Doctor of Philosophy / Department of Biology / Samantha Wisely / Emerging infectious diseases play an increasingly critical role in many biological fields, including conservation biology and public health. Many emerging diseases originate in wildlife, most are caused by viruses, and often emergence is due to adaptation to and amplification in a new host, frequently in areas where ecological transformation is occurring. These emergence patterns suggest that the complex interactions among host, virus, and landscape drive disease emergence. Terrestrial rabies in striped skunks (Mephitis mephitis) in the central Great Plains is an excellent model system to investigate the interactions among the components of disease emergence: host ecology, pathogen properties, and landscape features. Striped skunks are not only numerous in the central Great Plains, they are also the reservoir for two genetically distinct rabies strains that co-occur in the region. Additionally, the landscape in the central Great Plains has undergone significant land use change over the last 70 years through increased urbanization and industrial agriculture practices. I used a combination of molecular and spatial techniques to investigate the interactions among host, pathogen, and landscape. Molecular epidemiology results indicated that rabies strains in the central Great Plains exhibit different epidemiological properties, while population genetic analyses indicated that striped skunks in the region are highly admixed and comprise a single population. Spatial analysis revealed that landscape features such as rivers are not a barrier to striped skunk dispersal, but differentially influence the movement of the two rabies strains. Because striped skunks are reservoirs for many diseases other than rabies and are ubiquitous throughout North America, I also examined the historical movements and distribution of striped skunks in North America using a phylogeographic approach. Results revealed that a combination of multiple Pleistocene dispersal events and Holocene admixture are responsible for the contemporary population structure of striped skunks in North America, and allowed me to place my regional-scale striped skunk rabies study into a larger biogeographic context. My results support the use of a holistic approach for studying emerging infectious diseases that includes studies of viral characteristics, host ecology and biogeography, and spatial features.

Master of Science / Department of Geology / Sambhudas Chaudhuri / Rare earth element (REE) concentrations were measured in a source (reference) clay Ca-smectite standard and in the roots, stems, and leaves of a species of poplar plant (Populus eugenei). The poplar plant was grown in the clay standard under controlled laboratory conditions during a period of about three months. REEs were shown to fractionate in the clay mineral and plant materials with greater fractionation observed in plant materials. The REE data provide insight into the process of weathering of clay minerals such as a Ca-bearing smectite and provide insight into the degradation of and the composition of clay minerals in the plant environment. The degradation process is not followed by significant interlayer ion exchange effect on remaining clay minerals in the root environment. REEs were found to be transported into complex forms, potentially as REE-carboxylic anion pair complexes. The plant materials in this study were in general heavy REE (HREE) enriched relative to the source clay minerals due to the complexation effect. The REE anomalies observed in this study, in addition to the Ce and Eu anomalies, may be explained by the selective uptake by the plant by an enzyme effect rather than due to the influence of oxidation-reduction. The enzyme influence was more evident in the REE distribution when compared among the plant organs. These REE characteristics described for the plants may eventually be incorporated with data from numerous other studies and also used as a guide in the assessment of the contribution of plant materials to dissolved REE content in surface water and groundwater.

Master of Science / Department of Entomology / Srinivas Kambhampati / Termites (Isoptera) are separated into morphologically and behaviorally specialized castes of sterile workers and soldiers, and the reproductive alates. Previous research on eusocial insects has indicated that caste differentiation has a genetic basis. Although much has been studied about the genetic basis of caste differentiation and behavior in the honey bee, Apis mellifera, termites remain comparatively understudied. Therefore, my objective was to compare the gene expression patterns of different castes of the termite Reticulitermes flavipes based on EST analyses and a microarray. Soldier, worker, and alate caste and two larval life stage cDNA libraries were constructed, and ~15,000 randomly chosen clones were sequenced to compile an EST database. Putative gene functions were assigned based on a BLASTX Swissprot search. Categorical expression patterns for each library were compared using the in silico methods of BLAST2GO and r-statistics. I chose 2,240 unique-ESTs based on their putative function and sequence quality, which I used to fabricate a Combimatrix microarray. I used the microarray to compare expression levels between workers and soldiers from Kansas and Florida populations. Seventy to ninety percent of the sequences from the ESTs of each caste and life stages had no significant similarity to those in existing databases. All libraries contained sequences with putative reproductive functions, which was unexpected in the non-reproductive soldier and worker castes. Sequences of interest that showed a putative bias among castes include a viral protein in soldiers and a possible chemosensory protein in alates, which may be involved in termite reproductive functionality or communication. The microarray showed increased expression in the soldier caste of a sequence that matched tropomyosin and an increased expression level of a sequence that matched a PDZ-domain containing protein in some worker samples. This study leads to several candidate genes of potential caste specific function, which can be further tested using functional analysis and between the different castes and life stages of R. flavipes. These genes include the sequences similar to pebIII and RGS-GAIP. I have also expanded upon the available sequences for this termite and utilized the r-statistic in silico method for the first time to putatively compare gene expression in the different castes of a eusocial insect. The in silico analysis allowed us to identify several genes which may show biased expression patterns in the different cDNA libraries and which may reveal caste-specific expression controlling important functions after further analysis. These candidates include: an alate-biased gene, which had a predicted function of neurotransmitter secretion and cholesterol absorption as well as a late larval-biased gene which was predicted to be involved in protein biosynthesis and ligase activity.

