The drive to exercise voluntarily likely results from complex interactions between genes in many organ systems and various psychological parameters, such as motivation and the perception of fatigue. Reproducible variations in exercise intensity and duration are well established in laboratory rodents, but the genes responsible remain largely unknown. Also, to date, studies addressing the adaptive changes to exercise that might prevent dietary-induced obesity have focused primarily on energy intake and nutrient oxidation/partitioning, as opposed to genetics. We hypothesize that increased voluntary physical activity may be a normal mechanism in certain rodent strains to deter dietary-induced obesity and that in an inbred strain of mice, environmentally sensitive genes must be responsible for observed differences in individual voluntary exercise performance. To study this theory, we have designed a set of experiments that establish an animal model to address whether different gene expression profiles can be detected using microarrays and confirmed with quantitative real-time PCR (qRT-PCR) in distinct exercise phenotypes. We also used the model to address whether dietary manipulations affect voluntary exercise performance in a single strain of inbred mice susceptible to dietary-induced obesity. We determined that animals weaned onto high fat diet exercise at levels significantly higher than those weaned onto low fat diet. These animals were able to maintain body weight and decrease body fat after three weeks of exercise. We also report the results and validation of three microarray comparisons using pooled RNA from the hippocampi of exercising animals. These data suggest that several genes from the HSP 70 family, specifically several molecular chaperones localized to the endoplasmic reticulum, are differentially regulated in running versus sedentary animals at several exercise time points. We suggest that increased voluntary physical activity may be an adaptive response in male C57Bl/6J mice that prevents dietary-induced obesity on high fat diets, and we demonstrate that differential gene expression profiles related to exercise could be identified in the brain using microarrays and qRT-PCR. We conclude that genes from the molecular chaperone family, a well-described environmentally sensitive gene family, are differentially regulated in response to voluntary exercise in an inbred mouse strain.

The SARS-Coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. Investigations, described herein, have focused on the genetic manipulation of the SARS-CoV S glycoprotein in order to delineate functional domains within the protein. This was accomplished by incorporating single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl terminal truncations into the protein and investigating these mutants in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or adjacent but distinct regions, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulate the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The cysteine rich domains were further studied by constructing cysteine cluster to alanine mutants in order to ascertain their importance in the function of the protein. Results showed that the two cysteine clusters proximal to the transmembrane region were vital in the functioning of the spike protein in mediating cell-to-cell fusion. Mutagenesis of the acidic amino acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino acid motif may play a functional role in the retention of S at cell-surfaces. A panel of truncations for Bovine Coronavirus (BCoV) S was also constructed and compared to truncations made for the SARS-CoV S glycoprotein. It was found that the two sets of truncations had very little comparable effects on protein function when compared to one another. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression and cell fusion.

Increased expression of cytokines and chemokines in the central nervous system (CNS) is closely associated with the development of retroviral-induced neurological diseases such as HIV-associated dementia, as well as other neuropathologies such as Alzheimers Disease and Multiple Sclerosis. The specific functions of many of these pro-inflammatory factors have yet to be elucidated in the disease process, and it is unclear whether the nature of their effects is protective, pathogenic, or both. Additionally, current models of chemokine function have inherent limitations, with direct injection resulting in a brief response that doesnt accurately represent the effects of chronic production, and transgenic mice constitutively expressing chemokines by a large percentage of intrinsic brain cells from inception. This study takes the first step in developing a more representative in vivo system by which the direct of effects of the over-expression of individual CNS chemokines may be studied. C17.2 neural stem cells (NSC) were transduced with retroviral vectors containing the genes for CCL2, CCL7, CCL12, or the vector alone, and were inoculated into mice to generate a more accurate representation of the limited chemokine producing cell population that is seen with disease. The in vivo data generated herein suggests that this system is capable of consistently expressing comparable levels of these three chemokines, and characterizes the migration patterns of these cells into the major regions of the CNS. As no significant glial activation was seen concurrent with CCL over-expression, this study provides support for idea that the CCL chemokines may be involved in priming the CNS immune response, rather than its direct initiation. Additionally, ancillary histopathological data suggests a possible role for CCL12 in the development of spongiform lesions in the presence of recombinant retrovirus infection.

