A capacidade de preservar a viabilidade do sêmen pelo emprego das biotecnologias, como refrigeração e criopreservação, oferece muitas vantagens a equideocultura. O estresse oxidativo é um dos principais entraves para estas biotecnicas e advêm do ataque de distintas espécies reativas de oxigênio (EROs). Assim, o objetivo do presente estudo foi verificar o impacto das diferentes EROs sobre os espermatozoides com e sem plasma seminal. Foram colhidos ejaculados de 13 garanhões Mangalarga Marchador de fertilidade conhecida. Os ejaculados foram divididos em duas frações (A e B). As frações foram centrifugadas (2200g/10min) e o pellet com os espermatozoides foi ressuspendido em solução salina fisiológica ou plasma seminal de modo que as duas frações apresentassem a mesma concentração final (sptz/mL). Ambas as frações (A e B) tiveram 4 alíquotas de 400µL retiradas e submetidas a 3 sistemas distintos de produção de EROs (xantina + xantina oxidase que produz anion superóxido; peróxido de hidrogênio; ferro + vitamina C que produz radical hidroxil) e malondialdeído. Após a incubação, as amostras A e B foram avaliadas de acordo com os testes funcionais (coloração de eosina-nigrosina para membranas, fast-green/rosa bengala para acrossomos, coloração 3-3'diaminobenzidina para atividade mitocondrial e SCSA™ para susceptibilidade a denaturação de cromatina) e avaliação do índice de peroxidação lipídica (TBARs).Todas as análises estatísticas foram realizadas pelo programa SAS System for Windows. A susceptibilidade do espermatozoide equino ao estresse oxidativo é variável de acordo com a espécie reativa de oxigênio empregada na incubação; as espécies mais deletérias no presente experimento foram a malondialdeído e principalmente o radical hidroxil o que já era esperado. Houve um efeito benéfico quanto a manutenção das funções celulares e status oxidativo em amostras cujo plasma seminal foi preservado. A proteção atribuída ao plasma seminal influenciou parâmetros como atividade mitocondrial, susceptibilidade a denaturação acida da cromatina e índice de peroxidação lipídica. O plasma seminal exerceu mais pronunciada proteção aos eventos desencadeados pela malondialdeído / Biotechnologies such as chilling and cryopreservation of equine semen have been largely used in horse breeding due to its importance on storage and propagation of genetic material. The oxidative stress is one of the main causes of poor results related to the fertility of processed stallion sperm. Oxidative injuries are mainly due to the attack of different reactive oxygen species (ROS). Thus, the aim of the present study was to evaluate the impact of distinct ROS attack on equine spermatozoa with and without seminal plasma. The collection of one ejaculate of thirteen Mangalarga fertile stallions were performed. Ejaculates were divided in two aliquots (A and B). These aliquots were centrifuged (600G /10 min) and pellets were resuspended in saline solution (0,9%) or seminal plasma certifying that each sample had the same concentration (sptz/mL). Both aliquots (A and B) were divided in four samples of 400 µL each and incubated with four ROS generating systems (superoxide anion; hydrogen peroxide; hydroxil radical and malondialdehyde). After the 30 incubation, all the samples were evaluated for membrane integrity (eosin-nigrosin stain), acrosome integrity (simple stain of fast-green/ Bengal rose), 3-3'diaminbenzidine for mitochondrial activity and SCSA™ for the chromatin suceptibility to the acid denaturation. Lipid peroxidation was accessed by TBARs assay. Statistical analysis was performed using SAS System for Windows. The susceptibility of equine sperm to the oxidative stress is different in each ROS generation system; but the most deleterious ROS were hydroxyl radical and malondialdehyde. A positive effect of seminal plasma on the sperm viability was observed which may be linked to its protective role. The protection occurred in mitochondria, chromatin and against lipid peroxidation. Higher beneficial effect of seminal plasma was observed in samples treated with malondialdehyde

