Frost assays carried out on winter barley (Hordeum vulgare cv Igri) showed that a single (10ml I) application of liquid seaweed extract (LSE) marginally increased the frost resistance of non-acclimated (NA) plants by 2.3% compared to NA controls and cold-acclimated (CA) plants by 2.1% compared to CA controls. Three applications of LSE increased the frost resistance of NA plants by 16% compared to NA controls and CA plants by 7.5% compared to CA controls. These observations were durable in a small scale field trial where LSE increased plant dry weights (control 0.55, single LSE, 0.611 and multiple LSE 0.621 log dry weight), but rain following LSE application reduced LSE mediated frost resistance. Glasshouse growth trials illustrated that LSE enhanced tiller production (control 2.8, one LSE 3.8 and three LSE 4.5 tillers) and dry weight gain, but where precipitation followed LSE application, up to 3 days post application, the LSE mediated effect was not sustained. Protein analysis demonstrated that cold-acclimation and LSE treatments increased the total soluble protein content of winter barley. A single application of LSE increased the soluble protein content of NA plants by 36.7% and three applications of LSE to NA plants increased protein concentration by 86.5%. There was not a significant increase in the soluble protein concentration of LSE treated CA plants. There was a significant increase in the number of high molecular weight proteins and the up-regulation of a 118kDa and a 57kDa protein when plants were treated with LSE. However precipitation following LSE application adversely affected LSE mediated protein expression. A tentative immunological identification of the up-regulated proteins suggested that the 118kDa protein is a dehydrin. There was a 2 fold decrease in plant water potential of NA plants treated with three applications of LSE compared to controls and a similar decrease in plant water potential was observed in cold-acclimated plants. The duration of LSE mediated decline in water potential lasted for 6 days, post LSE application. However there was no significant reduction in the percentage water content of cold-acclimated and LSE treated plants. Differential scanning calorimetry demonstrated that both cold-acclimated and LSE treated plants had significantly less frozen water in their crown tissue compared to non-acclimated controls. Further thermal analysis (infrared thermography and thermocouple data) showed that both cold-acclimation and LSE treatments reduced the speed of water removal from plant cells to the extracellular ice (NA 4.06, NA3LSE 13.4, CA 15.7 and CA3LSE 19.31 minutes). It is hypothesised that both CA and LSE treatments are modifying plant water status, so that water becomes more structured at the physico-chemical level, and thus alters the osmotic behaviour of cellular water. This higher level of water structuring reduces frost damage by conserving the cellular water environment and thus reducing protein denaturation and membrane damage.

In this thesis I present complex experimental data on the physiological and molecular adaptations during diapause development and overwintering in a linden bug, Pyrrhocoris apterus (Heteroptera, Pyrrhocoridae). I focus on adjustments of the enzymatic complement, which is involved in the biosynthesis of cryoprotectants, and heat shock proteins, which are expressed in response to temperature stress.

