Indentification and characterization of a novel CA^(2+)-binding protein in avian erythrocytes

A novel Ca^(2+)-binding protein with Mr of 23 K (designated p23) has been identified in avian erythrocytes and thrombocytes. p23 localizes to the marginal bands (MBs), centrosomes and discrete sites around the nuclear membrane in mature avian erythrocytes. p23 appears to bind Ca^(2+) directly and its interaction with subcellular organelles seems to be modulated by intracellular [Ca^(2+)]. However, its unique protein sequence lacks any known Ca^(2+)-binding motif. Developmental analysis reveals that p23 association to its target structures occurs only at very late stages of bone marrow definitive erythropoeisis. In primitive erythroid cells, p23 distributes diffusely in the cytoplasm and lacks any distinct localization. It is postulated that p23 association to subcellular structures may be induced in part by decreased intracellular [Ca^(2+)]. In vitro and in vivo experiments indicate that p23 does not appear to act as a classical microtubule-associated protein (MAP) but p23 homologues appear to be expressed in MB-containing cells of a variety of species from different vertebrate classes. It has been hypothesized that p23 may play a regulatory role in MB stabilization in a Ca^(2+)-dependent manner.

Binucleated (bnbn) turkey erythrocytes were found to express a truncated p23 variant (designated p21) with identical subcellular localization as p23 except immunostaining reveals the presence of multi-centrosomes in bnbn cells. The p21 sequence has a 62 amino acid deletion at the C-terminus and must therefore have an additional ~40 amino acids at the N-terminus. In addition, p21 seems to have lost the ability to bind Ca^(2+) and its supramolecular interactions are not modulated by intracellular [Ca^(2+)]. These apparent differences between p23 and p21 raised the possibility that the p23/p21 allelism could be the Bn/bn genotype. However, genetic analysis suggested that p23/p21 allelism had no absolute correlation with the Bn/bn genotype.

Synthesis and potency of long end-associative polymers for mist control

Long linear polymers that are end-functionalized with associative groups were studied as additives to hydrocarbon fluids to mitigate the fire hazard associated with the presence of mist in a crash scenario. These polymers were molecularly designed to overcome both the shear-degradation of long polymer chains in turbulent flows, and the chain collapse induced by the random placement of associative groups along polymer backbones. Architectures of associative groups on the polymer chain ends that were tested included clusters of self-associative carboxyl groups and pairs of hetero-complementary associative units.

Linear polymers with clusters of discrete numbers of carboxyl groups on their chain ends were investigated first: an innovative synthetic strategy was devised to achieve unprecedented backbone lengths and precise control of the number of carboxyl groups on chain ends (N). We found that a very narrow range of N allows the co-existence of sufficient end-association strength and polymer solubility in apolar media. Subsequent steady-flow rheological study on solution behavior of such soluble polymers in apolar media revealed that the end-association of very long chains in apolar media leads to the formation of flower-like micelles interconnected by bridging chains, which trap significant fraction of polymer chains into looped structures with low contribution to mist-control. The efficacy of very long 1,4-polybutadiene chains end-functionalized with clusters of four carboxyl groups as mist-control additives for jet fuel was further tested. In addition to being shear-resistant, the polymer was found capable of providing fire-protection to jet fuel at concentrations as low as 0.3wt%. We also found that this polymer has excellent solubility in jet fuel over a wide range of temperature (-30 to +70°C) and negligible interference with dewatering of jet fuel. It does not cause an adverse increase in viscosity at concentrations where mist-control efficacy exists.

Four pairs of hetero-complementary associative end-groups of varying strengths were subsequently investigated, in the hopes of achieving supramolecular aggregates with both mist-control ability and better utilization of polymer building blocks. Rheological study of solutions of the corresponding complementary associative polymer pairs in apolar media revealed the strength of complementary end-association required to achieve supramolecular aggregates capable of modulating rheological properties of the solution.

Both self-associating and complementary associating polymers have therefore been found to resist shear degradation. The successful strategy of building soluble, end-associative polymers with either self-associative or complementary associative groups will guide the next generation of mist-control technology.

Intramolecular conflict : conformation and self-assembly of architecturally complex macromolecules in solution

The solution behavior of linear polymer chains is well understood, having been the subject of intense study throughout the previous century. As plastics have become ubiquitous in everyday life, polymer science has grown into a major field of study. The conformation of a polymer in solution depends on the molecular architecture and its interactions with the surroundings. Developments in synthetic techniques have led to the creation of precision-tailored polymeric materials with varied topologies and functionalities. In order to design materials with the desired properties, it is imperative to understand the relationships between polymer architecture and their conformation and behavior. To meet that need, this thesis investigates the conformation and self-assembly of three architecturally complex macromolecular systems with rich and varied behaviors driven by the resolution of intramolecular conflicts. First we describe the development of a robust and facile synthetic approach to reproducible bottlebrush polymers (Chapter 2). The method was used to produce homologous series of bottlebrush polymers with polynorbornene backbones, which revealed the effect of side-chain and backbone length on the overall conformation in both good and theta solvent conditions (Chapter 3). The side-chain conformation was obtained from a series of SANS experiments and determined to be indistinguishable from the behavior of free linear polymer chains. Using deuterium-labeled bottlebrushes, we were able for the first time to directly observe the backbone conformation of a bottlebrush polymer which showed self-avoiding walk behavior. Secondly, a series of SANS experiments was conducted on a homologous series of Side Group Liquid Crystalline Polymers (SGLCPs) in a perdeuterated small molecule liquid crystal (5CB). Monodomain, aligned, dilute samples of SGLCP-b-PS block copolymers were seen to self-assemble into complex micellar structures with mutually orthogonally oriented anisotropies at different length scales (Chapter 4). Finally, we present the results from the first scattering experiments on a set of fuel-soluble, associating telechelic polymers. We observed the formation of supramolecular aggregates in dilute (≤0.5wt%) solutions of telechelic polymers and determined that the choice of solvent has a significant effect on the strength of association and the size of the supramolecules (Chapter 5). A method was developed for the direct estimation of supramolecular aggregation number from SANS data. The insight into structure-property relationships obtained from this work will enable the more targeted development of these molecular architectures for their respective applications.