Doctor of Philosophy / Department of Biology / Mark Ungerer / Elucidating the molecular basis of adaptive phenotypic variation represents a central aim in evolutionary biology. Using the model plant species Arabidopsis thaliana, I studied the intra-specific variation in freezing tolerance among natural accessions across its native range. Considerable variation in freezing tolerance among 71 selected accessions was observed both with and without a prior cold acclimation treatment, suggesting that both differences in cold-acclimation capacity and in intrinsic physiology contribute to this variation. A highly significant positive relationship was observed between freezing tolerance and latitude of origin of these accessions. This clinal pattern of variation is found to be attributable, at least in part, to relaxed purifying selection on CBF/DREB1 genes in the species’ southern range. These CBF/DREB1 genes encode transcriptional activators that play a critical role in the ability of A. thaliana plants to undergo cold acclimation and thereby achieve maximum freezing tolerance. Relative to accessions from northern regions, accessions of A. thaliana from the southern part of their geographic range exhibit significantly higher levels of nonsynonymous polymorphisms in coding regions of CBF/DREB1 genes. Relaxed selection on the CBF/DREB1s in southern accessions also has resulted in mutations in regulatory regions that lead to abrogated expression. These mutations in coding and regulatory regions compromise the function of CBF/DREB1 transcriptional activators during the cold acclimation process, as determined by reductions in rates of induction and maximum levels of expression in the downstream genes they regulate. These mutations could be selective neutral or beneficial in southern accessions depending on whether there is an allocation cost associated with cold acclimation. The fitness benefit and possible allocation cost of cold acclimation was examined in freezing and freezing-free environments using natural accessions exhibiting contrasting abilities of cold acclimation as well as transgenic CBF gene over-expression or knockdown/knockout lines. The extent to which cold acclimation benefits the plant in presence of freezing temperature is revealed, but a cost of cold acclimation wasn’t detected in the absence of freezing temperature under our experimental design, which suggests that these mutations in CBF genes in southern accessions might be neutral to natural selection.

Doctor of Philosophy / Department of Biology / Brett K. Sandercock / Sexual selection via female choice and male-male aggression leads to elaboration of male traits. If male traits correlated with reproductive success are honest signals of male quality, survival costs may be associated with the expression of those traits. Testosterone (hereafter ‘T’) may enhance male breeding success, but T can also reduce immunocompetence and survival. Socially monogamous male birds with higher circulating T experience reproductive advantages, but the role of T in lek mating systems is largely unknown. To address these issues, I individually marked and conducted focal behavioral observations of greater prairie-chickens (Tympanuchus cupido) at five lek sites over a 5-year period. Females were fitted with radio-telemetry to monitor nesting success and survival. I examined the relationship between male traits and mating success using multinomial discrete choice models, a statistical method not previously applied to studies of sexual selection. Male mating success was highly skewed at greater prairie-chicken leks with 18.5% of males obtaining 87.2% of all successful copulations (n = 108 males; 85 copulations). Mating success was influenced most by male behavior, followed by several morphological attributes. The role of T was quantified using blood samples and by experimentally implanting a subset of males with T. T did not consistently affect mating success. Non-territorial males had lower T levels than territorial males. Among territory holders, T was unexpectedly negatively correlated with mating success. However, the odds of receiving a copulation were 4.3 times (0.42 to 45.3) greater for T-implanted males than males with sham implants. Future work should explore the interactions among the immune system, parasite load, and mating success of prairie-chickens. Annual survival of male prairie-chickens was not related to mating success, behavior, age or T level, suggesting there is no cost of increased male mating success. Like males, reproductive success of females was also highly skewed because < 10% of nests successfully hatched young. Comparisons of seasonal and annual survival rates indicate that females experience increased mortality during the breeding season relative to the nonbreeding season. Synthesis of field estimates of demographic parameters indicates prairie-chicken populations will decline without changes in rangeland management to reduce predator numbers or provide more nesting cover.

