Browsing by Subject "pathogenesis"
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Item Analysis of the Fungal Virulence of Cryptococcus and Exploration of Novel Antifungals Against Cryptococcosis(2014-08-01) Zhai, BingCryptococcosis is one of the leading causes of the deaths among AIDS patients. The high mortality rates of cryptococcosis are mainly due to inadequate information of its major causative agent Cryptococcus neoformans and the limitations of current therapies. Cryptococcus neoformans is an unconventional dimorphic fungus that can grow either as a yeast or in a filamentous form. To study this dimorphism that is critical to the pathogenicity of many fungi, we constructed the congenic a and ? strains for XL280(?), a strain with robust ability to undergo the yeast-hyphal transition. We compared the congenic strains in different in vivo models and found they are equivalent in virulence. Furthermore, deletion or overexpression of a known transcription factor Znf2 in XL280 abolished or enhanced filamentation and biofilm formation, consistent with its established role. Therefore, the congenic strains provide a new resource for the study of morphogenesis and the related virulence of Cryptococcus. Meanwhile, we searched for novel antifungals by screening of a clinical compound library. Two hits from the screen, the antibiotic polymyxin B and the antidepressant sertraline are all potently fungicidal against Cryptococcus. Polymyxin B works synergistically with the azoles both in vitro and in vivo, thus it may serve as an adjunctive therapy with fluconazole in clinic. Our investigation on sertraline has implicated its unique advantage in treating cryptococcal infections since it is able to traverse the blood brain barrier and reduce the fungal burden in brains. We further examined the fungal target of sertraline and found that this compound inhibits the fungal protein synthesis. Taken together, the studies in this thesis facilitate further research on the pathogenesis of Cryptococcus and provide new antifungal drug candidates with clinical value.Item Extensive investigation of reticuloendotheliosis virus in the endangered Attwater's prairie chicken(Texas A&M University, 2007-09-17) Bohls, Ryan LanierReticuloendotheliosis virus (REV) is a retrovirus that causes a neoplastic disease in a wide range of avian hosts including chickens, turkeys, and ducks. In 1993, REV was detected in the endangered Attwater's prairie chicken (Tympanachus cupido attwateri), a subspecies of Tympanachus cupido. Subsequent infections of this prairie chicken have been identified at captive breeding facilities throughout Texas. The implications of these infections have severely hindered repopulation efforts by these facilities. This study focused on investigating REV infection of captive Attwater'????????s prairie chicken in order to better understand the disease affecting these endangered birds. The overall objective was to develop a means of eliminating this threat to the repopulation of the Attwater's prairie chicken. Several aspects of virus infection were investigated. Reagents capable of recognizing prairie chicken IgY and viral gag polypeptides were developed for use in assays for detection of antibody responses and titration of viral concentrations. Sequencing data of genomes collected from isolates of Texas prairie chickens and domestic chickens, as well as three REV prototype viruses, were compared to determine relationships among strains and identify the potential origin of the REV infecting Attwater'????????s prairie chicken. Additionally, a flow cytometry technique of segregating the lymphocyte population from peripheral blood mononuclear cells (PBMC) using a pan leukocyte monoclonal antibody was developed to more accurately measure changes within lymphocyte populations. This technique combined with intracellular labeling was used to deduce the target cells of REV infection. A nested polymerase chain reaction (PCR) test was developed for greater sensitivity in detecting infection in birds than the previous method of single amplification PCR. This greater sensitivity results in earlier identification of the virus in infected birds, which allows for earlier removal of infected birds to minimize transmission of the virus throughout the flock. The sensitivity of the nested PCR diagnostic test was determined in a dose response pathogenesis study, which was conducted on hybrid greater/Attwater's prairie chicken to observe the experimental development of disease in these birds. Finally, a vaccine was developed using plasmid DNA with REV encoded genes and tested on naturally infected prairie chickens to determine its efficacy in reducing viral load. Although no reduction in viral load was detected, the vaccine may be effective in providing prophylactic protection in future studies.Item PROPERTIES OF THE TOMBUSVIRUS MOVEMENT PROTEIN AND RNAi SUPPRESSOR THAT INFLUENCE PATHOGENESIS(2010-01-16) Hsieh, Yi-ChengTomato bushy stunt virus (TBSV) provides a good model system to investigate molecular virus-host interactions in plants. P22 and P19 proteins encoded by TBSV contribute to multiple invasion-associated functions. Green fluorescence-mediated visualization of TBSV invasion in this study suggests that virus exit from inoculated epidermal cells is a crucial event. Close examination of one P22 mutant showed that it had lost the capacity to move between epidermis and mesophyll which was possibly due to an altered subcellular localization. P19 is a potent suppressor of RNA interference (RNAi) in various systems by forming dimers that bind 21-nucleotide (nt) duplex siRNAs (short interfering RNAs), to affect the programming of the RNA-induced silencing complex (RISC). P19 is attractive for biotechnological and research purposes to prevent RNAi of certain value-added genes in plants. To obtain a good plant-based expression platform, a suppression-active mutant P19 was expressed in transgenic N. benthamiana lines. This is the first example of P19 accumulating to detectable levels in a transgenic plant and initial results suggest it is actively suppressing RNAi. Furthermore, to investigate the correlation between siRNA binding of P19 and its various biological roles, predicted siRNA-interacting sites of TBSV P19 were modified, and the corresponding TBSV mutants were used to inoculate plants. Substitutions on siRNA-contact sites on the central domain of P19 resulted in more severe symptoms in N. benthamiana compared to those affecting peripheral regions. All tested combinations of siRNA-binding mutations were associated with reduced accumulation of total TBSV-derived siRNAs, and loss of siRNA sequestration by P19. Additionally, some modifications were found to cause RNAi-mediated disappearance of viral and host materials in N. benthamiana but not in spinach. In conclusion, exit out of epidermal cells is a key host range determinant for TBSV and particular amino acids on P22 may influence this by regulating the proper subcellular localization. Mutant P19 transgenic plants were successfully established with minor physiological effects to be applied as a platform to study RNAi and to over-express proteins. Finally, a compromised P19-siRNA binding impacts symptom development, systemic invasion, integrity of virus plus host RNA and proteins, and that all in a hostdependent manner.