published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

No description available.

The goal of my dissertation research is to elucidate the population structure of two understudied but increasingly important fungal pathogens of humans. Coccidioides immitis and C. posadasii cause the disease coccidioidomycosis (Valley fever). These fungi occur in the soil of the desert regions of North and South America. Although studied for over 100 years, the primary host, ecological niche, and sexual cycle of Coccidioides spp. still remain unknown. Understanding the population structure of these fungi will permit identification of fundamental aspects of their ecology and allow researchers to identify potential hosts. Assessing genotypic diversity of pathogens is one step to understanding the population structure and evolutionary potential of organisms, and is the focus of this dissertation. The first appendix focuses on developing and evaluating methods to obtain environmental samples, and comparison of genotypes found in soil vs. human patients. Direct inoculation of mice proved to be the most reliable method of obtaining environmental strains. Environmental isolates from Tucson group with Arizona patient isolates. Comparing genotypes of human, environmental and non-human host strains of Coccidioides may help to determine if gene flow occurs over long distances and provide some indication of the population structure of C. posadasii in the environment, and is the focus of the second appendix. Finally, whole-genome sequencing and resequencing has been completed for 20 strains of C. immitis and C. posadasii. The resulting data provide greater insight into variation between and within species. In particular, the final appendix provides evidence for hybridization and gene flow between species. Data show that a region of C. posadasii origin is found at a higher frequency among the C. immitis southern California and Mexico patient isolates, and is found rarely among patient isolates from the San Joaquin Valley. Of particular interest is the fact that there is a conserved border region for all instances of introgression, and the gene immediately adjacent to this border is a metalloproteinase gene. Together these studies provide insight into the population biology of two human pathogenic fungi: gene flow is limited between species and populations, but genetic exchange occurs at all levels.

Listeria monocytogenes is a human and animal pathogen responsible for severe and sometimes fatal infections. Several outbreaks have been associated with contaminated commercial foodstuffs such as raw milk, soft cheese, fresh and frozen milk, poultry, seafood, fruits and vegetable products. Currently, the official method recognized by the Government of Canada for the detection and isolation of L. monocytogenes can take up to six days without confirmation, which can require two more days. An approach based on molecular beacons that fluoresce upon hybridization was developed and tested to detect L. monocytogenes and the genus Listeria in food. Two different beacons were created: one specific to species L. monocytogenes (MG1) and another for the genus Listeria (MG2). Each of these molecular beacons was used with two separate sets of primers: MG1-1f/MG1-2r, MG1-7f/MG1-4r, MG2-2f/MG2-2r and MG2-3ft/MG2-2r. (Abstract shortened by UMI.)

Candidate virulence genes were sought through the genetic analyses of two strains of Mycobacterium tuberculosis, one virulent, H37Rv, one attenuated, H37Ra. Derived from the same parent, H37, genomic differences between strains were first examined via two-dimensional DNA technologies: two-dimensional bacterial genome display, and bacterial comparative genomic hybridisation. The two-dimensional technologies were optimised for mycobacterial use, but failed to yield reproducible genomic differences between the two strains. Expression differences between strains during their infection of murine bone-marrow-derived macrophages were then assessed using Bacterial Artificial Chromosome Fingerprint Arrays. This technique successfully identified expression differences between intracellular M. tuberculosis H37Ra and H37Rv, and six candidate genes were confirmed via quantitative real-time PCR for their differential expression at 168 hours post-infection. Genes identified to be upregulated in the attenuated H37Ra were frdB, frdC, and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE, and Rv1571. Further qPCR analysis of these genes at 4 and 96h post-infection revealed that the frd operon (encoding for the fumarate reductase enzyme complex or FRD) was expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 was higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of culture when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Furthermore, inhibition of the fumarate reductase complex in intracellular bacteria resulted in a significant reduction of intracellular growth. Microarray technology was also applied in the expression analysis of intracellular bacteria at 168h post-infection. Forty-eight genes were revealed to be differentially expressed between the H37Ra and H37Rv strains, and a subset were further analysed via qPCR to confirm and validate the microarray data. phoP was expressed at a lower level in the attenuated M. tuberculosis H37Ra, whereas members of the phoPR regulon were up-regulated in the virulent H37Rv. Additionally, a group of genes (Rv3616c-Rv3613c) that may associate with the region of difference 1 were also up-regulated in the virulent H37Rv.

