In the field of mammalian reproduction, the success rate of fertilization largely relies on spermatozoa fertility potential. Total proteins change to a large degree during gametogenesis to activate gametes. There are some techniques that are most popular for proteomic studies of fertilization, including SDS-PAGE, 2D-DIGE, Western Blotting and Immunocytochemistry, as well as Mass Spectrometry. Proteins and their cofactors play important roles at different stages of gametogenesis, fertilization and embryo development. However, insufficiencies in the construction mode of fertility hinder the techniques in determination the sperm fertility. This study focused on identifying and tracking crucial proteins for fertility based on comparison between high- and lowertility sperms by means of 2D-DIGE and immunoblotting. We identified 18 proteins that varied significantly, including Outer dense fiber 2 (ODF2) of sperm tails and Manganous superoxide dismutase (MnSOD). Differences in these proteins suggest that posttranslational modification and deoxydation of sperm proteins might be associated with fertility.

Background: Oxidative stress, generating from betel quid (BQ) chewing, cigarette smoking, and alcohol drinking; regulating by antioxidant-oxidant enzymes and dietary antioxidants seems to play a role in oral carcinogenesis. Objective: We aimed to examine the association between antioxidant-oxidant gene polymorphisms (CYBA, MnSOD, MPO, GPX1 and CAT), oral habits, and dietary antioxidants with the risk of oral squamous cell carcinoma (OSCC). Design: A total of 381 pathologically proved primary OSCC cases and 598 healthy controls matched for age and sex were recruited between July 2003 and February 2008 in the hospital-based case-control study. Another 200 cancer-free controls frequency matched to 200 case patients on sex, age (¡Ó5 years), and pack-years of betel quid chewing. All subjects were interviewed to collect the data on socio-demographic variables, histories of BQ-chewing, tobacco smoking, alcohol drinking, and dietary antioxidant intake. Then, TaqMan assay were used to identify the genotype of functional or common allele tagging SNPs of each gene. The plasma total antioxidant capacities were measured by colorimetric assay. Results: Higher intakes of vitamin C, vitamin E and lycopene together with gene polymorphisms (SOD2, GPX1, and CYBA) were associated with a decreased risk for OSCC in a trend-related manner. The risk of OSCC associated with CYBA genotype was modified by alcohol (Pinteraction = 0.04). Significant interactions were observed between BQ-chewing and SOD2 V16A (Pinteraction = 0.001), MPO G-463A (Pinteraction = 0.006) and CAT C3261T (Pinteraction = 0.002). GPx1 polymorphism interact with vitamin C and lutein/zeaxanthin to modify the risk of OSCC, respectively (Pinteraction = 0.023 and 0.006). In the combined analysis, a preventive relation appeared with subjects with seven ¡§at risk genotype¡¨ (AOR, 0.62; 95% CI, 0.36-1.04) and those with three to six ones (AOR, 0.55; 95% CI, 0.33-0.94) compared with 8-9 ones in a trend-related manner (Ptrend = 0.042). It showed an interaction effect between BQ-chewing and the combination of antioxidant-oxidant gene polymorphisms with OSCC risk (Pinteraction = 0.001). The dose-dependent protective effect was related to the decreased numbers of ¡§at risk genotypes¡¨ in lower intake of vitamin E (AOR, 0.54; 95% CI, 0.27-1.11 for 7 ¡§at risk genotype¡¨; AOR, 0.44; 95% CI, 0.21-0.90 for 3-6 ¡§at risk genotype¡¨; Ptrend = 0.035), and in higher intake of vitamin C (AOR, 0.33; 95% CI, 0.13-0.82 for 7 ¡§at risk genotype¡¨; AOR, 0.31; 95% CI, 0.12-0.73 for 3-6 ¡§at risk genotype¡¨; Ptrend = 0.047) and lycopene (AOR, 0.48; 95% CI, 0.20-1.14 for 7 ¡§at risk genotype¡¨; AOR, 0.38; 95% CI, 0.16-0.93 for 3-6 ¡§at risk genotype¡¨; Ptrend = 0.049). In stratification of the numbers of ¡§at risk genotypes¡¨ of XRCC1 (XRCC1 R194W, R180H and R399B) for two groups (0-1 and 2-3 ¡§at risk genotype¡¨ of XRCC1), the decreased risk of OSCC was observed with the decreasing number of ¡§at risk genotype¡¨ in the antioxidant-oxidant genes (AOR, 0.34; 95% CI, 0.14-1.83 for 7 ¡§at risk genotype¡¨; AOR, 0.31; 95% CI, 0.14-0.74 for 3-6 ¡§at risk genotype¡¨; Ptrend = 0.032) among those with 0-1 ¡§at risk genotype¡¨ of XRCC1. Significant interactions between MPO G-463A and alcohol consumption (Pinteraction 0.035), as well as between CYBA