Polycystic ovarian syndrome (PCOS) is a multi-factorial infertility disorder whose etiology and pathogenesis is not completely understood. Although there is an association between dysregulation of mitochondrial fission and fusion to cell death (apoptotic and autophagic) and the pathogenesis of various diseases, this had not been reported in the context of granulosa cell death, follicular growth arrest and PCOS. Follicle-stimulating hormone (FSH), a granulosa cell survival factor, is used during gonadotropin therapy to assist in follicular maturation and ovulation in women with PCOS. Whether FSH can modulate the possible dysregulation of granulosa cell mitochondrial dynamics and cell death in PCOS had not been elucidated. Chemerin is an adipokine that has been associated with PCOS and granulosa cell apoptosis in follicle cultures. Its role in cell death (apoptotic and autophagic) of primary granulosa cells had not been confirmed. In this thesis, we investigated the dysregulation of granulosa cell mitochondrial dynamics in PCOS pathogenesis by using an androgenized rat model, and its modulation by exogenous gonadotropin. The mechanisms involved in gonadotropic regulation were investigated using primary granulosa cells. Our data suggest that increased mitochondrial fission leads to early antral granulosa cell death, follicular growth arrest and anovulation in women with PCOS. FSH can regulate the phosphorylation of mitochondrial fission protein Drp1, which may lead to its suppression of mitochondrial fission and apoptosis in PCOS. Finally, chemerin had no effect on cell death in granulosa cell cultures. These findings provide a greater understanding of the processes involved in PCOS pathogenesis and the regulatory role of FSH in granulosa cells, laying the foundation for future study into the development of potential biomarkers and new treatment strategies.

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Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: Mitochondria in AD & PD models. Includes bibliographical references (p. 146-159). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 167-186).

Mitochondrial Complex I (CI) is composed of 44 distinct subunits that are assembled with eight Fe- S clusters and a single flavin mononucleotide. Mitochondria is highly enriched in the flight muscles of Drosophila melanogaster, however the assembly mechanism of Drosophila CI has not been described. We report that the mechanism of CI biogenesis in Drosophila flight muscles proceeds via the formation of ~315- , ~550-, and ~815 kDa CI assembly intermediates. Additionally, we define specific roles for several CI subunits in the assembly process. In particular, we show that dNDUFS5 is required for converting the ~700 kDa transient CI assembly intermediate into the ~815 kDa assembly intermediate, by stabilizing or promoting the incorporation of dNDUFA10 into the complex. Our findings highlight the potential values of Drosophila as a suitable model organism and resource to study the CI biogenesis in vivo, and to address questions relevant to CI biogenesis in humans. CI biogenesis is regulated by transient interactors known as CI assembly factors (CIAFs). To date, about half of CI disorders are attributed to the mutations in the CI subunits and the known CIAFs. The cause for the other half remains to be discovered, warranting the investigation for additional regulators of CI biogenesis such as novel CIAFs. To identify novel regulators, we cataloged interactors of a core subunit, NDUFS3, knocked each one down by RNAi in the Drosophila flight muscle, and analyzed its effect in the stability of CI by blue-native PAGE. We identified the Drosophila Fragile X Mental Retardation protein (dFMRP) to destabilize the holoenzyme of CI and cause it to misassemble. Therefore, we report dFMRP as a novel regulator of CI biogenesis, and demonstrate the utilization of Drosophila as an effective model system to uncover the mysteries of CI biogenesis.

Nitrate is a progenitor of nitric oxide found in high concentrations in diets rich in fruits and vegetables, such as the Mediterranean. Following enterosalivary circulation, nitrate is converted by salivary bacteria to nitrite and then nitric oxide. Nitrite is a bioactive metabolite of nitric oxide hypothesised to play a role in several metabolic pathways. We explored the effect of nitrate and nitrite in skeletal muscle metabolism and mitochondrial function in mice and humans. Our aims were to assess whether the mouse model is appropriate for use to ascertain the effects of nitrate supplementation, to determine whether these effects are nitrate or nitrite dependent and to investigate whether 7 days nitrate supplementation with nitrate in humans affects metabolism and mitochondrial function. Nitrate supplementation in the mouse, increased plasma nitrate concentration but did not elicit any changes in skeletal muscle metabolism or mitochondrial function at rest. Nitrite supplementation increased plasma nitrate and nitrite concentrations and achieved an increase in metabolic efficiency manifested as a reduction in complex I-linked leak respiration at rest. Nitrate supplementation in humans for 7 days increased plasma nitrate but not nitrite concentration. Skeletal muscle metabolism and mitochondrial function after nitrate did not differ from the placebo. However, blood perfusion of muscles consisting predominantly of type I fibres was increased. Our work suggested that nitrate metabolism is similar between mice and humans and the mouse could be a good model to investigate the effects of nitrate supplementation on skeletal muscle metabolism. This work also suggests that increases in plasma nitrite concentration may be responsible for the changes observed in mitochondrial efficiency. Finally, nitrate supplementation in humans did not elicit any metabolic changes. Deliberate nitrate supplementation in order to increase performance requires further research and any recommendations on increased consumption of nitrates for this purpose may not be accurate.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

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