Glycogen synthase kinase-3 (GSK-3) alpha and beta are central regulators of key developmental pathways, including Wnt, Hedgehog and Notch, which control stem cell activities and cellular differentiation. Both forms of GSK-3 are also regulated by receptor tyrosine kinases via the PI3K/Akt growth-promoting pathway and are involved in feedback mechanisms that maintain signaling homeostasis. These signaling systems have critical functions in mammary gland development and aberrations in them have been implicated in breast cancer. However, the role of GSK-3 in breast oncogenesis is unclear. Here, I provide evidence that maintenance of appropriate GSK-3 activity is necessary for normal acinar morphogenesis of mammary cells in the ductal/alveolar epithelium. Inadequate GSK-3 levels result in enlarged structures that often lack hollow lumens and resemble early premalignant breast cancer lesions. A potential contribution for PI3K signaling to this phenotype was identified as a PI3K inhibitor partially reversed the observed morphological defects. Mammary epithelial cell (MEC) identity also requires modulation of signals through the Wnt/beta-catenin pathway. GSK-3-depleted mammary glands not only transdifferentiate into squamous epithelium but also develop highly proliferative adenosquamous carcinomas characterized by activated beta-catenin. Furthermore, beta-catenin appears to be required for both cell fate changes and tumorigenesis in the absence of GSK-3 function. Mammary tissues engineered to enable conditional deletion of beta-catenin in a GSK-3-null background also assumed an epidermoid cell fate with ensuing tumor formation albeit with a significantly longer latency and different histopathology. The metaplastic nature of tumors observed is similar to a rare yet aggressive form of human breast disease, metaplastic breast carcinomas (MBCs). Mammospheres (MS) generated from GSK-3 depleted MECs exhibited a less compact morphology compared to those with activated beta-catenin, which also exhibited an expansion of the CD24:CD49f double positive progenitor population and enhanced self-renewal. No MS were formed by MECs lacking GSK-3 and beta-catenin. ErbB2/Neu-mediated mammary tumor progression has been associated with Wnt/beta-catenin pathway activation. Loss of beta-catenin delayed tumor onset in a constitutively active ErbB2 mouse model but did not alter either the luminal characteristics of the ensuing tumors nor their metastatic potential. Collectively these studies indicate GSK-3 plays important roles in mammary gland function thereby suppressing mammary tumor formation.

Master of Science / Department of Entomology / David C. Margolies / Yoonseong Park / Phytoseiulus persimilis (Acari: Phytoseiidae) is a specialist predator on tetranychid mites, especially on the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). The foraging environment of the predatory mites consists of prey colonies distributed in patches within and among plants. Quantitative genetic studies have shown genetic variation in, and phenotypic correlations among, several foraging behaviors within populations of the predatory mite, P. persimilis. The correlations between patch location, patch residence, consumption and oviposition imply possible fitness trade-offs. We used molecular techniques to investigate genetic variation underlying the foraging behaviors. However, these genetic studies require a sufficiently large amount of DNA which was a limiting factor in our studies. Therefore, we developed a method for obtaining DNA from a single mite by using a chelex extraction followed by whole genome amplification. Whole genome amplification from a single mite provided us with a large quantity of high-quality DNA. We obtained more than a ten thousand-fold amplified DNA from a single mite using 0.01ng as template DNA. Sequence polymorphisms of P. persimilis were analyzed for nuclear DNA Inter Transcribed Spacers (ITS1 & ITS2) and for a mitochondrial 12S rRNA. The sequence comparisons among individuals identified a number of polymorphisms in the 12S sequences. The foraging gene (for) associated with rover-sitter behavioral strategies of Drosophila is known to have role in feeding behaviors of honeybee and other arthropods. We surmised that the same or a similar gene may be present in P. persimilis. Among the foraging behavior(s) exhibited by this predatory mite, we were particularly interested in resource/prey-dependent dispersal behavior. We isolated a partial sequence that is presumed to be the orthologue of the foraging (for) gene. We named the putative foraging gene as Ppfor (for Phytoseiulus persimilis foraging gene). We used a fragment of Ppfor gene as a molecular marker between populations and among individuals and, further, to help understand behavioral phenotypes.

