Thesis (Ph.D.) -- University of Adelaide, Dept. of Physiology, 2001. / Errata pasted onto back page. Bibliography: leaves 193-228.

Thesis (M.C.S.)--Regent College, Vancouver, BC, 1992. / Abstract and vita. Includes bibliographical references (leaves 256-294).

Thesis (M.Sc. (Molecular genetics-genetic engineering )) -- Mahidol University, 1999.

Thesis--Copenhagen. / "Translated from the Danish by Elisabeth Aagesen." "Dansk résumé": p. 253-256. Bibliography: p. 257-266.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 203-218).

Thesis--Copenhagen. / "Translated from the Danish by Elisabeth Aagesen." "Dansk résumé": p. 253-256. Bibliography: p. 257-266.

Background: Lung cancer is the leading cause of cancer-related death and one of the most common cancer types worldwide. Epidermal growth factor receptor (EGFR) has been shown to be an important therapeutic target in non-small cell lung cancer. Kirsten rat sarcoma viral oncogene homologue (KRAS) is a downstream signalling molecule in the EGFR pathway. Lung cancer patients with EGFR mutations respond to tyrosine EGFR inhibitor therapy, in contrast, patients with KRAS mutations do not benefit of such treatment. Methods: This study investigates the frequency of EGFR and KRAS mutations in non-small cell lung cancer patients. Fifty-one lung cancer patients with primary non-small cell lung cancer diagnosed between 1995 and 2005 in the Uppsala-Örebro region were analysed by Sanger sequencing and Pyrosequencing to determine the mutation status of these genes. Results: Five EGFR mutations were found in four patients (8%), two deletions in exon 19, one point mutation in exon 20 and two point mutations in exon 21. KRAS mutations were found in 12 patients (24%), ten codon 12 mutations and two codon 61 mutations. Conclusions: This study confirms previous observations regarding the frequency of EGFR and KRAS mutations in non-small cell lung cancer. Mutations in EGFR and KRAS were mutually exclusive, indicating that both mutations present relevant tumorigenic genomic aberrations.

The integrity and barrier properties of intestinal epithelium are determined by specialized adhesive structures known as intercellular junctions; composed of adherens junctions (AJs), tight junctions (TJs) and focal adhesions that mediate cell-cell and cell matrix interactions, respectively. These two types of epithelial cell adhesions regulate each other during disruption and restitution of the epithelial barrier. Inflammatory cytokines such as interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) are elevated during intestinal inflammation. The most notable effects of IFNγ and TNFα on intestinal epithelial homeostasis involve disruption of apical junctions and attenuation of cell migration. Although molecular mechanisms underlying these effects remain poorly understood, expressional downregulation of different adhesion proteins may play a major role in the cytokine-dependent disruption of the intestinal epithelial barriers. This thesis is based on the hypothesis that inhibition of the protein translation initiation machinery promotes the disruption of the intestinal epithelial barrier and attenuates epithelial restitution during mucosal inflammation. This study was focused on two eukaryotic translation initiation factors, eIF4G1 and eIF4G2, which play essential roles in the regulation of cap-dependent protein translation. Expression of both translation initiation factors was dramatically downregulated in model intestinal epithelial cell monolayers treated with IFNγ and TNFα in parallel to cytokine-induced disruption of the epithelial barrier. siRNA or shRNA-mediated downregulation of either eIF4G1, or eIF4G2 increased permeability of well-differentiated SK-CO15 intestinal epithelial cell monolayers and decreased expression of different adherens junction and tight junction proteins. Furthermore depletion of these translation initiating factors inhibits different modes of migration (wound healing and transfilter migration) of stem-cell like and well-differentiated intestinal epithelial cells. These findings suggest that eukaryotic translation initiation factors of the eIF4G family play unique roles in regulating integrity and restitution of the intestinal epithelial barrier. Downregulation of these translation initiating factors may mediate disruption of the intestinal epithelial barriers during mucosal inflammation.

Rare gene-disrupting variants and common regulatory variants play key roles in rare and common disease, respectively. These variants are of great interest for investigation into genetic contributions to disease, but experimental methods to validate their impact on gene expression levels are lacking. In this study, we utilized CRISPR/Cas9 genome editing to validate regulatory variants including cis-eQTLs, rare stop-gained variants in healthy and disease cases and one immune-response trans-eQTL master regulator. For investigation into common and rare regulatory variants within transcribed regions, we developed a scalable CRISPR-based polyclonal assay for experimental assessment. First, we applied this assay to nine rare stop-gained variants found in the general population, in GTEx. After editing, the stop-gained variants show a significant allele-specific depletion in transcript abundance, as expected. Next, we utilized the assay to validate 33 common eQTLs found in GTEx. After editing, the eQTL variants show higher variance in effect size than control variants, indicating a regulatory effect. Finally, we applied the polyclonal editing approach to clinical and new stop-gained variants in two disease-associated genes. The results follow the expected trend, with NMD being triggered by variants upstream of the NMD threshold but not by those beyond. This method demonstrates scalable experimental confirmation of putative causal regulatory variants, and improved interpretation of regulatory variation in humans. Next, we sought to experimentally validate an immune-response eQTL for IRF1 in cis and many genes in trans under LPS stimulation. We used CRISPRi to repress the enhancer locus and found that the enhancer is active in our immune cell system. Next, we used CRISPR-Cas9 genome editing and isolation of monoclonal cell lines to target this variant locus. After LPS stimulation, we performed RNA-sequencing on wild type and edited clones, showing that the effect size of the genes which are associated with the trans-eQTL are correlated with differential expression between the edited and wild type cell lines for the same genes. Additionally, we find that the differential expression between edited clones is correlated with CRISPRi repression of the IRF1 promoter and enhancer. In this way, we were able to identify a common genetic variant which modifies the transcriptomic immune response to LPS and validate the trans-eQTL signal.

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