Dissertations / Theses on the topic 'Cryobiology'

Thesis (Ph. D.)--University of Missouri-Columbia, 2007.
"May 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Preservation of tissue structure, morphology and biomarkers is of utmost importance for pathological examination of biopsy specimens for diagnostic and therapeutic purposes. However current methods employed to evade tissue degradation and preserve biomarkers have several shortcomings that include irreproducibility, morphological artifacts and altered biomarker antigenicity. These artifacts may affect the analysis and subsequent diagnosis of the tissue pathology. This creates need for developing improved preservation methods that reproducibly maintain tissue morphology and biomarker antigenicity and are simple, rapid and inexpensive. Experiments conducted for testing the hypothesis that cryopreservation procedures yield high quality morphology and antigenicity showed that cryopreservation maintains tissue structure, morphology and antigenicity at equivalent or better levels compared to standard freezing techniques. In order to understand the mechanisms of osmotic transport in cellular systems upon exposure to multi-component solutions that are prevalent in virtification protocols, experimental studies were undertaken using microfluidics for single cell manipulation. The experimental data yielded permeability parameters in binary and ternary solutions for MC3T3-E1 murine osteoblasts for the first time. The hydraulic conductivity (L(p)) decreased with increasing concentrations but the solute permeability either increased or decreased with increasing solution concentration. The changes in hydraulic conductivity were consistent with previously published trends and conform to a functional relationship in the form of Arrhenius type relationship between L(p) and solution concentration. Further a theoretical model was developed from principles of linear irreversible thermodynamics to simulate multi--‐‑component mass transport across membrane. The model was successfully validated by comparison with experimental data for murine osteoblasts and showed good agreement between the numerical predictions and experimental observations. The modeling approach can be used to investigate the transport mechanisms, which show that in multicomponent osmotic transport response, the dynamics is dictated by slower moving solute.

Freeze-injury at the plasma membrane level has been identified as being crucial for the survival of living matter. Since plasma membranes consist of several micro domains that make the structure rather complex, this study attempted to use simple model membranes to investigate the changes of phospholipid bilayers at sub-zero temperatures. Egg yolk L-α-phosphatidylcholine (EPC) and 1, 2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC) that mimic plasma membranes in their unique ways were used to prepare large unilamellar vesicles (LUV), which were the model membranes of this study. At cooling rates of 0.5 and 10�C/min, LUV were freeze-concentrated in the unfrozen matrix as a result of the advancing extraliposomal ice front and the decreasing phase volume of the unfrozen matrix, both of which led to membrane lesion. At the slow cooling rate of 0.5�C/min, an additional freezing stress imposed by the osmotic gradient across the bilayers, due to the increase of solute concentration in the unfrozen matrix, promoted leakage of LUV. The gel-liquid crystal phase transition temperature of phospholipids played an important role in determining if the LUV could withstand freezing stress when the LUV were held at a defined sub-zero temperature for a given period of holding time. EPC LUV were more leaky than DPPC LUV when they were held at the high sub-zero temperatures and their leakage increased with increasing holding time. The leakiness of EPC LUV could be related to the fluid and deformable nature of the EPC above its phase transition temperature. In contrast, DPPC LUV with a higher gel-liquid crystal phase transition temperature compared to EPC may become increasingly fragile at lower sub-zero temperatures, which led to the increase of leakage when the DPPC LUV were held at the lower sub-zero temperatures. These results indicated that the determination of the fatty acid profile of the plasma membranes was essential to aid in developing the most suitable holding temperature and time during the cryopreservation of biological specimens. Adding to the integrity of LUV that depended on the gel-liquid crystal phase transition temperature of phospholipids, intraliposomal ice formation also depended on the phase transition temperature of phospholipids. Intraliposomal ice formation was only observed for DPPC LUV but not for EPC LUV. In addition to the extraliposomal ice formation, other physical changes such as the eutectic crystallization of sodium chloride (NaCl) and ice mixture on the stability of LUV were also investigated. The eutectic crystallization of NaCl/ice mixture was governed by the intra- and extraliposomal distribution of NaCl and was more likely to occur at the physiological NaCl concentrations compared to lower NaCl concentrations. The eutectic crystallization of NaCl/ice mixture further increased the leakage of LUV. The understanding of the freezing behaviour and the mechanisms of freeze-injury of LUV allowed the use of the current model membranes for further investigations of the cryoprotective actions of cryoprotective agents (CPA). Partial phase diagrams of sugar-salt-water, dimethyl sulfoxide (DMSO)-salt-water and ethylene glycol (EG)-salt-water systems that resembled extraliposomal solute compositions were constructed and the phase volume of ice and unfrozen matrix was estimated from the freezing curves. Ice reduction was the major mechanism by which the non-permeable and permeable CPA protected the LUV from freeze-injury. Other cryoprotective mechanisms of the non-permeable and permeable CPA through the dilution and spacing out of the LUV in the unfrozen matrix as well as the suppression of the eutectic crystallization of NaCl/ice mixture were not ruled out. Non-permeable CPA were more effective in preventing leakage of DPPC than EPC LUV. Unlike the non-permeable CPA, permeable CPA were more effective for EPC than DPPC LUV that had been subjected to freezing and thawing processes. At room temperature, however, DMSO and EG were detrimental to the stability of DPPC LUV. The choice of CPA is strictly dependent on the type of phospholipids that varied in their acyl chain length and phase transition temperature. In summary, this study provides insights of the freeze-injury of LUV and the cryoprotective mechanisms of the non-permeable and permeable CPA which are beneficial to the field of cryopreservation that often depends on empirical trial and error methods. By integrating a comprehensive molecular-based understanding, an optimal cryopreservation procedure could be designed.

With emerging trends of new cellular therapies, the need for quick access to cellular components is necessary. For most applications genetically compatible biological components are essential to prevent adverse immune responses and graft-versus host disease (GVHD). Since these biological components have a limited window to be used, techniques for long-term storage are needed. Cryopreservation is essential for this in the field of biobanking and regenerative medicine to allow for long-term storage of cell products. During this process, ice recrystallization is the major contributor to cell death and decreased cell viability post-thaw. Due to this, controlling ice growth and recrystallization is imperative to increasing cell survival and function. The Ben lab is focused on the synthesis of small molecule, carbohydrate-based cryoprotectants that function as ice recrystallization inhibitors (IRIs). Previously, many IRIs have been synthesized showing varying degrees of ice recrystallization inhibition (IRI). Through the structure-function work, a delicate balance between hydrophobic and hydrophilic portions on the same molecule must be met. These compounds are believed to disrupt hydrogen bonding networks present in the formation of ice, and control ice growth. While numerous types of functional groups on carbohydrate derivatives have been explored, many highly solvated functional groups (amines, sulfates, phosphates, etc.) have not been thoroughly investigated. Highly solvated functional groups should disrupt hydrogen bond networks due to their solvation and in theory, should illicit an IRI response. Sulfate groups have not previously been studied, but are present in several different biological processes, such as immune response and blood coagulation. This suggests that sulfated carbohydrates should be well tolerated biologically. Sulfate groups can also be easily installed on existing IRI active molecules through orthogonal protecting group chemistry. The first part of this thesis is focused on the synthesis and IRI activity of sulfated carbohydrates based upon previously synthesized, IRI active pyranose derivatives. When compared to their parent compounds, most of the sulfated derivatives were less active, but all compounds were incredibly soluble in aqueous media. These derivatives did not show much promise as new IRIs due to the length of their synthesis and reduced IRI activity compared to their parent compounds. The Ben lab has also developed a new class of IRI active carbohydrates: aldonamide derivatives. These compounds are open-chain carbohydrates with an amide bond, arising from the ring opening of a carbohydrate lactone with a substituted amine. While many of these compounds displayed high degrees of IRI activity, many were incredibly insoluble in aqueous systems (many with solubility limits under 50 mM). Since sulfate groups were able to greatly increase solubility with some derivatives retaining IRI activity, installing sulfate groups on existing aldonamide-based IRIs should increase their solubility. Additionally, since many of these derivatives display high degrees of IRI activity, a reduction in IRI activity can be tolerated. Similarly, to the sulfated pyranose derivatives, the presence of a sulfate group reduced the IRI activity compared to the parent compounds in most derivatives. Though some sulfated derivatives possessed a higher degree of IRI activity, all the derivatives experienced a drastic increase in solubility (over 200 mM in PBS). Some of the sulfated aldonamide derivatives were assessed for their ability to protect red blood cells (RBCs) during freezing with reduced glycerol concentrations (15% glycerol), although none of thew tested derivatives showed an improvement over existing IRIs explored by the Ben lab. Since the introduction of sulfate groups to existing IRIs drastically increased solubility in aqueous systems, but resulted in reduced IRI activity in most compounds, focus was switched to the addition of different hydrophilic functional groups. Amino functional groups were briefly explored with galactose-based pyranose IRIs, aldonamide derivatives had not been explored. Amino groups are present on many biological carbohydrates and should be well tolerated biologically. The addition of amino groups to aldonamide derivatives should increase solubility, with the amino derivatives ideally retaining some IRI activity. The amino aldonamide derivatives synthesized had high solubilities (>500 mM in PBS), but did possess lower degrees of IRI activity. Due to the high solubility these derivatives were initially assessed in the cryopreservation of RBCs with reduced glycerol concentrations. Initial experiments showed improvements over current IRIs, and the compounds were assessed in a number of other biological cryopreservation scenarios including articular cartilage, platelets, and hematopoietic stem/progenitor cells (HSPCs). While the compounds showed toxicity in these cell types, more studies need to be conducted for the cryopreservation of RBCs.

