Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

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The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated ... continued below

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3272 Kilobytes pages

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Gruenhagen, Jason Alan December 12, 2003.

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The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca{sup 2+} imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca{sup 2+} signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K{sup +} and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized CdS monocrystals. Aggregates of nanospheres were bathed in imaging solution, and ATP bioluminescence was monitored to investigated the release kinetics of the nanosphere drug delivery systems. Addition of disulfide bond-cleaving molecules induced uncapping of the nanospheres and subsequently, the release of ATP. Increasing the concentration of the uncapping molecule decreased the temporal maximum and increased the magnitude of release of encapsulated ATP from the nanospheres. Furthermore, the release kinetics from the nanospheres varied with the size of the particle aggregates.

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3272 Kilobytes pages

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OSTI as DE00822057

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  • Other Information: TH: Thesis (Ph.D.); Submitted to Iowa State Univ., Ames, IA (US)

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  • Report No.: IS-T 2604
  • Grant Number: W-7405-Eng-82
  • Office of Scientific & Technical Information Report Number: 822057
  • Archival Resource Key: ark:/67531/metadc784808

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Office of Scientific & Technical Information Technical Reports

Reports, articles and other documents harvested from the Office of Scientific and Technical Information.

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  • December 12, 2003

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  • Dec. 3, 2015, 9:30 a.m.

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  • Jan. 9, 2018, 9:54 a.m.

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Gruenhagen, Jason Alan. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence, thesis or dissertation, December 12, 2003; Ames, Iowa. (digital.library.unt.edu/ark:/67531/metadc784808/: accessed October 17, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.