published_or_final_version / Zoology / Master / Master of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (doctor of medicine)--Københavns Universitet, 1979.

Type IV pili, or fimbriae, are long, thin proteinaceous appendages found on the surface of many well-known pathogens. They mediate a variety of important virulence functions for the organism, such as twitching motility, biofilm formation, uptake of genetic material and host cell recognition and adhesion. Pili are formed by the rapid polymerization and de-polymerization of the pilin subunit, and this is orchestrated by a complex macromolecular machine which spans the bacterial cell envelope, requiring a variety of gene products. The type IV pilus biogenesis system is closely related to the bacterial type II secretion system, one of six designated multi-protein cell envelope complexes which are dedicated to the specific secretion of exotoxins and virulence factors. Many of these secretion systems also produce fimbrial structures to facilitate the extrusion of their substrates or to communicate with the host. As they form crucial virulence factors, the secretion systems and the type IV pilus biogenesis system have become attractive potential antimicrobial targets and obtaining structural and functional information for the components of these systems is an important first step towards achieving this.Type IV pili appear on the surface of bacteria through an outer membrane pore, PilQ, which is a member of the secretin family. Secretins are also found in the type II and III secretion systems, but the way in which they are regulated remains unclear. PilQ forms a dodecameric chamber in the outer membrane with a large vestibule which reaches into the periplasm, composed of its N-terminal domains. In this project, N-terminal domains from PilQ were produced in recombinant form and their structures determined by NMR. One of these domains revealed an eight-stranded beta-sandwich structure which appears to be unique to type IV pilus secretins and has not been structurally characterized before. Another revealed an alpha/beta type fold which is common to secretins of other systems. In the second part of this project, the interaction formed between the N-terminal alpha/beta domains of PilQ and an essential inner membrane-anchored lipoprotein, PilP, was probed by NMR chemical shift perturbation. Based on changes to the 15N-HSQC spectra the binding site was mapped onto each protein to produce a computational model for the complex formed between the two. Using a recent cryo-EM structure for the Neisseria PilQ dodecamer determined by colleagues, it was possible to model the PilQ N-terminal domains in complex with PilP into the electron density map. This produced a model for the trans-periplasmic assembly formed by PilQ and PilP in the type IV pilus biogenesis system, and led to the conclusion that the PilQ dodecamer needs to disassemble considerably at the base to accommodate a pilus fibre. The novel beta-domains might therefore function to gate or open the secretin, and PilP may play a role in stabilizing the secretin during this and serve to connect the outer and inner membrane system components.