Le specie animali, sia domestiche che selvatiche, rappresentano un patrimonio di biodiversità genetica di inestimabile valore in tutto il mondo. Le loro caratteristiche intrinseche contribuiscono a sostenere la prosperità economica e il benessere umano, nonostante i profondi cambiamenti in atto a livello globale e locale. Conoscere, valorizzare e conservare queste preziose risorse genetiche è un dovere sociale, culturale, scientifico ed economico e rappresenta la chiave dell’adattamento e della sopravvivenza in un ambiente dominato dall’uomo. L’Italia, essendo caratterizzata da una grande varietà di paesaggi ambientali e climi estremamente diversi, è considerata uno dei paesi europei più ricchi di biodiversità animale con il maggior numero di endemismi. Tuttavia, l’effetto combinato di azioni antropiche dannose e l’introduzione dei moderni sistemi di produzione intensiva, associati all’aumento del reddito, della popolazione umana e all’urbanizzazione, hanno portato ad un aumento drammatico del numero di specie autoctone in via di estinzione, guidando il paese verso un irreversibile perdita di risorse genetiche. In questo scenario, il numero di razze avicole e cunicole italiane, così come le naturali popolazioni autoctone di Trota Mediterranea, sono attualmente in allarmante declino. Pertanto, negli ultimi due decenni, queste preziose risorse genetiche sono diventate il fulcro di importanti programmi di conservazione, finalizzati esclusivamente al mantenimento delle popolazioni in situ. Secondo l’Organizzazione delle Nazioni Unite per l’alimentazione e l’agricoltura (FAO), questa strategia di conservazione è considerata prioritaria per la salvaguardia delle popolazioni a rischio di estinzione. Tuttavia, a causa della complessità del mantenimento degli animali vivi, questo tipo di approccio risulta spesso troppo costoso ed il raggiungimento degli obiettivi genetici procede ad un ritmo relativamente lento. Di conseguenza, la strategia di conservazione ex situ in vitro sta ottenendo sempre più maggiore attenzione nell’ambito della gestione e conservazione delle risorse genetiche animali, come supporto integrato alla strategia in situ. In particolare, la possibilità di utilizzare il seme congelato nella pratica dell’inseminazione artificiale (AI), rappresenta un fattore chiave per assicurare la conservazione a lungo termine della diversità genetica, attraverso l’istituzione di una criobanca del seme. In questo contesto, l’urgente bisogno di istituire azioni nazionali volte a supportare l’attuazione di programmi di conservazione ex situ in vitro, ha recentemente promosso il finanziamento di tre progetti, di cui uno europeo 1) “Recovery of S. macrostigma: Application of innovative techniques and participatory governance tools in the rivers of Molise” (Life Nat.Sal.Mo); e due italiani 2) “Tutela della Biodiversità nelle razze Avicole Italiane” (TuBAvI); 3) “La Cunicoltura del Futuro: benessere e sostenibilità degli allevamenti cunicoli italiani” (Cun-Fu). I presenti progetti, sono caratterizzati da uno scopo comune, ossia quello di preservare e valorizzare le razze avicole e cunicole italiane (TuBAvI e Cun-Fu) e le popolazioni native di Trota Mediterranea (Life Nat.Sal.Mo), attraverso l’uso combinato di strategie di conservazione in situ ed ex situ. Pertanto, nell’ambito di questi progetti, la creazione delle prime criobanche del seme italiane finalizzate alla salvaguardia di questo prezioso patrimonio di biodiversità animale, ha rappresentato un importante pietra miliare. A tal riguardo, sono divenuti necessari studi sistematici dei principali fattori in grado di influenzare il successo delle procedure di crioconservazione del seme di tacchino, coniglio e Trota Mediterranea, al fine di ridurre il danno cellulare provocato dal processo di congelamento/scongelamento. Alla luce di questa premessa, le attività di ricerca incluse nella presente tesi di dottorato sono parte integrante dei tre progetti più estesi sopramenzionati, il cui scopo è stato quello di sviluppare efficaci procedure di riferimento per la crioconservazione del seme di queste preziose risorse genetiche italiane, al fine di garantirne la conservazione a lungo termine. Inoltre, in linea con il raggiungimento dei presenti obiettivi, è stato effettuato anche uno studio di proteomica comparativa che ha avuto come scopo quello di confrontare i pattern proteici del seme di coniglio fresco e crioconservato. La logica di questa ricerca è stata quella di svelare, per la prima volta, i principali meccanismi coinvolti nel danno criogenico del seme nella specie cunicola. I risultati di questi studi sono presentati sotto forma di sei manoscritti pubblicati e una bozza di articolo, opportunamente ripartiti in quattro sessioni come segue: • la sessione 1 include tre studi finalizzati ad ottimizzare e identificare una procedura di riferimento per la crioconservazione del seme di Trota Mediterranea, ed i risultati ottenuti dall’avvio della prima criobanca del seme per questa specie; • la sessione 2 include due studi il cui scopo è stato quello di identificare e standardizzare una procedura di riferimento per il congelamento del seme di tacchino, attraverso la valutazione della sensibilità al congelamento in vitro e della capacità fertilizzante in vivo. Inoltre, sono riportate nel dettaglio le attività svolte ed i risultati ottenuti dalla creazione della prima criobanca italiana del seme di razze avicole autoctone; • la sessione 3 include uno studio il cui obiettivo è stato quello di sviluppare una procedura di riferimento per la crioconservazione del seme di coniglio, attraverso la comparazione di due diluenti e tre differenti dosi di inseminazione. Inoltre, sono riportati i risultati ottenuti dall’avvio della prima criobanca italiana del seme di razze cunicole autoctone; • la sessione 4 include i risultati sperimentali ottenuti dalla prima analisi comparativa del profilo proteico del seme di coniglio fresco e congelato, finalizzato a valutare i principali meccanismi fisiologici e biochimici influenzati dalla procedura di criocoservazione del seme nella specie cunicola.
Both domestic and wild native animal species represent a genetic biodiversity heritage of inestimable value all over the world. Their valuable intrinsic properties contribute to support economic prosperity and human well-being, despite the deep changes that are “globally” and “locally” taking place. Knowing, valorizing and conserving these precious genetic resources is a social, cultural, scientific and economic imperative that is the key to adaptation and survival in a human dominated environment. Italy appears to be one of the richest countries in animal biodiversity within the European Union. Because of the presence of extremely different environments, it has the highest number and density of animal species characterized by a high rate of endemism. However, the combined effect of harmful anthropic actions and the introduction of modern intensive production methods, associated to increasing income, human population and urbanization, has led to a dramatic increase in the number of endangered native species, resulting in an irreversible loss of genetic resources. In this scenario, Italian biodiversity of local poultry and rabbit breeds, as well as native Mediterranean trout populations, is currently in an alarming decline. As result, over the last two decades, these valuable genetic resources becoming the focus of important national conservation programs, exclusively aimed at the maintenance of live populations. According to the Food and Agriculture Organization of the United Nations (FAO), the in situ conservation strategy is in fact considered a priority for the safeguard of endangered populations. However, this approach, is often too expensive, and the achievement of genetic goals is going on at a relatively slow pace. Therefore, the ex situ in vitro strategy, as integrated support to the in situ strategy, is getting more and more attention. In particular, the use of artificial insemination (AI) with cryopreserved semen has been recognized as the most widespread method for ensuring the long-term conservation of genetic diversity, through the establishment of semen cryobanks. In this context, the urgent need to establish national actions aimed at promoting ex situ conservation programs, has recently favored the financing of three important projects, one of which is an European project: 1) Recovery of S. macrostigma: Application of innovative techniques and participatory governance tools in the rivers of Molise - (Life Nat.Sal.Mo); and two national projects 2) Tutela della Biodiversità nelle razze Avicole Italiane – TuBAvI (MiPAAF); 3) La Cunicoltura del Futuro: benessere e sostenibilità degli allevamenti cunicoli italiani - Cun-Fu (MiPAAF). These projects have a common aim, which is to preserve and valorize the local poultry and rabbit breeds (TuBAvI and Cun-Fu projects), and native Mediterranean trout populations (Nat.Sal.Mo project), through the combined use of in situ and ex situ conservation strategies. Accordingly, milestones of the present projects are the creation of semen cryobanks for the safeguard of these precious Italian heritage. In this regard, systematic studies of the main factors affecting the cryopreservation procedures of turkey, rabbit and Mediterranean trout semen have become necessary, in order to reduce cell damages caused by freezing/thawing process. In the light of this, the present doctoral thesis is an integral part of the three more extended projects mentioned above. The research activities were focused on developing effective reference procedures in order to ensure the long-term conservation of this valuable Italian genetic diversity. In accordance with these goals, a comparative proteomic study on fresh and frozen rabbit semen was also performed in order to provide new knowledge concerning the main mechanisms involved in cryogenic sperm damage in this species. The results of these studies are presented in the form of six published manuscripts and one draft article divided into four sessions, as follows: • session 1 includes three studies designed to optimize and identify a reference procedure for semen freezing of native Mediterranean trout and the results obtained from the start-up of the semen cryobank for this species; • session 2 includes two studies aimed to identify and standardize a reference procedure for freezing turkey semen through the evaluation sensitivity to freezing in vitro and the fertilizing ability in vivo. Moreover, the activities performed for the implementation of the first Italian avian semen cryobank were also reported in detail; • session 3 includes one study designed to develop a reference procedure for rabbit semen cryopreservation by comparing two extenders and three different inseminating doses, followed by the results achieved from the creation of the first national semen cryobank for Italian rabbit breeds; • session 4 includes one draft article aimed to evaluate, for the first time, the changes in rabbit semen proteins induced by cryopreservation process, trought comparative analysis of the differential expression of proteins in fresh and frozen semen.

