Cryopreservation is a promising approach to long-term biopreservation of living cells, tissues and organs. The use of cryoprotective agents (CPAs) in combination with extremely low temperatures is mandatory for optimum biopreservation. CPAs (e.g., glycerol, trehalose, dimethyl sulphoxide (DMSO)), however, are relatively cytotoxic and compromise biopreserved cell quality. This is usually resultant in oxidative damage, diminishing cell functionality and survival rate. The growing market of cell therapy medicinal products (CTMPs) demands effective cryopreservation with greater safety, of which the currently available CPAs are unable to provide. The present study was aimed at developing cryomedia formulation to enhance the cryopreservation of nucleated and anucleated mammalian cells. Here, eleven compounds of a polyol nature were selected and examined for their cryoprotective properties. These compounds are derived from plants and honey, thereby ensuring their safety for human consumption. The selection was based on their molecular structure and chemical properties. Here, the presented study is divided into three main phases: 1) Screening the compounds panel for cryo-additive effects on cells during and post-cryopreservation and optimising the dose response and time course for trehalose and glycerol with and without the novel compounds; 2) Assessing the influence of biophysical criteria on biospecimen cryopreservation (e.g., biosampling procedure, cell age, donor age); 3) Establishing the mechanisms of action underpinning the modulatory effect of novel CPAs on biological pathways during cryopreservation. For the stated purposes, red blood cells (RBCs) obtained from sheep and humans were used to screen the compounds for novel cryo-additive agents. Cryosurvival rate was employed as an indication of the compounds' cryoprotective performance. Cellular biochemical profiles, including lipid and protein oxidative damage as well as key redox enzymatic activities (e.g., lactate dehydrogenase (LDH), glutathione reductase (GR)) were measured. The study revealed that nigerose (Nig) and salidroside (Sal) were significantly effective in protecting cells during the freeze-thaw cycle and recovery phases. Both compounds promoted the activity of GR and reduced oxidative stress mirrored by diminished LDH activity. This was also reflected in the protein and lipid oxidation levels, which was limited to a comparable level with the cells' prior freezing. Further studies on human leukaemia (HL-60) were carried out to elucidate the molecular and biological pathways associated with cryodamage and the modulatory effects of adding novel CPAs. The proteome profile and the corresponding biological functions were evaluated and iii showed that Nig and Sal protected cells against cryodamage. The additive compounds (Nig and Sal) demonstrated a unique and overlapping modulation effect pattern. Nig was found to highly influence proteins engaged with metabolic and energetic pathways, whereas Sal greatly affected nuclear and DNA-binding proteins. The current study concluded that novel CPAs have high potency in protecting cells and each compound has a unique effect on the cellular proteome. These features can be applied to designing cryomedia formulae with higher protective efficiency for targeted applications in cell based therapy and biopharmaceutical industries.

O pão produzido de massa congelada tem sido aprimorado pelos avanços tecnológicos e diferentes formulações, porém ainda apresenta problemas como fermentação prolongada, baixo volume, textura e desempenho variados. O efeito do congelamento pode ser minimizado através do uso de aditivos e ingredientes adequados para a elaboração da massa. Entretanto o mecanismo de fortalecimento devido aos aditivos como, por exemplo, o emulsificante não é completamente conhecido. A análise térmica é uma ferramenta útil para pesquisa, desenvolvimento e controle de qualidade de alimentos, pois permite o estudo das alterações na sua estrutura durante um tratamento térmico. O objetivo do trabalho foi estudar a influência do uso de aditivos de panificação sobre o comportamento da massa de pão francês após ciclos de congelamento e descongelamento. O trabalho experimental foi dividido em quatro etapas: na primeira, diferentes formulações foram produzidas com os componentes estearoil-2-lactil lactato de cálcio (CSL), polisorbato 80 (PS80) e gordura vegetal hidrogenada (GVH); na segunda etapa foi realizado um projeto de mistura com três componentes (CSL, PS80 e ésteres de ácido diacetil tartárico de mono e