Doctor of Philosophy, The Ohio State University, 2016, Human Ecology: Human Nutrition

Abstract Diet is one environmental factor contributing to the etiology of cancer [1], and our ability to make effective public health recommendations depends on the expert committees appointed to evaluate a rapidly growing body of literature. To this end, we trust experts to review, evaluate, and synthesize the published, peer-reviewed food and nutrition research to develop guidelines which provide the basis for our federal food and nutrition policy and education initiatives [2]. In recent decades, much of the data used to define guidelines are derived from large epidemiological cohort studies. The value of such studies depends upon the accurate quantification of their dietary and nutritional exposures using dietary assessment tools. The food frequency questionnaire (FFQ) is one of the most commonly used nutrition assessment tools because of its low cost compared to other dietary assessment tools, as well as its ability to reach a wide demographic and to simultaneously assess a multitude of dietary variables. There is significant data to suggest that carotenoid rich foods may play a role in human health and disease prevention. For example, tomato derived lycopene is hypothesized to be beneficial for the prevention of prostate cancer, and some epidemiologic studies using FFQs have shown a risk reduction associated with this carotenoid intake, while others have found no relationship. These inconsistencies have prevented public health guidelines from being developed and the FDA from approving a submitted “health-claim” for lycopene-rich foods in men for prostate cancer prevention [3]. In this dissertation, we will address the question of whether there is a difference in the ability of the most commonly used FFQs to estimate carotenoid intake and whether a carotenoid-specific FFQ can be developed for improved carotenoid intake estimates compared to conventional FFQs. Our findings may help explain why diverse results have been reported in large cohort studies usin (open full item for complete abstract)

Committee: Steven Clinton MD, Ph.D. (Advisor) Subjects: Nutrition

Master of Science, The Ohio State University, 2008, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 2008, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 2008, Graduate School

Committee: Not Provided (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2021, Health and Rehabilitation Sciences

A plant-based dietary pattern is associated with improved health outcomes and is integrated within guidelines to promote public and individual health, such as the Dietary Guidelines for Americans (DGA) and the WCRF/AICR Cancer Prevention Guidelines. The number of cancer survivors has increased dramatically, and efforts are focusing upon promoting healthy survivorship free of cancer and enhancing longevity. Data suggests that diets of cancer survivors are inadequate in plant foods including fruits, vegetables, and whole grains. There is a critical need for studies focusing upon improving survivor dietary patterns. The development and validation of tools to assess the complex dietary intake of humans and biomarkers of exposure are crucial to this effort. Further, assessment of individual response to dietary intake remains complex and impacted by interacting components in the diet as well as genetic influences on digestion, absorption, and metabolism. Aligning dietary intake data with biomarkers of individual exposure and metabolism is critical for advancing knowledge. The goals of these studies were to explore select phytochemicals as biomarkers of dietary exposure within human trials by: (a) Defining changes in carotenoid status as an indicator of overall exposure to fruits and vegetables following an intervention with a healthy dietary pattern in free living cancer survivors; (b) Developing, evaluating, and validating a targeted LCMS flavonoid profile in 24-hour urine that is reflective of human intake and exposure to (poly)phenol-rich fruits and vegetables; and (c) Examining the relationships between a targeted (poly)phenol profile in the urine and/or dietary patterns and indicators of cardiometabolic health in a clinical trial of a healthy dietary pattern in cancer survivors. To achieve these goals, we conducted a clinical trial in cancer survivors (n=80) with overweight/obesity. Individuals enrolled in a lifestyle intervention with a primary goal of improving mod (open full item for complete abstract)

Committee: Colleen Spees PhD, MEd, RDN, LD, FAND (Advisor); Steven Clinton MD, PhD (Committee Member); Fred Tabung PhD, MSPH (Committee Member); Xiaokui Mo PhD, MAS (Committee Member); Ken Riedl PhD (Committee Member) Subjects: Behavioral Sciences; Food Science; Health Sciences; Nutrition; Oncology

