4 experimentos. No experimento 1 foram utilizados 16 animais divididos em 2 grupos (n= 8/grupo): controle e Hcy. Estes animais receberam tratamento crônico do 6° dia ao 28° dia de vida com doses crescentes de Hcy e foram mortos 1 hora após a última dose.No experimento 2 foram utilizados 30 ratos, divididos em 4 grupos: Salina (n=8); Folato (n=6); Hcy (n=9) e Folato+Hcy (n=7). Estes animais receberam ácido fólico e/ou Hcy do 6° ao 28º dias de vida e foram mortos aos 80 dias de vida. No experimento 3, foram utilizados 64 animais divididos em 6 grupos (n= 8/grupo): NAIVE; NAIVE+Hcy; Sham; Sham+Hcy; castrada e castrada+Hcy. Estes animais foram castrados no 50° dia de vida e, após uma semana, receberam tratamento agudo com Hcy de 8 em 8 horas por 72 horas e foram mortos 1 hora após a última dose. No experimento 4, foram utilizados 32 animais divididos em 4 grupos (n= 8/grupo): controle, castrado, metionina e castrado+metionina. Estes animais foram castrados no 70° dia de vida, receberam metionina na água de beber por 30 dias e foram mortos logo após o final do tratamento. No modelo de homocistinúria (experimento 1), não foram observadas alterações na lipoperoxidação (LPO) cardíaca nos ratos com 28 dias. No entanto, as atividades das enzimas antioxidantes SOD e GST estavam aumentadas no grupo Hcy. Como este é um tratamento crônico, provavelmente estas enzimas estão aumentadas de forma a minimizar o dano oxidativo causado pela Hcy. Já no experimento 2, avaliou-se o efeito da Hhcy a longo prazo, em animais com 80 dias. Houve aumento na LPO dos animais tratados com Hcy que foi previnida com a administração de folato. A redução da LPO na presença do folato confirma sua capacidade de minimizar o dano causado pela Hcy. Observamos ainda adiminuição na atividade das enzimas GST e catalase nos animais tratados com Hcy o que resultaria em aumento da concentração de peróxido de hidrogênio no tecido cardíaco. O tratamento com folato previniu a atividade das enzimas antioxidantes. A partir dos resultados encontrados, podemos sugerir que os níveis de Hcy podem ser reduzidos com folato, uma vez que altas doses de folato reduziram consideravelmente os níveis de estresse oxidativo gerado pela Hcy. No modelo de HHcy aguda (experimento 3), o estresse oxidativo cardíaco aumentou em função da administração de Hcy no grupo sem estrogênio. Este resultado se correlaciona positivamente com a pressão arterial (PA), ou seja, os animais com maior LPO também apresentaram uma maior PA. Este efeito não foi observado nos grupos com níveis estrogênicos fisiológicos. Acredita-se que estes resultados sejam devidos à proteção antioxidante oferecida pelo estrogênio. Além disso, observamos uma diminuição na atividade da GST nos animais castrados+Hcy, o que pode estar contribuindo para o dano oxidativo observado. Já no modelo de HHcy causado pelo consumo de metionina (experimento 4), observamos um aumento na PDFVE no grupo castrado+metionina que se correlaciona negativamente com os metabólitos do NO. Este resultado mostra que os animais que tiveram disfunção ventricular apresentaram uma menor biodisponibilidade do NO. Neste modelo observamos também um aumento no dano oxidativo cardíaco em função da administração de metionina no grupo sem estrogênio. Este resultado se correlaciona positivamente com a PDFVE, ou seja, os animais com maior LPO também apresentam uma maior pressão ventricular diastólica, indicando uma possível participação do estresse oxidativo na disfunção ventricular. Estes animais ainda apresentaram um aumento na atividade das enzimas antioxidantes GST e GPx no grupo castrado+metionina, sugerindo que o tratamento crônico levou a uma adaptação do sistema antioxidante enzimático na ausência do estrogênio. / It is known that high concentrations of homocysteine (Hcy) are associated with the increase of risk of cardiovascular disease and of cellular damage caused by the formation of reactive oxygen species (ROS). It is also known that the estrogen acts as a non enzymatic antioxidant involved in cardiovascular protection. In this work we evaluated the effect of homocystinuria on myocardial oxidative stress parameters, and the effect of hyperhomocysteinemia (HHcy) on the same parameters and also on hemodynamics in rats with and without estrogen. Four experiments were performed. In experiment number one, sixteen animals were divided in 2 groups (n=8/group): control and Hcy. These animals received chronic treatment from the 6th to the 28th day of life with increasing doses of Hcy and were killed one hour after the last dose. In the second experiment, thirty rats were divided into four groups: Saline (n=8); Folate (n=6); Hcy (n=9) and Folate+Hcy (n=7).These animals had received folic acid and/or Hcy from the 6th to the 28th day of life and had been killed with 80 days of life. In the third experiment, fourty eight animals were divided in 6 groups (n=8/group): NAIVE; NAIVE+Hcy; Sham; Sham+Hcy; castrated and castrated+Hcy. These animals were castrated in the 50th day of life and, after one week, they received