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

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.

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.

In this thesis computational chemistry has been used to accelerate experimental discovery in the fields of ice recrystallization inhibitors for cryopreservation and ultra-microporous MOFs for carbon dioxide capture and storage. Ice recrystallization is one of the leading contributors to cell damage and death during the freezing process. This occurs when larger ice crystal grains grow at the expense of smaller ones. Naturally occurring biological antifreeze molecules have been discovered but only operate up to -4oC and actually exasperate the problem at temperatures lower than this. Recently, the group of Dr. Robert Ben have been successful in synthesizing small organic molecules which are capable of inhibiting the growth of ice crystals during the freezing process. They have built a library of diverse compounds with varying functionalities and activity. Chemical intuition has been unsuccessful in finding a discernible trend with which to predict activity. Herein we present work where we have utilized a quantitative structure activity relationship (QSAR) model to predict whether a molecule is active or inactive. This was built from a database of 124 structures and was found to have a positive find rate of 82%. Proposed molecules that had yet to be synthesized were predicted to active or inactive using our method and 9/11 structures were indeed active which is strikingly consistent to the 82% find rate. Our efforts to aid in the discovery of these novel molecules will be described here. Metal organic frameworks (MOFs) are a relatively new class of porous materials which have taken the academic community by storm. These three-dimensional crystalline materials are built from a metal node and an organic linker. Depending on the metals and organic linkers used, MOFs can possess a vast range of topologies and properties that can be exploited for specific applications. Ultra-microporous MOFs possess relatively small pores in the range of 3.5 Å to 6 Å in diameter. These MOFs have some structural advantages compared to larger pored MOFs such as molecular sieving, smaller pores which promote strong framework-gas interactions and cooperative effects between guests, and longer shelf-life due to small void volumes and rigid frameworks. Here we present newly synthesized ultra-microporous MOFs based on isonicotnic acid as the organic linker with Ni and Mg as the metal centre. Despite having such small pores, Ni-4PyC exhibits exceptionally high CO2 uptake at high pressures. Furthermore, Mg-4PyC exhibits novel pressure dependent gate-opening behaviour. Computational simulations were employed to investigate the origin of high CO2 uptake, predict high pressure (>10bar) isotherms, quantify CO2 binding site positions and energies, and study uptake-dependent linker dynamics. This work hopes to provide experimentalists with some explanation to these interesting unexplained phenomena and also predict optimal properties for new applications.

Induced pluripotent stem cells (iPSCs) are an attractive cell source for various applications in regenerative medicine and cell-based therapies given their unique capability to differentiate into any cell type of the human body. However, human iPSCs are highly vulnerable to cryopreservation with post-thaw survival rates of 40-60%; this is due to cryoinjury resulting from ice recrystallization when using conventional slow cooling protocols. Ice recrystallization is a process where the growth of large ice crystals occurs at the expense of small ice crystals. Ice recrystallization inhibitors (IRIs) are designed to inhibit the growth of intracellular ice crystals, increasing post-thaw viability. In this study, we tested a panel of four IRIs to determine if the inhibition of ice recrystallization can decrease cellular damage during freezing and improve viability post-thaw of iPSC colonies. We supplemented commercially available and serum-free cryopreservation medium mFreSR, routinely used for the cryopreservation of iPSCs, with a class of N-aryl-D-ß-gluconamide IRIs. A 2-fold increase in post-thaw viability was observed, in a dose dependent response, for N-(4-methoxyphenyl)-D-gluconamide (PMA) at 15 mM, N-(2-fluorophenyl)-D-gluconamide (2FA) at 10 mM, and N-(4-chlorophenyl)-D-gluconamide (4ClA) at 0.5 mM over mFreSR controls. After testing the panel of four IRIs, 2FA frozen iPSCs showed an increase in cell viability, proliferation, and recovery. The addition of ROCK inhibitor (RI), commonly used to increase iPSC viability post thaw, further enhanced the survival of the iPSCs frozen in the presence of 2FA and is used routinely in research. This additive effect increased cell recovery and colony formation post thaw, resulting in increased proliferation with no adverse effects on iPSC pluripotency or differentiation capabilities. The development of improved cryopreservation strategies for iPSCs is key to establishing master clonal cell banks and limiting cell selection pressures, all while maintaining high post-thaw viability and function. This will help ensure sufficient supplies of high-quality iPSC required to meet the cell demands for cell and regenerative based therapies. Since iPSCs hold promise as a potentially unlimited cell source for a plethora of cell-based therapies, improving cryopreservation is essential to the successful deployment of iPSC-derived therapeutic cell products in the future.