Master of Science, The Ohio State University, 2020, Animal Sciences

Cull cows represent a significant percentage of the revenue received from the U.S. beef industry, however, cull cows are often heavily price discounted at time of slaughter due to decreased body condition scores, carcass yield and quality and increased yellow fat color as a result of a prolonged high vitamin A diet. This study's objective is to evaluate different feeding strategies and their effects on body condition score, external fat color, carcass yield and quality traits in cull cows. The central hypothesis is feeding a high energy diet, with low levels of Vitamin A, for 56 days will improve animal performance, carcass yield and quality traits in addition to capturing the point (rate) of the conversion of yellow to white external fat. In the present study 86 Angus crossbreed cows, culled from two Ohio State University farms were utilized. Cows were fed either low vitamin A diet (LVA) consisting of whole shelled corn, soybean hulls, soybean meal and a mineral-vitamin supplement or a high vitamin A diet (HVA) diet, formulated using whole shelled corn, fescue hay, DDGS and a mineral-vitamin supplement for 56 days. During the 56 day feeding period, weights, body condition scores, and subcutaneous adipose samples were collected every 14 days. On days 56, cattle were slaughtered; 48 hours postmortem carcass characteristics and objective color scores were recorded and a sample of the longissimus dorsi lumborum was collected. Subcutaneous adipose tissue samples were utilized to record subjective color scores and then ground to be analyzed via HPLC for beta-carotene concentration. The longissimus dorsi lumborum samples were sliced into 2.54cm steaks, one of which was utilized to perform WBSF testing, the other was ground and utilized for pH testing. After results were gathered, data was analyzed via a proc mixed model of SAS. Feeding cull cows a low vitamin A diet resulted in significant differences in external carcass fat color (P = 0.01) as well as objective b* val (open full item for complete abstract)

Committee: Lyda Garcia Dr. (Advisor); Stephen Boyles Dr. (Committee Member); Alejandro Relling Dr. (Committee Member); Kopec Rachel Dr. (Committee Member) Subjects: Agriculture; Animal Sciences; Animals; Food Science; Livestock

Master of Science, The Ohio State University, 2020, Health and Rehabilitation Sciences

In cost-offset community supported agriculture (CO-CSA), low-income households receive subsidized shares of locally-grown produce with the aim of improving their access to and consumption of fruits and vegetables (FV). Social cognitive theory (SCT) is a commonly leveraged behavior change theory and informed the design of a CO-CSA plus nutrition education intervention trial for low-income households. The objective of the present study is to model the cross-sectional relationships at baseline between SCT-aligned variables and FV intake among CO-CSA enrollees. We hypothesized that nutrition-related attitudes, beliefs, and self-efficacy as well as perceived FV availability and accessibility would be significant predictors of FV intake. In Spring 2016 and 2017, adult enrollees (n=305) completed a baseline questionnaire containing study-specific and validated questions to measure nutrition-related psychosocial (knowledge, attitudes, and beliefs), environmental (FV availability and accessibility), and behavioral factors (self-efficacy) in relation to self-reported and proxy measures of FV intake (National Cancer Institute's All-Day FV Screener and resonance Raman spectroscopy to assess dermal carotenoid score). Descriptive statistics and multiple regression models were completed in SPSS. Models controlled for demographic characteristics. Self-efficacy to prepare and eat FV and in-home FV availability were significant predictors of daily FV intake. In-home FV availability, accurate knowledge of FV recommendations, and perceived geographic access to FV were significant predictors of carotenoid score. Among a set of SCT-informed variables expected to influence FV intake, knowledge of FV recommendations, geographic access to FV, in-home availability of FV, and self-efficacy to prepare and eat FV were significant predictors. However, these only explained 22-23% of outcome variability among a sample of CO-CSA adult enrollees. SCT alone may not be the most appropriate theory thro (open full item for complete abstract)

