Thesis (Ph. D.)--University of Michigan. 1931. / "By Alfred L. Ferguson. Richard Hitchens and Kenneth Van Lentes." From Transactions of the Electrochemical society, v. 71, 1937.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 91-102).

The glycine receptor (GlyR) is a ligand-gated ion channel and a member of the cys-loop receptor family. Like other members of this family, the GlyR is a target for many drugs of abuse, including alcohol. While the effects of alcohol on these receptors have been well-characterized, the contribution of each receptor subtype to the overall physiological and behavioral effects of alcohol use are unclear. This is partially due to the limited pharmacology of the GlyR, which limits the ability to isolate GlyR function within a complex system. One method for identifying compounds that bind to and modulate a given target is phage display. This approach uses bacteriophage to screen a large number of peptide sequences for affinity at a given target. We developed a phage selection protocol to identify peptides that bind to the GlyR. These peptides were then tested for functional effects at the GlyR using two-electrode voltage clamp physiology. We identified several peptides that were able to modulate GlyR function. Peptide D12-116 showed specificity for the GlyR over two closely related γ-aminobutyric acid (GABA) channels. In addition, this method is easily adapted for the selection of peptides that bind to any cell-expressed target, increasing the utility of phage display in the neurobiology field. Another shortcoming in GlyR pharmacology is the lack of modulators with specificity for a single GlyR subtype. We next adjusted our selection protocol to search for peptides that can distinguish between the different Gly R α subtypes. We identified several promising lead peptides that show subtype preference. Finally, we found that trifluoroacetic acid (TFA), a common peptide contaminant, also modulates GlyR function. This finding has important implications for both previously reported peptide modulators and the pharmacology of several volatile anesthetics, for which TFA is the major metabolite / text

This work describes the development of a microfluidic platform that can be used to study suspension stability and crystallization with in droplets as a function of time and concentration. Techniques for monodisperse droplet formation, droplet trapping and storage, and droplet dehydration are developed and used to design a microfluidic platform that can be adapted for the applications of interest. A geometric model is developed to predict the droplet shape and emulsion structure generated by microfluidic nozzles. However, droplet volume and structure spacing cannot be independently controlled using microfluidic nozzles, and a design consisting of an array of traps is considered to achieve the desired structure for stable, extended droplet observation. The dehydration of aqueous droplets stored in the array is characterized as a function of relative humidity, and is shown to be reasonably estimated as a species diffusing from a sphere into an infinite medium. The microfluidic platform for droplet dehydration is combined with particle tracking to show that the stability of particle suspensions can be probed as a function of salt concentration. The flocculation behavior observed in the trapped droplets agrees well with corresponding macroscale measurements as well as with previously published studies. The platform is also used to generate substantial sample sizes to measure nucleation statistics and crystal growth rates of glycine as a function of initial concentration, environmental conditions, and the presence of additives. These applications show proof of concept that the microfluidic platform is a useful tool for the analysis of the behavior observed during particle aggregation and crystallization.

In the catalytic cycle of the glycine cleavage system (GCS), the major physiological pathway for glycine degradation, the lipoic acid-containing hydrogen carrier protein (H-protein) plays a pivotal role as a mobile carrier of reaction intermediates. and as a regulator for glycine decarboxylase (P-protein). Defects in the GCS lead to the accumulation of glycine in all body tissues, resulting in the genetic disease glycine encephalopathy (GE). Defects in P-protein lead to more than 80% of reported cases of GE, therefore. routine biochemical analysis only tests P-protein for activity. Unlike other amino acid decarboxylases, P-protein is itself inactive, and requires H-protein for biochemical activity. Currently. researchers use H-protein purified from chicken liver extracts for the P-protein activity assay. However. extraction and purification of chicken H-protein is laborious. costly. and has poor yield, making the expression and purification of H-protein from an alternate source desirable. We have overexpressed recombinant human H-protein in the methylotropic yeast Pichia pastoris by utilizing the inducible alcohol oxidase (AOX1) promoter and the Saccharomyces cerevisiae a-mating factor secretion signal. Enhanced green fluorescent protein (EGFP) was used as a fusion partner to aid detection of H-protein during expression and purification. Secreted H-protein was detected from 24-96 hours post induction, and constituted the major protein species in the culture medium. H-protein was purified to apparent homogeneity in a single step using nickel-chelating affinity chromatography, and lipoylated using lipoate protein ligase from E. coli. Functional analysis of holo-H-protein using the NAD+ lipoamide dehydrogenase assay demonstrated biochemical activity with the artificial substrate, suggesting that human H-protein produced in P. pastoris may be an appropriate replacement for the chicken H-protein currently used in the biochemical diagnosis of GE.

