Thesis (M. Phil.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 64-70).

Universiẗat, Diss., 2002--Mainz.

The occurrence and biological activity of ecdysterone (insect moulting hormone) was examined in a number of plant species. A simple method was developed for the semiquantitative analysis of ecdysterone in plant extracts. The procedure consists of repeated washing of an aqueous methanolic extract of the plant with petroleum ether, followed by gradient elution of the freeze-dried aqueous extract on a SepPak (CIS reverse-phase cartridge), and high-performance liquid chromatography. Crude extract was purified on Sephadex LH20 for spectral analysis. Twelve species of ferns, three species of gymnosperms, and five species of angiosperms were examined for the presence of ecdysterone. Ecdysterone was found in a number of plant species, the chemistry of which has not previously been examined with respect to phytoecdysteroids. These include four species of ferns (Aspidotis densa (Brackenr.) Lellinger., Cryptogramma crispa (L.) R.Br., Blechnum spicant (L.) Roth, Polypodium glycyrrhiza D.C. Eat); two species of gymnosperms {Taxus brevifolia Nutt., Taxus canadensis Marsh.); and two species of angiosperms (Trillium cernuum L, Trillium ovatum Pursh.). Applied ecdysterone had no effect in a cytokinin bioassay, but elicited a slight increase in elongation of mung bean epicotyls (GA₃ bioassay) and elongation of excised dwarf pea hypocotyl hooks (auxin bioassay). / Science, Faculty of / Botany, Department of / Graduate

The physiological responses involved in mediating adaptive change due to varying environmental conditions or social interactions are complex and involve integration of numerous signaling pathways. With Drosophila melanogaster, I can investigate the responses to varied environmental and social stimuli through quantification of signaling activity, stress resistance, and changes in gene expression and behavior. My work focuses on investigating signaling pathways that adult insects use to regulate homeostasis. The steroid hormone 20- hydroxyecdysone (ecdysone) and its receptor (EcR/USP) are vital during arthropod development for coordinating molting and metamorphosis. However, recent adult studies in Drosophila melanogaster indicate that the hormone and receptor influence many processes. I characterized the wild-type expression patterns and activity of ecdysone receptors in individual tissues during early adult life. I found that receptor components EcR and usp were expressed in numerous adult tissues, but receptor activation varied depending on tissue type and adult age. EcR/USP activity did not detectably change in response to environmental stimuli but is reduced when a constitutively inactive ecdysone receptor is present. The current state of our understanding of this signaling system is reviewed with reference to my findings. I discuss future directions focusing on identifying locations of hormone synthesis, metabolism, and storage, isoform-specific roles of EcR, and functional roles of gene repression and activation to link hormone receptor activity with physiological responses. Adult physiology is also regulated by interactions between adipose tissue and the central nervous system. Genes expressed in the insect fat body are involved in regulating nutrient homeostasis, stress resistance, immunity, reproduction, and behavior. Of particular interest is female-specific independent of transformer (fit). Several studies indicate that fat body expression of fit may influence responses to environmental change by altering adult behavior or physiology. Our lab created fit mutants that I used to assess the effects of these mutations on adult Drosophila physiology and behavior. I found that fit mutant adults survive longer without food, have increased nutrient levels, are more active, and feed extensively. My findings indicate that the fat-biased gene fit influences multiple aspects of adult physiology that affect appetite modulation, metabolism, and behavior.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 140-159). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 140-159). Also available on the Internet.

Undergraduate honors paper--Mount Holyoke College, 2006. Dept. of Biological Sciences. / Includes bibliographical references (leaves 75-82).

This dissertation is a study in how heme facilitates biology using two heme proteins as examples. I write about my mechanistic studies on Cimex nitrophorin and preliminary studies on Ecdysone inducible protein 75, respectively. Nitrophorins are salivary heme proteins used by bloodfeeding insects to deliver NO to the victim, leading to vasodilation and antihemostasis. The bedbug nitrophorin cNP, a thiolate heme protein accomplishes this via an unusual heme-assisted S-nitrosation reaction, requiring proximal ligand cleavage. This dissertation explores this mechanism through mutational, crystallographic and transient kinetic approaches. I present the detailed investigation of the two NO binding events, one at the heme and the other at the proximal cysteine. The heme nitrosyl shows marked pH dependence arising out of the apparent protonation of the proximal cysteine ligand, a feature crucial to cNP function. The structures and spectroscopy of cNP mutant proteins reveal the SNO modification to be regulatory in nature. Laser flash photolysis measurements and the structures of mutant proteins reveal the negative influence of steric hindrance on SNO stability.Studies of insect embryogenesis and metamorphosis reveal the regulatory role of the hormone ecdysone via its target, the ecdysone receptor. Ecdysone triggers expression of several nuclear receptors in a time and tissue dependant fashion, which in turn carry out gene regulation. Ecdysone inducible protein 75 (E75), a nuclear receptor and an early ecdysone responsive gene product, regulates a subset of the developmental activities attributed to ecdysone. We are investigating E75 from Aedes aegypti to uncover its role in ecdysone signaling in mosquitoes. I have expressed and partially purified the full length protein using the baculovirus driven expression in SF9 cells, and purified to homogeneity the heme binding domain resolubilized from inclusion bodies obtained by expression in E. coli. Preliminary characterization of the proteins using UV-visible spectroscopy indicates that E75 has a b type heme with a low spin six-coordinate ferric iron. In the E75 heme binding domain, the heme exhibits an unstable ferrous state and only binds NO and CO at high non-physiological levels. These data place into doubt the suggested roles for E75 as a gas regulated transcription regulator.

The fruit fly Drosophila melanogaster is recognised as the most widely established genetic model of immunity of the contemporary scientific era, exhibiting a high degree of conservation between Drosophila and mammalian innate immunity genes. However, whilst the majority of Drosophila immunity studies have previously been performed in adults and larvae, the embryo has recently emerged as a potentially viable model system; aiding in vivo studies and providing a more amendable system to undertake live imaging, hence evading many of the caveats associated with current immunity models. This project aimed to further develop the Drosophila embryo as a more potent and insightful immunity model, focusing on the immune response to bacterial infection. Initial results demonstrated that the Stage 15 Drosophila embryo was able to mount a relatively robust immune response to bacterial infection. This included induction of antimicrobial peptide (AMP) genes upon a range of bacterial stimuli; a response which was able to effectively discriminate between differential types of bacterial infection via the characterised Drosophila systemic immunity pathways. Live-imaging studies also showed that the cellular immune response was functional within the Stage 15 embryo. Subsequently, immune competence was shown to arise at approximately mid-embryogenesis, under the control of 20-hydroxyecdysone (20-HE) signalling, as demonstrated by the partial rescue of AMP expression and bacterial clearance in early stage embryos upon 20-HE co-administration with infective agents. Further analysis of the global transcriptional response of the Drosophila embryo to infection and damage via microarray studies confirmed the immune potential of this system, but also permitted the identification of genes upregulated uniquely upon Gram-positive or Gram-negativ infection. Moreover, wounding via sterile laser ablation induced significant upregulation of a subset of AMP genes an a network of cuticular genes, providing an insight into the embryonic damage response. Parallel analysis of the hemocyte transcriptional profile upon infection and damage elucidated that these immune cells may play a role in the regulation of the immune response via 20-HE signalling and production of ROS, although this remains subject to further validation. As such, transcriptional profile analysis of the embryo has been successful in identifying candidate genes for further validation and study.

published_or_final_version / Zoology / Master / Master of Philosophy