One of the largest problems in current medicine is the shortage of organs for transplant due to technological limitations in the storage of organs for any length of time. A possible solution to this problem would involve cryopreservation. However, current cryopreservatives such as sucrose or DMSO have concerning cytotoxic issues that limit their possible applications. A major cause of cryoinjury is the uncontrolled recrystallization of inter and intra-cellular ice crystals that occurs during the thawing process leading to mechanical damage and dehydration. The Ben lab has thus been interested in the design of compounds that are capable of inhibiting this process but do not possess other undesirable properties found in the native compounds. These synthetic analogues have been shown to increase cellular viability post-thaw. A series of mixed α/β glycopeptides are prepared and analyzed for antifreeze properties. The results of this study imply that it is not the gross conformation of the glycopeptide that is responsible for activity, but rather that intramolecular relationships may be responsible for disrupting the reorganization of ice. A technique was devised for the incorporation of triazoles into the analogues to investigate the importance of the linker and to greatly simplify the synthesis of a library of glycoconjugates. It was found that the IRI activity of glycopeptides is very sensitive to the distance between carbohydrate and peptide backbone. The electron density at the anomeric oxygen is an important parameter with respect to intramolecular networks. A series of substituted galactosides is presented that modify the electronics of the anomeric oxygen. The results demonstrate that decreasing electron density at this position appears to improve IRI activity in a predictable manner. To better understand the remarkable IRI activity of a key analogue, it was systematically truncated. This study led to the serendipitous discovery of a series of very highly IRI active analogues that do not contain a peptide backbone. These compounds represent the first non-glycopeptides that can show very significant IRI activity even at very low concentrations. The final portion of the thesis reports the efforts towards the preparation of a carbasugar analogue of AFGP-8.

Cryopreservation of human red blood cells (RBCs) extends the storage time from 42 days (hypothermic storage limit) to a maximum of 10 years. While this reduces the possibility of RBC shortages in emergency situations, this preservation method is currently limited to individuals with rare blood phenotypes, patients who require autologous blood transfusions, and military applications. Furthermore, cryopreservation is associated with a high degree of cellular damage, which can subsequently reduce the viability of cells post-thaw. The cellular damage incurred upon cryopreservation is primarily attributed to the process of ice recrystallization. To reduce the degree of cellular damage, cryoprotective agents (CPAs) are used. Currently, 10 % dimethyl sulphoxide (DMSO) and 40 % glycerol are used for the cryopreservation of hematopoietic stem cells (HSCs) and human RBCs respectively. Unfortunately, these CPAs do not provide protection against ice recrystallization. The biological antifreezes (BAs) consisting of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) were identified as the first inhibitors of ice recrystallization. Consequently, the Ben laboratory is interested in synthesizing small molecule carbohydrate-based inhibitors of ice recrystallization that can be used as an alternative to glycerol or DMSO for the cryopreservation of various cell types. Therefore, this thesis focuses on elucidating important structural features of carbohydrate-based derivatives that are responsible for IRI activity. The first part of this study examines the importance of the anomeric oxygen atom of aryl glycosides for IRI activity. Our laboratory previously demonstrated that the O-linked aryl glycosides are effective inhibitors of ice recrystallization. However, the influence of stereoelectronic effects at the C1 position of aryl glycosides on IRI activity has not been investigated. As a result, N- and S-linked aryl glycosides were synthesized in this study and their IRI activities were compared to that of the O-linked aryl glycosides. These results suggest that a stronger exo-anomeric effect exhibited by the C1 nitrogen derivatives reduces the IRI activity of aryl glycosides. The second part of this study focuses on the synthesis of AFGP disaccharide analogs. While the β-(1,3) glycosidic linkage found in native AFGP-8 was previously assessed for its influence on IRI activity, an extensive structure-function analysis of AFGP disaccharide analogs has not yet been performed. As a result, an AFGP disaccharide analog was designed whereby a para-methoxyphenyl (PMP) substituent was incorporated. This was done to assess whether the PMP substituent could enhance the lack of IRI activity exhibited previously with AFGP disaccharide analogs. Although the synthesis of this disaccharide target was not completed, a number of advantageous developments have been made regarding the glycosylation of N-acetyl-D-glycosamine derivatives. In addition, the PMP-GlcNAc intermediate encountered in disaccharide synthesis was assessed for its IRI activity, confirming that the acetamido (NHAc) function is not required for IRI activity.

The cryopreservation of cells and tissues requires the addition of a cryoprotectant in order to prevent cellular damage caused by ice. Unfortunately, common cryoprotectants such as DMSO and glycerol exhibit significant toxicity which makes their use unfeasible for many clinical procedures. Our laboratory is interested in the development of alternative, non-toxic cryoprotectants which possess ice recrystallization inhibition (IRI) activity. Potent IRI activity has recently been discovered in certain small molecules, but the structural features required for this process are unclear. Herein we report the development of a library of O-aryl glycosides in order to probe the importance of electron density and hydrophobic moieties for IRI activity. It was found that the degree of electron density at the anomeric oxygen does not correlate with IRI ability in para-substituted aryl glycosides, nor does changing the position of the aryl substituent impart a predictable effect on activity. However, the addition of hydrophobic alkyl or acyl chains was beneficial for IRI activity; generally, increasing chain length was found to correlate with increasing activity. In some instances, an optimal alkyl chain length was identified, after which continued lengthening results in a loss of potency. We conclude from this study that a certain extent of hydrophobic character is beneficial for the IRI activity of aryl glycosides, and that a balance between hydrophobicity and hydrophilicity is required for optimum IRI ability. It is hoped that these findings will aid future efforts towards the rational design of novel cryoprotectants.