and lycopene intake in relation to OSCC risk (Pinteraction 0.036) respectively were found in those matched on BQ-chewing. Different from general population, the significant decreased risk of OSCC was observed among 2-3 ¡§at risk genotypes¡¨ of XRCC1 with less ¡§at risk genotype¡¨ (1-4) in the antioxidant-oxidant genes (AOR, 0.45; 95% CI, 0.25-0.82). In addition, we observed that subjects with seven to nine ¡§at risk genotype¡¨ had significantly lower TAS level than those with less than 7 (P = 0.024) ones. Conclusion: Antioxidant-oxidant genes and dietary antioxidants play an important role in cancer prevention. Dietary antioxidant intakes, alcohol and BQ-chewing may modify the protective magnitude of antioxidant genes. The synergistic effect of dietary antioxidant intakes and antioxidant-oxidant gene polymorphisms may decrease the impact of smoking, drinking or BQ-chewing on susceptibility to OSCC.

Abstract In this work the extent of apoptosis was studied in a set of 504 benign and malignant breast lesions to elucidate its role in breast tumor development and progression. Also the correlation of apoptosis with estrogen and progesterone receptor positivity, cell proliferation and patients' prognosis was studied. The breast lesions were also analyzed immunohistochemically with antibodies to apoptosis regulating proteins bcl-2 and bax, and caspases 3, 6 and 8. In addition, the immunohistochemical expression of NO• synthesizing enzyme iNOS in relation to apoptosis and angiogenesis was studied. Furthermore, the expression of the antioxidative enzyme MnSOD was studied in relation to apoptosis and cell proliferation. According to the results, the apoptotic index was lowest in benign breast lesions. It was higher in in situ carcinomas, where a gradual increase in the extent of apoptosis from grade I to III in situ carcinoma was seen. The apoptotic index in invasive carcinomas was higher than in in situ carcinomas, and also in invasive carcinomas there was a gradual increase in apoptosis from grade I to III carcinomas. The apoptotic index was highest in recurrent carcinomas. Strong bcl-2 expression was usually found in benign breast lesions but the immunoreactivity decreased in in situ and invasive carcinomas. There was a significant inverse association between bcl-2 immunoreactivity and the extent of apoptosis. Low bcl-2 immunoreactivity also associated with estrogen- or progesterone receptor negativity. In contrast, bax expression did not show any significant association with apoptosis, hormone receptors or the histologic types of tumors. Strong cytoplasmic caspase 3, 6 and 8 immunoreactivity was found in most carcinomas. It was weaker in in situ carcinomas and only weak immunoreactivity could be seen in benign breast lesions. There was a significant association between the extent of apoptosis and caspase immunoreactivity. iNOS expression was found in both tumor and stromal cells. iNOS expression in tumor cells was more frequently found in invasive than in in situ carcinomas. Its expression correlated significantly with a high apoptotic index and high vascularization of the lesion. There was significantly less MnSOD immunoreactivity in invasive breast carcinomas compared to in situ carcinomas or benign hyperplasias. MnSOD immunoreactivity did not associate with the extent of apoptosis, but there was a marginal inverse association between cell proliferation and MnSOD expression. Increased apoptosis was significantly associated with a high cell proliferation, and inversely associated with a positive estrogen status. A high apoptotic index (< 0.50%) was associated with a decreased survival of the patients. The results of this study show that apoptosis plays a decisive role in the development and progression of breast carcinoma. It is influenced not only by apoptosis regulating proteins, such as bcl-2 and caspases, but also by the estrogen receptor status. Apoptosis was also associated with iNOS positivity, the effect of which is mediated through increased NO• production. In line with the suggested role of MnSOD as a tumor suppressor gene, its expression was downregulated in invasive breast carcinoma. In conclusion, the association of apoptosis with patient survival in breast carcinoma may be secondary to its association with tumor cell proliferation and high tumor grade, not necessarily suggesting any causal association between apoptosis and survival.

