In considering the concept of surface-immobilised liposomes as a drug release system, two factors need to addressed, the interfacial surface density of the liposomes for maximum drug loading and the stability of these liposomes to allow for controlled drug release. This thesis investigates a multilayer system for the affinity immobilisation of liposomes and their stability to various applied stresses. In the work presented here an allylamine monomer was used to create plasma coatings that were stable, thin and amine-rich. The aging studies using AFM showed these films to rapidly oxidise on exposure to water. The freshly deposited films were used for further surface modifications, by the covalent grafting of PEG layers of different interfacial densities under the conditions of varying polymer solvation. The AFM was used to measure the interaction forces between the grafted PEG layers and modified silica interfaces. It was found that the polydispersity of the PEG species resulted in bridging interactions of ???brush???-like PEG layers with the silica surface. These interactions were screened minimised by increasing the ionic strength of the solution. Although the densely grafted PEG layers were found to be highly protein-resistant by the XPS and QCM-D some minor protein-polymer adhesions were observed by the AFM. The densely anchored biotinylated PEG chains served as an optimum affinity platform for affinity-docking of NeutrAvidinTM molecules, which assembled in a rigid, 2-D layer as confirmed by the QCM-D. The submonolayer surface density of NeutrAvidin, as determined by Europium-labelling, was attributed to steric hindrance of the immobilised molecules. The final protein layer enabled specific binding of biotin-PEG-liposomes as a highly dissipative, dense and stable layer verified by tapping mode AFM and QCM-D. We found that these liposomes were also stable under a range of stresses induced by the shearing effects of water, silica probe and HSA layer at increased loads and velocities. The frictional response of the liposome layer also demonstrated the viscoelasticity and stability of these surface immobilised liposomes. Finally, the minimal adhesive interaction forces, as measured by the AFM, demonstrated the repellency of these liposomes to commonly found proteins, such as HSA.

Cellulosic biomass is the major organic matter produced in the biosphere. The biodegradation of this cellulosic material is achieved by enzymatic activities of the cellulose degrading microorganisms. These organisms usually express a complex extracellular or a membrane bound cellulolytic system comprising combination of several cellulase enzymes. Cellulases are the group of hydrolytic enzymes capable of hydrolysing insoluble cellulose to glucose. Phlebia gigantea is an aggressive white rot basidiomycete with ability to tolerate resinous extracts on freshly cut wood and higher growth rate. This helps the fungus to colonise the sapwood preventing other fungi from becoming established. Early research on the cellulase system of this organism reported the presence of a cellulase system composed of P-glucosidase, endoglucanase and a cellobiohydrolase. Based on these unpublished studies, our aim was to obtain a complete sequence of putative cellobiohydrolase I (CbhI) from this organism. Attempts to identify and isolate the cellulase gene resulted in an incomplete cDNA sequence of I 154 bp. To understand the cellulase system, expression and regulation of the cellulase enzymatic activity was examined for incubation of P. gigantea on substrates glucose, xylose, Avicel, carboxymethyl cellulose and cellobiose. The pH, total protein and biomass production results indicated that the capacity of P. gigantea to degrade cellulose is dependent upon the nature of the carbon source and the regulation of the cellulase synthesis is repressed in the presence of simple sugars like glucose and xylose. The study employed the highly effective method of purification by affinity adsorption and purified cellulase complex in large quantity. Characterisation of the kinetic properties of this cellulase complex revealed that the rate of cellulase catalysis were optimum at pH 5.0 and temperature 50GC. The purified complex was comprised of multiple proteins and demonstrated significant CMCase and CBHase activity on zymogram analysis. The purified cellulase complex was characterised by 2D gel electrophoresis and by peptide mass finger printing using MALDI-TOF massspectrometry analysis. The 2D gel analysis of the purified cellulase complex showed 15 spots within the range of pI 3.5 to pI 7 and the molecular weight between 20KDa to 100KDa. Three protein spots were selected based on the IEF and SDS zymogram and identified using MALDI-TOF MS analysis. These proteins were identified based on the peptide mass data belonging to the 6-phospho-a-glucosidase, p-glucosidase and glycosyl hydrolase family 13 a-amylase or pullulanases, suggesting the divergent evolution of specific cellulase proteins. This study showed P. gigantea as a potential cellulase source and the cellulase complex secreted by the induction of substrate, comprises a variety of enzymes related to hydrolysis of cellulose biomass. It is evident from this and previous studies that P. gigantea cellulase complex comprises of a specific set of enzymes that possess the ability to degrade crystalline cellulose and is one of the first organisms to colonise freshly cut wood. Further studies on the cellulase system of this primary colonist may open up the prospects to utilise this organism as the potential onsite bioreactor agent, pre-treating the biomass and increasing the economic feasibility of the industrial bioenergy processes.

