Thesis (M.S. in computer science)--Washington State University, August 2009. / Title from PDF title page (viewed on Aug. 12, 2009). "School of Electrical Engineering and Computer Science." Includes bibliographical references (p. 80-87).

Thesis (M.Sc.)(Bioinformatics)--University of Pretoria, 2008. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.

Thesis (M.S.)--University of Nevada, Reno, 2008. / "May, 2008." Includes bibliographical references (leaves 38-40). Library also has microfilm. Ann Arbor, Mich. : ProQuest Information and Learning Company, [2008]. 1 microfilm reel ; 35 mm. Online version available on the World Wide Web.

Thesis (M.Phil.) - University of Queensland, 2005. / Includes bibliography.

Thesis (M.S.)--Duquesne University, 2008. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 79-83) and index.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (site viewed on September 12, 2006). PDF text of dissertation: 172 p. : ill. (some col.) ; 2.15Mb. UMI publication number: AAT 3209964. Includes bibliographical references. Also available in microfilm, microfiche and paper format.

The feasibility of using the ascomycetes filamentous fungus Aspergillus oryzae as a host organism for foreign protein expression and secretion was assessed. For this purpose, human interleukin-6 (hIL-6) and the Simian virus 5 haemagglutinin-neuraminidase (HN) were used as model foreign proteins. Expression and secretion of the human interleukin 5 gene have been achieved using vector construction containing the entire A.niger glaA coding region linked to the hIL-6 cDNA through a KEX2-motif encoding DNA. hIL-6 containing expression vectors were introduced into A.oryzae nitrate non-utilising mutant strains nlaD14, niaD200, niaD500 (niaD-) and arginine auxotrophic strain 1560-6 (argB-) by means of genetic cotransformation technology. The A.oryzae niaD gene or the A.nidulans argB or amdS genes were used as the transfer and selection system. A.oryzae nitrate and acetamide prototrophic transformants, containing the hIL-6 DNA sequence, failed to secrete free hIL-6 molecules. Recombinant hIL-6 protein was detected in culture filtrates from A.oryzae 1560-6 arginine prototrophic transformants T1560-gla15 and T1560-gpd1. The results suggested that A.oryzae proteases recognized the KEX2-motif yielding free hIL-6 molecules. However, hIL-6 was found to be selective degraded by A.oryzae proteases compared with the heterologous glucoamylase (GLAA). The incubation time for hIL-6 detection has been shown to vary with the host strain and growth conditions. Hybrid GLAA/hIL-6 protein was also found to be secreted. Improvement in hIL-6 yields were obtained by generating 2-deoxy-D-glucose resistant mutants of transformant T1560-gla15 and by manipulating the host growth conditions including the use of microbial solid state culture. The maximum level of secreted hIL-6 was found to be approximately 4 mg/1 as judged from SDS-PAGE polyacrylamide gels and western blots analysis. The Simian virus 5 Haemagglutinin-neuraminidase (HN) glycosylation, folding and oligomerization characteristics have been well studied in mammalian cells. Therefore, HN is an ideal model protein, a priori, for studying secretion / processing in foreign hosts including filamentous fungi. For such studies the entire HN cDNA sequence (designated HN-DNA fragment) was generated by PCR technology. However, as the HN protein is a membrane bound glycoprotein type II, HN might not be secreted due to its anchor-domain, thus a deletion of the first 108 bp of its cDNA sequence, corresponding to the hydrophobic N-terminal region, was carried out in a second PCR reaction (designated ASSHN-DNA fragment). Both DNA fragments, encoding HN or ASSHN protein sequences, were inserted in-frame downstream of the A.niger glaA coding region in plasmid vector pAN56-30, generating pAN56-HN and pAN56-ASSHN. Additionally, both DNA fragments were inserted in-frame downstream of the A.oryzae amy3 gene present in vector pSTA1210, generating pSTA1210-HN and pSTAX210-ASSHN. These expression cassettes were introduced into A.oryzae 1560- 6, an amylase over-producing industrial strain, by means of cotransformation. The A.nidulans argB and amdS genes were used as transfer and selection systems. The HN protein was found in culture filtrates of arginine prototrophic transformants harbouring the glaA/ASSHN and amy3/ASSHN DNA sequences. HN protein was mainly detected by immunoprecipitation of 50 ml of culture filtrate. Such low expression level do not seem to be related to gene copy number, but rather to proteolytic degradation. Another possibility is that HN was not translated efficiently. Improvement in HN yield was achieved by cultivation on solid state culture in which HN protein was detected in 1 ml aliquot of culture filtrate as determine by western blotting. Nevertheless, the yield of HN protein extracted with 50 ml of sterile distilled water from 10 g rice solid state culture is estimated to be less than 1μg per ml as judged by the western blots.