This study aimed to analyze the association between the genetic polymorphism of the manganese-dependent superoxide dismutase (Ala16Val MnSOD) and the oxidative and inflammatory markers in hypercholesterolemic and control individuals. Cholesterol levels in the control group were 104 to 178 mg/dL (2.69 4.61 mmol/L), while the hypercholesterolemic group presented levels 250 to 529 mg/dL (6.47 13.70 mmol/L).. The following biomarkers were also investigated: cholesterol-LDL oxidized (ox-LDL), antibodies anti-LDL oxidized (Anti-ox-LDL), ultra-sensitive C reactive protein (us-CRP), thiobarbituric acid reactive substances (TBARS), carbonyl protein, thiol groups, glutathione (GSH), Vitamins C and E, as well as the superoxide dismutase antioxidant enzymes (SOD) and catalasis (CAT). Additionally, we evaluated the levels of ischemia-modified albumin (IMA), as well as the lipid profile. IMA levels were higher in the hypercholesterolemic group and a significant association between hypercholesterolemia and ox-LDL, Anti-ox-LDL, IMA and us-CRP was observed. A negative correlation between HDL and us-CRP was observed as well. Ala16Val polymorphism influenced the oxidative and inflammatory markers and HDL cholesterol levels were lower in the hypercholesterolemic individuals with the allele V (VV + AV). The present study demonstrated a positive correlation between the total cholesterol levels, TBARS, carbonyl protein and thiol groups. In the hypercholesterolemic individuals there was a reduction in the GSH levels and in the SOD activity, probably due to the enzyme inactivation caused by the protein oxidation. The CAT activity significantly increased probably to partially compensate the oxidative stress. An increase in the Vitamin E serum levels was also observed in the hypercholesterolemic individuals. The group with hypercholesterolemia presented an increase of the oxidative stress, especially for the individuals with a VV genotype to Ala16Val MnSOD polymorphism. TBARS levels, carbonyl protein, thiols groups, Vitamin E and the catalasis activity were significantly higher in the hypercholesterolemic individuals with a VV genotype while GSH and SOD were lower in these individuals. Functionally, the Val MnSOD variant reduces the MnSOD efficiency thus increasing the probability of development of endothelial dysfunction and contributing to the increase in the risk of cardiovascular events, especially when associated to hypercholesterolemia states. / Este estudo analisou a associação entre o polimorfismo genético da superóxido dismutase dependente de manganês (Ala16Val MnSOD) e biomarcadores oxidativos e inflamatórios em sujeitos hipercolesterolêmicos e controles. Os níveis de colesterol no grupo controle foram de 104 a 178 mg/dL (2.69 4.61 mmol/L) e hipercolesterolêmicos foram de 250 a 529 mg/dL (6.47 13.70 mmol/L). Os biomarcadores estudados foram: colesterol-LDL oxidado (ox-LDL), anticorpos anti- LDL oxidado (Anti-ox-LDL), proteína C reativa ultra-sensível (PCR-us), TBARS, proteína carbonil, grupos tióis, glutationa (GSH), vitamina C e E, bem como enzimas antioxidantes [superóxido dismutase (SOD) e catalase (CAT)]. Adicionalmente foram avaliados os níveis da albumina modificada na isquemia (IMA), bem como o perfil lipídico. Os resultados mostraram uma associação significante entre hipercolesterolemia e altos níveis de ox-LDL, Anti-ox-LDL, IMA e PCR-us. Esses resultados mostraram uma correlação positiva entre IMA e Anti-ox-LDL, e uma correlação negativa entre HDL e PCR-us associado a hpercolesterolemia. A influência do polimorfismo Ala16Val nos biomarcadores oxidativos e inflamatórios mostrou que os níveis de HDL foram mais baixos em hipercolesterolêmicos com o alelo V (VV + AV). A presente investigação destacou uma correlação positiva entre níveis de colesterol total, TBARS, proteína carbonil e grupos tióis. Nos sujeitos hipercolesterolêmicos ocorreu uma redução dos níveis de GSH e da atividade da SOD, provavelmente devido à inativação da enzima causada pela oxidação da proteína. A atividade da CAT foi aumentada provavelmente para compensar parcialmente o estresse oxidativo. Um aumento nos níveis sorológicos da Vitamina E também foi observado em sujeitos hipercolesterolêmicos. Baseado nos resultados encontrados, sugere-se uma correlação significante entre hipercolesterolemia e biomarcadores inflamatórios e de estresse oxidativo. Estudos prévios tem demonstrado a interação entre o genótipo VV e biomarcadores oxidativos como oxi-LDL. Contudo, a redução nos níveis de HDL é relevante em hipercolesterolêmicos VV quando comparados com outros grupos. Funcionalmente, a variante Val MnSOD reduz a eficiência MnSOD aumentando a probabilidade de disfunção endotelial e quando associado a hipercolesterolemia pode contribuir para aumentar o risco de eventos cardiovasculares.