This thesis describes the development and use ofnon-immunoglobulin affinity proteins denoted affibodies asalternatives to antibodies in different immunotechnologyapplications. A 58 aa IgG Fc binding three-helix bundle domainZ, derived from staphylococcal protein A has been used asframework for library constructions, in which the face of themolecule involved in the native binding activity has beenengineered by combinatorial protein engineering. Recruting 13surface-located positions for simultanenous substitutionmutagenesis, using degenerated oligonucleotides for libraryassembly at the genetic level, two libraries differing in thechoice of codons were constructed to serve as general sourcesof novel affinity proteins. The libraries were adapted fordisplay onE. colifilamentous phage particles allowingin vitroselection of desired variants capable ofbinding a given target molecule. In selections using human IgAas target, several new IgA specific affibodies could beidentified. One variant ZIgA1, was further investigated and showed binding toboth IgA1 and IgA2 human subclasses as well as to secretoryIgA. This variant was further demonstrated uesful as ligand inaffinity chromatography purification for recovery of IgA fromdifferent samples including unconditioned human plasma.Affibodies of different specificities were also fused to otherprotein domains to construct fusion proteins of relevance forimmunotechnology applications. Using Fc of human IgG as genefusion partner, "artificial antbodies" could be produced inE. colias homodimeic proteins, where the antigenbinding was confered by N-terminally positioned affibodymoieties of different valencies. One area of application forthis type of constructs was demonstrated through specificdetection of the target protein by Western blotting. Exploitingthe uncomplicated structure of affibody affinity proteins, genefusions between affibodies and the homotetrameric reporterenzyme β-galactosidase were constructed, which could beproduced as soluble proteins intracellularly inE. coli. The potential use of such recombinantimmunoconjugates in immunotechnology was demonstrated in ELISAdot-blot and immunohistochemistry, where in the latter case IgAdepositions in the glomeruli of a human kidney biopsy could bespecfically detected with low background staining ofsurrounding tissues. In a novel format for sandwich ELISA, thepossible advantage of the bacterial origin of the affibodyclass of affinity proteins was investigated. As a means tocircumvent problems associated with the presence of humanheterophilic antibodies in serum, causing bakground signals dueto analyte-independent crosslinking of standard capture anddetection antibody reagents, assay formats based oncombinations of antibody and affibody reagents for capture anddetection were investigated and found to be of potentialuse. <b>Keywords:</b>phage display, combinatorial, affinity, IgAligand, immunohistochemistry, affibody-fusions

This thesis investigates in what way trade flows in Europe have been altered and differ for countries belonging to a preferential trade agreement as well as a common currency area. More specifically, how exports among the European countries are affected by memberships with the European Union and the EMU. A total of 72 countries have been chosen which represents the main trading partners between the EU and the rest of the world. Out of these 72 countries, 25 represent EU members which include 12 EMU member countries. The econometric analysis employ a gravity model with 18 variables in order to determine their impact on trade flows. This is done through a regression with a log-log equation where the dependent variable is export. The other variables included are chosen to explain export flows among the EU members as well as their trade with EMU countries and the rest of the world. Furthermore, variables representing trade affinities are included to determine whether or not they have a significant effect on trade. The regression is divided into four time periods in order to more easily determine how the trade pattern in Europe have altered from the establishment of the EU and the EMU. The first time period represent an early state of EU membership, the second a mature state of EU membership, the third when EU was reformed and the fourth an early state of EMU membership. The regression results illustrate that the majority of the selected variables are significant but most importantly that the trade affinity variables are proven to have an impact on trade flows. The results also show that trade has increased and that in the case of EU membership it is more profitable to join than to remain outside. Moreover, the result show in par-ticular that countries that belong to the EMU have a stronger orientation of their exports to the rest of the world then other EU countries. For the latter, the European market is of prime importance.

The need for an effective clustering algorithm for Vehicle Ad Hoc Networks (VANETs) is motivated by the recent research in cluster-based MAC and routing schemes. VANETs are highly dynamic and have harsh channel conditions, thus a suitable clustering algorithm must be robust to channel error and must consider node mobility during cluster formation. This work presents a novel, mobility-based clustering scheme for Vehicle Ad hoc Networks, which forms clusters using the Affinity Propagation algorithm in a distributed manner. This proposed algorithm considers node mobility during cluster formation and produces clusters with high stability. Cluster performance was measured in terms of average cluster head duration, average cluster member duration, average rate of cluster head change, and average number of clusters. The proposed algorithm is also robust to channel error and exhibits reasonable overhead. Simulation results confirm the superior performance, when compared to other mobility-based clustering techniques.

The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifications. Modification of a protein with a Ubl can alter its localization, activity and/or half-life. SUMO and Rub1p/Nedd8 are two Ubls that play important roles in a number of critical cellular processes, yet their specific cellular functions remain poorly understood. To better understand these important Ubls, we developed a robust affinity purification-mass spectrometry (AP-MS) technique to generate protein-protein interaction maps for the Ubl systems. Each bait was systematically expressed as a C-terminal HA-tagged fusion protein in S. cerevisiae. A standardized method in which affinity purification via the HA epitope, followed by mild washing and mass spectrometric analysis, was performed and the data generated were used to build interaction maps. Affinity purification of the Rub1p E3 ligase Dcn1p identified a novel interaction with the AAA ATPase Cdc48p. This interaction was further studied to determine its biological significance.