Cryopreservation of biological materials has many useful applications and is currently the most effective long term storage method used across a variety of fields. The success of freezing products or biological materials, however, varies because of the process' complexity and related cryo-injuries. One of the primary issues is the ice recrystallization induced development of extracellular and intracellular ice throughout the freezing and thawing process. Ice recrystallization is a significant contributor to freezing damage, ultimately reducing post-thaw viability and function. To address this issue, the Ben laboratory has developed and synthesized a variety of classes of small molecule carbohydrate-based ice recrystallization inhibitors (IRIs). These compounds act as supplements or alternatives to current cryoprotectants, such as trehalose, DMSO, or glycerol, which do not address ice recrystallization and can be cytotoxic. This thesis focuses on the comprehensive chemical property assessment of N-Aryl-β-D-aldonamides and N-Benzyl-β-D-gluconamides, as well as optimization of biopreservation protocols for tissue products and freeze-dried proteins. Utilizing a 5-minute modified splat cooling assay, dose-response curves of five N-Benzyl-β-D-gluconamides were generated. All compounds produced ice recrystallization inhibition active IC50 values comparable to previously investigated active compounds such as, N-Octyl-β-D-gluconamide and N-4-Bromophenyl-β-D-glucopyranoside. Furthermore, validation that the dose-response curves follow a 4-parameter logistic (4PL) or 5PL sigmodal trend depending on symmetry was obtained. In addition, all tested compounds had lower cytotoxicity than N-4-Bromophenyl-β-D-glucopyranoside and higher solubility than N-Octyl-β-D-gluconamide. Overall, N-Benzyl-β-D-gluconamides proved to be a promising class of compounds with the para derivatives being the most IRI active. The second part of this work involved the examination of IRIs' ability to cryopreserve two different biological materials using different biopreservation protocols. The first being proteins and master mix (enzymes and oligonucleotides) during RT-qPCR after the freeze-drying process. The data showed that the IRIs did not interfere and were effective during both the lyophilization and qPCR processes. When compared to most effective concentration of the current industry standard, N-4-Bromophenyl-β-D-glucopyranoside increased the protein activity by ~30%, reducing the number of cycles to reach threshold value. The most significant contribution of this work was the discovery that carbohydrate-based small molecules may be working in more than one mechanism, as both cryoprotectants and lyoprotectants. In addition to proteins, the ability of IRIs to cryopreserve tissue products was investigated. Cell media supplemented with IRIs indicated that they can increase viability and reduce mortality in both cell suspension and single dermal sheets. With N-4-Methylbenzyl-β-D-gluconamide and N-Octyl-β-D-gluconamide being the most effective at reducing the damage associated with freezing and increasing recovery of the cells within the system of a simple one cell type thin tissue matrix.

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultant: Doc. PharmDr. Zdeňka Šklubalová, Ph.D. Student: Eliška Macáková Title of Thesis: Formulation of freeze dried tablets for oromucosal administration of peptides Freeze-drying is one out of the important methods for stabilization of active substances, particularly peptides, in pharmacy. The formulation of freeze-dried buccal/sublingual tablets for administration of peptides into the oral cavity is the main target of this thesis. The aim is to propose the combination of appropriate excipients and their concentration to achieve the suitable organoleptic properties and disintegration time of the product cake. The measurement of pH, osmolality, the thermal properties of substances, as well as the evaluation of mechanical quality of tablets and their disintegration were used. In conclusion, the combination of excipients for the composition of matrix for the futher development.

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Mentor: Doc. PharmDr. Zdeňka Šklubalová, Ph.D. Student: Ngoc Lien Vuová Title of Thesis: Formulation of freeze dried tablets for buccal application of vaccines Mucosal vaccines represent an attractive way of vaccination with an advantage of inducing both systemic and local immunity. The aim of this diploma thesis was designing a composition of freeze-dried tablets for buccal administration of a model Bordetella pertusis vaccine. The easy removal from blister, firmness and aesthetic appearance as well as the appropriate taste and mucoadhesivity were the required product quality parameters. The excipients were characterised by differential scanning calorimetry, osmolality and pH measurement; the mechanical properties and disintegration time of freeze-dried tablets were evaluated. Among the variety of studied excipients and their combinations, dextran 40 as the main component of the preparation provided the best results. For the further modification of properties, the addition of fish gelatin, iota carrageenan and macrogol 300 is suitable. Freeze-dried tablets containing trehalose, mannitol and povidon 25 did not achieve the desirable quality parameters. Freeze- dried tablets containing iota carrageenan...

The first project in this work used classical molecular dynamics to study the ice recrystallization inhibition potential of a series of carbohydrates and alcochols, using the hydration index, partial molar volumes and isothermal compressibilities as parameters for measuring their cryogenic efficacy. Unfortunately, after 8 months of testing, this work demonstrates that the accuracy and precision of the density extracted from simulations is not sufficient in providing accurate partial molar volumes. As a result, this work clearly demonstrates that current classical molecular dynamics technology cannot probe the volumetric properties of interest with sufficient accuracy to aid in the research and development of novel cryoprotectants.The second project in this work used molecular dynamics simulations to evaluate the Gibbs free energy change of substituting CO2 in sI clathrate hydrates by N2,CH4, SO2 and H2S flue gas impurities under conditions proposed for CO2 capture (273 K, 10 bar). Our results demonstrate that CO2 substitutions by N2 in the small sI cages were thermodynamically favored. This substitution is problematic in terms of efficient CO2 capture, since the small cages make up 25% of the sI clathrate cages, therefore a significant amount of energy could be spent on removing N2 from the flue gas rather than CO2. The thermodynamics of CO2 substitution by CH4, SO2 and H2S in sI clathrate hydrates was also examined. The substitution of CO2 by these gases in both the small and large cages were determined to be favorable. This suggests that these gases may also disrupt the CO2 capture by sI clathrate hydrates if they are present in large concentrations in the combustion flue stream. Similar substitution thermodynamics at 200 K and 10 bar were also studied. With one exception, we found that the substitution free energies do not significantly change and do not alter the sign of thermodynamics. Thus, using a lower capture temperature does not significantly change the substitution free energies and their implications for CO2 capture by sI clathrate hydrates.

