Directed evolution and structural analysis of an OB-fold domain towards a specifc binding reagent

Interactions between proteins are a central concept in biology, and understanding and manipulation of these interactions is key to advancing biological science. Research into antibodies as customised binding molecules provided the foundation for development of the field of protein “scaffolds” for molecular recognition, where functional residues are mounted on to a stable protein platform. Consequently, the immunoglobulin domain has been describes as “nature’s paradigm” for a scaffold, and has been widely researched to make engineered antibodies better tools for specific applications. However, limitations in their use have lead to a number of non-immunoglobulin domains to be investigated as customisable scaffolds, to replace or complement antibodies. To be considered a scaffold, a protein domain must show an evolutionarily conserved hydrophobic core in diverse functional contexts. The study presented here investigated the oligosaccharide/oligonucleotide-binding (OB) fold as scaffold, which is a 5-standed β-barrel seen in diverse organisms with no sequence conservation. The term “Obody” was coined to describe engineered OB-folds. This thesis examined a previously engineered Obody with affinity for lysozyme (KD = 40 μM) in complex with its ligand by x-ray crystallography (resolution 2.75 Å) which revealed the atomic details of binding. Affinity maturation for lysozyme was undertaken by phage display directed evolution. Gene libraries were constructed by combinatorial PCR incorporating site-specific randomised codons identified by examination of the structure in complex with lysozyme, or by random generation of point mutations by error-prone PCR. Overall a 100-fold improvement in affinity was achieved (KD = 600 nM). To investigate the structural basis of the affinity maturation, two further Obody-lysozyme complexes were solved by x-ray crystallography, one at a KD of 5 μM (resolution 1.96 Å), one at 600 nM (resolution 1.86 Å). Analysis of the structures revealed changes in individual residue arrangements, as well as rigid-body changes in the relative orientation of the Obody and lysozyme molecules in complex. Directed evolution of Obodies as protein binding reagents remains a challenge, but this study demonstrates their potential. The structures presented here will contribute invaluable insights for the future design of improved Obodies

The Open Set Condition and Neighbor Maps in Fractal Geometry

Neighbor maps are a new development in fractal geometry that can be used to determine if an iterated function system (IFS) obeys the open set condition (OSC). Neighbor maps also describe the topology of the fractal attractor. In this thesis, the de nition of the set of proper neighbor maps is generalised to any IFS comprising contractive similitudes. It is proven, an IFS of similitudes obeys the OSC if and only if the identity map is not in the closure of the set of proper neighbor maps. The extended de nition of the set of proper neighbor maps is used to calculate several neighbor graphs of the generalised Sierpinski triangles. It is then proven, if a generalised Sierpinski triangle has scaling factors that obey the algebraic condition, then its neighbor graph is of nite type. The converse is proven to only hold for the Sierpinski triangle and the Steemson triangle. Neighbor maps are also applied to fractal tiling theory to discuss the prototile set

Numerical modelling of steady state continuous crystallization processes using piecewise cubic spline functions

An efficient technique for computing the steady state crystal size distributions from continuous crystallization units is developed using piecewise cub The technique is simple, explicit and offers considerable computation savings over other available techniques. Solution techniques are developed for cr dependent growth and size dispersion. The numerical results are very satisfactory when compared with other techniques or exact analytical results

Post-prandial glycaemic reduction by an alpha-glucosidase inhibitor in type 2 diabetic patients with therapeutically attained basal normoglycaemia.

Post-prandial glucose excursions remain elevated in most patients with diabetes even when normal fasting plasma glucose levels have been achieved. In 39 patients with type 2 diabetes who had attained basal normoglycaemia by therapy with diet alone, a sulphonylurea, a basal insulin supplement or basal plus prandial insulin the mean glycosylated haemoglobin (HbA1) values were at the upper end (mean +/- 1SD, 8.1 +/- 1.1%) of the normal range (5.0-8.2%). Miglitol, an alpha-glucosidase inhibitor, given in a dose of 50 mg three times a day was studied in a double blind randomized crossover study. In diet and sulphonylurea treated patients, a mean 25% reduction of the post-prandial plasma glucose excursions was obtained whereas in ultralente treated patients miglitol appeared to reduce basal plasma glucose levels (p < 0.006). Side effects were limited to minor gastrointestinal disturbances, usually ameliorating after the first week of therapy. Alpha-glucosidase inhibition to prevent post-prandial glycaemia may have a role in patients in whom sulphonylurea or diet therapy has been used to obtain normal basal glucose concentrations