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Serous ovarian cancer is the 5th leading cause of cancer-related deaths in women, with a 30% survival rate when spread into the highly hypoxic and visceral peritoneal cavity. Despite efforts to treat this highly metastatic disease, traditional chemotherapeutic and cytoreductive therapies are unable to diminish or induce cell death of circulating metastases from colonizing secondary sites due to their genetic and histologic heterogeneity and development of drug resistance. The dissemination route for primary metastasis, however, is most often conserved to the peritoneal cavity, which is low in nutrients and hypoxic (1-2% O2). Cells exfoliated from the primary tumor will aggregate during migration, which elicits a survival signal to maintain viability in this environment. The underlying cellular and molecular changes involved with aggregation have yet to be determined. We have previously found that aggregation of murine ovarian surface epithelial (MOSE) cells present a more suppressed metabolic phenotype upon aggregation. My research sought to identify how the mitochondria were internally regulated to support malignant transformation, migration, and invasion through modulation of quality control, mitochondrial dynamics, mitophagy, and mitobiogenesis. We have shown that aggregation of cancer cells supports increased mitochondrial fragmentation localized to the hypoxic core of our spheroid models. Further, aggregation supports enhanced viability through an upregulation of cancer genetic pathways associated with cell death, proliferation, stemness, and epithelial mesenchymal transition (EMT). Nutrient deprivation during migration further enhanced mitochondrial fragmentation and induction of mitophagy to prevent activation of apoptosis. Additionally, we have identified a phenotypic switch from enhanced mitophagy during peritoneal dissemination that supports survival of ovarian cancer cell aggregates to mitochondrial biogenesis during secondary tissue colonization that enables proliferation upon invasion. We have associated these changes with an increased bioenergetic proliferative niche through inhibition of proliferation, migration, and mitochondrial translation. This research has contributed to the understanding for the role of mitophagy as a survival rather than apoptotic signal in cancer cells as adaptation to nutrient-deprived environments, while also identifying how these processes can be reversed upon adhesion to support invasion and metastatic capacity during secondary colonization. This research is significant because it will identify molecular adaptations associated with the viability of disseminating cancer metastases as well as promote novel preventative therapeutics that can be used to limit the mortality of highly aggressive ovarian cancer in women. / Doctor of Philosophy / Ovarian cancer continues to be one of the highest contributors of gynecologic cancer-related deaths in women. This is due to limited symptomology, biomarker availability, and screenings for patients. Women are mostly diagnosed when the disease has already spread throughout the abdominal cavity which makes treatment much more difficult and, accordingly, the survival rate is much lower. Ovarian metastases mostly spread throughout the peritoneal cavity. Interestingly, this cavity has been identified to being limited in nutrients and oxygen that are essential for survival thus suggesting that these cancer cells must adapt to these harsh conditions to remain viable. We have previously observed that the cancer cells are able to clump together, and form 3D structures known as spheroids which have drastically reduced their proliferation and appear highly resistant tor treatment than single cells. In this project, we wanted to determine how the mitochondria (primary energy producers) were structurally changing in response to the formation of these spheroids and in nutrient- and oxygen-starved conditions. We have found that these organelles become much smaller and circular in low-oxygen conditions, especially in the center of the spheroids. Further, we found changes in cancer- and mitochondrial-related pathways during spheroid formation which could further support survival. Finally, we found that key functions related to the mitochondrial quality control and enhanced mitochondrial content and activity are switched when changing nutrient availability from low oxygen and nutrient conditions to oxygenated and nutrient-rich conditions and generate conditions that allow the spheroids to attach to abdominal organs and form secondary tumors. This research is important because it suggests new possible markers that can be used as therapeutic targets to prevent these aggressive functions associated with more terminally staged disease.

