Master of Science / Department of Grain Science & Industry / Xiuzhi Susan Sun / Bio-based protein adhesives derived from renewable resources, especially soy protein are becoming more significant due to the concerns about environment and health related issues and the limit of petroleum recourses. However, the relatively poor water resistance of soy-based protein adhesives limits its wide applications. The goal of this study was to improve the water resistance performance of soy-based protein adhesives by chemical modification. The specific objectives are 1) to modify soy protein with calcium carbonate (CaCO[subscript]3) and sodium bisulfite (NaHSO[subscript]3) as the denaturing agents; 2) to investigate the effects of calcium carbonate (CaCO[subscript]3) concentrations, curing time and curing temperatures on adhesion performance of the modified soy-based protein adhesives. In this study, the co-effects of NaHSO[subscript]3 and CaCO[subscript]3 on adhesion properties of soy protein adhesives were investigated. NaHSO[subscript]3 was added to soy flour slurry at constant concentration 6g/L, while concentration of CaCO[subscript]3 was chosen in the range of 0 to 23g/L. Soy protein adhesives modified with 4g/L and 16g/L CaCO[subscript]3 were selected to characterize the adhesion performance on 3 ply yellow pine plywood using the Response Surface Method (RSM). The effects of curing temperature and curing time on the adhesion properties were also studied. The major findings are 1) 4g/L CaCO[subscript]3, 6g/L NaHSO[subscript]3 modified soy protein adhesives (MSPA) had better adhesion performance (both dry and wet) than 16g/L CaCO[subscript]3, 6g/L NaHSO[subscript]3 MSPA; 2) Higher temperature (170°C) resulted in higher wet shear adhesion strengths; 3) Longer hot press time had positive impact on wet adhesion shear strength; and 4) 4g/L CaCO[subscript]3, 6g/L NaHSO[subscript]3 MSPA showed better adhesion shear strength after 2 weeks storage than 16g/L CaCO[subscript]3, 6g/L NaHSO[subscript]3 MSPA. In general, 4g/L CaCO[subscript]3, 6g/L NaHSO[subscript]3 MSPA, under longer hot press time and higher temperature would lead to a better adhesion performance.

Doctor of Philosophy / Division of Biology / Carolyn J. Ferguson / The genus Lappula Moench is a diverse group of herbaceous plants in the large, cosmopolitan family Boraginaceae. Over sixty species are recognized in Eurasia; many are found in cold deserts, steppes, and semi-deserts of Central Asia. Fewer species were described from western North America, and compared with the Asian species they are poorly known. Various North American taxa have been placed into synonomy under Eurasian species, and complex patterns of variation have made species circumscription challenging. The goal of this dissertation was to explore phylogenetic relationships between North American and Eurasian species and to revise the taxonomy of the North American species. A molecular phylogenetic study was initiated in order to infer patterns of relationships among the North American species relative to Eurasian diversity. Samples were collected from throughout the western United States and from Siberia. Additional samples of Eurasian species were taken from herbarium specimens. Sequences were generated for three DNA regions (the ITS region of the nuclear ribosomal DNA, and chloroplast intergenic spacers trnS-trnG and rpl32-trnL) and phylogenies were generated using parsimony and Bayesian analyses. Results were in general agreement among all analyses. The genus Lappula was recovered as a monophyletic group, with the exception of the morphologically anomalous L. sessiliflora (Boiss.) Gürke, which was sister to the genus Rochelia Rchb. The native North American species of Lappula and L. redowskii (Hornem.) Greene formed a clade. Samples of L. squarrosa (Retz.) Dumort., a Eurasian steppe plant with a wide introduced range in North America, grouped with samples from Eurasia. Sampled species of Hackelia Opiz, a genus sometimes treated as part of Lappula, formed a clade separate from the Lappula species sampled here. Herbarium and field studies resulted in a revised taxonomy for the North American Lappula. Nomenclatural problems were resolved; nine native species and four varieties were recognized along with the introduced Eurasian species L. squarrosa.