diglicerídio - DATEM); na terceira foi estudada a influência do ácido ascórbico sobre as características reológicas e os eventos determinados através de Calorimetria Exploratória Diferencial (DSC) afim de otimizar o uso destes aditivos em massas congeladas. A análise térmica mostrou-se adequada aos eventos de congelamento e descongelamento, porém não em relação aos eventos relativos aos aquecimentos. A resistência à extensão bem como a extensibilidade da massa foram influenciadas pelo tempo de armazenamento congelado. Na quarta etapa do projeto, foram produzidos pães franceses à partir de massas com a mistura (0,3 % de PS80 e 0,2 % de DATEM sobre a farinha de trigo) e dois níveis de ácido ascórbico (0 e 200 ppm). Volume específico, perfil de textura, conteúdo de água e medida da produção de gás e tolerância da massa durante a fermentação foram determinados. Os pães produzidos com a adição de ácido ascórbico apresentaram maiores volumes específicos e menores valores para os parâmetros de textura dos pães (firmeza, elasticidade, coesividade e mastigabilidade). / Bread produced from frozen dough has been improved due to technological advances and formulation, however it still presents problems like long proofing, low specific volume, varied texture and performance. The effect of freezing can be minimized by use of additives and adequate ingredients. The mechanism of dough strengthening due to use of additives, such as emulsifiers, is not fully understood. Thermal analysis is a useful tool for food research, development and quality control, because it provides information on food structural changes during thermal treatment. The goal of this research is to study the influence of additives in bread making on French bread dough after a freeze-thaw cycles. The experimental part consists of four parts: in the first, different formulations were produced with the components calcium stearoyl-2-lactylate (CSL), polysorbate 80 (PS80) and vegetable shortening (VGH); in the second, a mixture design with three components (CSL, PS80 and diacetyl tartaric esters of mono and digliceride – DATEM) was made; in the third, influence of ascorbic acid was studied on rheological measurement and events determined by differential scanning calorimetry (DSC) to optimize the use of additives on frozen dough. Thermal analysis was adequate for the freeze-thaw events, however not for the heating events. Resistance to extension and dough extensibility were influenced by storage time. In the fourth part, French breads from frozen dough were produced with the mixture (0,3 % of PS80 and 0,2 % of DATEM in flour basis) and two levels of ascorbic acid. Specific volume, texture profile, water content, gas production and mass tolerance during proofing were determined. Breads made with ascorbic acid addition presented a higher specific volume and lower values for texture profile (firmness, springiness, cohesiveness and chewiness).

Este estudo investiga o Módulo de Young (Módulo de Elasticidade) em amostras de osso cortical oriundas de uma mesma região da tíbia bovina depois de submetidas a congelamentos e descongelamentos sucessivos. As amostras foram coletadas da face caudal do terço médio da diáfise de tíbias bovinas e depois submetidas a testes de flexão em três pontos e ensaios de ruptura, obedecendo à norma ASTMD790M86ε1. Nesse experimento, 20 pares de amostras de ossos corticais foram retirados de 20 tíbias bovinas, cada amostra medindo 2 mm x 4 mm x 40 mm, aproximadamente. Todos os testes foram realizados à temperatura controlada (21 graus Celsius), e as condições de ensaio foram as mesmas durante os testes. As amostras foram identificadas e divididas em dois grupos experimentais: Grupo A: as amostras foram conservadas (em sacos plásticos) a - 20 graus Celsius (submetidos a congelamentos e descongelamentos sucessivos por 50 vezes: uma vez ao dia durante 50 dias). Esses descongelamentos produziram 15 ensaios de flexão, sendo que o primeiro foi realizado com as amostras ainda frescas mantidas em geladeira a 4 graus Celsius. Após os 50 descongelamentos foram levados até a ruptura. Grupo B: as amostras foram conservadas frescas (em sacos plásticos) a 4 graus Celsius em geladeira aguardando o tempo para que os ensaios do grupo A fossem concluídos e, posteriormente fez-se o teste de ruptura. Importantes cuidados foram tomados. Depois de cada descongelamento, as amostras foram analisadas e comparadas com as amostras frescas tanto para os ensaios de flexão em três pontos como para os ensaios de ruptura. Observamos que o congelamento não alterou de modo