Ovarian cancer is the fifth most lethal cancer in women (1) and the most lethal gynecological malignancy. In 2018, there were approximately 22,240 new diagnosed cases of ovarian cancer and 14,070 deaths in the United States alone (2). The lifetime risk for developing ovarian cancer in the United States is 1.3% or approximately 1 in 78 women. The five-year survival rate for women with ovarian cancer is a grim 47.6% (2) while the average five year survival rate for all cancers is about 68%. This dismal prognosis for ovarian cancer patients indicates the critical need for improved treatment options, efficient early detection methods and effective preventative measures for ovarian cancer (1). The objective of this study was to determine if DHA causes a reduction in cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) by blocking the activation of NF-κB regulated transcription in the ovary. DHA is a 22 carbon long-chain omega-3 polyunsaturated fatty acid that is biologically derived from Alpha-linolenic acid (ALA) found in flaxseed. COX-2 is an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. Prostaglandin E2 (PGE2) is a key regulator of inflammation which has been shown to be highly associated with ovarian cancer development and progression. Our laboratory studies ovarian cancer in the laying hen because it is the only known animal model to naturally develop ovarian cancer that both pathologically and histologically matches that of the human form of the disease. Dietary flaxseed is one of the richest vegetable sources of omega-3 polyunsaturated fatty acids. Our previous studies have shown that in laying hens, a long-term flaxseed supplemented diet reduces the incidence and severity of ovarian cancer and decreases COX-2 and PGE2. It was hypothesized that DHA, derived from ALA found in flaxseed, decreases inflammation in the ovaries by suppressing the activation of COX-2 and the production of PGE2 through inhibition of the NF-κB pathway. For this study, an NF-κB reporter plasmid was transfected into HEK293 cells. The reporter plasmid (“met-luc”) produces a secreted luciferase allowing sequential analysis of media from DHA and TNF-α treated cells to assess changes in NF-κB transcriptional activation. Tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB was used as a positive control. NF-κB activation was also assessed by measuring its nuclear translocation and cytoplasmic accumulation through immunocytochemistry (ICC) and western blot analysis. In a parallel study, immortalized ovarian surface epithelial (IOSE) cells were challenged with the same treatments of DHA and TNF-α. In these cells, COX-2 mRNA was assessed through RT-qPCR and COX-2 protein expression was analyzed through ICC and western blot.Our results indicate that DHA acts in a cell specific manner to reduce inflammation associated with cancer. We have found that in HEK293 cells DHA reduces TNFα induced NF-κB reporter activity. In contrast, ALA does not affect NF-κB reporter activity. HEK293 cells treated with TNFα alone indicated a dose-dependent increasing trend in nuclear translocation of the NF-κB p65 subunit and a decreasing trend in cytoplasmic p65, suggesting potential increased pathway activation. ICC suggests DHA treatment causes increased cytoplasmic sequestration of the NF-κB p65 subunits indicating inhibition of TNFα induced NF-κB activation. Western blot data also indicates a decreasing trend in nuclear NFκB p65 when cells are pretreated with DHA and subsequently challenged with TNF. The IOSE cells, were the only cells out of the cell lines tested (BG1, HEYC2, TOV112D, SKOV3, HEK293) to express COX-2. In these IOSE cells, TNFα alone showed a dose-dependent increasing trend in COX-2 protein (analyzed through ICC and western blot) and mRNA levels (analyzed through RT-qPCR). ICC analysis revealed that DHA reduces TNF induced COX-2 protein expression. However, the western blot did not further support this observation. Only a slight non-significant reduction with DHA treatment was observed. In addition, both DHA and TNFα, while also not significant, seemed to increase mRNA levels of COX-2 compared to control. This slight decreasing trend in COX-2 protein expression and increase in mRNA, could indicate a possible post-transcriptional mechanism of regulation of COX-2 by DHA independent of NF-κB in the IOSE cells. These data suggest that DHA could act via distinct mechanisms in a cell specific manner to potentially reduce COX-2 and subsequently PGE2 levels. DHA can act at the transcriptional level by reducing the nuclear translocation of NF-κB and transcriptional activation of NF-κB target genes such as COX-2 in some cell types. DHA also has the potential to work via a post-transcriptional mechanism to inhibit COX-2 and in turn reduce PGE2 levels. Both mechanisms ultimately have the potential to decrease the inflammation associated with ovarian cancer. This study describes the anti-inflammatory action of dietary flaxseed consumption, making flaxseed supplementation a promising preventive measure for reducing the risk of ovarian carcinogenesis.

