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<p>It has been suggested that environmental factors substantially contribute to the increased prevalence of peanut allergy in industrialized countries. Specifically, the role of disrupted barrier integrity in the gastrointestinal tract has been implicated in the development of food allergy. The use of non-steroidal anti-inflammatory drugs (NSAIDs), which increase intestinal permeability, for the treatment of pain and fever is prevalent in industrialized countries. Therefore, the first aim of this study was to determine whether treatment with indomethacin, a prototypical NSAID, would act in an adjuvant like manner to facilitate sensitization to co-administered peanut protein. Furthermore, we investigated whether indomethacin increases susceptibility to anaphylaxis following oral challenge with peanut in sensitized mice.</p> <p>First, a short model of cholera toxin-mediated sensitization to peanut was developed. Mice were given 1 mg of peanut protein and 5 μg of cholera toxin by oral gavage for 10 consecutive days. This resulted in a robust anaphylactic response and increased peanut-specific IgG1, but not IgE, two weeks following treatment. Mice exposed to peanut during a 10-day treatment with indomethacin (5 mg/kg on alternating days or 3.5 mg/kg daily) did not develop peanut-specific immunoglobulins or anaphylaxis following systemic challenge with peanut protein. Furthermore, treatment with two 5 mg/kg doses of indomethacin 24 and 1 hour before oral challenge did not facilitate anaphylaxis in peanut-sensitized mice. Therefore, we concluded that NSAID treatment is unlikely to play a role in the increased prevalence of peanut allergy, and that NSAID treatment does not increase susceptibility to peanut-induced anaphylaxis in sensitized mice.</p> <p>The second part of this study aimed to develop a short model of epicutaneous sensitization, and address the impact of epicutaneous exposure to peanut during infancy. We investigated the role of site of exposure, duration of exposure, epidermal integrity, strain and age in epicutaneous sensitization. 10 consecutive days of epicutaneous exposure to 20 μg of peanut protein through tape stripped skin induced robust anaphylaxis following i.p. challenge. Neither 7 days of exposure through tape stripped skin, nor 10 days of exposure through intact skin facilitated sensitization. The strength of sensitization was strain-dependent; peanut-specific IgG1 was increased equally in both C57BL/6, and BALB/c mice, and to a greater extent in C3H/HeJ mice. Peanut-specific IgE was increased in both BALB/c and C3H/HeJ mice three weeks following peanut exposure. Epicutaneous exposure beginning one day after birth did not facilitate the development of either peanut-specific IgE or IgG1 in BALB/c mice, or anaphylaxis following systemic challenge. Beginning exposure at 2 weeks of age resulted in peanut-specific IgE and IgG1 production, as well as a robust anaphylaxis following i.p. challenge. Therefore, the development of peanut allergy through epicutaneous exposure is age dependent, and exposure during the neonatal period results in a hyporesponsive response.</p> / Master of Science (MSc)

<p>BACKGROUND: The ontogeny of peanut allergy (PA) is poorly understood, and the treatment of its most severe manifestation, peanut-induced anaphylaxis (PIA), remains limited to rescue epinephrine. We argued that an untargeted metabolomic analysis would be a useful hypothesis-generating tool to identify novel biomarkers, mediators and possibly therapeutic targets in PA and PIA.</p> <p>METHODS: Models of PA and PIA used in this thesis involved either the oral administration of peanut along with cholera toxin or the topical application of peanut on tape-stripped skin. Liquid-chromatography mass-spectrometry (LC-MS) was performed to identify chemical changes in the serum of mice undergoing sensitization and anaphylaxis. Flow cytometry as well as <em>in vivo</em> gain-of-function and loss-of-function immunological studies were used to determine the biological significance of particular molecules in sensitization.</p> <p>RESULTS: LC-MS followed by multivariant analysis showed that the purine metabolism pathway was altered with elevated levels of uric acid (UA) in sensitized mice. UA depletion using allopurinol and uricase fully prevented the development of the allergic and anaphylactic phenotype. Conversely, administration of UA crystals, instead of cholera toxin or tape stripping along with peanut induced a typical allergic and anaphylactic phenotype. The effects of UA and UA crystals are likely a consequence of effects on the activation of resident dendritic cells. Post-challenge metabolic analysis also revealed a distinct metabolic signature in sensitized mice, highlighted by an increase in several metabolites such as histamine. Likewise, peanut allergic patients display a distinct metabolic profile after oral peanut challenge.</p> <p>CONCLUSION: We identified UA, released after damage to the mucosa and/or skin, as a critical alarmin that facilitates the development of Th2 immunity, specifically PA and PIA. Metabolomics analyses of either mice undergoing anaphylaxis or peanut allergic children subjected to a peanut oral challenge provided an extensive overview of metabolomic changes underlying these conditions. Further studies may lead to the identification of novel biomarkers and mediators.</p> / Master of Science in Medical Sciences (MSMS)