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.

Foi estudado a influência das estações não reprodutiva e reprodutiva, dos crioprotetores glicerol e etilenoglicol e do processo de congelação e descongelação sobre a motilidade, o vigor, a morfologia e o complexo DNA-proteína de espermatozóides em garanhões, comparando o sêmen fresco, o exposto a congelação e descongelação sem crioprotetores, o exposto aos crioprotetores sem congelação e o exposto aos crioprotetores com congelação e descongelação. Foram utilizados seis garanhões da raça Mangalarga Paulista, colhendo 12 ejaculados de cada animal na estação não reprodutiva (1) e 12 ejaculados na estação reprodutiva (2), analisando a motilidade, o vigor, a concentração, a morfologia e a patologia do complexo DNA-Proteína. A patologia do complexo DNA-Proteína foi avaliada em sêmen fixado com etanol-ácido-acético glacial 3:1 (v/v), tratado com HCL 4N a 25oC e corado com azul de toluidina a 0,025% em tampão Mcllvaine, empregando microscopia óptica com aumento de 1000x. Os resultados mostraram que a motilidade, o vigor, a morfologia e a anomalia do complexo DNA-Proteína dos espermatozóides diferem entre os grupos congelado e não congelado (P<0,05). Para o grupo fresco, a taxa de patologia do complexo DNA-Proteína apresentou diferença significativa (P<0,05) entre as estações 1 e 2. O processo de congelação e descongelação exerce influência negativa sobre o complexo DNA-Proteína de espermatozóides em garanhões. / To study the effects of freezing and thawing on DNA-protein complexes in spermatozoa, 12 ejaculate were collected from each of 6 stallions (Mangalarga Paulista breed) in each of two consecutive breeding seasons. Motility, vigor, concentration, morphology and incidence of the DNA-protein complex pathologies of spermatozoa were evaluated and compared among fresh semen, semen expose to freezing and thawing without cryoprotectants and semen exposed to the cryoprotectors glycerol and ethylene glycol left at room temperature or frozen. Incidence of the DNA-protein complex pathologies was evaluated in semen smear fixed in na ethanol-acetic-acid mixture (3:1, v/v) for 1 minute, washed in 70% ethanol for 3 minutes and air dried. To induce nuclear metachromasy, smears were treated with 4N HCL at 25ot for 20 minutes followed by rinsing in distilled water. Preparation were stained with a 0.025% toluidine blue solution in Mailvaine buffer for 15 minutes. Frequency of spermatozoa exhibiting nuclear metachromasy was determined in 1000 cells/animal by light microscopy at 1000X magnification. Motility, vigor, morphology and DNA-protein complex pathologies of spermatozoa were different for semen frozen compared to not frozen (P<0.05). For fresh semen, there were effects of seasons on the incidence of DNA-protein complex pathologies (P<0.05) For semen frozen without cryoprotectants there were significant effects seasons (P<0.05) The process of cryopreservation and thawing influences negatively the DNA-protein complexes in stallion spermatozoa.

