Thesis (Ph. D.)--Lehigh University, 2003. / Includes vita. Includes bibliographical references (leaves 150-165).

Onchocerciasis, or river blindness, has historically represented one of the significant neglected tropical diseases on the planet in terms of socio-economic impact. The discovery that ivermectin was a safe and effective treatment for onchocerciasis, together with the decision of the manufacturer to donate the drug for the treatment of this disease became the basis for several large international programs to control and eventually eliminate the infection. These programs have managed to virtually eliminate transmission of the parasite causing Onchocerca volvulus from many foci in Africa and the Americas. Verifying that transmission has been halted requires sensitive and specific assays to detect the presence of the parasite. The gold standard to accomplish this has been to employ a PCR assay targeting a specific repeated sequence family encoded in the genome of O. volvulus to screen for the presence of the parasite in pools of vector black flies. While this assay is highly sensitive, obtaining the high specificity required to document an absence of transmission requires an independent confirmatory assay. To meet this need, an independent PCR assay targeting the cytochrome B (cytB) gene of the O. volvulus mitochondrion was developed. This assay could detect O. volvulus mitochondrial DNA purified by magnetic bead capture using the primers for the cytB gene and from the nuclear encoded repeated sequence DNA targeted in the primary assay. These preliminary data suggest that the mitochondrial PCR assay may be employed as a confirmatory assay to detect O. volvulus in pools of vector flies.

The ability to quickly and accurately quantify fissile constituents in bulk materials remains essential to many aspects of nuclear forensics and for safeguarding nuclear materials and operations. This often entails the analysis of trace quantities of nuclear debris or effluents, and typically requires bulk sample digestion followed by actinide separation and mass spectrometry. Because destructive methods are time and labor intensive, efforts have been made to develop alternative nondestructive methods for this type of analysis. This work, performed at Oak Ridge National Laboratory at the High Flux Isotope Reactor (HFIR), seeks to utilize delayed neutron activation analysis on samples of interest containing multiple fissile constituents. Based on the variances in the fission product yields of individual fissile nuclides, this work utilizes methods of linear regression to derive a technique that allows for such analysis, forgoing chemical separation and using only a single irradiation and counting step. / text

Ranaviruses are large dsDNA viruses that are considered emerging pathogens, and they are known to cause mortality events in amphibian and fish populations. This research utilizes experimental and genomic data to elucidate the mechanisms driving the evolution and spread of ranaviruses, with a focus on host switching within the genus. In Chapter 1, we utilize virus challenge assays to examine potential transfer of ranaviruses between cultured juvenile largemouth bass (M. salmoides) and bullfrog tadpoles (Rana catesbeiana). Additionally, a commonly used antiparasitic treatment containing malachite green and formalin (MGF) was utilized to suppress the immune system of largemouth bass to assess the susceptibility of immunocompromised fish to ranaviruses. The results indicate that tadpoles are not susceptible to Largemouth Bass Virus (LMBV), but that bass are susceptible to ranaviruses isolated from amphibians. Furthermore, immunocompromised fish were more susceptible to both LMBV and FV3 infections than immunocompetent fish. In Chapter 2, we used eight sequenced ranavirus genomes and two selection-detection methods (site-based and branch-based) to identify genes that exhibited signatures of positive selection, potentially due to the selective pressures at play during host switching. We found evidence of positive selection acting on four genes via the site-based method, three of which are newly-acquired genes unique to ranavirus genomes. Our results suggest that the group of newly acquired genes in the ranavirus genome may have undergone recent adaptive changes that have facilitated interspecies and interclass host switching. In Chapter 3, we annotated and analyzed the nearly complete genomic sequence of LMBV to determine its taxonomic classification. The available genomic content and phylogenetic evidence suggests that LMBV is more closely related to amphibian-like ranaviruses (ALRVs) than grouper ranaviruses, and this is further supported by greater genomic collinearity between LMBV and ALRVs. This data suggests that the classification of LMBV as a ranavirus is warranted. The results presented here will help to clarify the taxonomic relationships of ranaviruses, and will also be useful in developing management strategies to limit interspecific and intraspecific viral spread. The information garnered from this research will have far-reaching implications in studies of amphibian conservation, disease evolution, and virology. / text

