This work endeavors to delineate modern paradigms for crystal engineering, i.e. the design and supramolecular synthesis of functional molecular materials. Paradigms predicated on an understanding of the geometry of polygons and polyhedra are developed. The primary focus is on structural determination by single crystal x-ray crystallography, structural interpretation using a suite of graphical visualization and molecular modeling software, and on the importance of proper graphical representation in the presentation and explanation of crystal structures. A detailed analysis of a selected series of crystal structures is presented. The reduction of these molecular networks to schematic representations that illustrate their fundamental connectivity facilitates the understanding of otherwise complex supramolecular solids. Circuit symbols and Schlafli notation are used to describe the network topologies, which enables networks of different composition and metrics to be easily compared. This reveals that molecular orientations in the crystals and networks are commensurate with networks that can be derived from spherical close packed lattices. The development of a logical design strategy for a new class of materials based on our understanding of the chemical composition and topology of these networks is described. The synthesis and crystal structure of a series of new materials generated by exploitation of this design strategy is presented, in addition to a detailed analysis of the topology of these materials and their relationship to a parent structure. In summary, this dissertation demonstrates that molecular polygons can self-assemble at their vertexes to produce molecular architectures and crystal structures that are consistent with long established geometric dogma. The design strategy represents a potentially broad ranging approach to the design of nanoporous structures from a wide range of chemical components that are based on molecular shape rather than chemical formula. In effect, this work represents another example of the molecular meccano approach to self-assembled structures. Most importantly, given that these materials are designed from first principles, they offer materials scientists the ability to control the chemical nature of the constituent components and therefore influence the bulk physical properties of materials.

Degeneration of the lumbar intervertebral disc, a condition widely implicated in the cause of low back pain among adult humans, is typically characterised by progressive biochemical and structural changes to the extracellular matrix. Comprehensive descriptions of the structural and functional inter-relationships within the extracellular matrix are therefore critical to understanding the degenerative process and developing effective treatments. In the anulus fibrosus, this matrix has a complex, hierarchical architecture comprised of collagens, proteoglycans, and elastic fibres. Elastic fibres are critical constituents of dynamic biological structures that functionally require elasticity and resilience. Studies to date of elastic fibre network structure in the anulus fibrosus have been qualitative and limited in scope. Additionally, there is poor understanding of the structural and functional associations between elastic fibres and other matrix constituents such as collagen, and, critically, there have been no studies directly examining the nature and magnitude of the contribution made by elastic fibres to anulus fibrosus mechanical behaviour. In this thesis, multiple experimental studies are described that specifically examine each of these areas. Novel imaging techniques were developed and combined with histochemistry and light microscopy to facilitate the visualisation of elastic fibres at a level of detail not previously achieved. Examination of elastic fibre network structure revealed architectural differences between the intralamellar and interlamellar regions, suggesting that elastic fibres perform functional roles at distinct levels of the anulus fibrosus structural hierarchy. The density of elastic fibres within lamellae was found to be significantly higher in the lamellae of the posterolateral region of the anulus than the anterolateral, and significantly higher in the outer regions than the inner, suggesting it may be commensurate with the magnitude of the tensile strains experienced by each region of the disc in bending and torsion. The nature of the structure-function associations between elastic fibres and collagen was then examined with respect to the reported structural mechanisms of collagen matrix tensile deformation. Histological assessment of collagen crimp morphology in specimens from which elastic fibres had been enzymatically removed revealed no observable differences when compared with controls, suggesting that any contribution made by elastic fibres to maintaining crimp is minimal. Elastic fibres in anulus fibrosus specimens subjected to radial tensile deformations exhibited complex patterns of re-arrangement, suggesting that they maintain cross-collagen fibre connectivity. Elastic fibres were also observed to maintain physical connections between consecutive lamellae undergoing relative separation. Finally, the nature and magnitude of the contribution made by elastic fibres to anulus fibrosus mechanical properties at the tissue level was investigated using a combination of biochemically verified enzymatic treatments and biomechanical tests. Targeted degradation of elastic fibres resulted in a significant reduction in both the initial modulus and the ultimate modulus, and a significant increase in the extensibility, of radially oriented anulus fibrosus specimens. Separate treatments and mechanical tests were used to account for any changes attributable to non-specific degradation of glycosaminoglycans. These results suggest that elastic fibres enhance the mechanical integrity of the anulus fibrosus extracellular matrix in the direction perpendicular to the plane containing the collagen fibres. In summary, the results of the studies presented in this thesis provide important new insights into the structure and function of the anulus fibrosus elastic fibre network, and highlight its potential importance as a contributing or ameliorating factor in the progression of the structural and mechanical changes associated with intervertebral disc degeneration. Additionally, these results establish an improved framework for the development of more accurate analytical and finite element models to describe disc behaviour. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317006 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2008

