Theses and Dissertations at Montana State University (MSU)
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Item The genetic characterization of a nil polyphenol oxidase (PPO) trait for the improvement of end product quality in wheat (Triticum aestivum)(Montana State University - Bozeman, College of Agriculture, 2014) Hystad, Steven Michael; Chairperson, Graduate Committee: Michael J. GirouxWheat (Triticum aestivum) polyphenol oxidase (PPO) contributes to the time dependent discoloration of Asian noodles. Wheat contains multiple paralogous and orthologous PPO genes expressed in wheat kernels, Ppo-A1, Ppo-D1, Ppo-A2, Ppo-D2, and Ppo-B2. To date, wheat improvement efforts have focused on breeding cultivars containing Ppo-D1 and Ppo-A1 alleles conferring reduced PPO activity. A major impediment to wheat quality improvement is a lack of additional PPO alleles conferring reduced kernel PPO. Thus, the discovery of novel low or null PPO alleles is critical to further reduce PPO and improve noodle color stability. The objective of this study was to 1) Characterize mutations present in a low PPO line 07OR1074 and create molecular markers associated with newly characterized alleles; 2) assess the allelic impact of the newly characterized alleles on kernel PPO activity and upon Chinese white salted noodle quality. To characterize additional mutations present and develop molecular markers, genomic clones were created for each known PPO gene and sequenced. Previously reported very low PPO line, 07OR1074, was found to contain a novel allele at Ppo-A2 and null alleles at Ppo-A1 and Ppo-D1 loci and molecular markers were created that encompassed each mutation. To determine the allelic impact of the new alleles on kernel PPO activity, F3:4 lines were genotyped with the novel molecular markers, grown in the field in Bozeman, Montana, and assayed for kernel PPO activity. Evaluation of lines indicated a substantial genotypic effect on PPO with Ppo-A1 and Ppo-D1 loci contributing significantly (P <.001) to total PPO. To determine the effect of the null PPO alleles upon noodle quality Chinese white salted noodles were produced from both refined white flour and whole wheat flour. The results clearly demonstrate that the null PPO alleles at Ppo-A1 and Ppo-D1 loci produce noodles that are brighter (greater L*), more red (greater a*), and more yellow (greater b*) at 24 and 48 hours. These results show that mutations in Ppo-A1 and Ppo-D1 genes are important to lowering overall wheat kernel PPO activity producing a more desirable and marketable product.Item Lymphocyte responses to the polyphenol, oenothein B, and the influence of age on these responses(Montana State University - Bozeman, College of Agriculture, 2014) Ramstead, Andrew Gordon; Chairperson, Graduate Committee: Mark Jutila; Igor A. Schepetkin, Mark T. Quinn, and Mark A. Jutila were co-authors of the article, 'Oenothein B, a cyclic dimeric ellagitannin isolated from Epilobium angustifolium, enhances IFNgamma production by lymphocytes' in the journal 'PLoS ONE' which is contained within this thesis.; Igor A. Schepetkin, Kimberley Todd, James Loeffelholz, Mark T. Quinn and Mark A. Jutila were co-authors of the article, 'Aging influences the response of T cells to stimulation by the ellagitannin, oenothein B' which is contained within this thesis.; Mark A. Jutila was a co-author of the article, 'Complex role of gamma delta T cell-derived cytokines and growth factors in cancer' in the journal 'Journal of interferon and cytokine research' which is contained within this thesis.Innate lymphocytes, such as NK cells, play an important role in a number of human diseases. Therefore, they have become popular target cells for new therapeutics. Studies to identify materials that stimulate these cells have identified a number of molecules from nutritional supplements which can activate and/or inhibit their activity. While the use of nutritional supplements has potential to enhance the activity of innate lymphocytes to help treat and prevent disease, there is still much which is currently unknown about the activity of these compounds. Previous work has identified oenothein B, an ellagitannin isolated from Epilobium angustifolium, to be a stimulant for myeloid cells. In this study, we further examined the immunomodulatory effects of oenothein B and identified that it also stimulates lymphocytes. We found that oenothein B induces the production of IFN gamma by NK cells and both alpha beta and gamma delta T cells. In addition to inducing the production of IFN gamma by NK cells alone, oenothein B also enhances IFN gamma production in response to secondary stimuli, such as IL-18 or a tumor cell line, in a synergistic manner. We also identified a novel effect of aging on the lymphocyte response to oenothein B. The induction of IFN gamma, as well as GM-CSF, production by T cells in response to oenothein B increases in adults compared to young individuals. The predominant producers of IFN gamma in response to oenothein B also shift from the NK cell population in young individuals to the T cell population in adults. The increase in cytokine production in adult T cells correlates with increasing numbers of CD45RO+ T cells, which produce more cytokines in response to oenothein B than