Zhang Jie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 54-74). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / ABSTRACT (Chinese) --- p.iv / ABBREVIATION --- p.vi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Overview of the study --- p.1 / Chapter 1.2 --- Secretin --- p.3 / Chapter 1.2.1 --- Discovery / Chapter 1.2.2 --- Molecular biology / Chapter 1.2.3 --- Biosynthesis and localization / Chapter 1.2.4 --- Function / Chapter 1.3 --- Secretin receptor --- p.8 / Chapter 1.3.1 --- Molecular biology / Chapter 1.3.2 --- Localization / Chapter 1.3.3 --- Signal transduction pathway / Chapter 1.4 --- Secretin and autism --- p.13 / Chapter 1.5 --- AMPA receptor --- p.15 / Chapter 1.5.1 --- Molecular biology / Chapter 1.5.2 --- Localization / Chapter 1.5.3 --- Pharmacological property / Chapter 1.5.4 --- Function / Chapter 1.6 --- Cerebellum --- p.20 / Chapter 1.6.1 --- Structure of the cerebellar cortex / Chapter 1.6.2 --- Neurons of the cerebellar cortex / Chapter 1.6.2.1 --- Granule cells / Chapter 1.6.2.2 --- Purkinje cells / Chapter 1.6.2.3 --- Basket and stellate cells / Chapter 1.6.2.4 --- Golgi cells / Chapter 1.6.3 --- Intrinsic circuitry of the cerebellar cortex / Chapter CHAPTER 2 --- METHODS AND MATERIALS --- p.25 / Chapter 2.1 --- Brain slice preparation and maintenance --- p.25 / Chapter 2.2 --- Experimental set-up --- p.26 / Chapter 2.2.1 --- Visualization of neurons / Chapter 2.2.2 --- Electrophysiological recordings / Chapter 2.2.3 --- Evoked stimulation / Chapter 2.2.4 --- Drug preparation and administration / Chapter 2.3 --- Data analysis --- p.29 / Chapter 2.3.1 --- Construction of dose-response curve / Chapter 2.3.2 --- Analysis of synaptic currents / Chapter 2.3.3 --- Statistics / Chapter CHAPTER 3 --- RESULTS --- p.31 / Chapter 3.1 --- Basic characteristics of IPSCs recorded from PCs --- p.31 / Chapter 3.1.1 --- Spontaneous IPSCs / Chapter 3.1.2 --- Miniature IPSCs / Chapter 3.1.3 --- Evoked IPSCs / Chapter 3.1.4 --- Rundown of IPSCs / Chapter 3.2 --- Electrophysiological effects of secretin --- p.33 / Chapter 3.2.1 --- Effects of secretin on evoked IPSCs and EPSCs / Chapter 3.2.2 --- Effects of secretin on spontaneous IPSCs / Chapter 3.2.3 --- Effects of secretin on miniature IPSCs / Chapter 3.3 --- Mechanisms of secretin as a neuropeptide --- p.37 / Chapter 3.3.1 --- Non-involvement of a postsynaptic site of action / Chapter 3.3.2 --- Non-involvement of calcium influx / Chapter 3.3.3 --- Involvement of cAMP second messenger / Chapter 3.3.4 --- Involvement of presynaptic AMP A receptors / Chapter 3.3.4.1 --- Glutamate-mediated action of secretin / Chapter 3.3.4.2 --- Effects of AMPA on miniature IPSCs / Chapter 3.3.4.3 --- Pharmacological evidence / Chapter CHAPTER 4 --- DISCUSSION --- p.45 / Chapter 4.1 --- Secretin as a novel neuropeptide --- p.45 / Chapter 4.2 --- Mechanisms of secretin --- p.46 / Chapter 4.3 --- Physiological role of secretin in the cerebellum --- p.52 / Chapter 4.4 --- Secretin and autism --- p.52 / REFERENCES --- p.54

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Peptide hormones are critical modulators of physiology and development in humans and have been well characterized for their effects on humans and other mammals. The question of the origin of the many families of peptide hormones in mammals is pressing, as it gives us a window into the evolution of important systems in all extant animals and their common ancestors. The focus of this thesis was to examine the origin of a select group of peptide hormone families including the secretin superfamily, reproductive neuropeptides, insulin and the insulin-like peptides, and stanniocalcin. The evolution of the secretin superfamily was found to have originated with the vertebrates, and new information from the genomes of basal vertebrates like the lamprey Petromyon marinus and elephant shark Callorhinchus milii allows us to better piece together the gene duplications that produced the current hormone family in humans and fish. The reproductive hormones, including gonadotropin-releasing hormone (GnRH), vasopressin/oxytocin, and kisspeptin were examined, with a focus on the evolution of their G protein-coupled receptors. GnRH was found to have originated in the early bilaterians, and its receptors clearly belong to a superfamily also containing receptors of the related neuropeptides adipokinetic hormone and corazonin, which have only been found in protostome invertebrates. Vasopressin/oxytocin receptors share a common ancestor with the GnRH receptors, although their peptides are not structurally related, and evolved at a similar time. Kisspeptin evolved later, within the vertebrates, however its receptors are closely related to an orphan receptor in protostome invertebrates, GPR54, with an unknown ligand. Insulin family members from the tunicate Ciona intestinalis and the amphioxus Branchiostoma floridae were identified, isolated and characterized to determine the nature of the insulin superfamily at the origin of the chordates, and it appears this family was well-developed already. Finally, the calcium-regulator stanniocalcin was identified, isolated and characterized in C. intestinalis and compared with the vertebrate and amphioxus stanniocalcins. A group of stanniocalcins were also discovered in a wide range of both protostomes and unicellular eukaryotes, indicating this ancient group of neurohormones appeared early in eukaryotic evolution. / Graduate

Thesis (M. Phil.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 61-74).