Understanding the ecology and epidemiology of tick-borne diseases requires detailed knowledge of the complex interactions among the tick vector, the microorganisms they carry and the vertebrate hosts used by ticks, as well as the environmental conditions experienced by all three groups of organisms in this triad. In this thesis, I addressed questions relating to the biology and vector ecology of the Rocky Mountain wood tick (Dermacentor andersoni) and the American dog tick (Dermacentor variabilis). Comparisons were made of the distribution of both tick species, the vertebrate hosts used by immature ticks, and the types and prevalence of bacteria in individual ticks from multiple localities near the northern extent of their geographic ranges in western Canada. The results revealed that the distributions of both D. andersoni and D. variabilis have expanded since the 1960s, and there is now a broad zone of sympatry in southern Saskatchewan. In this zone of sympatry, D. andersoni and D. variabilis immatures were found to use the same species of small mammals as hosts and, in some cases, the same host individuals. This provides for the possibility of cross-transmission of bacteria from one tick species to the other. Bacteria of several genera (e.g. Rickettsia, Francisella, Arsenophonus and Anaplasma) were detected in D. andersoni and/or D. variabilis, some of which represented new tick-bacteria associations. However, most bacterial species were highly host (tick)-specific, except for three examples of apparent host switching from one tick species to the other at localities where the two tick species occurred in sympatry. The findings of this thesis provide a basis for understanding microbial transmission, the structure of tick-borne microbial communities, the risk of tick-borne disease in humans and animals, and the vector potential of D. andersoni and D. variabilis in geographical areas where they have not been studied previously.

A new paradigm for the treatment of infectious disease is through the modulation of innate immune responses. In this capacity, host defense peptides (HDPs) and synthetic Toll-like receptor 9 (TLR9) ligands have the greatest demonstrated potentials. The work presented here considers mechanisms for the improvement of these treatments through optimization, or in the case of HDPs the minimization, of the interactions of these ligands with sensory receptors.<p>Toll-like Receptor 9 activates the innate immune system in response to microbial DNA or immune-modulating oligodeoxynucleotides. While cell stimulation experiments demonstrate the preferential activating ability of CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions regarding the sequence-specificity of TLR9 ligand binding. To address this discrepancy the characterization of human TLR9 ligand binding properties is reported. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity of the receptor is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. <p>Host defense peptides are among the leading candidates to combat antibiotic resistant bacterial strains. Recently, HDPs have been demonstrated to function as ligands for the bacterial sensory kinase PhoQ resulting in the induction of virulence and adaptive responses. Thus, concerns have been raised regarding therapeutic applications of HDPs. Here a methodology is described that permits discrimination and quantification of the distinct, but related, peptide behaviors of direct antimicrobial activity and PhoQ ligand potential. Utilizing peptide derivatives of the model HDP Bac2A it is demonstrated that antimicrobial efficiency is significantly, and inversely, related to PhoQ ligand efficacy. This provides a rational basis for HDP selection with greater therapeutic potential and minimized potential for initiation of bacterial resistance.

Understanding the ecology and epidemiology of tick-borne diseases requires detailed knowledge of the complex interactions among the tick vector, the microorganisms they carry and the vertebrate hosts used by ticks, as well as the environmental conditions experienced by all three groups of organisms in this triad. In this thesis, I addressed questions relating to the biology and vector ecology of the Rocky Mountain wood tick (Dermacentor andersoni) and the American dog tick (Dermacentor variabilis). Comparisons were made of the distribution of both tick species, the vertebrate hosts used by immature ticks, and the types and prevalence of bacteria in individual ticks from multiple localities near the northern extent of their geographic ranges in western Canada. The results revealed that the distributions of both D. andersoni and D. variabilis have expanded since the 1960s, and there is now a broad zone of sympatry in southern Saskatchewan. In this zone of sympatry, D. andersoni and D. variabilis immatures were found to use the same species of small mammals as hosts and, in some cases, the same host individuals. This provides for the possibility of cross-transmission of bacteria from one tick species to the other. Bacteria of several genera (e.g. Rickettsia, Francisella, Arsenophonus and Anaplasma) were detected in D. andersoni and/or D. variabilis, some of which represented new tick-bacteria associations. However, most bacterial species were highly host (tick)-specific, except for three examples of apparent host switching from one tick species to the other at localities where the two tick species occurred in sympatry. The findings of this thesis provide a basis for understanding microbial transmission, the structure of tick-borne microbial communities, the risk of tick-borne disease in humans and animals, and the vector potential of D. andersoni and D. variabilis in geographical areas where they have not been studied previously.

