M.S.

The development of effective methods to cryopreserve precious cell types has had tremendous impact on regenerative and transfusion medicine. Hematopoietic stem cell (HSC) transplants from cryopreserved umbilical cord blood (UCB) have been used for regenerative medicine therapies to treat conditions including hematological cancers and immodeficiencies. Red blood cell (RBC) cryopreservation in blood banks extends RBC storage time from 42 days (for hypothermic storage) to 10 years and can overcome shortages in blood supplies from the high demand of RBC transfusions. Currently, the most commonly utilized cryoprotectants are 10% dimethyl sulfoxide (DMSO) for UCB and 40% glycerol for RBCs. DMSO is significantly toxic both to cells and patients upon its infusion. Glycerol must be removed to <1% post-thaw using complicated, time consuming and expensive deglycerolization procedures prior to transfusion to prevent intravascular hemolysis. Thus, there is an urgent need for improvements in cryopreservation processes to reduce/eliminate the use of DMSO and glycerol. Ice recrystallization during cryopreservation is a significant contributor to cellular injury and reduced cell viability. Compounds capable of inhibiting this process are thus highly desirable as novel cryoprotectants to mitigate this damage. The first compounds discovered that were ice recrystallization inhibitors were the biological antifreezes (BAs), consisting of antifreeze proteins and glycoproteins (AFPs and AFGPs). As such, BAs have been explored as potential cryoprotectants, however this has been met with limited success. The thermal hysteresis (TH)activity and ice binding capabilities associated with these compounds can facilitate cellular damage, especially at the temperatures associated with cryopreservation. Consequently, compounds that possess “custom-tailored” antifreeze activity, meaning they exhibit the potent ice recrystallization inhibition (IRI) activity without the ability to bind to ice or exhibit TH activity,are highly desirable for potential use in cryopreservation. This thesis focuses on the rational design of potent ice recrystallization inhibitors and on elucidating important key structural motifs that are essential for potent IRI activity. While particular emphasis in on the development of small molecule IRIs, exploration into structural features that influence the IRI of natural and synthetic BAs and BA analogues is also described as these are some of the most potent inhibitors known to date. Furthermore, this thesis also investigates the use of small molecule IRIs for the cryopreservation of various different cell types to ascertain their potential as novel cryoprotectants to improve upon current cryopreservation protocols, in particular those used for the long-term storage of blood and blood products. Through structure-function studies the influence of (glyco)peptide length, glycosylation and solution structure for the IRI activity of synthetic AFGPs and their analogues is described. This thesis also explores the relationship between IRI, TH and cryopreservation ability of natural AFGPs, AFPs and mutants of AFPs. While these results further demonstrated that BAs are ineffective as cryoprotectants, it revealed the potential influence of ice crystal shape and growth progression on cell survival during cryopreservation. One of the most significant results of this thesis is the discovery of alkyl- and phenolicglycosides as the first small molecule ice recrystallization inhibitors. Prior to this discovery, all reported small molecules exhibited only a weak to moderate ability to inhibit ice recrystallization. To understand how these novel small molecules inhibit this process, structure-function studies were conducted on highly IRI active molecules. These results indicated that key structural features, including the configuration of carbons bearing hydroxyl groups and the configuration of the anomeric center bearing the aglycone, are crucial for potent activity. Furthermore, studies on the phenolic-glycosides determined that the presence of specific substituents and their position on the aryl ring could result in potent activity. Moreover, these studies underscored the sensitivity of IRI activity to structural modifications as simply altering a single atom or functional group on this substituent could be detrimental for activity. Finally, various IRI active small molecules were explored for their cryopreservation potential with different cell types including a human liver cell line (HepG2), HSCs obtained from human UCB, and RBCs obtained from human peripheral blood. A number of phenolic-glycosides were found to be effective cryo-additives for RBC freezing with significantly reduced glycerol concentrations (less than 15%). This is highly significant as it could drastically decrease the deglycerolization processing times that are required when RBCs are cryopreserved with 40% glycerol. Furthermore, it demonstrates the potential for IRI active small molecules as novel cryoprotectants that can improve upon current cryopreservation protocols that are limited in terms of the commonly used cryoprotectants, DMSO and glycerol.