Master of Science / Department of Biology / Theodore J. Morgan / The organismal response to temperature represents one of the most ubiquitous processes that occur in the natural world, and this response is critical for survival in most habitats. Increased attention should be focused on how organisms cope with temperature extremes, either through adaptation, plasticity, or a combination of both, as climate models predict increased variations in temperature accompanied by novel thermal extremes. Drosophila melanogaster is an excellent resource for answering questions pertaining to how organisms persist in environmental extremes because they originated in central tropical Africa and have since colonized nearly the entire globe, exposing them to many novel thermal stressors. In this work I elucidated regions of the genome contributing to phenotypic variation in cold tolerance and thermal plasticity. A quantitative trait locus (QTL) approach was used, which involved phenotyping roughly 400 recombinant inbred lines (RILs) of D. melanogaster from the Drosophila Synthetic Population Resource (DSPR). The DSPR captures genetic variation from around the globe, allowing for precision mapping of cold tolerance and thermal plasticity QTL, while simultaneously determining the frequency of the QTL alleles. Upon development at both 18°C and 25°C, RILS were measured for a common cold tolerance metric, chill-coma recovery time (CCR), and a plasticity value was derived as the change in CCR between environments. Analysis of variance revealed significant effects of sex, line (RIL), treatment (temperature), and line by treatment interaction (GxE). Mapped QTL for chill-coma recovery time at 18°C and 25°C spanned the same regions as several studies previously reported, validating the automated phenotyping method used and the mapping power of the DSPR. QTL between CCR at 18°C and 25°C overlapped significantly, and QTL for thermal plasticity shared the similar regions as QTL for CCR, but also exhibited two non-overlapping QTL on the left arm of the third chromosome. This study demonstrated the tremendous amount of variation present in cold tolerance phenotypes and identified candidate regions of the genome that contribute to thermal plasticity and require further investigation.

Master of Science / Department of Biology / Kathrin Schrick / Steryl glucosides (SGs) are a common conjugate of sterols found in the plasma membranes of most plants and fungi, but their cellular functions remain largely unknown. Glycosylation of the C3 hydroxyl group of the sterol nucleus is catalyzed by UDP-glucose:steryl glucosyltransferase 80 (UGT80) enzymes. Two genes encoding UGT80A2 and UGT80B1 are responsible for most SG production in Arabidopsis thaliana, while UGT80C1 presents a putative third enzyme. In Arabidopsis, seed imbibition signals the epidermal seed coat cells to secrete an encapsulating mucilage that consists primarily of hydrated polysaccharides. Cellulose has been identified in the inner layer of the mucilage, providing a convenient model to study cellulose synthesis since seed mucilage is dispensable for viability and pectin and cellulose staining dyes are readily available. A reverse genetics and biochemical approach was used to characterize the role of UGT80 enzymes and their impact on cellulose synthesis in seed mucilage. ugt80B1 mucilage was found to have elongated cellulosic rays, but no defects in pectin synthesis. A double mutant of ugt80B1 and mum3-1, a mutant allele of CELLULOSE SYNTHASE 5 (CESA5), displays a novel phenotype with irregular cellulose patterning and extreme shedding of the pectinaceous layer surrounding the seed coat. The observed mucilage defects may be indicative of disrupted cellulose synthesis and a mechanistic relationship between SGs and the cellulose synthase machinery. UGT80A2 and B1 demonstrate glycosylation activity with a multitude of plant sterols. The two enzymes do display some substrate specificity, however, with UGT80A2 producing the large majority of sitosterol and stigmasterol glucoside compared to B1. UGT80C1 shows little or no sterol glucosyltransferase activity in vitro or in vivo and likely has evolved a different function from the two other genes. GFP:UGT80C1 expressed either from the constitutive 35S or from its native promoter was localized to lipid droplets and possibly chloroplasts as well, creating a new perspective on the role of the protein in plant lipid metabolism. This study extends the currently limited view of SGs as ubiquitous components of the plasma membrane to active regulators of cellulose synthesis in seeds. Evidence presented here changes the perceived role of the plant conserved protein, UGT80C1, from a putative steryl glucosyltransferase enzyme to having a function in intracellular lipid droplets.

Master of Science / Department of Anatomy and Physiology / Lei Wang / Jishu Shi / Interleukin-1 beta (IL-1β) is an important mediator of inflammatory response, and the elevated expression of IL-1β is correlated with tumor growth and metastasis. Epithelial-mesenchymal transition (EMT) is a reversible transition between epithelial phenotype and mesenchymal phenotype. Usually, EMT can be identified by its unique morphology change and expression of EMT markers. In our study, we have found after treated HCT-116, a colon cancer cell line, and human primary colon cancer cells with IL-1β, cells began to display mesenchymal phenotype with highly down-regulated E-cadherin expression and up-regulated ZEB factors expression. For colon cancer cells, sphere formation assay in serum free medium (SFM) with the presence of growth factors is used to identify cancer stem cell population. We have shown that IL-1β can induce colon cancer stem cell proliferation and express stem cell markers (Bmi1, Nanog, and Nestin). In addition, besides the stem cell markers, we also found ZEB factors were highly up-regulated in spheroid cells as well. We silenced Zeb1 expression and investigated the effect of IL-1β on shZeb1 HCT-116 cells. The results indicated Zeb1 knockdown not only inhibited IL-1β-induced EMT but also reduced proliferation of spheroid cells and inhibited Bmi1 expression. Therefore, ZEB factors must play an important role in both EMT process and cancer stem cell development. From our data, we conclude that IL-1β promotes epithelial-mesenchymal transition and a stem cell phenotype in colon cancer via ZEB factors.