Doctor of Philosophy / Department of Biology / Susan J. Brown / All arthropods share a segmented body plan. Detailed studies on segmentation mechanisms in the long-germ insect Drosophila melanogaster identified a segmentation hierarchy composed of maternal, gap, pair-rule, and segment polarity genes. In this hierarchy, pair-rule genes play an important role to translate gradients of regional information from maternal and gap genes into segmental expression of segment polarity genes. However, our understanding of the role of pair-rule genes in other short-germ insects and basally branching arthropods is still limited. To gain insights into the role of pair-rule genes in short-germ segmentation, I analyzed genetic interactions as well as expression patterns and functions of homologs of Drosophila pair-rule genes in the short-germ insect Tribolium castaneum. Interestingly, despite the pair-rule like expression patterns of Tribolium homologs of almost all eight canonical Drosophila pair-rule genes, only five have a segmentation function. Knock-down of primary pair-rule genes caused asegmental and truncated phenotypes while knock-down of secondary pair-rule genes caused typical pair-rule phenotypes. Epistatic analysis between the genes revealed that primary pair-rule genes form a gene circuit to prepattern a two-segmental unit, and secondary pair-rule genes are downstream targets of the gene circuit. The typical pair-rule phenotypes observed in secondary pair-rule gene RNAi embryos led to a detailed comparative analysis of the role of paired (prd) and sloppy-paired (slp) between Drosophila and Tribolium. This study revealed that prd is functionally conserved while the functional parasegmental register for Tribolium slp is opposite that of Drosophila slp. The fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning. Despite this flexibility in pair-rule patterning between Drosophila and Tribolium, segmental expression of engrailed (en) and wingless (wg) at parasegmental boundaries is conserved in both insects. Analysis of double and triple RNAi for pair-rule genes in Tribolium revealed that the primary pair-rule genes even-skipped and runt are redeployed to directly regulate en and wg with prd or slp at parasegmental boundaries. This redeployment of primary pair-rule genes seem to compensate for the apparently fewer number of functional secondary pair-rule genes in Tribolium segmentation.

Doctor of Philosophy / Department of Plant Pathology / Bikram S. Gill / The wild relatives of crop plants are sources of useful genes, but such genes when transferred to agricultural crops are often associated with deleterious traits. Because most of the recombination and the disease resistance genes are localized towards the ends of wheat chromosomes, cryptic terminal alien segments, carrying rust resistance genes, were transferred from Aegilops geniculata (UgMg) and Ae. triuncialis (UtCt) into common wheat without the usual linkage drag. The alien segment with the leaf rust and stripe rust resistance genes Lr57 and Yr40 in translocation T5DL•5DS-5MgS(0.95) was found to be less than 3.3 cM in genetic length and spans less than four overlapping BAC/PAC clones of the syntenic rice chromosome arm 12L. The alien segment with leaf rust resistance gene Lr58, transferred from Ae. triuncialis, was found to be less than 5% of the chromosome arm 2BL of wheat in T2BS•2BL-2tL(0.95), further suggesting that it is feasible to transfer small alien segments with disease resistance genes. Resistance genes Lr57, Yr40 and Lr58 were transferred to Kansas hard red winter wheat cultivars by backcrossing and marker assisted selection. Tillering, a key component of grain yield, and seed color which influences seed dormancy and pre-harvest sprouting in wheat, are agronomically important domestication traits in wheat. A tiller inhibition mutant with monoculm phenotype was isolated and the mutated gene (tin3) was mapped on the distal region of chromosome arm 3AmL of T. monococcum. As a first step towards isolating candidate gene(s), the tin3 and the seed color gene (R-A1) of chromosome 3A were mapped in relation to physically mapped ESTs and STS markers developed based on synteny with rice. Physically mapped wheat ESTs provided a useful framework to identify closely related rice sequences and to establish the most likely syntenous region in rice for the wheat tin3 and R-A1 region. Comparative genomic analysis of the tin3 and R-A1 genomic regions with the corresponding region in rice localized the tin3 gene to a 324 kb region spanned by two overlapping BACs and the R-A1 gene was mapped to a single BAC of the colinear rice chromosome arm 1L.

Master of Science / Department of Entomology / Srinivas Kambhampati / Termites (Isoptera) are eusocial insects and exhibit highly complex eusocial behavior. Eusociality is characterized by the presence of castes (workers, soldiers, reproductives), polyphenisms (same genotype exhibiting multiple phenotypes), flexible developmental pathways, complex communication, cooperative brood care, construction and maintenance of complex nests, and division of labor. Previous studies on honey bees implicated several genes in caste-specific behavior; here, we investigate if orthologs of such genes are present in termites and if so, whether they are expressed differentially among the castes. A candidate gene approach using degenerate primers was used to amplify three candidate genes in the termite Reticulitermes flavipes. Quantitative real time PCR analysis revealed differential expression among termite workers, soldiers, and alates, with a general pattern of higher expression in alates. These results provide information on three novel genes in the termite R. flavipes.