Peanut (Arachis hypogaea L.) is second major legume crop grown after soybean in the United States, and its productivity is often limited by drought stress. Drought negatively impacts the yield and quality of peanut. Drought stress in peanut causes an annual loss of approximately $520 million in the United States. Improving peanut yield under water deficit conditions is crucial for peanut growers to maintain their profitability in the market. To achieve this, it is essential to either breed or adopt already available drought tolerant cultivars that can produce higher yield under water deficit conditions. Therefore, the objectives of this research were to (1) evaluate five commercially available virginia and runner type peanut cultivars for pod yield stability using multilocation trials by studying G x E interaction across 13 environments including year, location, and irrigation regime. Linn and Binns, AMMI, Shukla, Wricke's, Finlay and Wilkinson stability models were used to determine pod yield stability. Bailey and Sullivan showed higher stability and adaptability across all stability indices whereas Wynne and TUFRunner presented high mean productivity with lesser stability across environments reflecting specific adaptation to just a few environments. Bailey and Sullivan are recommended for sustainable production across the growing region of Virginia and Carolinas. The second objective (2) was identification of drought tolerance related quantitative trait loci (QTL) and genetic markers to facilitate the development of drought tolerant cultivars. Three diverse recombinant inbred line (RIL) populations, derived from crossing lines N05006 x N04074FCT (Pop-1), line N05006 x Phillips, an old virginia-type cultivar (Pop-2), and lines N08086olJCT x PI 585005 (Pop-3) were phenotyped for the Normalized Difference Vegetation Index (NDVI), Canopy Temperature Depression (CTD), SPAD-meter relative chlorophyll content of the leaves (SPAD) and wilting for QTL mapping. Mapping identified 27 minor QTL on eight chromosomes for all physiological characteristics, i.e NDVI, CTD, SPAD and wilting, with logarithmic of odds values ranging from 2.5 to 38.5 and the phenotypic variance explained by these traits from 1.04 to 11.46 %. There were 4 loci on chromosome 2 associated with NDVI in Pop-1 and Pop-3, explaining 1.8 to 10.38% of the phenotypic variation. These genomic regions may be important resources in peanut breeding programs to improve drought tolerance. Further research is needed to increase the marker density in order to fine map the identified QTL and validate markers linked with these regions. / Doctor of Philosophy / Peanut is a multi million-dollar industry in the United States, but water limitations have a detrimental impact on yield, quality, and grower income. Drought along with aflatoxin contamination are two major challenges faced by U.S peanut industry. Annual losses to peanut caused by drought are around $520 million in the United States. Irrigation can alleviate water shortage in drought prone regions, but around 65% of peanut production in U.S is under rainfed condition, meaning that only rainfall can satisfy peanut crop water requirements. The most feasible and economical solution to peanut growers under these circumstances is to adopt drought tolerant varieties. In this research, our goal was to facilitate breeding drought tolerant cultivars through identification of molecular markers associated with drought tolerance and to identify already available drought tolerant peanut cultivars that could be a game changer for the producers. Therefore, the objectives of my research were to (1) evaluate in multiple environments five commercially available virginia and runner type peanut cultivars for pod yield stability and grade factors. In this study, we considered 13 environments, including 4 years, 4 locations, and 2 water regime. Statistical tools including Linn and Binns, AMMI, Shukla, Wricke's, and Finlay and Wilkinson were used to determine pod yield stability. These stability indices showed that Bailey and Sullivan are more stable and adaptable across different locations in terms of yield, whereas Wynne and TUFRunner presented high mean yield with lesser stability showing specific adaptation to only few environments. Based on stability analysis, Bailey and Sullivan are recommended for sustainable production across different growing region of Virginia and Carolinas. The second objective (2) was to identify drought tolerance related genomic regions using three mapping populations. Phenotyping and genotyping of three diverse recombinant inbred line (RIL) populations, derived from crossing lines N05006 x N04074FCT (Pop-1), lines N05006 x Phillips, an old virginia-type cultivar (Pop-2), and lines N08086olJCT x PI 585005 (Pop-3) were done to find quantitative trait loci (QTL) for drought related traits. These population were phenotyped for the Normalized Difference Vegetation Index (NDVI), Canopy Temperature Depression (CTD), SPAD-meter relative chlorophyll content of the leaves (SPAD) and wilting for QTL mapping. These surrogate traits are related to trait of interest for drought tolerance. NDVI is effective in predicting biomass and yield. Similarly, CTD is associated with transpiration efficiency and carbon dioxide assimilation. Mapping identified 27 minor QTL on eight chromosomes for all physiological characteristics, i.e NDVI, CTD, SPAD and wilting with logarithmic of odds values range from 2.5 to 38.5 and the phenotypic variance explained by these traits ranging from 1.04 to 11.46 %. There were 4 loci on chromosome 2 associated with NDVI in Pop-1 and Pop-3, explaining 1.8 to 10.38% of the phenotypic variation. These genomic regions may be important resources in peanut breeding programs to improve drought tolerance. Further research is needed to increase the marker density in order to fine map the identified QTL and validate markers linked with these regions.

