The Golgi body is responsible for the modification and sorting of proteins and lipids in the secretory pathway. The Golgi must coordinate with other endomembrane compartments in order to target cargo to the correct destination. While our understanding of Golgi function is vast, we can extend our knowledge base by examining the functions of Golgi-associated proteins in developing animals. Lava lamp (Lva) is a Golgi-associated protein and a Drosophila golgin. Previously, Lva was shown to facilitate efficient membrane secretion required for cleavage furrow formation in early embryos. By acting as an adaptor molecule between Golgi and microtubule motility factors, Lva is thought to position Golgi bodies for targeted secretion during cellularization, the Drosophila cleavage stage of development. Here, I further characterize the role of Lva during animal development. I demonstrate that Lva is required for animal viability, and gamete production in females but not males. While Lva is expressed in many tissues, adult fat body cells are the most sensitive to decreased Lva activity, resulting in the disorganization of endomembrane compartments. Furthermore, this disruption in adult fat body cells correlates with a defect in neuroendocrine signaling, altering the activity of juvenile hormone. I propose that Lva activity in adult fat body cells is important for recognizing and/or processing juvenile hormone in order to support Drosophila oogenesis. Lva’s role in cellularization, which is a specialized form of cytokinesis in early embryos, provided insights into the combined processes of actomyosin-based contraction and membrane secretion. While some proteins have been implicated in cellularization, there are thought to be many more that have yet to be identified. In an effort to isolate additional genes involved in animal cell cytokinesis, we screened a unique collection of temperature sensitive (ts) mutations on the X-chromosome of Drosophila melanogaster. At the restrictive temperature, we identified five mutants that displayed a cellularization phenotype. For one of the mutants, fs(1)ts242, we narrowed the mutation to a region on the X chromosome consisting of 17 possible gene candidates. Identification of the gene should provide further elucidation of the mechanisms controlling actomyosin-based contraction and membrane secretion. / text

published_or_final_version / Zoology / Master / Master of Philosophy

Gonadotropins were used to stimulate luteal function, as determined by progesterone secretion, in both in vitro and in vivo systems. LH and hCG were capable of significantly stimulating progesterone secretion in the in vivo systems. Stimulation of progesterone secretion by hCG was greater than that for LH. PMSG failed to increase progesterone production at any level of treatment. hCG was also used to stimulate progesterone production by the corpus luteum in mares during early gestation. hCG administration resulted in a significant (p < 0.10) increase in peripheral progesterone levels in treatment mares through day 14 post-estrus. Peripheral progesterone concentrations were also higher in hCG treated mares for days 15 through 30 post-estrus in mares that conceived. hCG treatment had no influence on anterior pituitary release of LH.

Allatostatins (ASTs) are neuropeptides that inhibit the biosynthesis and release of juvenile hormone from the corpora allata (CA) of various insects including the cockroach Diploptera punctata. We hypothesized that a similar allatostatin receptor would exist in the cockroach D. punctata that may regulate the numerous physiological effects that this family of peptides exerts on a range of target tissues. Using polymerase chain reaction (PCR) strategies successful in isolating other insect allatostatin receptor sequences utilized primers designed to known mammalian somatostatin receptors and Drosophila allatostatin receptor (AlstR). Once an internal PCR fragment was proven to be consistent with the sequence of an allatostatin receptor (AstR) then the sequence was completed by rapid amplification of cDNA ends (RACE). The putative allatostatin-like receptor sequence encoding 425 amino acid residues was isolated from a cDNA library prepared from corpora allata of D. punctata. We show that dsRNA targeting the allatostatin receptor gene of D. punctata injected into freshly moulted adult cockroaches produced a long-lasting reduction in the mRNA levels in midgut tissues. The effect lasted up to 6 days. Following dsRNA injection, the juvenile hormone (JH) titers in the corpora allata were clearly raised suggest that the putative inhibition of receptor RNA expression may increase JH production. The receptor is expressed in brains, corpora allata, abdominal ganglion, midguts, ovaries, and testes. We have examined these same tissues with regard to changes in expression levels of Dippu-AstR. JH biosynthesis peaks on day 5 post-emergence in mated females. In mated females, Dippu-AstR mRNA is expressed at the highest levels on day 6 post-emergence in brain and corpora allata and day 2 post-emergence in midgut. Dippu-AstR is likely responsible for the decline in JH biosynthesis after day 5 post-emergence. Virgin females midgut and CA Dippu-AstR mRNA expression dramatically elevated on days 6 and 7, respectively. Expression of Dippu-AstR was similar in the abdominal ganglia of mated or virgin females. Ovarian Dippu-AstR expression declines to low levels by day 4. Testes also express peaks of Dippu-AstR expression on days 4 and 7. A role for Dippu-AST in testes is yet unknown. / Thesis (Ph.D, Biology) -- Queen's University, 2008-04-22 15:21:55.107