an acute treatment with Hcy for 72 hours at each eight hours and were killed one hour after the last dose. In the fourth experiment, thirty two animals were divided into four groups (n=8/group): control, castrated, methionine and castrated+methionine. These animals had been castrated in the 70th day of life, and received methionine in drinking water for 30 days and were killed at the end of treatment. In the homocystinuria model (experiment number one), there were no signs of alterations in lipid peroxidation (LPO) in 28 day rats. However, antioxidant enzyme activities of SOD and GST were increased in Hcy group. As this is a chronic treatment, probably these enzymes are increased to minimize the oxidative damage caused by Hcy. In the second experiment, the effect of the homocystinuria was evaluated in animals with 80 days. It was observed an increase in LPO in the animals that had received Hcy, but it returned to control values with folate administration. The reduction of LPO in the presence of folate confirms its capacity of minimize the damage caused by Hcy. We also observedreduction in the enzyme activities of GST and catalase in the animals receiving Hcy, which also returned to the control values with the administration of folate. It is possible that Hcy increases the hydrogen peroxide concnetration in the myocardium of these animals. From the results obtained, we can suggest that the levels of Hcy can be reduced with folate, since high doses of folate had significantly reduced the levels of oxidative stress caused by Hcy. In the acute model of HHcy (experiment number three), myocardial oxidative stress increased due to the administration of Hcy in the group without estrogen. This result has a positive correlation with mean arterial pressure (MAP), that is, the higher LPO, the higher MAP. This effect was not observed in groups with physiological estrogens levels. It is possible that these findings are related to the antioxidant protection offered by estrogen. Moreover, we observed a reduction in the activity of GST in the group castrated+Hcy, which can be contributing for the oxidative damage observed. In the HHcy model caused by the consumption of methionine (experiment number four), we observedThis result has a negative correlation with the nitric oxide metabolites (Nox) showing that the animals that had an increased ventricular diastolic pressure had presented lesser NO bioavailability. In this model we also observed an increase in the myocardial oxidative stress due to the administration of methionine in the group without estrogen. This result has a positive correlation with LVEDP, that is, the animals with enhanced LPO also presented high ventricular diastolic pressure, indicating a possible participation of oxidative stress in ventricular dysfunction. These animals also presented an increase in the activities of GST and GPx in group castrated+methionine, suggesting that chronic treatment with methionine leads to an adaptation of the enzymatic antioxidant system in the absence of the estrogen.

A Anemia Falciforme (AF) é uma doença autossômica recessiva e, dentre as hemoglobinopatias, é a mais comum das alterações hematológicas hereditárias conhecidas no homem. Sua distribuição é ampla, abrangendo todos os continentes. AF foi a primeira doença caracterizada em nível molecular. É causada por uma mutação no gene beta da globina, produzindo uma alteração estrutural na molécula. No gene da globina beta S, há a substituição de uma base nitrogenada do códon GAG para GTG, resultando na substituição do ácido glutâmico (Glu) pela valina (Val) na posição número seis da globina beta. Essa troca dos aminoácidos que resulta na HbS, altera estruturalmente a molécula e, sob determinadas condições, ocorre a polimerização, trazendo graves conseqüências ao indivíduo sintomático. As espécies reativas de oxigênio (ERO) podem causar profundas lesões em eritrócitos, diminuindo seu período de vida útil, em especial nos pacientes com AF. Acredita-se que, os eritrócitos falcizados estejam sob constante estresse oxidativo e, assim, liberem produtos de degradação da HbS, contribuindo para a progressão da doença. Dessa forma, o dano oxidativo agrava a fisiopatologia dos doentes falciformes. Utilizando técnica espectrofotométrica, foram determinadas as atividades das enzimas antioxidantes catalase (CAT), glutationa peroxidase (GPx) e superóxido dismutase (SOD) e quantificada a glutationa total (GSH) nos eritrócitos dos pacientes. Também determinou-se o dano oxidativo nas proteínas plasmáticas e no hemolisado celular pelo método do carbonil a 360 nm. Os níveis da peroxidação lipídica (MDA) e da vitamina C foram determinados por cromatografia líquida de alta performance (HPLC). Quantificou-se, por fim, os níveis de proteína C reativa ultra-sensível (CRPus) por técnica turbidimétrica. Os participantes da pesquisa (30 HbAA, 28 HbAS e 20 HbSS) foram selecionados junto ao Centro de Apoio do Portador de Anemia Falciforme (CAPAF/RS) e/ou cadastrados no Laboratório de Hematologia da Faculdade