The turtle is a unique model of anoxic survival. The turtle's brain can tolerate total oxygen deprivation for hours to days as well as prevent high levels of mitochondrial-derived free radicals upon re-oxygenation. Because of its ability to prevent elevated free radical generation, the turtle has also become recognized as a model of exceptional longevity. We are employing the turtle model for an investigation into the regulation of a key antioxidant enzyme system - methionine sulfoxide reductases (Msrs), primarily MsrA and MsrB. The Msr system is capable of reversing oxidation of methionines in proteins and Msr subtypes have been implicated in protecting tissues against oxidative stress, as well as, enhancing the longevity of organisms from yeast to mammals. Preliminary data, unpublished results, indicate that MsrA protein and transcripts are elevated by anoxia. A recent study on Caenorhabditis elegans demonstrated that FOXO is involved in activation of the MsrA promoter. Using the turtle MsrA promoter sequence we worked to determine which regions in the promoter are necessary for activation by anoxia. The results of the present study were 1) to prepare a TAT-FOXO3a fusion protein which could penetrate animal cells and 2) to construct a FOXO3a expression vector for transcription studies on MsrA expression. / by Diana Navarro. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

The mitochondrion is the powerhouse of the cell. Therefore, it is critical to the homeostasis of the cell that populations of mitochondria that are damaged or in excess are degraded. The process of targeted elimination of damaged or excess mitochondria by autophagy is called mitophagy. In this report, analysis of the mitophagy regulators PINK1/PARKIN and BNIP3L and their roles are assessed in the lens. PARKIN, an E3 ubiquitin ligase, has been shown to play a role in directing damaged mitochondria for degradation. While BNIP3L, an outer mitochondrial membrane protein, increases in expression in response to excess mitochondria and organelle degradation during cellular differentiation. We have shown that PARKIN is both induced and translocates from the cytoplasm to the mitochondria in human epithelial lens cells upon oxidative stress exposure. In addition, our findings also show that overexpression of BNIP3L causes premature clearance of mitochondria and other organelles, while loss of BNIP3L results in lack of clearance. Prior to this work, PARKIN mediated mitophagy had not been shown to act as a protective cellular response to oxidative stress in the lens. This project also resulted in the novel finding that BNIP3L-mediated mitophagy mechanisms are required for targeted organelle degradation in the lens. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Oxidant stress and injury is inherent in many human diseases such as ischemic vascular and respiratory diseases, heart failure, myocardial infarction, stroke, perinatal and placental insufficiencies, diabetes, cancer, and numerous psychiatric and neurodegenerative disorders. Finding novel therapeutics to combat the deleterious effects of oxidative stress is critical to create better therapeutic strategies for many conditions that have few treatment options. This study used the anoxia-tolerant fruit fly, Drosophila melanogaster, to investigate endogenous cellular protection mechanisms and potential interactions to determine their ability to regulate synaptic functional tolerance and cell survival during acute oxidative stress. The Drosophila larval neuromuscular junction (NMJ) was used to analyze synaptic transmission and specific motor axon contributions. Drosophila Schneider 2 (S2) cells were used to assess viability. Acute oxidative stress was induced using p harmacological paradigms that generate physiologically relevant oxidant species: mitochondrial superoxide production induced by sodium azide (NaN3) and hydroxyl radical formation via hydrogen peroxide (H2O2). A combination of genetic and pharmacological approaches were used to explore the hypothesis that endogenous protection mechanisms control cellular responses to stress by manipulating ion channel conductance and neurotransmission. Furthermore, this study analyzed a group of marine natural products, pseudopterosins, to identify compounds capable of modulating synaptic transmission during acute oxidative stress and potential novel neuromodulatory agents. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

by Rebecca McGreal. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web. / The central premise of this dissertation is that the small heat shock protein (sHSP), (Sa(BB-crystallin is essential for lens and retinal pigmented epithelial (RPE) cell function and oxidative stress defense. To date, the mechanism by which it confers protection is not known. We hypothesize that these functions could occur through its ability to protect mitochondrial function in lens and RPE cells. To test this hypothesis, we examined the expression of (Sa(BB-crystallin/sHSP in lens and RPE cells, we observed its localization in the cells, we examined translocation to the mitochondria in these cells upon oxidative stress treatment, we determined its ability to form complexes with and protect cytochrome c (cyt c) against damage, and we observed its ability to preserve mitochondrial function under oxidative stress conditions in lens and RPE cells. In addition to these studies, we examined the effect of mutations of (Sa(BB-crystallin/sHSP on its cellular localization and translocation patterns under oxidative stress, its in vivo and in vitro chaperone activity, and its ability to protect cyt c against oxidation. Our data demonstrated that (Sa(BB-crystallin/sHSP is expressed at high levels in the mitochondria of lens and RPE cells and specifically translocates to the mitochondria under oxidative stress conditions. We demonstrate that (Sa(BB-crystallin/sHSP complexes with cyt c and protects it against oxidative inactivation. Finally, we demonstrate that (Sa(BB-crystallin/sHSP directly protects mitochondria against oxidative inactivation in lens and RPE cells. Since oxidative stress is a key component of lens cataract formation and age-related macular degeneration (AMD), these data provide a new paradigm for understanding the etiology of these diseases.