The development of effective methods to cryopreserve precious cell types has had tremendous impact on regenerative and transfusion medicine. Hematopoietic stem cell (HSC) transplants from cryopreserved umbilical cord blood (UCB) have been used for regenerative medicine therapies to treat conditions including hematological cancers and immodeficiencies. Red blood cell (RBC) cryopreservation in blood banks extends RBC storage time from 42 days (for hypothermic storage) to 10 years and can overcome shortages in blood supplies from the high demand of RBC transfusions. Currently, the most commonly utilized cryoprotectants are 10% dimethyl sulfoxide (DMSO) for UCB and 40% glycerol for RBCs. DMSO is significantly toxic both to cells and patients upon its infusion. Glycerol must be removed to <1% post-thaw using complicated, time consuming and expensive deglycerolization procedures prior to transfusion to prevent intravascular hemolysis. Thus, there is an urgent need for improvements in cryopreservation processes to reduce/eliminate the use of DMSO and glycerol. Ice recrystallization during cryopreservation is a significant contributor to cellular injury and reduced cell viability. Compounds capable of inhibiting this process are thus highly desirable as novel cryoprotectants to mitigate this damage. The first compounds discovered that were ice recrystallization inhibitors were the biological antifreezes (BAs), consisting of antifreeze proteins and glycoproteins (AFPs and AFGPs). As such, BAs have been explored as potential cryoprotectants, however this has been met with limited success. The thermal hysteresis (TH)activity and ice binding capabilities associated with these compounds can facilitate cellular damage, especially at the temperatures associated with cryopreservation. Consequently, compounds that possess “custom-tailored” antifreeze activity, meaning they exhibit the potent ice recrystallization inhibition (IRI) activity without the ability to bind to ice or exhibit TH activity,are highly desirable for potential use in cryopreservation. This thesis focuses on the rational design of potent ice recrystallization inhibitors and on elucidating important key structural motifs that are essential for potent IRI activity. While particular emphasis in on the development of small molecule IRIs, exploration into structural features that influence the IRI of natural and synthetic BAs and BA analogues is also described as these are some of the most potent inhibitors known to date. Furthermore, this thesis also investigates the use of small molecule IRIs for the cryopreservation of various different cell types to ascertain their potential as novel cryoprotectants to improve upon current cryopreservation protocols, in particular those used for the long-term storage of blood and blood products. Through structure-function studies the influence of (glyco)peptide length, glycosylation and solution structure for the IRI activity of synthetic AFGPs and their analogues is described. This thesis also explores the relationship between IRI, TH and cryopreservation ability of natural AFGPs, AFPs and mutants of AFPs. While these results further demonstrated that BAs are ineffective as cryoprotectants, it revealed the potential influence of ice crystal shape and growth progression on cell survival during cryopreservation. One of the most significant results of this thesis is the discovery of alkyl- and phenolicglycosides as the first small molecule ice recrystallization inhibitors. Prior to this discovery, all reported small molecules exhibited only a weak to moderate ability to inhibit ice recrystallization. To understand how these novel small molecules inhibit this process, structure-function studies were conducted on highly IRI active molecules. These results indicated that key structural features, including the configuration of carbons bearing hydroxyl groups and the configuration of the anomeric center bearing the aglycone, are crucial for potent activity. Furthermore, studies on the phenolic-glycosides determined that the presence of specific substituents and their position on the aryl ring could result in potent activity. Moreover, these studies underscored the sensitivity of IRI activity to structural modifications as simply altering a single atom or functional group on this substituent could be detrimental for activity. Finally, various IRI active small molecules were explored for their cryopreservation potential with different cell types including a human liver cell line (HepG2), HSCs obtained from human UCB, and RBCs obtained from human peripheral blood. A number of phenolic-glycosides were found to be effective cryo-additives for RBC freezing with significantly reduced glycerol concentrations (less than 15%). This is highly significant as it could drastically decrease the deglycerolization processing times that are required when RBCs are cryopreserved with 40% glycerol. Furthermore, it demonstrates the potential for IRI active small molecules as novel cryoprotectants that can improve upon current cryopreservation protocols that are limited in terms of the commonly used cryoprotectants, DMSO and glycerol.

