Hydroxyapatite (HAP) coatings are used in orthopaedic surgery for bone regeneration. Current methods of phase quantification of HAP coatings suffer from drawbacks. A novel methodology of quantitative phase analysis of HAP coatings has been devised and validated. This method, based on whole pattern fitting with a fundamental parameters approach, incorporates amorphous calcium phosphate (ACP) and apatite phases into structural refinements. A comparison of the structural and chemical properties of plasma sprayed (PS) and novel electrodeposited (ED) HAP coatings has been conducted. ED coatings contained less ACP and more preferred orientation than the PS coatings, although the stoichiometry was similar. In vitro investigations of PS and ED coatings in simulated body fluid and foetal calf serum revealed that both are bioactive. A carbonated apatite layer produced on the ED coatings was -0.7μm thick with a stoichiometry and chemical constituents similar to that of natural bone apatite. PS coatings produced a nanocrystalline carbonated apatite layer (-4μm). For the first time it has been possible to model crystalline HAP and nanocrystalline apatite as independent phases and obtain accurate lattice parameters for each. A positive linear correlation has been made between microstrain and the solubility of HAP and carbonated apatites. Dissolution studies have shown that the behaviour of HAP and carbonated apatite is dominated by crystallite size at low undersaturation and by crystallite size and microstrain at high undersaturation for crystallites between -30OA- 1000A. Metastable equilibrium occurred for crystallites <_400A at low undersaturation. Carbonate content did not affect the solubility or dissolution behaviour. A novel technology for coating polymeric tape with HAP for potential use in anterior cruciate ligament reconstruction has been devised. Mechanical tests have demonstrated that no adverse properties are induced by the coating technology. Cell culture studies have shown that the HAP layer is capable of enhanced attachment, proliferation and differentiation of osteoblast cells compared to uncoated tape.

Synaptic plasticity, in its broadest sense, can be defined as the ability of synapses to be modified structurally and functionally in response to various internal and external factors. Growing evidence has established that at the very core of these modifications are alterations in the cytoskeletal architecture. This discovery has led to the unearthing of a number of signaling pathways that might be involved in cytoskeletal regulation and also in the regulation of other aspects of synapse development and plasticity. In this regard, polarity proteins and secreted morphogens such as the Wnt proteins, typically involved in embryonic development, are emerging as critical determinants of synaptic growth and plasticity. However, their mechanism of action at synapses needs further investigation. Additionally, not much is known about how these morphogens are secreted or transported across synapses. Using the Drosophila larval NMJ as a model system, I have addressed aspects related to the issues mentioned above in the subsequent body of work. In the first half of my thesis, I have uncovered a role for the aPKC/Baz/Par-6 polarity protein complex in the regulation of the postsynaptic actin cytoskeleton in conjunction with the lipid and protein phosphatase PTEN. In the second half of my thesis, I have contributed to the elucidation of mechanisms underlying the secretion of Wg, the Drosophila Wnt homolog. Our findings suggest that Wnts might be secreted via a previously unidentified mechanism involving the release of exosome like vesicles from the presynapse and this process requires Evi/Wntless (Evi), a protein dedicated to Wnt secretion. Alterations in signaling pathways and aberrant cytoskeletal regulation lead to a variety of neurological disorders. The body of work in this thesis will provide a deeper understanding of the mechanisms involved in synaptic plasticity and provide a basis for uncovering similar pathways in the context of vertebrate synapses.

