The cloning, characterisation and engineering of an IGF-I-BINDING single chain Fv

This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) and the utilisation of this scFv as a model protein for the study of the application of DNA shuffling and ribosome display to antibody engineering. The variable domain genes were isolated from the hybridoma cell line producing the monoclonal antibody and successfully joined by PCR for the construction of the scFv, named anti-GPE. Sequencing of the gene revealed an unusually short heavy chain CDR2 region. The cloned scFv was expressed in E. coli and purified. Expression levels were low and the protein has poor solubility, most likely due to a reduction in folding efficiency caused by the abbreviated CDR2. The purified monomeric form of the protein was analysed for binding to IGF-I using surface plasmon resonance on the BIAcore 1000 with the specificity of the IgG version of the antibody for the three N-terminal residues of IGF-I - Gly-Pro-Glu - reproduced. The scFv's calculated dissociation constant of 3.68 µM is a low affinity for an antibody and is approximately 36-fold weaker than was calculated for the Fab version of the antibody, but it is concluded that the calculated affinity for the scFv was an apparent affinity that may be an underestimation of true affinity due to the presence of non-functional or misfolded scFv species within the gel-filtration purified monomer peaks. A mutant version of anti-GPE with residues inserted in the CDR2 to restore it to normal length produced a protein with improved expression and solubility characteristics while retaining IGF-I-binding. It was concluded that the short CDR2 was due to deletions generated during the somatic mutation process and a model for this is described. A ribosome display method using a rabbit reticulocyte lysate as a source of ribosomes was developed for specific selection of anti-GPE against IGF-I. Error prone PCR was used to produce a random point mutated library of anti-GPE (EPGPE). This was taken through several cycles of display and selection but selection for non-specifically binding scFvs was commonly observed. This was probably due to poor folding of ribosome-displayed proteins in the system used, possibly caused by the presence of DTT in the lysate and/or the low capacity of the anti-GPE framework to tolerate mutation while retaining stability. It is assumed misfolds, exposing hydrophobic regions, would have a tendency to non-specifically interact with the selection surface. Of the 64 EPGPE clones screened from four rounds of display and selection, many were shown to have poor or non-specific binding, but one scFv was characterised that was affinity matured 2.6-fold over anti-GPE wild type affinity for IGF-I. A DNA shuffling method was developed to produce libraries of chimaeric scFvs between anti-GPE and NC10 (anti-neuraminidase scFv) with the objective of isolating functional IGF-I-binding chimaeras. The NC10 scFv had its CDRs replaced with the anti-GPE CDRs prior to the shuffling to increase the likelihood of isolating IGF-I binders. Ribosome display was used for selection from the chimaera libraries. Selection strategies included elution of specific binders by GPE peptide and a GPE 10-mer peptide. Selection was also performed using IGF-I immobilised on a BIAcore sensorchip as a selection surface. Again, much non-specific selection was observed as seen for display of EPGPE, for what was expected to be the same reasons. Selected scFvs were genuinely chimaeric but with poor expression and solubility and mostly non-specific in their binding. One characterised selected chimaera, made up of three segments of each of the parental scFvs, was shown to bind specifically to IGF-I by BIAcore. Steps to improve the efficiency of the ribosome display system have been identified and are discussed

Trade Openness: An Australian Perspective

Australia’s external trade is relatively low compared with the size of its economy. Indeed, Australia’s openness ratio (exports plus imports as a proportion of GDP) in 2002 was the third-lowest among the 30 OECD countries. This paper seeks to understand Australia’s low openness by analysing the empirical determinants of aggregate country trade. We begin by estimating a standard gravity model of bilateral trade. Although the model appears to fit the bilateral data very well, it does a relatively poor job at fitting countries’ aggregate trade levels, with different methodologies sometimes providing highly conflicting results. The focus of the paper is an equation for country openness. Our equation explains a substantial amount of the variation in how much countries trade using a small number of explanatory variables. We find that the most important determinants of openness are population and a measure of distance to potential trade partners. Countries with larger populations trade less, as do countries that are relatively more remote. Furthermore, after controlling for trade policy there is little evidence of a positive correlation between openness and economic development. While gravity models suggest Australia trades much more than expected, the openness equation suggests that its level of trade is relatively close to what would be expected. The most important factors in explaining Australia’s low openness ratio are its distance to the rest of the world, and to a lesser extent its large geographic size.trade; outward orientation; economic geography; trade liberalisation

A semi-invertible Oseledets Theorem with applications to transfer operator cocycles