Reactive oxygen species are formed internally through cellular metabolism and through external sources including radiation and pollutants. They play an important role in physiologic functions; however, when reactive oxygen species exceed our body’s antioxidant defense system, oxidative stress can occur. Oxidative stress has been implicated in aging and aging-related diseases including cancer and cardiovascular disease. Numerous oxidative stress genes produce antioxidative enzymes to mitigate the effects of reactive oxygen species. Single nucleotide polymorphisms within these genes may impact the functionality of antioxidant enzymes produced leaving the body more susceptible to damage from oxidative stress. The Iowa 65+ Rural Health Study was one of the four study populations in the Established Population for Epidemiologic Studies of the Elderly (EPESE) project initiated by the intramural Epidemiology, Demography and Biometry Program of the National Institute on Aging in 1980. The Iowa cohort was comprised of Iowa county and Washington county residents aged 65 and older at the time of the baseline interview in 1982. Participants completed three in-person interviews and five telephone interviews over eight years which collected data on habits, lifestyle and disease. During the in-person Year 06 interview participants were asked to donate a blood sample. The DNA extracted from the samples was used in each of the three aims of this project. The first aim evaluated single nucleotide polymorphisms in selected oxidative stress genes and their association with lifespan while controlling for aging-associated risk factors such as body mass index, comorbidity, alcohol consumption, smoking, and physical activity. Multivariable linear regression models were fit in the framework of the co-dominant genetic model. The oxidative stress genes selected for this project included the sirtuin family of genes (SIRT1-7), two of the forkhead box genes (FOXO1 and FOXO3), superoxide dismutase 2 and 3 (SOD2 and SOD3), glutathione peroxidase (GPX1), AKT, TP53, and CAMK4. A model was fitted with the risk factors before assessing the impact of each single nucleotide polymorphism. The q-value was used to control for the multiple hypothesis tests. Significant associations were detected between human lifespan and SNPs in genes SIRT3, SIRT5, SIRT6, FOXO3, and SOD3; gender modified the effect of SNPs in SIRT3, SIRT5, and AKT1. The second aim of this project evaluated single nucleotide polymorphisms in selected oxidative stress genes and their association with blood pressure measures while controlling for known risk factors including body mass index, alcohol consumption, smoking, and physical activity. Blood pressure was measured at the baseline and Year 06 interviews. Systolic pressure and diastolic pressure were used to calculate mean arterial pressure and pulse pressure at baseline and Year 06. Multivariable linear regression was used within the co-dominant genetic framework to determine if single nucleotide polymorphisms in SIRT1-7, FOXO1, FOXO3, SOD2-3, GPX1, AKT, TP53, and CAMK4 were associated with systolic, diastolic, mean arterial, or pulse pressure at baseline or Year 06. To examine longitudinal effects, the difference between each measure (i.e., Year 06 systolic – baseline systolic) was calculated for each individual and used to evaluate if any of the single polymorphisms was associated with change in blood pressure measures over time. Significant associations were detected between SIRT1 and SIRT3 and for males in SIRT1 and various blood pressure measures for females. Gender modified the effect of SIRT1, SIRT3, SIRT6, and FOXO1 variants. The third aim of this project evaluated if these genetic variants were associated with incident stroke while controlling for known risk factors including blood pressure, diabetes, body mass index, alcohol consumption, smoking, and physical activity. Multivariable logistic regression within the framework of the co-dominant genetic model was used. Individuals with the GPX1 genotype TT had 2.76 times the risk of an incident stroke compared to the CC genotype. This project identified several associations between single nucleotide polymorphisms within oxidative stress genes and lifespan, blood pressure measures, and incident stroke. Gender modified the association of several single nucleotide polymorphisms and lifespan as well as blood pressure measures. These results suggest genetic variation within oxidative stress genes may play a role in aging, blood pressure and incident stroke.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21837号 / 農博第2350号 / 新制||農||1069(附属図書館) / 学位論文||H31||N5209(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 田中 千尋, 教授 本田 与一, 准教授 刑部 正博 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