Intracellular ice formation (IIF), a major cause of cryoinjury in biological cells, is significantly more pronounced during freezing of tissue than during freezing of suspended cells. While extensive studies of IIF have been conducted for single cells in suspension, few have investigated IIF in tissue. Due to the increased complexity that arises from both cell-substrate and cell-cell interactions in tissue, knowledge of cryobiology of isolated cells cannot simply be extrapolated to tissue. Different theories have been hypothesized for the mechanisms of IIF in tissue, but none have been conclusively proven. Towards the goal of developing mathematical models to accurately predict the probability of IIF in tissues of one or more cell types, we have developed a novel high-speed video cryomicroscopy system capable of image acquisition at sampling rates up to 32,000 Hz. Specifically, the effects of cell adhesion to the substrate and cell-cell interactions were investigated with experimental (micropatterned endothelial cell constructs) and mathematical models (Monte Carlo simulations). We have reported the first direct observations of the IIF process recorded at unprecedented sub-millisecond and sub-micron resolution. For the majority of our experiments, IIF nucleation was determined to occur preferentially at the cell perimeter. This observation was not consistent with the commonly accepted hypotheses of ice nucleation in suspended cells and suggests that an alternative mechanism of IIF initiation is dominant in adherent cells. In addition, the kinetics of ice nucleation were shown to be influenced by time in culture, attached cell perimeter, fibronectin coating density, and degree of cell-cell contact. Moreover, an additional phenomenon, paracellular ice penetration was identified, and the frequency of formation was correlated with focal adhesion formation. The data and mathematical models presented in this thesis bring closer the goal of elucidating the primary mechanisms contributing to IIF in tissue; providing important contributions to both the fields of cryopreservation (minimizing IIF) and cryosurgery (maximizing IIF).

This study examined the extent to which physiological and psychological concomitants of aerobic terrestrial performance were affected by body cooling of varying degrees induced by cold water immersion (CWI). Thirteen male and 13 female subjects underwent three randomly assigned 30 min treadmill runs: a control run without prior manipulation of the subjects' thermal status and the same exercise after "central" (core temperature 1°C below pre-immersion) and "peripheral" cooling (skin heat loss 100kcal.m⁻².h⁻¹). During treadmill runs core temperature was measured, together with chest, leg, arm and hand temperatures, from which mean skin temperature (T [subscript]s[subscript]k) and mean body temperature (T[subscript]b) were calculated. Heart rate, oxygen consumption (VO₂,), carbon dioxide production (VCO₂), minute ventilation (V₂ (BTPS)), breathing frequency (f), cadence and ratings of perceived exertion (RPE) and thermal sensation (PTS) were also measured. Both central and peripheral cooling resulted in significantly reduced T[subscript]r[subscript]e (males : control 37.9±0. 3°C; central cooling : 36.8±0.5°C; peripheral cooling: 37.5±0.4°C; females: control: 37.9±0.4°C; central cooling: 37.2±0.5; p<0.05) during subsequent treadmill running, except following peripheral cooling for females (37.9±0.3°C) . For males and females T[subscript]s[subscript]k was lower following peripheral cooling than control values and lowest after central cooling (males: control: 30.0±1.3°C; central cooling: 36.8±0.5°C; peripheral cooling: 37.5±0.4°C; females: control: 30.5±1.2°C; central cooling: 25.9±1.8°C; peripheral cooling: 26.9±1.9°C; p<0.05). Female subjects experienced significantly higher T[subscript]r[subscript]e than males following central and peripheral cooling and a lower T[subscript]s[subscript]k following central cooling. Females experienced less of an increase in heart rate than males during exercise following central and peripheral cooling (control: l57.7±23.7b.min⁻¹; central cooling: 143.5±20.5b.min⁻¹; peripheral cooling 151.7±16.7b.min⁻¹; p<0 .05). Male responses were the same following central cooling but higher for peripheral cooling than control values (control: 139.1±7.3b.min⁻¹; central cooling 134.7±17.5b.min⁻¹; peripheral cooling: 145.0±16.4b.min⁻¹; p<0.05). These data indicate a depression in cardiovascular function for females following peripheral cooling that was not apparent for males. The VO₂ was not different between tests for males; only peripheral cooling resulted in a raised VO₂ of 28.6±3 .3ml.kg⁻¹.min⁻¹ (p<0 .05) for females compared to 27.6±2.6ml.kg⁻¹.min⁻¹ (control). A biphasic response was evident for VO₂ VCO₂ and V[subscript]B (BTPS). For both sexes overall RPE was lower for peripheral cooling (males: 9.4±1.9; females: 8.7±1.3; p<0 .05) than for control and central cooling. Central RPE was only changed for females following peripheral cooling. Changes in cadence and step length together with the effect of low skin and leg temperatures resulted in higher local RPE for females after central cooling (9.6±1.2; p<0.05) than control (9.4±1.9) and peripheral cooling (8.9±1.2 ). Males and females rated the same ambient temperature during the same exercise lower after peripheral cooling (males: 4.6±1.5; females : 5.3±1.3) than control values and lower still after central cooling (males: 3. 8±1.8; females: 2 .7±l. 5) In this study T[subscript]s[subscript]k was the primary determinant of PTS after precooling.
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Cryopreservation in suspension is commonplace for a variety of cell types. However, cryopreservation of adherent cells has achieved limited success. This research aimed to cryopreserve adherent nerve cell networks in vitro in a manner that preserved network morphology and physiology. Successful implementation would enable long term storage of adherent neuronal networks on microelectrode arrays and on-demand access for use in pharmacological and toxicological testing. Based upon morphological assessments, excellent post-thaw preservation was obtained and post-thaw cultures survived in a transitional medium for up to 3.5 hours. However, transitions to native culture medium post-thaw presented difficulties, ultimately resulting in necrosis. A discussion of methods to supplement the current research and increase post-thaw viability is included in the thesis.