Alzheimer’s disease (AD) is a multifactorial, progressive, age-related neurodegenerative disease. Oxidative stress hypothesis is most prevalent and is gaining significant support. Inspite of the progress achieved on oxidative stress related damages in AD brain; the modification occurring on the various cellular antioxidant enzymes antioxidant has not been identified. Tyrosine nitration, a marker for peroxynitrite induced oxidative damage to protein is widespread in AD brain and Manganese superoxide dismutase (MnSOD), primary mitochondrial antioxidant enzyme is prone to peroxynitrite induced nitration and inactivation. Nitration of proteins involved in energy metabolism has been demonstrated in AD brain, which may explain the altered glucose metabolic status existing in AD brain. In the present study, we investigated the effect of tyrosine nitration of MnSOD on energy metabolism by the use of AD mouse model and cultured neuronal cells. The AD mouse model was generated from a double homozygous knock-in mouse, designated as APP/PS-1 mice, by incorporating the Swedish familial AD mutations in APP and P264L familial AD mutation in PS – 1. These animals develop age dependent increase in Aβ deposition beginning at 6 months along with an increase in insoluble Aβ1-40/Aβ1-42 levels. Genotype and age associated increase in nitration of MnSOD without any change in protein levels was also observed. MnSOD activity and mitochondrial respiration was decreased in APP/PS-1 mice. There was also concomitant increase in levels of lactate, an index of glycolytic activity in APP/PS-1 mice. To directly investigate the role of MnSOD inactivation in mitochondrial function and subsequent alteration in glycolytic activity, SH-SY5Y neuroblastoma cells line was used and treated with peroxynitrite. Enhanced nitration and reduction in the activity of MnSOD was observed upon peroxynitrite treatment. Peroxynitrite treatment also induced mitochondrial dysfunction, but MnSOD was inactivated at a concentration of peroxynitrite 10 times lower than that required to inhibit mitochondrial respiration. Mitochondrial dysfunction was alleviated by SOD mimetic and reproduced by MnSOD siRNA. The decline in mitochondrial function did not result in decreased ATP levels but was accompanied by an up-regulated glycolysis signified by high levels of lactate and lactate dehydrogenase activity but decreased activity of pyruvate dehydrogenase. These changes were prevented by SOD mimetic and were promoted by MnSOD siRNA. Specific reduction of MnSOD in MnSOD heterozygous knock-out mice resulted in decreased RCR and complex I activity with increased lactate levels. Taken together, these data demonstrate a critical role of MnSOD in influencing the mitochondrial function and thereby the switch in the energy metabolism switch that might occur under the pathological condition of MnSOD deficiency.

UVB radiation is a known environmental carcinogen that causes DNA damage and increase ROS generation in mitochondria. Accumulating evidence suggests that mtDNA damage and increased ROS generation trigger mitochondrial translocation of p53. Within mitochondria, p53 interacts with nucleoid macromolecular complexes such as mitochondrial antioxidant MnSOD, mitochondrial DNA polymerase Polγ, and mtDNA. Mitochondria are considered to be a potential source for damage-associated molecular patterns (DAMPs) such as mtDNA, cytochrome C, ATP, and formyl peptides. Intracytoplasmic release of DAMPs can trigger inflammasome formation and programmed cell death processes. Autophagic clearance of mitochondria with compromised integrity can inhibit inflammatory and cell death processes. In this study we investigated whether and how MnSOD plays a protective role in UVB-induced mitochondrial damage. The possibility of MnSOD participating in the mtDNA repair process was addressed in vivo using transgenic and pharmacological approaches. The results demonstrate that MnSOD functions as a fidelity protein that maintains the activity of Polγ by preventing UVB-induced nitration and inactivation of Polγ and that MnSOD coordinates with p53 to prevent mtDNA damage. We also investigated whether autophagy is an adaptive response mechanism by which skin cells respond to mitochondrial injury, using mouse keratinocytes (JB6 cells) and C57/BL6 mice as in vitro and in vivo models. The results demonstrate that UVB induces autophagy initiation in murine skin tissues and that down regulation of AKTmTOR levels triggers initiation of autophagy processes. These results suggest that autophagy may play a role in scavenging damaged mitochondria. Taken together, the results from these studies suggest that MnSOD plays a protective role against UVB-induced mitochondria injury beyond its known antioxidant function. Within the mitochondrial matrix, MnSOD acts as an antioxidant and fidelity protein by prevention of UVB-induced nitration of Polγ. The functions of MnSOD may be to enhance mitochondrial membrane integrity and to prevent the genesis of oxidatively damaged mitochondrial components and subsequent intracytoplasmic spillage. Activation of autophagy serves as an additional response that scavenges damaged mitochondria.