The work leading to this thesis has provided additional information and novel knowledge concerning structure-function relationship in the potato epoxide hydrolase. Epoxide hydrolases are enzymes catalyzing the hydrolysis of epoxides to yield the corresponding vicinal diols. The reaction mechanism proceeds via a nucleophilic attack resulting in a covalent alkylenzyme intermediate, which in turn is attacked by a base-activated water molecule, followed by product release. Epoxides and diols are precursors in the production of chiral compounds and the use of epoxide hydrolases as biocatalysts is growing. The promising biocatalyst StEH1, a plant epoxide hydrolase from potato, has been investigated in this thesis. In paper I the active site residue Glu35, was established to be important for the formation of the alkylenzyme intermediate, activating the nucleophile for attack by facilitated proton release through a hydrogen bond network. Glu35 is also important during the hydrolytic half reaction by optimally orienting the hydrolytic water molecule, aiding in the important dual function of the histidine base. Glu35 makes it possible for the histidine to work as both an acid and a base. In paper II a putative proton wire composed of five water molecules lining a protein tunnel was proposed to facilitate effective proton transfer from the exterior to the active site, aiding in protonation of the alkylenzyme intermediate. The protein tunnel is also proposed to stabilize plant epoxide hydrolases via hydrogen bonds between water molecules and protein. Enzyme variants with modified enantiospecificity for the substrate (2,3-epoxypropyl)benzene have been constructed by in vitro evolution using the CASTing approach. Residues lining the active site pocket were targeted for mutagenesis. From the second generation libraries a quadruple enzyme variant, W106L/L109Y/V141K/I155V, displayed a radical shift in enantioselectivity. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the quadruple variant showed a 15:1 preference for the R-enantiomer.

Heme is the major iron source for the deadly human pathogen, Group A Streptococcus (GAS). During infection, GAS lyses host cells releasing hemoglobin and other hemoproteins. This dissertation aims to elucidate the general mechanism by which GAS obtains and utilizes heme as an iron source from the host hemoproteins. GAS encodes a heme relay system consisting of Shr, Shp and the SiaABC transporter. We specifically determine the role of Shr in the heme uptake process, by conducting a detailed functional characterization of its constituent domains. We also undertake to solve the long-standing mystery surrounding the catabolism of heme in streptococci. The studies presented herein established Shr as a prototype of a new family of NEAT-containing hemoproteins receptors. They demonstrate its importance in heme acquisition by GAS and provide a molecular model for heme scavenging and transfer by the protein. We show that Shr modulates heme uptake depending on heme availability by a mechanism where NEAT1 facilitates fast heme scavenging and delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface. Finally, we identified and characterized for the first time, a heme oxygenase (HO) in the Streptococcus genus which was named HupZ. Sequence comparison between HupZ and several HOs from different structural families indicates that this enzyme is unrelated to any of the previously characterized HOs. However, orthologs of the protein are found in other important pathogens. The structure and the catalytic mechanism of HupZ suggest that it is the representative of a new family of flavoenzymes capable of degrading heme using their reduced flavin cofactor as a source of electrons. Overall, this work contributes significant knowledge to the topic of heme utilization by pathogens and importantly, provides new direct evidence that associates flavins with heme metabolism in bacteria. Thus it sets a new direction in the field and lays the ground for future fundamental and applied discoveries.

Active galactic nuclei (AGN) are known to vary in brightness in all regions of the electromagnetic spectrum and over a wide range of timescales. Many methods have been utilized to transform this observed variability into meaningful information about the central engines of AGN. One such technique, adapted from time series analysis of galactic x-ray binary systems, has been used to detect a characteristic break timescale in the power density spectra of x-ray variability in Seyfert galaxies. This timescale, thought to be related to instabilities in the accretion disk, appears to scale with black hole mass over many orders of magnitude. This dissertation performs similar time series analyses with the optical data of eight blazars. The majority of these objects also display a characteristic break timescale. In cases where a black hole estimate is known, the timescales are in good agreement with the relationship observed for galactic x-ray binary systems and Seyfert galaxies. For objects of unknown mass, this relationship can be used to provide a mass estimate of the supermassive black hole. Comparisons are made between the structure function and power density spectrum for each object, and the implications for the connection between the accretion disk and the relativistic jet in AGN are discussed.

