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This abstract sent to supervisor 5/4/12 Tyrosine hydroxylase is the enzyme which controls the ratelimiting step in the synthesis of noradrenaline. In order to discover whether the activity of tyrosine hydroxylase might serve as an indicator of noradrenergic function in the brain, two preparations for the direct measurement of TH activity in rat brain regions by a tritium-release method were employed: synaptosomal suspensions prepared from pinchedoff nerve terminals, and partially solubilised enzyme preparations prepared from frozen homogenates or synaptosomal suspensions and assayed at saturating concentrations of cofactor and tyrosine. There was evidence of an increase in tyrosine hydroxylase activity in hippocampal synaptosomes of rats killed immediately after a mild electrical footshock. Activation of synaptosomal enzyme activity was also found after single doses of clonidine and parachloroamphetamine, and after repeated handling; and single doses of morphine and of yohimbine appeared to lower tyrosine hydroxylase activity. Repeated administration of drugs such as clonidine, desipramine and 2-deoxyglucose, however, did not affect tyrosine hydroxylation rate. A preliminary finding , suggesting differences in synaptosomal tyrosine hydroxylase activity related to experience with different reinforcement schedules (continuous reward vs. partial reward) in a runway experiment, was not substantiated in later experiments; nor was there any difference between the synaptosomal tyrosine hydroxylase activity of naive controls and rats given repeated daily shocks for a week. The saturated TH assay performed on solubilised enzyme was, as predicted, unresponsive to the short term stimulation effects detected with the synaptosomal assay. However, other changes, such as a reduced maximal hydroxylation rate after repeated desipramine administration, and an increased rate several weeks after a course of electrical stimulation of the septal area, were established with the saturated assay. Although the changes in stimulated rats were associated with increased behavioural tolerance to stress, e.g. resistance to extinction of a running response in a runway, other experiments in which the behavioural stress-tolerance was induced by behavioural methods alone showed no accompanying changes in TH activity. Measures of synaptosomal and saturated soluble TH activity appear to constitute independent indicators of noradrenergic function. It seems that synaptosomal tyrosine hydroxylase activity is not, as anticipated, controlled by the firing rate of locus coeruleus neurones; but it may be subject to local regulation in noradrenergic terminals. The results are discussed in the context of theoretical aspects of the regulation of noradrenaline synthesis in the brain, and the mechanisms underlying physiological responses to stress and behavioural tolerance to stress.

Thesis (Ph.D.)--George Mason University, 2008. / Vita: p. 221. Thesis director: Ancha Baranova. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biosciences. Title from PDF t.p. (viewed Aug. 28, 2008). Includes bibliographical references (p. 168-220). Also issued in print.

Indiana University-Purdue University Indianapolis (IUPUI) / The proteasome is responsible for breaking down the majority of the proteins in the cell. However, a complete understanding of how this large multi-subunit protease is assembled is currently lacking. Proper and timely assembly of the proteasome is critical for the functioning of the ubiquitin-proteasome pathway, defects in which have been associated with several different cancers. A recently discovered heterodimeric proteasome assembly chaperone, Pba1p-Pba2p, has been suggested to prevent the assembly process from straying off path. Pba1p-Pba2p associates with proteasomal assembly intermediates via C-terminal HbYX motifs. The HbYX motif is a tri-peptide sequence containing a hydrophobic residue (Hb) followed by a tyrosine (Y), then any amino acid (X). This motif was originally identified in proteasomal activators, and shown to mediate the association of activators with the proteasome by inserting into intersubunit pockets on either end of the proteasome. There are seven unique intersubunit binding pockets, located between neighboring α subunits on the proteasome, to which a HbYX-containing protein can bind; which of these pockets Pba1p-Pba2p binds to remains elusive. I attempted to identify where Pba1p and Pba2p bind via a crosslinking approach. Specific residues were mutagenized to cysteines on Pba1p, Pba2p, and the individual α subunits in order to generate crosslinkable species. By exposing yeast cells expressing these crosslinkable proteins to mild oxidizing conditions, I attempted to trap the Pba1p and Pba2p α intersubunit pocket interactions. In order to optimize crosslinking conditions, the assay was modified several ways. Additionally, measures were taken to increase detection of the crosslinked species via immunoblotting. Despite the efforts to improve the crosslinking and detection, I was unable to successfully detect a crosslinked species. However, crosslinking is a reasonable method to identify the Pba1p and Pba2p proteasomal binding sites, having been successfully used to identify binding sites for other HbYX-motif-containing proteins; further assay optimization should yield Pba1p and Pba2p proteasomal crosslinks.