PCB126 is the most potent agonist of the aryl hydrocarbon receptor (AhR) in the whole family of chlorinated biphenyls (PCBs), a class of persistent organic pollutants that are classified as probable human carcinogens. It exerts systematic toxicities in animals mainly through the AhR pathway and generates cellular oxidative stress which might damage cellular macromolecules. The goal of this thesis is to elucidate the mechanisms of PCB126 toxicity in the aspect of AhR-related oxidative stress. Reactive superoxide radicals could be generated as the result of electron leakage in the catalytic cycles of cytochrome P450 (CYP) enzymes which are super-induced by sustained AhR activation in response to PCB126. In addition, the disruption of mitochondrial electron transport chain caused by AhR ligands also contributes to the production of superoxide radicals. Correspondingly in this thesis, the impact of PCB126 on the expression of the recently-discovered, TCDD-inducible microsomal CYP2S1 enzyme was studied. Then the regulation of mitochondrial antioxidant enzyme MnSOD in response to PCB126 was investigated. Lastly, a microarray study on classic AhR ligands of β-naphthoflavone and 3-methylcholanthrene was conducted to explore possible mechanisms of AhR-associated toxicities and to find explanations for the regulation of CYP2S1 as well as MnSOD after PCB126 exposure. It was found that CYP2S1 was only weakly or not at all regulated by PCB126 in rats. As for MnSOD, although its messenger and protein levels were induced by PCB126, its enzymatic activity was significantly reduced in the liver, probably through post-translational mechanisms. Although dietary manganese supplementation did not reverse the loss of MnSOD activity due to PCB126 exposure, it significantly protected against liver hypertrophy caused by PCB126. Microarray study results were consistent with previous findings and indicated that in addition to changed expression of a number of CYPs, metal homeostasis as well as mitochondria functions were also affected by AhR ligands. Overall, both CYP enzymes and the mitochondria contribute to the AhR-mediated toxicities of PCB126 that are associated with a disturbance of cellular redox homeostasis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The deficit of estrogen that accompanies menopause may be involved in the metabolic changes and increased oxidative stress during non-reproductive female life. Hormone replacement therapy (HRT) has been used to attenuate the menopausal symptoms. It is prescribed either as the replacement of estrogen alone or the combination of estrogen with progestins. Superoxide dismutase (SOD) is a key enzyme in the control of reactive oxygen species levels and SOD modulators may have potential use as therapeutic agents in oxidative stress-associated disorders. The objectives of this study were to evaluate: i) the effects of natural and synthetic estrogens and progestins on the activity of SOD from human blood in vitro; and ii) the effect of the hormone therapy with estrogen or estrogen plus progestin on the markers of oxidative stress in the blood of postmenopausal women and the relationship among these markers and the serum levels of estradiol and progesterone. The in vitro effect of steroid hormones (17 β-estradiol 17-acetate, progesterone, β-estradiol 3-benzoate and medroxyprogesterone 17-acetate) was evaluated in the enzyme purified from human erythrocytes (CuZnSOD) (Sigma) and in samples of erythrocytes (cytosolic CuZnSOD) and platelets-rich plasma (PRP) (MnSOD and cytosolic and extracellular CuZnSOD) obtained from healthy men and women. Hormones caused a dose-dependent stimulation of erythrocyte CuZnSOD activity at low concentrations (physiological), but this effect was abolished at higher concentrations. The combination of an estrogen with a progestin had a synergic effect on the erythrocyte CuZnSOD activity. In the PRP the activity of MnSOD was not affected by hormones, whereas the CuZnSOD activity was modulated only by the natural, but not by the synthetic hormone derivatives. Four groups of women were selected to evaluate blood markers of oxidative stress: premenopausal women (n=24), postmenopausal women without hormone therapy (HT) (n=31), postmenopausal women with estrogen-only HT (ET) (n=12) and estrogen plus progestin HT (EPT) (n=16). The levels of protein carbonyl, lipid peroxidation and the activity of catalase and glutathione peroxidase did not differ among groups. However, the activities of SOD isoforms (CuZn and MnSOD) and total plasma antioxidant power (FRAP) were significantly higher in postmenopausal