mPIP is a mouse homologue of human PIP/GCDFP-15 which is an established marker of both malignant and benign pathological conditions of the mammary gland. mPIP gene expression has been identified in both lacrimal and salivary glands of healthy mice and the mPIP protein has been detected in saliva. The mPIP protein has been found to bind oral bacteria, showing the highest affinity for streptococci, suggesting a potential function of mPIP in the non-immune host defense in the mouse oral cavity. Since the exact functions of mPIP are still unknown, we examined the roles of mPIP through both in vitro and in vivo studies, specifically to address the possible role of this protein in non-immune host response through modulating the oral flora. The in vitro studies were primarily focused on elucidation of the consequences of interaction between mPIP and oral bacteria, in particular to examine whether mPIP plays a role in bacterial aggregation. The in vivo studies addressed the roles of mPIP through the analysis of an mPIP knockout mouse model generated in our laboratory. Following confirmation of the null mutation, the delineating the phenotype of this model was pursued through morphopathological analysis as well as examination of the impact of the lack of mPIP on the mouse oral flora. The null mutation in the mPIP knockout mice was confirmed by both the gene and protein analysis. Histological analysis revealed lymphocytic proliferation in both the submaxillary and prostate glands of the mPIP knockout mice. In addition, both quantitative and composition differences in the oral flora of mPIP knockout mice were identified when compared with wild-type controls. Specifically, a higher proportion of the oral bacteria of mPIP knockout mice were found to belong to genus Streptococcus and certain genera were found to be absent from the oral cavity of these mice. The effect of knockout mouse saliva, which lacks mPIP, on the aggregation of oral bacteria was compared to wild-type mouse saliva. Our data suggests that mPIP contributes to saliva-induced bacterial aggregation. While oral flora has multiple functions, including protection against infection, mPIP might play a role in the non-innate host defense through modulating the resident oral flora in the mouse. The identification of lymphocytic proliferation in submaxillary and prostate glands of mPIP knockout mice suggests that mPIP might also interfere with lymphocyte activity, playing a possible immunomodulatory role. / May 2008

The emergence of pathogens resistant to available drug therapies is a pressing global health problem. Antimicrobial peptides (AMPs) may potentially form new therapeutics to counter these pathogens. AMPs are key components in the mammalian innate immune system and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. This thesis describes computational methods for the identification of novel natural and synthetic AMPs. A bioinformatic resource was constructed for classification and discovery of gene- coded AMPs, consisting of a database of clustered known AMPs and a set of hidden Markov models (HMMs). One set of 146 clusters was based on the mature peptide sequence, and one set of 40 clusters was based on propeptide sequence. The bovine genome was analyzed using the AMPer resources, and 27 of the 34 known bovine AMPs were identified with high confidence and up to 69 AMPs were predicted to be novel peptides. One novel cathelicidin AMP was experimentally verified as up-regulated in response to infection in bovine intestinal tissue. A chemoinformatic analysis was performed to model the antibacterial activity of short synthetic peptides. Using high-throughput screening data for the activities of over 1400 peptides of diverse sequence, quantitative structure-activity relation (QSAR) models were created using artificial neural networks and physical characteristics of the peptide that included three-dimensional atomic structure. The models were used to predict the activity of a set of approximately 100,000 peptide sequence variants. After ranking the predicted activity, the models were shown to be very accurate. When 200 peptides were synthesized and screened using four levels of expected activity, 94% of the top 50 peptides expected to have the highest level of activity were found to be highly active. Several promising candidates were synthesized with high quality and tested against several multi- antibiotic-resistant pathogens including clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. These peptides were found to be highly active against these pathogens as determined by minimal inhibitory concentration; this serves as independent confirmation of the effectiveness of high-throughput screening and in silico analysis for identifying peptide antibiotic drug leads.

On the Florida peninsula, the soapberry bug (Jadera haematoloma) has been able to colonize the golden rain tree, Koelreuteria elegans, since the introduction of this invasive tree only a few decades ago. The populations feeding on the new host have been rapidly differentiating from the native populations. Possibly the most dramatic differentiation is that of the beak (mouthpart) length. Derived populations showed shorter beaks more appropriate for feeding on the flattened pods of the new host. Previous studies have shown that the divergence of the beak length has a genetic basis and involves non-additive genetic effects. However, to date, the soapberry bug divergence has not been studied at the molecular level. In the current study, I have generated a three-generation pedigree from crossing the long-beaked and short-beaked ecomorphs to construct a de novo linkage map and to locate putative QTL controlling beak length and body size in J. haematoloma. Using the AFLP technique and a two-way pseudo-testcross design I produced two parental maps. The maternal map covered six linkage groups and the paternal map covered five; the expected number of chromosomes was recovered and the putative X chromosome was identified. For beak length, QTL analyses revealed one significant QTL. Three QTL were found for body size. Interestingly, the most significant body size QTL co-localized with the beak length QTL, suggesting linkage disequilibrium or pleiotropic effects of related traits. Through single marker regression analysis, nine single markers that could not be placed on the map were also found to be associated with either trait. However, I found no evidence for epistasis. Overall, my findings support an oligogenic model of genetic control on beak length and body size, and the underlying genetic architectures were complex. This study is the first to look at the molecular basis underlying adaptive traits in the soapberry bug, and contributes to understanding of the genetic changes involved in early stages of ecological divergence.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), characterized by lesions formed due to demyelination. MS is a complex disease thought to be triggered by environmental factors in genetically predisposed individuals. The strongest associated susceptibility allele is HLA-DRB1*1501. Environmental factors include smoking, latitude and previous infection of Epstein-Barr virus (EBV), a common herpes virus. There is no cure for MS, but several inhibitor and symptomatic drugs. Tysabri® (natalizumab) is the most effective drug, but it may lead to progressive multifocal leukoencephalopathy (PML), a rare but often fatal disease caused by reactivation of JC virus. The aim of this thesis was to replicate previous findings from a genome-wide association study and to find host genetic factors influencing JCV seropositivity and EBNA1 IgG titers in Swedish MS patients and healthy controls. Samples from the EIMS and IMSE studies were genotyped by TaqMan® OpenArray™ PCR, an end-point SNP genotyping analysis. 1143 cases and 556 healthy controls were genotyped. Due to poor call rates, genotype data from an Immunochip study was added. A total of 3408 samples (1664 cases and 1744 controls) were analyzed. EBNA1 IgG antibodies were previously measured as a detection of EBV infection and increased MS risk, and JCV IgG antibodies were measured to find patients potentially at risk for PML. One significant result was found, gene 105 (p = 0.01674, OR 0.68, CI 95% 0.49-0.93), with a protective effect in MS. More significant results might have been found with better loading of the plate, or with a different genotyping method.