Peanut (Arachis hypogaea L.) is an important food crop in the USA and worldwide with high net returns but yield in excess of 4500 kg ha-1 is needed to offset the production costs. Because yield is limited by biotic and abiotic stresses, cultivars with stress tolerance are needed to optimize yield. The U.S. peanut mini-core germplasm collection is a valuable resource that breeders can use to improve stress tolerance in peanut. Phenotyping for plant height, leaf area, and leaf wilting have been used as proxies for the desired tolerance traits. However, proximal data collection, i.e. measurements are taken on individual plants or in the proximity, is slow. Remote data collection and machine learning techniques for analysis offer a high-throughput phenotyping (HTP) alternative to manual measurements that could help breeding for stress tolerance. The objectives of this study were to 1) develop HTP methods using aerial remote sensing; 2) evaluate the mini-core collection in SE Virginia; and 3) perform a detailed physiological analysis on a sub-set of 28 accessions from the mini-core collection under drought stress, i.e. the sub-set was selected based on contrasting differences under drought in three states, Virginia, Texas, and Oklahoma. To address these objectives, replicated experiments were performed in the field at the Tidewater Agricultural Research and Extension Center in Suffolk, VA, in 2017, 2018, and 2019, under rainfed, irrigated, and controlled conditions using rainout shelters to induce drought. Proximal data collection involved physiological, morphological, and yield measurements. Remote data collection was performed aerially and included collection of red-green-blue (RGB) images and canopy reflectance in the visible, near infra-red, and infra-red spectra. This information was used to estimate plant characteristics related to growth and drought tolerance. Under objective 1), we developed HTP for plant height with 85-95% accuracy, LAI with 85-88% accuracy, and wilting with 91-99% accuracy; this was done with significant reduction of time as compared to proximal data collection. Under objectives 2) and 3), we determined that shorter genotypes were more drought tolerant than taller genotypes; and identified CC650 less wilted and with increased carbon assimilation, electron transport, quantum efficiency, and yield than other accessions. / Doctor of Philosophy / Peanut is a profitable food crop in the USA but has high input costs. Pod yield over 4500 kg ha-1 is required for a profitable production, which is challenging in dry and hot years, and under disease pressure. Varieties tolerant to dry weather conditions (drought) and disease presence are required to sustain production. A collection of 112 peanut varieties is available for researchers to study the mechanisms of tolerance to drought and disease, and identify tolerant varieties to these stresses. Plant characteristics including height, leaf area, and leaf wilting can be used as proxies to estimate stress tolerance and yield, and identify tolerant varieties. How to measure these characteristics is very important. We think that using images collected by a drone and automated analysis by specific computer programs is the easiest, fastest, and most accurate way. Therefore, the objectives of my study were to 1) use drones and cameras to collect images, and computer programs to derive plant characteristics from these images, 2) evaluate the peanut collection to identify varieties with tolerance to drought and disease, and 3) evaluate in depth a sub-set of 28 varieties from this collection under controlled drought conditions to further learn about peanut mechanisms of tolerance to drought and diseases. Field experiments were conducted in 2017, 2018, and 2019, at the Tidewater Agricultural Research and Extension Center in Suffolk, VA. For some tests, we used rainout shelters to mimic drought. We measured plant height, leaf area, color, and wilting, canopy temperature, photosynthesis, and pod yield. From a drone, we collected images in the visible and invisible radiation and, using specific computer programs, estimated plant characteristics with 95% accuracy for height, 88% for leaf area, and 91% for leaf wilting under drought. We concluded that taller varieties were more susceptible to drought than shorter varieties. Peanut varieties CC650 and CC068 had higher end of season yield. The study showed that drought reduced several key mechanisms of photosynthesis including electron transport; and reduced the end of season yield. Variety CC650 performed better under drought than other varieties of the collection.