The incidence of type 2 diabetes is considered an epidemic in Western countries, and its prevalence is more common in the Hispanic population than in non-Hispanic whites. Postprandial hyperglycemia has been associated with oxidative stress (OS), thus; reducing postprandial glycemia and/or OS through dietary consumption of resistant starch (RS) may be one approach to help modulate glucose and insulin responses. The purpose of this study was twofold: 1) to evaluate the physicochemical and sensory properties of cereal food products supplemented with RS. 2) to compare the effects of a single ingestion of granola bars with high (~18 grams of RS) and low (~0 grams of RS) RS compositions on the postprandial glucose and insulin responses (n=14) and oxidative stress parameters (cellular glutathione peroxidase, F2- isoprostanes, and oxygen radical absorbance capacity) in Hispanic women (n=9). Granola bars and cereals were developed to provide 2 levels (10% and 15%) of RS; isocaloric (0% RS) control samples were prepared with readily digestible (high amylopectin) starch. Samples were stored for up to 4 weeks at 20 °C. Mean composition of the high RS granola bars was 6% protein, 15% moisture, and 18% lipid. RS levels slightly increased from 14 to 16 g/serving after 4 weeks of storage, supporting published research that RS increases with storage due to retrogradation and crystallization of amylose chains. Color became lighter as the level of RS increased (p<0.001). Granola bars containing RS were less brittle (p=0.0043) than control granola bars. Sensory results indicated granola bars/cereals were acceptable. RS-supplemented granola bars were then used for the evaluation of RS ingestion in humans. There was no difference in postprandial glucose and insulin responses after a single ingestion of a RS-supplemented (18 g) granola bar. No differences were found in the oxidative stress parameters measured. In a subgroup of subjects (n=9), a lower glucose response 30 minutes after RS consumption was found (p=0.0496). Thus, RS consumption may lower fluctuations in blood glucose, which may help manage glucose levels in individuals at risk of type 2 diabetes. Further studies of short term RS consumption are warranted to elucidate its benefits in glucose management. / Ph. D.

The objective of this study was to determine the photothermal and photochemical tissue response to carbon nanotube inclusion in laser therapy using experimental and computational methods. In this study, we specifically considered varying types and concentrations (0.01-1 mg/ml) of carbon nanotubes (CNTs), e.g., multi-walled carbon nanotubes (MWNTs), single-walled carbon nanotubes (SWNTs), and single-walled carbon nanohorns (SWNHs). In order to determine the photothermal effect of CNT inclusion, the thermal conductivity and optical properties of tissue representative phantoms with CNT inclusion were measured. Thermal conductivity of tissue phantoms containing CNTs was measured using the hot wire probe method. For identical CNT concentrations, phantoms containing MWNTs had the highest thermal conductivity. Optical properties (absorption and reduced scattering coefficients) of solutions and tissue phantoms containing carbon nanotubes were measured with spectrophotometry and determined by the inverse adding doubling (IAD) method. Inclusion of CNTs in phantoms increased light absorption with minimal effect on scattering and anisotropy. Light absorption of MWNTs was found to be higher than SWNTs and SWNHs. The photochemical response to laser irradiation (wavelength 1064 nm) of CNTs was measured with spin-trap electron paramagnetic resonance (EPR) spectroscopy. Only SWNHs appeared to produce significant levels of ROS production in response to laser excitation in the presence of NADH. We detected the predominant presence of trapped hydroxyl radical (•OH) with a trace of the trapped super oxide (O2•-) radical. These free radicals are highly reactive and could be utilized to cause targeted toxicity to cancer cells. The distribution of CNTs at the cellular level, in phantoms, and in kidney tumors was measured using transmission electron microscopy (TEM) imaging. Samples were imaged following various time periods (2-48h) of incubation and CNTs were observed inside the cell cytoplasm, nucleus, vacuole, and outside cells for the above mentioned time periods. CNTs in phantoms and tumor tissue were randomly and uniformly distributed in the entire volume. Computational model geometries were developed based on CNTs distribution in cells, tissue phantoms, and kidney tumor tissue. In the computational part of this research the temperature response to laser irradiation alone or with CNT inclusion was determined using Penne's bioheat equation which was solved by finite element methods. Experimentally measured thermal conductivity and absorption and reduced scattering coefficients were used as input parameters in Penne's bioheat equation. The accuracy of the model predicted temperature distribution was determined by comparing it to experimentally measured temperature in tissue phantoms and kidney tumors following CNT inclusion and laser therapy. The model determined temperature distribution was in close correspondence with the experimentally measured temperature. Our computational model can predict the effectiveness of laser cancer therapy by predicting the transient temperature distribution. / Ph. D.