In an effort to minimize the harmful effects of intracellular ice formation (IIF) during cryopreservation of confluent tissues, computer simulations based on Monte Carlo methods were performed to predict the probability of IIF in confluent monolayers during various freezing procedures. To overcome the prohibitive computational costs of such simulations for large tissues, the well-known Johnson-Mehl-Avrami (JMA) model of crystallization kinetics was implemented as a continuum approximation of IIF in tissues. This model, which describes nucleation, growth, and impingement of crystals in a supercooled melt, is analogous to the process of intracellular ice formation and propagation in biological tissues. Based on the work of Weinberg and Kapral (1989), the JMA model was modified to account for finite-size effects, and was shown to predict accurately the results of freezing simulations in 1-D tissue constructs, for various propagation rates and tissue sizes. An initial analysis of IIF kinetics in 2-D tissues is also presented. The probability of IIF in 2-D liver tissue was measured experimentally during freezing of HepG2 cells cultured in monolayers, and compared to Monte Carlo simulations and predictions of the continuum model. The Avrami coefficient and exponent for IIF in HepG2 tissue were estimated to be k = 0.19 and n = 0.45.

Plusieurs composés présents dans les solutions de cryoconservation pour embryons sontsource de préoccupations : soit du point de vue sanitaire à cause de leur origine animale, soitdu fait de leur rôle potentiellement mutagène, notamment pour certains cryoprotecteurspénétrants. Leur retrait ou leur substitution par des composés chimiquement définis pourraitdonc constituer une amélioration sensible des techniques de cryoconservation des embryons.C’est dans ce cadre de réflexion que s’inscrit notre travail.Souvent, la conception et la mise en oeuvre des protocoles de cryoconservation sontempiriques. Il en résulte de nombreuses variations entre les études qui rendent lacomparaison des résultats obtenus d’autant plus difficile. Dans notre démarche, deuxapproches complémentaires ont été associées :•La première, physique, s’appuie sur l’utilisation de la calorimétrie différentielle à balayage(DSC) pour standardiser la comparaison des différentes solutions de congélation lente.Ainsi, les propriétés thermodynamiques de solutions contenant un substituant défini ontété caractérisées et comparées à celles de solutions contenant des produits de référence(sérum de veau foetal ou albumine bovine sérique) ;• La seconde, biologique, a consisté à congeler des embryons de lapins produits in vivo etdes embryons bovins produits in vitro, puis à analyser les taux de survie après culture invitro et/ou transferts. Cette approche a permis d’objectiver les propriétés biologiques dessolutions ainsi définies.Nos résultats confirment qu’il est judicieux d’utiliser une approche thermodynamiquepour sélectionner des molécules chimiquement différentes des composés habituellementutilisés
Several compounds in embryo cryopreservation solutions are a source of concern:products of animal origin because of the sanitary risks, and the permeating cryoprotectantsbecause of their potential mutagenic effect. Removing or substituting these compounds withchemically defined products might improve embryo cryopreservation technics.Conception and use of cryopreservation protocols are often empirical. This empiricismleads to many variations between the studies which make a comparison between results allthe more difficult. In our study, two complementary approaches were associated:• The first approach (physical) consisted of using the differential scanning calorimetry tostandardize the comparison between different slow-freezing solutions. So, thethermodynamic properties of solutions containing a potential substitute were characterizedand compared to those obtained with solutions containing reference products (fetal calfserum and bovine serum albumin) ;• The second approach (biological) consisted of using freezing of in vivo-produced rabbitembryos or freezing of in vitro-produced bovine embryos in order to evaluate survival

Des solutions aqueuses contenant un polyalcool lineaire ou ramifie a quatre carbones ont ete etudiees a basse temperature par calorimetrie differentielle et par diffraction de rayons x pour trouver de nouveaux solutes pour la cryoconservation d'organes par vitrification totale

O Transplante de Células Tronco Hematopoiéticas (TCTH) baseia-se no princípio de infusão de Células Tronco Hematopoiéticas (CTH) CD34 + num receptor condicionado. Sabe-se que tais células são capazes de gerar uma nova hematopoiese bem como que o benefício do aumento de dose de CD34 + estende-se de aproximadamente 2 x 106 CD34/Kg a 5 x 106 CD34/Kg do receptor. A determinação do número de CD34 baseia-se no número total de CD34, porém sabe-se que apenas uma pequena porção celular do grupo de CD34 está associada à recuperação medular. Uma maneira de refinar a detecção desse grupo de células quando comparado com a quantificação do CD34 total, é a análise citométrica dos padrões de tamanho e granularidade citoplasmática. No contexto do TCTH autólogo e algumas vezes no TCTH alogênico, onde a infusão das células precursoras é feita com intervalo superior a três dias após a sua coleta, o congelamento das CTH CD34 +as faz-se necessário para que as células permaneçam viáveis. O agente crioprotetor mais comumente usado é o Dimetilsulfóxido (DMSO), que em temperaturas extremamente baixas protege as CTH da morte celular, porém em temperatura ambiente torna-se tóxico. As reações adversas do receptor no momento da infusão são atribuídas, em geral, ao DMSO. Portanto existem diferentes protocolos de congelamento de CTH que usam diferentes concentrações desse agente, com o intuito de reduzir as reações adversas e, ao mesmo tempo, evitar a morte celular decorrente de temperaturas baixas. Alguns estudos concluem que a viabilidade das CTH congeladas pode ser mantida com concentrações baixas de DMSO ou com celularidade elevada nos produtos congelados. Além do DMSO, a albumina e o Hidroxetilamido (HES) são usados como adjuvantes na proteção celular ao frio. Alguns protocolos de criopreservação usam apenas o DMSO enquanto outros indicam a necessidade do uso dos três agentes juntos. Não há consenso a respeito do protocolo de criopreservação ideal. Um expressivo número de trabalhos sugere o uso do DMSO 5 a 10% juntamente com a albumina 20% e HES 6% em enxertos com celularidade até 3 x 108 células nucleadas/ml, porém existem sugestões de protocolos diferentes desse padrão que usam o DMSO como agente único assim como concentrações menores de DMSO. Com relação ao efeito tóxico do DMSO, os eventos adversos do paciente no momento da infusão são atribuídos diretamente a ele. Porém evidências recentes apontam outras possíveis causas: citocinas liberadas durante o período de armazenamento e infundidas com o enxerto e micro-agregados leucocitários decorrentes da elevada celularidade do enxerto. O presente estudo pretendeu verificar a viabilidade das CTH através da medida da Anexina-V e 7-AAD pela técnica de citometria de fluxo usando-se diferentes concentrações de DMSO, bem como comparou tal desfecho entre amostras que foram congeladas apenas com DMSO e amostras congeladas com DMSO, HES e albumina. Além disso, foram verificados os níveis de citocinas inflamatórias nos enxertos e sua relação com a concentração de DMSO.
Bone Marrow Transplantation (BMT) is based on the principle of hematopoietic CD34 stem cells infusion in a conditioned recipient. It is known that such cells are capable of generating a new hematopoiesis, as well as known that the benefit of the increase of CD34 dose is goes best when it is between from 2 and to 5 x 106 CD34 by patient’s body weight. The CD34 determination is based on the total CD34 number. However it is known that only a small portion of the CD34 population is related to the marrow repopulating capacity. An alternative way of detecting such cells when compared to total CD34 dose is the cytometric analysis of citoplasmatic granularity and size patterns. In the autologous BMT context and, sometimes in the allogeneic type where the graft infusion occurs more than three days after the graft harvest, the CD34 cells freezing is necessary to keep their viability. The crioprotective agent most commonly used is the DMSO, which at extremely low temperatures this agent protects the CD34 cells from dying. However at temperatures above 30°C it became toxic and causing adverse events in the patient receiving them. The adverse reactions at the infusion moment in general are linked to DMSO, thus there are some different cryopreservative protocols using different DMSO concentrations aiming to reduce the incidence of collateral damage and at the same time avoid the CD34 from dying at low temperatures. Some studies conclude that CD34 viability can be maintained with the use of high mononuclear cellularity in the graft or with low DMSO concentrations. Besides DMSO, albumin and Hydroxietilstarch (HES) are cryoprotectants adjuvants. Some Cryopreservation protocols only use DMSO as cryoprotective agent, while others indicate the three agents together. There is no consensus about the ideal cryopreservation protocol. An important number of studies suggests the use of DMSO 5 to 10% with albumin 20% and HES 6% in grafts with cellularity number of no more than 3 x 108 cells/ml, however there are some new different protocols that use DMSO alone and at lower concentrations. As to adverse events during the infusion moment, it is known that DMSO causes the effects. However, there are evidences that other causes could cause these events: cytokines released during the storage freezing time and infused with the graft and leukocyte micro-clots caused by the high cellularity in the graft. The present study aimed to study the viability of CD34 cells using the Annexin-V and 7-AAD apoptosis reagents through the flow cytometry technique in different DMSO concentrations as well as compared such outcome among the samples only frozen with DMSO and samples frozen with DMSO, HES and albumin. Besides, it was verified the inflammatory cytokine levels and their relationship with the DMSO concentration.