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

Introduction: The desire to have a biological child transcends race, religion and socio-economic status. However for those faced with infertility, the financial resources needed to conceive are often not available. Current research in assisted reproduction has gravitated towards cost reduction to address restricting financial factors, without compromising quality of treatment. One such initiative is the development of a low-cost embryo culture method by The Walking Egg foundation. This method utilizes a standard chemical reaction and simple equipment to equilibrate culture media pH and to regulate temperature; both aspects were investigated in this study. An exploration into the insemination concentration to achieve oocyte fertilization was also undertaken. Methods: Quality control of temperature regulation on six different heating devices, including a comparison of inter- and intra-variations was carried out. The utilization of citric acid and bicarbonate of soda for carbon dioxide production, which subsequently facilitate setting of pH values, was tested by injecting increasing citric acid volumes (1.2 ml – 3.0 ml in 0.2 ml increments) into set volumes of bicarbonate of soda. Further investigation evaluated gas production at various temperatures (37°C, 25°C and 15°C), at increasing intervals (16 – 30 hours) of equilibration and these were compared by measuring pH of the culture media. The influence of altitude on pH was explored by repeating the chemical reaction experiment at five different locations in South Africa. Furthermore, the addition of water to citric acid before gas generation was explored. The minimal insemination concentration needed for fertilization was determined by the addition of decreasing numbers of spermatozoa to non-fertilized bisected oocytes. The experiment was repeated with a selected sperm insemination number in 1 ml or 50 μl culture media to compare the tested culture system with conventional culture. Spermatozoa bound to the hemi-zonae were counted with the aid of an inverted phase contrast microscope. Hemi-zonae with bound sperm were also stained with ethidium homodimer and evaluated using a confocal laser-scanning microscopy system. After removal of hemi-zonae, the spermatozoa in culture were isolated for deoxyribonucleic acid fragmentation analyses and reactive oxygen species presence in the culture media was measured. Additionally, reactive oxygen species generation in simulated culture was measured over time. Results: All the equipment tested bar one, the warming oven, proved useable with the simplified Walking Egg in vitro fertilization culture system. By decreasing the citric acid volumes, it was indicated that 1.8 ml citric acid, diluted with 1.2 ml water, is the optimal volume to facilitate the required culture media pH. Omitting the water dilution from citric acid volumes affected the culture media pH adversely, however reducing the temperature during gas equilibration did not. A change in altitude had no effect on culture media pH. Lower insemination numbers resulted in decreased sperm binding, with 2 x 103 motile sperm insemination providing the lowest number to still obtain sufficient sperm–zona binding (≥20 sperm bound). Incubation in 1 ml vs. 200 μl culture media indicated decrease in sperm bound. Sperm deoxyribonucleic acid fragmentation and the presence of reactive oxygen species in the culture media were similar in both the test and control groups. A comparison over time revealed less reactive oxygen species in 1 ml culture media, from the simplified Walking Egg in vitro fertilization culture system after three days of culture, than 200 μl culture media drops under oil, from conventional culture after 18 hours, however the results were not statistically significant. Discussion: Purpose-made heating devices provide superior stabilization of culture media temperature. When selecting a heating device, intra-variations should be considered. Culture media can be manipulated to the required pH by carbon dioxide production, with meticulous attention paid to the citric acid volumes used. However, if gas generation is performed at room temperature, equilibration time must be increased. In conventional culture, the minimum insemination number can be reduced to 2 x 103 motile sperm. Due to lower binding of sperm in large volumes of culture media, 2 – 5 x 103 motile sperm should be considered for the simplified culture system, depending on a holistic consideration of all sperm parameters. Extended culture for at least three days with the simplified culture system can be performed without increasing reactive oxygen species present in culture media. Further research of this novel culture method should include the application of the culture method in a South African environment. / Dissertation (MSc)--University of Pretoria, 2017. / Obstetrics and Gynaecology / MSc / Unrestricted

Vitamine E analogues α-tocopheryl succinate (α-TOS) and mitochondrially targeted vitamine E succinate (MitoVES) are anti-cancer agents from the group of "mitocans", the compounds acting via mitochondria which present a promising invariant target for cancer cell therapy. α-TOS and MitoVES induce apoptosis selectively in various cancer cell types involving generation of reactive oxygen species (ROS). Generated superoxid anion radicals in response to α-TOS and MitoVES are believed to be converted into hydrogen peroxide that is known to activate Mammalian sterile 20-like kinase (Mst1), the central component of Hippo signalling pathway, that presents an universal size control mechanism in all metazoans and its deregulation is linked to tumourigenesis. MitoVES and α-TOS were both reported to activate Mst1 that phosphorylates Forkhead box O1 (FoxO1) transcription factor resulting in its transport to nucleus where induce the expression of pro-apoptotic genes, including NOXA, and thus promote apoptosis. The target of Hippo signalling pathway is transcriptional co- activator Yes-associated protein (Yap) which was found in Drosophila melanogaster to regulate the expression of transcription factor c-Myc which is known as the most prominent human oncogene. This thesis focused on involvement of Hippo signalling...