Crude glycerol is a major byproduct if the biodiesel industry. Biodiesel manufacturers are currently facing the challenges of appropriate disposal of this waste material. Crude glycerol is expensive to purify for use in food, cosmetic, and pharmaceutical industries and therefore, alternative methods for use of this crude glycerol are needed. A promising alternative is to use this crude glycerol as a carbon source for microalgae fermentation. In this project, we investigated the use of crude glycerol as a less expensive substrate for the fermentation of the microalgae <i>Schizochytrium limacinum</i> and <i>Pythium irregulare</i> which are prolific producers of omega-3 polyunsaturated fatty acids. Omega-3 fatty acids have many beneficially effects on treating human diseases such as cardiovascular diseases, cancers, and neurological disorders. In addition, the omega-3 fatty acids docosahexaenoic acid (DHA) has been shown to be an important factor in infant brain and eye development. The first part of this study focused on the continuous fermentation of <i>S. limacinum</i>, a prolific producer of DHA. The objective of this study was to examine the algal cellular physiology and maximize its DHA productivity. Two important parameters used in continuous fermentation were studied: dilution rate (D) and feed glycerol concentration (S₀). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day⁻¹ and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day⁻¹ and S₀ = 90 g/L. The cells had a true growth yield of 0.283 g/g, a maximum specific growth rate of 0.692 day⁻¹, and a maintenance coefficient of 0.2216 day⁻¹. The second part of this study focused on morphology issues with <i>P. irregulare</i>, a prolific producer of eicosapentaenoic acid (EPA). <i>P. irregulare</i> has a filamentous morphology, which can make fermentation difficult. The mycelium can stick to the agitation blades resulting in mechanical problems. In addition, this filamentous morphology prevents adequate amounts of oxygen from reaching some cells resulting in decreased productivities. The focus of this research was to control the fermentation conditions to make the algae grow in small pellets, a morphology more suitable for fermentation. In flask culture studies, pellets were formed at an agitation speed of 110 rpm in both regular and baffled flasks. Baffled flasks resulted in pellet formation at 90 and 130 rpm as well. Fermentation studies resulted in pellet formation at agitation speeds of 150 and 300 rpm. Pellets were better able to form when a baffle was not in place. In addition, agitation speed influenced pellet size, with smaller pellets forming at the higher agitation speed. Overall, this study showed that crude glycerol can be used as a carbon source for the continuous fermentation of <i>S. limacinum</i> with high DHA productivity and the morphology of <i>P. irregulare</i> could be controlled by manipulating culture conditions, mainly agitation speed. These results show the potential for scale-up studies for both algal species. / Master of Science

No description available.

Doctor of Philosophy / Department of Human Nutrition / Mark D. Haub / Mark D. Haub / Psychological problems and human error are leading causes of death and disability among military service members. Strategies to improve the psychological health and cognitive performance of those in the military are much needed. Recent advances in neuroscience suggest that omega-3 fatty acids may play an important role in the psychological well-being of those in the military. The purpose of this research was to explore the relationship between omega-3 status and psychological outcome variables among soldiers deploying to combat. Data collection was preceded by the development and reliability testing of a novel food frequency questionnaire (FFQ) designed to capture intake from contemporary sources of omega-3 fatty acids including functional foods and supplements. Based on the instrument assessment study (Chapter 2) conducted among university students (n = 165), this FFQ appears to be a comprehensive and reliable (n = 54, ρ = 0.86, p < 0.001) instrument for measuring docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) intakes in young adults. As described in Chapter 3, intake of EPA + DHA as estimated by the FFQ was positively correlated (r = 0.39, p < 0.001) with biomarker measurements of omega-3 status. Primary data were obtained from a volunteer sample of soldiers (n = 272) scheduled for deployment to Iraq. Preliminarily analyses revealed relationships between attention deficit hyperactivity disorder (ADHD) screening scores and psychological outcome variables (Chapter 4). Primary analyses (Chapter 5) indicated intake of EPA + DHA was not significantly correlated with mood, nor were omega-3 exposure variables correlated with cognitive performance based on the required p value (< 0.001) calculated using the Bonferroni correction for multiple tests. Among participants with EPA + DHA intakes at or below the median, omega-3 HUFA was related (p < 0.002) to happiness (β = -0.46), depression (β = 0.44), and fatigue (β = 0.43). Although exploratory in nature, the results of this study suggest a relationship between omega-3 fatty acids and mood. Given the current concerns regarding the psychological health of those in the military, additional research is warranted.

