Call number: LD2668 .T4 1951 W45 / Master of Science

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The effects of intragastrically administered tryptophan and tyrosine, which are direct precursors of brain neurotransmitters affecting food intake, were investigated. Differences in sensitivities and/or responses in relatively fast-growing and slow-growing strains of chickens were also tested. The amino acids were intubated intragastrically in a series of four experiments. Food consumption following tryptophan intubation decreased in both the slow and fast-growing strains. Increased food intake was observed in the slow-growing birds as a result of tyrosine treatment suggesting that food consumption can be altered by manipulation of dietary amino acids. Tyrosine treatment did not stimulate food intake in the fast-growing strain, implying that chickens in this population were in a state of maximal or near maximal stimulation, and thus relatively incapable of increasing food intake. To determine whether the changes observed in feeding behavior were perhaps due to some general inhibition or stimulation of the central nervous system rather than the result of mediation of feeding mechanisms in the brain, the effects of intragastrically administered tyrosine and tryptophan on tonic immobility (TI) were also tested. Duration and susceptibility of TI were unaffected by tyrosine and tryptophan, suggesting that these amino acids acted upon mechanisms specifically involved in the regulation of food intake. / Master of Science

The metabolism of tyrosine is essential for many critical biochemical events including catecholamine synthesis, melanogenesis and insect cuticle sclerotization. These pathways are highly regulated in both insects and mammals by many well-characterized enzymes including dopa decarboxylase and tyrosine hydroxylase. On the other hand, there are still many enzymes involved in these processes that we know very little about. Dopachrome tautomerase (DCT), dopachrome conversion enzyme (DCE) and α-methyldopa resistant protein (AMD) fall into the category of the less characterized enzymes. Dopachrome is a pivotal intermediate in melanogenesis. Mammalian DCT and insect DCE both use dopachrome as a substrate. DCE catalyzes a decarboxylative structural rearrangement of dopachrome to 5,6-dihydroxyindole (DHI), whereas DCT mediates the isomerization/tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). DHI is oxidized easily, leading to the production of melanin, as well as reactive oxygen species (ROS). DHICA is less reactive, relative to DHI, and consequently produces less toxic byproducts during melanogenesis; therefore DCT plays an important role in detoxification of DHI and ROS. Purification and MS analysis of DCE and DCT determined that N-glycosylation is a primary post-translational modification. Q-TOF mass spectrometry was used to determine N-glycosylation patterns from Aedes aegypti DCE and MALDI-TOF/TOF was used to determine multiple glycosylation sites in DCT. N-glycosylation is critical for the folding and trafficking of secreted proteins in the endomembrane system. The analysis of glycosylation sites in DCE and DCT therefore is essential toward achieving a comprehensive understanding of their structure and function. Like DCT, AMD also plays a protective role. The AMD protein was originally identified in Drosophila mutants hypersensitive to α-methyldopa, an inhibitor of dopa decarboxylase (DDC). Production of dopamine by DDC is critical for developing insects because dopamine conjugates are used as crosslinking agents for cuticle sclerotization. Although there has been much discussion into the function of AMD, what exactly this protein does has been unknown. AMD shares 48% sequence identity with DDC, however we have found that AMD is an enzyme, which possesses a different catalytic activity. GC-MS analysis of AMD enzymatic reaction components revealed that AMD catalyzes the oxidative decarboxylation of L-DOPA to DOPAL, and also the oxidative decarboxlation of α-methyldopa to 3,4-dihydroxyphenylacetone. In summary, multiple N-glycosylation sites were characterized in DCT and DCE, furthermore a new protein function has been demonstrated for AMD. These experiments were performed using classical biochemistry techniques in combination with mass spectrometry. / Ph. D.

The metabolic stability of these tracers is, in addition to its affinity and selectivity, an important factor for a successful disease diagnosis. PET tracers and all other drugs are subject to biotransformation which can form metabolites as a part of the inactivation or detoxification process of the human body. These metabolites may result in a higher background which has a detrimental influence on the PET image quality or can even make imaging impossible. The aim of this work was to investigate whether [18F]fluorocyclobutyl rings can be introduced into biologically active small molecules to improve metabolic stability of the PET tracer while maintaining or improving the binding affinity and lipophilicity. To test this hypothesis, the tyrosine model compound, O-(3-[18F]fluorocyclobutyl)-L-tyrosine (L-3-[18F]FCBT), was chosen to be investigated. Precursors for the indirect and direct radiolabeling as well as the non-radioactive L- and D-3-FCBT were successfully synthesized. The radiolabeled of L-3-[18F]FCBT were produced via the indirect and direct method in sufficient yield and activity for the biological evaluation. In the biological characterization, L-3-[18F]FCBT showed good tumor uptake in human lung carcinoma cell lines (A549) and was able to be blocked by both non-radioactive L-3-FCBT and non-radioactive FET. In the biodistribution study, the tracer demonstrated tumor uptake and high metabolic stability due to non accumulation of activity in bone. These results were consistent with the animal-PET imaging where L-3-[18F]FCBT showed good tumor uptake and no accumulation of activity in the bone. The D-isomer in comparison with the L-isomer was found to give lower tumor uptake and very low accumulation in the pancreas. The in vitro stability of the L-3[18F]FCBT in human and rat plasma was excellent over 120 minutes. In vivo stability in mice showed very little metabolites and L-3-[18F]FCBT is considered to be stable in vivo. These results have shown that the new [18F]fluorocyclobutyl group has the potential for the preparation of metabolically stable radiotracers and the application looks very promising.