Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIP2) is a kinase which modulates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2. It is known that activation of RIP2 by engaging NOD receptors increases the production of pro-inflammatory cytokine and lipid mediators. We have some data indicating that RIP2 may also be involved in specialized pro-resolution lipid mediator (SPM) production. However, the molecular mechanisms by which RIP2 is involved in lipid mediator biosynthesis, are currently unknown. Understanding this process may have significant implications for RIP2-targeted therapies, which may not only inhibit pro-inflammatory cytokine and lipid mediator production but may also disrupt SPM production and resolution programs. This thesis aims to demonstrate that RIP2 is involved in promoting the activation of ALOX5 in a transient overexpression setting but also in an endogenous setting using relevant bacterial stimuli. These aims were accomplished through the optimization of a fluorescent assay to assess ALOX5 enzymatic activity, by optimization of ALOX5 enzyme purification and through molecular cloning of ALOX5 into a retroviral vector followed by viral transduction of the THP-1 human monocytic cell line. We find that co-expression of RIP2 with ALOX5 significantly enhances the enzymatic activity of ALOX5. We have successfully cloned NTAP-tagged ALOX5 into the pBABE retroviral vector and are currently selecting transduced cells so that we might test if this effect also occurs endogenously. Understanding the mechanisms underlying the production and regulation of SPMs would provide greater insight into potential new therapeutic approaches to promote resolution in chronic inflammatory diseases.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:honorstheses-2027 |
| Date | 01 January 2021 |
| Creators | Sia, Marianne |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Honors Undergraduate Theses |
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