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 Entomology / Kun Yan Zhu / Chitin metabolism represents an attractive target site for combating insect pests as insect growth and development are strictly dependent on precisely toned chitin synthesis and degradation and this process is absent in humans and other vertebrates. However, current understanding on this process and the involved enzymes is rather limited in insects. In this study, two chitin synthase genes (AgCHS1 and AgCHS2 or AgCHSA and AgCHSB), and 20 chitinase and chitinase-like genes (groups I-VIII) presumably encoding the enzymes for chitin biosynthesis and degradation, respectively, were identified and characterized in African malaria mosquito, Anopheles gambiae. Immunohistochemistry analysis and developmental stage- and tissue-dependent transcript profiling by using reverse transcription PCR, real-time quantitative PCR, and in situ hybridization revealed new information on these genes. Current understanding on chitin synthases is extended by the expression profiles such as the localization of AgCHS1 and AgCHS2 transcripts in eggs, AgCHS2 transcripts in the posterior larval midgut, AgCHS1 and AgCHS2 proteins in the compound eyes, and AgCHS2 enzyme in pupal inter-segments. Chitinase and chitinase-like genes are highly diverse in their gene structure, domain organization, and stage- and tissue-specific expression patterns. Most of these genes were expressed in several stages. However, some genes are stage- and tissue-specific such as AgCht8 mainly in pupal and adult stages, AgCht2 and AgCht12 specifically in foregut, AgCht13 exclusively in midgut. Functional analysis of each chitin synthase gene was conducted by using the chitosan/dsRNA nanoparticle-based RNA interference (RNAi) through larval feeding. The repression of the AgCHS1 transcripts which are predominantly expressed in carcass initiated from the mosquito larval feeding of dsRNA suggests the systemic nature of RNAi in mosquito larvae. In addition, silencing of AgCHS1 increased larval susceptibilities to diflubenzuron, whereas silencing of AgCHS2 enhanced the peritrophic matrix disruption and thus increased larval susceptibilities to calcofluor white or dithiothreitol. Furthermore, a non-radioactive method was adapted and optimized to examine the chitin synthase activity in mosquitoes. By using this method, diflubenzuron and nikkomycin Z show limited in vitro inhibition on chitin synthase at high concentration in cell free system, whereas no in vivo inhibition was observed.

Doctor of Philosophy / Department of Anatomy and Physiology / Chris R. Ross / The immediate antiviral defense residing in the innate immune system of multicellular organisms critically determines the outcome of viral infection. This dissertation presents a study of the "effectors" and "receptors" of porcine innate immunity in infection caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is the most devastating pathogen impacting the swine industry. In the first investigation, eleven novel porcine host defense peptides (HDPs), [Beta]-defensins (pBDs), were identified and characterized. All of these peptides have a consensus [Beta]-defensin motif and phylogenetically are similar to orthologs from other species. A differential expression pattern for these 11 newly identified genes was found. For example, pBD-2 and pBD-3 were expressed in bone marrow, lung, skin and other lymphoid tissues. pBD-2 and pBD-3 were further characterized for their gene structure, and antimicrobial activity of synthetic peptides. The second study was conducted to evaluate PRRSV-induced differential expression of porcine HDPs and direct antiviral activity of selected HDPs against PRRSV. In vitro incubation of PRRSV with synthetic pBD-3 or protegrin-4 (PG-4) significantly inhibited viral infectivity. Using nine protegrin-derived peptides, it was determined that cyclization of PG-4 increased anti-PRRSV activity and mutation of some residues in PG-4 diminished some of the activity. These findings suggest the potential role of porcine HDPs as a group of innate antiviral effectors. In the third and fourth investigations, porcine Toll-like receptor (TLR) 3 and TLR7 were identified and functionally expressed. Increased expression of TLR3 was observed in PRRSV-infected porcine lungs. Stimulation of porcine alovelar macrophages with poly (I:C), a synthetic TLR3 ligand, increased expression of interferon-[Beta] and suppressed PRRSV infectivity. Activation of porcine TLR3 overexpressed in a PRRSV-sensitive cell line, elicited antiviral responses to PRRSV infection. Partial silencing of TLR3 in PAMs resulted in increased PRRSV infection. In summary, these data provide molecular information on porcine TLR3 and TLR7, and their involvement in PRRSV pathogenesis, which may elicit new strategies to prevent this costly swine disease.