Os n-3 e n-6 são duas famílias de ácidos graxos poli-insaturados. Os ácidos graxos de cadeia longa como o ácido araquidônico (AA) e docosahexaenoico (DHA) apresentam importantes funções no desenvolvimento e funcionamento do cérebro. Os produtos de oxidação dos ácidos graxos poli-insaturados estão presentes ou aumentados ao longo do desenvolvimento de doenças neurodegenerativas. A caracterização de tais produtos é crítica para o estudo que busca entender o seu papel fisiopatológico no desenvolvimento de tais doenças. No presente trabalho, buscou-se o desenvolvimento de uma ferramenta analítica sensível e específica para a detecção e quantificação dos hidroperóxidos e hidróxidos do AA (HpETE e HETE), do seu precursor, o ácido linoleico (HpODE e HODE) e do DHA (HpDoHE e HDoHE). Estes hidroperóxidos foram sintetizados por fotooxidação e os hidróxidos correspondentes foram obtidos através da redução com o NaBH4. Os isômeros isolados foram caracterizados por LC-MS/MS. Os íons produto específicos de cada isômero foram escolhidos para a construção do método de monitoramento de reação selecionada (selected reaction monitoring - SRM) para a realização da análise quantitativa dos analitos de interesse. Cabe salientar que os dados obtidos poderão ser utilizados em bibliotecas de análise lipidômica e oxi-lipidômica pois serão essenciais para a identificação e quantificação dos analítos de interesse do presente estudo em diversas doenças. Utilizando o método padronizado, buscamos investigar o papel dos hidroperóxidos e hidróxidos do DHA, LA e AA em um modelo animal para a esclerose lateral amiotrófica (ELA), uma doença neurodegenerativa que acomete neurônios motores. Foi observado um aumento nos níveis de 13-HpODE, 9-HpODE e 12-HETE no córtex motor dos animais avaliados. Adicionalmente, foram observadas alterações nas taxas lipólica e lipogênica no tecido adiposo para os animais ELA em relação aos respectivos controles. Em conjunto, os dados apresentados no presente trabalho corroboram com os trabalhos da literatura que associam alteração dos níveis dos produtos de oxidação dos ácidos graxos poli-insaturados em doenças neurodegenerativas e o metabolismo energético alterado em ELA. Futuramente é necessária uma investigação mais ampla dos níveis dos hidroperóxidos e hidróxidos lipídicos em diferentes tecidos e do metabolismo lipídico, e os conhecimentos gerados poderão ser uma importante fonte de novas opções terapêuticas para os pacientes portadores de ELA. / The n-3 and n-6 are two olyunsaturated fatty acids families. The long chain fatty acids such as arachidonic (AA) and docosahexaenoic acid (DHA) have important roles in the development and function of the brain. Polyunsaturated fatty acids (PUFAs) oxidation products are present or increased during the progression of neurodegenerative diseases. The characterization of DHA oxidation products is critical to understand their roles in the development of such diseases. In the present study, we sought to develop a sensitive and specific analytical tool for the detection and quantification of AA hydroperoxides and hydroxides (HPETE and HETE), its precursor linoleic acid (HPODE and HODE) and DHA (HpDoHE and HDoHE). These hydroperoxides were synthesized by photooxidation and the corresponding hydroxides were obtained by reduction with NaBH4. The isolated isomers were characterized by LC-MS/MS, and unique and specific fragment ions were chosen to construct a selected reaction monitoring (SRM) method for the targeted quantitative analysis. It should be emphasized that the data obtained - in the form of lipidomics and oxy-lipidomics libraries - may be used to assist in several diseases. Using the standardized method, we investigated the role of hydroperoxides and hydroxides of DHA, LA and AA in an animal model of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects motor neurons. Increased levels of 13-HPODE, 9-HPODE and 12-HETE were observed in the animals motor cortex. Additionally, results show changes in lipogenic and lipolytic rates in adipose tissue for ALS animals when compared to their respective controls. Altogether, the data presented herein corroborate with the literature by linking altered levels of PUFAs oxidation products in neurodegenerative diseases with altered energetic metabolism in ALS. In the future, a more extensive investigation of the hydroperoxide and hydroxide level in different tissues as well as the lipid metabolism must be done, which could lead to new therapeutic options for ALS patients