Lipid oxidation is one of the leading causes of food quality degradation. Manufacturers typically add antioxidants or purge a product's package of oxygen to inhibit oxidation and the resulting off-flavors. Synthetic antioxidants (e.g. BHT, BHA) and some natural antioxidants (e.g. α-tocopherol) have found widespread use in this application. Unfortunately, the public views synthetic additives in a negative light and the current natural antioxidants have been unable to match the protection afforded by the synthetic antioxidants. The search for underutilized and natural antioxidants has led scientists to investigate many different plant-based extracts for use in food and in the treatment and prevention of disease. The objectives of this research were (1) to use ORAChromatography to identify peanut root extract fractions with high antioxidant capacity, (2) identification of compounds in peanut root extracts using HPLC and mass spectrometry, (3) test for the presence of aflatoxins in the extracts, (4) test peanut root extract in food model system for oxidation reduction capabilities, and (5) Testing peanut root extract's ability to decrease protein oxidation in cell culture. Crude peanut root extracts have high antioxidant activities that do not vary by cultivar. The ORAC activities of the peanut root fractions separated by HPLC with a C18 column varied (600.3 – 6564.4 μM TE/g dry extract), as did the total phenolic contents (23.1 – 79.6 mg GAE/g dry extract). Peanut root fractions had aflatoxins contamination well above the 20 ppb limit. Peanut root extracts and the known antioxidants tested were found to have no significant effect in inhibiting oxidation of peanut paste or HBMEC. Peanut root extracts were not shown to have any positive effects, but further research is necessary to eliminate peanut root extracts as a possible food ingredient and health supplement. / Ph. D.

Folate, also known as vitamin B9, is an essential dietary vitamin that provides the donor group for one carbon transfer reactions. Deficiency in folate is associated with neural tube birth defects (NTDs), cancer, cardiovascular disease, and anemia. In the US enriched food products including bread, pasta, and cereal are fortified with folic acid, the synthetic analog of folate. While effective in reducing NTDs, this practice is costly and not economically practical in developing countries. Folate biofortification, increasing the natural folate level in foods by metabolic engineering, has been proposed as a sustainable alternative to food fortification with folic acid. To increase folate levels in peanut seed, GTP cyclohydrolase I from Arabidopsis thaliana (AtGCHI) was introduced into peanut by biolistic transformation. Plant transformation vectors were constructed using publicly available or licensable vector components to avoid intellectual property restrictions that hinder commercialization. Thirteen peanut cultivars were evaluated for transformation efficiencies and regeneration potential. Expression levels of the AtGCHI transgene were determined by quantitative real-time PCR. The endogenous peanut GCHI (AhGCHI) was isolated and sequenced. Studies were conducted to test whether heterologous over-expression of AtGCHI altered expression of the endogenous AhGCHI. Seed-specific expression of AtGCHI does not affect AhGCHI transcript accumulation. For validation of the proposed folate biofortification strategy, vitamin quantification will be required. A liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed to identify and quantify the different forms of folate. However, additional work will be needed to determine sensitivity of the instrument, to optimize vitamin extraction, and to increase sufficient seed for vitamin extraction and analysis. Peanut products derived from folate biofortified peanut kernels will have a niche market in the United States, but there is a larger global implication as a mechanism for sustainable delivery of essential vitamins to populations that can not adopt synthetic vitamin supplementation/fortification. Successful demonstration of increased folate in peanut will result in better vitamin availability for populationssonsuming peanut based foods as a dietary staple. / Ph. D.