Generation of reactive oxygen species (ROS) by mammalian host cells is a double-edged sword. ROS are clearly beneficial in directly killing pathogens and as a signaling molecule to alert macrophages and neutrophils to the site of infection. However, ROS are also capable of damaging host cells by destroying DNA, oxidizing proteins and lipids, inactivating enzymes, and eliciting apoptosis. Therefore the balance of ROS generation and clearance is essential for homeostasis. Although multiple mechanisms can contribute to the generation of ROS, NADPH oxidase (Nox) is a primary producer. In terms of clearance, several ROS scavenging enzymes are induced by Nrf2, a sensor of excessive ROS. The mechanisms behind the skewing of this balance toward prolonged accumulation of ROS under chronic inflammatory conditions are not well understood. Lipopolysaccharide (LPS), a major component of the Gram-negative bacteria cell wall, is specifically recognized by Toll-like receptor 4 (TLR4). LPS triggers robust activation of Nox and ROS production through TLR4, while also activating Nrf2 and ROS clearance. Intracellular pathways regulating ROS generation and clearance mediated by TLR4 are not well defined. Since interleukin-1 receptor associated kinase 1 (IRAK-1) is a key downstream component of TLR4, we test the hypothesis that IRAK-1 may play a critical role in maintaining the balance of LPS triggered ROS generation and clearance. Using wild type and IRAK-1 deficient murine embryonic fibroblasts, we tested the dynamic induction of Nox1 (a key NADPH oxidase) and Nrf2 by varying dosages of LPS. Our data confirm that high dose LPS (as seen in acute bacterial infection) induced both Nox1 and Nrf2. The generation of Nox1 is IRAK-1 dependent. Low dose LPS (as seen in chronic metabolic endotoxemia) fails to induce Nrf2 and induces mild and prolonged expression of Nox1. Cells pre-challenged with low dose LPS are primed for more robust expression of ROS following a second LPS challenge. The conclusions and implications generated by this study are that chronic low dose endotoxemia (prevalent in adverse health conditions) may skew the balance of ROS generation and clearance to favor prolonged ROS accumulation, and that IRAK-1 represents a potential therapeutic target to treat chronic inflammatory diseases. / Ph. D.

Cerium oxide nanoparticles have the unique ability to accept and donate electrons, making them powerful antioxidants. Their redox nature is due to oxygen defects in the lattice structure, which are more abundant at the nanoscale. Reactive oxygen species (ROS) are pro-oxidants whose presence is increased during periods of inflammation in the body. ROS damage tissues and cellular function by stripping electrons from proteins, lipids, and DNA. We investigated the ability of nanoceria to quench ROS in vitro and in vivo, and examined the biodistribution and biocompatibility of nanoceria in murine models. Nanoceria was internalized in vitro by macrophages, is non-toxic at the concentrations we investigated, and proteins, mRNA, and oxidative markers of ROS were abated with nanoceria pretreatment in immune stimulated cells as measured by western blot, real time RT PCR, and Greiss assay respectively. In vivo, nanoceria was deposited in the spleen and liver, with trace amounts in the lungs and kidneys as determined by ICP-MS. Using IVIS in vivo imaging, it appeared that nanoceria deposition occurred in lymph tissue. Histology grades show no overt pathology associated with nanoceria deposition, although white blood cell (WBC) counts were generally elevated with nanoceria treatment. Nanoceria suspect particles were seen in lysosomes from kidney samples of IV injected mice in HRTEM images. Lastly, IV nanoceria treatment appears to reduce markers of oxidative stress in mice treated with carbon tetrachloride (CCl4) to induce ROS production. Taken together, our data suggest that nanoceria treatment has the potential to reduce oxidative stress. / Master of Science