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.

Orientador: Eugênio José Zoqui / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-24T12:59:54Z (GMT). No. of bitstreams: 1 Campo_KaioNiitsu_M.pdf: 7361224 bytes, checksum: c6570a6666ed0e98563f7345bc5c6734 (MD5) Previous issue date: 2014 / Resumo: Este trabalho investigou o uso da extrusão em canal angular (ECA) na produção de matéria-prima para tixoconformação visando à obtenção de uma rota simples e eficiente na formação de microestruturas globulares. Para tanto, uma liga de alumínio A356 foi submetida a um único passe ECA e, em seguida, reaquecida ao estado semissólido a 580°C. Dessa forma, pôde-se determinar a evolução morfológica e os mecanismos envolvidos na formação da pasta metálica, a influência do tratamento isotérmico na evolução microestrutural no estado semissólido, o comportamento de deformação durante a compressão a quente e o comportamento mecânico em tração do material tixoconformado. Os resultados indicaram que apenas um passe ECA foi suficiente para gerar uma pasta metálica refinada e globular apenas com o reaquecimento da matéria-prima. O engrossamento da microestrutura durante o tratamento isotérmico foi controlado principalmente pelo mecanismo de Ostwald ripening, fato comprovado pelo baixo valor da constante taxa de engrossamento K, o que mostrou a estabilidade dessa pasta no estado semissólido. As amostras exibiram comportamento tixotrópico típico com baixos valores de viscosidade aparente, na faixa de 10^2 a 10^3 Pa.s para as taxas de cisalhamento testadas. Por fim, as amostras tixoconformadas apresentaram valores de ductilidade e resistência à tração superiores aos das amostras fundidas e deformadas por um passe ECA / Abstract: This work investigated the use of equal channel angular pressing (ECAP) for the production of raw materials for thixoforming in order to obtain a simple and efficient processing route to generate globular microstructures. Therefore, an A356 aluminum alloy was processed by one ECAP pass, and then reheated to the semisolid state up to 580°C. Thus, it was possible to determine the morphological evolution and mechanisms involved in the formation of the semisolid slurry, the influence of isothermal treatment on microstructural evolution in the semisolid state, the deformation behavior during hot compression tests and the tensile properties of the thixoformed material. The results indicated that a single ECAP pass was sufficient to promote a refined and globular semisolid slurry. Microstructure coarsening during the isothermal treatment was mainly controlled by Ostwald ripening, which was verified by the low rate constant K, showing the stability of the reheated material in the semisolid state. The samples exhibited typical thixotropic behavior with low apparent viscosity ranging from 10^2 to 10^3 Pa.s over the applied shear rates. Finally, the thixoformed samples exhibited values of ductility and tensile strength superior to the as-cast and ECAPed samples / Mestrado / Materiais e Processos de Fabricação / Mestre em Engenharia Mecânica

Ce mémoire présente une étude sur la caractérisation des propriétés mécaniques dépendantes du temps par nanoindentation instrumentée à différentes températures.Ce sujet de recherche porte sur l’adaptation des méthodes d’indentation classiques aux problématiques de la caractérisation à hautes températures. Les méthodes développées dans ces travaux ont pour premier objectif d’apporter une meilleure compréhension des phénomènes dépendants du temps et de la température par une approche locale. Le second objectif est d’apporter des éléments de comparaison entre le comportement à l’échelle microscopique et macroscopique.Les méthodes proposées sont principalement fondées sur l’essai de relaxation en indentation. Ses développements et applications aux matériaux polymères et métalliques sont étudiés de manière analytique puis expérimentale. L’étude analytique nous montre que ce type d’essais en indentation peut être directement comparé à un essai uniaxial. Elle montre également que la cinétique de chargement a une grande influence sur la qualité des résultats obtenus en relaxation.L’étude expérimentale proposée, en température, permet d’extraire la sensibilité à la vitesse de déformation ainsi que l’énergie d’activation des phénomènes visqueux. Cependant, la dérive thermique limite la durée des essais – la durée maximum du segment de relaxation reste inférieure à quelques minutes. Une autre étude réalisée à température ambiante ouvre la voie à des durées de caractérisation plus longues. Celle-ci se fonde sur l’équivalence entre aire et raideur de contact pour un matériau homogène. Un maintien de l’aire de contact constante pendant 10 heures est effectué sans signes de dérive.Finalement, l’application de la nanoindentation à hautes températures à la caractérisation in situ des changements microstructuraux pour un alliage d’aluminium est étudiée. Les résultats de l’étude montrent qu’il est possible d’obtenir la cinétique de recristallisation avec un seul échantillon et en un temps limité. / This manuscript presents a study on the mechanical properties’ characterization of time dependent phenomena using instrumented nanoindentation at different temperatures.This research subject treats the development of methods dealing with the adaptation of classical indentation methodologies to high temperature characterizations. Bringing a better understanding of time and temperature dependent phenomena at a local scale is the first aim of the methods developed. The second objective is to compare materials behaviors measured at micro and macro-scale.The proposed methods are based on indentation relaxation tests. Their development and applications to polymers and metals characterization are studied analytically and experimentally. The analytical study shows that the indentation relaxation test is equivalent to the uniaxial one. This study also highlights the great influence of loading kinetics on the measured relaxation behavior.The proposed experimental study in temperature permits the extraction of the strain rate sensitivity and the activation energy of the viscous phenomena. However, thermal drift limits the characterization duration – i.e. the maximum experimental time remains limited to a couple of minutes. Another experimental study configuration, at room temperature, opens the way to longer test durations. It is based on the equivalence of contact area and stiffness for a homogeneous material. With this configuration, we successfully hold the contact area constant for 10 hours without any evidences of drift.Eventually, the high temperature nanoindentation application to in situ microstructural changes characterization of an aluminum alloy is studied. Measurements and limitations are carefully discussed for a better understanding of the studied phenomenon. The results show that the recrystallization kinetics can be successfully described with reduced test duration and samples’ set.

