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The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na+ and Cl- binding on [H-3]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (K-d) for [H-3]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.

The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na+ and Cl- binding on [H-3]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (K-d) for [H-3]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.

Fundamental understanding of biological systems requires elucidation of biological pathways and processes that can then be exploited to advance new approaches to treat and diagnose diseases. Continuous development of new analytical tools (e.g. biosensors) with enhanced sensitivity and selectivity with spatial and temporal resolution is important to better probe biological systems. Ideal sensor platforms should offer minimal perturbation and be unimpaired by the biological system being probed. Nanoparticle-based platforms offer advantages in sensor technologies, including miniaturization, compartmentalization for multifunctionality, high surface-to-volume ratio, and amenability to surface modification. The work presented in this dissertation describes efforts in fabrication and functionalization of polystyrene-core silica-shell nanoparticles for applications in radioisotope-sensing and O2-sensing. Scintillant-doped polystyrene-core silica-shell nanoparticles (nanoSPA) were prepared and functionalized with various recognition elements for applications in scintillation proximity assay (SPA). In model systems, nanoSPA showed enhanced scintillation response due to binding but suffered from non-specific adsorption. Phospholipid coating was subsequently used to minimize non-specific adsorption and to facilitate insertion of membrane-based receptors. Model binding systems were used to investigate the utility of phospholipid-coated nanoSPA for studying ligand-receptor interactions in a simple separation-free format. GM1-doped phospholipid-coated nanoSPA was also applied in a competitive binding assay to quantify CTB in a complex shrimp extract matrix. The polystyrene-core silica-shell nanoparticle architecture was subsequently used to prepare O2-responsive core-shell nanoparticles (OxyReCS) for O2 detection. OxyReCS were doped with O2-sensitive dye and reference dye for ratiometric measurement of intracellular O2. The in vitro response of OxyReCS was characterized. OxyReCS w

The development of analytical tools for investigating biological pathways on the molecular level has provided insight into diseases and disorders. However, many biological analytes such as glucose and inositol phosphate(s) lack the optical or electrochemical properties needed for detection, making molecular sensing challenging. Scintillation proximity assay (SPA) does not require analytes to possess such properties. SPA uses radioisotopes to monitor the binding of analytes to SPA beads. The beads contain scintillants that emit light when the radiolabeled analytes are in close proximity. This technique is rapid, sensitive and separation-free. Conventional SPA beads, however, are large relative to the cells and made of hydrophobic organic polymers that tend to aggregate or inorganic crystals that sediment rapidly in aqueous solution, thus limiting SPA applications. To overcome these problems, polystyrene core-silica shell nanoparticles (NPs) doped with pTP and dimethyl POPOP were fabricated to produce scintillation NPs that emit photons in the blue region of visible light. The developed scintillation particles are approximately 250 nm in diameter (i.e. 200 nm of core diameter and 10-30 nm of shell thickness), responsive to β-decay from tritium (³H) and have sufficient stability in the aqueous media. DNA hybridization-based SPA was performed to determine whether the scintillation NPs could be utilized for SPA applications. A 30-mer oligonucleotide was immobilized on the polystyrene core-silica shell NPs to give approximately 7.6 x 10³ oligonucleotide molecules per NP and ³H-labeled complementary strand was annealed to the immobilized strand. At the saturation point, increases in scintillation signal due to oligonucleotide binding to the NPs were about 9 fold compared to the control experiments in which no specific binding occurred, demonstrating that the scintillation NPs can be utilized for SPA. Along with the improved physical properties including smaller size and be