women under EPT compared with postmenopausal women without HT, whereas ET increased only the activity of CuZnSOD in postmenopausal women. The duration of HT and serum E2 levels were positively correlated with the activity of CuZnSOD and the total antioxidant power of plasma (FRAP levels), whereas progesterone levels were positively correlated with the activity of CuZnSOD and negatively correlated with protein carbonyl levels. The total antioxidant power of plasma was positively correlated to the CuZnSOD activity and to the GPx activity. The present study demonstrated for the first time, that the natural and synthetic steroid hormones have a direct biphasic effect on CuZnSOD activity of human erythrocytes in vitro. We also observed that the hormone replacement therapy increase the antioxidant status of postmenopausal women due to an increase of the enzymatic antioxidant defenses and this effect is more remarkable with the combined hormone therapy (estrogen plus progestin). / O déficit de estrogênio, que acompanha a menopausa pode ser relacionado às alterações metabólicas e ao aumento do estresse oxidativo, observados na fase não reprodutiva feminina. A terapia de reposição hormonal é utilizada para atenuar os sintomas da menopausa. Ela é prescrita como reposição de estrogênio ou uma combinação de estrogênio com progestina. A superóxido dismutase (SOD) é uma enzima chave no controle dos níveis de espécies reativas de oxigênio e, moduladores da SOD podem ser úteis como agentes terapêuticos em desordens associadas ao estresse oxidativo. Os objetivos deste estudo foram avaliar: i) os efeitos, in vitro, de estrógenos e progesterona, naturais e sintéticos, sobre a atividade da SOD presente em sangue humano, e ii) o efeito da terapia hormonal com estrogênio ou estrogênio mais progestinas sobre os marcadores de estresse oxidativo no sangue de mulheres na pós-menopausa e a relação entre esses marcadores e os níveis séricos de estradiol e progesterona. O efeito, in vitro, de hormônios esteroides (acetato de 17β-estradiol (E2), progesterona, 3-benzoato de 17β-estradiol e 17-acetato de medroxiprogesterona) foi avaliado na enzima purificada a partir de eritrócitos humanos (CuZnSOD) (Sigma), e em amostras de eritrócitos (CuZnSOD citosólica) e de plasma rico em plaquetas (PRP) (CuZnSOD, citosólica e extracelular, e MnSOD, mitocondrial), obtidas a partir de homens e mulheres saudáveis. Os hormônios, em concentrações baixas (fisiológica), causaram uma estimulação, dose dependente, da atividade da CuZnSOD eritrocitária, embora, este efeito tenha sido suprimido em concentrações mais elevadas. Ademais, a combinação de um estrogênio com uma progestina apresentou um efeito sinérgico sobre a atividade da CuZnSOD eritrocitária. No PRP a atividade da MnSOD não foi afetada por hormônios, enquanto que a atividade da CuZnSOD foi modulada apenas pelos esteroides naturais. Quatro grupos de mulheres foram selecionados para avaliar marcadores sanguíneos de estresse oxidativo: mulheres na pré-menopausa (n = 24), mulheres na pós-menopausa sem terapia hormonal (TH) (n = 31), mulheres na pós-pausa utilizando TH, composta apenas de estrogênio (ET) (n = 12), ou de uma combinação de estrogênio mais progestinas (EPT) (n = 16). Os níveis de proteína carbonilada, peroxidação lipídica e da atividade de catalase e glutationa peroxidase (GPx) não diferiram entre os grupos. No entanto, as atividades das isoformas da SOD (CuZn e MnSOD) e o poder antioxidante total do plasma (FRAP) foram significativamente maiores em mulheres na pós-menopausa sob EPT em comparação com mulheres na pós-menopausa sem TH, enquanto que a ET aumentou apenas a atividade da CuZnSOD em mulheres na pós-menopausa. A duração da TH e os níveis séricos de E2 foram positivamente correlacionados com a atividade da CuZnSOD e com o poder antioxidante total do plasma (FRAP), enquanto que os níveis de progesterona foram positivamente correlacionados com a atividade da CuZnSOD e negativamente correlacionados com os níveis de proteína carbonilada. O poder antioxidante total do plasma foi positivamente correlacionada com a atividade da CuZnSOD e da GPx. O presente estudo demonstrou, pela primeira vez, que os hormônios esteroides, naturais e sintéticos, têm um efeito direto e bifásico na atividade da CuZnSOD eritrocitária humana in vitro. Também foi observado que a terapia de reposição hormonal aumenta a capacidade antioxidante de mulheres na pós-menopausa devido a um aumento das defesas antioxidantes enzimáticas (SODs) e que este efeito é ainda mais pronunciado com o uso de terapia hormonal combinada (estrogênio e progestinas).