Doctor of Philosophy / Department of Biology / Michael Herman / Soil nematode communities are important components of the micro fauna in grassland ecosystems and their interaction with soil microbes affects important ecological processes such as decomposition and nutrient recycling. To study genetic mechanisms underlying ecologically important traits involved in the response of nematode communities to soil microbes, we employed genomic tools available for the model nematode, Caenorhabditis elegans. Previous work identified 204 C. elegans genes that were differentially expressed in response to growth on four different bacteria: Bacillus megaterium, Pseudomonas sp., Micrococcus luteus and Escherichia coli. For many of the genes the degree of differential gene expression between two bacterial environments predicted the magnitude of the effect of the loss of gene function on life-history traits in those environments. Mutations can have differential effects on fitness in variable environments, which can influence their maintenance in a population. Our fitness assays revealed that bacterial environments had varying magnitude of stress, defined as an environment in which the wild-type has a relatively low fitness. We performed fitness assays as part of a comprehensive analysis of life history traits on thirty five strains that contained mutations in genes involved in the C. elegans response to E. coli, B. megaterium, Pseudomonas sp. We found that many of the mutations had conditionally beneficial effects and led to increased fitness when nematodes bearing them were exposed to stressful bacteria. We compared the relative fitness of strains bearing these mutations across bacterial environments and found that the deleterious effects of many mutations were alleviated in the presence of stressful bacteria. Although transcriptional profiling studies can identify genes that are differentially regulated in response to environmental stimuli, how the expressed genes provide functional specificity to a particular environment remains largely unknown. We focused on defense and metabolism genes involved in C. elegans-bacterial interactions and measured the survivorship of loss-of-function mutants in these genes exposed to different bacteria. We found that genes had both bacteria-specific and bacteria-shared responses. We then analyzed double mutant strains and found bacteria-specific genetic interaction effects. Plasticity in gene interactions and their environment-specific modulation have important implications for host phenotypic differentiation and adaptation to changing environments.

Doctor of Philosophy / Biology / Michael A. Herman / Nematodes play an important role in various habitats where numerous factors serve to shape their communities. One such factor is the potentially pathogenic nematode-prey interaction. This project is focused on the elucidation of the genes that the bacterivorous nematode Caenorhabditis elegans employs to respond to the emerging nosocomial bacterial pathogen Stenotrophomonas maltophilia. A virulent S. maltophilia strain JCMS requires the action of several C. elegans conserved innate immune pathways that serve to protect the nematode from other pathogenic bacteria. However, insulin-like DAF-2/16 signaling pathway mutants that are typically pathogen resistant are susceptible to JCMS, and several DAF-2/16 regulated genes are not significantly differentially expressed between JCMS and avirulent E. coli OP50. We have determined the complete set of mRNA transcripts under different bacterial treatments to identify genes that might explain this JCMS specific DAF-2/16 pathway evasion. The identified set included 438 differentially expressed transcripts among pairwise comparisons of wild-type nematodes fed OP50, JCMS or avirulent S. maltophilia K279a. Candidate genes were nominated from this list of differentially expressed genes using a probabilistic functional connection model. Six of seven genes that were highly connected within a gene network generated from this model showed a significant effect on nematode survival by mutation. Of these genes, C48B4.1, mpk-2, cpr-4, clec-67 and lys-6 are needed for combating JCMS, while dod-22 was solely involved in K279a response. Only dod-22 had a documented role in innate immunity, which merits our approach in the identification of gene candidates. To a lesser extent, we have also focused on the identification of virulence factors and the mode of action employed by S. maltophilia. JCMS virulence requires rpfF, xps and involves living bacteria that accumulate in the intestinal lumen. Additionally, the bacterial secretion encoding genes cs, p773, p1176, pi1y1 and xdi are involved in JCMS evasion of daf-2. In summary, we have discovered a novel host-pathogen interaction between C. elegans and S. maltophilia JCMS, revealed genes that are involved in each partner of the interaction, and established a new animal model for the study of S. maltophilia mode of action.