Brugia infections occur via the bite of an infected mosquito. Third stage infective larvae (L3) deposited on the skin during feeding migrate into the bite wound, through skin and into the lymphatic system. It is hypothesized that L3 excretory/secretory products (ES) are important in this initial phase of the infection. A model for these early migrations has been established by inoculating L3s into the dermis (ID) of the permissive gerbil host. In this model, most L3s injected ID in the louer hind limb travel to the popliteal lymph node by 3 days post infection. Adult parasites are located primarily in the spermatic cord lymphatics by 28 days post infection. L3s injected into the peritoneal cavity (IP) do not migrate, thus ES may play a different role in these infections. Knowledge is lacking on the role of L3 ES in B. pahangi migration and establishment. Proteins in 24 hour L3 ES may facilitate early L3 migration and antibodies to ES may inhibit migration and/or worm establishment. Migration inhibition was assayed in vivo by immunizing gerbils with either 24 hour L3 ES in RIBI adjuvant or RIBI alone. Gerbils were subsequently challenged either ID or IP with 100 L3s and euthanized at 3 and 106 days post infection. Western blot analysis indicates that antibodies in prechallenge sera are produced against ES and share homology with antigens in other B. pahangi stages. ES immunization increased L3 recovery in both ID and IP infected animals at 3DPI. No difference was noted at 106DPI. ES immunization also reduced L3 migration in ID infected gerbils at 3DPI. At 106DPI, immunized animals showed fewer circulating microfilaria and intralymphatic thrombi. At 3DPI, the increase in worm recoveries following immunization may be associated with a decrease in larval migration. The results also suggest that antibody to ES is insufficient to provide protection at both 3DPI and 106DPI. Nonetheless, this response appears to limit the fecundity of adult worms and subsequent formation of intralymphatic thrombi.

In herd health studies, the mixed effects logistic regression model with random herd effects are commonly used for modeling clustered binary data. These models are well developed and widely used in the literature, among which is the logistic-normal regression model. In contrast to the rich literature in modeling methods, the sample size/power analysis methods for such mixed effects models are sparse. The sample size/power analysis method for the logistic-normal regression model is not readily available. This study is to develop a power analysis/sample size estimation method for the logistic-normal regression model. Extended from the sample size method for the likelihood ratio test in the generalized linear models (Self et al., 1992), a power analysis method for the logistic-normal model is developed based on a noncentral chi-square approximation to the distribution of the likelihood ratio statistic. The method described in this dissertation can be applied to both exchangeable and non-exchangeable responses. The power curves are presented with respect to the change of each of the planning values while holding other planning values fixed for two examples of the logistic-normal model containing one random cluster effect. The results from this proposed sample size/power analysis method for the logistic-normal model were compared to the results from the method for the fixed effects logistic regression model. For a given total sample size and the same applicable planning values, the power for the logistic-normal regression model is smaller than that for the fixed effects logistic regression model, suggesting that the minimum required sample size calculated from using the method for the fixed effects model is too small to achieve the desired power when the logistic-normal model is to be used in data analysis.