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-09-26T18:59:48Z No. of bitstreams: 1 EMANUELLE DAL PONTE.pdf: 853027 bytes, checksum: 3383ad493f7b637d87265a5e9064c8f0 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-09-26T19:00:18Z (GMT) No. of bitstreams: 1 EMANUELLE DAL PONTE.pdf: 853027 bytes, checksum: 3383ad493f7b637d87265a5e9064c8f0 (MD5) / Made available in DSpace on 2018-09-26T19:00:18Z (GMT). No. of bitstreams: 1 EMANUELLE DAL PONTE.pdf: 853027 bytes, checksum: 3383ad493f7b637d87265a5e9064c8f0 (MD5) Previous issue date: 2017-07-03 / O traço falciforme (HbAS) é o heterozigoto da anemia falciforme, portador do gene S assintomático e considerado, ainda, clinicamente normal. Estudos epidemiológicos mostram sua alta prevalência na população brasileira sendo que, em determinadas cidades do Rio Grande do Sul, estima-se uma frequência de 1:75 habitantes. Novos estudos têm apontado que o portador do traço falciforme, por apresentar aproximadamente 40% de HbS, tem envolvimento com estresse oxidativo, o qual pode atuar na progressão da doença. A Superóxido Dismutase (SOD) é uma enzima antioxidante que pode ter sua atividade diminuída em algumas patologias. O polimorfismo mais comum da MnSOD é o Ala16Val, que é resultado de uma mutação que substitui o códon original GCT (alanina) para GTT (valina). O alelo valina (V) dificulta o transporte e a entrada da enzima para dentro da mitocôndria diminuindo, assim, a capacidade antioxidante do indivíduo e favorecendo as vias de estresse oxidativo. O objetivo deste trabalho foi analisar a presença do polimorfismo Ala16Val MnSOD em portadores do traço falciforme e associá-lo com biomarcadores de estresse oxidativo. Os participantes do estudo foram recrutados junto ao Banco de Sangue do Hospital da Santa Casa de Caridade de Uruguaiana. Ao total obteve-se 102 indivíduos, sendo 50 do grupo controle e 52 do grupo traço falciforme (HbAS). Após a assinatura do termo de consentimento livre e esclarecido, foi realizada a coleta de sangue venoso. Dois tubos contendo EDTA foram coletados para as análises hematológicas, genômicas e de estresse oxidativo. Os resultados encontrados apontam que os portadores do traço falciforme apresentam as atividades da catalase, superóxido dismutase e glutationa peroxidase significativamente diminuídas (p<0,001) em relação ao grupo controle. O mesmo também foi observado com os níveis de compostos antioxidantes como a glutationa, vitamina C e status antioxidante total (p<0,001). Entretanto, os portadores do traço falciforme apresentam aumento estatisticamente significativo (p<0,001) no dano oxidativo a biomoléculas como carbonilação de proteínas, lipídeos e o DNA. A análise do polimorfismo Ala16Val MnSOD mostrou 100% do genótipo AV no grupo controle e 21% do genótipo AA e 79% do genótipo AV no grupo HbAS. O presente estudo não conseguiu, todavia, associar o polimorfismo com os biomarcadores de estresse oxidativo. Sugere-se, dessa forma, que o portador do traço falciforme possui aumento do estresse oxidativo e o polimorfismo Ala16Val MnSOD parece não estar associado aos parâmetros analisados. Entretanto, por esse estudo ser o primeiro a ser relatado, espera-se que em uma população maior de HbAS seja possível ampliar o conhecimento acerca dessa alteração genética. / Sickle cell trait (SCT) is the heterozygote of sickle cell anemia (HbAS), which carries the asymptomatic S gene and is considered clinically normal. Epidemiological studies show its high prevalence in the overall Brazilian population, and in certain cities such as Rio Grande do Sul, where a frequency of 1:75 is estimated. Recent studies have pointed out that the SCT because it represents approximately 40% of hemoglobin S (HbS), is involved with oxidative stress, which can act in the progression of the disease. Superoxide dismutase (SOD) is an antioxidant enzyme that may have decreased activity in some pathologies. The most common MnSOD polymorphism is Ala16Val, which is the result of a mutation that replaces the original GCT (alanine) codon for GTT (valine). The valine allele (V) hinders the transport and entry of the