Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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Item Growth and development in Toxoplasma gondii(Montana State University - Bozeman, College of Agriculture, 2002) Guerini, Michael NicholasItem Toxoplasma inhibitory factor and interleukin 2 : assessment of their effects on toxoplasma gondii replication within a continuous macrophage-like cell line(Montana State University - Bozeman, College of Agriculture, 1982) Hagemo, Anita SusanItem Proteomic host response to Toxoplasma gondii strains of distinct viruence phenotypes(Montana State University - Bozeman, College of Letters & Science, 2014) Tanaka, Naomi; Chairperson, Graduate Committee: Sandra HalonenToxoplasma gondii is an obligate intracellular protozoan parasite that can infect all warm-blood animals. In humans, it is estimated that one-third of world's population is infected with T. gondii. Most T. gondii strains can be classified into three genotypes; type I, II and III. Type I strains (e.g. RH) are virulent strains with features of rapid growth. Type II (e.g. Me49) and type III strains (e.g. CTG) are avirulent, with slower growth and more readily form cysts in the brain in the chronic infection. In infected host cells, the parasite modulates the host immune system for their successful replication and dissemination. To investigate the host response to three different virulence phenotypes, we proposed to use a two dimensional-differential gel electrophoresis (2D-DIGE) based proteomic approach. Though preliminary studies, it was concluded that Me49 was not appropriate for this study due to their significantly slower growth rate within 24 hours p.i.. Subsequent studies were then done comparing the type I RH strain vs. the type III CTG strain. Using the RH strain, kinetic analysis of the host cell response to infection was done, analyzing samples at 2, 12 and 24 hours p.i.. Few protein changes were observed at 2 and 12 h p.i., and thus only 24 hours p.i. was analyzed via 2D-DIGE analysis. For 2D-DIGE analysis, quadruplicates of protein extracts were separated in 2DDIGE based on pH and molecular weight. For RH strain, we found a total of 439 protein spots were found to be significantly affected by infection, with 247 spots with increased expression and 187 spots with decreased expression and fold-changes ranging from 0.16 to 13.1. For CTG straina total of 175 protein spots were found with significant expression changes. Among them, 48 spots were increased and 127 were decreased with expression changes ranging between 0.66 and 2.41. Interestingly, many decreased spots were seen in trains of spots indicating these may be due to a post-translational modification such as phosphorylation. For the further studies, the identification of protein spots is necessary to elucidate the interactions between host cell and T. gondii strains of distinct virulence phenotypes.Item Studies of gene expression control in the apicomplexan parasite Toxoplasma gondii(Montana State University - Bozeman, College of Agriculture, 2008) Behnke, Michael Sean; Chairperson, Graduate Committee: Michael W. White.Our understanding of global gene expression patterns and control of both developmental and strain specific aspects of Toxoplasma gondii has broadened in the past few years. A global approach was initially undertaken to construct the "transcriptome" for the Toxoplasma intermediate life cycle using serial-analysis-of-gene expression (SAGE). From this analysis, we confirmed the increased expression of known as well as novel mRNAs associated with the tachyzoite-to-bradyzoite transition. Accumulation of bradyzoite specific mRNAs in the bradyzoite SAGE libraries raises the possibility that transcriptional mechanisms play a key role in the developmental switch. To investigate this question, we adapted the dual luciferase model to recombinational cloning in order to construct a high throughput model for testing and mapping multiple bradyzoite promoters. Expression of luciferase from constructs matched previously published results indicating that developmental gene expression in Toxoplasma is controlled by elements contained in the 5'-flanking regions upstream of the protein coding regions. Promoter cis-elements from two genes, BAG1 and B-NTPase, have been fine mapped to 6-8 bp. EMSA assays confirm that these elements form sequence specific protein/DNA complexes. These data suggest that gene proximal cis-elements are required to initiate developmental gene expression, most likely by the binding of gene-specific trans-acting factors. Although strategies to identify these putative trans factors such as yeast one-hybrid are ongoing, we describe here a more global approach to identifying controllers of gene expression. We conducted an expression Quantitative Trait Locus (eQTL) mapping project that combines the power of Affymetrix microarray technology and QTL mapping to explore the genetic basis of differences in stage-specific gene expression in Type I versus III parasite strains and in F1 progeny from the I X III cross. Gene expression QTLs discovered in this analysis were either local or non-local to the associated transcriptional change and reflect proximal cis-mutations, transcription factors or central mechanisms that co-regulate gene expression during tachyzoite differentiation. Additionally, we identified segmental duplication events in various parents and progeny of the Type I X III cross that have a gene dosage effect on the level of gene expression in those parasites.