Foi estudado a influência das estações não reprodutiva e reprodutiva, dos crioprotetores glicerol e etilenoglicol e do processo de congelação e descongelação sobre a motilidade, o vigor, a morfologia e o complexo DNA-proteína de espermatozóides em garanhões, comparando o sêmen fresco, o exposto a congelação e descongelação sem crioprotetores, o exposto aos crioprotetores sem congelação e o exposto aos crioprotetores com congelação e descongelação. Foram utilizados seis garanhões da raça Mangalarga Paulista, colhendo 12 ejaculados de cada animal na estação não reprodutiva (1) e 12 ejaculados na estação reprodutiva (2), analisando a motilidade, o vigor, a concentração, a morfologia e a patologia do complexo DNA-Proteína. A patologia do complexo DNA-Proteína foi avaliada em sêmen fixado com etanol-ácido-acético glacial 3:1 (v/v), tratado com HCL 4N a 25oC e corado com azul de toluidina a 0,025% em tampão Mcllvaine, empregando microscopia óptica com aumento de 1000x. Os resultados mostraram que a motilidade, o vigor, a morfologia e a anomalia do complexo DNA-Proteína dos espermatozóides diferem entre os grupos congelado e não congelado (P<0,05). Para o grupo fresco, a taxa de patologia do complexo DNA-Proteína apresentou diferença significativa (P<0,05) entre as estações 1 e 2. O processo de congelação e descongelação exerce influência negativa sobre o complexo DNA-Proteína de espermatozóides em garanhões. / To study the effects of freezing and thawing on DNA-protein complexes in spermatozoa, 12 ejaculate were collected from each of 6 stallions (Mangalarga Paulista breed) in each of two consecutive breeding seasons. Motility, vigor, concentration, morphology and incidence of the DNA-protein complex pathologies of spermatozoa were evaluated and compared among fresh semen, semen expose to freezing and thawing without cryoprotectants and semen exposed to the cryoprotectors glycerol and ethylene glycol left at room temperature or frozen. Incidence of the DNA-protein complex pathologies was evaluated in semen smear fixed in na ethanol-acetic-acid mixture (3:1, v/v) for 1 minute, washed in 70% ethanol for 3 minutes and air dried. To induce nuclear metachromasy, smears were treated with 4N HCL at 25ot for 20 minutes followed by rinsing in distilled water. Preparation were stained with a 0.025% toluidine blue solution in Mailvaine buffer for 15 minutes. Frequency of spermatozoa exhibiting nuclear metachromasy was determined in 1000 cells/animal by light microscopy at 1000X magnification. Motility, vigor, morphology and DNA-protein complex pathologies of spermatozoa were different for semen frozen compared to not frozen (P<0.05). For fresh semen, there were effects of seasons on the incidence of DNA-protein complex pathologies (P<0.05) For semen frozen without cryoprotectants there were significant effects seasons (P<0.05) The process of cryopreservation and thawing influences negatively the DNA-protein complexes in stallion spermatozoa.

The first project in this work used classical molecular dynamics to study the ice recrystallization inhibition potential of a series of carbohydrates and alcochols, using the hydration index, partial molar volumes and isothermal compressibilities as parameters for measuring their cryogenic efficacy. Unfortunately, after 8 months of testing, this work demonstrates that the accuracy and precision of the density extracted from simulations is not sufficient in providing accurate partial molar volumes. As a result, this work clearly demonstrates that current classical molecular dynamics technology cannot probe the volumetric properties of interest with sufficient accuracy to aid in the research and development of novel cryoprotectants.The second project in this work used molecular dynamics simulations to evaluate the Gibbs free energy change of substituting CO2 in sI clathrate hydrates by N2,CH4, SO2 and H2S flue gas impurities under conditions proposed for CO2 capture (273 K, 10 bar). Our results demonstrate that CO2 substitutions by N2 in the small sI cages were thermodynamically favored. This substitution is problematic in terms of efficient CO2 capture, since the small cages make up 25% of the sI clathrate cages, therefore a significant amount of energy could be spent on removing N2 from the flue gas rather than CO2. The thermodynamics of CO2 substitution by CH4, SO2 and H2S in sI clathrate hydrates was also examined. The substitution of CO2 by these gases in both the small and large cages were determined to be favorable. This suggests that these gases may also disrupt the CO2 capture by sI clathrate hydrates if they are present in large concentrations in the combustion flue stream. Similar substitution thermodynamics at 200 K and 10 bar were also studied. With one exception, we found that the substitution free energies do not significantly change and do not alter the sign of thermodynamics. Thus, using a lower capture temperature does not significantly change the substitution free energies and their implications for CO2 capture by sI clathrate hydrates.