During hemostatic and inflammatory responses, cell adhesion molecules play a major role in regulating the leukocytes and platelets adhesion to vascular surfaces under the hydrodynamic environment of the circulation. Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Using atomic force microscopy (AFM), the kinetics of three mutants L-selectin interacting with surrogates of PSGL-1 and PNAd, is compared with those of wild-type L-selectin. The interaction between glycoprotein Ib (GPIb) and von Willebrand Factor (VWF) mediates platelet translocation at the vascular vessel damage sites, which plays a critical role in initiating the platelets adhesion and thrombus formation. Translocation of platelets on VWF requires a shear threshold, suggesting a possible catch bond at work there. We characterized the kinetics of GPIbα interacting with VWF A1 domain, confirming the catch bond existed. Two type 2B VWD A1 mutants eliminated the catch bond and gave longer low force lifetimes. The prolonged lifetimes at low force resulted in more agglutination of platelets with A1 coated microspheres in flow. During the process of hemostasis, the size of prothrombotic ULVWF affects the affinity of VWF to platelets bearing GPIbα on the membrane. ADAMTS13 has been identified and characterized as a multi-domain metalloprotease that regulate the size of ULVWF. We studied how force regulated the binding and cleavage of ADAMTS13 on VWF. We found the cleavage effects could only be observed after the catastrophic structural change of A1A2A3. The unfolding exposed the ADAMTS13 cleavage site and favored the cleavage. Two protocols using different stretching molecules (GPIbα and CR1) and A1A2A3 immobilization methods revealed the cleavage effects diminished with increasing stretching force. This study elucidated mechanisms of the binding kinetics of L-selectin with different structure components from PSGL-1 and PNAd by structural variants. It also provided new insights into our current knowledge of the dynamic adhesion and regulation of GPIbα-VWF interaction in vivo. Using single molecule method, the chemical catalytic reaction between enzyme and substrate has been targeted. These results help us understand this important enzyme-substrate interaction involved in the hemostasis.

The science and technology of catalysis is more important today than at any other time in our history due to the grand energy and environment challenges we are facing. With the explosively growth of computation power nowadays, computer simulation can play an increasingly important role in the design of new catalysts, avoiding the costly trail-and-error attempts and facilitating the development cycle. The goal to inverse design of new materials with desired catalytic property was once far off, but now achievable. The major focus of this dissertation is to find the general rules that govern the catalytic performance of a nanoparticle as the function of its structure. Three types of multi-metallic nanoparticles have been investigated in this dissertation, core-shell, random alloy and alloy-core@shell. Significant structural rearrangement was found on Au@Pt and Pd@Pt nanoparticle, which is responsible for a dramatic improvement in catalytic performance. Nonlin- ear binding trends were found and modeled for random alloy nanoparticles, providing a prescription for tuning catalytic activity through alloying. Studies of ORR on Pd/Au random alloy NP and hydrogenation reaction on Rh/Ag random alloy NP revealed that binding on individual ensemble should be in- vestigated when large disparity of adsorbate affinity is presented between two alloying elements. In the alloy-core@shell system, I demostrated a general linear correlations between the adsorbate binding energy to the shell of an alloy-core@shell nanoparticle and the composition of the core. This relation- ship allows for interpolation of the properties of single-core@shell particles and an approach for tuning the catalytic activity of the particle. A series of promising catalysts were then predicted for ORR, HER and CO oxidation. As a first attempt to bridge the material gap, bimetallic nano clus- ter supported on CeO₂(111) was investigated for CO oxidation. A strong support-metal interaction induces a preferential segregation of the more reac- tive element to the NC-CeO₂ perimeter, generating an interface with the Au component. (Au-Cu)/CeO₂ was found to be optimal for catalyzing CO oxida- tion via a bifunctional mechanism. O₂ preferentially binds to the Cu-rich sites whereas CO binds to the Au-rich sites. A method called distributed replica dynamics (DRD) is proposed at last to utilize enormous distributed computing resources for molecular dynamics simulations of rare-event in chemical reac- tions. High efficiency can be achieved with an appropriate choice of N [subscript rep] and t [subscript rep] for long-time MD simulation. / text

The homologous p10 fusion-associated small transmembrane (FAST) proteins of the fusogenic avian (ARV) and Nelson Bay (NBV) reoviruses are the smallest known proteins capable of mediating syncytiogenesis. Their extremely small size precludes them from following the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. I exploited the sequence conservation/divergence and differential syncytiogenic rates between ARV and NBV to define functional motifs in the p10 ectodomains. Using chimeric p10 constructs, I determined the 40-residue ectodomain (sizes refer to ARV) comprises two distinct functional motifs essential for syncytiogenesis. Cellular syncytiogenic and surface biotinylation assays identified an indivisible, 25- residue, N-terminal ectodomain motif required for cystine loop fusion peptide formation. I further determined the roles of this cystine loop in promoting lipid binding and cholesterol-dependent lipid destabilization. Immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies identified a second motif comprising the 13 membrane-proximal ectodomain residues (MPER). This motif governs the reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. I demonstrate that ARV and NBV homomultimers segregate to separate foci in the plasma membrane, and the four juxtamembrane residues present in the multimerization motif dictate species- specific homomultimerization. I also discovered the novel codependency of p10 multimerization and cholesterol-dependent microdomain localization. The majority of enveloped virus membrane fusion proteins function as stable multimers, which nonetheless must undergo dramatic, irreversible, tertiary structure rearrangements to mediate membrane fusion. Cholesterol-rich membrane microdomains have also been implicated in the function of several enveloped virus fusion proteins, and a limited number of studies have investigated the role of cholesterol in multimerization. My results reveal cholesterol-dependent p10 homomultimerization is an essential aspect of p10- mediated syncytium formation, and I identify the motifs responsible for this process. The reversible nature of p10 cholesterol-dependent multimerization at the plasma membrane is in line with several other studies suggesting that the dynamic clustering and dispersion of cholesterol microdomains, as well as protein transitioning from multimeric to monomeric intermediates, are essential phenomena of protein mediated membrane fusion.