Tissue ischemia is a major cause of morbidity contributing to disease processes such as cardiovascular diseases, stroke, cancer, and traumatic injury and may lead to death. Failure to quickly reestablish flow to ischemic tissue results in tissue death. However, even timely return to normal flow has a downside in that the reintroduction of oxygen to ischemic tissue results in ischemia/reperfusion (I/R) injury that produces an oxidant stress. This pathological process requires new therapeutic strategies and agents to reduce the personal, social and economic loss. One of the most generally accepted mechanisms for the pathology of I/R injury is the production of the reactive oxygen species (ROS), suggesting antioxidants may ameliorate I/R injury. Caffeic acid phenethyl ester (CAPE), a plant derived polyphenolic compound, has been shown to protect organs from I/R induced damage in vivo, and this effect has been attributed to its antioxidant activity. To better understand the mechanism of CAPE protection, a model using menadione-induced oxidative stress in human endothelial cells to simulate I/R injury in vitro was developed. Gene expression analysis was performed with microarrays undergoing cytoprotection with CAPE. The dose-dependent cytoprotection of CAPE has been related to its induction of heme oxygenase-1 (HO-1). With the aim of improving the beneficial effect of CAPE and understanding structure activity relationship, six new catechol ring-fluorinated CAPE derivatives were synthesized and evaluated in the menadione-endothelial cell model. The data suggest good cytoprotective effects of CAPE and some analogues and indicate important structural features for cytoprotection. Further investigation of the mechanism of cytoprotection showed that cytoprotection profiles of CAPE and derivatives correlate better to their ability to induce HO-1 in human endothelial cells than free radical scavenging activity. One CAPE derivative (FCAPE) with cytoprotective effects similar to CAPE in vitro exhibited better stability in rat plasma. A validated ultra-performance liquid chromatography/tandem mass spectrometric method was developed that allowed for quantification of CAPE and FCAPE in plasma samples. Pharmacokinetic studies in male Sprague Dawley rats following intravenous bolus administration of 5, 10, and 20 mg/kg CAPE and 20 mg/kg FCAPE were performed. The results indicate that dose proportionality for CAPE does not exist in the dose range studied. Although the elimination half life was found not to be significant different between CAPE and FCAPE, significant difference was observed between the total body clearance of FCAPE and CAPE which may due to the difference in volume of distribution.

The vertebrate eye lens functions to focus light onto the retina to produce vision. The lens is composed of an anterior monolayer of cuboidal epithelial cells that overlie a core of organelle free fiber cells. The lens develops and grows throughout life by the successive layering of lens fiber cells via their differentiation from lens epithelial cells. Lens developmental defect and damage to the lens are associated with cataract formation, an opacity of the lens that is a leading cause of visual impairment worldwide. The only treatment to date for cataract is by surgery. Elucidating those molecules and mechanisms that regulate the development and lifelong protection of the lens is critical toward the development of future therapies to prevent or treat cataract. To determine those molecules and mechanisms that may be important for these lens requirements we employed high-throughput RNA sequencing of microdissected differentiation statespecific lens cells to identify an extensive range of transcripts encoding proteins expressed by these functionally distinct cell types. Using this data, we identified differentiation state-specific molecules that regulate mitochondrial populations between lens epithelial cells that require the maintenance of a functional population of mitochondria and lens fiber cells that must eliminate their mitochondria for their maturation. In addition, we discovered a novel mechanism for how lens epithelial cells clear apoptotic cell debris that could arise from damage to the lens and found that UVlight likely compromises this system. Moreover, the data herein provide a framework to determine novel lens cell differentiation state-specific mechanisms. Future studies are required to determine the requirements of the identified molecules and mechanisms during lens development, lens defense against damage, and cataract formation. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Oxidative stress is the major driving force behind the aging process and many age-related diseases. However, direct experimental evidence of whether antioxidants, such as ascorbate (AA) and lipoic acid (LA) can slow the progression of aging process and/or reduce risks of developing degenerative disease is largely absent. This suggests a better understanding of the precise mechanism of how dietary micronutrient affect parameters of involved in cellular redox balance and aging are warranted. In this dissertation, young and old rats were used as our model to understand potential pro-oxidant events that contribute to increases in oxidative stress in various tissues and how antioxidants such as ascorbate and lipoic acid influence these events. Our major findings are that the age-related impairment of mitochondria and increased deposition of iron contribute significantly to heighten levels of oxidative stress, as evidenced by the resultant increases in the rates of oxidant appearance and in the levels of oxidative damage to DNA, lipids and proteins. We find that AA and LA strongly protected against transition metal-ion dependent increases in oxidative stress. AA effectively inhibited transition metal-mediated lipide peroxidation in human plasma. LA in its reduced form effectively binds iron and copper in a redox inactive manner and reversed chronically elevated levels of iron in the brain without removing enzyme bound transition metal ions. LA also significantly attenuated the age-related increase in oxidative stress associated with mitochondrial decay in the heart, as evidenced by the improvements in AA levels and glutathione redox status. The declines in tissue GSH levels in aged rats were strongly associated with the diminished γ-GCL activity (in parallel with decreased expression of the catalytic and modulatory subunits), and lowered Nrf2 expression and binding to ARE sequence in rat liver. Remarkably, all these events were effectively reversed by the administration of LA, modulating the parameters to return to the observed in young animals. The implications of this work open new avenues not only for further understanding of the aging process but also for possible strategies in its modulation by the micronutrients. / Graduation date: 2004

Graduation date: 2004