La cryopréservation, dans le domaine de la reproduction médicalement assistée, constitue depuis de nombreuses années une branche suscitant beaucoup d’intérêts et d’espoirs. En effet, de nombreuses équipes de recherche se sont attelées à mettre au point et à améliorer des protocoles permettant de conserver les gamètes, les embryons et les tissus reproducteurs.

Malgré le fait que la cryopréservation soit une technique très attractive, elle peut avoir des effets délétères sur les cellules. Les protocoles expérimentaux visent donc à minimiser ces effets afin d’augmenter la survie et la compétence cellulaire après décongélation.

Les deux méthodes les plus utilisées, la congélation lente et la vitrification, présentent chacune des avantages et des inconvénients. En effet, la première ne permet pas d’éliminer la cristallisation intracellulaire. Quant à la seconde, elle empêche la formation de cristaux de glace mais pourrait provoquer une toxicité due à la forte concentration des cryoprotecteurs.

Cette thèse de doctorat propose plusieurs objectifs :

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Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

A numerical model for describing the kinetics of intracellular water transport during cryopreservation was developed. As ice is formed outside the cell, depleting the extracellular liquid of water, the cell will experience an osmotic pressure difference across its membrane, which causes cell dehydration and concomitant shrinkage. Although Mazur (1963) has previously modeled this phenomenon as a two-compartment system with membrane limited transport, the assumption of well-mixed compartments breaks down at large Biot numbers. Therefore, we have developed a numerical solution to this moving-boundary problem, including diffusive transport in the intracellular liquid, in addition to the osmotically driven membrane flux. Our model uses a modified Crank-Nicolson scheme with a non-uniform Eulerian-Lagrangian grid, and is able to reproduce predictions from Mazurs model at low Biot numbers, while generating novel predictions at high Biot numbers. Given that cell damage may result from excessive water loss, our model can be used to predict freezing methods that minimize the probability of cell injury during the cryopreservation process.

De facon a optimiser la production et la commercialisation du parasite encarsia formosa, des experiences ont ete realisees concernant: le changement de substrat des pupes noires: il ne perturbe pas le potentiel biotique du parasite et l'ajout de sucre permet d'augmenter l'efficacite de l'encarsia; le stockage au froid: il a un effet nocif sur encarsia; son taux de mortalite s'accroit avec la duree de stockage et avec l'abaissement des temperatures. Malgre des taux d'emergence performants, les parasites issus de ces pupes sont affaiblis en ce qui concerne la fecondite et la longevite. L'apport de sucre permet de pallier l'effet nocif du froid. L'acclimatation d'encarsia aux basses temperatures a ete mesuree par dosage du glycerol en chromatographie en phase gazeuse, en fonction des differents types de stockage au froid et a trois epoques de l'annee. Il n'existe pas de variation significative dans la synthese de cette substance en fonction de la duree de stockage mais cette synthese diminue significativement avec l'abaissement des temperatures uniquement pour les pupes elevees en jours longs. Par ailleurs, l'action de la photoperiode est predominante au niveau des potentialites biotiques de cet insecte mais le stress du au froid aplanit cette variation, lorsque le stockage se prolonge (20 jours a 9c)

Des bourgeons apicaux de vitroplants ont été utilisés comme source d'explant pour la régénération par embryogenèse somatique du palmier dattier (Phoenix dactylifera L. ). Le recyclage de vitroplants permet de disposer en permanence et à moindre coût du matériel végétal nécessaire au clonage. L'effet de la qualité de la lumière, lors de la phase d'induction de l'embryogenèse somatique, a été étudiée avec cette nouvelle source d'explant. Des éclairements de lumière rouge clair peuvent favoriser l'embryogenèse somatique à condition qu'ils soient appliqués à un certain stade de développement des cals, après que ceux-ci aient été cultivés à l'obscurité. La période de sensibilité aux éclairements rouge clair est à déterminer pour chaque génotype. Les effets de la lumière rouge clair peuvent en partie être réversés par la lumière rouge sombre, ce qui est en faveur de l'implication du phytochrome dans ce processus. La culture en milieu liquide avec immersions temporaires a été testée grâce au système RITA R. Les résultats sont très variables selon le stade de développement des embryons somatiques. Dans les conditions utilisées, ce mode de culture ne favorise pas l'embryogenèse lors de l'étape d'induction. Durant la phase de multiplication des embryons somatiques, avec deux immersions d'une minute par jour, on observe une augmentation de la quantité de matière fraîche accompagnée de la maturation des embryons. Une méthode de cryoconservation a été mise au point dans le but de disposer d'un outil de gestion de la production et d'un mode de conservation des ressources génétiques pour le palmier dattier. Cette méthode repose sur la déshydratation osmotique de massifs d'embryons somatiques lors d'une préculture sur un milieu enrichi en saccharose (0,75 M), un réchauffement rapide puis un post-traitement sur des milieux progressivement appauvris en saccharose et enrichis en 2,4-D. Les massifs avant cryoconservation ont une teneur en eau voisine de 3gH2Og-1MS. Ce protocole n'est applicable que sur les massifs en multiplication. Seul le saccharose parmi d'autres hydrates de carbone testés (mono, di, triholosides et polyols) permet de conférer une résistance à la cryoconservation aux massifs d'embryons. La préculture induit une synthèse de raffinose au niveau des tissus. La cristallisation du milieu intracellulaire ne s'est pas révélée létale pour les cellules.

Très répandus sur terre, les biotopes «froids» sont habités, notamment par des bactéries appelées psychrophiles (« qui aiment le froid »). Afin d'étudier l’adaptation au froid, nous comparons la bactérie Escherichia coli incapable de croître en dessous de 8°C («mésophile») aux deux -protéobactéries bactéries psychrophiles Colwellia psychrerythraea et Pseudoalteromonas haloplanktis qui, elles, se développent encore en dessous de 0°C. Actuellement, on ne sait pas comment les activités biologiques clef impliquant l’ARN sont adaptées au froid. La plupart d’entre elles nécessitent des interconversions de structures qui, en l’absence de catalyse, deviendraient très lentes à froid. D'ailleurs, chez E. Coli plusieurs des ARN hélicases, qui sont supposées catalyser ces interconversions, sont facultatives à 37°C mais essentielles à 20°C. Nous avons comparé les propriétés enzymatiques de certaines de ces hélicases à celles de leurs orthologues provenant de C. Psychrerythraea et P. Haloplanktis. Nous observons que les hélicases psychrophiles ont des énergies d'activation plus basses et donc sont de meilleurs catalyseurs à froid. Cette propriété, entre autres, participerait à l'adaptation au froid des organismes psychrophiles. Nous observons qu’à 10°C, l'élongation de la traduction et la dégradation des ARNm sont fonctionnelles chez ces trois organismes, mais que le démarrage de la traduction est bloqué chez E. Coli. Nous avons examiné certaines causes de ce blocage, qui semble responsable de l’arrêt de sa croissance à basse température.