This thesis deals with influences of graded deformations, temperatures and holding times on the grains and subgrains size in steel 08Cr18Ni10T. In literary part of the thesis there are described static and dynamic annealing treatments, then also austenitic stainless steels and some selected types of their processing. In experimental part there is studied grain size on differently deformed samples annealed in temperature range 1050–1200 °C for 10, 50 and 100 minutes. Analysis of present structures units was perform by EBSD. Except of that, there were realized chemical analysis of selected inclusions and also hardness test were realised. Achieved measurement shown, that grain size of chosen steel is depended on all 3 variable values.

Analysis of quartz c-axis fabrics and microstructures from ductily deformed rocks allows for the examination of the kinematics associated with crustal deformation. This thesis expands on the current knowledge of the kinematic evolution of the Himalayas and Scottish Caledonides, by examining samples from the Main Central Thrust (MCT) (Himalayas) and the Sgurr Beag Thrust (SBT) (Scottish Caledonides).  Metamorphic temperatures (Tm) associated above the MCT are inverted; chapter one attempts to test if deformation temperatures (Td) correlate to Tm, indicating that ductile shearing occurred during peak Tm. In the Scottish Caledonides, Td and Tm increase from foreland to hinterland, potentially indicating a right way up thermal structure;  chapter two presents Td and Tm associated with the region around the SBT. Above the MCT, quartz c-axis fabrics yield Td ranging between 500-650 "C, corresponding to the temperatures of dynamic recrystallization for subgrain rotation (SGR) and grain boundary migration (GBM). Up to 1000m above the MCT, Td and Tm are within error of each other, suggesting that shearing occurred during peak Tm; while further away from the MCT  Tm is significantly hotter than Td, suggesting that shearing continued past Tm. Deformation associated with the upper part of the Moine thrust sheet and the SBT yields quartz c-axis fabrics with Td ranging between 395-583 "C, corresponding to the regional dynamic recrystallization. Tm calculations original to this study yield pressure-temperature constraints of 4.8-5.8 kbar and 586-625 "C. Tm is within error of Td, suggesting that deformation and metamorphism were synchronous. / Master of Science

The last seven hot rolling passes of the ferritic stainless steels (FSS) AISI 430 and AISI 433 (the latter an Al-added variant of 430) were simulated on Gleeble-1500D® and Gleeble-3800TM® thermo-mechanical simulators to investigate the effect of temperature, strain rate and inter-pass time on the development of texture in these steel grades and its subsequent influence on ridging. The compression tests were carried out over a wide range of strain rates (0.1 s-1 to 5 s-1, 25 s-1 and 50 s-1) and temperatures (1100 to 820 oC) with different inter-pass times (2 s, 10 s, 20 s and 30 s). The transition temperature from dynamic recrystallization (which may introduce a texture change) to dynamic recovery (in which no texture changes are expected) was determined by examining the relationship between the mean flow stress and the deformation temperature in multi-pass tests. Both macrotexture (XRD) and microtexture (EBSD) analyses were employed to characterise and study the texture present in these steels. It was found that the texture in the central layer of the compressed sample is a strong recrystallization-type. The through-thickness textural and microstructural banding was found to be responsible for ridging in these grades of stainless steels. Dynamic recrystallization which promotes the formation of the desired ã-fibre texture leading to high ductility, formability and eventually reduction or elimination of ridging, was found to occur in both AISI 430 and AISI 433 at high temperatures, low strain rates and longer inter-pass times with multi-pass testing. Generally AISI 433 has a stronger gamma texture developed than the AISI 430 when hot rolled under similar conditions, which leads to improved ductility and less ridging in AISI 433 than AISI 430. / Dissertation (MSc)--University of Pretoria, 2012. / Materials Science and Metallurgical Engineering / unrestricted

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