Oseledets' celebrated Multiplicative Ergodic Theorem (MET) is concerned with the exponential growth rates of vectors under the action of a linear cocycle on R^d. When the linear actions are invertible, the MET guarantees an almost-everywhere pointwise splitting of R^d into subspaces of distinct exponential growth rates (called Lyapunov exponents). When the linear actions are non-invertible, Oseledets' MET only yields the existence of a filtration of subspaces, the elements of which contain all vectors that grow no faster than exponential rates given by the Lyapunov exponents. The authors recently demonstrated that a splitting over R^d is guaranteed even without the invertibility assumption on the linear actions. Motivated by applications of the MET to cocycles of (non-invertible) transfer operators arising from random dynamical systems, we demonstrate the existence of an Oseledets splitting for cocycles of quasi-compact non-invertible linear operators on Banach spaces.Comment: 26 page

Time Series Data Mining Algorithms for Identifying Short RNA in Arabidopsis thaliana

The class of molecules called short RNAs (sRNAs) are known to play a key role in gene regulation. Th are typically sequences of nucleotides between 21-25 nucleotides in length. They are known to play a key role in gene regulation. The identification, clustering and classification of sRNA has recently become the focus of much research activity. The basic problem involves detecting regions of interest on the chromosome where the pattern of candidate matches is somehow unusual. Currently, there are no published algorithms for detecting regions of interest, and the unpublished methods that we are aware of involve bespoke rule based systems designed for a specific organism. Work in this very new field has understandably focused on the outcomes rather than the methods used to obtain the results. In this paper we propose two generic approaches that place the specific biological problem in the wider context of time series data mining problems. Both methods are based on treating the occurrences on a chromosome, or “hit count” data, as a time series, then running a sliding window along a chromosome and measuring unusualness. This formulation means we can treat finding unusual areas of candidate RNA activity as a variety of time series anomaly detection problem. The first set of approaches is model based. We specify a null hypothesis distribution for not being a sRNA, then estimate the p-values along the chromosome. The second approach is instance based. We identify some typical shapes from known sRNA, then use dynamic time warping and fourier trans-form based distance to measure how closely the candidate series matches. We demonstrate that these methods can find known sRNA on Arabidopsis thaliana chromosomes and illustrate the benefits of the added information provided by these algorithms

Geometric Satake, Springer correspondence, and small representations II

For a split reductive group scheme $G$ over a commutative ring $k$ with Weyl group $W$, there is an important functor $Rep(G,k) \to Rep(W,k)$ defined by taking the zero weight space. We prove that the restriction of this functor to the subcategory of small representations has an alternative geometric description, in terms of the affine Grassmannian and the nilpotent cone of the Langlands dual group to $G$. The translation from representation theory to geometry is via the Satake equivalence and the Springer correspondence. This generalizes the result for the $k=\mathbb{C}$ case proved by the first two authors, and also provides a better explanation than in that earlier paper, since the current proof is uniform across all types.Comment: Version 4: minor revisions; 73 page

Isothermal models of combustion chamber flows

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Spectroscopic studies of some iron complexes

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Comparison of Volumetric Analysis Methods for Machine Tools with Rotary Axes

Confidence in the ability of a production machine to meet manufacturing tolerances requires a full understanding of the accuracy of the machine. However, the definition of “the accuracy of the machine” is open to interpretation. Historically, this has been in terms of linear positioning accuracy of an axis with no regard for the other errors of the machine. Industry awareness of the three-dimensional positioning accuracy of a machine over its working envelope has slowly developed to an extent that people are aware that “volumetric accuracy” gives a better estimation of machine performance. However, at present there is no common standard for volumetric errors of machine tools, although several researchers have developed models to predict the effect of the combined errors. The error model for machines with three Cartesian axes has been well addressed, for example by the use of homogenous transformation matrices. Intuitively, the number of error sources increases with the number of axes present on the machine. The effect of the individual axis geometric errors can become increasingly significant as the chain of dependent axes is extended. Measurement of the “volumetric error” or its constituents is often restricted to a subset of the errors of the Cartesian axes by solely relying on a laser interferometer for measurement. This leads to a volumetric accuracy figure that neglects the misalignment errors of rotary axes. In more advanced models the accuracy of the rotary axes are considered as a separate geometric problem whose volumetric accuracy is then added to the volumetric accuracy of the Cartesian axes. This paper considers the geometric errors of some typical machine configurations with both Cartesian and non-Cartesian axes and uses case studies to emphasise the importance of measurement of all the error constituents. Furthermore, it shows the misrepresentation when modelling a five-axis machine as a three-plus-two error problem. A method by which the five-axis model can be analysed to better represent the machine performance is introduced. Consideration is also given for thermal and non-rigid influences on the machine volumetric accuracy analysis, both in terms of the uncertainty of the model and the uncertainty during the measurement. The magnitude of these errors can be unexpectedly high and needs to be carefully considered whenever testing volumetric accuracy, with additional tests being recommended