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Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 1992.

Thesis (M.Phil.)--University of Hong Kong, 2002. / Includes bibliographical references. Also available in print.

Ice recrystallization during thawing after cryopreservation results in extensive cellular damage that ultimately leads to cell death and decreased cell viabilities. This is a significant problem particularly with cryopreserved cells utilized in various regenerative medicine therapies. Given the success of these therapies to treat spinal cord injury, cartilage lesions, and cardiacdisease, the development of new and improved cryprotectants that minimize cell damageduring freeze-thawing and improve cell viability post-cryopreservation are urgently required. The current cryopreservative dimethyl sulfoxide, DMSO, is associated with cytotoxicity in clinical settings and is not an optimal cryopreservative. Our laboratory is interested in synthesizing small molecules that possess the property of ice recrystallization inhibition (IRI) activity that can be utilized as cryopreservatives without the cytotoxic effects associated with DMSO. This thesis focuses on the development of small molecule ice recrystallization inhibitors and elucidating the structural features of disaccharides and surfactants that are responsible for potent IRI activity. The first part of this study examines simple disaccharide derivatives mimicking those found in the native AFGP to determine whether disaccharide structure influences IRI activity. Towards this end, the (1,6)-linked AFGP disaccharide analogue was synthesized, assessed for IRI activity using a splat-cooling assay, and compared to the native (1,3)- and (1,4)-linked AFGP disaccharide analogues. The change in linkage was found to have a profound affect on IRI activity. The second part of the study focuses on surfactants and gelators as ice recrystallization inhibitors. Our laboratory has demonstrated that carbohydrate-based hydrogelators can be potent inhibitors of ice recrystallization. While our studies have indicated that a delicate balance between hydrophobic and hydrophilic interactions is crucial for ice recrystallization inhibition (IRI) activity, the essential structural features necessary for potent IRI activity remain unknown. To address this issue, structurally diverse amino acid-based surfactants/gelators, anti-ice nucleating agents, and glycoconjugates were synthesized and assessed for IRI activity. The results indicate that long alkyl chains and increased hydrophobicity are important for potent IRI activity and iii that the position of these alkyl chains is essential. Also, the counterion of these compounds affects the IRI activity and is related to the counterion degree of hydration. These compounds were assessed for their ability to cryopreserve human liver cells (Hep G2) and human bone marrow cells (Tf-1α) in cell-based assays. Additionally, the best IRI assay solution was determined, which involved studying how the salts of the phosphate buffered saline (PBS) solution modulated IRI activity. Finally, small molecule ice recrystallization inhibitors were assessed for their ability to protect the viral vectors vaccinia virus, vesicular stomatitis virus, and herpes simplex-1 virus at various storage conditions. This will aid in developing improved preservation protocols for vaccines and viruses utilized in cancer therapy (oncolytic viruses).

Over the past few decades, there has been an increase in the development of new cellular therapies used for the treatment of various conditions. Thus, the rapid development of therapies requiring transfusion and transplantation of cells has resulted in a need to preserve these cellular therapy products. Cryopreservation is the only currently used method for the long-term storage of cells. The most commonly used cryoprotectants are 10% dimethyl sulfoxide (DMSO) for hematopoietic stem cells (HSCs) and 40% glycerol for red blood cells (RBCs). DMSO fails to protect the functionality of HSCs after cryopreservation and therefore, up to 20% of HSC transplantations fail to engraft. The glycerol in thawed RBC units must be removed to <1% to prevent intravascular hemolysis which is time-consuming. Thus, there is an urgent need to develop improved cryoprotectants for HSCs and RBCs. DMSO and glycerol are unable to control ice recrystallization which is a major source of cellular injury during cryopreservation. Therefore, compounds with the ability to inhibit ice recrystallization could represent a new class of cryoprotectant with a novel mechanism of action. This thesis focuses on the rational design of small-molecule ice recrystallization inhibitors. The key structural attributes required for ice recrystallization inhibition (IRI) activity are investigated. The amphiphilic balance required for IRI activity is explored. Furthermore, two new classes of small-molecule IRIs containing aromatic rings were rationally designed. As a result, several very highly IRI active molecules were discovered. The use of IRIs to improve the cryopreservation of HSCs and RBCs was explored. A number of IRIs improved the post-thaw functionality of HSCs. Supplementation of the current cryoprotectant solution with IRIs resulted in an increase in CFU recovery and frequency of multipotent progenitors. This would reduce the percentage of engraftment failure and allow for a larger proportion of cord blood banks’ inventory to provide an adequate dose for patients requiring transplants. Several IRIs were found to be effective cryoprotectants for RBCs with reduced amounts of glycerol. This could reduce the deglycerolization time for RBCs. These results demonstrate the potential of small-molecule IRIs to improve the current cryopreservation procedures for important cellular therapy products.

