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.

This thesis explores aspects pertinent to the phenotypic classification of paediatric patients with inflammatory bowel disease (IBD). In the current era it has never been more important to have rigourous phenotypic classification to facilitate genotype-phenotype correlation studies as well as to optimize design of clinical trials of emerging therapies, where frequently response may differ according to phenotype of disease. The first study examined the reliability of the Montreal Classification for classifying paediatric IBD patients. This is the first study exploring the reliability of phenotypic classification in a paediatric population. The reliability of assigning an overall diagnosis of type of IBD was good, but not excellent. Amongst Crohn’s disease patients, reliability of assigning disease behaviour was excellent, while the reliability of assigning disease location categories varied from fair to good. The percentage agreement when describing disease extent for ulcerative colitis was high. The second study described the evolution of disease phenotype in a cohort of paediatric IBD patients. Similar to observations in adult-onset IBD, disease location was found to be relatively stable, while Crohn’s disease behaviour evolves from an inflammatory to a stricturing and/or penetrating phenotype in 20% of patients by 5 years of follow-up. The final study explored the association between 2 polymorphisms in the NOD2 gene with the requirement for intestinal resection (a surrogate marker of complicated disease) in models that included and excluded disease duration. Although no difference was found, this may have been influenced by data quality, which was suboptimal. In conclusion, this thesis has demonstrated that imprecision exists in the phenotyping of paediatric IBD patients, in whom phenotypic characteristics evolve over time. It is pertinent that disease duration be considered in any study attempting to make phenotypic correlations.

This thesis explores aspects pertinent to the phenotypic classification of paediatric patients with inflammatory bowel disease (IBD). In the current era it has never been more important to have rigourous phenotypic classification to facilitate genotype-phenotype correlation studies as well as to optimize design of clinical trials of emerging therapies, where frequently response may differ according to phenotype of disease. The first study examined the reliability of the Montreal Classification for classifying paediatric IBD patients. This is the first study exploring the reliability of phenotypic classification in a paediatric population. The reliability of assigning an overall diagnosis of type of IBD was good, but not excellent. Amongst Crohn’s disease patients, reliability of assigning disease behaviour was excellent, while the reliability of assigning disease location categories varied from fair to good. The percentage agreement when describing disease extent for ulcerative colitis was high. The second study described the evolution of disease phenotype in a cohort of paediatric IBD patients. Similar to observations in adult-onset IBD, disease location was found to be relatively stable, while Crohn’s disease behaviour evolves from an inflammatory to a stricturing and/or penetrating phenotype in 20% of patients by 5 years of follow-up. The final study explored the association between 2 polymorphisms in the NOD2 gene with the requirement for intestinal resection (a surrogate marker of complicated disease) in models that included and excluded disease duration. Although no difference was found, this may have been influenced by data quality, which was suboptimal. In conclusion, this thesis has demonstrated that imprecision exists in the phenotyping of paediatric IBD patients, in whom phenotypic characteristics evolve over time. It is pertinent that disease duration be considered in any study attempting to make phenotypic correlations.

No description available.

In the first part of this thesis, a general one dimensional theory of strategically determined phenotypes is developed and applied to biological signalling games. Abstract modular modelling techniques are utilised to solve hitherto analytically intractable problems including error-prone signalling, and how to incorporate genetic features into optimization models. Links are drawn between previous biological models, such as the War of Attrition and Strategic Handicap mod- els. Mistakes in previous biological models are recognised and, where possible, rectified. A number of novel insights into biological phenomena arising from the models are presented, including analyses of: when free signals are possible; honest signalling of future paternal investment; dimorphic signals; the effects of the mechanisms of female discrimination in sexual selection on male signalling strategies; and the effects of relatedness on the magnitude and stability of equi- librium signalling strategies. It is argued that Zahavi's proposed demarcation between signal selection and natural selection is unjustifiable from a theoretical perspective. The second part of the thesis concerns the epistatic handicap process of sex- ual selection. Unlike the conditional and revealing handicap mechanisms, the epistatic or 'Zahavian' handicap mechanism of sexual selection has hitherto found scant support in the theoretical literature, as it appeared to function only under the most extreme conditions. A continuous game theory model, a quantitative genetics model, and a three locus major gene model are presented which show that the epistatic handicap mechanism can function, independent of the Fisher process of sexual selection, under reasonable assumptions. More- over, the game theory model illuminates the connection between the strategic and epistatic handicap mechanisms. The quantitative genetics and major gene models, together with a fourth model, are also used to show that a general argu- ment concerning indirect genetic correlations, which has appeared in a number of papers on sexual selection, is specious. Finally, a general theorem on games that satisfy the single-crossing condition (also known as the sorting, Spence-Mirrless, or constant sign condition) which underlies many of the results presented in the first part of the thesis is proven in appendix C. Applying a limit result to this general theorem provides a new proof of, and extensions to, Nash's existence result for equilibria to strategic- form games without having to resort to Kakutani's fixed point theorem.