The focus of the research described in this section of the thesis is the synthesis of compounds expected to bind strongly to both the estrogen β and α receptors and act as estrogen agonists. Based on earlier results in our group and docking studies we prepared a series of A-CD analogs, compounds 1, in which the usual 13-methyl group was replaced by an ethyl group. Docking studies also indicated that substituents at C8 could lead to enhancement of binding to the estrogen receptor. With this in mind two such derivatives, compounds 2 were prepared. A major concern in the use of estradiol in hormone replacement therapy is its potential metabolism of dangerous ortho-quinones. The 1,2-naphthalenediol derivatives 3 avoid this possibility. They were predicted to be potent binders to the estrogen receptors with the naphthalene diol portion serving as rings A and B and the hydroxyl group taking the place of the 17-OH group of estradiol. The preparation of several derivatives of 2 is reported. The estrogen receptor binding [ERB] relative to estradiol as standard has been determined at the University of Illinois for a number of the compounds prepared in this thesis. Unfortunately, the results were not as encouraging as expected. Importantly, all of the 13-ethyl derivatives tested showed lower binding affinity compared to the 13-methyl analogs. Similarly, the derivatives with substituents at C8 do not show higher activity than those having only hydrogens at C8. Finally, the situation with the naphthalene derivatives is, at this stage, still not completely resolved. The binding for the compounds thus tested is quite low, but it must be admitted that the structures thus far synthesized have a much lower LogP than estradiol, a factor known to greatly decrease the binding constants to the estrogen receptors.

Osterix (Osx) is a transcription factor required for osteoblast differentiation and bone formation. We previously demonstrated that continuous parathyroid hormone (PTH) treatment inhibited Osx expression in murine calvaria and osteoblastic UMR106-01 cells through the regulation of two regions on the Osx promoter. Mutational analysis of transcription factor elements within these regions revealed two "Sp-sites" were vital for Osx promoter activity. Blockage of these Sp-sites with Mithramycin A demonstrated their importance for Osx expression. Osx bound to its own promoter at these sites, while PTH treatment inhibited this association. PTH regulation of Osx expression in vivo was investigated in mice by: daily injection of PTH for 3 days, continuous infusion of PTH from osmotic pumps for 14 days, or mice fed a calcium-deficient diet for 21 days. Osx expression was decreased by daily injection, while Osx expression was stimulated in mice receiving continuous PTH infusion and mice fed a calcium-deficient diet.

Osterix (Osx) is a transcription factor required for osteoblast differentiation and bone formation. We previously demonstrated that continuous parathyroid hormone (PTH) treatment inhibited Osx expression in murine calvaria and osteoblastic UMR106-01 cells through the regulation of two regions on the Osx promoter. Mutational analysis of transcription factor elements within these regions revealed two "Sp-sites" were vital for Osx promoter activity. Blockage of these Sp-sites with Mithramycin A demonstrated their importance for Osx expression. Osx bound to its own promoter at these sites, while PTH treatment inhibited this association. PTH regulation of Osx expression in vivo was investigated in mice by: daily injection of PTH for 3 days, continuous infusion of PTH from osmotic pumps for 14 days, or mice fed a calcium-deficient diet for 21 days. Osx expression was decreased by daily injection, while Osx expression was stimulated in mice receiving continuous PTH infusion and mice fed a calcium-deficient diet.

Taste is detected by cells of taste buds in the oral cavity. Mammalian taste buds contain three types of cells: receptor, presynaptic, and glial-like. Of these three, glial-like cells are the least studied. Their only known function is that they clear neurotransmitters from the extracellular space. The present work describes two previously undocumented properties of glial-like cells. First, Oxytocin receptor (OXTR) mRNA was detected by RT-PCR in taste tissue of mice. In the taste buds of Oxtr-YFP knockin mice, YFP was seen in glial-like taste cells and other cells immediately outside the taste bud, but no other cells in oral epithelium. Oxytocin (OXT) elicited Ca2+ responses from cells that resemble glial-like taste cells (by criteria including gene expression and lack of excitability). The EC50 for OXT in these cells was 33 nM, and responses saturated at 1 µM. 500 nM L-371,257 (an OXTR antagonist) significantly inhihited the responses to OXT. In a semi-intact preparation of lingual slices, OXT did not alter bitter tastant-evoked Ca2+ responses. Further, in behavioral studies, OXT (10 mg/kg i.p.) did not alter the responses of mice to aversive salty (NaCl), bitter (quinine), or sour (citric acid) solutions. In contrast, OXT (0.1 mg/kg i.p.) significantly decreased taste behavioral responses to low-to-intermediate concentrations of sucrose. My data suggest that OXT may modulate sweet taste sensitivity in vivo by acting on glial-like cells in taste buds. Second, Renal Outer Medullary K channel (ROMK) mRNA was also detected by RT-PCR in taste buds . Immunostaining revealed that ROMK is localized to the apical tips of glial-like taste cells. In the kidney, ROMK, apically localized in nephron epithelium facilitates a unidirectional flow (i.e. excretion) of K+. I suggest that, analogous to glia in the central nervous system, glial-like taste cells homeostatically redistribute extracellular [K+ ] within taste buds to maintain their sensitivity. The results of this study reveal that glial-like taste cells resemble nervous system glia in more ways than simply clearing neurotransmitters. They may also modulate the sensory output of the taste bud and buffer the extracellular [K+]. A more active role for glial-like cells in the functioning of the taste bud should be investigated.

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