Thesis (Ph. D.)--University of Wisconsin--Madison, 1992. / Typescript. Description based on print version record. Includes bibliographical references (leaves 135-142).

Thesis (M.S.)--University of Wisconsin--Madison, 1984. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 84-94).

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 272-279).

Thesis (Ph. D.)--Oregon State University, 2006. / Printout. Includes bibliographical references (leaves 191-192). Also available on the World Wide Web.

Dissertation (Ph.D.)--University of Toledo, 2005. / Typescript. "A dissertation [submitted] as partial fulfillment of the requirements of the Doctor of Philosophy degree in Engineering." Bibliography: leaves 106-120.

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The protozoan parasite Neospora caninum is among the most frequently diagnosed infectious causes of bovine abortion thus causing significant economic losses, production inefficiency and welfare concern to the cattle sector worldwide. The control of bovine neosporosis relies on management techniques within which the identification of infected animals by serological testing plays a key role. However, the reliable diagnosis of the disease is hindered by the complex host-parasite interactions and the intrinsic limitations of the serological diagnostic tools currently available; as a result, some infected animals may not be detected. At the herd, regional and national levels, this can potentially undermine efforts for the control of the disease. The work presented in this thesis was undertaken to further investigate avenues to improve the diagnosis of N. caninum infection in cattle. It has been shown previously that recombinant antigens expressed by the rapidly proliferating tachyzoite stage and the slowly multiplying bradyzoite stage of N. caninum can be successfully employed for the detection of specific antibody responses during acute and persistent infections respectively. Following the establishment of persistent infections, sustained by the bradyzoite stage, the antibody levels against the tachyzoite stage may decline below the detection limits of currently available diagnostic tests which are exclusively based on antigens expressed by the tachyzoite. Consequently, the use of bradyzoite antigens for the development of serological diagnostic tests, may enhance the identification of infected animals. Novel antigens putatively expressed by the quiescent bradyzoite stage of N. caninum have been identified, expressed as recombinant proteins and assayed for the detection of specific antibodies. The recognition of recombinant tNcSRS12A-B and tNcSRS44- A by specific antibodies in sera from persistently N. caninum-infected cattle suggested that these proteins could be used for the detection of persistently infected animals. Indirect ELISAs based on previously characterised N. caninum antigens, such as the tachyzoite surface protein rNcSRS2, the immunodominant dense granule protein rNcGRA7 and the bradyzoite specific surface antigens rNcSAG4, rNcBSR4 and rNcSRS9, as well as a commercial test using whole tachyzoite lysate as antigenic preparation, were evaluated within a cross-sectional study to estimate the seroprevalence of bovine neosporosis in British dairy cattle. Moderate, but not high, agreement was found amongst the tests based on whole tachyzoite lysate, rNcSRS2 and rNcGRA7, and amongst the bradyzoite-specific antigen-based iELISAs. In contrast, only slight agreement was observed when each test detecting antibody responses indicative of acute infection (whole tachyzoite lysate, rNcSRS2 and rNcGRA7) was compared with each test detecting antibody responses indicative of persistent infection (bradyzoite-specific antigen-based iELISAs). Most N. caninum antibody-positive cattle samples showed detectable antibodies only against either antigens predominantly expressed by the tachyzoite or bradyzoite antigens thus suggesting that the exclusive use of one type of test may result in the misclassification of a proportion of animals, which test negative despite harbouring the parasite. This may result in the underestimation of the seroprevalence. Consequently, the combination of multiple tests in parallel, both tachyzoite and bradyzoite antigen-based, would improve the diagnosis of bovine neosporosis. Molecular tools for the genetic discrimination of different N. caninum isolates were also investigated. A novel multilocus fragment typing (MLFT) tool based on twelve highly polymorphic microsatellite markers was developed and applied to the analysis of DNA samples obtained from laboratory-maintained N. caninum isolates and tissues collected from bovine foetuses aborted due to N. caninum. The locus-specific nested PCRs associated with automated fragment analysis by capillary electrophoresis enabled to evaluate the markers in terms of typeability and discriminatory power. Overall, the typing tool was characterised by good typeability and discriminatory power and enabled to provide information on the genetic diversity amongst the laboratory-maintained and clinical N. caninum isolates studied. The MLFT tool may help to investigate the likely source of infection within abortion outbreaks and aid the study of the association between the genetic heterogeneity of N. caninum and the diverse biological features in vitro and in vivo. Furthermore, the loci characterised by the highest discriminatory power and typeability may be used alongside already established microsatellite markers for the development of an improved typing tool which could be proposed at the inter-laboratory level. Finally, current perceptions and common veterinary practice related to the diagnosis and control of bovine neosporosis were studied by developing a questionnaire for cattle practitioners in the United Kingdom. The survey highlighted the awareness of the limitations of current serological techniques and the demand for additional tools in terms of diagnostics and vaccines to tackle the economic losses and animal welfare implications related to N. caninum in cattle.