Food protein-derived peptides have increasingly become important sources of ingredients for the formulation of therapeutic products. The main aim of this work was to study the in vitro and in vivo bioactive properties of structurally diverse group of peptides produced through enzymatic hydrolysis of flaxseed proteins (FP). Hydrolysis of FP with seven proteases followed by fractionation into low-molecular-weight (LMW) cationic fractions yielded multifunctional peptides that inhibited angiotensin converting enzyme (ACE) and renin activities, which are molecular targets for antihypertensive agents. The LMW peptides also exhibited antioxidant properties by scavenging free radicals and inhibiting amine oxidase activity. The peptide fractions showed inhibition of calmodulin-dependent phosphodiesterase, an enzyme that has been implicated in the pathogenesis of several chronic diseases. Moreover, FP hydrolysis with thermolysin and pronase followed by mixing with activated carbon yielded branched-chain amino acids (BCAA)-enriched multifunctional peptide mixture (Fischer ratio of 23.65) with antioxidant properties and in vitro ACE inhibition; Fischer ratio of 20.0 is considered minimum for therapeutic purposes. The BCAA-enriched peptide product can be used in clinical nutrition to treat muscle wasting symptoms associated with hepatic diseases. Furthermore, an arginine-rich peptide mixture (31% arginine versus 11% in the original flaxseed protein) was produced by hydrolysis of FP with trypsin and pronase followed by separation using electrodialysis-ultrafiltration. Arginine plays important physiological roles especially as precursor to vasodilator, nitric oxide. The arginine-rich peptide mixture exhibited in vitro ACE and renin inhibition and led to decreased systolic blood pressure (–17.9 and –11.7 mmHg, respectively at 2 and 4 h) after oral administration to spontaneously hypertensive rats. For the first time in the literature, we showed that arginine peptides have superior physiological effects when compared to the amino acid form of arginine. Lastly, quantitative structure-activity relationship studies using partial least squares (PLS) regression yielded two predictive models for renin-inhibiting dipeptides with z-scales amino acid descriptors. The PLS models indicated that hydrophobic and bulky side chain-containing amino acids contribute to renin inhibition if present at the amino- and carboxyl-terminal of dipeptides, respectively. Based on this study, Ile-Trp was discovered as potent renin-inhibiting dipeptide, and may serve as a useful template for the development of potent antihypertensive peptidomimetics.

Postural control incorporates multiple neural and mechanical systems at various levels of the motor control system, yet the question of how all these systems interact remains unanswered. This dissertation describes development of a biologically based, three-dimensional mathematical model of the forelimb of the domestic cat that integrates skeletal anatomy, muscular architecture, and neural control. Previous work has shown that muscle architecture profoundly affects its function. However, even though the forelimbs of quadrupeds contribute to posture and locomotion differently from hindlimbs, most models of quadruped motion are based upon hindlimb mechanics. The proposed work consists of three main steps: (1) architectural and anatomical characterization, which involves acquisition of muscle attachment data, measurement of whole muscle and muscle fiber properties, and estimation of limb kinematic parameters; (2) model development and implementation, wherein the data will be integrated into a mathematical model using special-purpose software; and (3) model validation, including verification of model estimates against experimentally obtained measurements of muscle moment arms, and prediction of limb kinetics, namely end-point forces arising from perturbations to the limb. It was found that the forelimb does indeed possess structure, particularly at the shoulder and antebrachium, that allows for more diverse movements. The neural wiring in these regions is more complex than in the hindlimb, and there exists substantial muscular structure in place for non-sagittal motion and object suppression and retrieval. Other results showed that the kinematics of the limb alone produce a restorative response to postural disturbance but that the magnitude is reduced, indicating that neural input acts as a modulatory influence on top of the intrinsic mechanism of limb architecture. Furthermore the model demonstrated many of the essential features found in the experiments. This study represents the implementation of the first forelimb model of the cat incorporating mechanical properties and serves as a key component of a full quadruped model to explore posture and locomotion.