Doctor of Philosophy / Department of Biology / Kimberly A. With / The loss, fragmentation, and degradation of grasslands have resulted in widespread declines in grassland birds. Nest predation is the leading cause of avian reproductive failure; therefore minimizing nest predation can lessen the severity of bird declines. Snakes are important predators of bird nests, but little is known about how snakes may enhance predation risk. To address this issue, I studied the habitat use, movement behavior, population genetic structure, and connectivity of snakes in the grasslands of northeastern Kansas. I addressed the connectivity of eastern yellowbelly racer (Coluber constrictor flaviventris) populations by using a landscape genetics approach at a broad scale (13,500 km2). I also radio-tracked the yellowbelly racer and Great Plains ratsnake (Pantherophis emoryi) at Konza Prairie Biological Station to understand their spatial ecology while simultaneously evaluating nest survival in grassland birds. Individual racers had limited dispersal (<3 km), but substantial admixture occurred within 30 km and populations were in migration-drift equilibrium and had high allelic diversity; therefore, racers must be abundant and continuously distributed for gene flow to be fluid throughout the region. Racers may be more likely to encounter bird nests, as they had more frequent movements and traversed greater distances on average than ratsnakes, which exhibited long periods of inactivity between directed movements. As for grassland birds, nest survival rates decreased with increasing shrubs and decreasing vegetation height. Discriminant function analysis revealed that successful nests were likely to occur in tall vegetation but reduced shrub cover, whereas higher shrub cover characterized snake habitats. Because snakes often use shrubs, nests in areas of increased shrubs may be at higher risk of predation by snakes. Targeted removal of shrubs may increase nest success by minimizing the activity of predators attracted to shrubs. Although predator removal is often a strategy for protecting bird populations, it may not be feasible in this instance, especially since snakes are a native component of the grassland community. Efforts to reduce snake predation on grassland bird nests should therefore focus on managing habitat within grasslands (i.e., shrubs) that influence snake activity, as no natural or anthropogenic habitat barriers currently limit snake movement across the landscape.

Beaked whales (family Ziphiidae) are one of the least known of all mammalian groups. The majority of species have been described from only a handful of specimens. Found in deep ocean waters, these species are widespread and often sexually dimorphic. Little is known of intra-specific variation in morphology, and many species are very similar in external appearance. A reference database of mitochondrial DNA sequences was compiled for all 20 recognised ziphiid species to aid in species identification. All reference sequences were derived from validated specimens, which were often represented only by bone or teeth. DNA was obtained from this ‘historic’ material using ‘ancient’ DNA methods. For three species, holotypes were sampled. Phylogenetic analyses using this database led to the discovery of a new, previously unrecognised species of beaked whale (Mesoplodon perrini), new specimens of Longman's beaked whale (Indopacetus pacificus), a species known previously from only two partial skulls and the synonymy of a third (M. traversii = M. bahamondi). Phylogenetic reconstructions based on sequence data from three mitochondrial and two nuclear loci (total, 2815 bp) using neighbour joining, parsimony, and maximum likelihood methods, resolved many of the sister-species relationships in this group. Inferred relationships among Mesoplodon beaked whales indicated that cranial and tooth morphology may be far more variable between closely related species than previously assumed. No support was found for a linear-progression of tooth form as suggested by Moore (1968) in his phenetic evaluation of relationships among the Ziphiidae. The geographic distribution of Mesoplodon species with similar or divergent tooth morphology is likely due to a combination of sexual selection and selection for species recognition. Both hypotheses predict similar patterns, such as dissimilar tooth morphology among species with sympatric or parapatric distributions. However, only sexual selection appears to offer an explanation for why there are so many Mesoplodon beaked whales. Investigation of mtDNA diversity among a number of beaked whale species indicated that nucleotide diversity was generally lower in this group than in other wide-ranging oceanic cetaceans. The cause of this low diversity was not clear but may be indicative of overall low abundance. Particularly low levels of diversity were found in Baird's beaked whale Berardius bairdii , Arnoux's beaked whale B. arnuxii and the northern bottlenose whale Hyperoodon ampullatus. Strong geographic structure in haplotype frequencies was observed among a worldwide sample of Cuvier's beaked whales Ziphius cavirostris. / Subscription resource available via Digital Dissertations only.

The doctoral studies described in this thesis involve the phenotypic characterization of the tremor rat, an animal model of Canavan disease, and a proof of principle gene transfer study in this model. The phenotype of the tremor rat is examined at the genetic, molecular, cellular, neurochemical, physical and behavioural levels, and tremor mutants are described within the context of Canavan disease. Tremor mutants appear to share many phenotypes with both human patients and to the knock-out mouse model. The deletion of aspartoacylase results in a total loss of the capacity to metabolize N-acetyl-aspartate to acetate and aspartate in brain, leading to elevations in brain N-acetyl-aspartate levels, changes in cell and tissue morphology, and physical and behavioural deficits including mild akinesia and loss of normal motor coordination and balance. Parallel to this work was the development of a gene transfer approach to treat Canavan disease, involving Adeno-associated virus mediated delivery of aspartoacylase to the mammalian central nervous system. Gene transfer was undertaken in tremor rat mutants, and analysis was made of gene expression and function as well as the effect of aspartoacylase expression on improving the phenotypic deficits observed in mutant animals. Gene expression was observed at the RNA and protein level, with recombinant protein observed in cell soma and processes. Although not significant the data suggested a trend of decreased NAA levels after aspartoacylase transfer in comparison to animals injected with a vector encoding green fluorescent protein. Improvement was noted in the rotorod phenotype with mutant animals receiving aspartoacylase gene transfer performing better at tests of balance and coordinated locomotion than animals receiving a control vector. The study provided evidence that Adeno-associated virus mediated aspartoacylase gene transfer to the brain improves some of the deficits in tremor mutants, and supports the rationale of human gene transfer for Canavan disease. / Subscription resource available via Digital Dissertations only.