Extracts of adult Haemonchus contortus were purified and used as a vaccine against the blood feeding parasite in goats as previously studied in sheep. The proteins used were H11 and H-gal-GP, hidden gut antigens from the microvillar membrane of the gut of the worm and combined with Quil A as adjuvant, then administered to a group of goats kept on concrete then artificially infected with H. contortus. The control group received Quil A injections and also infected. This study was performed to analyze the effects of the H11/H-gal-GP vaccine when given to goats as compared to sheep. The trial showed that IgG levels peaked three weeks after the first vaccine and remained high throughout the remaining booster series but began to wane after artificial infection. However, the IgG levels remained significantly higher than in the controls throughout the entire study. Overall mean fecal egg counts (FEC) were significantly higher in the controls and packed cell volume levels were significantly higher in vaccinated goats compared to controls from Week 3 post infection to the end of the study. A booster vaccine given Week 7 pi caused a sharp increase in IgG levels, elimination of worm burdens and decrease in FEC in the vaccinated group. 96% fewer H. contortus adults recovered at necropsy in vaccinated group compared to controls and >96% reduction in FEC after booster vaccine given during established infection. This study shows that the H11/H-gal-GP vaccine was sufficient in protecting goats after challenge infections but is shorter lived than when given under the same conditions in sheep. Booster vaccine given when infection levels are rising are effective in eliminating infections, reducing FEC and therefore may be used in place of an anthelmintic to control haemonchosis in goats as in sheep.

Racial and socioeconomic disparities persist throughout the country regardless of which specific disparity is studied; however, some geographic regions experience more significant racial disparities. East Baton Rouge Parish, Louisiana has a large racial disparity among birth outcomes. Many factors impact the degree of racial disparities, some of which include racial segregation, isolation, or centralization, access to and quality of medical care, and factors of the neighborhood environment. EBRP has undergone dramatic shifts in the demographics of its residents. The purpose of this dissertation was to study the demographic changes in the population; as well as to study the disparities among the birth outcomes of infant mortality, low birthweight, and preterm delivery. To conduct this study, GIS and multilevel analysis were heavily utilized. Census data from 1970, 1980, 1990, and 2000 census periods were used to determine demographic changes in the parish of East Baton Rouge. The analysis found that EBRP is becoming more racially and economically segregated as the surrounding parishes have increased in population and become flourishing suburbs of the city of Baton Rouge. EBRP has experienced all five dimensions of segregation: unevenness, isolation, clustering, centralization, and concentration. Throughout the thirty year study period, the inner-city area of EBRP has continuously become more populated by poor black residents, and the area directly surrounding the inner city has also changed from a predominately white middle-class area to a predominately black middle-class area. A total of 75,170 birth certificates records from the years 1990 through 2001 were available for analysis. Eight separate multilevel regressions were conducted using data from two census periods at the block group and tract geographic area for both preterm delivery and low birthweight. The multilevel regressions showed that the amount of racial and socioeconomic segregation in the block group or tract were significant in the model. The higher the percentage of black residents in the census area, the more likely a woman is to deliver preterm or a low birthweight infant. A correlation analysis found that high poverty and high percentage of black residents found the two variables to be highly correlated.

West Nile virus (WNV), a member of the genus Flavivirus transmitted by mosquitoes, first appeared in the New York in 1999. Within five years WNV was detected throughout the contiguous 48 states causing disease in reservoirs and accidental hosts alike. In Louisiana, WNV was first detected in 2001 with one human case, ten equine cases, and six dead birds reported. The introduction of WNV into Louisiana presented an unique opportunity to observe an emerging disease unfold, so a study was launched to gain insight into the epidemiology of WNV in Louisiana. The first component, an environmental predictive model for West Nile virus, was developed using geographic information systems and remote sensing in relationship to the prevalence of human cases and the percent of WNV positive dead birds by parish for 2002 and 2003. Linear regression analysis showed a 13 variable model with environmental and human factors for the 2003 human dataset to be the best model. This model was able to explain 74% of the variation in human WNV prevalence by parish. The results of the model along with one-way chi-square analysis of categorical variables indicated largely urban cycle when the mosquito-bird transmission cycle reaches high levels as the main mode of WNV transmission with spillover to humans, and other accidental hosts. A serosurvey of wild birds in East Baton Rouge Parish was conducted from November 2002 to October 2004. A total of 1287 samples were tested by the plaque reduction neutralization test. Overall, 222/1287 (17.25%; CI: 15.19-19.31) tested positive. Species, location, sex, age, and monthly differences were detected. The study identified Northern cardinals (Cardinalis cardninalis) as a statistically significant host for WNV in Louisiana. Mediterranean house geckos (Hemidactylus turcicus) were assessed as a potential reservoir for West Nile virus. Geckos were inoculated orally with West Nile virus and a field study was conducted to determine the prevalence of WNV in naturally infected geckos. Results obtained through virus culture and RT-PCR indicated that geckos could become infected with an oral inoculation of WNV, but that naturally infected geckos do not produce high enough viremias to act as a reservoir.