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.

Doctor of Philosophy / Cystic fibrosis (CF) is the most common inherited lethal disorder among Caucasian populations. Chronic pulmonary infections, particularly from Pseudomonas aeruginosa, are the major determinant of the morbidity and mortality of people with CF. It is generally accepted that people with CF acquire this pathogen independently from their surrounding environment, and that individual CF patients carry unique strains different from others. The spread of this pathogen from patient to patient is thought to be rare and occurs particularly among closely contacted cases such as CF siblings. However, over the past decade, there have been several reports of an emergence of clonal P. aeruginosa strains commonly found infecting a number of CF patients. One such report is from the CF paediatric clinic at the Royal Children’s Hospital in Melbourne in which more than half of the patients were infected with a single strain or clone, subsequently called Australian epidemic strain 1 or AES-1. A preliminary survey showed that AES-1 had spread extensively along the Australian eastern seaboard among CF patients attending other CF centres in Melbourne, Sydney and Brisbane, including adult patients at the Royal Prince Alfred Hospital (RPAH), Sydney. Another clonal strain, subsequently called AES-2, was identified in both CF adults and children at the Prince Charles Hospital and the Royal Children’s Hospital, in Brisbane. The total extent of prevalence of the AES-1 and AES-2 strains at the RPAH as well as the clinical status of patients who carried these strains was unknown. Moreover, the pathogenicity of these two clonal strains had not been investigated. The studies presented in this thesis investigated the prevalence of these clonal strains among CF patients attending the adult CF clinic at RPAH, Sydney by using pulsed-field gel electrophoresis. Overall, 50% of 112 patients with P. aeruginosa were found to be infected with clonal strains. The AES-1 and AES-2 strains were identified in 38% and 5% of the patients respectively. Two new clonal strains, called Sydney-1 and Sydney-2, were also identified. Patients with clonal strains had a significant increase in their number of exacerbations and hospitalisation days, and tended to have lower pulmonary functions when compared to patients infected with non-clonal strains. By using a variety of bioassays to examine the pathogenicity of the clonal and non-clonal strains, it was found that both AES-1 and AES-2 produced more virulence factors and were more resistant to antibiotics when compared to the non-clonal strains. AES-1 and AES-2 were associated with increased production of proteases, including elastase, alkaline protease and protease IV. Overall the results presented in this thesis suggest that there may be a link between virulence and transmissibility of this pathogen. The studies presented in this thesis also compared the biofilm forming capacities of the AES-1 and non-clonal isolates. AES-1 was shown to have greater biofilm-forming capacity than the non-clonal strains, when they were grown on a glass surface, suggesting a possible association between clonality and biofilm formation. A model for the study of bacteria grown in conditions similar to CF sputum was also developed. P. aeruginosa grown in this model was found to develop into clumps which may be comparable to the biofilm structure in the CF lung. This model was shown to be beneficial for transcriptomic and proteomic studies which are underway within the research group. AES-1 was also found to have phenotypic variations between isolates. By applying the amplified fragment length polymorphism technique, more subtypes of this clone were revealed. However, these detected subtypes did not correlate with the different phenotypes, suggesting minor mutations such as single point polymorphisms may be responsible for the phenotypic diversity within the clone. The final part of this thesis was devoted to examining the safety of a novel CF treatment: hypertonic saline (HS) inhalation. HS was shown to increase airway mucociliary clearance, while increased osmolarity associated with the use of HS was also shown to have an inhibitory effect on the formation of biofilms. Findings in this study proved that there was no evidence of strain selection in patients who received the long-term treatment with HS. The study also demonstrated that AES-1 was significantly more persistent in the CF lung than the non-clonal strains. The present thesis not only defines the clonal strains of P. aeruginosa and their implications for infected patients, but also provides a general understanding into the pathogenesis of both clonal and non-clonal strains infecting CF lungs.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 6 uppsatser.

Thesis (Ph. D.)--State University of New York at Binghamton, Department of Biological Sciences, 2007. / Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.