New methods for the study of physicochemical processes relevant to the agrochemicals industry

This thesis concerns the application of modern analytical techniques for the study of a range of systems relevant to the agrochemicals industry, with a focus on adapting and further developing these technologies to the systems of study. The major systems involved include the study of bioadhesion relevant to plant root cells and their local environment, and the further study of the root cell surface. Evanescent wave cavity ring-down spectroscopy (EW-CRDS) is the first major technique employed. For these studies a range of mimetic surfaces are designed to represent the plant cell surface and soil environment. These studies are then used to study various chemical functionalities, in the form of functional groups on polymer molecules, for adsorption properties on such surfaces. Kinetic information on the adsorption rates is measured in the form of initial rate constants. These studies are the extended to examine the cell membrane via the use of supported lipid bilayer (SLB) membranes by the combination with an impinging jet flow cell. Confocal laser scanning microscopy (CLSM) is then used to measure microparticle adsorption rates to functionalised surfaces. These studies then employ two different delivery systems, resulting in the development of a microscale vertical flow cell on the scale of individual plant cells. Understanding of the characteristics of this flow system are enhanced by the use of finite element modelling to examine the movement of the particles and flow rates. Finally, a range of electrochemical scanned probe microscopy (ECSPM) techniques are used to study the root cell surface and local environment, with a focus on assessment of each for such applications. Scanning ion conductance microscopy (SICM) is found to be the most applicable and initial work on flux imaging is performed

Lectin based glycoprotein analysis

Many of the biopharmaceutical therapeutics entering the market and currently in clinical trails are recombinant glycoprotein molecules, the glycan moieties of which have a significant impact on efficacy and immunogenicity. The cell culture techniques required to produce these glycoproteins often result in products that are heterogeneous with respect to glycan content. This inconsistency ultimately leads to increased production costs and restricts patient accessibility to these therapeutics. To overcome these difficulties novel analytical platforms facilitating rapid in-process monitoring and product quality control are essential. Work undertaken within the Centre for Bioanalytical Sciences (CBAS) seeks to exploit the microbial world as a source of novel biorecognition elements to produce such platforms

Production of lectin-affinity matrices for process-scale glycoprotein purification

A selection of prokaryotic lectins with a variety of glycan specificities and affinities have been identified, cloned, expressed in Eschericia coli and characterised. The aims of this project are to: - express the lectins at 1L scale to produce sufficient quantities for immobilisation studies (~100 mg) - immobilisethelectinsonSepharose - evaluate lectin performance on column by monitoring their ability toreproducibly capture and elute glycoprotein glycoforms

Exploiting prokaryotic chitin-binding proteins for glycan recognition

• The cloning, expression and characterisation of prokaryotic chitin-binding proteins from Serratia marcescens, Pseudomonas aeruginosa, Photorhabdus luminescens Microfluidics and Photorhabdus asymbiotica • Development of an assay to assess the activity of chitin-binding proteins • Mutagenesis of chitin-binding proteins to alter glycan recognition pattern

Genetically enhanced recombinant lectins for glyco-selective analysis and purification

- Generation of a library of recombinant prokaryotic lectins (RPL’s) through random mutagenesis of the carbohydrate binding sites of bacterial lectins. - Characterisation of mutant lectins with respect to structure and specificity - Provision of mutant RPL’s with enhanced affinity and/or altered specificity, alongside wild-type RPL’s, for glycoprotein analysis and purificatio

Adaptive gain and filtering circuit for a sound reproduction system

Adaptive compressive gain and level dependent spectral shaping circuitry for a hearing aid include a microphone to produce an input signal and a plurality of channels connected to a common circuit output. Each channel has a preset frequency response. Each channel includes a filter with a preset frequency response to receive the input signal and to produce a filtered signal, a channel amplifier to amplify the filtered signal to produce a channel output signal, a threshold register to establish a channel threshold level, and a gain circuit. The gain circuit increases the gain of the channel amplifier when the channel output signal falls below the channel threshold level and decreases the gain of the channel amplifier when the channel output signal rises above the channel threshold level. A transducer produces sound in response to the signal passed by the common circuit output