Intakes of the highly unsaturated fatty acids (HUFA, ≥20 Carbons, ≥3 double bonds) eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3, DHA) greater than 0.25 g/d are currently recommended for health benefits. Targets for omega-3 blood biomarkers have also been proposed based on associations with protection against coronary heart disease mortality. The relationship between diet intakes and blood biomarkers is not well defined, particularly differences between men and women. North American intakes and blood biomarkers of EPA and DHA are typically below recommendations and targets. To address this disparity, adherence to dietary advice strategies to increase EPA + DHA intake was investigated over one year. Adherence was sustained up to 12 weeks and long-term adherence was well characterized by the % of DHA in erythrocytes. For women, n-3 HUFA blood biomarkers increased following nutraceutical or combined strategy dietary advice but not seafood or functional food advice. To assist in the assessment of EPA + DHA intakes, food sources of EPA and DHA in Canada were incorporated into a semi-quantitative, nutrient-specific food frequency questionnaire (FFQ) and validated. The FFQ is an adequate tool for estimating habitual EPA and DHA intake and ranking Canadian adults by their intakes. The blood biomarker response to recommended intakes of 0.25, 0.5 and 1 g/d EPA + DHA was also characterized in adult men and women. Blood n-3 HUFA biomarkers increased in a dose-dependent manner and aligned with blood targets associated with primary cardiac arrest risk reduction. Sex differences in the DHA:EPA ratio in blood observed with low intakes at baseline disappeared following 0.25 g/d EPA + DHA. These findings are applicable towards informing achievable dietary guidelines for EPA + DHA intake and improving measurement of EPA + DHA intake in relation to blood n-3 HUFA biomarkers.

This thesis investigated the effects of omega-3 supplementation (2.0 g/day EPA + 1.0 g/day DHA) for 12 weeks on human skeletal muscle sarcolemmal and mitochondrial membrane fatty acid (FA) composition and whole body energy expenditure in young healthy males. Supplementation resulted in significant incorporation of EPA and DHA into sarcolemmal and mitochondrial membranes, with an increase in total unsaturation of mitochondrial membranes. The incorporation profile of the sarcolemma and mitochondria differed, with the mitochondria mimicking changes in whole muscle. There were no changes in the protein content of mitochondrial and selected proteins involved in energy metabolism, except for a significant increase in the long form of UCP3. Despite changes in membrane FA compositions, there were no changes in whole body substrate oxidation at rest or during exercise. These data demonstrate that omega-3 supplementation for 12 weeks altered the FA composition of sarcolemmal and mitochondrial membranes in human skeletal muscle.

Intrauterine growth restriction (IUGR) reduces bone mass by 10-30% and impairs arachidonic (AA) and docosahexaenoic (DHA) acid status in infants. Because AA and DHA enhance neonatal bone mass, the aim of this study was to determine the effects of dietary 0.5% AA and 0.2% DHA (w/w) prior to weaning on bone and growth. 40 guinea pigs were randomized to either a control (C) or low-protein diet (LP) during pregnancy and the C diet or the C diet with AA+DHA during lactation. Measurements included bone mass, metabolism, and strength, and erythrocyte lipid of sows and offspring from birth to 16 wk post-partum. The LP diet induced IUGR, while the AA+DHA increased bone mass by 5-20% in sows and offspring and corrected growth and bone mass in IUGR pups. Thus, AA+DHA provided in lactation rescues the growth trajectory in an IUGR state and is beneficial to maternal and neonatal bone mass.