significativo as propriedades mecânicas destes ossos, pois não foi encontrada nenhuma diferença significativa entre o comportamento dos dois grupos testados. Isso implica que, congelando e estocando mesmo por longos períodos, as propriedades mecânicas não se alteram tanto nos ensaios de flexão em três pontos como nos ensaios de ruptura. Com respeito às diferenças observadas no dia a dia, as alterações do primeiro dia até o último dia não foram significativas / The present study was conducted to investigate the Young Module (Elasticity Module) in cortical bone samples submitted to successive freezing and thawing and then assayed in flexion tests at three points and in rupture tests according to the norms of ASTMD790M86ε1. Twenty pairs of cortical bone samples, each measuring approximately 2 x 4 x 40 mm, were collected from the same region of the caudal surface of the middle third of the diaphysis of 20 bovine tibiae and divided into two experimental groups: Group A: consisting of 20 cortical bone samples stored at – 20 Celsius degrees in plastic bags and submitted to successive freezing and thawing 50 times once a day for 50 days. These thawing episodes produced 15 flexion assays. After the 50 thawing episodes, the samples were tested until rupture. Group B: consisting of 20 cortical bone samples stored at 4 Celsius degrees in plastic bags in a refrigerator until the time when the assays of group A were concluded and then submitted to the rupture test. No statistically significant variation in the Young Module was observed after the flexion test at three points performed for Group A. The values of rupture tension also did not differ significantly between groups A and B. We observed that freezing did not cause a significant change in the mechanical properties of these bones, indicating that freezing and storage of these bones even for long periods of time does not alter their mechanical properties in flexion tests at three points or in rupture assays

A criopreservação de sêmen é de fundamental importância para a expansão das técnicas reprodutivas como a inseminação artificial, a transferência de embriões e a fecundação in vitro, entretanto é responsável pelo estresse oxidativo da célula espermática. Para incrementar a sobrevivência celular após a congelação, a membrana plasmática deve ter adequada fluidez que é garantida pelos ácidos graxos poliinsaturados e suporte de defesa antioxidante para proteger contra a peroxidação lipídica. No intuito de tornar os espermatozoides mais resistentes à criopreservação, 48 touros da raça Nelore foram confinados (três animais por baia) e designados a quatro tratamentos de acordo com a adição de gordura protegida da degradação ruminal e/ou antioxidantes à dieta. Aos primeiros 30 dias, todos os touros receberam a mesma dieta de adaptação (bagaço de cana, polpa cítrica, farelo de glúten de milho, ureia e sal mineral). Posteriormente, durante 75 dias, a mesma dieta foi oferecida, diferindo na adição de: G) Gordura protegida da degradação ruminal (rica em ácido linoleico, Megalac-E®, 1,5% na MS; n = 12); A) antioxidante (fonte de vitamina C e selênio, EconomasE®, 3g/cabeça/dia; n = 12; GA), Megalac-E® e EconomasE® (n = 12) ou C) controle (n = 12). Colheitas e congelação de sêmen foram realizadas ao 0, 15, 30, 45, 55, 65 e 75 dias. Os dados foram analisados como medidas repetidas pelo Glimmix do SAS. O peso corporal e o perímetro escrotal não diferiram entre os tratamentos (P = 0,37). A atividade da glutationa peroxidase no plasma sanguíneo e seminal foi maior para as dietas contendo antioxidantes, especialmente aos 75 dias. O volume, turbilhonamento, vigor, porcentagem de cabeças aglutinadas do sêmen fresco, foram similares entre os grupos. Entretanto, dietas contendo gordura reduziram o número total de espermatozoides (P = 0,07) e a porcentagem de espermatozoides normais (P = 0,09). A peroxidação lipídica e a cinética espermática do sêmen descongelado foram similares entre as dietas experimentais. Por outro lado, dietas não contendo gordura aumentaram a porcentagem de espermatozoides com membrana plasmática (MP) íntegra e membrana acrossomal (MA) não reagida (62,2 ± 2,87 vs 53,3 ± 2,87%; P < 0,05). Além disso, dietas sem antioxidantes provocaram aumento no número de células espermáticas com MP lesada e MA reagida. Aos 65 dias de suplementação, espermatozoides de touros alimentados com dietas com gordura apresentaram maior estabilidade da MP, em relação aos outros tratamentos (60,4 ± 2,62 vs 52,7 ± 2,62%; P = 0,05), porém, aumentou o número de espermatozoides mortos (P = 