There is an emerging threat of Neisseria gonorrhoeae strains that are resistant to all antibiotics. Because of this, the purpose of this research is to isolate, analyze, and partially characterize a new inhibitor(s) of N. gonorrhoeae. Since there is an unknown molecule secreted by Candida albicans that inhibits N. gonorrhoeae, this molecule can be partially characterized using 1H NMR Spectroscopy to assist in the development of a new antibiotic compound. It was hypothesized that quorum-sensing molecules, trans, trans- farnesol, tyrosol, phenylethyl alcohol, and tryptophol, could be possible candidates for the inhibitor. Because of this, 1H NMR spectra for these quorum-sensing molecules were obtained to serve as controls. Column chromatography and fractionation was used to isolate the inhibitor in large scale from C. albicans grown in salts-based media. Attempts to isolate the inhibitor in large scale, however, was unsuccessful since no inhibition of N. gonorrhoeae was observed. Because of this, analysis of growth media was conducted to test the media effect on producing the inhibitor. C. albicans was grown in liquid chocolate, liquid white chocolate, salts-based, and YPD media in aerobic and candle jar environments. Analysis of growth media in different environments suggests that liquid chocolate and salts-based media retain the inhibitory activity. 1H NMR spectra were obtained for the isolated molecule in liquid chocolate and salts-based media in both aerobic and candle jar environments. Analysis of this 1H NMR suggested that the inhibitor could be isolated from either the aerobic or candle jar environment for both liquid chocolate and salt-based media because a clear peak between 3.5 and 4.0 ppm was observed in all spectra. Comparison of 1H NMR spectra from quorum-sensing molecules with spectra from the isolated molecule suggests that the inhibitor is not a quorum-sensing molecule. The peaks represented by the inhibitor cannot be fully characterized and thus, either correspond to a single molecule or a complex molecular structure. It can be concluded that the inhibitor secreted by C. albicans to inhibit N. gonorrhoeae is a new unknown compound.

Xu, Xiao-ping. / "March 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 152-166). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an attaching and effacing pathogen that upon attachment to host cells, induce characteristic attaching and effacing lesions and formation of F-actin rich pedestals beneath sites of bacterial attachment. EHEC harbors a Type III secretion system through which it delivers dozens of effectors into the host cell. The two secreted effectors critical for EHEC-mediated actin pedestal formation are the translocated intimin receptor (Tir) and EspFU. EspFU consists of an N-terminal secretion signal and a C-terminus containing six tandem 47-residue proline-rich repeats, each of which can bind and activate the actin nucleation promoting factor N-WASP. Structural and functional analyses described here have identified the mechanism of N-WASP activation by EspFU and the minimal domains and specific residues required for this activity. While EspFU and Tir are the only bacterial effectors required for F-actin pedestal formation, recruitment of EspFU to Tir is mediated by an unidentified putative host factor. To identify the host factor responsible for linking these two effectors, a combination of in vitro and functional assays were used to identify the host factor, IRTKS and the residues required for these interactions were defined. Further, the presence of at least two 47-residue repeats in all characterized clinical isolates of canonical EHEC strains led us to address the minimal requirements for EspFU functional domains to promote recruitment to Tir and N-WASP activation. Here we show that two proline-rich elements of EspFU are required for recruitment of EspFU by IRTKS to sites of bacterial attachment. Furthermore, once artificially clustered at the membrane, a single N-WASP binding element of EspFU can induce actin pedestal formation.

