AMPA RECEPTOR ALLOSTERISM: MEASUREMENT OF THE CONFORMATIONAL CHANGES IN THE LIGAND BINDING DOMAIN OF A FUNCTIONAL RECEPTOR

Ionotropic glutamate receptors are important excitatory neurotransmitter receptors in the mammalian central nervous system that have been implicated in a number of neuropathologies such as epilepsy, ischemia, and amyotrophic lateral sclerosis. Glutamate binding to an extracellular ligand binding domain initiates a series of structural changes that leads to the formation of a cation selective transmembrane channel, which consequently closes due to desensitization of the receptor. The crystal structures of the AMPA subtype of the glutamate receptor have been particularly useful in providing initial insight into the conformational changes in the ligand binding domain; however, these structures are limited by crystallographic constraint. To gain a clear picture of how agonist binding is coupled to channel activation and desensitization, it is essential to study changes in the ligand binding domain in a dynamic, physiological state. In this dissertation, a technique called Luminescence Resonance Energy Transfer was used to determine the conformational changes associated with activation and desensitization in a functional AMPA receptor (ÄN*-AMPA) that contains the ligand binding domain and transmembrane segments; ÄN*-AMPA has been modified such that fluorophores can be introduced at specific sites to serve as a readout of cleft closure or to establish intersubunit distances. Previous structural studies of cleft closure of the isolated ligand binding domain in conjunction with functional studies of the full receptor suggest that extent of cleft closure correlates with extent of activation. Here, LRET has been used to show that a similar relationship between cleft closure and activation is observed in the “full length” receptor showing that the isolated ligand binding domain is a good model of the domain in the full length receptor for changes within a subunit. Similar LRET investigations were used to study intersubunit distances specifically to probe conformational changes between subunits within a dimer in the tetrameric receptor. These studies show that the dimer interface is coupled in the open state, and decoupled in the desensitized state, similar to the isolated ligand binding domain crystal structure studies. However, we show that the apo state dimer interface is not pre-formed as in the crystal structure, hence suggesting a mechanism for functional transitions within the receptor based on LRET distances obtained.

Measurement and correlates of physical activity behavior among older minority women

Research has shown that physical activity serves a preventive function against the development of several major chronic diseases. However, studying physical activity and its health benefits is difficult due to the complexity of measuring physical activity. The overall aim of this research is to contribute to the knowledge of both correlates and measurement of physical activity. Data from the Women On The Move study were used for this study (n = 260), and the results are presented in three papers. The first paper focuses on the measurement of physical activity and compares an alternate coding method with the standard coding method for calculating energy expenditure from a 7-day activity diary. Results indicate that the alternative coding scheme could produce similar results to the standard coding in terms of total activity expenditure. Even though agreement could not be achieved by dimension, the study lays the groundwork for a coding system that saves considerable amount of time in coding activity and has the ability to estimate expenditure more accurately for activities that can be performed at varying intensity levels. The second paper investigates intra-day variability in physical activity by estimating the variation in energy expenditure for workers and non-workers and identifying the number of days of diary self-report necessary to reliably estimate activity. The results indicate that 8 days of activity are needed to reliably estimate total activity for individuals who don't work and 12 days of activity are needed to reliably estimate total activity for those who work. Days of diary self-report required by dimension for those who don't work range from 6 to 16 and for those who work from 6 to 113. The final paper presents findings on the relationship between daily living activity and Type A behavior pattern. Significant findings are observed for total activity and leisure activity with the Temperament Scale summary score. Significant findings are also observed for total activity, household chores, work, leisure activity, exercise, and inactivity with one or more of the individual items on the Temperament Scale. However, even though some significant findings were observed, the overall models did not reveal meaningful associations. ^

Understanding the Influence of Photon Energy on 6MV Non Reference Dosimetry and CT Dosimetry using TLD and OSLD

Measurement of the absorbed dose from ionizing radiation in medical applications is an essential component to providing safe and reproducible patient care. There are a wide variety of tools available for measuring radiation dose; this work focuses on the characterization of two common, solid-state dosimeters in medical applications: thermoluminescent dosimeters (TLD) and optically stimulated luminescent dosimeters (OSLD). There were two main objectives to this work. The first objective was to evaluate the energy dependence of TLD and OSLD for non-reference measurement conditions in a radiotherapy environment. The second objective was to fully characterize the OSLD nanoDot in a CT environment, and to provide validated calibration procedures for CT dose measurement using OSLD. Current protocols for dose measurement using TLD and OSLD generally assume a constant photon energy spectrum within a nominal beam energy regardless of measurement location, tissue composition, or changes in beam parameters. Variations in the energy spectrum of therapeutic photon beams may impact the response of TLD and OSLD and could thereby result in an incorrect measure of dose unless these differences are accounted for. In this work, we used a Monte Carlo based model to simulate variations in the photon energy spectra of a Varian 6MV beam; then evaluated the impact of the perturbations in energy spectra on the response of both TLD and OSLD using Burlin Cavity Theory. Energy response correction factors were determined for a range of conditions and compared to measured correction factors with good agreement. When using OSLD for dose measurement in a diagnostic imaging environment, photon energy spectra are often referenced to a therapy-energy or orthovoltage photon beam – commonly 250kVp, Co-60, or even 6MV, where the spectra are substantially different. Appropriate calibration techniques specifically for the OSLD nanoDot in a CT environment have not been presented in the literature; furthermore the dependence of the energy response of the calibration energy has not been emphasized. The results of this work include detailed calibration procedures for CT dosimetry using OSLD, and a full characterization of this dosimetry system in a low-dose, low-energy setting.