Master of Science / Department of Anatomy and Physiology / Mark L. Weiss / When mouse embryonic stem cells (ESCs) were grown on mitotically inactivated rat umbilical cord-derived stromal cells (RUCs) in the absence of leukemia inhibitory factor (LIF) and 2-mercaptoethanol (2-MCE), the ESCs showed alkaline phosphatase (AP) staining. ESCs cultured on RUCs maintain expression of the following pluripotency genes, Nanog, Sox2 and Oct4 and grow at a slower rate when compared with ESCs grown on mitotically inactivated mouse embryonic fibroblasts (MEFs). Differences in gene expression for the markers of pluripotency Oct4, Sox2 and Nanog, AP staining and ESC growth rate were also observed after LIF and 2-MCE were removed from the co-cultures. Reverse transcriptase polymerase chain reaction (RT-PCR) suggested differences in Sox2 and Nanog mRNA expression, with both genes being expressed at higher levels in the ESCs cultured on RUCs in the absence of LIF/2-MCE as compared to ESCs cultured on MEFs. Semi-quantitative RT-PCR indicated that Nanog expression was higher when ESCs were grown on RUCs in the absence of LIF and 2-MCE as compared to MEFs in the same treatment conditions. Bisulfite-mediated methylation analysis of the Nanog proximal promoter suggested that the maintenance of Nanog gene expression found in ESCs grown on RUCs after culture for 96 hours in the absence of LIF/2-MCE may be due to prevention of methylation of the CpG dinucleotides in the Nanog proximal promoter as compared to ESCs grown on MEFs. Thus, RUCs may release factors into the medium that maintain the pluripotent state of mouse ESCs in the absence of LIF and 2-MCE.

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.

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.

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.

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.

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.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / Gerald E. Wilde / Analysis of transcriptomes from salivary glands and midgut of the Hessian fly [Mayetiola destructor (Say)] identified a diverse set of cDNAs that were categorized into five groups, group I – V, based on their phylogenetic relationship. All five of these groups may encode putative protease inhibitors based on structural similarity with known proteins. The sequences of these putative proteins among different groups are highly diversified. However, sequence identity and structural analysis of the proteins revealed that all of them contained high cysteine residues that were completely conserved at their respective positions among these otherwise diversified proteins. Analysis of bacterial artificial chromosome (BAC) DNA for two groups, group I (11A6) and group II (14A4), indicated that group I might be a single copy gene or genes with low copy number whereas group II exists as multiple copies clustered within the Hessian fly genome. To test the inhibitory activity and specificity of these putative proteins, recombinant proteins were generated. Enzymatic analysis of the recombinant proteins against commercial and insect gut proteases demonstrated that recombinant proteins indeed are strong inhibitors of proteases with different specificities. Northern analysis of the representative members of five groups revealed that the group I-IV genes were expressed exclusively in the larval stage with variations among groups at different larval stages. The group V (11C4) genes were expressed in the late larval and pupal stage. Tissue specific gene expression analysis revealed that group I-IV genes were predominantly expressed in malpighian tubules whereas the group V genes were abundantly expressed in the salivary glands. Localization experiments with the antibody for representative members from group II (14A4) demonstrated that the protein was predominantly localized in the malpighian tubules and in low amounts in the midgut, suggesting that malpighian tubules are the primary tissue of 14A4 inhibitor synthesis. The overall results indicated that the Hessian fly contains a complex network of genes that code for protease inhibitors which regulate protease activities through different developmental stages of the insect.