dc.contributor.advisor | Ye, Shui Qing, 1954- | |
dc.contributor.author | Shortt, Katherine Anne | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018 Spring | |
dc.description | Title from PDF of title page viewed May 10, 2019 | |
dc.description | Dissertation advisor: Shui Qing Ye | |
dc.description | Vita | |
dc.description | Includes bibliographical references (pages 154-165) | |
dc.description | Thesis (Ph.D.)--School of Biological Sciences and School of Medicine. University of Missouri--Kansas City, 2018 | |
dc.description.abstract | Acetaminophen (APAP) is a commonly used analgesic responsible for over 56,000
overdose-related emergency room visits annually. A long asymptomatic period and limited
treatment options result in a high rate of liver failure, generally resulting in either organ
transplant or mortality. The underlying molecular mechanisms of injury are not well
understood and effective therapy is limited. Identification of previously unknown genetic
risk factors would provide new mechanistic insights and new therapeutic targets for APAP
induced hepatocyte toxicity or liver injury.
This study used a genome-wide CRISPR/Cas9 screen to evaluate genes that are
protective against or cause susceptibility to APAP-induced liver injury. HuH7 human
hepatocellular carcinoma cells containing CRISPR/Cas9 gene knockouts were treated with
15mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on
the 1) top screening hits, 2) overlap with gene expression data of APAP overdosed human
patients, and 3) biological interpretation including assessment of known and suspected
APAP-associated genes and their therapeutic potential, predicted affected biological
pathways, and functionally validated candidate genes.
This screen is the first genome-wide CRISPR/Cas9 knockout screen of APAP
induced hepatocyte toxicity. The top hits from this screen included numerous genes
previously not linked to liver injury. We further demonstrated the implementation of
intermediate time points for the identification of early and late response genes. A negative
selection screen identified genes involved in fundamental processes, including NAAA,
ATG2B, and MYOZ3. A positive selection screen identified numerous genes potentially
involved in pathogenic processes, including LZTR1, PGM5, and EEF1D. A top essential
pathway at 24 hours of APAP treatment was Regulation of Skeletal Muscle Contraction.
We additionally identified 6 genes, 3 novel and 3 known, that have drug-gene interactions
favorable for re-purposing existing therapies to treat APAP-induced hepatotoxicity.
Collectively, this line of research has illustrated the power of a genome-wide CRISPR/Cas9
screen to systematically identify novel genes involved in APAP induced hepatocyte
toxicity and to provide potential new targets to develop novel therapeutic modalities. | eng |
dc.description.tableofcontents | Introduction -- Review of the literature -- Research question -- Methods -- Results and discussion part 1: CRISPR/Cas9 screen -- Results and discussion part 2: our screen in the context of other acetaminophen data sets -- Results and discussion part 3: acetaminophen-associated single nucleotide polymorphisms in the literature -- Results and discussion Part 4: validation of top candidate genes -- Conclusions and future directions -- Appendix A. Supplementary figures -- Appendix B. Supplementary tables | |
dc.format.extent | xiv, 166 pages | |
dc.identifier.uri | https://hdl.handle.net/10355/67995 | |
dc.publisher | University of Missouri -- Kansas City | eng |
dc.subject.lcsh | Acetaminophen -- Toxicology | |
dc.subject.lcsh | Liver -- Failure -- Gene therapy | |
dc.subject.mesh | Acetaminophen -- Toxicology | |
dc.subject.mesh | Liver Failure | |
dc.subject.mesh | Genetic Therapy | |
dc.subject.other | Dissertation -- University of Missouri--Kansas City -- Biology | |
dc.subject.other | Dissertation -- University of Missouri--Kansas City -- Medicine | |
dc.title | Identification of Novel Regulatory Genes in Acetaminophen Induced Hepatocyte Toxicity by a Genome-Wide CRISPR/Cas9 Screen | eng |
dc.type | Thesis | eng |
thesis.degree.discipline | Cell Biology and Biophysics (UMKC) | |
thesis.degree.discipline | Bioinformatics (UMKC) | |
thesis.degree.grantor | University of Missouri--Kansas City | |
thesis.degree.level | Doctoral | |
thesis.degree.name | Ph.D. (Doctor of Philosophy) | |