Beaked whales (family Ziphiidae) are one of the least known of all mammalian groups. The majority of species have been described from only a handful of specimens. Found in deep ocean waters, these species are widespread and often sexually dimorphic. Little is known of intra-specific variation in morphology, and many species are very similar in external appearance. A reference database of mitochondrial DNA sequences was compiled for all 20 recognised ziphiid species to aid in species identification. All reference sequences were derived from validated specimens, which were often represented only by bone or teeth. DNA was obtained from this ‘historic’ material using ‘ancient’ DNA methods. For three species, holotypes were sampled. Phylogenetic analyses using this database led to the discovery of a new, previously unrecognised species of beaked whale (Mesoplodon perrini), new specimens of Longman's beaked whale (Indopacetus pacificus), a species known previously from only two partial skulls and the synonymy of a third (M. traversii = M. bahamondi). Phylogenetic reconstructions based on sequence data from three mitochondrial and two nuclear loci (total, 2815 bp) using neighbour joining, parsimony, and maximum likelihood methods, resolved many of the sister-species relationships in this group. Inferred relationships among Mesoplodon beaked whales indicated that cranial and tooth morphology may be far more variable between closely related species than previously assumed. No support was found for a linear-progression of tooth form as suggested by Moore (1968) in his phenetic evaluation of relationships among the Ziphiidae. The geographic distribution of Mesoplodon species with similar or divergent tooth morphology is likely due to a combination of sexual selection and selection for species recognition. Both hypotheses predict similar patterns, such as dissimilar tooth morphology among species with sympatric or parapatric distributions. However, only sexual selection appears to offer an explanation for why there are so many Mesoplodon beaked whales. Investigation of mtDNA diversity among a number of beaked whale species indicated that nucleotide diversity was generally lower in this group than in other wide-ranging oceanic cetaceans. The cause of this low diversity was not clear but may be indicative of overall low abundance. Particularly low levels of diversity were found in Baird's beaked whale Berardius bairdii , Arnoux's beaked whale B. arnuxii and the northern bottlenose whale Hyperoodon ampullatus. Strong geographic structure in haplotype frequencies was observed among a worldwide sample of Cuvier's beaked whales Ziphius cavirostris. / Subscription resource available via Digital Dissertations only.

The doctoral studies described in this thesis involve the phenotypic characterization of the tremor rat, an animal model of Canavan disease, and a proof of principle gene transfer study in this model. The phenotype of the tremor rat is examined at the genetic, molecular, cellular, neurochemical, physical and behavioural levels, and tremor mutants are described within the context of Canavan disease. Tremor mutants appear to share many phenotypes with both human patients and to the knock-out mouse model. The deletion of aspartoacylase results in a total loss of the capacity to metabolize N-acetyl-aspartate to acetate and aspartate in brain, leading to elevations in brain N-acetyl-aspartate levels, changes in cell and tissue morphology, and physical and behavioural deficits including mild akinesia and loss of normal motor coordination and balance. Parallel to this work was the development of a gene transfer approach to treat Canavan disease, involving Adeno-associated virus mediated delivery of aspartoacylase to the mammalian central nervous system. Gene transfer was undertaken in tremor rat mutants, and analysis was made of gene expression and function as well as the effect of aspartoacylase expression on improving the phenotypic deficits observed in mutant animals. Gene expression was observed at the RNA and protein level, with recombinant protein observed in cell soma and processes. Although not significant the data suggested a trend of decreased NAA levels after aspartoacylase transfer in comparison to animals injected with a vector encoding green fluorescent protein. Improvement was noted in the rotorod phenotype with mutant animals receiving aspartoacylase gene transfer performing better at tests of balance and coordinated locomotion than animals receiving a control vector. The study provided evidence that Adeno-associated virus mediated aspartoacylase gene transfer to the brain improves some of the deficits in tremor mutants, and supports the rationale of human gene transfer for Canavan disease. / Subscription resource available via Digital Dissertations only.