de Farmácia (UFRGS). Os doentes falciformes foram identificados por HPLC e confirmados por estudo molecular, utilizando a reação da polimerase em cadeia (PCR). Todos os indivíduos assinaram o termo de consentimento livre e esclarecido e foram submetidos a um questionário nutricional. Os dados obtidos foram expressos como médias ± desvio padrão e analisados utilizando-se o Teste ANOVA de uma via com posterior teste ad hoc. Os resultados do trabalho mostram que os indivíduos traço falciforme (HbAS) apresentam atividade significativamente elevada da CAT em relação aos indivíduos controle. Por outro lado, os doentes falciformes possuem maior atividade da GPx e SOD. O nível de GSH foi proporcionalmente maior nos HbSS seguido dos HbAS e HbAA. Observamos também, dano oxidativo em proteínas plasmáticas, mas não no hemolisado celular. Os HbSS possuem dano oxidativo em proteínas plasmáticas significativamente maior que nos demais grupos. Um aumento significativo da produção de MDA no soro dos HbSS foi observado, como um indicativo do aumento da auto-oxidação dos lipídios sob condições de estresse oxidativo. Os níveis séricos da vitamina C foram significativamente maiores nos HbSS que nos indivíduos controle. A CRPus apresentou-se significativamente elevada nos HbSS em relação aos HbAA. Esses resultados reforçam a idéia de que os pacientes com AF estão sujeitos a um estresse oxidativo crônico, o que contribui para a progressão das complicações dessa anemia hemolítica. Já, os indivíduos traço possuem elevada atividade das defesas antioxidantes capazes de reduzir o dano oxidativo em biomoléculas como proteínas e lipídios. / Sickle cell anaemia (SCA) is an autossomal recessive disease and, among the hemoglobinopaties, is the most common of the known hereditary hematologic alterations in man. Its distribution is ample, enclosing all the continents. Sickle cell anaemia (SCA) was the first disease to be characterized on the molecular level. It is basically a red blood cell (RBC) disorder in which the gene encoding the human β-globin subunit presents a mutation with the resulting replacement of β6 glutamic acid (Glu) by valine (Val). This exchange of the amino acids that results in the HbS modifies the molecule structurally, and under determined conditions, the polymeration occurs, bringing serious consequences to the symptomatic individual. The reactive oxygen species (ROS) can cause deep injuries in erythrocytes, diminishing its period of useful life, specially in patients with sickle cell anaemia. Sickled erythrocytes are under constant oxidative stress and thus liberate products of degradation of the HbS, contributing for the progression of the disease. Because of the oxidative damage aggravates the pathophysiology of sickle cell patients. Using spectrophotometrically technique, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), were determined the activities of antioxidant enzymes and total glutathione (GSH) quantified in the erythrocytes of the patients. We also determined oxidative damage of plasma proteins and in hemolysate using carbonyl assay at 360 nm. The levels of lipid peroxidation (MDA) and vitamin C were determined by high-performance liquid chromatography (HPLC). Finally, we quantified the levels of high sensitivity Creactive protein (hsCRP) using turbidimetry technique. The participants of the research (30 HbAA, 28 HbAS and 20 HbSS) were selected from Centro de Apoio ao Portador de Anemia Falciforme (CAPAF/RS) and/or registered in cadastre in Laboratório de Hematologia da Faculdade de Farmácia (UFRGS). Sickle cell patients had been identified by HPLC and confirmed by molecular study, using the polymerase chain reaction amplification (PCR). All individuals signed the term of free and clarified assent and were submitted to a nutricional questionnaire. Data were expressed as average ±SD and analyzed using ANOVA test of one way via with the post ad hoc test. Results show that sickle cell trait subjects (HbAS) had significantly high activity of CAT when compared to healthy controls. On the other hand, sickle cell patients had greater activity of GPx and SOD. The GSH levels was proportionally higher in the followed HbSS of the HbAS and HbAA. We also observe oxidative damage in plasma proteins, but not in the cellular hemolysate. HbSS have significantly higher oxidative damage in plasma proteins than other groups. A significant increasing of the production of MDA in the serum of the HbSS was observed as an indicative of the increasing of the auto-oxidation of the lipids under oxidative estress. Serum vitamin C levels in HbSS were significantly higher than healthy control. The hsCRP was presented significantly higher in HbSS than HbAA. These results reinforce the idea that patients with SCA are subjected to chronic oxidative stress, that contributes for the progression of this hemolytic anaemia. On the other hand, sickle cell trait subjects have higher antioxidant defenses that are able to reduce oxidative damage in biological macromolecules such as proteins and lipids.

