In helminths, including the free-living nematode, Caenorhabditis elegans, serotonin (5-HT) acts as an important neuroactive agent and is associated with carbohydrate metabolism, glucouse utilization, motility, feeding and reproductive behaviour. In mammals and other organisms, 5-HT is synthesized through the action of tryptophan hydroxylase (TPH). TPH is the rate limiting enzyme in the biosynthesis of 5-HT, and as such sets the pace for the formation of 5-HT. TPH is a member of a family of enzymes that hydroxylate aromatic amino acids and have an absolute requirement for the pterin cofactor, tetrahydrobiopterin (BH4). It is unknown if this same enzyme catalyzes the synthesis of 5-HT in C. elegans and other helminths. / Based on sequence information from the C. elegans Genome Data Base and RT-PCR, we have cloned a full-length C. elegans TPH cDNA (CeTPH) that shows high homology to mammalian TPH. The predicted coding sequence of CeTPH was subcloned into the prokaryotic expression vector, pET-15b, and the resulting construct was introduced into E. coli (BL21 DE3 pLys strain) for IPTG-inducible expression of CeTPH protein. Results show that CeTPH expressed in E. coli has TPH activity and also shows an absolute requirement for the cofactor, BH4, just as shown previously for the mammalian enzyme. It has been well established that 5-HT is present and is biologically active in the tissues of C. elegans. By way of characterizing furthur CeTPH, we examined the localization of TPH in whole mounts of C. elegans by immunofluoresence using a polyclonal antibody against TPH. / Taken together, the results of this thesis characterize at the structural, functional and in situ levels one of the most primitive forms of TPH enzyme ever cloned.

Glutamate-gated chloride channels (GluCl) belong to then icotinic ligand-gated ion channel family and are thus assumed to be heteropentamers. Each subunit contains a large extracellular N-terminal domain, four transmembrane domains (TM1--TM4), and an extracellular C terminal. Caenorhabditis elegans expresses various GluCl channels formed by alpha1, alpha2, alpha3, alpha4 and beta subunits. The best understood GluCl channel is expressed in pharyngeal muscle cells where it mediates response to the M3 motor neuron. alpha2 forms this channel, probably in association with beta. The alpha2 mutant lacks M3 neurotransmission which can be rescued by pharynx-specific alpha2 expression. My results show that alpha1 and alpha3 subunits cannot substitute for alpha2. Formation of chimeric constructs of alpha1, alpha2 and alpha3 pinpoints the M1--M3 transmembrane region of alpha2 as the minimal rescuing domain. This region may therefore be important for localization or, in association with another subunit, in the formation of the active channel.

Cell migration and extension is essential for development. The ability of a cell or cell extension to reach its target is dependent on spatial cues and receptors that translate positional information into directed plasma membrane extension. For example, the UNC-40/DCC receptor is required to direct circumferential migrations towards the source of the ligand, UNC-6/Netrin, expressed at the ventral midline. To better understand the process of cell extension, I used a specialized process called muscle arms as a model system. In C. elegans, body wall muscles (BWMs) extend membrane projections called muscle arms to the nearest nerve cord at the midline. These muscle arms harbor the postsynaptic element of the neuromuscular junction and extend in a stereotypical and regulated manner. In a screen for muscle arm development defective (Madd) mutants, I isolated madd-2, a novel component of the UNC-40 pathway. MADD-2 is a C-1 TRIM protein and functions cell-autonomously to direct numerous muscle and axon extensions to the ventral midline of worms. In a striking analogy, mutations in a human homologue of MADD-2, MID1, cause numerous ventral midline defects that culminate as Opitz Syndrome. How MID1 regulates midline development is unclear. MADD-2 enhances UNC-40 pathway activity by facilitating the physical interaction between UNC-40 and the downstream Rho-GEF, UNC-73. It is possible that MID1 may mediate the function of a DCC-like pathway at the ventral midline of humans. This work provides the first indication that C1-TRIM proteins may have a conserved biological role of regulating midline-oriented development events and may provide key insights into the role of MID1 in the pathogenesis of Opitz syndrome.

Ubiquinone (UQ) is a lipid found in all cellular membranes. It is involved in multiple cellular processes, either directly or through its effect on the redox status of the cell. clk-1 encodes a highly conserved hydroxylase required for UQ biosynthesis. In C. elegans, mutations in clk-1 result in the accumulation of an UQ precursor, DMQ, and a pleiotropic phenotype in the mutants characterized by the slowing down of development, behaviors and aging. Additionally, in the absence of dietary UQ, clk-1 mutants also show a transient growth arrest and are sterile. Mutants that can suppress both sets of phenotypes in the point mutant clk-1(e2519) have been isolated. Their suppression patterns indicate that various aspects of the clk-1 phenotype can be uncoupled from each other. Furthermore, the analysis of their quinone content suggests that the phenotypes on UQ-producing bacteria are caused by the inability of dietary UQ to completely substitute for endogenous UQ. These suppressors carry mutations in tRNA genes, and thus to our knowledge, they are the first tRNA missense suppressors found in any metazoan.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "December 1996." Typescript. Vita. Includes bibliographical references (leaves 196-206). Also available on the Internet.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 116-123). Also available in electronic version. Access restricted to campus users.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 215-242).

Thesis (Ph. D.)--University of North Texas, Dec., 2008. / Title from title page display. Includes bibliographical references.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2005. / Includes bibliographical references (leaves 143-153). Also available in electronic version.