Committee: Jennifer Garner (Advisor) Subjects: Health Sciences; Nutrition; Public Health

Master of Science (M.S.), University of Dayton, 2019, Chemistry

The projects described in this thesis were focused on studying two aspects of singlet oxygen. The first is the ability of singlet oxygen to be generated by photosensitizers for use in photodynamic therapy and the second is the ability of singlet oxygen to be quenched with β-carotene. Photodynamic therapy (PDT) is a medical technique which utilizes a photosensitizing drug, light of a certain wavelength and molecular oxygen to generate singlet oxygen, a toxic oxidizing species. When present, singlet oxygen will rapidly react with surrounding biomolecules, causing cellular damage that ultimately leads to cell death. To the ends of creating a photosensitizer for PDT, a new pi-extended dipyrrin containing isoquinolpyrrole has been synthesized by solvent free reactions with trifluoroacetic acid (TFA) as a catalyst. The borondipyrrin (Bodipy) of the isoquinolpyrrole was synthesized by standard procedures followed by synthesis of the bis-ruthenium(II) Bodipy analog. The spectroscopic properties of this complex show the typical intra-ligand charge transfer transitions (ILCT) along with the Ru(π) to ligand(π*) metal to ligand charge transfer (MLCT) transitions. An intense transition at 608 nm with molar absorptivity greater than 100,000 M-1 cm-1 associated with the ππ* transition of the Bodipy core is observed. In acetonitrile solutions the bis-Ru(II)-Bodipy complex generates significant singlet oxygen when irradiated with low energy light. In aqueous solutions the complex is capable of photo-nicking plasmid DNA when irradiated within the photodynamic therapy (PDT) window of 600 to 850 nm. β-carotene (βC) is an orange pigment present in the photosynthetic reaction center (PRC) of green plants, where it plays a vital role in photosynthesis: It quenches singlet oxygen before it damages chlorophyll and other components of the PRCs. During photosynthesis, βC temporarily converts from its native orange–450 state to a pink–515 state via the so–called 515nm Effect. Because of (open full item for complete abstract)

Committee: Shawn Swavey PhD (Committee Co-Chair); Mark Masthay PhD (Committee Co-Chair); Jeremy Erb PhD (Committee Member) Subjects: Chemistry; Inorganic Chemistry; Physical Chemistry

Master of Science, The Ohio State University, 2019, Environment and Natural Resources

Worldwide, a major threat to aquatic systems is increased sediment runoff, which can lead to elevated levels of turbidity. In an increasingly variable world, the ability for animals to respond rapidly to environmental disturbance can be critical for survival. Chronic and acute turbidity exposure can have both indirect and direct effects on fish across large and small spatial scales. Indirect impacts include alteration of the sensory environment of fishes (disrupting communications) and shifts in prey availability; while direct impacts include damage to respiratory organs or eliciting physiological compensatory mechanisms that influence fitness-related traits associated with reproduction and survival. I used a combination of field and laboratory studies to examine the effects of elevated turbidity on an African cichlid fish (Pseudocrenilabrus multicolor victoriae). This sexually dimorphic species is widespread across the Nile River basin and is found across extreme environmental gradients (e.g. dissolved oxygen, turbidity). I investigated if within-population variation in diet and male nuptial coloration are associated with turbidity on a microgeographic spatial-scale. Diet was investigated because many cichlid fish depend on dietary carotenoids (red and yellow pigments) for their reproductive displays and other physiological mechanisms associated with health. I found that fish from mostly clear waters ate a higher proportion of plant material and males were more colorful than fish found at more turbid locations. This could indicate that male reproductive traits are plastic across environmental extremes. In the laboratory study, I used a split-brood rearing experiment to investigate the effects of turbidity level (high/low) and dietary carotenoid concentration (trace/low) on reproductive traits in P. multicolor. I found that chronic turbidity and carotenoid diets had differential effects on males and females: nuptial coloration and gonadosomatic index were higher in (open full item for complete abstract)