A deficiência de ferro é o resultado de um longo período de balanço negativo do ferro, culminando na exaustão do estoque de ferro do organismo. Isto é revelado quando a concentração de hemoglobina declina para valores abaixo dos limites normais e então aparece a anemia, que ocorre devido à deficiente síntese de hemoglobina, com diminuição da proliferação eritrocitária. A anemia ferropênica é considerada o maior problema de saúde no idoso e está associada com várias complicações, incluindo doença cardiovascular, disfunção cognitiva, aumento no risco de morte, e principalmente tem um significante efeito na qualidade de vida. Estudos revelam que a anemia por deficiência de ferro causa maior susceptibilidade aos agentes oxidantes. Utilizando técnica espectrofotométrica, foram determinadas as atividades das enzimas antioxidantes catalase (CAT), glutationa peroxidase (GPx) e superóxido dismutase (SOD) e quantificada a glutationa total (GSH) nos eritrócitos dos pacientes. Também determinou-se o dano oxidativo nas proteínas plasmáticas e no hemolisado celular pelo método do carbonil a 360 nm. Os níveis da peroxidação lipídica (MDA) e da vitamina C foram determinados por cromatografia líquida de alta performance (HPLC). Os participantes da pesquisa foram selecionados de Ambulatórios de Atenção Básica de Saúde da Região Norte do Rio Grande do Sul, sendo que 17 indivíduos apresentaram anemia ferropênica e hipertensão primária e 18 apenas hipertensão primária devido à dificuldade de selecionar pacientes sem nenhuma patologia associada. Todos os indivíduos assinaram o termo de consentimento livre e esclarecido. Os dados obtidos foram expressos como médias ± desvio padrão e analisados utilizando-se o Teste t-Student. Os resultados do trabalho mostram que os indivíduos com anemia ferropênica apresentam atividade significativamente elevada da SOD e da CAT em relação aos indivíduos controle, mas não apresentaram diferença na atividade da GPx nem da GSH. Observamos também, dano oxidativo em proteínas plasmáticas, mas não no hemolisado celular. Os pacientes com anemia ferropênica mostraram dano oxidativo em proteínas plasmáticas significativamente maiores, assim como um aumento significativo da produção de MDA no soro, como um indicativo do aumento da auto-oxidação dos lipídios sob condições de estresse oxidativo. Os níveis séricos da vitamina C não mostraram diferença significativa entre os grupos. Estes dados revelam o envolvimento de espécies reativas de oxigênio no agravamento da anemia carencial. Sendo assim, reforça-se a necessidade de diagnosticar e tratar esses pacientes precocemente, diminuindo a magnitude dos efeitos da anemia na saúde do idoso. / Iron deficiency is the outcome of a long period of negative iron balance, culminating in exhaustion of the body’s iron stores. This is revealed when hemoglobin concentration declines to values below normal levels and anaemia is detected, due to a deficient synthesis of hemoglobin, with decrease in eritrocitary proliferation. Iron deficiency anaemia is considered the most important health problem in elderly and it is associated with various complications, including cardiovascular disease, cognitive dysfunction, increased risk of death and, mostly, it has a meaningful effect in the quality of life. Studies reveal that iron deficiency anaemia cause higher susceptibility to oxidant agents. Using spectrophotometric techniques, the activities of antioxidant enzymes, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and total glutathione (GSH) were quantified in the erythrocytes of the patients. We also determined oxidative damage of plasma proteins and in hemolysate using the carbonyl assay at 360 nm. The levels of lipid peroxidation (MDA) and vitamin C were determined by highperformance liquid chromatography (HPLC). Participants in this study were selected in Ambulatories of Basic Health Attention in the North of the State of Rio Grande do Sul. 17 individuals presented iron deficiency anaemia and primary hypertension and 18 only primary hypertension, because it was difficult to select patients without any associated deficiency. All individuals signed the term of free and clarified consent to participate in this study. Data were analyzed using t- Student Test. Results show that individuals with iron deficiency anaemia show significantly higher activity of SOD and CAT when compared with those from the control group, but they do not show difference in the activity of GPx or GSH. Oxidative damage was also observed in plasma proteins, but not in the cellular hemolysate. Patients with iron deficiency anaemia showed oxidative damage in plasma proteins significantly higher when compared with the control group. And they also showed a significant increase in the production of MDA in the serum as an indicative of the increased auto-oxidation of lipids under oxidative stress. Serum vitamin C levels did not show significant difference between the groups. These data reveal the involvement of the reactive oxygen species in carential anaemia. Therefore, we reinforce the need to diagnose and treat these patients early to reduce the magnitude of the effects of anaemia on the heath of the elderly.