Show full item record

Author: Zilliox, Michael
Abstract: The sequencing of the human genome ushered in the era of high-throughput biology. Rapid, whole cell analysis is replacing the molecular reductionist approach of the last century. Microarrays are one of the main high-throughput technologies in this field, allowing researchers to query the whole transcriptome of the cell in one experiment. Learning to harness the power of this technology is a significant research problem that requires expertise in biology, statistics and computation. Towards that goal, this work examines three projects exploring the role of microarray analysis in biological research: an in vitro infection model, acute disease in humans and vaccination in mice. To test the technology in a highly controlled system, an in vitro infection model was developed where monocyte-derived dendritic cells were infected with measles virus and RNA was extracted over 24 hours. There were 1553 significantly regulated genes during this time, with nearly 60% of them down regulated. The results were compared to other in vitro infection systems, which highlighted a group of genes that formed a core response to all the pathogens, including 2’5’ oligoadenylate synthetase, Mx and interferon response factors 1 and 7. The analysis also showed that measles virus is the only pathogen that does not induce dsRNA-dependent protein kinase above its constitutive expression level. Measles also induced a robust interferon-α response in contrast to the other pathogens. These results showed that microarray analysis could provide a modular view of the immune response. Since microarrays worked well in vitro, they were tested in vivo on peripheral blood mononuclear cells from children with acute measles. We found 13 up regulated genes and 206 down regulated genes in children at discharge from the hospital and at 1- iii month follow-up. All of the up regulated genes peaked at discharge but did not return to baseline by the time of follow-up. The same pattern was found in the down regulated genes. A number of immune factors were up regulated including interleukin-1β, interleukin-8, TNF-α, CXCL2 and CCL4. The down regulated genes were involved in three main biological processes: transcription, signal transduction and the immune response, but also included the chemokine receptors CCR2 and CCR7. Finally, a vaccine model in mice compared a formalin-inactivated measles vaccine (FIMV) with an alphavirus replicon particle vaccine expressing the H protein (VCR-H) from measles virus. Although both vaccines induced comparable antibody titers to measles virus, VCR-H induced many more interferon-γ producing cells. Gene expression analysis found many genes significantly regulated at day 4 post-vaccination in CD8+ T cells from VCR-H vaccinated mice, while FIMV vaccinated mice did not show any gene regulation until day 28. Many of the genes regulated by both vaccines were involved in transcription and signal transduction. High-throughput techniques are changing the nature of biological research by providing a new view of the cell. This increased data load requires more computational power and statistical expertise to effectively manage the information and extract knowledge. As scientists learn to harness the power of these new technologies, basic understanding of the cell and the fight against disease will benefit.
Date: 2007-03-26
Subject: Microarrays
Dendritic cells
Measles virus

Files in this item

Files Size Format Download
Dissertation.pdf 3.912Mb application/pdf Download
Supplemental_Data.tar 14.77Mb application/octet-stream Download

The following license files are associated with this item:

This item appears in the following Collection(s)

Show full item record