Antifreeze proteins (AFPs) help cold-adapted organisms survive below 0 ◦C by binding to and inhibiting the growth of ice crystals. In this way, AFPs depress the freezing point of aqueous fluids below the melting point of ice (thermal hysteresis; TH). They also have the ability to inhibit ice recrystallization in the frozen state (ice recrystallization inhibition; IRI). Some AFPs show an order of magnitude higher TH activity than others, and are termed ‘hyperactive’. One of the objectives of this thesis was to see if IRI activities of the hyperactive AFPs are also an order of magnitude higher than the moderately active AFPs. Using a capillary-based assay for IRI, the activities of three hyperactive and three moderately active AFPs were determined. There was no apparent correlation between hyperactivity in TH and high IRI activity. However, mutations of residues on the ice-binding face (IBF) of both types of AFP reduced IRI and TH activities to a similar extent. In this way, the use of IBF mutant AFPs showed that the IBF responsible for an AFP’s TH activity is also responsible for its IRI activity. Analysis of the diverse AFP structures solved to date indicate that their IBFs are relatively flat, occupy a significant proportion of the protein’s surface area and are more hydrophobic than other surfaces of the protein. The IBFs also often have repeating sequence motifs and tend to be rich in alanine and/or, threonine. The de novo design of an ice-binding protein was undertaken using these features to verify the underlying physicochemical requirements necessary for a protein’s interaction with ice. Using site-directed mutagenesis, a total of sixteen threonine substitutions were made on one of the four faces of a cyanobacterial protein with no endogenous TH activity. The inclusion of eight paired threonines on one face of this quadrilateral helix gave the engineered protein low levels of TH activity, but at the cost of destabilizing the structure to some extent. The results of this study have validated some of the properties needed for the ice-binding activity of AFPs. / Thesis (Master, Biochemistry) -- Queen's University, 2010-01-29 17:37:24.322

Microbes have a remarkable ability to adapt to a host of environmental stressors, including low temperature, high pressure and osmotic stresses. The adaptations of resistant microbes to low temperatures are varied, and may include the accumulation of solutes to maintain osmotic balance, the production of antifreeze proteins (AFPs) or ice nucleation proteins (INPs) to manipulate ice growth or formation. AFPs depress the freezing point, inhibit ice recrystallization, and have been reported to inhibit or delay the growth of gas hydrates. Conversely, INPs precipitate ice formation at relatively high subzero temperatures. Collectively, these activities can be described as ‘ice-association’ activities. Here, ice-affinity and/or freeze-thaw cycling were used to either select for isolates with ice association properties or to assess the low temperature resistance of microbial consortia derived from various environments. Ice-affinity successfully selected psychrotolerant microbes from cultured temperate and boreal soils, some of which had been previously reported in glaciers and Arctic/Antarctic sites. Many of the recovered microbes demonstrated ice-association activities. Freeze-thaw selection also greatly decreased the abundance and diversity of consortia from distinct sites, and allowed the recovery of individual isolates, many of which demonstrated ice-association. Freeze-thaw selection was also used to assess the role of cross-tolerance between osmotic and freeze-thaw stresses, based on the common challenge of desiccation. Microbial consortia from lakes with varying degrees of salinity were subjected to freeze-thaw stress, and the consortia from more saline lakes tended to show greater low temperature resistance. While few of the recovered microbes demonstrated ice-association activities, those from the more saline lakes tended to contain a higher intracellular solute concentration and were more likely to form biofilms. This underscores the diversity of resistance strategies and supports the notion of cross-tolerance. To determine if these selective regimes would have applications for hydrate growth inhibition, microbes derived from an oil well sample were subjected to freeze-thaw stress. Selection reduced microbial abundance, shifted the diversity, and resulted in the recovery of microbes with some ice-association activity. Taken together, this thesis demonstrates that the application of low temperature stress can be used to successfully investigate stress resistance mechanisms within microbial communities from distinct environments. / Thesis (Ph.D, Biology) -- Queen's University, 2010-09-21 15:58:14.932

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Cryopresevation is the process of cooling biological materials to low sub-zero temperatures for storage purposes. Numerous medical and technical applications, such as hematopoeitic stem cell transplantation and sperm banking, sometimes require the use of cryopreserved cells. Cryopreservation, however, can induce cell injury and reduce the yields of viable functional cells. Ice recrystallization is a mechanism of cryopreservation injury, but is rarely addressd in strategies to optimize cell cryopreservation. The results from this thesis demonstrate an association between the potency of carbohydrate-mediated ice recrystallization inhibition used in the cryopreservation of umbilical cord blood and recovery of viable non-apoptotic cells and hematopoietic progenitor function. Furthermore, increased numbers of apoptotic cells in hematopoeitic stem cell grafts were associated with reduced hematopoietic function and delayed hematopoietic recovery in patients undergoing blood stem cell transplantation. These findings provide a basis for pursuing further studies assessing ice recrystallization inhibition as a strategy for improving cell cryopreservation.

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Constant strain rate compression and torsion tests were carried out isothermally at temperatures of 875 to 1075(DEGREES)C on a series of six steels. The base steel had a composition of 0.06% C and 1.43% Mn and the others contained one of the following sets of additions: (i) 0.035% Nb; (ii) 0.035% Nb + 0.115% V; (iii) 0.035% Nb + 0.30% Mo; (iv) 0.035% Nb + 1.90% Mn; (v) 0.115% V. The tests were conducted to determine the effects of Mn, Mo, Nb and V, singly and in combination, on the high temperature flow and recrystallization behavior of the materials. The dynamic precipitation kinetics for Nb(CN) and VN were determined by the Weiss method. The two PTT curves were similar, with the nose of the VN curve being situated at a slightly lower temperature (885 vs. 900(DEGREES)C) and at a somewhat longer time (26 vs. 16 s), in agreement with the lower equilibrium solution temperature of VN. The dynamic precipitation kinetics of Nb(CN) were retarded by the addition of Mn, V or Mo. This retardation is attributed to the increased carbonitride solubility that follows the addition of these elements because of the way in which they decrease the C and N activity coefficients. / RTT curves were constructed for dynamic recrystallization in the six steels investigated. These were derived from the peak strains of the compression flow curves, as determined at a strain rate of 3.7 x 10('-2)s('-1). Recrystallization occurred earliest in the plain C steel followed fairly quickly by the 0.115% V steel. All of the Nb bearing steels recrystallized considerably later, with the greatest retardation being noted in the 0.3% Mo steel, where it was nearly twice that due to Nb addition alone. This very large effect, and the retardation due to each of the transition elements, is explained in terms of the electronic differences between iron and the particular element. The effect of the atomic size differences with respect to iron is also considered. / The strengthening due to the presence of Mn, Mo, Nb and V in solution was determined from the yield strengths of these steels. The increment in yield strength over that of the plain C steel was determined as 70% and 7% per 0.1 at.% of Nb and V when each is added singly. The strengthening increased to 80% and 8% respectively for these elements when present jointly in austenite. The strength increments were 9% for Mo and 1.3% for Mn per 0.1 at.% when added to a 0.035% Nb steel. The rank order of these effects is also explained in terms of the electronic and atomic size differences, and a possible reason for the synergistic effect (e.g. in the case of Nb and V in a Nb-V steel) is proposed.

Thesis (M.S.)--University of Missouri--Rolla, 2007. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed November 29, 2007) Includes bibliographical references.

Thesis (PhD.(Metallurgy)--University of Pretoria, 2007. / Includes bibliographical references.