A new paradigm for the treatment of infectious disease is through the modulation of innate immune responses. In this capacity, host defense peptides (HDPs) and synthetic Toll-like receptor 9 (TLR9) ligands have the greatest demonstrated potentials. The work presented here considers mechanisms for the improvement of these treatments through optimization, or in the case of HDPs the minimization, of the interactions of these ligands with sensory receptors.<p>Toll-like Receptor 9 activates the innate immune system in response to microbial DNA or immune-modulating oligodeoxynucleotides. While cell stimulation experiments demonstrate the preferential activating ability of CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions regarding the sequence-specificity of TLR9 ligand binding. To address this discrepancy the characterization of human TLR9 ligand binding properties is reported. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity of the receptor is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. <p>Host defense peptides are among the leading candidates to combat antibiotic resistant bacterial strains. Recently, HDPs have been demonstrated to function as ligands for the bacterial sensory kinase PhoQ resulting in the induction of virulence and adaptive responses. Thus, concerns have been raised regarding therapeutic applications of HDPs. Here a methodology is described that permits discrimination and quantification of the distinct, but related, peptide behaviors of direct antimicrobial activity and PhoQ ligand potential. Utilizing peptide derivatives of the model HDP Bac2A it is demonstrated that antimicrobial efficiency is significantly, and inversely, related to PhoQ ligand efficacy. This provides a rational basis for HDP selection with greater therapeutic potential and minimized potential for initiation of bacterial resistance.

Dicyemid mesozoans are endoparasites inhabited in benthic cephalopod molluscs. Previous studies of dicyemids were mostly concentrated on their taxonomy. The life cycle remains a mystery since Cavolini observed this animal in 1787. The purpose of this study is to investigate further on the dicyemid life cycle building on the limited knowledge from previous studies, and investigate the infection of dicyemid in octopuses in tropical waters, using the area in the southwestern Taiwan as a model. A total of 324 octopus hosts was collected¡Aincluding 3 genera and 13 species from DungGang Fish Market from 2001 to 2002. There were 9 host species detected with dicyemid mesozoans including 14 species within 2 genera. Moreover, there is no dicyemid species overlap among the octopus species indicating high host-specificity. There is large range of the dicyemid infection prevalence among octopod hosts. Cistopis indicus has the highest prevalence (91%) and Octopus luteus the lowest (20%) in this study. In C. indicus, the infected mechanism correlated with season. The infusoriform larvae of dicyemid mesozoan dispersal from the host to sea water in the autumn and winter, then entering host in the spring and summer. The change of dicyemid life cycle stage in the C. indicus kidney correlates with the sharp decrease of infection intensity. However, the dicyemid infection in Octopus marginatus correlates with body size, not with season. Besides, there is significant difference of dicyemid development between two kidneys of the same individual, both in the analysis of C. indicus and O. marginatus. It indicates the separated way of dicyemid infection into two sides of kidneys.

In this study, we focus on the electric optical characteristic of guest/host polymer system. The charge transfer chromophore Wu182 which contain fluorine atom in it was mixed with polymer PMMA and solved in chloroform, while DR1/PMMA served as reference sample. The wu182/PMMA and DR1/PMMA thin films were produced by spin coating. The thin film was poled at the temperature above glass transition temperature until the second harmonic generation signal was present. The poled thin film was placed in one arm of Mach-Zehnder interferometer. Voltage modulation applied to the polymer films can cause phase changes in the interferometer from that we estimate the electro-optic coefficient. From our experiments, we found that modulating frequency had significant influence on the electro-optic effect. The result shows that Wu182 possess large nonlinear optical coefficient. The valid electro-optic coefficient r13 was obtained to be 2.620 pm/V when the frequency was above 6 kHz.