Cryopreservation is the preservation of biomaterials at extremely low temperatures. It is the only alternative for long-term storage of high quality biomaterials, with applications to biobanking and transplant medicine. Cryopreservation success revolves around the control of ice formation, which is known to be harmful. Ice formation is a path-dependent phenomenon, affected by the thermal history and presence of nucleation promotors. Cryoprotective agents (CPAs) are commonly added to the biomaterial to be preserved, in order to suppress ice formation and inhibit its growth during the cryopreservation protocol. Ice-free cryopreservation can be achieved in large-size systems when the biomaterial is loaded with a high CPA concentration solution and cooled rapidly, in a process that is known as vitrification (vitreous means glassy in Latin). During vitrification, the CPA viscosity increases exponentially with decreasing temperature, while the material is cooled to deep cryogenic temperatures faster than the typical time scale for crystallization. The material can potentially be stored indefinitely at such low temperatures. Large-size vitrification is associated with three competing needs on the CPA concentration. Since the cooling rate at the center of the specimen decreases with the increasing specimen size due to the scaling conductive resistance, higher CPA concentrations may be required to suppress crystallization in larger specimens. Higher CPA concentration generally requires lower cooling rates to avoid ice crystallization. On the other hand, since CPAs are potentially toxic, the lowest possible CPA concentration is required to maintain viability and facilitate functional recovery. The decrease in CPA concentration combined with an increase in cooling rates may intensify thermo-mechanical stress due to non-uniform thermal contraction to the point of structural destruction. Essentially, successful cryopreservation represents the outcome of an optimization problem on the composition and concentration of the CPA cocktail. The work presented in this thesis combines an experimental study on the thermal conductivity of relevant materials, and a theoretical study to identify the effects of the measured values on cryopreservation protocols. The unique contributions presented as the initial stage of the experimental study are: (i) the modification of the cryomacroscope and creation of an experimental program to make thermal conductivity measurements of CPA based on the existing transient hot wire technique, (ii) to develop a protocol for making thermal conductivity measurements during rewarming portion of the cryoprotocol, and (iii), to begin generating a data bank of thermal conductivity of CPA and materials used in cryopreservation. Thermal conductivity measurements are presented for the CPA Dimethyl Sulfoxide (DMSO), over a concentration range of 2M to 10M, in a temperature range of -180°C to 25°C. Samples of 2M to 6M DMSO were found to crystallize at quasi-steady cooling rates, while samples of 7.05 to 10M were found to vitrify. Thermal conductivities of the crystallized and vitrified material reach a tenfold difference at -180°C. The quality of measurements using the presented technique has been verified theoretically by means of finite element analysis (FEA) using the commercial code ANSYS. This experimental study is expanded to the study of thermal conductivity of the CPA cocktail DP6--a mixture of 3M DMSO and 3M propylene glycol, which has drawn significant attention in the cryobiology community in recent times. The unique contributions are the first thermal conductivity measurements reported in literature of the combined effect of DP6 with synthetic ice modulators (SIMs), including 6% 1,3Cyclohexanediol, 6% 2,3Butanediol, and 12% PEG400. Results of this study demonstrate that the thermal conductivity may vary by three fold between the amorphous and crystalline phases of DP6 below the glass transition temperature. Results of this study further demonstrate the ability of SIMs to decrease the extent of crystallization in DP6, even at subcritical cooling and rewarming rates. The accompanying theoretical investigation focuses on cryopreservation in a kidney model, in effort to explore how the thermal history is affected by variations in the measured thermal conductivity. This analysis is based on FEA using the commercial code ANSYS. In particular, the unique contributions of this study are: (i) thermal analysis of a vitrifying rabbit kidney based on an established rabbit-kidney cryopreservation protocol, and (ii), exploring scale-up thermal effects to a human-size organ. This represents a 21-fold increase in organ size. Results indicate that even in the case of the human kidney, cooling rates remain high enough in all parts of the kidney to prevent ice formation at temperatures above -100oC.

