Genome-wide studies have suggested that NRF1 regulates transcription of ~5-6% of human genes, including nuclear genes encoding mitochondrial products. My thesis focus is in neural systems in which NRF1 is a master regulator. Prader-Willi syndrome (PWS) results from genetic loss of function of an imprinted domain in human chromosome 15q11.2. I confirmed NRF1 regulation of ~83% of PWS-region genes using chromatin immunoprecipitation (ChIP). Further studies focused on evolution of this region. Uniquely in marsupials, SNRPN and the ancestral SNRPB gene are adjacent each with an intronic snoRNA paralog. Based on molecular phylogenetics, a model is proposed for origin of each PWS snoRNA from a single ancestral snoRNA. Thus, most extant eutherian PWS genes originated by stepwise duplication and divergence over the past ~180 million years. Circadian rhythms regulate organismal physiology in a 24 hour day-night cycle. Functional NRF1 binding sites in promoters/enhancers were found for ~56% of circadian regulatory genes using bioinformatics, ChIP, NRF1 siRNA assays, and luciferase reporter constructs having significantly reduced transcriptional activity on mutation of NRF1 sites. Further, co-immunoprecipitation showed that NRF1 and the phosphorylated, active form of CLOCK interact in a molecular complex. In serum-induced NIH3T3 cells with circadian oscillations of Dbp and Nr1d1 mRNA, Nrf1 mRNA and protein levels show ultradian oscillations. Hence, NRF1 regulates numerous circadian regulatory genes and interacts with CLOCK, suggesting multiple roles in circadian biology. Additional studies included finding that NRF1 regulates ~45% known hereditary spastic paraplegia (HSP) genes, that NRF1 activates its own transcription, and that the number of NRF1 sites determine the degree of transcriptional activation. In summary, NRF1 is a master regulator in PWS, circadian rhythms, and HSP. Identification of NRF1 target genes and mechanisms will lead to an understanding of the evolution, functions, disease processes, and therapeutic targets within gene regulatory networks involving NRF1. Circadian rhythms are disrupted by travel, shift-work, and in illness, including infection, psychiatric and sleep disorders, obesity, diabetes, and cancer. Consequently, understanding body clocks will provide insights into the pathogenesis of these disorders and potentially lead to improved treatment and prevention options, which will have enormous public health impact.

Simply defined, therapeutic gene transfer involves the transfer of genetic material into the cells with the aim of correcting a disease and/or slowing down the progression of a disease. The public health significance is that therapeutic gene delivery carries the hope of curing currently incurable diseases such as cystic fibrosis and Duchenne muscular dystrophy (DMD), or providing better solutions to current treatments for conditions such as chronic pain. There is high expectation in this new generation of medicine that therapeutic gene transfer would rid the patients of the painful conditions and lead them to better lives. One key component of this procedure is the vehicle that has the capability to deliver the therapeutic gene into different tissues efficiently and safely. A number of vectors based on retroviruses, lentiviruses, adenoviruses and adeno-associated viruses have been developed and their efficacy and safety evaluated in animal models and clinical trials. Possessing several advantages, such as its large genome, the ability to transduce both dividing and non-dividing cells, HSV-1 based vectors represent promising candidates for gene delivery. Three types of HSV-based vectors have been developed: HSV amplicons, replication competent vectors and replication defective vectors. Replication defective vectors based on multiple essential immediate early (IE) gene deletions carrying different transgenes have been constructed. However, HSV vectors have disadvantages. The most prominent one is toxicity. To reduce toxicity, more viral genes are deleted in addition to the essential IE genes. One such gene is the one encoding viral regulatory protein ICP0. However, the deletion of ICP0 gene renders the vector highly inefficient in transgene expression although the toxicity is lowered. Thus, how to restrict the toxicity while retaining the expression is one of the issues that needs to be addressed in HSV vector development. One possible solution is to lower ICP0 level through mutations in the ICP0 promoter region. To test this method, activities of reporter gene driven by mutated ICP0 promoter were assayed and the results showed that deletions in the promoter region did reduce ICP0 expression. However, the optimal level to meet the goal could not be determined by reporter gene assays. Thus, constructs carrying mutant promoters driving ICP0 coding sequence were created. During the construction of these viruses, the positive control virus, later named JDTOZHERO, was found to carry an unexpected deletion in the 5'-UTR. This deletion gave this virus some unique features that fulfilled our goal, i.e., low toxicity and reasonable expression. This virus is characterized in detail in this thesis.