Recent advances in the clinical diagnosis and treatment of diseases using cell transplantation have emphasized the urgent need to cryopreserve many types of cells. In transfusion medicine, red blood cell (RBC) transfusions are used to treat anemia and inherited blood disorders, replace blood lost during or after surgery and treat accident victims and mass casualty events. In regenerative medicine, mesenchymal stem cell (MSC) therapy offers promising treatment for tissue injury and immune disorders. Current cryoprotective agents (CPAs) utilized for RBCs and MSCs are 40% glycerol and 10% dimethyl sulfoxide (DMSO), respectively. Although glycerol is required for successful cryopreservation of RBCs, it must be removed from RBCs post-thaw using costly and time-consuming deglycerolization procedures to avoid intravascular hemolysis. Unfortunately, while DMSO prevents cell damage and increases post-thaw MSC viability and recovery, recent reports have suggested that MSCs cryopreserved in DMSO display compromised function post-thaw. As a result, improvements to the current cryopreservation protocols such as reducing post-thaw RBC processing times and improving MSC function post-thaw are necessary in order to meet the increasing demands of emerging cellular therapies. Ice recrystallization has been identified as a significant contributor to cellular injury and death during cryopreservation. Consequently, the ability to inhibit ice recrystallization is a very desirable property for an effective CPA, unlike the conventional CPAs such as DMSO and glycerol that function via a different mechanism and do not control or inhibit ice recrystallization. Over the past few years, our laboratory has reported several different classes of small molecules capable of inhibiting ice recrystallization such as lysine-based surfactants, non-ionic carbohydrate-based amphiphiles (alkyl and aryl aldonamides) and O-linked alkyl and aryl glycosides. The use of these small molecule ice recrystallization inhibitors (IRIs) as novel CPAs has become an important strategy to improve cell viability and function post-thaw. With the overall goal to identify highly effective inhibitors of ice recrystallization, the first part of this thesis examines the IRI activity of three diverse classes of small molecules including carbohydrate-based surfactants bearing an azobenzene moiety, fluorinated aryl glycosides and phosphate sugars. While the majority of the carbohydrate-based surfactants and fluorinated aryl glycosides were not effective inhibitors of ice recrystallization, this work revealed that monosaccharides possessing a phosphate group could be effective IRIs. Our laboratory has previously demonstrated that small molecule IRIs β-PMP-Glc and β-pBrPh-Glc can protect human RBCs from cellular injury during freezing using reduced concentrations of glycerol (15% w/v). This was significant as reducing the concentration of glycerol can drastically decrease deglycerolization times. Consequently, structure- function studies were conducted on β-PMP-Glc and β-pBrPh-Glc to elucidate key structural features that further enhance their IRI activity and may increase their cryoprotective ability. In particular, the influence of an azido moiety on the IRI activity of β-PMP-Glc and β-pBrPh-Glc was investigated and it was determined that the position of the azide substituent on the pyranose ring is crucial for effective inhibition of ice recrystallization. Furthermore, the presence of an azido group at C-3 was found to increase the IRI activity of β-PMP-Glc and β-pBrPh-Glc. Despite the discovery that β-PMP-Glc and β-pBrPh-Glc are beneficial additives for the freezing of RBCs, a significant amount of cellular damage occurred during deglycerolization, resulting in very low cell recoveries. Thus, IRI active azido aryl glucosides were explored for their cryopreservation potential in RBCs to determine whether they could function as effective additives that reduce cellular damage post-thaw and improve cell recovery. One of the most significant results of this thesis is the discovery that azido aryl glucosides can successfully cryopreserve RBCs in the presence of 15% glycerol with significantly improved cell recovery. This thesis also explores the use of small molecule IRIs to improve the cryopreservation of MSCs. In particular, the addition of an N-aryl-aldonamide (2FA) to the standard 10% DMSO solution was found to enhance the proliferative capacity of MSCs post-thaw. Lastly, the ability of small molecule IRIs to cross the cell membrane and behave as permeating CPAs was evaluated in two different cell models, RBCs and human umbilical vein endothelial cells (HUVECs). These studies demonstrated that small molecule IRIs are capable of permeating the cell membrane and controlling intracellular ice recrystallization.

The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.

The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.

Antifreeze proteins (AFPs) are produced by a variety of organisms to either protect them from freezing or help them tolerate being frozen. Recent structural work has shown that AFPs bind to ice using ordered surface waters on a particular surface of the protein called the ice-binding site (IBS). These 'anchored clathrate' waters fuse to particular planes of an ice crystal and hence irreversibly bind the AFP to its ligand. An AFP isolated from the perennial ryegrass, Lolium perenne (LpAFP) was previously modelled as a right-handed beta helix with two proposed IBSs. Steric mutagenesis, where small side chains were replaced with larger ones, determined that only one of the putative IBSs was responsible for binding ice. The mutagenesis work also partly validated the fold of the computer-generated model of this AFP. In order to determine the structure of the protein, LpAFP was crystallized and solved to 1.4 Å resolution. The protein folds as an untwisted left-handed beta-helix, of opposite handedness to the model. The IBS identified by mutagenesis is remarkably flat, but less regular than the IBS of most other AFPs. Furthermore, several of the residues constituting the IBS are in multiple conformations. This irregularity may explain why LpAFP causes less thermal hysteresis than many other AFPs. Its imperfect IBS is also argued to be responsible for LpAFP's heightened ice-recrystallization inhibition activity. The structure of LpAFP is the first for a plant AFP and for a protein responsible for providing freeze tolerance rather than freeze resistance. To help understand what constitutes an IBS, a non-ice-binding homologue of type III AFP, sialic acid synthase (SAS), was engineered for ice binding. Point mutations were made to the germinal IBS of SAS to mimic key features seen in type III AFP. The crystal structures of some of the mutant proteins showed that the potential IBS became less charged and flatter as the mutations progressed, and ice affinity was gained. This proof-of-principle study highlights some of the difficulties in AFP engineering. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2012-07-18 15:24:42.082