Doctor of Philosophy / Department of Biology / Stephen K. Chapes / Silvia Mora Fayos / My work focused on understanding adipocyte function and regulation because of the importance to diabetes. In addition to being a fat storage depot, adipose tissue is an endocrine tissue. Adiponectin and leptin are two adipokines that control insulin sensitivity and energy balance. In spite of their importance, there are still questions about their secretion. I hypothesized that leptin and adiponectin follow different secretory routes. I found adiponectin localized in Golgi and the trans Golgi Network, while leptin mostly localized in ER during basal metabolisms. Common requirements for their secretion were the presence of class III Arf proteins and an intact Golgi apparatus, since BFA treatment inhibited secretion of both adiponectin and leptin. I found that trafficking of adiponectin is dependent on GGA1 coated vesicles. Endosomal inactivation significantly reduced adiponectin, but not leptin, secretion in both 3T3L1 and isolated rat adipocytes. Also, adiponectin, but not leptin, secretion was reduced in cells expressing non- functional form of Rab11 and Rab5 proteins. However, secretion of leptin, but not adiponectin was inhibited in cells expressing mutants of Protein Kinase D1. These results suggest that leptin and adiponectin secretion involve distinct intracellular compartments and pathways. Insulin resistance is associated with macrophage infiltration into adipose tissue and elevated levels of IL-6, TNF-alpha and IL-1beta Therefore, the second part of my dissertation tested the hypothesis that the interaction of macrophages and adipocytes causes insulin resistance. To test this hypothesis, I co-cultured macrophages and adipocytes. I found that mouse elicited peritoneal macrophages significantly decreased insulin-stimulated GLUT4 translocation to the plasma membrane in a contact-independent manner. IL-6 was the most inhibitory cytokine in reducing GLUT4 translocation, GLUT4 expression, Akt phsphorylation and reducing adipocyte differentiation compared to TNF-alpha and IL-1beta. These data suggest that IL-6 is the most effective cytokine secreted by macrophages involved in insulin resistance. Lastly, I tested the impact of adipocytes on macrophage differentiation in vitro and in vivo. I found that C2D macrophages isolated from the peritoneal cavity had increased IL-6 transcript levels after co-culture with 3T3L1 adipocytes in vitro. After i.p. injection, C2D macrophages isolated from WAT increased expression of mature macrophage surface markers and transcript levels of proinflammatory cytokines compared to C2D cells in vitro. However, macrophages isolated from BAT expressed low levels of cytokines and macrophage surface markers.

Master of Science / Department of Biology / Peter P. Wong / Abstract Water purification methods that remove pathogens and harmful or distasting molecules make water potable. Recently, silver loaded ion-exchange resins have demonstrated a strong role in removing microbes. The goal is to make an effective silver ion-based water purifier that is portable, environmentally stable, and cost efficient. The project was conducted as a collaborative effort with Safewater A/S, an up and coming entrepreneurial business located in Denmark that is interested in developing novel water purifiers for developing nations, adventurers and military personnel. Purolite, a prominent business in ion-exchange resins located in Whales, designed and provided Safewater A/S and our research team with experimental resins for water purification, which will be discussed in the body of this thesis. The data reveals critical issues that may render this tool unavailable for commercial production in some countries due to the mode of action for killing the bacteria and the amount of silver leaching. Tests were conducted using Escherichia coli K12 and Enterococcus faecalis OG1SSp as model fecal organisms using different silver ion-exchange resins. Surveillance of leached silver ions, pH changes, and total dissolved solids (TDS) were also monitored to find correlations with capacity (liters of purified water produced) and effectiveness of microbicidal action. Overall, one resin was found to contain properties consistent with the stated objectives; however its use in some countries as a water purifier for human consumption will be nullified due to extensive silver leaching. Although this resin could be used in the United States of America since it passes the Environmental Protection Agency (EPA) standards, Safewater A/S is interested in further developing it for countries with stricter regulatory constraints before mass production. The goal of the present thesis report is to address the stated objectives in the development of a water purifier.