Committee: Suzanne Gray Dr. (Advisor); Lauren Pintor Dr. (Committee Member); Roman Lanno Dr. (Committee Member); Lauren Chapman Dr. (Committee Member) Subjects: Freshwater Ecology; Physiology

Doctor of Philosophy, Case Western Reserve University, 2018, Pharmacology

Vitamin A homeostasis control on different levels is essential to maintain retinoid function in peripheral tissues and to ensure optimal vision and requires a well-coordinated team of metabolic enzymes and transport proteins. The production of vitamin A from a chemically diverse set of over 60 putative provitamin A precursors poses a metabolic challenge. We scrutinized enzymatic properties of the two carotenoid cleavage dioxygenases (CCDs), BCO1 and BCO2, which catalyze the metabolism of carotenoids. Recombinant BCO1 split substrates at the C15,C15' position adjacent to a canonical but not substituted ß-ionone ring site to vitamin A aldehyde, and was not impeded by removal of methyl groups from the substrate backbone. BCO2 split substrates at the C9,C10 position adjacent to assorted ionone ring sites. Kinetic analysis revealed a higher catalytic efficiency of BCO2 with substrates bearing non-canonical ionone rings. Accordingly b- cryptoxanthin, with one canonical and one non-canonical ionone ring site, was xix exclusively converted by both CCDs to retinoids in mice. Homology modeling and site directed mutagenesis identified amino acid residues at the entrance of the BCO1 substrate tunnel which determined its substrate specificity. Thus, selectivity of CCDs for distinct ionone ring sites guarantees production of vitamin A. To exert its effects, vitamin A must be distributed and transported across cell membranes, a process mediated by the STRA6 protein. Impaired vitamin A transport and homeostasis are associated with type 2 diabetes and Matthew Wood Syndrome. STRA6's role in vitamin A homeostasis is defined. Stra6-/- mice were raised on diets sufficient or insufficient for vitamin A, with heterozygous sibling controls. Vitamin A levels of eyes, brain and testes, which highly express Stra6, as well as lung and fat, were measured, and consequences of STRA6 deficiency on retinoid-dependent processes were determined. Furthermore, effects of STRA6 deficiency on retinoid (open full item for complete abstract)

Committee: Johannes von Lintig PhD (Advisor); Marcin Golczak PhD (Committee Chair); Timothy Kern PhD (Committee Member); Joseph LaManna PhD (Committee Member); Derek Taylor PhD (Committee Member) Subjects: Biochemistry; Pharmacology

Master of Science, The Ohio State University, 2018, Food Science and Technology

The objective of this study was to develop reliable, fast and non-destructive protocols for analyses of carotenoids in tomato paste by Raman spectroscopy combined with pattern recognition analyses. Tomato paste samples were kindly provided by tomato processing companies in California and different carotenoid varieties of tomatoes were obtained from the OSU Tomato Genetics and Breeding Program at Wooster, Ohio. Spectra of tomato paste were directly collected, without any sample preparation steps, with a handheld Raman spectrometer equipped with a 1064nm laser. Lycopene concentration in red tomato paste was determined by UV-Vis spectrophotometry at 503nm. HPLC equipped with a photodiode array detector was utilized for carotenoid profiling. Raman spectra were evaluated by pattern recognition analyses, including quantification by partial least square regression (PLSR) and classification by soft independent modeling of class analogy (SIMCA). Raman spectra of red tomatoes showed unique marker bands at 1510 cm-1 (C=C stretching), 1156cm-1 (C-C stretching), and 1002cm-1 (C-CH3 in-plane rocking) that are characteristic of the long conjugated polyene chain of lycopene. Cross-validated (leave-one-out) PLSR model (3 latent variables) predicted lycopene content with a standard error of cross-validation (SECV) of 4.7 mg/100g and a coefficient of determination of 0.96. Screening of tomato paste from varieties that accumulate a diverse carotenoid profile showed a unique region associated with C=C stretching that changed according to the dominant carotenoid: all-trans-lycopene at 1511 cm-1, ß-carotene at 1521 cm-1, and tetra-cis-lycopene at 1525 cm-1. The unique Raman spectral signatures allowed SIMCA to classify the tomato paste into four classes based on their carotenoid profile without any misclassification, producing tight and well-separated clusters that can be used for predicting unknown samples. Raman spectroscopy is positioning as an attractive fingerprinting technique for t (open full item for complete abstract)

