Ceramide, a sphingolipid, is an important lipid second messenger that is involved in regulating a number of cellular processes, including programmed cell death, cell growth and differentiation, as well as cellular responses to stress stimuli. Many of the biological effects of ceramide are linked to its ability to modulate the biophysical properties of membranes and cause clustering of signalling molecules in ceramide-rich domains, which allows for more efficient signal transmission in the cell. However, the specific roles of different ceramide species in these signaling pathways have yet to be clearly established. Assessing the effects of long N-acyl chain ceramides in cells involves some limitations due to their poor solubility and their low membrane permeability. Caging these molecules with photolabile protecting groups allows for their delivery into cells where photochemical uncaging of the biologically active compound can be achieved with spatial and temporal control.
A series of coumarinyl-caged ceramides has been prepared in order to probe the biological effects of ceramide in cells. This unique series of compounds was used to investigate the dependence of these cellular effects on N-acyl chain length. Hereafter, I describe the photophysical and photochemical characterization of these novel caged ceramides, assess their uptake and measure the biological effects of the different ceramides which are generated photochemically in HeLa cells. The caged ceramides were shown to be taken up by the cells and to cause a decrease in viability, with UV irradiation, that can be detected after 24 hours of treatment. An investigation of the mechanism of cell death induced by coumarinyl-caged ceramides in HeLa cells revealed that cell death proceeds in a caspase-independent manner and involves the mitochondria. The role of the mitochondria in this cell death pathway, however, remains to be studied further. RIP1 kinase activity, which was also probed in the cells, was determined to not be implicated in cell death caused by photochemically generated ceramide. Intracellular ROS generation, however, was shown to occur in this system, but results primarily from UV irradiation of the free coumarin. Overall, the results from this study have provided insight into the signalling pathways triggered by treatment of HeLa cells with the bioactive lipid ceramide using coumarin photocages.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/32336 |
| Date | January 2015 |
| Creators | Day, Jenna |
| Contributors | Johnston, Linda |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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