Trimethylamine N-oxide (TMAO) is a metabolite derived from dietary choline, betaine, and carnitine via intestinal microbiota metabolism. In several recent studies, TMAO has been shown to directly induce inflammation and reactive oxygen species (ROS) generation in numerous cell types, resulting in cell dysfunction. However, whether TMAO will impact stem cell properties remains unknown. This project aims to explore the potential impact of TMAO on mouse embryonic stem cells (mESCs), which serve as an in vitro model of the early embryo and of other potent stem cell types. Briefly, mESCs were cultured in the absence (0mM) or presence of TMAO under two different sets of treatment conditions: long-term (21 days), low-dose (20µM, 200µM, and 1000µM) treatment or short-term (5 days), high-dose (5mM, 10mM, 15mM) treatment. Under these treatment conditions, mESC viability, proliferation, and stemness were analyzed. mESC properties were not negatively impacted under long-term, low-dose TMAO treatment; however, short-term, high-dose treatment resulted in significant reduction of mESC viability and proliferation. Additionally, mESC stemness was significantly reduced when high-dose treatment was extended to 21 days. To investigate an underlying cause for TMAO-induced loss in mESC stemness, metabolic activity of the mESCs under short-term, high-dose TMAO treatment was measured with a Seahorse XFe96 Analyzer. TMAO treatment significantly decreased the rate of glycolysis, and it increased the rate of compensatory glycolysis upon inhibition of oxidative phosphorylation (OxPHOS). It also significantly increased the rate of OxPHOS, maximal respiratory capacity, and respiratory reserve. These findings indicate that TMAO induced a metabolic switch of mESCs from high glycolytic activity to greater OxPHOS activity to promote mESC differentiation. Additionally, TMAO resulted in increased proton leak, indicating increased oxidative stress, and elucidating a potential underlying mechanism for TMAO-induced loss in mESC stemness. Altogether, these findings indicate that TMAO decreases stem cell potency potentially via modulation of metabolic activity. / Master of Science / Trimethylamine N-oxide (TMAO) is a metabolite that is produced by the bacteria in the gut after the consumption of specific dietary ingredients (e.g., choline, carnitine, betaine). These ingredients are commonly found in meat and dairy products, and thus make up a large part of the average American diet. Recently, it was discovered that high TMAO levels in the bloodstream put people at an increased risk for heart disease, neurodegenerative diseases (e.g., Alzheimer's Disease), diabetes, stroke, and chronic kidney disease. At the cellular level, there is evidence that TMAO increases inflammation and the production of oxygen radicals, which causes cells to lose their function and promotes the onset of disease. TMAO has been well studied in adult cell types; however, no one has investigated whether TMAO will impact cells of the early embryo. This project aims to explore the impact of TMAO on mouse embryonic stem cells (mESCs), which are cells that represent the early stage of embryonic development and are critical for proper development of the final offspring. In addition, mESCs may also help to provide insight into how TMAO impacts other stem cell types, some of which are present throughout the entire human lifespan and play an important role in the body's ability to repair itself and maintain overall health. My project demonstrated that TMAO does not impact the overall health of mESCs under normal conditions, which signifies that TMAO generated by a pregnant mother may not directly impact the early embryonic stage of development. Further studies should be conducted to determine the potential impact of TMAO on late stages of embryonic and fetal development. Next, to simulate diseased conditions, the mESCs were treated with extremely high concentrations of TMAO in order to determine what concentration of TMAO will negatively impact these cells. It was found that at 5mM TMAO, mESCs begin to lose their basic properties and become dysfunctional. They are impaired in their viability, growth, ability to become other cell types, and in their metabolic activity. These mESC properties are shared with several types of adult stem cells, and therefore, these findings help to provide insight into how TMAO may impact stem cells found in the adult body which are exposed to a lifetime of high TMAO levels. In the future, we would like to further explore the impact of TMAO on mESCs at the molecular level as well as examine the direct impact of TMAO on other stem cell types.