CD45RO- T cells. This influence of aging on the immune response to oenothein B may be applicable to numerous nutritional supplements which stimulate innate immunity and could be an important variable in the effectiveness of these compounds in improving human health. In this dissertation, we identify age as an important factor which should be taken into account when examining the effect of polyphenols on innate immunity and could have important implications for optimizing the benefit of immunomodulatory polyphenols.Item The nutritional value of six barleys and their proanthocyanidin-free mutants(Montana State University - Bozeman, College of Education, Health & Human Development, 1986) Heintzman, Kimberly BolinItem Molecular and biochemical characterization of wheat (Triticum aestivum. L) polyphenol oxidases (PPOs)(Montana State University - Bozeman, College of Agriculture, 2005) Jukanti, Aravind Kumar; Chairperson, Graduate Committee: Andreas Fischer.Polyphenol oxidases (PPOs) from several plant species, including wheat, have been implicated in the undesirable brown discoloration of food products. Wheat (Triticum aestivum L.) represents an interesting system to advance our understanding of plant PPO function for two important reasons, namely (1) the large size an complexity of its (allohexaploid) genome, and (2) its economic importance. Prior to this study, the molecular and biochemical properties of wheat PPOs were largely unknown. To remedy this situation, we have performed several BLAST searches of expressed sequence tag (EST) databases, using a known wheat PPO sequence as a search tool. This study suggested the presence of at least six PPO genes in hexaploid wheat, falling into two different phylogenetic clusters of three genes each. Presence of a wheat PPO multigene family was confirmed by Southern blotting. A combination of biochemical (enzyme purification and mass spectrometric analysis) and molecular (Northern) approaches confirmed that members of one cluster are not expressed in the developing kernels and senescing flag leaves, while regulation of one or several members of the other gene cluster controls PPO activity in these tissues. Our data, including immunoblotting and enzyme activity studies, further indicated that wheat PPOs are synthesized as inactive precursor (early kernel development) which are proteolytically processed and activated as the kernels mature. Activation of PPO precursor proteins was also demonstrated in vitro, in presence of purified trypsin. In these experiments, PPO activity increased during the first four hours and remained stable thereafter, indicating that the protein domains responsible for catalytic activity are quite stable. Research performed as a part of this dissertation has also demonstrated that wheat PPO activity is influenced by strong anionic detergents such as SDS and N-lauroylsarcosine. The corresponding experiments indicated that these detergents influenced both enzyme extraction and activity, at least in high-PPO wheat varieties. This work also has practical aspects, making PPO assays (as used by breeders for germplasm screening) more robust. In conclusion, as a result of this dissertation, wheat PPOs have emerged as a fascinating example of a plant multigene family with complex transcriptional and posttranscriptional regulation.Item Select Procyanidins induce gammadelta T cell activation and proliferation(Montana State University - Bozeman, College of Agriculture, 2008) Holderness, Jeffrey Scott; Chairperson, Graduate Committee: Mark Jutila.Many pharmaceutical drugs in use today were originally identified in plants from traditional medicine. However, there remain many plants in traditional medicine that produce confusing immune responses and are therefore unlikely candidates for pharmaceutical drugs. The effects of some of the traditional medicines that induce these confusing immune responses may now be explained by recent advances in the characterization of our immune system, namely in our understanding of the unique functions of the gammadelta T cell. These gammadelta T cell functions include tissue repair and homeostasis, cancer infiltration and clearance, pathogen detection and cytokine response, and antigen presentation. Although there are currently therapies being studied to increase the effector function of gammadelta T cells, these techniques are only active on a limited population of gammadelta T cells, the human Vdelta2 subset. Although these cells are potent effectors against pathogens and some cancers, Vdelta2 T cells demonstrate a restricted tissue distribution and limited effector function in other gammadelta T cell host defense responses. As such, we screened compound libraries and traditional medicines for agonists with activity encompassing alternative gammadelta T cell subsets. Tannins derived from select plant species are able to fulfill this role as demonstrated by the activation and expansion of gammadelta T cell subsets not responsive to current gammadelta T cell expansion therapies. The ability of tannins to expand these gammadelta T cell populations will potentially increase the therapeutic range of gammadelta T cells and may be used as treatments for wound healing as well as in the clearance of solid tumor cancers.