Cardiovascular diseases are the number one cause of death in Western societies. Endothelial dysfunction is an early event in the pathology of atherosclerosis, which is an underlying cause in the majority of cardiovascular events. Exposure to persistent environmental pollutants, such as polychlorinated biphenyls (PCBs), is a risk factor for the development of atherosclerosis. First, we tested a hypothesis that coplanar PCBs, dioxin-like chemicals with affinity for aryl hydrocarbon receptor (AhR), can stimulate up-regulation of monocyte chemoattractant protein-1 (MCP-1), an endothelium-derived chemokine that attracts monocytes into sub-endothelial space in early stages of atherosclerosis. Coplanar PCBs 77 and 126 increased expression of MCP-1 in endothelial cells, and this effect was dependent on activation of AhR and increased levels of cytochrome P450 monoxygenases. Subsequent rise in the levels of reactive oxygen species (ROS) led to a downstream stimulation of redox-sensitive kinases and transcription factors. Lipid rafts, and particularly caveolae, are enriched in endothelial cells, and down-regulation of caveolin-1, a key structural protein of caveolae, decreases the progression of atherosclerosis. Studies using deletion of caveolin-1 in vitro and in vivo demonstrated that intact caveolae were required for up-regulation of MCP-1 and pro-inflammatory interleukin-6 (IL-6) by PCB77. Nutrition can modulate adverse outcomes of human exposure to environmental chemicals. Fish oil-derived long-chain omega-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6ω-3), can alleviate inflammatory responses and the risk of cardiovascular disease. Cyclopentenone metabolites produced by oxidation of DHA contribute to these protective effects. Endothelial cells were pre-treated with oxidized DHA (oxDHA), prepared by incubation of the fatty acid with a free radical generator. Subsequent up-regulation of MCP-1 by coplanar PCB77 was markedly reduced. DHA-derived cyclopentenones increased nuclear translocation and DNA binding of a transcription factor NF-E2-related factor-2 (Nrf2), as well as expression levels of its target, antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO1). This stimulation of antioxidant responses prevented ROS production and inflammatory responses induced by PCB77. These data support the concept that nutrition prevents toxicity caused by environmental pollutants; thus, nutrition and can be a sensible approach to alleviate chronic pathologies associated with these chemicals.

Diseases, such as cardiovascular disease (CVD), are linked to chronic low levels of inflammation. This inflamed state is the product of risk factors including exposure to environmental pollutants, such as polychlorinated biphenyls (PCBs), which are correlated with increased risk for CVD and diabetes. In response to this health risk, our research addresses the mechanisms by which coplanar PCBs elicit an inflammatory response and the mitigation of PCB-induced inflammation through dietary intervention using docosahexaenoic acid (DHA), an omega-3 lipid. Investigators from the University of Kentucky Engineering Department are developing remediation technologies that detoxify PCBs through dechlorination. We studied the cellular toxicity of coplanar PCB 77 remediation products in primary vascular endothelial cells. The dechlorination products elicited different toxicological responses, which were less than the parent compound and contributed to the overall inflammatory response. The presence of PCB 77 at any concentration was sufficient to promote an inflammatory response, which was attenuated with complete dechlorination. PCB 77 is a good model for coplanar PCB-induced toxicity, but in environmental and human samples, coplanar PCB 126 is detected more frequently. Using different doses of PCB 126, we determined that acute exposure to 5 μmol PCB 126/kg mouse was sufficient to produce an inflammatory response without inducing a toxic wasting phenotype. PCB-induced inflammation was attenuated in vitro by DHA-derived neuroprostanes. Applying this information, we fed mice a DHA-enriched diet and exposed them to PCB 126. Liver and adipose lipid profiles confirm an increase in omega-3 fatty acid composition and DHA metabolites, and changes in gene expression indicate a heightened anti-oxidant response in the presence of PCB-induced inflammation. These data provide an overview of the in vivo response to a PCB-induced inflammation after DHA dietary feeding. We have demonstrated that PCB-induced endothelial dysfunction is propagated through lipid domains called caveolae. Caveolae are also signaling domains for toll-like receptor 4 (TLR4), and receptor for lipopolysaccharide (LPS). Similar to PCBs, TLR4 signaling is inhibited by DHA. We compared the caveolae-associated signaling response after exposure to coplanar PCB 126 or LPS. The domain localization of caveolae was altered by both PCB 126 and LPS. Our study determined that PCB 126-induced inflammation was not inhibited by a TLR4-specific inhibitor, but caveolae-based signaling was critical to both PCB 126- and LPS-induced inflammation. Environmental pollutants, such as coplanar PCBs, are risk factors in the development of chronic diseases. Here we investigate possible signaling pathways associated with environmental toxicity and apply potential dietary interventions with omega-3 lipids.