Yes / Platinum drugs are widely used for cancer treatment. Other precious metals are promising, but their clinical progress depends on achieving different mechanisms of action to overcome Pt-resistance. Here, we evaluate 13 organo-Os complexes: 16-electron sulfonyl-diamine catalysts [(η6-arene)Os(N,N′)], and 18-electron phenylazopyridine complexes [(η6-arene)Os(N,N’)Cl/I]+ (arene = p-cymene, biphenyl, or terphenyl). Their antiproliferative activity does not depend on p21 or p53 status, unlike cisplatin, and their selective potency toward cancer cells involves the generation of reactive oxygen species. Evidence of such a mechanism of action has been found both in vitro and in vivo. This work appears to provide the first study of osmium complexes in the zebrafish model, which has been shown to closely model toxicity in humans. A fluorescent osmium complex, derived from a lead compound, was employed to confirm internalization of the complex, visualize in vivo distribution, and confirm colocalization with reactive oxygen species generated in zebrafish. / Wellcome Trust (grant no. 107691/Z/15/Z), ERC (grant nos. 247450, 324594), Science City (AWM and ERDF), WCPRS and Bruker Daltonics (Studentship for JPCC), Mike and Enfys Bagguley, and EPSRC (Studentship for HEB, and grant no. EP/F034210/1).

Dravet Syndrome (DS) is a pediatric epilepsy disorder. Individuals with DS are at increased risk of Sudden Unexpected Death in Epilepsy (SUDEP). One mechanism implicated in the pathology of SUDEP is cardiac arrhythmias. A central element involved in cardiac regulation is mitochondria. In the heart, mitochondria maintain cardiomyocyte energy (ATP) generation and ion homeostasis but also produce harmful reactive oxygen species (ROS) byproducts. We hypothesized that deficits in cardiac mitochondria could underlie arrhythmias and SUDEP in two independent mouse models of DS. Mitochondria produce ATP through the mitochondrial respiratory chain, also called electron transport chain, comprised of a series of large, multimeric complexes (labeled I-IV) coupled to the activity of an ATP synthase. We first simultaneously analyzed electron transport chain activity and ROS production via Complex I- and Complex II-linked respiratory pathways in cardiac mitochondria isolated from DS mouse hearts. ROS produced as a byproduct of ATP generation is scavenged by cellular antioxidant systems, primarily glutathione in the heart. Therefore, we next subjected isolated cardiomyocytes to diamide, which oxidizes thiol-based antioxidants, to determine ROS scavenging ability in DS hearts. Furthermore, isolated hearts were also perfused with diamide via Langendorff assays to test for arrhythmia susceptibility in the presence of oxidative stress. Another essential function of mitochondria is buffering ions like Ca2+. While fatal at high concentrations, Ca2+ stimulates mitochondrial ATP production at physiological levels. We determined if cardiac mitochondria from DS hearts have altered levels of Ca2+ uptake (via the mitochondrial Ca2+ uniporter) and retention. We also investigated if mitochondrial energetic capacity was impacted by Ca2+ dysregulation. Furthermore, due to its impacts on ATP, mitochondrial Ca2+ sequestration is an essential physiological mechanism linking the sympathetic drive to the heart with cardiac output. We tested the response of DS hearts to norepinephrine, a sympathetic agonist, to determine if these conditions caused increased vulnerability to arrhythmias. Overall, our results indicate that these models of DS have distinct energetic and antioxidant phenotypes that may also be impacted by biological sex. Considering these differences may be necessary when tailoring treatment and preventing SUDEP in the clinical practice of DS patients with different etiologies.

The aim of this thesis is to investigate molecular mechanism of reactive oxygen species production by flavin dehydrogenases mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH). Together, they represent important source of reactive oxygen species in mammalian mitochondria, but the mechanism of electron leak is still poorly understood. Because mechanisms of reactive oxygen species production by other complexes of respiratory chain are better characterized, they can serve as case studies to get insight into mechanisms of reactive oxygen species by flavin dehydrogenases. Relevant knowledge is therefore summarized in the first part of the thesis. To study the production of reactive oxygen species by the isolated flavin dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements, hydrogen peroxide production studies by Amplex UltraRed fluorescence and luminol luminescence revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q binding site as the site of reactive oxygen species production in the case of mGPDH. Distinct mechanism of this production by the two dehydrogenases is also apparent from induction of reactive oxygen species...