The purpose of this study was to observe and compare the blood glucose response of individuals with documented noninsulin dependent diabetes (NIDDM) and controls after they consumed a peanut butter and cracker snack product (Austin "Toasty" crackers). Twenty-one subjects with NIDDM (15 with current NIDDM and 6 with previously abnormal glucose tolerance [prevAGT]) and 11 controls participated. Height, weight, body mass index (BMI) and age of subjects were recorded and statistically compared. Subjects tested their own blood glucose using the One Touch II glucometer, and participated on two separate occasions which were designated session 1 and session 2. Blood glucose was measured before eating the test food, every 15 (+3) minutes for two hours after eating, and at 150 and 180 minutes. Mean peak blood glucose value and the time at which glucose peaked were identified and compared among the groups. Repeated measures ANOV A was used to compare blood glucose response curves of the NIDDM group, the control group, and the PrevAGT group; slope of the line after the peak was of interest. The null hypothesis was that the groups would not differ in their blood glucose responses. / Master of Science

This study concerns the competitive position of peanuts and cashews in the salted nut trade of the United States. The effect of the price support and production control program for peanuts is evaluated in regard to the demand and price relationships between salted peanuts and cashews. This research analyses demand and price relationships for salted peanuts and cashews, compiles the available information on the world cashew nut industry, which has not heretofore been readily available to the public, and summarizes relevant information about the United States nut trade in terms of supplies, consumption, and price of peanuts and tree nuts with emphasis on the salted nut trade. The results of this research, in its present stage, do not suggest conclusively that salted peanut consumption is likely to be affected in a manner detrimental to the peanut industry, in the foreseeable future, by moderate price increases, or by competition from cashew nuts, as a result of the peanut price support and production control program. However, a substantial expansion of world cashew production has occurred in the recent past. Prospects for further expansion appear bright in view of the encouragement currently provided by India's plans for total economic growth. African production has also demonstrated capability of substantial expansion. Accordingly, a further increase in cashew imports by the United Stats might reasonably be expected. / Master of Science

<i>Sclerotinia minor</i> Jagger is a fungal pathogen of cultivated peanut (<i>Arachis hypogaea</i> L.) that can cause crop losses in excess of 50%. Fungicides are not completely effective at controlling the disease and can cost up to $311 per hectare for three applications. The ability to produce oxalic acid is necessary for the pathogenicity of some <i>Sclerotinia</i> spp. With little to no naturally occurring resistance to Sclerotinia blight in <i>Arachis</i> spp., a biotechnological approach was used to confer resistance to the disease. Peanut plants were transformed with a gene from barley encoding oxalate oxidase, an enzyme that degrades oxalic acid. Transformed peanuts showed resistance to S. minor and increased yields under disease pressure compared to the parental lines. Before the resistant varieties can be marketed, they must be reviewed and approved by the governmental regulatory system. Responsibility for regulation of transgenic plants in the U.S. is shared among the U.S. Department of Agriculture (USDA) through the Animal and Plant Health Inspection Service (APHIS), the Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA). These agencies require several different data sets including molecular characterization and field studies before each transformation event can be commercialized. This project was designed to characterize three different transformation events, N70, P39 and W171. Molecular characterization included determination of insertion number, copy number, intactness of the expression cassette and stable inheritance of the transgene. N70 was found to have two insertions and two copies while W171 had one insertion with one copy. The P39 event has two insertions and two or more copies. Each of the three events was stable over multiple generations. Phenotypic comparisons of each transgenic line to the parent cultivar were carried out in field studies. Characteristics such as oxalate oxidase expression, yield and quality, hay quality, disease occurrence, aflatoxin content and plant height were assessed. Transgenic peanuts showed few differences from the parent cultivar other than resistance to Sclerotinia blight and yield under disease pressure. Outcrossing studies were completed to determine the rate and distance of cross pollination. Outcrossing rates in N70, P39 and W171 were less than 2.5% and occurred up to 19 rows or 17.4 m from the nearest transgenic row. The molecular characterization and field performance of N70, P39 and W171 have been assembled into a document to petition APHIS for determination of non-regulated status. / Master of Science in Life Sciences