Couramment utilisées dans les laboratoires du monde entier, les techniques de cryoconservation issues des recherches en cryobiologie sont aujourd’hui devenues des outils incontournables dans plusieurs domaines tels que la biomédecine et l’industrie agroalimentaire. À partir de l’étude des écrits du biologiste anglais Alan Sterling Parkes (1900-1993), ce mémoire propose une reconstruction historique et épistémologique des avancées en cryobiologie. Pour ce faire, nous examinerons, dans un premier temps, les premières observations qui ont avivé l’intérêt pour l’étude des effets du froid sur les organismes vivants. Nous prendrons comme point de départ les découvertes fascinantes des naturalistes du XVIIe siècle. Nous verrons que leurs constats sont souvent étroitement associés à d’autres épisodes majeurs de l’histoire de la pensée biologique. Nous aborderons ensuite la période plus prolifique concernant les avancées en biologie aux basses températures. La découverte des propriétés protectrices du glycérol par l’équipe du National Institute of Medical Research (NIMR) au milieu du siècle dernier marque un point de rupture dans l’histoire de cette spécialité. Ce cryoprotecteur permet en effet d’élargir considérablement la gamme de tissus et de cellules pouvant être cryoconservés. Les développements orchestrés par les chercheurs anglais ont, à cet égard, contribué à l’émergence d’une nouvelle discipline scientifique : la cryobiologie. Enfin, nous étudierons dans une dernière section de ce mémoire le contexte sociohistorique dans lequel ont eu lieu les premières applications pratiques des méthodes de cryoconservation. Cela nous conduira à analyser, en particulier, certains enjeux soulevés par l’implantation de ces techniques dans le champ de la médecine reproductive. Ce mémoire aura non seulement permis de retracer certains évènements qui ont jalonné l’histoire de la cryobiologie au fil du temps, mais aussi de mettre en valeur les contributions fondatrices de Parkes et son équipe tout en montrant le rôle de premier plan qu’elles ont joué dans la structuration de cette discipline.
With a panoply of applications ranging from therapeutics to everyday processes in the biomedical and agricultural industry, cryobiology-derived techniques have become essential to many present-day human activities. In this master’s thesis, I explore the history and epistemology of cryobiology through the lens of English biologist Alan Sterling Parkes (1900-1990). I will start by looking at the very first observations made by naturalists in the XVIIth century on the effects of low temperatures on living organisms and how these have been essential to the field of cryobiology. Those observations, as we will see, are also linked to other major episodes in the history of biology. From there, I will trace the early days of cryobiology, with a particular focus on a major scientific breakthrough: the discovery of protective properties of glycerol by Parkes and his colleagues at the National Institute of Medical Research (NIMR) at the end of World War II. This new cryoprotective agent enabled the preservation of living tissues and cells, thus revolutionizing the field of biology at low temperatures as well cementing cryobiology as a brand-new research field of its own. Finally, I will analyze the socio-historical context of cryobiology’s first practical applications, most notably in the field of reproductive medicine. This work aims at reconstructing the history of cryobiology and highlights the contribution of Parkes and his NIMR colleagues while also showing the role these played into structuring the emerging field.

Ces travaux concernent l'étude cristallographique de complexes ternaires de la Glycéraldéhyde-3-phosphate déshydrogénase phosphorylante de B. Stearothermophilus. Cette enzyme catalyse la phosphorylation oxydative du Glycéraldéhyde-3-phosphate en présence d'un cofacteur, le NAD, et de phosphate inorganique. Pour fixer les groupements phosphates de ces deux substrats, l'enzyme possède deux sites de reconnaissance anionique, dénommés respectivement Pi et Ps. Cependant, la façon dont ces deux sites contribuent à la fixation des deux groupements phosphate reste encore soumise à controverse et différents modèles ont été proposés. Afin de tester la validité des ces modèles, il semblait essentiel de pouvoir disposer d'évidences structurales directes. Deux mutants ont été étudiés : le mutant C149A, inactif et le mutant C149S, très faiblement actif. Des cristaux de ces deux mutants ont été obtenus dans des conditions originales, exemptes de tout anion. Ces nouvelles conditions de cristallisation ont conduit à de nouveaux empilements cristallins. L'analyse des structures des complexes binaires met en évidence des sites Pi libres de tout anion. Par contre, les sites Ps sont occupés par un anion sulfate issu probablement de la purification. Ceci montre que le site Ps a une meilleure affinité que le site Pi vis-à-vis des anions. Ce résultat a été confirmé par l'analyse des acides aminés impliqués dans la formation des sites Pi et Ps et par le fait que le potentiel électrostatique local est plus important au niveau du site Ps qu'au niveau site Pi. Les complexes ternaires que nous avons obtenus constituent la première structure tridimensionnelle d'une GAPDH en complexe avec son substrat physiologique. L'analyse de ces complexes a permis d'apporter des informations quant aux interactions enzyme-substrat. Nous montrons sans ambigüité que dans les deux complexes obtenus, le groupement phosphate du G3P est localisé dans le site Ps. De même, les facteurs moléculaires responsables de la stéréosélectivité vis-à-vis du D-G3P ont pu être élucidés. Cependant, le débat reste ouvert concernant, d'une part, la contribution de la boucle 205-210 à la catalyse et d'autre part, la conséquence de la formation de la liaison covalente entre l'enzyme et le substrat sur le positionnement des intermédiaires réactionnels dans le site actif.

L'objectif de ce travail est d'analyser la mortalité cellulaire au cours de la congélation en intégrant la vitesse de refroidissement. Après le développement de techniques de refroidissement sur une gamme de vitesses s'étendant de 5 à 30000°C/min. , la viabilité cellulaire a été déterminée, puis le volume des cellules a été mesuré par analyse d'image et microscopie électronique après cryosubstitution. Enfin les milieux de congélation ont été caractérisés par analyse enthalpique différentielle. Ce travail a été mené essentiellement sur S. Cerevisiae et a été confirmé en utilisant d'autres modèles cellulaires : levure (C. Utilis) , bactéries (E. Coli, L. Plantarum) et cellules humaines (cellules leucémiques, globules rouges). Il a été montré que la cristallisation de l'eau est à l'origine de la mort des cellules : (i) Pour des vitesses de refroidissement allant jusqu'à 1800°C/min, la cristallisation est exclusivement extracellulaire, ce qui entraîne une déshydratation des cellules. La forte viabilité cellulaire mesurée dans cette gamme de vitesse de refroidissement correspond à une vitesse de déshydratation optimale. (ii) ) Pour des vitesses de refroidissement supérieures à 1800°C/min, une cristallisation se produit dans la cellule. Celle-ci est léthale si elle coïncide avec des mouvements d'eau transcellulaire. Pour des vitesses de refroidissement ultrarapides qui induisent de très faibles variations de volume cellulaire par la cristallisation et/ou vitrification quasi-instantanée des cellules, la viabilité cellulaire est préservée. Dans ce cas, la vitesse de réchauffement doit être optimisée afin d'éviter la recristallisation intracellulaire et préserver la survie cellulaire.

Geotrichum candidum, micro-organisme ubiquiste, est un champignon levuriforme thallosporé utilisé essentiellement comme ferment d’affinage et d’aromatisation en industrie fromagère. Notre projet a consisté en l'étude de la sensibilité à la congélation de G. Candidum, afin de développer un modèle d'étude de la cryoconservation de la biodiversité intra-espèce des micro-organismes d’intérêt laitier et d’optimiser la congélation comme technologie de pérennisation. Cette espèce polymorphe, peut présenter un aspect filamenteux de type moisissure, un aspect levuriforme, majoritairement constitué d’arthrospores ou un aspect intermédiaire. Nos travaux ont permis de mettre en évidence une variabilité au niveau du comportement physiologique (croissance et cryotolérance) liée aux variabilités structurelles associées aux aspects phénotypiques de G. Candidum. Le rapport UFT x ml-1 / DO600nm apporte à ce sujet une indication originale sur la capacité de sporulation des souches de G. Candidum, tant au niveau quantitatif que temporel. L’étude de la cryorésistance a nécessité la mise au point d’un protocole de refroidissement afin de contrôler le comportement stochastique de la congélation. Ce protocole associe l’addition d’un agent nucléateur (Snomax®) aux échantillons expérimentaux, à un procédé de refroidissement par effet Peltier. L'addition du nucléateur conduit à trois effets indépendants mais complémentaires : (i) la synchronisation du déclenchement de la nucléation, entraînant une congélation homogène des échantillons testés, (ii) l’augmentation, dose dépendante, de la température correspondant au pic de surfusion et (iii) la diminution de la mort cellulaire provoquée par les cycles de congélation/décongélation. La variabilité intra-espèce au niveau moléculaire de la réponse au stress à basse température positive a été évaluée en se basant sur une approche protéomique comparative. Nous avons utilisé pour cela une méthode d’analyse protéomique différentielle sur gel (2D DIGE), basée sur le marquage des protéines avec des cyanines fluorescentes. Nos résultats ont permis de mettre en évidence des différences significatives entre les profils protéomiques des souches soumises à un stress hypothermique. Ces protéines analysées par spectrométrie de masse n’ont pas montré d’homologie avec les séquences répertoriées dans les banques de données
Geotrichum candidum, ubiquitous microorganism, is a filamentous yeast-like fungus mainly used as ripening starter in cheese industry. Our project has consisted to study the crytolerance of G. Candidum used as a model (i) to develop our knowledge on the cryopreservation of the intraspecies microbiodiversity in dairy science and (ii) to optimize the frozen storage technology. This polymorphic species show different morphotypes: "yeast" type strains that produce numerous arthrospores, "mould" type strains characterized by a preponderance of hyphae, and "intermediate" type strains. Our results have led to highlight a physiological variability (growth and cryotolerance) related to the structural specificities which are associated to the phenotypic aspects of G. Candidum. The TFU x ml-1 /OD600nm ratio provide interesting informations on the sporulation of G. Candidum strains, quantitatively and temporally. The cryotolerance experiments have required the development of a cooling protocol in order to control the stochastic behaviour of freezing. This protocol combines the addition of a nucleator (Snomax®) with a cooling process using Peltier effect. The nucleator addition led to three independent but complementary effects: (i) synchronization of the different samples nucleation, leading to a homogeneous and earlier freezing, (ii) increase of the freezing point temperature, (iii) significant decrease of the lethality of G. Candidum subjected to a freezing-thawing cycles challenge. Intraspecies variability of the molecular response to cold stress was studied using a comparative proteomic approach by two-Dimensional Differential In Gel Electrophoresis (2D DIGE). This technology is based on the labelling of proteins by specific fluorescent CyDyes. Our results showed significant differences between the proteomic profiles of the G. Candidum strains subjected to cold stress. Selected proteins analyzed by mass spectrometry did not show any homology with the sequences indexed in protein databases