Adaptive noise reduction circuit for a sound reproduction system

A noise reduction circuit for a hearing aid having an adaptive filter for producing a signal which estimates the noise components present in an input signal. The circuit includes a second filter for receiving the noise-estimating signal and modifying it as a function of a user's preference or as a function of an expected noise environment. The circuit also includes a gain control for adjusting the magnitude of the modified noise-estimating signal, thereby allowing for the adjustment of the magnitude of the circuit response. The circuit also includes a signal combiner for combining the input signal with the adjusted noise-estimating signal to produce a noise reduced output signal

Regions of the Cry1Ac toxin predicted to be under positive selection are shown to be the carbohydrate binding sites and can be altered in their glycoprotein target specificity

The cry gene family, is a large family of homologous genes from Bacillus thuringiensis. Studies have examined the structural and functional relationships of the Cry proteins. They have revealed several residues in domains II and III that are important for target recognition and receptor attachment. In 2007 Wu, Jin-Yu et al employed a maximum likelihood method to detect evidence of adaptive evolution in Cry proteins. They identified positively selected residues, which are all located in Domain II or III. Figure 1 shows a protein sequence alignment between domain II and III of Cry1Ac and Cry1Aa. This highlights the areas which are thought to be under positive selection. Cry1Ac and Cry1Aa are structurally very similar and they both bind to a variety of N-aminopeptidases (APN’s) in different insect species. However Cry1Aa has a higher specificity for the cadherin like receptor HevCalP and Cry1Ac binds to N-acetylgalactosamine (GalNAc) on the surface of APN’s. Differences in the binding of the two toxins has been shown in an in-direct toxin-binding assay where GalNAc completely abolished toxin binding of Cry1Ac but had no effect on the binding of Cry1Aa. The binding site has been shown to be located in the third domain of Cry1Ac. Some of these sites correlate with the positively selected residues found by Wu et al 2007 in Cry1Aa. Our aim was to use the comparison of the toxins to analyse the potential to alter the binding specificity of Cry1Ac and its domains. In this work we identified critical amino acid residues for this objective

The investigation of a recombinant GalNAc binding protein from bacillus thuringiensis as a tool for glycan analysis and detection

Changes in the structures of glycans on the surfaces of eukaryotic cells can be important biomarkers for developmental or disease states. Improved methods are needed for the detection and analysis of alterations in glycan structures. Carbohydrate binding proteins such as lectins have potential for the recognition of changes in glycan structure. Host-pathogen interactions frequently involve the recognition of host carbohydrates by proteins of bacteria or viruses. Many bacterial toxins have evolved to interact with host cell receptors or with a specific tissue due to lectin like properties. The toxins from Bacillus thuringiensis have been shown to have carbohydrate binding abilities, in particular N-Acetylgalactosamine (GalNAc) has been shown to inhibit the binding of the toxin Cry1Ac. GalNAc has been shown to be an important marker in many diseases such as breast cancer and colon carcinogenesis. Moreover, changes in GalNAc glycosylation have been identified in many disorders such as cystic fibrosis, neuromuscular disorders and nephropathy. Here we describe the purification of a GalNAc binding protein of bacterial origin that may have potential in the development of diagnostic assays

Exploitation of siderophores for the speciation of iron

Iron is essential for life. It acts as an electron donor/acceptor in metabolic processes facilitated by its variable valency. Although vital, it is toxic at high levels due to Fe2+ oxidation. Iron toxicity is a concern as it can affect growth and product yields in animal cell culture. Siderophores are high affinity Fe3+ chelators produced by microorganisms. This affinity gives them the potential to be used as a basis in platforms to detect and speciate iron in industrial cell culture. Rhizobactin 1021 is of interest due to its decanoic acid “tail” that is not involved in chelation which makes it an ideal target for immobilisation