enzyme into the mitochondria, thereby both reducing the antioxidant capacity of the individual and favoring oxidative stress pathways. The aim of this study is to analyze the presence of the Ala16Val MnSOD polymorphism in patients with SCT and to associate it with biomarkers of oxidative stress. The study participants were recruited from the Blood Bank of the Santa Casa de Caridade Hospital of Uruguaiana. A total of 102 individuals were enrolled, 50 in the control group and 52 in the SCT group. After obtaining written informed consent, venous blood was collected. For each one, two tubes containing EDTA were collected for hematological, genomic, and oxidative stress analyses. The results showed that sickle cell carriers exhibit significantly decreased (p < 0.001) catalase, superoxide dismutase, and glutathione peroxidase activities compared with the control group. The same was also observed for the levels of antioxidant compounds such as glutathione, vitamin C, and total antioxidant status (p < 0.001). However, patients with SCT presented a statistically significant increase (p < 0.001) in oxidative damage to biomolecules such as protein carbonylation, lipid peroxidation, and DNA. Analysis of the Ala16Val MnSOD polymorphism showed 100% of the AV genotype in the control group, and 21% of the AA genotype and 79% of the AV genotype in the HbAS group. Although the current study could not associate the polymorphism with oxidative stress biomarkers, it suggested the SCT increased oxidative stress, and the Ala16Val MnSOD polymorphism was not associated with the parameters analyzed. However, because this study is the first to report these findings regarding the Ala16Val MnSOD polymorphism, it is expected that in a larger HbAS population, it will be possible to gain a better understanding of this genetic alteration.

Breast cancer is a heterogeneous disease with multiple phenotypes that specify both treatment options and prognosis. The Luminal A-type breast cancers, characterized by high levels of estrogen receptor (ER) expression and transcriptional activity, have a stable of hormone-related treatment options and a favorable prognosis. Recent efforts to identify mechanisms governing the Luminal A phenotype have identified transcription factor activator protein 2C (TFAP2C) as a critical regulator of both ER and ER-associated gene expression, making it a prime target for manipulation in breast cancer therapy. To that end, we sought to establish specific contributions of TFAP2C in both normal development and cancer progression, with the overarching hypothesis that TFAP2C is an integral transcription factor in maintaining a luminal differentiation and expression pattern in human breast cancer. To address this, we utilized several parallel approaches to identify potential TFAP2C-related contributions to carcinogenesis. In the first approach, we identified that tissue-specific abrogation of the mouse homolog TCFAP2C in mammary epithelium produced a reduction in luminal populations and a concomitant increase in basal populations, producing mild phenotypic alterations in gland structure. In the second, we stably manipulated TFAP2C expression in the established Luminal A model cell line MCF-7, establishing that TFAP2C loss promotes a loss of luminal differentiation characteristics and a gain of basal / mesenchymal traits, mirroring the results found in the mouse model. As a final approach, we examined the role of a TFAP2C target gene, manganese superoxide dismutase (MnSOD), in modulating redox biological and epigenetic parameters that may contribute to carcinogenesis. We found that loss of MnSOD in the murine liver, chosen because of its utility as a model system, promoted subtle changes in the redox buffering capacity and identified preliminary changes in the epigenome, suggesting that MnSOD modulation by TFAP2C could play a role in cancer development. Overall, these results further establish a role of TFAP2C in the genesis of Luminal A breast cancer, and serve as a foundation for more comprehensive future work evaluating specific contributions in carcinogenesis.