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La température est le paramètre abiotique qui influence de façon majoritaire les traits d’histoire de vie des espèces ectothermes. Pour appréhender les relations entre physiologie, environnement et histoire évolutive et leur influence respective sur la délimitation des aires de distribution des espèces, ce travail exploite les caractéristiques thermiques du milieu souterrain. Les résultats réfutent l’hypothèse de sténothermie des deux organismes hypogés étudiés Niphargus rhenorhodanensis et Proasellus valdensis et prouvent que l’histoire évolutive, la dispersion ou la compétition sont des paramètres importants dans l’établissement des aires de distribution des espèces souterraines. En outre, ce travail caractérise pour la première fois chez des organismes souterrains, un gène codant pour des protéines de choc thermique et montre l’importante sensibilité cellulaire de N. rhenorhodanensis face à une augmentation de température / Temperature is the abiotic factor that most influences the life-history traits of ectothermic organisms. In order to study the relationships between physiology, environment and evolutionary history and their respective role in the determination of species distribution areas, this work takes advantage of the thermal caracteristics of subterranean aquatic biotopes. Our results refuted stenothermy in both studied hypogean organisms Niphargus rhenorhodanensis and Proasellus valdensis and they showed that evolutionary history, dispersal and competition are important factors that determine the distribution of subterranean species. Moreover, this work characterized for the first time in subterranean organisms a gene encoding heat shock proteins and demonstrated the high cellular sensitivity of N. rhenorhodanensis to increased water temperature

La thérapie cellulaire et les médicaments de thérapie innovante sont des solutions prometteuses pour la régénération des tissus ou organes présentant des défauts fonctionnels ou organiques. Avant le stade de l'insuffisance cardiaque terminale (stade IV NYHA) suite à un infarctus du myocarde, l'implantation d'un patch de fibrine cellularisé avec des progéniteurs myocardiques sur le site de nécrose de l'infarctus, est l'une des perspectives qui permettrait de régénérer un muscle cardiaque fonctionnel et apparait comme étant une alternative nouvelle avec notamment un essai clinique de phase I en cours (ESCORT : NCT02057900). Cependant, cette thérapie innovante présente de réelles contraintes, parmi lesquelles, un protocole nécessitant, i) une utilisation pour la production de cellules progénitrices myocardiques CD15+, de DMSO, de sérum foetal bovin, de trypsine porcine, de fibroblastes murins pouvant être la source d'une contamination chimique ou microbiologique, ii) une caractérisation importante des cellules produites, pour déterminer leur viabilité, leur pureté, leur état de différenciation, iii) d'implanter le patch de fibrine cellularisé dans un délai limité avant l'obtention des résultats de stérilité et d'endotoxines, iv) d'inciser le péricarde et de former une poche, geste chirurgical très invasif, afin d'implanter le patch cellularisé. Avec l'objectif de limiter ces contraintes et de renforcer la sécurisation pharmaceutique de ce médicament de thérapie innovante, les différents axes de ce travail ont porté sur i) l'ajout, juste avant l'implantation, d'une étape de cryoconservation des cellules dans un milieu sans sérum et sans DMSO, mais avec des agents cryoprotectants de qualité pharmaceutique. L'avantage apporté par la cryoconservation étant de rendre possible une production par lot, et la réalisation des contrôles sans contrainte de temps avant l'implantation, ii) la vectorisation des cellules par une encapsulation dans des microsphères formant une suspension injectable et permettant une implantation directement au travers du péricarde et immédiatement après la décongélation, iii) l'utilisation de polymères bioadhésifs afin de maintenir les microsphères au site d'implantation. Dans un premier temps, ce travail a permis d'identifier l'alginate de sodium de faible viscosité à 1,2% comme polymère pour réaliser l'encapsulation à l'aide d'une buse vibrante de 120 µm de diamètre. La nature et la concentration d'agents cryoprotectants ont également été définies. Les agents cryoprotectants ont été sélectionnés parmi les oses (glucose, saccharose, tréhalose), les polyols (glycérol, mannitol, sorbitol) et l'urée, à une concentration permettant d'atteindre une osmolarité totale de 500 mOsm/L pour abaisser le point de congélation de l'eau. Enfin le chitosane de faible viscosité à 0,5% a été utilisé comme polymère bioadhésif de surface pour maintenir les propriétés mécaniques et la forme des microsphères après la congélation. Dans un second temps, une évaluation biologique a permis de mesurer l'impact des étapes du procédé d'encapsulation et de cryoconservation, sur des cellules souches mésenchymateuses humaines utilisées comme modèle. Il a ainsi été possible d'optimiser le protocole ce qui a eu pour effet d'augmenter la viabilité, évaluée après encapsulation et congélation par une analyse en cytométrie de flux avec le 7AAD, de moins de 5% à environ 35%. / Cell therapy and advanced therapy medicinal products are promising solutions for the regeneration of tissues or organs with functional or organic defects. Before the terminal heart failure stage (stage IV NYHA) following a myocardial infarction, the implantation of a cellularized fibrin patch with myocardial progenitors at the location of the infarct