Microorganisms have developed ways to preserve cellular functions under low temperature conditions using a variety of biochemical adaptations including the modification of ice formation. In order to conduct a limited survey of microbial ice-associating strategies, a bacterial community associated with frost-exposed leaves was assessed by the construction of a 16S rDNA library, followed by the characterization of some isolates. Fifteen different species were identified based on their 16S rDNA. Among these, Pseudomonas syringae J6 had ice nucleation activity (INA), which promoted ice formation close to 0ºC, whereas Erwinia billingiae, Flavobacterium sp. and Sphingobacterium kitahiroshimense inhibited the recrystallization of small ice crystals at temperatures close to melting. The Erwinia billingiae isolate showed adhesive and swarming behaviour, which can be associated with biofilm formation. Visualization using negative staining, transmission electron microscopy and scanning electron microscopy confirmed the presence of flagella in addition to the presence of slimy biofilm architecture in these Erwina billingiae cultures. Subsequent purification of the extracellular polymeric substance followed by mass spectrometry allowed the identification of a putative outer membrane protein A, which may be involved in the protection of this bacterium to freeze-thaw cycles. To further explore bacterial ice nucleation activity, an ice nucleation protein was cloned from Pseudomonas borealis, a bacterium originating from tundra soil, using degenerative PCR and chromosome walking. The sequence of the putative ice nucleation protein gene (inaPb) was cloned and expressed in Escherichia coli, and its identification was confirmed in the recombinant cells. Although the INPPb was more divergent than other plant-related bacterial INPs, it retained the highly conserved, repetitive core region. The protein may fold so that it has two flat faces, one for protein-protein interactions and the other for ice binding. Expression of the INPPb coding region fused to jelly fish green fluorescent protein showed a temperature-dependent polarized distribution of the recombinant protein in E. coli. In summary, results from this thesis suggests that low temperature survival may be associated with a number of ice-associating adaptations including the presence of biofilm formation in Erwina billingiae amongst other bacteria, INA in P. borealis and INA-expressing recombinant E. coli. / Thesis (Ph.D, Biology) -- Queen's University, 2010-01-27 11:47:02.385

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

Mémoire (M.Eng.)--Université du Québec à Chicoutimi, 1999. / Document électronique également accessible en format PDF. CaQCU

Fishes of the perciform suborder Notothenioidei are found in Antarctic and sub-Antarctic waters that are separated by the Antarctic Polar Front (APF), with some species being distributed on both sides of this front. In this wide latitudinal range, these fishes are exposed to different temperatures ranging from -2 °C in the High Antarctic regions to 12 °C in the sub-Antarctic regions. To survive in icy Antarctic waters, the Antarctic notothenioid species have evolved antifreeze glycoproteins (AFGPs) that prevent their body fluids from freezing. The findings of past research on the AFGP attributes of several notothenioid species inhabiting ice-free sub-Antarctic environments have presented a complex picture. Furthermore, previous taxonomic studies split widely distributed notothenioids into different species and/or subspecies, with other studies disagreeing with these splits. To understand the response of the sub-Antarctic notothenioids to warmer, ice-free environments, it is necessary to have a good understanding of their antifreeze biology and systematics. Therefore, this study aimed to determine the association, if any, between the antifreeze attributes of sub-Antarctic notothenioid fishes and their taxonomic status. And more...

Advances in molecular biology have revolutionized the field of biotechnology and allowed the development of recombinant protein production as an alternative to harvesting proteins from their natural sources. Production of target proteins in controllable host organisms offers scalable and economic approaches to meet market needs. Current host cell expression systems vary, and each has advantages and disadvantages. Photoautotrophic organisms, like microalgae, represent alternatives to fermentative microbes with the promise of recombinant protein production from sustainable inputs like carbon dioxide as a carbon source. In this thesis, a Chlamydomonas reinhardtii strain that was recently developed for phototrophic high- density cultivation and nuclear transgene expression was used to express target recombinant proteins from its plastid genome as a demonstration of possibilities for expansion of its potential value. Here, sequences of two anti-freeze proteins, the insect Choristoneura fumiferana (CfAFP) and grass Lolium perenne ice binding protein (LpIBP), and a yellow fluorescent protein (YFP, mVenus) were adapted to algal chloroplast genome expression plasmids, transformed, and protein titers characterized under various nutrient and growth regimes in alga. Rather than antibiotic selection, transformants were selected based on photosynthesis restoration in a knock-out recipient strain. LpIBP and mVenus expression were detected by Western blot and in gel fluorescence and estimated to be expressed up to ~7.65% and ~8.41% total soluble protein, respectively, whereas expression of CfAFP was not observed in any transformant. This work forms the basis of further investigation of recombinant protein expression in C. reinhardtii in high-density antibiotic-free culture and may influence feasibility assessments of scale up processes.

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