Committee: Luis Rodriguez-Saona (Advisor); Christopher Simons (Committee Member); Rafael Jimenez-Flores (Committee Member) Subjects: Food Science

Master of Science, The Ohio State University, 2017, Allied Medicine

Cancer survivors are at risk for suboptimal nutrition due to therapy, inadequate dietary patterns, and susceptibility to unproven dietary advice. Effective biobehavioral interventions aimed at increasing adherence to evidence-based survivorship recommendations are key in reducing cancer recurrence, comorbidities, and all-cause mortality. Data from a six-month phase II non-randomized trial were analyzed to determine pre- to post- changes in: (1) dietary patterns; (2) skin and plasma carotenoids; and (3) red blood cell fatty acid (RBC FA) composition. Overweight cancer survivors (N=29) were provided weekly produce harvesting, semi-monthly group education, remote motivational interviewing, and access to a secure web portal. Data were collected at baseline and post-intervention. Dietary patterns were assessed via 30-day food frequency questionnaires (FFQs) and Healthy Eating Index 2010 (HEI-2010) scores. Skin carotenoids were measured by resonance Raman spectroscopy, plasma carotenoids by high performance liquid chromatography, and RBC FAs by gas chromatography. Increases were documented in HEI-2010 total diet (p=0.006), total fruit (p=0.003), and fatty acids (p=0.007) scores. HEI-2010 scores for total vegetables also trended positively (p=0.054). As assessed by FFQ, survivors increased total dietary intakes of carotenoids by 66% (p<0.001) including increased individual intakes of all five carotenoids analyzed. Mean total dietary fat intakes decreased by 12.37 g (p=0.010), with survivors also decreasing intakes of saturated, unsaturated, and trans fats. Plasma concentrations of total carotenoids increased by 35% (p<0.001), as did as did plasma alpha-carotene, beta-carotene, and lycopene (p<0.001, p<0.001, p=0.017, respectively). Total skin carotenoids increased (p=0.015) and were highly correlated with total plasma carotenoids (r=0.728, p<0.001). Despite changes in dietary intakes of FAs, RBC FA composition was largely unchanged. However, relative abundance of omega-3 F (open full item for complete abstract)

Committee: Colleen Spees PhD, MEd, RDN, LD, FAND (Advisor); Martha Belury PhD (Committee Member); Rachel Kopec PhD (Committee Member) Subjects: Health Education; Nutrition

Doctor of Philosophy, The Ohio State University, 1964, Graduate School

Committee: Not Provided (Other) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2002, Graduate School

Committee: Not Provided (Other) Subjects: Agriculture

Doctor of Philosophy, The Ohio State University, 2016, Ohio State University Nutrition

