The release of cells from a biofilm to the surrounding environment is poorly understood and the importance of this stage of biofilm development has only recently been realized. A key part of this process is the generation of phenotypic variants in the biofilm dispersal population. This thesis reports on the characterization of biofilm development of Serratia marcescens MG1, the analysis of the biofilm dispersal population, and the identification of the conditions that trigger phenotypic diversification. Furthermore, it provides an insight into the molecular understanding of how phenotypic variation is being generated, and demonstrates the clinical and environmental implications of phenotypic diversification during bacterial pathogenesis and bacterial persistence. Characterization of the microcolony biofilm development of S. marcescens revealed that the S. marcescens biofilm develops through a process involving microcolony formation, hollowing of mature microcolonies, and a sudden biofilm expansion within a very short period (&lt 24h) resulting in an increase in biofilm biomass with a radiation of biofilm structures at days 3 to 4. The biofilm expansion phase consistently correlated to an increase in the number of dispersal variant morphotypes. Studies of variant induction in planktonic cultures and biofilm flow cells demonstrated that phenotypic diversification in S. marcescens is not only a biofilm-specific phenomenon, but also involves biofilm-specific morphotypes. These morphological variants can only be isolated from the microcolony biofilm morphotype and not from the filamentous biofilms, leading to the hypothesis that there is a strong diversifying selection that is specific to the microcolony biofilms. To further explore how these variants were generated, molecular analyses revealed that exopolysaccharides and lipopolysaccharides are important moieties that are involved in phenotypic variation in S. marcescens biofilms. The etk gene, encoding a tyrosine protein kinase within the exopolysaccharide biosynthesis operon, was found to contain single nucleotide polymorphisms (SNPs) that were present in the 'sticky' variants but not in the 'non-sticky' wild-type or the 'non sticky' small colony variants. Furthermore, infrequent-restriction-site PCR (IRS-PCR), BIOLOG metabolic profiling, and gene sequence analyses, suggest that phenotypic diversification in S. marcescens is likely to involve mutational hotspots in specific genes. The biofilm-derived morphotypic variants differed extensively in cell ultrastructure properties, and exhibited specialized colonization and virulence traits, such as attachment, biofilm formation, swimming and swarming motilities, protease production, and hemolysin production. It was also demonstrated that phenotypic diversification contributed to a varying degree of resistance to protozoan predation, and bacterial pathogenecity in Caenorhabditis elegans, highlighting the complexity of the dispersal populations from S. marcescens biofilms. Furthermore, mixed-culture experiments involving multiple variant isolates (with or without the parental wild-type) showed that the persistence and virulence potential of S. marcescens can be synergistically enhanced in the Acanthamoeba castellanii grazing model and in the C. elegans infection model, respectively. This indicates that the different bacterial morphotypes work in concert to provide S. marcescens with enhanced protection against environmental perturbations and a competitive edge during the infection process. It was proposed that phenotypic diversification is not only an integral part of S. marcescens biofilm life-cycle, but also represents an important strategy for bacteria to greatly enhance its survival and persistence in different environments, ranging from aquatic and soil ecosystems, to those of the infected hosts.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 164-176).

Thesis (Ph. D.)--Göteborg University, 2006. / Includes bibliographical references.

