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• W307029463 abstract "Sulfur Dioxide (SO2) is the most recently discovered gasotransmitter. As a result there is a significant gap in the literature compared to the other established gasotransmitters which have had several decades of research. Initially, SO2 was thought to be a toxic air pollutant and byproduct from the metabolism of sulphur containing amino acids. It is thus important to understand its mechanism of action and molecular targets of SO2. The pathways for metabolism of sulfur containing gasotransmitters (hydrogen sulfide and sulfur dioxide) in lower organisms was previously unknown. My thesis goal is to gain a better understanding of the function and metabolism of SO2. To better elucidate this function I have used the eukaryotic model organisms; Saccharomyces cerevisiae (S. cerevisiae) and Caenorhabditis elegans (C. elegans). Two high-throughput methods were developed as part of this research to improve detail and accuracy of analysis in C. elegans. The first method developed was WormScan, which allowed for automatic whole organism phenotyping. The second method developed was pHi nanoparticles, to enable the characterization of intracellular pH (pHi). These two C. elegans methods were published in PLOS ONE, 2012 and Analytical Biochemistry, 2014. WormScan is based on an affordable consumer grade flatbed scanner that facilitates the quantification of the four main toxicological endpoints of C. elegans and greatly reduces the user bias associated with manual counting. The light intensity produced by the scanner was sufficient to cause negative phototaxis equivalent to a physical stimulus for mortality determination. The affordability and high-throughput nature of WormScan also enable high-throughput whole animal drug screening in C. elegans. The pHi nanoparticles were generated through a modified Stober synthesis and easily calibrated externally to the C. elegans. The nanoparticles were taken up during feeding, and found to bypass the selective intestinal uptake and translocate to the primary organs and secondary organs of the reproductive tract. This was the first diagnostic use of nanoparticles in C. elegans. It was found that the hypoxia-inducible factor-1 (hif-1) transcription factor was required in order for C. elegans to survive exposure to SO2. HIF-1 is a regulator of both pHi homeostasis and metabolic rate. When C. elegans are exposed to a non- lethal concentration of SO2, it induced a reversible state of suspended animation, a condition that is characterized by metabolic suppression. A physiologically relevant drop of 0.15 pH units was observed in response to SO2 exposure. Where such a decrease in pHi is a characteristic of reduced metabolism. A forward genetic screen was carried out in C. elegans to identify mutations that conferred resistance to SO2. A resistance mutation was mapped to a region of Chromosome III using an as yet unpublished high-throughput technique (Mip-Map). Together with whole genome sequencing the gene F08F8.9 causing resistance was identified. The F08F8.9 gene is homologous to a gene that encodes a subunit of RNA polymerase II found in H. sapiens and S. cerevisiae. The involvement of the F08F8.9 gene in SO2 resistance was also validated in C. elegans using single nucleotide variants of F08F8.9 from the million-mutation project (MMP) library. Simultaneously, a complementary chemical genomic screen was undertaken in S. cerevisiae using the Yeast deletion collection (YDC). This screen identified RPO21, the closest homologue of the C. elegans F08F8.9 gene, as a major protein target of SO2. Thus, F08F8.9 was identified as a SO2 resistance factor in the forward genetic and confirmed in the model organism C. elegans and identified in chemical genomic screen in S. cerevisiae. This supports a role for RNA polymerase II subunit in SO2 resistance. Further investigation into how the RNA polymerase II subunit contributes to SO2 resistance is important. Gasotransmitters have significant biological functions and contribute to pathogenesis of human diseases. Additional investigation into SO2 mediated suspended animation may be of practical importance if it leads to developments that could help trauma victims or heart attack victims survive Ischemia/Reperfusion injury." @default.
• W307029463 created "2016-06-24" @default.
• W307029463 creator A5058554393 @default.
• W307029463 date "2015-02-12" @default.
• W307029463 modified "2023-09-24" @default.
• W307029463 title "Developing high-throughput methods for C. elegans to better understand the newly discovered gasotransmitter sulfur dioxide" @default.
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