The need for an effective clustering algorithm for Vehicle Ad Hoc Networks (VANETs) is motivated by the recent research in cluster-based MAC and routing schemes. VANETs are highly dynamic and have harsh channel conditions, thus a suitable clustering algorithm must be robust to channel error and must consider node mobility during cluster formation. This work presents a novel, mobility-based clustering scheme for Vehicle Ad hoc Networks, which forms clusters using the Affinity Propagation algorithm in a distributed manner. This proposed algorithm considers node mobility during cluster formation and produces clusters with high stability. Cluster performance was measured in terms of average cluster head duration, average cluster member duration, average rate of cluster head change, and average number of clusters. The proposed algorithm is also robust to channel error and exhibits reasonable overhead. Simulation results confirm the superior performance, when compared to other mobility-based clustering techniques.

The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifications. Modification of a protein with a Ubl can alter its localization, activity and/or half-life. SUMO and Rub1p/Nedd8 are two Ubls that play important roles in a number of critical cellular processes, yet their specific cellular functions remain poorly understood. To better understand these important Ubls, we developed a robust affinity purification-mass spectrometry (AP-MS) technique to generate protein-protein interaction maps for the Ubl systems. Each bait was systematically expressed as a C-terminal HA-tagged fusion protein in S. cerevisiae. A standardized method in which affinity purification via the HA epitope, followed by mild washing and mass spectrometric analysis, was performed and the data generated were used to build interaction maps. Affinity purification of the Rub1p E3 ligase Dcn1p identified a novel interaction with the AAA ATPase Cdc48p. This interaction was further studied to determine its biological significance.

KCN-1 is known to reduce tumor growth 6-fold in mice implanted with LN229 glioma cells. Although this inhibitor is effective, the mechanism of action for KCN-1 is not well understood. Based on preliminary studies, KCN-1 reduces tumor growth by disrupting the HIF 1 (hypoxia-induced factor-1) pathway. The binding target of KCN-1 needs to be investigated in order to develop KCN-1 or its analogs for therapeutic applications. In this research, a molecule was designed and synthesized for the identification of the binding target of KCN-1. Specifically, this molecule contains the inhibitor (KCN-1), a photocleavable linker, beads, and the affinity label (L DOPA). When UV light shines on the linker, the trans-alkene isomerizes to cis-alkene and undergoes intramolecular ring-closing reaction, which helps cleave the immobilized bead from the linker. The immobilized bead is used to separate the binding fragment attached to the photocleavable linker from the solution after enzyme digestion. The affinity label (L-DOPA) reacts with a nucleophile from the binding target and creates a covalent bond. If the design is successful, this method is able to analyze the mass of the peptide sequence and determine the binding target of KCN-1.

Ribonucleoside diphosphate reductase (RR) from vaccinia virus was recently cloned and overexpressed rn Escherichia coli. The amino acid sequence identities of the small and large subunits between the mouse and the vaccinia virus reductase are approximately 80 and 72 percent, respectively. In addition, vaccinia virus RR displays similar complex allosteric regulation to the mouse enzyme and other eukaryotic reductases. The overall activity of the enzyme, which has two subunits (Rl and R2), is regulated through binding to ATP, which activates the enzyme, and dATP which seryes as an inhibitor. Both nucleotides bind to the same allosteric site, called the activity site, on the large subunit of RR. The specificity of the enzyme towards the four ribonucleoside diphosphate substrates is regulated by the binding of ATP, dATP, dTTP and GTp. Each of these nucleotides affects the reduction of a specific nucleoside diphosphate. Although this enzyme's allosteric regulation is kinetically well understood it has not been possible so far to gain further structural information about the location of the activity site and specificity site. The use of deletion mutants and photoaffinity labeling of the large subunit to identify the location of the binding sites is the incentive for this thesis. With the introduction of 6xHis/Nickel Nitrilo-tri-acetic acid (Ni-NTA) chromatography, the purification of the large subunit was improved in the E. coli and vaccinia virus/T7 RNA polymerase hybrid system. The purification of several deletion mutant forms of the large subunit was also attempted, but it was not possible to purify any of them from either of the expression systems. The purified full-length large subunit obtained with the Ni-NTA-chromatography system was used for a photoaffinity labeling experiment with [³²P]dATP and [³²P]dTTP. The labeled proteins were proteolytically digested to find out about the specificity of the labeling experiment and also to map the binding site of the nucleotide. It was found that labeling of dATP yielded few discrete bands indicating specific binding, while a comparable experiment with dTTP indicated less specific binding, based on a larger number of labeled bands. In competition experiments with non-radioactive nucleotides, vaccinia virus R1 featured the same properties as the mouse and E. coli counterparts. This is consistent with data from kinetic experiments, which also establish the same kinetic properties between vaccinia virus RR with those of mouse and E. coli (RR). To identify the sequence of the fragments carrying the label the digests were subjected to mass spectrometric analysis. However, it was not possible to determine the sequence of the labeled fragment by mass spectrometry due to poor spectral resolution. / Graduation date: 1994