Thesis (Ph. D.)--Clark University, 1916. / Published also without thesis note. Bibliography: p. 3-7.

Thesis--PlanB (M.S.)--University of Wisconsin--Stout, 2000. / Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 84-91).

Thesis (M.A.)--Denver Seminary, 2000. / Includes bibliographical references (leaves 165-173).

Thesis (Ph. D.)--University of Chicago, 1911. / Includes the t.-p. of the original issue. Includes bibliographical references (p. 104) Also available on the Internet. Also issued online.

Not available / text

Mosquitoes are vectors of many human diseases. The vectorial capacity of mosquitoes is directly a consequence of olfactory driven, host seeking behaviors. Olfaction in mosquitoes is mediated by several gene families which encode the olfactory receptors present in the major chemosensory appendages (antenna and maxillary palp) of the adult mosquito. Here, we employ RNA-seq to elucidate the transcriptional profile of these appendages in the major Afrotropical vector mosquito Anopheles gambiae. By then integrating odorant receptor functional information with the quantitative transcriptional data, we construct a synthetic model of the chemoreceptive capacity of the entire mosquito antennae. This model is then assessed against known mosquito behaviors to add a phenotypic dimension to the models. In all we conclude that olfactory receptors in the peripheral olfactory system are subtly dynamic based upon the physiological state of the mosquito. Moreover, these subtle differences are also manifest when compared to a closely related sibling species, and the resulting model is in agreement with known phenotypic differences between the species. In all we have added further evidence to the generalizable notion that peripheral odor coding at the level of olfactory receptors is sufficient to explain many observable differences in olfactory-driven behaviors.

Cardiovascular disease (CVD) is the leading cause of death worldwide. The vast possibilities of interaction between genetic and environmental factors that contribute to CVD can be simplified by identifying conditions that favor the emergence of specific risk factor networks. If the relationships among CVD risk factors that give rise to these networks are under genetic control, then such relationships can be considered heritable phenotypes in themselves, amenable to genetic analysis. We characterized correlational networks of cardiovascular risk factors in a large cohort of urban and rural men and women in Ghana, and investigated how they may be perturbed by factors such as sex and urban lifestyle. We also assessed the comparative relevance of individual risk factors to thrombosis within and across networks, using as a proxy their association with an intermediate phenotype of CVD, plasminogen activator inhibitor type-1 (PAI-1). We found that the relationships between risk factors and PAI-1 were far more sensitive to differences in sex and environment than were the relationships among the risk factors themselves. <p> To lay the theoretical groundwork for our subsequent genetic analyses, we modeled multiple types of biological SNP-by-covariate interactions and derived the statistical parameters to which they should give rise. In doing so, we demonstrated that even the strongest gene-by-covariate interactions at the biological level could display weak statistical interactions using general linear models. Moreover, we quantified the expected strength of the interaction relative to the marginal effect, depending on the nature of biological interaction. We then developed the ordinal joint interaction model (OJIM), which can not only identify biological SNP-by-covariate interactions where they exist, but also pick up marginal effects and leverage the change in residual correlation induced by marginal effects. In our analyses of the Ghanaian study population, the OJIM had more power than univariate or bivariate analysis to detect lipid SNPs of known biological significance, indicating that context-dependent genetic effects are probably quite common, and that the OJIM can identify them where they exist. We also used the OJIM to interrogate exome-wide data of our Ghanaian study population, and identified genetic variants that may increase thrombotic risk by influencing the covariance between these risk factors and PAI-1.