In this thesis, I examined the inhibitory control of REM sleep motor activity using both a pharmacological rat model and a genetic mouse model. I characterized the role for GABA and glycine in mediating the REM-specific suppression of muscle activity as well as their involvement in regulating the phasic muscle twitches that punctuate this atonia. Based on four specific research objectives, the following conclusions were drawn: 1. REM atonia is not directly mediated by glycinergic or GABAA-mediated inhibition. These data refute the prevailing hypothesis that REM atonia is caused by glycinergic inhibition. These receptors are, however, important in the regulation of phasic muscle twitch activity. 2. GABAB receptors can modulate REM atonia but only when acting in concert with GABAA and glycine receptors. Blockade of all three receptor types results in a partial reversal of REM atonia, suggesting a functional interaction is occurring between these receptors during REM sleep. 3. The phasic glycinergic/GABAA-mediated inhibitory drive present in REM sleep regulates the temporal pattern of phasic twitch activity that is seen across this state. I hypothesize that this progressively decreasing inhibitory input counteracts a gradually increasing excitatory input to shape the temporal distribution of muscle twitches across REM sleep. 4. A loss of normal inhibitory function may play a causal role in the pathology of REM sleep behaviour disorder (RBD), the sleep disorder characterized by excessive motor activity in REM sleep.

N-Methyl-D-aspartate receptors (NMDARs) are a principal subtype of excitatory ligandgated ion channel with prominent roles in physiology and disease in the mammalian central nervous system (CNS). Activation of NMDARs requires binding of both glutamate and glycine. Apart from its co-agonist action, glycine can also prime NMDARs for subsequent internalization upon binding of both glutamate and glycine. However, the molecular basis responsible for mediating and regulating glycine priming and NMDAR endocytosis is largely unknown. In my thesis, I discovered the principle that although NMDAR gating and priming share a common requirement for glycine binding, the molecular constraints for gating are distinct from those for priming through two mutations of the glycine binding site in GluN1 subunit of the NMDAR that, while maintaining gating of NMDARs, eliminate glycine priming of the receptors. One of the molecular signatures of glycine priming is recruitment of the endocytic adaptor protein AP-2. I have characterized the two regions in GluN2 subunits required for enhanced AP-2 association. This unexpected result suggests binding of glycine initiates a conformational change transmitted from GluN1 to GluN2 allowing for docking of endocytic machinery. Furthermore, I have discovered that naturally occurring splice variants of GluN1 subunit, containing a 21 amino acid sequence in the N-terminus domain (N1) cassette, abrogate glycine stimulated AP-2 recruitment and glycine-primed NMDAR internalization. These findings imply that there are distinct populations of native NMDARs in the CNS – those lacking N1 that show glycine-primed internalization and those containing N1 that are not primable. Collectively, my thesis work demonstrates a dramatic all-or-none priming effect with splice variants of NMDARs, a highly unexpected discovery providing novel insight into the molecular mechanisms and physiological role of glycine priming. Ultimately, elucidating principles and mechanisms of glycine priming lay the foundation for new types therapeutic approaches for CNS disorders, approaches without the deleterious consequences of directly blocking NMDARs.

Bibliography: leaves [i]-xvi / xi, 132, xvi, [137] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1984

Thesis (Ph. D.)--Carleton University, 2004. / Includes bibliographical references (p. 118-146). Also available in electronic format on the Internet.

Thesis (Ph. D.)--University of Florida, 1988. / Description based on print version record. Typescript. Vita. Includes bibliographical references.