Habilitationsschrift--Mainz. / Bibliography: p. 163-174.

Habilitationsschrift--Mainz. / Bibliography: p. 163-174.

Thesis (M. Arch.)--University of Hong Kong, 1996. / Includes special report study entitled: Natural light, museum and underground space. Includes bibliographical references.

Thesis (M.Arch.)--University of Hong Kong, 1996. / Includes special report study entitled : Natural light, museum and underground space. Includes bibliographical references. Also available in print.

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The impacts of global change on ecosystems from climate change and invasive species are likely to be complex. Rising atmospheric CO2 concentrations, the associated climate forcing and greater frequency of extreme weather are serious challenges to natural ecosystems. In tandem with climate change, globalisation has led to the spread of invasive alien species around the globe that threaten to interrupt food web dynamics. Advancing understanding of the effects of global change on trophic interactions therefore requires study of interspecific and multi-trophic interactions. The aim of this thesis was to examine how host-plant heterogeneity, native–invasive species interactions and climate change effects (elevated atmospheric CO2 (eCO2) or drought) influence trophic interactions. An experimental approach was used which centred on a study system comprising the European raspberry (Rubus idaeus), the herbivorous large raspberry aphid (Amphorophora idaei) and coccinellid beetle predators (native species: Adalia bipunctata, Coccinella septempunctata; invasive alien species Harmonia axyridis). Under eCO2, R. idaeus resistance to A. idaei was unchanged for two cultivars (Glen Clova, Glen Ample) partially susceptible to A. idaei, but significantly reduced for another (Octavia) with complete resistance in ambient climatic conditions. The inclusion of a coccinellid predator, however, mitigated the reduction in the resistance of Octavia by reducing aphid abundance. Behavioural responses to predation by A. idaei were also impaired under eCO2 after feeding on Glen Ample. The role of natural enemies in controlling herbivore abundance in future climates is therefore crucial. Native coccinellid species are currently declining in much of Europe, attributed to the occurrence of the invasive species, H. axyridis. Despite the declines in native coccinellid species, it was found that behavioural modification to feeding by both native and invasive coccinellid species can, theoretically, result in coexistence. Plant resistance in a future climate is likely to be modified significantly. Reduced resistance to aphid herbivory demonstrated here mirrors previous studies, highlighting the future importance of natural enemies to control aphid abundance. Changes to the abundance and behaviour of aphid prey and intraguild predators will modify the effectiveness of native and invasive natural enemies. Further mechanistic research is required to understand multi-trophic interactions in dynamic environments.

Kinetochores are multi-protein machines that control chromosome movements by regulating the dynamics of attached microtubules. In human cells chromosome movements are orchestrated by the leading kinetochore tracking a shrinking microtubule whilst its sister tracks a growing microtubule. Directional switches occur when (both) kinetochore-attached microtubules fl ip between these two states, adaptive and coordinated switching then giving rise to the oscillations observed during metaphase. However the mechanisms (and rules) controlling directional switching are poorly understood. This work demonstrates that by tracking kinetochores with sub-pixel resolution in HeLa cells and fitting stochastic mathematical models that a sensor on the leading sister triggers switching when the tension across the centromeric spring connecting the sisters builds up sufficiently rapidly. Further it is shown that the trailing sisters polymerisation state is stabilised by high spring tension. These mechanisms pre-empt trail-first switching that would otherwise impose abnormal pulling forces between sister chromatids. As a consequence sister-switching is biased towards lead-first switching, switching of the trailing sister rapidly following as the spring tension falls, this removing the force dependent stabilisation of the trailing sisters K-fibre (kinetochore bound microtubules). This model explains how switching events are initiated and resolved, the centromeric spring tension providing a means for inter-sister communication and cross regulation that results in coordinated oscillations within a context of low spring tension. This study demonstrates that high throughput analysis and modelling pipelines can provide novel mechanistic insight into mechanochemical systems.

G protein-coupled receptors (GPCRs) are a superfamily of membrane proteins that bind to a diverse array of stimuli and are involved in a large number of physiological functions. The family A GPCRs are the largest and most comprehensively studied. The family B GPCRs are a small but important group of receptors (~15 members) that bind to peptide ligands and are involved in physiological processes that include vasodilation, stress, digestion and glucose homeostasis. The CGRP receptor is a unique member of this family as it is a heterodimer consisting of a GPCR subunit (calcitonin receptor-like receptor, CLR) and a single transmembrane accessory protein (receptor activity modifying protein, RAMP1). The extracellular loop two (ECL2) domain is involved in ligand binding and activation in a number of studied GPCRs. This makes it vital both with respect to receptor function and in the design of therapeutics. The main focus of this thesis is to study the structure and function of the ECL2 domain in the CGRP receptor. This was initially done through individual alanine substitutions of each ECL2 residue and measuring the effect of this on a number of receptor processes. Residues that were identified as important for receptor function through this investigation were selected for an extensive set of mutagenesis to identify the precise molecular interactions that were involved at each position. These experiments have shown that ECL2 is the most important domain of the CGRP receptor for ligand-based activation. The N-terminal half of ECL2 contains residues predicted to have structural function and the C-terminal half is predicted to be involved in direct ligand binding. These results have been used in collaboration to refine a computer model of receptor structure and ligand binding to predict specific ligand docking sites that can be used to design therapeutics for migraine, heart attack and hypertension. The final part of thesis produced preliminary data to support proof of concept for two techniques that can be used in the study of CGRP receptor function.

