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Title page for ETD etd-03282016-104058


Type of Document Master's Thesis
Author Sharick, Joseph Thomas
Author's Email Address joseph.t.sharick@vanderbilt.edu
URN etd-03282016-104058
Title Fluorescence Lifetime Imaging of NAD(P)H Distinguishes Pathways of Metabolic Inhibition
Degree Master of Science
Department Biomedical Engineering
Advisory Committee
Advisor Name Title
Melissa Skala Committee Chair
Henry Charles Manning Committee Member
Keywords
  • cancer
  • optical imaging
  • NAD(P)H
  • microscopy
  • fluorescence lifetime imaging
  • multiphoton
  • FAD
  • metabolism
Date of Defense 2016-03-30
Availability unrestricted
Abstract
Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (NAD(P)H) non-invasively probes metabolic protein-binding dynamics without the need for exogenous dyes. FLIM measurements are sensitive to changes in fluorophore microenvironment. NAD(P)H fluorescence lifetime signals are generally interpreted as a two exponential decay due to the free and protein-bound forms of the molecule, which have distinctly short and long lifetimes, respectively. However, the biochemical significance of shifts in the bound lifetime of NAD(P)H as a result of drug treatment remains unclear. An understanding of this biochemical source of contrast is needed in order to interpret FLIM studies of drug efficacy, and thus leverage FLIM as a powerful tool for drug screening and metabolic research. We hypothesized that changes in NAD(P)H bound lifetime are a result of conformational folding effects as the activity levels of its various enzyme binding partners change. The effects of two metabolic inhibitors which promote oxidative phosphorylation through different pathways were measured in cancer cell lines using this imaging technique. We demonstrate that FLIM of NAD(P)H is differentially sensitive to unique shifts in metabolic enzyme activities, and thus could be used to non-invasively quantify changes in flux through individual metabolic pathways.
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