The objective of this study is to evaluate material response to plastic deformation between aluminum alloys AA2219-T87 and AA2195-T87. Of particular interest are the deformation conditions that result in subsequent grain refinement or recrystallization. Although both alloys have a face centered cubic (FCC) microstructure, variations are expected in their plastic deformation behavior. During plastic deformation, dislocation motion results in faulted regions whose width can vary according to chemical composition of the alloy. These faulted regions, or stacking faults, influence whether dislocation entanglements form or annihilation occurs during the deformation process. In addition, differences in mechanical properties can affect the amount of adiabatic heating that occurs during deformation. Ultimately these differences can affect the uniformity of deformation and the stability of the microstructure.

<p> Recrystallization of aluminum alloys, which affects their mechanical properties, was examined by a variety of experimental techniques with electrical resistivity (ER) measurements being one of them. In this work, a new method based on in-situ electrical resistivity measurements has been originated and employed for characterization of recrystallization. In contrast to traditional method of resistivity measurements at either room or liquid N2 temperature on isothermally/isochronally heat-treated samples, a new and simpler approach was proposed. In this work, resistivity of a long sample with a small cross section area (usually, 70 x 2 x 0.2 mm3 strips were employed) was continuously measured while the sample was heated from room temperature to 400°C. In this case, there are several independent contributions to electrical resistivity including contributions from lattice vibrations, second-phase particles, presence of solutes and defects, etc. </p> <p> In order to separate precipitation and recrystallization, a new annealing cycle was proposed through thermodynamic modeling. It was demonstrated that for 5xxx AI alloys only contributions from phonons, Pr, and dislocations, pd, were important. By analyzing a temperature dependence of p , it was intended to separate the effect related to a dislocation density, i.e. to a degree of recrystallization. Since Pr >> pd, this effect is elusive. It can only be detected if a robust numerical differentiation is utilized. A novel spline-based method of robust numerical differentiation was developed and employed to reveal these elusive effects from p(T) (or R(T)) profiles. It was shown that a characteristic peak of the d p (T) / dT curves points to onset and end of "events" taking place in AI alloys and that peaks' positions depend on the degree of deformation and amount of alloying additions such as Fe and Mn. The onset and extent of recrystallization seen in the resistivity measurement experiments were confirmed by optical metallography and hardness measurements. Uniformity of deformation in samples was investigated by micro-hardness measurements followed by a detailed statistical analysis to prove that deformation is uniform throughout cross-section of the materials. </p> <p> Results suggested that increasing degree of deformation and alloying additions (Fe and Mn) lower the temperature of onset of recrystallization. In addition, it was observed that kinetics of recrystallization was accelerated by the increasing degree of deformation and alloying additions whereas recrystallized grain size decreases with increasing additions of Fe and Mn in Al-3% Mg alloys. </p> / Thesis / Master of Applied Science (MASc)

Inhibiting the formation of ice is an essential process commercially, industrially, and medically. Compounds that work to stop the formation of ice have historically possessed drawbacks such as toxicity or prohibitively high active concentrations. One class of molecules, ice recrystallization inhibitors, work to reduce the damage caused by the combination of small ice crystals into larger ones. Recent advances made by the Ben lab have identified small molecule carbohydrate analogues that are highly active in the field of ice recrystallization and have potential in the cryopreservation of living tissue. A similar class of molecules, kinetic hydrate inhibitors, work to prevent the formation of another type of ice – gas hydrate. Gas hydrates are formed by the encapsulation of a molecule of a hydrocarbon inside a growing ice crystal. These compounds become problematic in high pressure and low temperature areas where methane is present - such as an oil pipeline. A recent study has highlighted the effects of antifreeze glycoprotein, a biological ice recrystallization inhibitor, in the inhibition of methane clathrates. Connecting these two fields through the synthesis and testing of small molecule ice recrystallization inhibitors in the inhibition of methane hydrates is unprecedented and may lead to a novel class of compounds.