In order to identify patients with severe coronary artery disease (CAD) and at a higher risk of future cardiac events after uncomplicated myocardial infarction, 105 consecutive patients were studied prospectively. There were 93 men and 12 women with a mean age of 56 +/- 8.2 years. Treadmill testing, exercise radionuclide ventriculography, thallium-201 myocardial imaging and selective coronary arteriography were performed 6-8 weeks after infarction. Patients were grouped into those who had single and multiple vessel disease. Multiple regression analysis of 18 noninvasive indices was carried out using generalized linear interactive modelling (GLIM) and the results were compared with the severity of underlying CAD and the clinical outcome after a mean follow-up period of 18.8 +/- 3. 4 months. At the end of the follow-up period, patients were categorized into those who had no cardiac events, minor and major cardiac events. Multivariate analysis produced an algorithm from three factors found to be most predictive of the severity of CAD. These included ST-segment depression on exercise, total score of rest and exercise regional wall motion and the presence of significant redistribution on thallium-201 imaging. The sensitivity of this algorithm for predicting multiple vessel disease was 42%, with a specificity of 94%, and a predictive accuracy of 69%. However, the total score of regional wall motion abnormalities was the single most predictive factor of major cardiac events with a sensitivity of 94%, a specificity of 57%, and predictive accuracy of 63%. None of the other factors produced additional prognostic information. Therefore, exercise radionuclide ventriculography appears to be the investigation of choice in assessing prognosis after myocardial infarction.

It has been proposed that delusional thinking 'may be on a continuum with, normal behaviour and can be assessed by taking account of, factors such as the client's degree, of belief conviction or the extent of preoccupation with the belief. In the present research a number of -measures were employed to assess theý delusional thinking of people diagnosed as schizophrenic. - Two -interventions were eipployed: (i) a structured verbal challenge, and (ii) a reality test in which the belief was subject to an empirical test. The research offered support for the continuum view of delusional behaviour, and demonstrated that a number of aspects of delusional behaviour, including the degree of conviction with which the belief is held, are open to modification.

As a high-resolution wide-field near-surface microscopy, total internal reflection fluorescence microscopy (TIRFM) has been widely applied for the study of biomolecules. Unlike those costly, sample consuming and time consuming traditional detection assays, the application of TIRFM enable the direct quantification of biomolecules in a sample pretreatment and enrichment free fashion. Taking advantages of the TIRFM imaging system, in this thesis we have applied the TIRFM imaging system to directly quantify the content of different cancer associated biomarkers. Four different detection approaches for direct cancer biomarkers quantification with the aid of TIRFM were herein presented respectively. In Chapter 2, a direct quantification of nasopharyngeal carcinoma associated miRNAs was described. In the assay, five different miRNAs were chosen as the target analytes, which hybridized with the synthetic complementary LNA, probe in solution. The duplex was labeled with intercalating fluorescence dye YOYO-1 and the signal was then detected by the TIRFM-EMCCD imaging system. The LNA probe exhibited a high binding affinity towards the complementary target miRNAs and a limit of detection of 8 pM was achieved. Since the LOD is far below the reported concentration of miRNAs found in body fluids, this developed assay is of high potential to serve as a tool for non-invasive detection of miRNAs for early disease diagnosis. In Chapter 3, an advanced single-molecule based assay for direct circulating miRNAs detection was developed. The assay was demonstrated to be capable of differentiating the expression of a nasopharyngeal carcinoma (NPC) up-regulator hsa-mir-205 (mir-205) in serum collected from patients of different stages of NPC. To overcome the background matrix interference in serum, locked nucleic acid modified molecular beacon (LNA/MB) was applied as the detection probe to hybridize, capture and detect target mir-205 in serum matrix with enhanced sensitivity and specificity. A detection limit of 500 fM was achieved. The as-developed method was capable of differentiating NPC stages by the level of mir-205 quantified in serum with only 10 μL of serum and the whole assay can be completed in an hour. The experimental results agreed well with reported and while the quantity of mir-205 determined by our assay was found comparable to that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), supporting that this assay can be served as a promising non-invasive detection tool for early NPC diagnosis, monitoring and staging. In chapter 4, a self-assembled protein nanofibril based online pre-concentrating sensor was developed. This solution-based hybridization assay was applied to quantified the amount of target miRNAs, mir-196a. Biotinylated locked nucleic acid (LNA) of complimentary sequence was served as the probe to capture the target miRNA analyte. The target hybridization duplex was immobilized on the backbone of the nanofibril through the biotin-streptavidin interaction. The quantification was achieved by the fluorescence intensity measured with total internal reflection fluorescence microscopy. A detection limit of 1 pM was achieved with trace amount of sample consumption. This assay showed efficient single-base mismatch discrimination. The applicability of quantifying circulating mir-196a in both normal and cancer patient’s serums was also demonstrated. In chapter 5, a magnetic nanoparticles based sandwich immunosensor with carbazole-based cyanine as the fluorescence labeling dye for the direct quantification of prostate cancer related antigen, PSA, was developed. Taking benefit of the magnetic property of the nanoparticles, the target sandwich immunocomposites can be easily online separated from the sample matrix. The as-developed assay can efficiently discriminate the target PSA from other disease related antigens and achieve a LOD of 400 fM (13 pg/mL) and a LOQ of 2 pM (0.66 ng/mL). As the whole detection assay can be completed in 1 h with only 10 μL of sample, this assay is fast and cost effective and of high potential for early disease and cancer diagnosis, staging and monitoring