Thesis (Ph.D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (January 25, 2007) Vita. Includes bibliographical references.

Thesis (Ph. D.)--Illinois State University, 2005. / Title from title page screen, viewed September 26, 2006. Dissertation Committee: Anthony J. Otsuka (chair), Radheshyam Jayaswal, Kevin A. Edwards, David L. Williams, Hou Tak Cheung. Includes bibliographical references (leaves 110-124) and abstract. Also available in print.

Thesis (Ph. D.)--Illinois State University, 1996. / Title from title page screen, viewed May 26, 2006. Dissertation Committee: Anthony J. Otsuka (chair), Herman E. Brockman, Alan J. Katz, David F. Weber, Brian J. Wilkinson. Includes bibliographical references (leaves 177-191) and abstract. Also available in print.

München, Techn. Univ., Diss., 2004.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 111-119).

Master of Science / Division of Biology / Michael A. Herman / Stenotrophomonas maltophilia is an opportunistic bacterial pathogen found ubiquitously in the environment. Although S. maltophilia is an emerging pathogen associated with hospital-acquired infections in patients with respiratory diseases, particularly cystic fibrosis, very little is known about its mechanism of pathogenesis in any system. In addition, S. maltophilia isolates vary in pathogenicity to several hosts and are genetically diverse, including variation in virulence factors. In this thesis, I address the genetic basis of S. maltophilia pathogenesis from both host and bacterial perspectives. Our lab has previously developed Caenorhabditis elegans as a model for S. maltophilia infection. Stenotrophomonas is found in relatively high abundance in the microbiome of C. elegans, making it a suitable platform for studying S. maltophilia-host interactions. I performed a transcriptomic analysis to determine C. elegans responses to several S. maltophilia strains of varying pathogenicity. Treatments included K279a, an avirulent clinical isolate, JCMS, a virulent environmental strain isolated in association with nematodes near Manhattan, KS, and JV3, an even more virulent environmental isolate. Overall, I found that most genes (89%) that are differentially expressed in response to pathogenic S. maltophilia strains are upregulated, with many even further upregulated in response to the more virulent strain, JV3. Using information from a variety of transcriptomic datasets, I found that most of these genes are also commonly differentially expressed in C. elegans in response to other pathogens. Many more genes were differentially expressed specifically in response to JV3 when compared to all other strains (221 genes) than JCMS as compared to all other strains (14 genes), suggesting JV3 has unique virulence mechanisms that could explain its observed increased virulence. Candidate genes were chosen from the above differentially expressed gene sets (differentially expressed in response to both pathogenic S. maltophilia strains or in a strain-specific manner) for functional analysis. Mutational analysis of these candidate genes revealed that several mutants caused increased susceptibility of C. elegans to pathogenic S. maltophilia, regardless of the strain(s) that caused differential expression of that gene. Furthermore, many of these mutants also caused increased susceptibility to K279a, suggesting that K279a may also employ virulence mechanisms that wild-type C. elegans are able to defend against. To address the pathogen side of the interaction, we analyzed draft assemblies of the S. maltophilia strains, with the addition of another slightly pathogenic environmental strain, R551-3. We hypothesized that differences in observed pathogenicity and host responses to strains of S. maltophilia could be explained by differences in their genomes. When comparing draft assemblies to their respective reference genomes, few differences were observed. However, several genomic features were present in some strains and absent in others, including components of the CmeABC efflux pump and the Type IV secretion system, that might play a role in different virulence mechanisms. Genome-wide comparison of shared and unique genetic features across many S. maltophilia strains revealed that most S. maltophilia genes are strain-specific, suggesting that many potential virulence factors are unique and have yet to be functionally analyzed. Overall, variation in observed pathogenicity, differences in host transcriptional responses, and comparative genomics of S. maltophilia strains reveal that strain-specific mechanisms play important roles in S. maltophilia pathogenesis.

