dc.contributor.advisor | Koulen, Peter | |
dc.contributor.author | Gerdes, Bryan | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018 Summer | |
dc.description | Title from PDF of title page viewed September 20, 2019 | |
dc.description | Dissertation Advisor: Peter Koulen | |
dc.description | Vita | |
dc.description | Includes bibliographical references (pages 48-57) | |
dc.description | Thesis (Ph.D.)--School of Biological Sciences. University of Missouri--Kansas City, 2018 | |
dc.description.abstract | Worldwide there are an estimated 1 billion people that are affected by a form of
neurodegenerative disease. The most common forms of neurodegenerative disease are
Alzheimer’s disease and Parkinson’s disease, along with diseases that are attributed to
damage of the retina and optic nerve, such as macular degeneration and glaucoma. Nearly
five million people have been diagnosed with Alzheimer’s disease in the United States of
America alone (1). Another disease that effects 4 million people in the US is glaucoma, and
it is the second most common form of blindness after age-related macular degeneration
(AMD) (2). Alzheimer’s disease, AMD, and glaucoma are all neurodegenerative diseases
that are caused by oxidative stress. Neuroprotection and protection of cells supporting the
function of neurons could provide novel therapeutic approaches to treating and preventing
neurodegenerative diseases when therapies do not exist or existing therapies fail (3).
Nicotinamide phosphoribosyltransferase (Nampt) is an enzyme critical for cellular
energy metabolism, and it also functions as a proinflammatory cytokine and growth factor
with neuroprotective properties and in these contexts is referred to as Pre-B-Cell Colony
Enhancing Factor (PBEF) and Visfatin, respectively. Nampt/PBEF/Visfatin has been
implicated in a number of human diseases, including acute lung injury, rheumatoid arthritis,
vascular disorders, and diabetes. Based on previous evidence suggesting a protective role of
Nampt/PBEF/Visfatin in ischemia, the hypothesis was tested that Nampt/PBEF/Visfatin
exerts protective effects against oxidative stress in established in vitro models of
neurodegeneration and of cellular degeneration related to neurodegenerative diseases.
The PBEF gene was cloned into a prokaryotic expression vector, recombinantly
expressed as a Glutathione-S-transferase-fusion protein, and purified by affinity
chromatography. Optimization of a fluorescent enzyme activity assay was performed in order
to quantify the Nampt/PBEF/Visfatin-mediated conversion of nicotinamide to nicotinamide
mononucleotide, confirming enzymatic activity of recombinant Nampt/PBEF/Visfatin in
vitro. Testing of neuroprotective properties of recombinant Nampt/PBEF/Visfatin was
conducted in the human neuroblastoma cell line SH-SY5Y using the calcein-acetomethoxy
ester uptake assay, which measures cellular viability. Testing of siRNA transfection
mediated knockdown of endogenous Nampt was performed in the retinal pigment epithelial
cell line ARPE-19 and primary isolated optic nerve head astrocytes as models for cells
supporting the viability and function of retinal neurons. The use of siRNA in both ARPE-19
and ONHA cell lines demonstrated how the loss of endogenous Nampt affected each cell line
when treated with tBHP. Visual cell counts were performed after knockdown of Nampt and
treatment of tBHP, and rNampt was used as a neuronal protective in order to produce cellular
recovery.
Recombinant Nampt/PBEF/Visfatin was enzymatically active and applied to each cell
type extracellularly in the pre-treatment stage when fresh media was added to the cells. This
addition of rNampt reduced cell death resulting from chemically induced oxidative stress in
vitro by approximately 25% in SH-SY5Y cell line. Similar results were seen in both ARPE
19 cells and ONHA when oxidative stress was induced by ROS. Removal of endogenous
Nampt by pharmacological inhibition or siRNA-mediated knockdown had no effect on
general cell viability but cell lines ARPE-19 and ONHA became more sensitive to oxidative
stress and cells entered apoptosis earlier than WT. After knockdown of endogenous Nampt
cells were pre-treated with rNampt and shown to have increased cell survival from neuronal
protection from oxidative stress. An improved understanding of the mechanisms of action
underlying Nampt/PBEF/Visfatin-mediated neuro- and cellular protection has the potential to
contribute to the development of new therapies to treat neurodegenerative diseases. | eng |
dc.description.tableofcontents | Introduction -- Materials and methods -- Results -- Discussion -- Future directions | |
dc.format.extent | xii, 58 pages | |
dc.identifier.uri | https://hdl.handle.net/10355/69674 | |
dc.publisher | University of Missouri -- Kansas City | eng |
dc.subject.lcsh | Nervous system -- Degeneration -- Treatment | |
dc.subject.lcsh | Oxidative stress | |
dc.subject.other | Dissertation -- University of Missouri--Kansas City -- Biology | |
dc.title | Mechanism of Action of Nicotinamide Phosphoribosyltransferase Mediated Signaling in Oxidative Stress | eng |
dc.type | Thesis | eng |
thesis.degree.discipline | Cell Biology and Biophysics (UMKC) | |
thesis.degree.discipline | Molecular Biology and Biochemistry (UMKC) | |
thesis.degree.grantor | University of Missouri--Kansas City | |
thesis.degree.level | Doctoral | |
thesis.degree.name | Ph.D. (Doctor of Philosophy) | |