Les vertébrés ectothermes ont développé deux stratégies de tolérance au froid leur permettant de survivre aux contraintes thermiques imposées par l'hiver : la tolérance au gel et l'évitement au gel (par l'augmentation des capacités de surfusion). Cette étude s'articule sur deux axes principaux. D'une part, je me suis attaché à approfondir les connaissances physiologiques de tolérances au froid d'un vertébré ectotherme européen : le lézard vivipare (Lacerta vivipara) dont la répartition géographique dépasse le cercle polaire arctique et qui possède la capacité rare d'utiliser les deux stratégies au cours d'un même hiver. Les résultats apportent des éléments de compréhension quant à la balance métabolique de cet animal pendant l'hiver, sa tolérance au gel (la plus importante parmi les squamates et son métabolisme aérobie en état de surfusion et de congélation. Je me suis ensuite attaché à intégrer les stratégies de tolérance au froid dans un contexte plus écologique et évolutionniste. Afin d'apporter une tentative d'explication quant aux existence des stratégies, un modèle d'optimisation de fitness a été développé en tenant compte de différents paramètres physiologiques (comme les réserves énergétiques et le stress associé à chaque stratégie) et les conditions environnementales (température et nombre de jours de froid). Les résultats théoriques, discutés à la lumière de données physiologiques concernant différentes espèces d'ectothermes, permettent d'émettre des prédictions quant à l'influence de la physiologie dans l'évolution des stratégies de tolérance au froid des ectothermes et suggèrent des voies d'expérimentations, principalement liées à l'influence du stress sur l'évolution des stratégies.

La fertilité de garçons prépubères soumis à un traitement fortement gonadotoxique pourrait être préservée en ayant recours à une biopsie de tissu testiculaire en vue d'une transplantation autologue après guérison. Une telle stratégie n'est toutefois envisageable que si l'on dispose d'un protocole de cryoconservation permettant de préserver de façon satisfaisante l'intégrité structurale et fonctionnelle des tissus prélevés. Nous avons développé un modèle murin de congélation de tissu testiculaire immature. Nos travaux ont permis de démontrer que l'évaluation de l'intégrité structurale du tissu testiculaire et de la survie des cellules germinales après décongélation permettaient d'identifier rapidement les conditions de congélation les plus favorables. Différents paramètres tels que la nature du cryoprotecteur utilisé, sa concentration, le temps et la température d'équilibration du tissu avec le milieu de congélation ou le type de descente en température ont été évalués. Une préservation optimale du tissu a été obtenue en utilisant du DMS01,5M, un temps d'équilibration de 15 minutes et une descente en température contrôlée lente sans seeding. Ce protocole a ensuite été validé par une évaluation de l'intégrité fonctionnelle du tissu cryoconservé après 9 jours de culture organotypique, ou 4 à 5 mois de maturation in vivo sur le dos de souris ayant reçu une allogreffe. Après maturation in vitro, la prolifération cellulaire, la croissance des tubes séminifères, l'activité hormonale des cellules de Leydig et la différenciation de cellules germinales étaient comparables à celles du tissu frais. Pour ce qui est des tissus greffés, des taux de différenciation des cellules germinales comparables à ceux observés pour le tissu non congelé ont été obtenus. Des spermatozoïdes ont ainsi pu être produits après allogreffe de tissu immature cryonconservé. Les conditions optimales définies chez la souris ont ensuite été utilisées afin de préserver des fragments de tissu testiculaire provenant de patients suivis au sein de notre équipe. Une analyse histologique des prélèvements a été effectuée avant et après cryoconservation. La maladie et ou les traitements antérieurs semblent avoir un effet délétère plus marqué sur les tissus provenant des patients les plus âgés (entre 11 et 16 ans). Pour ces patients, une absence totale de différenciation des cellules germinales s'accompagne également d'une diminution de leur nombre, associée dans certains cas à un ralentissement de la croissance des tubes et à une fibrose péritubulaire et de l'espace interstitiel. La cryoconservation entraîne une faible augmentation des altérations morphologiques affectant les tubes séminifères. Plus de 75% d'entre eux demeurent intacts, même si l'on observe une diminution moyenne de leur taille de l'ordre de 11%. La maladie, les traitements et la cryoconservation peuvent ainsi être à l'origine d'altérations structurales du tissu testiculaire humain immature. Il conviendra, lorsque des cellules germinales sont présentes, d'en mesurer l'impact sur son intégrité fonctionnelle après maturation in vitro ou xénogreffe avant d'envisager une utilisation dans le cadre d'une application clinique. De nouveaux paramètres de congélation devront être évalués, chez l'animal, afin d'optimiser le protocole existant.

Ce travail est consacre a l'etude des transferts de chaleur et de masse dans un procede de congelation de cellules en suspension. Une approche macroscopique est utilisee pour la validation d'une methode simplifiee de congelation des cellules souches hematopoietiques. L'etude experimentale et numerique est entreprise afin d'identifier les parametres influencant la descente en temperature des poches de cellules placees dans un congelateur a 80\c. A partir des conclusions de cette etude, un protocole est propose. La deuxieme partie de ce travail porte sur le developpement d'un modele elements finis permettant d'etudier la reponse osmotique d'une ou plusieurs cellules lors de la propagation d'un front de solidification. A cette fin, les equations de la chaleur et de la diffusion sont resolues dans un domaine bidimensionnel avec une technique de suivi de front pour suivre les deplacements du front de solidification et de la membrane. L'interaction entre un front de glace et des cellules est etudiee en fonction des vitesses de refroidissement et de la position initiale des cellules.

Les embryons bovins produits in vitro sont plus sensibles à la cryoconservation que ceux produits in vivo, en partie à cause de leur contenu lipidique, triglycérides et phospholipides. L’objectif de ce travail visait à comprendre les mécanismes moléculaires responsables de cette différence. Le profil transcriptomique de gènes impliqués dans le métabolisme lipidique a été établi. Le niveau d'expression génique de l’adipophiline obtenu indique qu’il peut être un marqueur spécifique de l'accumulation des triglycérides et de la cryorésistance des embryons. Ainsi, l’accumulation des triglycérides pourrait être liée à une absence de dégradation des lipides et non à une synthèse de novo uniquement. L’ajout d’acides gras polyinsaturés, C18:2, C18:3 ou DHA dans le milieu de développement, a régulé l'expression génique de SCD1 et de FADS2, deux enzymes qui désaturent les lipides, et ce, probablement via la régulation de SREBP1, ce qui pourrait être en lien direct avec les modifications de la balance acides gras saturés / insaturés et jouer sur la fluidité membranaire et la cryorésistance
In vitro produced embryos are more sensitive to cryopreservation than those in vivo derived, partly because of their fat content, triglycerides and phospholipids. The objective of this work was to understand the molecular mechanisms responsible for this difference. mRNA expression of genes involved in lipid metabolism has been established. Results of adipophilin mRNA level indicates that it maybe a specific marker for triglycerides accumulation and embryo cryorésistance. Thus, triglyceride accumulation could be related to a lack of lipids degradation rather than new lipids synthesis only. Polyunsaturated fatty acids supplementation, C18: 2 C18: 3 or DHA in culture media regulated mRNA expression of SCD1 and FADS2, two enzymes involved in lipids desaturation, probably through SREBP1 regulation, which could be directly linked to changes in the balance of saturated / unsaturated fatty acids and could contribute to change membrane fluidity and embryo cryoresistance