Master of Agribusiness / Department of Agricultural Economics / Subramanyam Bhadriraju / Environmental concerns are growing as new information is being discovered as to what is harmful to the environment. The desire to help the environment along with improving fumigations is a big concern and effects many people. Ongoing research to improve fumigations without harming the environment has shown great promise in advancing technology and lowering the cost in protecting commodities consumed by the consumer. Methyl Bromide (MB) has been the major fumigant used to control stored-product insects for many commodities for many years. There has been a lot of concern surrounding MB because of health and environmental drawbacks. These concerns have caused MB to be reduced by 2005. With the total phase out of MB becoming critical, there is much anticipation as what will be the alternative(s). The research presented in this thesis describes two different and very effective methods of controlling stored product insects. Although there are many other methods of fumigating this thesis analyzes two forms; Sulfuryl Fluoride and Heat. The first presented alternative in this paper will be heat treatments. It has the attraction that chemical forms of treatments do not have by having pesticide-free products. The total cost of heat fumigation depends on the complexity of the lay-out/structure and the cost heaters and electricity. This cost can range anywhere from $15,000-25,000. The second alternative that will be discussed is sulfuryl fluoride. This fumigant has many positive aspects that counter act the negative aspects that have been a concern with methyl bromide treatments. These positive aspects include environmentally safe and fast off-gassing. Sulfuryl fluoride is also very efficacious with stored product insects. The labor costs per job, ProFume shows a cost of about $216.00 less than that of a methyl bromide treatment.

Master of Science / Department of Human Nutrition / Denis M. Medeiros / Copper deficiency has been linked with many health problems, among them cardiac hypertrophy. Because of its potential for causing oxidative damage, copper within the cell must be bound to chaperone proteins. In this thesis, we examined the role of dietary copper deficiency in the regulation of select copper chaperone proteins in cardiac tissue of rats. Sixteen weanling male Long-Evans rats were randomized into treatment groups, one group receiving a copper deficient diet (< 1 mg Cu/kg diet) and one group receiving a diet containing adequate copper (6 mg Cu/kg diet) for 5 weeks. Rats were sacrificed and a small blood sample was removed to determine hematocrit. Also, heart and liver tissues were removed for subsequent analysis. Rats fed the copper deficient diet had lower body weights but greater heart weights and heart:body weight. Hematocrit levels and liver copper concentrations were markedly decreased in copper deficient rats. These variables indicated that the copper deficient diet did in fact induce a copper deficiency in these animals. Non-myofibrillar proteins from the hearts were removed and separated by SDS-PAGE. Western Blotting was used to determine the concentrations of CTR1, CCS, Cox17, SCO1, Cox1 and Cox4. No changes were observed in the concentrations of CTR1 and Cox17. CCS and SCO1 were up-regulated as a result of copper deficiency, while Cox1 and Cox4 were both down-regulated. However, use of another antibody against Cox subunits suggested that only the nuclear encoded subunits including subunit IV were decreased, but not subunits I and II. These data provide new insight into the cardiac hypertrophy observed in copper deficiency, which suggests that select chaperone proteins may be up-regulated by a dietary copper deficiency.

Doctor of Philosophy / Agronomy / Allan K. Fritz / Bikram S. Gill / Gary M. Paulsen / Post-anthesis high temperature stress in wheat (Triticum aestivum L.) is a major cause of yield reduction. This process results in the loss of viable leaf area and a decrease in green leaf duration ultimately causing a yield loss. The objectives of this study were to (i) phenotype a recombinant inbred line population for heat tolerance traits, (ii) understand the genetic basis of heat tolerance by mapping quantitative trait loci (QTL) linked to yield-related traits under high temperature, (iii) model stay-green under high temperature stress and map the QTL linked to stay-green parameters, and (iv) validate the markers linked to QTL under field conditions. A filial6:7 (F6:7) recombinant inbred line (RIL) population was developed by crossing Ventnor, a heat-tolerant white winter wheat with Karl 92, a relatively heat susceptible hard red winter wheat. From 10 DAA to maturity, the treatments of optimum temperature or high temperature stress (30/25°C) were imposed on the RILs. The traits measured included grain filling duration (GFD), kernels per spike, thousand kernel weight (TKW), and grain filling rate (GFR). The stay-green traits calculated were: i) time between 75% and 25% green, ii) maximum rate of senescence, iii) time to maximum rate of senescence, and v) percent green at maximum senescence. Genetic characterization was performed using microsatellite (SSR), amplified fragment length polymorphism (AFLP) and a sequence tag site (STS) markers. GFD was positively correlated with TKW and negatively with GFR and maximum rate of senescence. Principle component analysis (PCA) showed kernels per spike, maximum rate of senescence, and TKW accounted for 98% of total variability among the genotypes for heat tolerance. The most significant QTL for yield traits co-localized with marker Xgwm296 for TKW, Xgwm356 for kernels per spike, and Xksum61 for GFR. The QTL for stay-green traits co-localized with markers P41/M62-107 on Chromosome 2A, Xbarc136 on Chromosome 2D, P58/MC84-146 on Chromosome 3B, P58/M77-343 on Chromosome 6A, and. P58/MC84-406 on Chromosome 6B. These results indicate that increased green leaf area duration has a positive effect on the grain yield under high temperature. Once the kernels per spike are established, GFD and TKW can be used as selection criteria for post-anthesis heat-tolerance.