Type of Document Master's Thesis Author Ross, Emily Jones Author's Email Address email@example.com URN etd-03262013-215640 Title Development of quantum dot-based live-cell pharmacological assays aimed at membrane transporters and receptors Degree Master of Science Department Chemistry Advisory Committee
Advisor Name Title Dr. Sandra J. Rosenthal Committee Chair Keywords
- LIVE-CELL PHARMACOLOGICAL ASSAYS
- QUANTUM DOT
Date of Defense 2013-03-25 Availability unrestricted AbstractDisorders of the central nervous system (CNS) continue to be among mankind’s most devastating illnesses. Along with causing enormous suffering for those affected, mental disorders place an extreme socioeconomic burden on the global community, with the annual cost estimated to be well over $2 trillion in North America and Europe alone. Despite recent progress in understanding the molecular mechanisms underlying various CNS disorders, the development of novel therapeutics to treat these disorders has been largely held back by the lack of appropriate screening platforms.
One strategy to improve the discovery process is fluorescence-based high-content assays, which can distinguish cell types and simultaneously measure the effects of a drug on multiple downstream pathways within a global cellular signaling network. Fluorescence-based assays provide a promising tool for drug discovery by enabling quantitative measurements with single-cell and single-molecule sensitivity as well as unparalleled specificity. Additionally, fluorescence-based assay platforms permit real-time monitoring of ligand-protein binding kinetics or protein-protein interactions.
Our strategy is based on the use of quantum dot (QD) conjugated probes for target-selective, high-content drug discovery assays.
Here, we describe probe design and our labeling approach utilizing a biotinylated, high-affinity dopamine transporter (DAT) antagonist beta-CFT (2-beta-carbo-methoxy-3-beta-(4-fluorophenyl)tropane) (IDT444) and serotonin transporter (SERT) agonist (5-mthoxy-3-(1,2,5,6-tethydro-4-pyridinyl)-1H-indole) (IDT318). To demonstrate and validate the utility of the DAT antagonist-conjugated QD probe or SERT agonist-conjugated QD probe, fluorescence-based techniques, flow cytometry and microscopy, were used to assay DAT and SERT activity in live cells in a multi-well plate format. Furthermore, we describe the development of a Qdot-based fluorescent somatostatin probe in hopes to enable specific targeting and imaging of somatostatin receptors.
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