Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Shiva M. Singh

Abstract

Fetal alcohol spectrum disorders (FASD) refers to the neurological, developmental, and behavioural abnormalities arising from in utero ethanol exposure. These abnormities included attention deficit, anxiety, and learning and memory impairment persisting into adulthood. The molecular mechanisms of such persistent behavioural changes remain unknown and are an area of intense research. In this thesis, mice were exposed to ethanol during the third trimester equivalent, the peak of synaptic development. Following this exposure, genome-wide epigenetic and gene expression and changes in the hippocampus were assessed in adult (70 day old) mice.

In the first experiment, genome-wide trimethylation of histone H3 at histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) were assessed using chromatin immunoprecipitation (ChIP) microarray (ChIP-chip). Cell-cell signalling genes were enriched for changes in both methylations. It included the protocadherin (Pcdh) genes, which confer neuronal identity and may be important for synaptic development. Changes in methylation also occurred at imprinted genes and lipid-metabolism genes

The second experiment assessed DNA methylation using methylated DNA immunoprecipitation (MeDIP) microarray (MeDIP-chip). The screen identified genes involved in peroxisome biogenesis, which metabolize lipids and generate free-radicals. This was also true when the histone and DNA methylation changes were considered together. Combined analysis of affected genes from each experiment implicated free-radical scavenging genes. Identification of this novel interplay between epigenetic and oxidative stress genes may provide insight into diagnostic or therapeutic interventions. In general, the results support a role of epigenetic mechanisms in long-term FASD phenotypes.

Finally, the third experiment examined gene expression and miRNA microarrays identified 59 and 60 differentially expressed genes and miRNAs between ethanol-exposed and control mice. These genes primarily affect free radical scavenging genes. Differential expression of five genes in this pathway was confirmed with droplet digital PCR (ddPCR), including the transcription factor Tcf7l2 and the apoptosis regulator Casp3. The affected genes also included other oxidative stress proteins, olfactory receptors, and biosynthetic enzymes that may contribute to FASD-related abnormalities.


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