Microglia/macrophage activation has been associated with the pathogenesis of various neurological diseases including human immunodeficiency virus encephalitis, transmissible spongiform encephalitis, and Alzheimer's disease (AD). In vitro studies have indicated a role for TNFα in activating these cells which leads to their migration, proliferation, and secretion of proinflammatory cytokines and chemokines that may potentially damage brain tissue. In the current study, we analyzed the phenotype of microglia and macrophages enriched from wild type and TNFα deficient mice infected with a neurovirulent murine retrovirus. Although TNF receptors CD120a and CD120b were expressed on both microglia and macrophage population, unaltered by either retrovirus infection or TNFα deficiency. To determine if hindering microglia/macrophage activation and TNFα expression during an established viral infection would impede the development of neurological disease, we treated mice with minocycline which has been reported to inhibit both microglia activation and TNFα production. Despite the decreased expression of certain genes involved in TNF signaling and microglia/macrophage activation, there was no delay in onset of neurological disease between PBS and minocycline treated EC infected mice. mRNA expression for accessory molecules involved in the TNF Superfamily signaling was significantly reduced with minocycline treatment. Understanding how to better manipulate these pathways could lead to ways to decrease the severity of neurological disease in not solely this model but others in which they has been directly linked to pathogenesis.

An Edwardsiella ictaluri strain with disruption of ureG was identified through the use of signature tagged insertion mutagenesis as being attenuated for virulence in the channel catfish host. Sequencing of the flanking regions surrounding the insert showed that the gene was part of a urease gene complex that included ureE, ureF, ureG, ureD, ureI, and an ammonium transporter homologue. The ureG gene encodes a GTP-binding accessory protein which is thought to function in energy-dependent urease assembly. The ureG mutant strain was found to be attenuated for mortality, persistence, and for the ability to establish infection in a competition challenge during co-infection with the wild type (WT) strain. In an experimental infection of channel catfish macrophages, the ureG mutant strain was attenuated for intracellular replication while the WT strain showed more than a ten-fold increase in numbers of viable organisms recovered at 12 hours post infection (PI), even though there were no significant differences in initial uptake of either strain. Light microscopy of prepared cell culture slides at 8 and 12 hours PI showed macrophages containing large numbers of WT bacteria, confirming the replication of the WT strain. A gentamicin exclusion assay performed with macrophages treated with 6 mM urea revealed that numbers of WT recovered from macrophages treated with urea was more than twice that recovered from macrophages without urea. Survival in macrophages requires the ability to tolerate or alter the acidic environment of the phagolysosome. Growth curves performed at acidic pH and survival assays following extreme acid shock indicate that E. ictaluri is naturally acid resistant and is able to utilize urea to enhance growth and replication at acidic pH by the neutralization of environmental pH. Two-dimensional gel analysis of WT cell lysates, prepared following growth at neutral and acidic pH, identified three urease proteins, UreA, UreC, and UreG, that were uniquely expressed during growth at acidic pH, indicating that expression of these proteins is acid inducible. The results presented here confirm the importance of the urease enzyme complex in virulence, intracellular survival, and acid resistance of E. ictaluri despite its "urease negative" characterization in traditional biochemical tests.