0,10). O potencial mitocondrial espermático não foi afetado pelos tratamentos. Conclui-se que, apesar da viabilidade espermática pré-congelação não ter sido afetada pelas dietas experimentais, efeito benéfico aos espermatozoides após criopreservação foi encontrado quando suplementou-se touros com dietas contendo antioxidantes, em relação a dietas contendo gordura. / Cryopreservation of semen is of fundamental importance to the expansion of reproductive techniques such as artificial insemination, embryo transfer and in vitro fertilization, however, it is responsible for the oxidative stress of the sperm cell. To enhance cell survival after freezing, the plasma membrane has to have sufficient fluidity that is guaranteed by polyunsaturated fatty acids and antioxidant defense to protect against lipid peroxidation. In order to make the sperm more resistant to cryopreservation, 48 Nelore bulls were confined (three animals per pen) and assigned to four treatments according to the addition of rumen protected fat and/or antioxidants to the diet. During 30 days, all bulls were fed the same adaptation diet (sugar cane bagasse, citrus pulp, corn gluten meal, urea and mineral salt). Thereafter, for 75 days, the same diet was offered, differing in the addition of: F) fat protected from rumen degradation (rich in linoleic acid; Megalac-E®, 1.5% DM, n = 12); A) Antioxidant (a source of vitamin C and selenium, EconomasE®, 3g/head/day, n = 12); FA) Megalac-E® and EconomasE ® (n = 12); or C) control group (n = 12). Semen collection and freezing were performed seven times: 0, 15, 30, 45, 55, 65 and 75 days of offering experimental diet period. Data were analyzed by repeated measures of GLIMMIX of SAS. Body weight and scrotal circumference did not differ between treatments (P = 0.37). The glutathione peroxidase activity in blood and seminal plasma was higher for diets containing antioxidants, especially at 75 days. The volume, mass motility, vigor, numbers of agglutinated spermatozoa in fresh semen were similar between groups. However, fat-containing diets reduced the total number of sperm (P = 0.07) and the percentage of normal spermatozoa (P = 0.09). Lipid peroxidation and the kinetics of thawed sperm were similar among diets. Moreover, diets not containing fat increased the percentage of intact sperm plasma membrane (PM) and unreacted acrosomal membrane (62.2 ± 2.87 vs 53.3 ± 2.87%, P < 0.05). In addition, diets without antioxidants caused an increase in the number of sperm cells with damaged PM and with Induction of sperm acrosome reaction. After 65 days of supplementation, sperm of bulls fed diets with fat had higher stability of the PM, compared to other treatments (60.4 ± 2.62 vs. 52.7 ± 2.62%, P = 0.05), however there was an increase the number of dead sperm (P = 0.10). The mitochondrial potential of spermatozoa was not affected by treatments. It is concluded that despite tha fact that the pre-freezing sperm viability was not affected by the experimental diets, beneficial effect to the sperm cells after cryopreservation was found when bulls were supplemented with diets containing antioxidants, compared to diets containing fat.

It is surprising to know the small amount of work that has been done on alternate freezing and thawing of seeds. Many experiments, however, have been conducted to see the effect of very low temperatures on seeds and to notice the effect it had on their vitality. Some experimenters subjected seeds to temperatures as low as liquid hydrogen with very interesting results but in very few cases, if any, was the effect of alternate freezing and thawing studied. The work that follows deals directly with the effects of alternate freezing and thawing on impermeable seeds of alfalfa and of dodder. It is known that this seed does not germinate readily; therefore it often lays over winter in the soil. Does this natural freezing and thawing aid or hinder this impermeable seed in it's later germination? this has been the main question kept in mind throughout this experiment.

Frequently, in evaluating plant deficiencies, one considers only those soil factors which are associated with the fertility of the soil-- mainly the levels of phosphorus, potash, nitrogen, and organic matter. A certain level of nutrition is essential for plant growth, but in order for the nutrients to exert their maximum influence on the productivity of the soil, it is equally important that a good soil environment be provided and maintained. A good environment requires that good physical conditions of soil, sir, and water be in balance.