Trimethylation of histone H3 lysine 27 (H3K27me3) marks facultative heterochromatin, containing silent genes. My research investigated factors that influence the distribution of H3K27me3 in the filamentous fungus Neurospora crassa. The H3K27 methyltransferase complex, PRC2, is well conserved in eukaryotes and consists of four core members: E(Z), EED, SUZ12 and P55. I showed that three of the PRC2 subunits (SET-7, the homolog of E(Z), EED and SUZ12) are required for H3K27me3 in Neurospora, while NPF, the homolog of P55, is only required for a subset of H3K27me3 domains. H3K27me3 is organized into large, gene-rich domains in Neurospora and normally does not overlap with constitutive heterochromatin, which is marked by both H3K9me3 and DNA methylation and bound by heterochromatin protein 1 (HP1). I discovered that loss of HP1 binding results in a genome-wide relocalization of H3K27me3. Specifically, it is lost from many of its normal domains while it becomes associated with much of the genome that is constitutive heterochromatin. This contrasts plant and mouse studies in which the loss of DNA methylation relocalizes H3K27me3. The DCDC complex is the H3K9-specific methyltransferase consisting of DIM-5, DIM-7, DIM-9, CUL4 and DIM-8. Separate deletions of DCDC subunits, with the exception of dim-7, relocalized H3K27me3 to constitutive heterochromatin, presumably due to the loss of HP1 binding. The deletion of dim-7 resulted in the loss of all H3K27me3, suggesting a novel role for dim-7. To look for a recruitment signal for PRC2, I moved large fragments contained within an H3K27me3 domain to loci devoid of H3K27me3, his-3 and csr-1. None of the fragments induced H3K27me3, demonstrating that a recruitment signal is not present within every fragment of H3K27me3-marked DNA. Large chromosomal rearrangements had profound effects on H3K27me3 domains, resulting in the loss of some H3K27me3 domains and the formation of others. In Drosophila and mammals, a subset of PRC2 complexes contains the histone deacetylase, Rpd3. A close homolog of Rpd3 in Neurospora, HDA-3, did not appear to be a member of PRC2 in Neurospora. This dissertation includes both previously published and unpublished co-authored material.

The recent advancement of whole-transcriptome gene expression quantification technology - particularly at the single-cell level - has created a wealth of biological data. An increasingly popular unsupervised analysis is to find one dimensional manifolds or trajectories through such data that track the development of some biological process. Such methods may be necessary due to the lack of explicit time series measurements or due to asynchronicity of the biological process at a given time. This thesis aims to recast trajectory inference from high-dimensional "omics" data as a statistical latent variable problem. We begin by examining sources of uncertainty in current approaches and examine the consequences of propagating such uncertainty to downstream analyses. We also introduce a model of switch-like differentiation along trajectories. Next, we consider inferring such trajectories through parametric nonlinear factor analysis models and demonstrate that incorporating information about gene behaviour as informative Bayesian priors improves inference. We then consider the case of bifurcations in data and demonstrate the extent to which they may be modelled using a hierarchical mixture of factor analysers. Finally, we propose a novel type of latent variable model that performs inference of such trajectories in the presence of heterogeneous genetic and environmental backgrounds. We apply this to both single-cell and population-level cancer datasets and propose a nonparametric extension similar to Gaussian Process Latent Variable Models.

Quantitative traits and disease risk in humans are affected by both genetic and environmental factors. Using genome-wide association studies (GWAS) over the past decade, researchers have been successful in finding common genetic polymorphisms that explain a proportion of the variation in many common phenotypes. Despite these significant leaps forward in our understanding, the heritable components of many traits remain largely unaccounted for. A number of explanations as to the “missing heritability” of complex traits and disease risk have been postulated. This thesis addresses some of the unexplained potential sources of heritable trait variation and explores two of its potential causes: low frequency and structural genetic variation. Chapter 1 provides a background to GWAS, what we have learned from them, discusses the different mechanisms of heritability and reviews the potential explanations for “missing heritability” in complex traits. The chapter then describes low frequency and structural genetic variation and how they fit into the spectrum of genetic variation. Chapter 2 describes a study that tests the extent to which low frequency association signals can be discovered through low pass whole genome sequencing when using well-powered gene expression and biomarker phenotypes as model traits. The study then compares these association signals to 1000 Genomes based imputation in the same individuals. Chapter 3 uses methods to detect the structural forms of the human amylase locus with whole-genome sequencing data. The study detects and validates multi-allelic copy number within this region and finds a lack of evidence of a previous association between structural variation of the amylase locus and obesity and body mass index. Chapter 4 scans for rare copy-number variation (CNV) using SNP microarray data from over 120 thousand individuals at 69 sites that were previously identified as being associated with developmental delay. The chapter aims to refine their prevalence in the general population and attempts to understand their relationship with developmental delay and complex traits. Chapter 5 aims to detect large deletions and duplications genome-wide using SNP microarray data in a sample of over 120 thousand individuals where we have power to detect rare copy number events. I used novel approaches to test their association with 204 clinically relevant complex traits to determine their role in the heritability of complex traits. Chapter 6 discusses the findings from the previous chapters within this thesis. I then continue by describing some limitations of this work and explore the potential further directions for future work in this area of study.