Hydroxyapatite (HAP) coatings are used in orthopaedic surgery for bone regeneration. Current methods of phase quantification of HAP coatings suffer from drawbacks. A novel methodology of quantitative phase analysis of HAP coatings has been devised and validated. This method, based on whole pattern fitting with a fundamental parameters approach, incorporates amorphous calcium phosphate (ACP) and apatite phases into structural refinements. A comparison of the structural and chemical properties of plasma sprayed (PS) and novel electrodeposited (ED) HAP coatings has been conducted. ED coatings contained less ACP and more preferred orientation than the PS coatings, although the stoichiometry was similar. In vitro investigations of PS and ED coatings in simulated body fluid and foetal calf serum revealed that both are bioactive. A carbonated apatite layer produced on the ED coatings was -0.7μm thick with a stoichiometry and chemical constituents similar to that of natural bone apatite. PS coatings produced a nanocrystalline carbonated apatite layer (-4μm). For the first time it has been possible to model crystalline HAP and nanocrystalline apatite as independent phases and obtain accurate lattice parameters for each. A positive linear correlation has been made between microstrain and the solubility of HAP and carbonated apatites. Dissolution studies have shown that the behaviour of HAP and carbonated apatite is dominated by crystallite size at low undersaturation and by crystallite size and microstrain at high undersaturation for crystallites between -30OA- 1000A. Metastable equilibrium occurred for crystallites <_400A at low undersaturation. Carbonate content did not affect the solubility or dissolution behaviour. A novel technology for coating polymeric tape with HAP for potential use in anterior cruciate ligament reconstruction has been devised. Mechanical tests have demonstrated that no adverse properties are induced by the coating technology. Cell culture studies have shown that the HAP layer is capable of enhanced attachment, proliferation and differentiation of osteoblast cells compared to uncoated tape.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) and enteropathogenic E. coli O127:H7 (EPEC) induce characteristic F-actin rich pedestals on infected mammalian cells. Each pathogen delivers its own translocated intimin receptor (Tir) to the host cell to act as a receptor for the bacterial outer membrane adhesin, intimin. Interaction of translocated Tir with intimin is essential for mammalian cell binding and host colonization, as well as to induce actin pedestal formation in vitro. In spite of these parallels, EHEC and EPEC Tir appear to generate actin pedestals by distinct mechanisms. Further, while the ability to form actin pedestals is a striking phenotype, the function of pedestals during infection remains unclear. To address these issues, a systematic and quantitative analysis of Tir-mediated actin assembly was conducted. We identified a three-residue Tir sequence involved in actin pedestal formation for both EHEC and EPEC, and developed evidence that the two pathogens trigger a common pathway for actin assembly. Further, the ability of these bacteria to promote actin assembly appears to promote both intimin-mediated bacterial binding in vitro and optimal colonization during experimental animal infection.

Myosin II and actin are two major components of the ingressing cortex during cytokinesis. However, their structural dynamics and functions during cytokinesis are still poorly understood. To study the role of myosin II in cortical actin turnover, dividing normal rat kidney (NRK) cells were treated with blebbistatin, a potent inhibitor of the non-muscle myosin II ATPase. Blebbistatin caused a strong inhibition of actin filament turnover and cytokinesis. Local release of blebbistatin at the equator caused inhibition of cytokinesis, while treatment in the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a global role. These observations indicate that myosin II ATPase is essential for actin turnover and remodeling during cytokinesis. To further study the mechanism of myosin II and actin recruitment to the cytokinetic furrow, equatorial cortex were observed with total internal reflection fluorescence microscope (TIRF-M) coupled with spatial temporal image correlation spectroscopy (STICS) and a new approach termed temporal differential microscopy (TDM). The results indicated at least partially independent mechanisms for the early equatorial recruitment of myosin II and actin filaments. Cortical myosin II showed no detectable directional flow toward the equator. In addition to de novo equatorial assembly, localized inhibition of disassembly appeared to contribute to the formation of the equatorial myosin II band. In contrast, actin filaments underwent a striking, myosin II dependent flux toward the equator. However, myosin II was not required for equatorial actin concentration, suggesting that there was a flux-independent, de novo mechanism. The study was then extended to retraction fibers found typically on mitotic adherent cells, to address the hypothesis that they may facilitate post-mitotic spreading. Cells with retraction fibers showed increased spreading speed in post-mitotic spreading compared to cells without retraction fibers. In addition, micromanipulation study suggested that retraction fibers may guide the direction of post-mitotic spreading. Focal adhesion proteins were present at the tips of retraction fibers, and may act as small nucleators for focal adhesions reassembly that help cell quickly respread and regrow focal adhesions. These findings may suggest a general mechanism utilized by adherent cells to facilitate post-mitotic spreading and reoccupy their previous territory.