Lutein and zeaxanthin are dietary carotenoids that selectively accumulate in the macula of the primate retina. Meso-zeaxanthin is a non-dietary carotenoid that concentrates in the fovea of the macula and is thought to form from lutein. Macular carotenoids may play a role in light absorption, protection from oxidative stress and inflammation, and perhaps influence visual performance. Strong evidence indicates that the macular carotenoids protect against the development of age-related macular degeneration (AMD), the leading cause of blindness in adults over 50 years of age in the United States. Understanding the mechanisms of selective accumulation of carotenoids and the influences on accumulation in retinal tissues may help in developing treatments for the prevention of AMD. To reach the retina, macular carotenoids must cross the highly selective retinal pigment epithelium (RPE). To better understand carotenoid accumulation in the retina, we studied the uptake of xanthophyll delivered by lipoproteins and human serum under different conditions using differentiated ARPE-19 cells, an RPE cell model. Our first objective was to determine uptake of ß-carotene, lutein, zeaxanthin, and meso-zeaxanthin in human serum or lipoproteins. Previous studies show that xanthophylls associate mostly with HDL in human serum while ß-carotene associates mostly with LDL. HDL binds to cells via a scavenger receptor class B1 (SR-B1)-dependent mechanism while LDL binds via the LDL receptor (LDLR). Therefore, we hypothesized that xanthophylls are delivered via HDL to the RPE in an SRB1-dependent manner. Carotenoid-enriched whole human serum and lipoproteins were added to ARPE-19 cells. A chemical inhibitor, BLT-1, and a biological inhibitor, serum amyloid albumin (SAA), were used to block binding of HDL to the SRB1 receptor. Carotenoids were extracted and measured using HPLC analysis. For lutein and ß-carotene, LDL delivery resulted in the highest rate and extent of uptake. HDL was (open full item for complete abstract)

Committee: Earl Harrison (Advisor); Martha Belury (Committee Member); Joshua Bomser (Committee Member); Richard Bruno (Committee Member) Subjects: Cellular Biology; Nutrition

Doctor of Philosophy, Case Western Reserve University, 2017, Pharmacology

Carotenoid cleavage oxygenases (CCOs) constitute a large group of evolutionarily conserved enzymes that metabolize a variety of carotenoid and apocarotenoid substrates, including retinoids, stilbenes, and related compounds. They typically catalyze the cleavage of non-aromatic double bonds by O2 to form aldehyde or ketone products. Their reaction products, denoted as apocarotenoids, serve critical functions in both prokaryotic and eukaryotic cells, including pigmentation, light harvesting, antioxidation, and cell signaling. An RPE65-subgroup of family members expressed in vertebrates catalyze a non-canonical reaction consisting of concerted ester cleavage and trans-cis isomerization of all-trans-retinyl esters, the product of which is essential for visual function. Our understanding of the biological functions of CCOs have progressed significantly in recent years. However, fundamental questions regarding to their catalytic mechanism remain largely unknown. In this project, we employed Synechocystis ACO that catalyzes canonical cleavage of carotenoids, and Novosphingobium NOV2 which catalyzes the cleavage of stilbene compound, through use of biochemical, structural, and biophysical methods to investigate the conserved catalytic mechanisms. In contrast to findings by others, our biochemical and crystallographic studies of ACO demonstrated that this prototypical CCO member is not an isomerase, as proposed previously. Rather, our results answered the important question of whether isomerase activity is a feature common to all CCOs. Our subsequent structure-directed mutagenesis studies of ACO then provided insights into substrate selectivity and regiospecificity regarding C-C cleavage during catalysis. Furthermore, our structure-function characterization of mutations of iron-coordination ligands demonstrated the biochemical and structural roles of the conserved 3-Glu, iron-outer sphere in metal coordination among CCOs. Finally, our isotope labeling studies of ACO and N (open full item for complete abstract)

Committee: Krzysztof Palczewski PhD (Advisor); Philip Kiser Pharm D, PhD (Advisor); Jason Mears PhD (Committee Chair); Johannes von Lintig PhD (Committee Member); Vivien Yee PhD (Committee Member); Matthias Buck PhD (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Biophysics; Pharmacology