<p>There is consistent research evidence that shows improvement of the engineering properties of subgrade soils treated with lime or cement. However, limited information is available on the effect of climatic changes on the subgrade stiffness. The thesis studies the effects of changes in soil moisture content and temperature on the resilient modulus of treated and untreated subgrades in Indiana. Two types of soils were tested: A-6 and A-7-6, from two locations in Indiana: Hartford City and Bloomington, respectively. When existing standards ASTM D559/559-15 and ASTM D560/560-16 for wetting/drying (WD) and freezing/thawing (FT) processes, respectively, were followed, the treated and untreated samples failed through the process of preparation due to the stringent procedures in the standards. Appropriate test conditions were investigated, as part of the research, to develop new protocols more appropriate to the field conditions in Indiana. Two new test protocols were developed and successfully applied to the treated soils. A total of 26 resilient modulus, M_R, tests were conducted following the standard AASHTO T307-99. The M_R results showed that the repeated action of WD and FT cycles reduced the stiffness of the chemically-treated soils down to values similar to or lower than those of the untreated soils. However, when the amount of chemical was doubled, with respect to the optimum, the M_R of the treated soils improved over that of the untreated soils, even after the wetting-drying cycles.</p>

La congélation des produits alimentaires est un procédé qui permet d'augmenter leur durée de conservation. Ceci est possible grâce à l'abaissement de la température mais surtout à la solidification de l'eau réduisant la disponibilité de l'eau nécessaire au développement des micro-organismes. Cependant, si les produits ne sont pas emballés ou si l'emballage n'est pas adhérent à leur surface pendant la congélation, un transfert d'eau a également lieu simultanément à la libération de chaleur. Ce transfert d'eau se traduit par la déshydratation des produits qui implique une perte de masse. Celle-ci a un impact direct sur la masse de produit congelé qui pourra être vendue et donc un coût économique pour les industriels.La cristallisation de l'eau est également un paramètre clé de la congélation. En effet, les caractéristiques des cristaux de glace formés pendant la congélation (nombre, taille et forme) peuvent provoquer des modifications de structure des aliments et des dommages irréversibles. Ceux-ci se traduisent par la modification des propriétés organoleptiques et nutritionnelles des produits une fois décongelés et une baisse de leur qualité.Que ce soit pour la déshydratation ou la cristallisation, les conditions opératoires de congélation (température, vitesse d'écoulement d'air et hygrométrie) ainsi que certaines caractéristiques des produits (température initiale, surface, épaisseur) ont une influence importante. L’objectif de la thèse est d’étudier et savoir estimer la perte en eau et les caractéristiques des cristaux formés dans un aliment non poreux en fonction des conditions opératoires afin de choisir une technologie de congélation adaptée.Le travail a été axé sur l'étude des transferts de chaleur et de matière (eau) à la surface et à l'intérieur du produit lors de la congélation à deux niveaux d'échelle : macroscopique pour la déshydratation et microscopique pour la cristallisation. Cette approche multi-échelles s’est appuyée à la fois sur l’expérimentation et sur la modélisation.Pour ce qui est de la déshydratation, un dispositif expérimental a été conçu à l’échelle du produit pour mesurer la variation de la masse et de la température de l’aliment au cours de sa congélation (conditions mécaniques ou cryogéniques).Parallèlement, un modèle (1D) de prédiction de la déshydratation et de la durée de congélation en fonction du produit (homogène et non poreux) et des conditions opératoires de congélation a été développé. Cette étude a été réalisée pour un produit modèle, le gel de méthyl-cellulose (Tylose®) et pour de la viande hachée de bœuf (5 % de matière grasse).Pour l’étude de la cristallisation, un modèle à l’échelle des cristaux (2D) a pour vocation de représenter les phénomènes liés au changement de phase de l’eau (apparition et croissance des cristaux) en fonction des conditions opératoires de congélation. Il simule la cristallisation de l’eau dans des solutions notamment en prenant en compte la diffusion des molécules de solutés dans la solution cryo-concentrée ainsi que la libération et la dissipation de la chaleur latente de solidification de l'eau.Des observations de la cristallisation au sein d'échantillons de viande hachée de bœuf ont été faites par micro-tomographie à rayon X et cryo-microscopie électronique à balayage sur des échantillons congelés mais aussi par microscopie électronique à balayage et microscopie optique sur des échantillons lyophilisés (après congélation en conditions mécaniques ou cryogéniques). / Freezing extends food shelf life by lowering the temperature and mainly thanks to water solidification which decreases water availability for micro-organism growth. Nevertheless, if the food is not packaged or if the packaging is not adherent to its surface, a water transfer occurs simultaneously with the heat transfer during freezing. This water transfer leads to product dehydration which