Dihydromyricetin (DMH) is a natural flavonoid compound with positive effects on the human organism. In traditional Chinese medicine, plants containing DMH were used to treat liver diseases and to reduce alcohol intoxication. The effects of DHM on ethanol metabolism are not yet completely understood. Effects of DHM during alcohol intoxication were studied on primary hepatocytes of rats. DCFDA and DHR probes were used to prove that DHM (depending on concentration) reduces the number of reactive oxygen and nitrogen species in primary hepatocytes. However, the hepatoprotective effects of DHM were not achieved when presence of the alanine aminotransferase (ALT) was used to measure the damage of cells exposed to alcohol. Further, the effects of DHM on alcohol metabolism were studied in vivo. Rats were administered with single dose of ethanol or ethanol combined with DHM. Measured blood levels of ethanol and acetaldehyde show that DHM has no effects on the rate or levels of alcohol metabolism. The effects of DHM were also studied with repeated alcohol administration. In the group that was administered also DHM, increased blood levels of ethanol were measured. This points that DHM slow down the metabolic rate of ethanol. Obtained results did not prove any positive effects of DHM on alcohol metabolism....

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Aging and kinase kinetics of Y77E11.A and Y17G7B.10 in C. Elegans Jacob Goodlaxson, Department of Biomedical Sciences, College of Medicine, East Tennessee State University, Johnson City, TN. The free radical theory of aging suggests that free radical induced oxidative damage may play a role in the pathogenesis of age-related neurological diseases. The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) protects against redox stress by providing reducing equivalents to antioxidants such as glutathione and thioredoxin. A measurement of the kinase kinetics of nicotinamide adenine dinucleotide demonstrated a decline in the rate of conversion from NAD into NADP. A homeostatic relationship of NADPH and NADP+ in mitochondria and cytosol may prevent the progression of aging due to the amelioration of the reactive oxygen species (ROS), and other charged particles. The NAPD+ cation is the chief acceptor of negatively charged particles which ionize and create free radical ions. Two previously uncharacterized proteins, Y77E11.A (NADK1) and Y17G7B.10 (NADK2), were studied for their possible kinetic role in producing and maintaining NADPH levels. The roles of NADK1 and NADK2 were determined by taking whole worm lysates of Caenorhabditis elegans deficient of these proteins, followed by supplementation with NAD, then monitoring of NADP levels. These two proteins were statistically important in the conversion of NAD to NADP+. These NADK’s displayed statistically significant reduction in NADP production which could lead to more redox stress. This research indicates that pro-longevity therapies should aim to maintain elevated levels of NADPH and NADP+ to hinder the aging process.

UCP1 is a member of the mitochondrial transmembrane anion carrier protein superfamily and is required to mediate adaptive thermogenesis in brown adipose tissue (BAT). Once activated, UCP1 uncouples mitochondrial respiration from ATP synthesis, thereby wasting the protonmotive force formed across the mitochondrial inner membrane as heat. It is hypothesized that proton leaks through UCP1 could be a molecular target to combat certain forms of obesity. Although it is well established that UCP1 is regulated by allosteric mechanisms, alternative methods such as post-translational modification still remain to be explored. The aims of the present study were to confirm the phosphorylation of UCP1 and the physiological relevance of this modification. Using isoelectric focusing, we confirmed that UCP1 displayed acidic shifts consistent with phosphorylation in BAT mitochondria isolated from cold exposed versus warm acclimated mice. A mouse model that ectopically expressed UCP1 in skeletal muscle was used to explore the link between the mitochondrial redox status and UCP1 function. Our results show that the expression of UCP1 in skeletal muscle led to decreases in body and tissues weights. In contrast, glucose uptake into skeletal muscle, food intake and energy expenditure was increased with the expression of UCP1. Finally, proton leaks through UCP1 were determined to be increased in isolated mitochondria from transgenic versus wild-type mice. Taken together these results indicate a complex interplay between mitochondrial redox status, post-translational modification and UCP1 function. Elucidation of novel mechanisms regulating UCP1 offers alternatives strategies that can be explored in order to modulate BAT thermogenesis.