Doctor of Philosophy, Case Western Reserve University, 2016, Biochemistry

Carotenoids are involved in a rich variety of physiological functions in nature and are essential as retinoid precursors. While the knowledge about retinoid homeostasis has increased over the past, the metabolism of carotenoids, the parent precursors of retinoids, is less well described. Retinoids, such as vitamin A, are produced from dietary carotenoids such as ß-carotene by centric cleavage via the enzyme BCO1. While BCO1 cleaves provitamin A carotenoids, a second enzyme, BCO2, is more promiscuous and metabolizes a veritable host of carotenoids, including nonprovitamin A carotenoids such as zeaxanthin into short and long chain apocarotenoids. We demonstrated in cell lines that human BCO2 is associated with the inner mitochondrial membrane. Different human BCO2 isoforms possess cleavable N-terminal leader sequences critical for mitochondrial import. Subfractionation of murine hepatic mitochondria confirmed the localization of the murine homolog to the inner mitochondrial membrane. Studies in knockout mice revealed that zeaxanthin accumulates in the inner mitochondrial membrane; in contrast, ß-carotene is retained predominantly in the cytoplasm. With the use of a mouse model lacking both BCO1 and BCO2 and applying a genome wide microarray analysis we assessed the effects of supplemented carotenoids on the liver transcriptome and documented changes in pathways for liver lipid metabolism and mitochondrial respiration. By genetically dissecting carotenoid and apocartenoid functions, we observed that ß-carotene accumulation resulted in an elevation of liver triglycerides and liver cholesterol, while zeaxanthin accumulation increased serum cholesterol levels. We further showed that carotenoids were predominantly transported within HDL particles in the serum of mice. Thus our findings were twofold; first we provided evidence for a compartmentalization of carotenoid metabolism which prevents competition between BCO1 and BCO2 for the precious ß-carotene and the production (open full item for complete abstract)

Committee: Johannes von Lintig (Advisor); Martin Snider (Committee Chair); Charles Hoppel (Committee Member); Jason Mears (Committee Member); John Mieyal (Committee Member); David Samols (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2016, Pharmacology

Animals endowed with the ability to detect light have evolved pathways to produce and maintain the continuous regeneration of the visual chromophore, 11-cis-retinal. This C20 retinoid metabolically derives from a C40 carotenoid precursor that is processed by successive oxidative cleavage and geometric isomerization reactions of double bonds. Vertebrates devote three structurally related proteins of the carotenoid cleavage enzyme (CCE) family to this task, whereas insects utilize only one. Expression and purification protocols of recombinant BCO1, the vitamin A forming enzyme, yielding highly active and a homogenous, monomeric protein was established. Enzyme activity assay screening with various detergents provided optimal conditions for kinetic characterization and the foundation for crystallization trials. Phase separation experiments indicate that despite BCO1's soluble state, the enzyme directly interacts with hydrophobic detergent micelles to extract substrates. Our studies on BCO2, the CCE responsible for asymmetrical conversion of carotenoids into apocarotenoids, established methods to isolate enzymatically active primate protein. Comparison tests to rodent BCO2 indicates that primate BCO2's enzymatic function is conserved and analyses suggest that the regulation of BCO2 gene expression is governed by induction of oxidative stress. We provide further evidence that this enzyme plays a role in carotenoid homeostasis, including the macular pigment which has been associated with eye health. Biochemical studies of the sole insect CCE, NinaB, revealed a bipartite substrate binding site that conveys regio- and stereo-selectivity and catalyzes the conversion of carotenoids into all-trans and 11-cis-retinoids via a dioxygenase mechanism. The described bipartite system recognizes key carotenoid features to optimally cleave and isomerize substrates that will produce the chromophore. The vertebrate retinoid isomerase, RPE65, converts all-trans-retinyl esters to 11-cis-r (open full item for complete abstract)

Committee: Johannes von Lintig Ph.D. (Advisor); Derek Taylor Ph.D. (Committee Chair); Michael Harris Ph.D. (Committee Member); Krzysztof Palczewski Ph.D. (Committee Member); Robert Bonomo Ph.D. (Committee Member); Jason Mears Ph.D. (Committee Member) Subjects: Biochemistry; Pharmacology

Master of Science, The Ohio State University, 2015, Human Ecology: Human Nutrition

