The recovery of the selected biological material in affinity-based separations relies on reversing the biological interaction responsible for the binding. General elution methods which are independent of the bioaffinity interaction have attracted increasing attention. The first three chapters of this thesis describe the development of a novel “click” functionalised azobenzene-based linker for affinity-independent elution protocols and the preliminary affinity studies using this linker. Ligands functionalised with a bioorthogonal propargyl label were readily attached to the terminal azide of the linker using the copper(I) catalysed Huisgen cycloaddition (or "click" reaction). Following separation, the linker was cleaved under mild non-denaturing conditions using Na2S2O4. In the last three chapters a novel approach towards the synthesis of the 4-methyl proline fragment of the cytotoxic natural product bisebromoamide (a potential affinity target) is proposed. For the pyrrolidine ring construction an enamide-olefin ring-closing metathesis (RCM) approach has been attempted. The installation of the required absolute stereochemistry has been achieved using a phase-transfer catalyst for the enantioselective alkylation of Schiff bases derived from glycine esters.

The ability of organisms to sense – and properly respond to – their environment is crucial to their survival. Higher organisms communicate with conspecifics to ensure the survival of the species. Nematodes, such as the roundworm Caenorhabditis elegans, are ubiquitous across all biomes, and rely on chemical communication to convey information with one another. The small molecules they utilize in this communication are called ascarosides. These modular pheromones are employed by all taxa, ranging from Caenorhabditis to Ascaris. The ascaroside, ascr#8, is release by hermaphroditic C. elegans to attract potential mates. Previous work has shown that a class of male specific neurons are required for sensation of this pheromone. Here, we show that these neurons initiate a neural circuit modulated by the FMRFamide-like neuropeptide, flp-3. This neuropeptide is sensed by a set of G protein-coupled receptors (GPCRs), NPR-10 and FRPR-16. Together, these components determine the behavioral valence of males to ascr#8. Within the male-specific sensory neurons, the CEM, we show that another group of GPCRs sense the ascr#8. Two of these receptors, DMSR-12 and SRW-97, are expressed in the cilia, suggesting their involvement in direct sensation of the cue. As a targeted approach to identifying and confirming receptors for ascr#8, we have developed a bioactive photoaffinity probe. We have also confirmed that the ability of ascr#8 to attract males is conserved across the genus. Together, these studies coalesce to deepen our understanding of sex-specific chemosensation and neuronal processing. These results can be used to better understand the defects that are seen in neurodegenerative diseases – many of which exhibit sex-specific defects in neuronal processing.

<p>The work presented in this thesis describes the use of polystyrene nanoparticles as model surfaces for bioanalytical work. Nanoparticles constitute convenient platforms for the attachment of bioactive agents, and receptor coated particles offer high local concentration of binding sites for specific ligands with minimal steric hindrance. However, it is not only the amount of bound protein that matters, the proteins must also be immobilized at the surface in such ways that they fully retain their activity, while at the same time protecting the surface from unspecific uptake of undesired components. The present work relates to the controlled immobilization of multiple types of active biomolecules onto nanoparticle surfaces to make them multifunctional. The surface expansion offered by the nanoparticles, in combination with the closeness between the reactants co-immobilized on the same particle, enables coupled reactions to be carried at a higher rate than otherwise possible. Thus, particle-decorated surfaces of this kind are highly suitable for miniaturized bioanalytical systems. Sensitive microarray systems are under development, including lectin-coated nanoparticles for glycoprotein mapping and a diagnostic device for Point-of-Care testing with a nanoparticle-based detection system.</p><p>The full evaluation of protein attachment to nanoparticles requires precise analytical techniques for particle characterization, both in bare and coated form. The mass-sensitive SdFFF technique occupies a prominent position for particle characterization, as it offers both accurate determination of particle size and a quantification of adsorbed layers on small particles, whether of synthetic or biopolymeric nature. Here, this analytical technique is developed to precisely characterize nanoparticles that are sequentially coated with different layers, each rendering the particles a specific functionality. The thesis demonstrates how precise mass uptakes can be determined for each specific layer, and how control over the exact surface composition of the modified particles can be established for optimization of biological activity.</p>

The work presented in this thesis describes the use of polystyrene nanoparticles as model surfaces for bioanalytical work. Nanoparticles constitute convenient platforms for the attachment of bioactive agents, and receptor coated particles offer high local concentration of binding sites for specific ligands with minimal steric hindrance. However, it is not only the amount of bound protein that matters, the proteins must also be immobilized at the surface in such ways that they fully retain their activity, while at the same time protecting the surface from unspecific uptake of undesired components. The present work relates to the controlled immobilization of multiple types of active biomolecules onto nanoparticle surfaces to make them multifunctional. The surface expansion offered by the nanoparticles, in combination with the closeness between the reactants co-immobilized on the same particle, enables coupled reactions to be carried at a higher rate than otherwise possible. Thus, particle-decorated surfaces of this kind are highly suitable for miniaturized bioanalytical systems. Sensitive microarray systems are under development, including lectin-coated nanoparticles for glycoprotein mapping and a diagnostic device for Point-of-Care testing with a nanoparticle-based detection system. The full evaluation of protein attachment to nanoparticles requires precise analytical techniques for particle characterization, both in bare and coated form. The mass-sensitive SdFFF technique occupies a prominent position for particle characterization, as it offers both accurate determination of particle size and a quantification of adsorbed layers on small particles, whether of synthetic or biopolymeric nature. Here, this analytical technique is developed to precisely characterize nanoparticles that are sequentially coated with different layers, each rendering the particles a specific functionality. The thesis demonstrates how precise mass uptakes can be determined for each specific layer, and how control over the exact surface composition of the modified particles can be established for optimization of biological activity.