Made available in DSpace on 2014-06-11T19:30:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-15Bitstream added on 2014-06-13T19:00:31Z : No. of bitstreams: 1 demant_car_dr_botfca.pdf: 567683 bytes, checksum: 020907ed10052d4741c5f2e1c5861dfb (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A mamona pode ser utilizada para mais de 700 finalidades diferentes, porém a sua toxidez é o limitante principalmente para o uso restrito da torta originada após a extração do óleo. Para as análises utilizou-se um organismo com o modo de reação a medicamentos e toxinas semelhante ao ser humano, o nematóide Caenorhabditis elegans, o qual tem sido amplamente utilizado pela indústria farmacêutica. O trabalho teve inicio com ensaios preliminares os quais se utilizaram culturas de C. elegans. Estas foram expostas às toxinas que foram colocadas em pequenas cavidades no meio de cultura, porém, devido à falta de praticidade do método, à difícil leitura e sua degradação pela E. coli, passou-se a realizar os testes em placas de 24 poços transparentes contendo apenas água, o nematóide e a toxina. Os resultados mostraram que o teste utilizando placas de 24 poços para exposição do nematóide, e a extração feita através da homogeneização, seguida por banho-maria seguida por centrifugação se mostrou eficiente para medir a ricina sendo semelhantes aos obtidos pelos testes de radioimunodifusão e Elisa, testes bastante específicos, porém de custo mais elevado. O presente trabalho teve por objetivo desenvolver um bioensaio para quantificar a ricina tóxica da mamona, pois a relação entre as análises realizadas por diferentes métodos de quantificação existentes, nem sempre refletem a toxidez real, podendo ocorrer que sementes analisadas por estes métodos, apresentem menor teor de ricina, embora sejam mais tóxicas do que outras sementes que tenham maior teor de ricina quando analisada pelo método. Isto se deve aos métodos empregados que medem além da ricina o RCA Ricinus Communis Aglutimina, um composto menos tóxico do que a ricina pura. / The castor oil can be used to 700 different products but its toxicity limits mainly the use of the castor bean cake, the product you have after extract the oil. The present reseach developed a bioassay to quantify the ricin content on castor bean seed because the relationship between the analyses performed by different quantifying methods not always reflect the actual toxicity, witch can occur that seeds analysed by these methods have lower ricin levels although are more toxic than other seeds, because when it test not only the ricin but also the complex ricin + RCA Ricinus Communis Algutimin is measured and the RCA is less toxic than the pure ricin. That is why it was used an organism that has similarities in the way the human body reacts to drugs and toxin, choosing to use the Caenorhabditis elegans that has been used to the pharmaceutical industry. This assay started with preliminary assays using C. elegans colonies that were exposed to the toxins placed in small holes did in the media , but it was hard to read and the E. coli degradation, the method changed to the 24 well plates with water, nematode and the ricin. The results showed the method is efficient to measure ricin and very similar to the radio-imuno-diffusion and the Elisa, very specific tests, but expensive with some operational problems.

Le domaine PDZ participe aux réseaux moléculaires à l’origine de fonctions cellulaires touchées lors de pathologies diverses. L’exploration de ce réseau par double hybride a permis d’attribuer de nouvelles fonctions putatives aux ligands protéiques des domaines PDZ du ver Caenorhabditis elegans. Les interactions ont laissé apparaitre une proportion inattendue de ligands atypiques interagissant par une séquence interne. Nous avons ensuite validé fonctionnellement in silico des groupes d’interactions de notre interactome qui forment des micro-réseaux co-exprimés par l’intégration de données de profils d’expression. Finalement, ce travail a permis la construction d’un outil exploratoire, le PIPE (PDZ Interacting Protein Explorer) qui permet de cribler l’ensemble des domaines PDZ du ver à la recherche d’interactions avec une protéine d’intérêt révélant déjà de nombreuses interactions supplémentaires entre domaines PDZ et ligands / PDZ domains allow the organization of molecular networks responsible for cellular functions essential for multicellularity as polarization or transduction of extracellular signals. Exploration of this network by two-hybrid revealed a functional diversity for ligands of Caenorhabditis elegans’s PDZ domains. New putative functions were being observed through GO-terms and an unexpected proportion of internal ligands appeared, confirmed by Co-IP. We then functionally validated in silico groups of interactions that form our interactome microarrays co-expressed by the integration of data from expression profiles. Finally, this work has enabled the construction of an exploratory tool, the PIPE (PDZ Interacting Protein Explorer) that allows screening of all PDZ domains looking for interactions with a protein of interest and had already showed many additional interactions between PDZ domains and ligands

It is well known that tight coordination of cell division timing is essential for proper cell fate specification and tissue growth during metazoan development. However, how cell divisions are coordinated in vivo is largely unknown. In this thesis, a high- content screening was conducted to identify genes responsible for temporal coordination of cell division during Caenorhabditis elegans embryogenesis. A total of 822 genes were depleted using RNA interference (RNAi). The genes were prioritized based on their degree of conservation in human, as well as their roles in development. In addition to RNAi, an experimental pipeline was established, including 3D time-lapse imaging of an RNAi perturbed C. elegans embryos followed by automated lineaging, which allows systematic quantification of division timing of each individual cells up to approximately 350-cell stage. To identify genes with a significant reduction in the asynchrony of division between sister cells (ADS) upon perturbation, average division timings of each cell between at least two replicate perturbed embryos was compared against the average division timings of 92 wild type embryos. It was found that cell fate determinants were not only important for maintaining fate asymmetries, but are also imperative for establishing ADS regardless of cellular context. Hence, the results demonstrate that fate and temporal asymmetries share a common genetic architecture. The temporal coordination appears to facilitate cell migration during fate specification or tissue growth. Given the observation that perturbations of signalling pathways, especially Wnt and Notch pathways, are frequently associated with the ADS, it would be essential to map the exact signalling event that takes place at cellular level which is responsible for a given ADS or fate asymmetry. To this end, a miniMos transposon-meditated transgenic technique was adopted to insert a fusion between GFP and a promoter derived from individual components of the two signalling pathways into the C. elegans genome. The resulting insertion was crossed into a strain expressing lineaging markers, which allows automated lineaging and gene expression profiling to map the expression of each component with single cell resolution. A combination of cellular expression and cell-cell contact data would lead to a comprehensive map of each signalling event during C. elegans embryogenesis. Our preliminary results on cell-cell contacts and cellular expression validated the existing signalling events and identified potential novel ones that are associated with either ADS and/or fate asymmetry.

During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1