Biofortification is defined as the enrichment of staple crops with essential micronutrients. At present, it is one of the strategies used to alleviate vitamin A deficiency (VAD) by breeding staple crops with β-carotene. Staple crops that have been successfully biofortified with β-carotene under the HarvestPlus program are cassava, maize (corn) and sweet potato. Recently, β-apocarotenoids have been identified and quantified in cantaloupe melons and orange-fleshed honeydew. These cleavage products of β-carotene are formed by chemical and enzymatic oxidations. However, there are no detailed analyses of these compounds in biofortified foods and little is known about their bioavailability and intestinal absorption. Hence, this research focused on the analysis of β-apocarotenoids in biofortified cassava and kinetics of cell uptake and metabolism of β-apocarotenoids. In the first study, we analyzed the β-carotene and β-apocarotenoids content of conventionally bred cassava biofortified with β-carotene. Using a previously described high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method, we identified and quantified β-apo-13-carotenone, β-apo-14'-carotenal, β-apo-12'-carotenal, β-apo-10'-carotenal, and β-apo-8'-carotenal in hexane/acetone extracts of raw, boiled, and fried roots of the two biofortified cassava varieties investigated. The levels of β-apocarotenoids in roots of non-biofortified cassava varieties were lower than those of biofortified varieties and some of these values approached the limit of detection (LOD) or limit of quantification (LOQ). The purpose of the second study was to determine kinetics of uptake and metabolism of β-apocarotenoids using Caco-2 intestinal cells as a model. We hypothesized that these compounds are directly absorbed from the diet similarly to β-carotene. Pure β-apocarotenoids were delivered to fully differentiated monolayers of Caco-2 cells using tween-40 micelles. Carotenoids were extracted from media (open full item for complete abstract)

Committee: Earl Harrison (Advisor); Steven Schwartz (Committee Member); Joshua Bomser (Committee Member) Subjects: Biochemistry; Nutrition

Master of Science, The Ohio State University, 2015, Food, Agricultural and Biological Engineering

Leafy green vegetables are gaining popularity due to the health benefit. However, the safety of fresh produce has been a major issue in recent years. So the elimination of microorganisms during the post-harvesting processing is significant. As a strong oxidizing agent, chlorine dioxide (ClO2) gas is an effective sanitizer for reducing bacteria on fresh produce. However, gaseous chlorine dioxide usually causes both the inactivation of bacteria and color damage of leafy greens. Consumers desire safe and high quality fresh produce with long shelf-life. Therefore, optimizing the conditions including ClO2 concentration and exposure time is critical. The effect of gaseous ClO2 at concentrations 2.05, 3.10, 4.73, and 5.86 mg/L on inactivating E. coli K-12 inoculated on spinach leaves were determined in this study. The inactivation of E. coli O157:H7 by ClO2 treatments with concentration 2.05 mg/L and exposure time 12 and 20 min was also studied. A kinetic model describing the inactivation of E. coli K-12 as a function of time and ClO2 concentration was developed. The morphological changes of inoculated bacteria caused by ClO2 treatment were obtained using scanning electron microscopy (SEM). The fresh spinach was dip-inoculated with E. coli K-12 resistant to nalidixic acid. After ClO2 treatments, the populations of survived bacteria were determined by plate counting and the survival ratios of bacteria, N(t)/N0, were calculated as a function of time. Two models were fitted to N(t)/N0 data. A semi-logarithmic survival model, log[N(t)/N0] =-k(C)t, with sanitizer concentration dependence k= k0 eP C was compared to the Weibull model, log [N(t)/N0] =-b(C) tn(C), with concentration dependence b(C) = a + g C. The optimal value of parameters in two models were obtained by using Matlab. Log reduction as high as 2.79 log CFU/g was achieved by the treatment with ClO2 concentration of 4.73 mg/L and exposure time of 14min. Weibull model was showed to describe the bacterial inactivati (open full item for complete abstract)

Committee: Kaletunc Gonul Dr. (Advisor) Subjects: Agricultural Engineering