Objective: The human immunodeficiency virus type 1 (HIV-1) currently infects over 30 million people worldwide. It uses one of two main co-receptors to infect cells. The primary objective of this thesis is to evaluate genotypic and phenotypic assays for co-receptor usage in the clinical setting and investigate approaches for improvement of these assays. Methods: The concordance of recombinant co-receptor phenotyping assays and the predictive ability of genotype-based methods including the ‘11/25’ rule, position specific scoring matrices (PSSMs), and support vector machines (SVMs) were evaluated in the clinical setting using patient-derived plasma samples. Samples and patient data were evaluated in cross-sectional analyses from a retrospective population-based cohort of HIV-infected individuals enrolled in the HIV/AIDS Drug Treatment Program in British Columbia, Canada. Results: Current implementations of HIV V3 region-based predictors for HIV co-receptor usage tested on patient derived samples are inadequate in the clinical setting, primarily due to low sensitivities as a result of difficult to detect minority species. Recombinant phenotype assays also show discordances when tested against each other on the same set of patient derived samples, raising doubts if any of these assays can truly be considered a ‘gold standard’. Significant associations between clinical progression, viral sequence-based predictors of co-receptor usage and the output of recombinant assays are observed, suggesting that sensitivity can be improved by incorporating CD4% into genotype-based predictors. This is verified with a SVM model which showed a 17% increase in sensitivity when CD4% was incorporated into training and testing. Conclusion: This work in this thesis has exposed the difficulty in determining the co-receptor phenotype in the clinical setting, primarily due to minority species. Although genotypic methods of screening for HIV co-receptor usage prior to the administration of CCR5 antagonists may reduce costs and increase turn-around time over phenotypic methods, they are currently inadequate for use in the clinical setting due to low sensitivities. Although the addition of clinical parameters such as CD4 count significantly increases the predictive ability of genotypic methods, the presence of low-levels of X4 virus continues to reduce the sensitivity of both genotypic and phenotypic methods.

Epithelial-mesenchymal transition (EMT) has a critical role in tumor progression and has been correlated with the basal-like subtype of human breast cancers. Here I report a novel β-catenin binding protein, which I have shown to be expressed in invasive breast cancer and hypothesized to have a role in breast tumor progression. In normal breast tissue, expression is restricted to the myoepithelium while in breast cancer the expression pattern is similar to smooth muscle actin and vimentin. I have demonstrated that silencing of this protein in breast tumor cells reduces migration by over 50 percent. Furthermore, I have identified this β-catenin binding protein as a target of the Snail EMT network and have demonstrated this protein to be a marker of basal-like carcinomas. These results define a role for this novel protein in EMT, as a marker for the basal subtype, and a promising therapeutic target for metastasis inhibition.

Epithelial-mesenchymal transition (EMT) has a critical role in tumor progression and has been correlated with the basal-like subtype of human breast cancers. Here I report a novel β-catenin binding protein, which I have shown to be expressed in invasive breast cancer and hypothesized to have a role in breast tumor progression. In normal breast tissue, expression is restricted to the myoepithelium while in breast cancer the expression pattern is similar to smooth muscle actin and vimentin. I have demonstrated that silencing of this protein in breast tumor cells reduces migration by over 50 percent. Furthermore, I have identified this β-catenin binding protein as a target of the Snail EMT network and have demonstrated this protein to be a marker of basal-like carcinomas. These results define a role for this novel protein in EMT, as a marker for the basal subtype, and a promising therapeutic target for metastasis inhibition.

 Why some species (or strains of a species) exhibit certain phenotypes (e.g. aerobic, anaerobic, pathogenic etc.) while the others do not is an important question to be answered. Apart from the conventional genomic study, studying the metabolism of the two groups of species may discover the corresponding pathways that are conserved in one group but not in the other. However, only a few tools provide functions to compare two groups of metabolic networks which are usually limited to the reaction level, not the pathway level. In this dissertation, a problem named DMP (Differentiating Metabolic Pathway) problem was formed. Given two groups of metabolic networks, it aims at finding conserved pathways exist in first group, but not the second group. The problem also captures the mutation in similar pathways and derives a measurement (p-value and e-score) for evaluating the significance of the pathways. An algorithm, DMPFinder, was developed to solve the DMP problem. Experimental results show that DMPFinder is able to identify pathways that are critical for the first group to exhibit a certain phenotype which is absent in the other group. Some of these pathways cannot be identified by other tools which only consider reaction level or do not take into account possible mutations among species. / published_or_final_version / Computer Science / Master / Master of Philosophy

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published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

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