necrosis, is one of the perspectives that would allow a functional heart muscle to regenerate and appears to be a new alternative, in particular, with an ongoing Phase I clinical trial (ESCORT : NCT02057900). However, this innovative therapy presents real constraints, among which, a protocol requiring, i) the use for the production of CD15+ myocardial progenitor cells of, DMSO, bovine fetal serum, porcine trypsin, and murine fibroblasts which may be the source of chemical or microbiological contamination, ii) an important characterization of the produced cells, to determine their viability, purity, and state of differentiation, iii) to implant the cellularized fibrin patch within a limited time frame before getting the results of sterility and endotoxins, iv) to incise the pericardium and to form a pouch, a very invasive surgical gesture, in order to implant the cellularized patch inside. With the objective of limiting these constraints and strengthening the pharmaceutical safety of this innovative therapy medication, different axes of this work have focused on i) the addition, just before the implantation, of a step of cryopreservation of the cells in a medium without serum and without DMSO, but with pharmaceutical-grade cryoprotectants. The advantages of cryopreservation is to allow production in batches, and controls to be carried out without time constraints before the implantation, ii) the vectorization of the cells by encapsulation in microspheres forming an injectable suspension and allowing direct implantation through the pericardium immediately after thawing, iii) the use of bioadhesive polymers to maintain the microspheres at the location of the implantation. This study initially enabled to identify a low-viscosity sodium alginate at 1.2% as a polymer being used for the encapsulation with the use of a vibrating nozzle which diameter is of 120 µm. The nature and the concentration of the cryoprotectants have also been defined. The cryoprotectants were selected from oses (glucose, sucrose, trehalose), polyols (glycerol, mannitol, sorbitol) and urea, at a concentration which achieves a total osmolarity of 500 mOsm/L in order to lower the freezing point of water. Finally, low viscosity chitosan at 0.5% was used as a bioadhesive polymer at the surface of the microspheres to maintain their shapes and mechanical properties after freezing. In a second step, a biological evaluation allowed to measure the impact of the encapsulation and the cryopreservation processes, on human mesenchymal stem cells used as a model. It was thus possible to optimize the protocol, which in return increased the viability ; evaluation made after encapsulation and freezing by a flow cytometry analysis with 7AAD ; from less than 5% to about 35%.

The thesis deals with ecophysiology of overwintering larvae of two butterfly genera, Colias and Erebia. It focuses on identification of supercooling point, survival of various low temperature regimes and composition of cryoprotective substances. Results are presented in the context of distributional limits of individual species, their habitat requirements and their potential endangerment due to environmental and habitat changes.

This thesis deals with the characterization of bacterial cells capable of polyhydroxyalkanoates (PHA) accumulation. The dissertation thesis is written in the form of a discussed published publications which are attached to the thesis as appendixes. The work develops a study of the current topic of the protective functions of PHA and clarifies protective mechanisms against selected stressors. Firstly, we focused on the protective effects of PHA granules against UV radiation and osmotic stress, specifically hypotonic conditions. In the case of UV exposition, the cells protected themselves by scattering UV radiation on the intracellular granules protecting especially nucleoid. When exposed to osmotic stress, the amorphous state of PHA granules is very important since it is capable of stabilization of cell membranes under hypertonic stress, afterwards, bacterial cells can maintain their integrity during the subsequent hypotonic challenge. In general, the amorphous state of PHA granules is key to ensure the proper biological functions of PHA whether as storage or protective polymer. Therefore, in the next part of this work, we focused on the core of the stabilization mechanism that protects native PHA granules from crystallization and thus the intracellular polymer maintains in a thermodynamically unfavorable amorphous phase state. Based on experimental work, we applied selected stresses because we proposed a new model of stabilization of the amorphous state of PHA granules in vivo. It consists of two mechanisms, where small volumes of PHA granules reduce the rates of crystallization and at the same time the water present in the granules plays the role of a low molecular plasticizer. Due to the metabolic apparatus of bacterial cells, PHA are simultaneously synthesized and degraded which leads to an increment of intracellular concentration of monomers that also figure in the protective effect of PHA. In this context, we aimed at the description of the mechanism of cryoprotective effects of 3-hydroxybutyrate, the monomer of the most common of PHA, poly(3-hydroxybutyrate). Hence, we constructed an equilibrium and non-equilibrium phase diagram of the 3HB-water system to prove that 3HB is a very effective cryoprotectant. This fundamental understanding of the protective properties of PHA monomers could be also used in the food industry or cryopreservation of biological samples.