In last decade, there has been a tremendous progress in scanning probe microscopies, some of which have achieved atomic resolution. However, there still exist some problems which have to be solved before the instrument can be used as a metrological measurement tool. The object of the project introduced in this thesis was to develop a scanning force microscope of metrological capability with the aim of making significant improvement in scanning force microscopy from the viewpoint of instrumentation. A capacitance based force probe has been studied theoretically and experimentally with the main concern being its dynamic properties, characterized by squeeze air film damping, which are believed to have direct effects on the fidelity of measurement. The optimization of design is investigated so as to achieve the results of both high displacement sensitivity and force sensitivity. An x-y scanning stage has been designed and built, which consists of a two axis linear flexure system of motion amplifying mode machined from a single aluminium alloy block. The stage is driven by two piezo actuators with two capacitance sensors monitoring the actual position of the platform to form a closed loop control system. The design strategy is introduced and the performances and characteristics of two commonly used types of flexure translation mechanisms, leaf spring and notch hinge spring system, are analyzed. The finite element analysis method is employed in the analysis and design of translation mechanism. Finally, a metrological scanning force microscope has been constructed, combining a constant force probe system, an x-y scanning stage and a 3D coarse positioning mechanism into a metrological system. The performance of the instrument system has been systematically evaluated and its measuring capability investigated on the. specimens of various properties and features. The results from this first prototype of the instrument demonstrated a subnanometer resolution with comparable stability and repeatability in all three axes.

Methanotrophic bacteria convert methane to methanol using a methane monooxygenase enzyme (MMO). Two types of MMO exist: a membrane bound enzyme (PMMO) and a cytoplasmic enzyme (sMMO). A system for the site-directed mutagenesis of residues in the active site of sMMO has recently been developed that uses a sMMO-minus strain of Methylosinus trichosporium OB3b as the expression host (Smith et al., 2002. Appl. Environ. Microbiol. 68:5265-5273); this strain, designated Mutant F, was created by disrupting the mmoX gene by marker-exchange mutagenesis. In this study a Ms. trichosporium OB3b strain was created in which all the sMMO structural genes were deleted or disrupted. This mutant was designated Ms. trichosporium SMDM. The recombinant expression of sMMO was performed in Ms. trichosporium SMDM using the same sMMO expression plasmid used for Ms. trichosporium Mutant F. The effect of sMMO expression in the absence of the enigmatic mmoD gene was investigated. Preliminary results indicate that mmoD is required for active expression of sMMO. The sMMO genes from Methylocella silvestris BL2T were sequenced and conjugated into Ms. trichosporium SMDM on a broad host range plasmid. No expression of Methylocella silvestris BL2 T sMMO was detected in Ms. trichosporium SMDM. A new system for the mutagenesis of the Ms. trichosporium OB3b sMMO a-subunit was created. Chimaeric sMMO mutants were created by introducing gene sequence from the alkene monooxygenase enzyme of Rhodococcus corallin us into the mmoX. The chimaeric sMMO enzymes appeared to be unstable in Ms. trichosporium Mutant F. An attempt was made to improve the stability of sMMO mutants in Ms. trichosporium Mutant F by disrupting the gene encoding the Lon protease. The Ms. trichosporium OB3b Ion gene was cloned and sequenced and attempts were made to disrupt the Ms. trichosporium OB3b Ion by marker-exchange mutagenesis. A mutant was not obtained, suggesting that Lon may be essential for vegetative growth of Ms. trichosporium OB3b. The diversity of sMMO in several environmental samples was investigated using PCR. The objective was to isolate novel mmoX sequences from uncultivated methanotrophs that could be used to design sMMO mutagenesis experiments. New PCR primers targeting the mmoX were developed. The primers were used to generate libraries from a blanket bog peat (UK) and from cave water (Romania). A group of sequences that did not cluster with the mmoX of any cultivated methanotroph was obtained from the cave water. The use of a PCR independent approach to clone methanotroph genes from environmental samEles was also investigated. This was performed by developing a method to clone 1 C-DNA from a stable isotope probing experiment with 13CH4 into a BAC vector. A library of 2300 clones was generated. Greater than 95 % of plasmids analysed contained inserts, which ranged in size from approximately 10 - 30 kb. The library was screened for mxaF, mmoX and pmoA by colony hybridization. A clone (15 kb) containing pmoA was completely sequenced. Other genes encoding proteins with (potential) roles in methylotrophy were contained on the clone, includingpmoC, pmoB, folP, folK, mptG and moxF.