Master of Science / Department of Biology / Stella Y. Lee / CLN5 is one of several proteins that when mutated result in the lysosomal storage disorder (LSD) Neuronal Ceroid Lipofuscinosis (NCL). CLN5 is a soluble lysosomal protein that has no known function at this time. Previously we showed that eight asparagine residues in CLN5 are N-glycosylated, and that this modification is important for the protein’s transport and function. Now, we have identified a link between the activation of autophagy and CLN5 deficiency. The autophagy-lysosomal protein degradation system is one of the major pathways the cell uses to degrade intracellular material and recycle cellular building blocks. It was recently shown that other CLN proteins affect the relative level of autophagy, indicating a potential link between the autophagy pathway and the NCLs. By knocking down endogenous CLN5 in HeLa we showed that, upon stress induction, cells responded with higher levels of autophagy activation. Consistent with these knockdown experiments, there is a higher level of the autophagy marker protein, LC3-II, in CLN5 patient cells that are naturally deficient for the CLN5 protein. Pharmaceutical induction of autophagy through different means also showed higher LC3-II levels compared to control, though patterns differed in the type of autophagy induced. In summary, we discovered that the autophagy pathway is altered in CLN5 deficient cells, indicating a potential role for CLN5 in autophagy. Further analyses of the autophagy pathway will shed light on where CLN5 is acting and the mechanism by which defective CLN5 causes NCL.

Master of Science / Biology / Keith B. Gido / Negative interactions with nonnative fish are often cited as a leading cause of declining native fish populations, but quantifying these interactions is difficult. Movement ecology and consumptive demand estimates of nonnative fish predators is needed to better understand potential impacts these organisms are having on native species. The objective of this thesis were to estimate the consumptive demand of Flathead Catfish Pylodictis olivaris on native fishes across an elevational gradient, and characterize the movement at hourly, daily, and seasonal scales of this introduced predator. This research was conducted in the upper Gila River basin of southwestern New Mexico. Bioenergetics modeling was used to estimate consumptive demand; model results were coupled with measured densities and size structure of Flathead Catfish populations, and water temperatures, to predict its predatory threat. Potential consumption was highest at lower elevation sites because of higher water temperatures, but actual consumption was highest at mid-elevation sites because of the prevalence of large-bodied individuals. Potential annual consumptive demand of Flathead Catfish on native fish across our nine sampling sites ranged from 0.0 to 3.1 g/m²/yr, which exceeded native fish productivity at one site. To characterize the movement of Flathead Catfish, we used radio telemetry and tracked individuals from May 2014 to June 2015. Movement behaviors varied among individuals with a majority moving <150 m from capture location and some more mobile, moving substantial distances (692-42,840 m). During the course of the study, activity was greatest in summer and fall, and individuals moving substantial distances moved downstream to warmer river reaches before the winter. Nightly movements only involved short distances (5 m) and no fish exceeded a single movement >80 m. Daily activity was greatest during evening but late afternoon activity was observed in summer and fall. Results from this study identify areas within the upper Gila River where introduced Flathead Catfish consumption is likely to negatively impact native fish populations and managers can use this information to understand potential overlap with native species, target future removal efforts in areas where these fish are concentrated, and avoid stocking native fishes in reaches where Flathead Catfish tend to aggregate.

Doctor of Philosophy / Department of Biology / Brett K. Sandercock / Samantha Wisely / Anthropogenic activities and climate change have dramatically altered landscapes worldwide. The ability of species to cope and adapt to ongoing changes is likely a function of their behavior, movements, and sensitivity to fragmentation. Greater Prairie-Chickens (GPC) are a lek mating grouse native to the Great Plains Landscape Conservation Cooperative (GPLCC), for which inbreeding depression and anthropogenic avoidance are a concern. The goals of my dissertation were to: 1) identify genetic correlates of male performance which may influence population viability under current land use practices, 2) identify GPC habitat characteristics and delineate areas of critical GPC habitat necessary for GPC conservation, and 3) identify the relative importance of distance and habitat quality for maintaining genetic connectivity among spatially structured populations. First, I found male reproductive success and survival to be positively associated with genetic diversity. Using multistate modeling in Program Mark, male survival across the observed range of variation in number of alleles (15-22) increased more than fourfold from 0.17 to 0.77. Second, I found 35-40% of Kansas, and 1.5 % (11,000 Km squared) of the GPLCC, were considered high-quality lek habitats. Top performing logistic models predicting lek presence (wi=0.95) included strong effects of grassland cover and avoidance of anthropogenic disturbance. When this model was applied to putative future landscapes based on climate change and current land use trends over a 70-year period, I found a 27-40% reduction in habitat area and a 137 Km southeast shift in habitat distribution. Under equilibrium conditions we expect isolation by distance (IBD) to explain the distribution of genetic diversity. However, if the landscape restricts dispersal, then we might observe isolation by resistance (IBR). I used model selection procedures to choose among competing IBR or IBD models to explain the distribution of genetic diversity among GPC populations across Kansas and the GPLCC. IBD was never supported (R-square<0.02, P>0.09). The best models for Kansas (R2=0.69, P<0.02) and for the GPLCC (R-square=0.46, P<0.02) indicated that human-mediated landscape changes have influenced landscape permeability for dispersal. The integration of behavioral, landscape, and genetic data provided new insights on prairie-chicken ecology, and is a powerful approach for developing conservation strategies for sensitive species.