Circulating tumor cells (CTCs) are the cells that are shed from a primary tumor into the vasculature and circulate in the bloodstream. CTCs may trigger cancer metastasis, which leads to most cancer-related deaths. CTCs are widely studied due to their value in cancer diagnosis, prognosis, and oncology studies. The major challenges with CTCs lie in their extremely low concentration in blood, thus requiring an effective enriching system to enable downstream analyses. The immunomagnetic assay has proved to be a promising CTC detection tool with high sensitivity and throughput. Key factors related to the immunomagnetic assay include the capture rate, which indicates the sensitivity, and distributions of target cells after capture, which impact the cell integrity and other biological properties. In this dissertation, we build a sedimentation model, a partial viscosity model, and a cell-tracking model to address the principle of the immunomagnetic cell separation. We examine the channel orientations and determine the favorable inverted condition. In addition, we develop a micromagnet approach to modulate the in-channel magnetic field toward enhanced cell detection and distribution. Through numerical studies, we calculate the magnetic field generated by the thin-film micromagnets, determine its effective ranges, and demonstrate its value in optimizing cell distribution. In the experimental demonstration, we present two types of micromagnets based on e-beam Ni deposition and inkjet printing technology, respectively. In the screening experiments, the Ni micromagnet integrated system achieves over 97% capture rate. It shows a 14% increase in capture rate, and a 14% improvement in distribution uniformity compared with plain slides. We also successfully isolate CTCs from metastatic cancer patients with the micromagnet assay. The inkjet-printed patterns yield a similarly high capture rate of 103%. With the pixel permanent magnet array, the inkjet patterns further increase the distribution uniformity for 20%. The proposed models lay the theoretical foundations for future modification of the immunomagnetic assay, and the micromagnet-integrated system provides a promising tool for translational applications in cancer diagnose and clinical cancer management. / text

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The properties of human immunodeficiency virus type 1 (HIV-1) and its simian counterpart SIV that enable persistent replication in the face of robust cellular, antibody, and innate immune responses have complicated efforts to develop a safe and effective vaccine. Vaccine protection against HIV-1 infection may require a combination of immune mechanisms. However, the types of immune responses that can be induced by vaccination to prevent HIV-1 infection remain unclear. The features of the viral envelope glycoprotein (Env) that confer inherent resistance to neutralization by antibodies also interfere with the development of antibody responses. We therefore vaccinated rhesus macaques with single-cycle SIV (scSIV) strains expressing Env proteins mutated to remove features that interfere with the induction of antibody responses. Antibodies capable of neutralizing Env-modified but not wild-type SIV were selectively enhanced. Identifying the immune responses underlying complete protection by live-attenuated SIV against pathogenic SIV challenge may provide guidance for HIV-1 vaccine design. To test the hypothesis that antibodies not measurable by assays for virus neutralization correlate with protection by live-attenuated SIV, we developed a novel assay for antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity increased progressively over time after inoculation, and was measurable against viruses expressing heterologous Env proteins from independent SIV isolates when neutralization was undetectable. Two separate pathogenic \(SIV_{mac}251\) challenge experiments took advantage of either the strain specificity or the time-dependent development of immunity to overcome complete protection by live-attenuated SIV. In both experiments, macaques inoculated with live-attenuated SIV that remained uninfected by \(SIV_{mac}251\) had significantly higher ADCC activity than those that became infected. We also measured ADCC for the primary immune correlates analysis of a recent HIV-1 vaccine clinical trial in Thailand (RV144) that reported modest vaccine protection (31%). There was a nonsignificant trend towards lower risk of infection among vaccinees with high versus low relative ADCC activity. However, Env-specific IgA correlated with risk, prompting an analysis stratified by IgA levels. Among vaccinees with low Env-specific IgA, there was lower risk of infection among those with higher ADCC activity. These observations suggest that antibodies that direct ADCC may contribute to vaccine protection against SIV and HIV-1 infection.

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