The androgen receptor (AR) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. Mutations in the androgen receptor are associated with androgen insensitivity syndrome (AIS), and a neurodegenerative disease, spinal bulbar muscular atrophy (SBMA). Most of the mutations causing AIS are loss-of-function missense mutations whereas SBMA is caused by a gain-of-function polyglutamine expansion in the N-terminal domain of the protein. Characterization of AR mutations has led to a better understanding of structure-function relationships of the AR and serves as a prototype for steroid receptors mechanisms of action. / In the first paper, we examine the role of an AR mutation in causing mild androgen insensitivity syndrome. We found that this mutation conferred reduced transactivation by AR through impaired interactions with the AR coactivator, TIF2, and impaired homodimerization. / In the second paper, we investigate the role of the AR polyGln expansion mutation in SBMA pathogenesis. Recent evidence has implicated proteolytic degradation of polyGln-expanded proteins and their subsequent intracellular aggregation in polyGn-expanded disease pathogenesis. We examined the role and composition of aggregates using fluorescently-tagged AR and found that proteolysis need not be a prerequisite for aggregation and that aggregation is not necessary for poly-Gln-induced cellular toxicity. / Finally, we characterize the novel heterodimerization of AR and ERalpha. We determined that this direct interaction has functional implications for the transactivational properties of both receptors.

Malaria is one of the most serious human infectious diseases. To date, the collection of studies suggest that the disease is determined by transmission dynamics and host age altogether with host genetics and immunological responses. The precise and direct contribution of parasite components to the activation of such immunological responses has not been fully unravelled. In addition to a role proposed for plasmodial GPI, different lines of evidence suggest that hemozoin (HZ) could also be a potential inflammatory agent. The role of HZ in the modulation of immune responses has remained a polemic subject, making it difficult to describe the contribution of this molecule in pathogenesis of malaria. However, our previous laboratory studies, suggest that HZ has a pro-inflammatory role. For this reason, our study was designed to further define the contribution of HZ to the pro-inflammatory events related to malaria immunopathology, and to identify the intracellular signals underlying the up-regulatory effects of HZ in the macrophage, one of the major sources of inflammatory mediators in malaria. In order to do that, we used a chemically characterized synthetic version of the native PfHZ, rcHZ; and evaluated its effects on macrophage nitric oxide (NO) production. Our first approach was to compare the effects of rcHZ with other morphologically different versions of this molecule (aHZ and scHZ) alone or in combination with IFN-gamma on macrophage NO production. In a second approach, we evaluated if the presence of serum proteins plays a role in the increased IFN-gamma induced-NO production by rcHZ. In the third part of our study, we explored if rcHZ is able to increase NO production by macrophages when incubated in combination with a molecule from another pathogen, such as gram-negative bacteria lipopolysaccaride (LPS). The present study is a functional study that uses a synthetic and morphologically identical version of the native PfHZ. Our results suggests that intrinsic physical characteristics, such as shape and size; presence of host serum proteins, and presence of other pathogenic molecules, are important determinants for the macrophage response to HZ in the context of NO production. Besides, it describes part of the signaling pathways that are involved, which may contribute in the future, not only to understand mechanisms of regulation; but also, to find new therapeutic targets against malaria.

The pathogenesis of the enteric protozoan parasite Entamoeba histolytica remains poorly understood. Moreover, the host responses during the early periods of interaction in the gut remain to be clarified. In this study I investigated the cell specific responses to the parasite and the importance of cross talk between epithelial-immune cells that could potentially influence the outcome of infection, with a central focus on Nuclear factor (NF)-kappaB. NF-kappaB is a ubiquitous transcription factor that plays a critical role in mucosal inflammation and its regulation by E. histolytica has not been studied so far. Gal-lectin is a well characterized parasite virulence factor and vaccine candidate. I first characterized the interactions between Gal-lectin and macrophages and found that several proinflammatory genes are upregulated as early as 2h. The Gal-lectin activated NF-kappaB and up-regulated Toll like receptor-2 expression in an NF-kappaB- and p38 Mitogen Activated Protein (MAP) kinase-dependent manner. As intestinal epithelial cells (IEC) form the first line of active host defense against mucosal pathogens, I determined the interaction between ameba soluble proteins and naive IEC. I observed that the parasite could elicit a chemokine response via activation of PI3 kinase and phosphorylation of p65 subunit to induce monocyte chemoattractant protein-1. The consequent recruitment of immune cells could be responsible for colonic inflammation. Finally, I made the novel observation that in macrophage-primed IEC, ameba proteins elicited a cytoprotective stress response. Using a combination of siRNA and over expression studies, I demonstrated that amebic proteins increased the expression and phosphorylation of Heat shock protein (Hsp) 27 thereby enhancing its association with and subsequent inhibition of Inhibitory kappaB kinase (IKK). The resulting inhibition of NF-kappaB could be a potential mechanism that explains the absence of inflammation in the majority of infected individuals. Taken together, the findings of this study open up a new facet in the pathogenesis of amebiasis and unravel a novel paradigm to study host-parasite interactions in the gut.

No description available.

No description available.