means weight loss. Food dehydration during freezing has consequences on the product weight and so has an economic cost for industrials.Water crystallization is also an important parameter of the freezing process. Indeed, ice crystal characteristics (number, size and shape) may induce food structure changes and irreversible damages. These modifications can alter organoleptic properties and food quality after thawing.Freezing operating conditions (temperature, flow velocity, hygrometry) and some food characteristics (initial temperature, surface, thickness) have an influence on both dehydration and water crystallization.The objective of this thesis is to study and to be able to estimate the water loss and crystal characteristics for non-porous food according to the freezing operating conditions in order to select the best freezing technology.The work was focused on the multi-scale study of heat and mass (water) transfers during freezing, at the product surface and throughout the product : on a macroscopic point of view for dehydration and on a microscopic point of view for crystallization. Each study is built on an experimental and a modelling work.For dehydration, an experimental device was developed to measure weight loss and product temperature kinetics during freezing (mechanical and cryogenic freezing conditions).Moreover, a predictive model (1D) was established for estimation of dehydration and freezing time according to the product and the freezing conditions. Dehydration was studied with a model material (methylcellulose gel-Tylose®) and with minced beef (5 % fat).For crystallization, a model at crystal scale (2D) aims to simulate phenomena related to the water phase change (nucleation and crystal growth) according to the freezing operating conditions. It takes into account water crystallization in solution considering the diffusion of the solute in the cryo-concentrated solution, the release and dissipation of the latent heat of water solidification.Crystallization observations were done in frozen samples of minced beef by X-ray micro-tomography and by cryo-scanning electron microscopy. Some visualizations were also carried out on freeze-dried samples by scanning electron microscopy and optical microscopy (after mechanical or cryogenic freezing).

A technique that will adequately describe the physical condition of water in the soil has long been desired by scientists. The method should include the effect of soluble materials on the osmotic pressure of the soil solution, and also the effect of surface force action between the soil and soil water. Parker (8) demonstrated that the freezing point was reduced in the presence of finely divided material. This suggested the possibility of using the cryoscopic procedure to measure the physical condition of water soil. More recently, the introduction of Thermistors for accurate temperature measurement have made it desirable to investigate in greater detail the freezing point of water in soil. The experimental work reported here deals with the freezing point of water in soil and its relation to soil moisture tension as determined on three soils in a puddled and unpuddled condition. Several freezing treatments were included in this study to investigate improvements in the freezing technique. The treatments were arranged in a standard split plot experimental design. Eighteen hundred freezing measurements were made and analyzed statistically.

Eleven experiments examined two late-developing learning and memory systems in rats: fear-potentiated startle (FPS) and the contextual regulation of latent inhibition and extinction. The first study was based on three previous developmental findings on FPS: (1) FPS to an odour CS emerges at postnatal day (PN) 23; Rats conditioned at PN16 to an odour CS express freezing but not FPS when tested at PN23, and (3) FPS to an odour CS trained at PN16 is activated if rats are also trained to a difference odour at PN23 (Yap, Stapinski, & Richardson, 2005). Yap et al. (2005) hypothesised that the activation effect only occurs if rats are given training to the second odour at an age when FPS has emerged. Study 1 assessed this hypothesis and trained the second odour CS at either PN23 or PN20. Contrary to expectations, the results of this study showed the activation effect for both groups of rats. Surprisingly, the results also revealed a significant FPS effect to the odour CS trained at PN20. Subsequent experiments examined this unexpected result, and found that learning to odour 1 at PN16 facilitated the age of onset for FPS at PN20. The results of Study 1 are discussed in relation to past findings on enrichment, cumulative learning, and neurobiological models of conditioned fear. The second section of this thesis (Studies 2 and 3) examined the context-specificity of two memory interference paradigms, latent inhibition and extinction, in developing rats. The studies found that both phenomena were context-specific at PN23-25 but not at PN16-18. Moreover, the results suggest that the context-specificity of both latent inhibition depended on the age of the rat during the second phase of training, but not their age during the first phase of training or their age at test. The implications of these findings for theoretical and neural models of learning, as well as the occurrence of latent inhibition and extinction during development are discussed.