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The metabolism and signaling of hydrogen sulfide: the role of CBS-related proteins in Caenorhabditis elegans
Vozdek, Roman ; Kožich, Viktor (advisor) ; Macůrková, Marie (referee) ; Jiráček, Jiří (referee)
Hydrogen sulfide (H2S) is a toxic gas that causes respiratory failure and death at high concentrations, but at low concentrations, it functions as a signaling molecule in vasodilation and neuromodulation, and it protects cells and tissues from reperfusion injury, hypoxia, hyperglycemia and endothelial dysfunction. Several model organisms have been used to study the physiological roles and signaling pathways of H2S. The roundworm Caenorhabditis elegans is a remarkable model for studying the physiology, developmental biology and signaling of H2S; however, the metabolism of H2S in this animal is largely unknown. Cystathionine beta-synthase (CBS) is one of three H2S-producing enzymes in mammals. Notably, C. elegans possesses 6 genes that encode proteins homologous to CBS, namely cbs- 1, cbs-2, cysl-1, cysl-2, cysl-3 and cysl-4. In this thesis we studied the roles of these genes in H2S metabolism and signaling. First, we identified cbs-1 as the gene encoding CBS in C. elegans; the recombinant purified CBS-1 protein exhibited canonical CBS activity, and RNA interference-mediated silencing of cbs-1 resulted in decreased CBS activity and increased homocysteine levels in worm extracts, recapitulating the phenotypes of CBS deficiency in mammals. Notably, the nematode and human enzymes differ in their domain...
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Making Transgenic \kur{C. elegans} with Polycistronic mCherry Vector
FARKA, Dominik
Creation of transgenic animals has become a popular method to analyse gene function. In the nematode Ceanorhabditis elegans transformation is widely used and can be achieved by microinjection. For functional analyses, transgene constructs typically contain a promoter driving the expression of the protein of interest that is fused to a fluorescent protein. However, as this fusion of proteins can lead to misfolding of the protein of interest and may not reflect proper function, a modification of the expression vector has been developed; introducing a short sequence of non-coding DNA in-between the sequences of the two proteins and making the construct compatible with a polycistronic operon system. In this study, four different polycistronic constructs were introduced into C. elegans by means of microinjection in order to provide new tools for the analyses of gene function. Tissue specific promoters wrt-2 (seam cells), grl-21 (hyp7), and egl-17 (vulval precursor cells) were used to over-express either NHR-25 or SMO-1 in the corresponding tissues and the expression was visualized by independently translated mCherry red fluorescent. 10 independent transformed C. elegans strains were established and corresponding tissue-specific promoter activities were confirmed. Furthermore, in some cases, ectopic behaviour was observed e.g. ectopic mCherry expression in different tissues or specific cell differentiation defects that was most likely caused by the overexpression of NHR-25 or SMO-1. This study was the first case in our laboratory to generate transformed C. elegans utilizing the polycistronic mCherry vector system. New genetic tools were introduced in the laboratory useful for further analyses of gene function.
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