During heart valve remodeling, and in many disease states, valvular interstitial cells (VICs) shift to an activated myofibroblast phenotype which is characterized by enhanced synthetic and contractile activity. Pronounced alpha smooth muscle actin (αSMA)-containing stress fibers, the hallmark of activated myofibroblasts, are also observed when VICs are placed under tension due to altered mechanical loading in vivo or during in vitro culture on stiff substrates or under high mechanical loads and in the presence of transforming growth factor-beta1 (TGF-β1). The work presented herein describes three distinct model systems for application of controlled mechanical environment to VICs cultured in vitro. The first system uses polyacrylamide (PA) gels of defined stiffness to evaluate the response of VICs over a large range of stiffness levels and TGF-β1 concentration. The second system controls the boundary stiffness of cell-populated gels using springs of defined stiffness. The third system cyclically stretches soft or stiff two-dimensional (2D) gels while cells are cultured on the gel surface as it is deformed. Through the use of these model systems, we have found that the level of 2D stiffness required to maintain the quiescent VIC phenotype is potentially too low for a material to both act as matrix to support cell growth in the non-activated state and also to withstand the mechanical loading that occurs during the cardiac cycle. Further, we found that increasing the boundary stiffness on a three-dimensional (3D) cell populated collagen gel resulted in increased cellular contractile forces, αSMA expression, and collagen gel (material) stiffness. Finally, VIC morphology is significantly altered in response to stiffness and stretch. On soft 2D substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates. Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. These studies provide critical information for characterizing how VICs respond to mechanical stimuli. Characterization of these responses is important for the development of tissue engineered heart valves and contributes to the understanding of the role of mechanical cues on valve pathology and disease onset and progression. While this work is focused on valvular interstitial cells, the culture conditions and methods for applying mechanical stimulation could be applied to numerous other adherent cell types providing information on the response to mechanical stimuli relevant for optimizing cell culture, engineered tissues or fundamental research of disease states.

Injury to bone is one of the most prevalent and costly medical conditions. Clinical treatment of volumetric bone loss or hard-to-heal bony lesions often requires the use of proper bone grafting materials, with or without adjuvant anabolic therapeutics. Despite significant problems associated with autografting (donor site morbidity, limited supplies) and allografting (disease transmissions, high graft failure rates) procedures, synthetic bone grafts remain the least utilized clinically. Existing synthetic orthopaedic biomaterials rarely possess a combination of bone-like structural and biochemical properties required for robust osteointegration, scalable and user-friendly characteristics indispensable for successful clinical translations. This thesis tests the hypothesis that by recapitulating key structural elements and biochemical components of bone in 3- and 2-dimensional biomaterials, scalable synthetic bone grafts can be designed to enable expedited healing of hard-to-heal volumetric bone loss. Specifically, FlexBone, a 3-dimensional hydrogel scaffold encapsulating 50 wt% of structurally well integrated nanocrylstalline hydroxyapatite, the main inorganic component of bone, was developed. The large surface area of nanocrystalline hydroxyapatite combined with its intrinsic affinity to proteins and its excellent structural integration with the hydrogel matrix enabled FlexBone to both sequester endogenous protein signals upon press-fitting into an area of skeletal defect and to deliver exogenous protein therapeutics in a localized and sustained manner. We demonstrated that FlexBone enabled the functional healing of critical-size long bone defects in rats in 8 – 12 weeks with the addition of a very low dose of osteogenic growth factor BMP-2/7. This promising synthetic bone graft is now being explored for the delivery of multiple growth factors to expedite the healing of diabetic bony lesions. In addition, a 2-dimensional electrospun cellulose fibrous mesh was chemically modified with sulfate residues to mimic sulfated polysaccharide ECM components of skeletal tissues to enabled progenitor cell attachment and differentiation as well as controlled retention and localized/sustained delivery of protein therapeutics. This sulfated fibrous mesh is currently explored as synthetic periosteum to augment the osteointegration of devitalized structural allografts. Finally, a rat subcutaneous implantation model developed to examine the biocompatibility of newly developed biodegradable shape memory polymer bone substitutes is also presented.