La thèse contient trois parties principales, la coordination du Cu, l'inhibition par le Zn, et le lointain infrarouge. Les vibrations de complexes de cuivre ont été étudiées en moyen et lointain infrarouge. Les spectres des complexes Cu- poly-L- histidine ont été enregistrés en fonction du pH en moyen et lointain infrarouge. De même, les vibrations métal- ligand ont été observées dans le lointain infrarouge. La coordination du Cu par l'amyloïde- beta16 est une étape déterminante dans l'apparition de la maladie d'Alzheimer. Les complexes cuivre- amyloïde- beta16 ont été étudiés dans le moyen infrarouge à différentes valeurs de pH. L'utilisation d'échantillons marqués isotopiquement pour cette étude a permis de déterminer les modes de coordination du cuivre. Dans la deuxième partie de la thèse, nous avons utilisé le moyen infrarouge pour étudier des protéines de la chaîne respiratoire. Les cations Zn2+ sont connus pour leur pouvoir inhibiteur vis-à-vis du pompage de protons par les enzymes respiratoires. Pour mieux comprendre l'effet de l'inhibition par le Zn sur les complexes III et IV, la spectroscopie différentielle IRTF induite par l'électrochimie a été utilisée. L'étude montre que le chélation du Zn par le complexe III a lieu via le résidu E295. L'inhibition du complexe IV se fait probablement dans deux sites de chélation. On a montré que le résidu E78 de la sous-unité II intervient dans la chélation du Zn. Enfin, le lointain infrarouge a été développé, y compris une approche électrochimique. Le domaine du lointain infrarouge offre un outil pour observer les vibrations métal- ligand, la signature amide VI et la signature des liaisons hydrogène
The thesis is constituted of three main parts, the Cu coordination, the Zn inhibition and the far infrared. The vibrations of the Cu-poly-L-Histidine complexes have been studied in the mid and far infrared ranges as a function of pH. The Cu coordination by the amyloid-beta 16 peptide is a critical step in the development of Alzheimer' s. La coordination du Cu par l'amyloïde- beta16 est une étape déterminante dans l' apparition de la maladie d'Alzheimer. The spectra of the Cu-amyloid-beta16 complexes have been recorded in the mid infrared domain and the use of isotopically labelled samples allowed revealing the coordination sphere of Cu within the amyloid-beta16. Ln the second part, the mid infrared domain was used to study the respiratory chain enzymes. Zn cations are known to inhibit the proton pumping by the respiratory complexes. To better understand the effect of the inhibition by Zn on the complexes III and IV, the FTIR difference spectroscopy was used. The data shows that the chelation of Zn by the complex III takes places via E295 residue. The inhibition of the complex VI takes place via two binding sites, one of theme corresponds to the E78 residuee of the subunit JI. Finally, the far infrared spectroscopy of proteins has been developed. This spectral domain offers a unique tool to observe the metal-ligand vibration, the amide VI band, as weil as the hydrogen bonding signature of proteins

Plusieurs composés présents dans les solutions de cryoconservation pour embryons sontsource de préoccupations : soit du point de vue sanitaire à cause de leur origine animale, soitdu fait de leur rôle potentiellement mutagène, notamment pour certains cryoprotecteurspénétrants. Leur retrait ou leur substitution par des composés chimiquement définis pourraitdonc constituer une amélioration sensible des techniques de cryoconservation des embryons.C'est dans ce cadre de réflexion que s'inscrit notre travail.Souvent, la conception et la mise en oeuvre des protocoles de cryoconservation sontempiriques. Il en résulte de nombreuses variations entre les études qui rendent lacomparaison des résultats obtenus d'autant plus difficile. Dans notre démarche, deuxapproches complémentaires ont été associées :*La première, physique, s'appuie sur l'utilisation de la calorimétrie différentielle à balayage(DSC) pour standardiser la comparaison des différentes solutions de congélation lente.Ainsi, les propriétés thermodynamiques de solutions contenant un substituant défini ontété caractérisées et comparées à celles de solutions contenant des produits de référence(sérum de veau foetal ou albumine bovine sérique) ;* La seconde, biologique, a consisté à congeler des embryons de lapins produits in vivo etdes embryons bovins produits in vitro, puis à analyser les taux de survie après culture invitro et/ou transferts. Cette approche a permis d'objectiver les propriétés biologiques dessolutions ainsi définies.Nos résultats confirment qu'il est judicieux d'utiliser une approche thermodynamiquepour sélectionner des molécules chimiquement différentes des composés habituellementutilisés.

Les milieux de cryopréservation d'embryons contiennent généralement des produits d'origine animale, qui présentent des inconvénients majeurs : une composition variable et insuffisamment définie, et un risque de transmission d'agents pathogènes. La substitution de ces produits par des composés synthétiques chimiquement définis pourrait contribuer à l’amélioration des procédures de cryopréservation d’embryons, en réduisant la variabilité de composition des milieux, et le risque de contamination des ressources conservées.L’objectif de ce travail était d’évaluer l’effet du remplacement de l’albumine sérique bovine utilisée dans les milieux de congélation lente ou de vitrification d’embryons cunicoles et ovins par un milieu synthétique formulé à base d’acide hyaluronique (STEM ALPHA.Cryo3 – « CRYO3 »). Dans un premier temps, les propriétés thermodynamiques des substituts potentiels ont été évaluées à l’aide de la calorimétrie différentielle à balayage. Parallèlement, nous avons optimisé les différents outils expérimentaux dont nous avions besoin pour cette étude. Nous avons adapté un protocole d’évaluation de l'activité mitochondriale (JC-1) pour complémenter l'évaluation morphologique in vitro des embryons de lapin, et nous avons évalué l’efficacité de différents protocoles de superovulation sur la production d’embryons chez la brebis. Dans un second temps, nous avons procédé à la substitution de l’albumine sérique bovine (BSA) utilisée dans des milieux de congélation lente et de vitrification d’embryons cunicoles et ovins par du CRYO3. Une approche in vitro a été utilisée pour les protocoles de congélation et de vitrification, puis complétée par une approche in vivo pour les protocoles de vitrification.Nos résultats confirment que la BSA peut être efficacement remplacée par le CRYO3 dans des protocoles de cryoconservation d‘embryons de lapin et d’embryons ovins, qu’il s’agisse de congélation lente ou de vitrification
Embryo cryopreservation media usually contain animal-derived products, such as bovine serum albumin (BSA). These products present two major disadvantages: an undefined variable composition and a risk of pathogen transmission. The substitution of animal products of embryo cryopreservation media by synthetical products may improve procedure standardization (by avoiding variability in media composition) and avoid sanitary concerns inherent to animal-derived products.We aimed to evaluate the effect of replacing BSA in rabbit and ovine embryo slow freezing and vitrification media with a synthetic animal products free medium composed of synthetic hyaluronic acid: STEM ALPHA.Cryo3 (« CRYO3 »).During the first part, we evaluated the substitution candidates through a thermodynamic approach, using differential scanning calorimetry. In paralel, we adapted a mitochondrial activity evaluation protocol (JC-1) to rabbit embryo, which allowed us to complement morphological in vitro evaluation, and evaluated ewe superovulation protocols.During the second part, we used a biological approach to evaluate the replacement of BSA with synthetical products (containing CRYO3) in rabbit and ovine embryo slow freezing and vitrification media, using in vitro (slow freezing and vitrification) and in vivo (vitrification) evaluation methods.Our results seem to demonstrate that the chemically defined substitute CRYO3 can successfully replace BSA during rabbit embryo and ovine embryo cryopreservation (slow-freezing and vitrification)

Thesis (Ph. D.)--University of Alberta, 2010.
Title from pdf file main screen (viewed on Jan. 15, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Engineering and Medical Sciences, Departments of Chemical and Materials Engineering and Medical Sciences - Laboratory Medicine and Pathology, University of Alberta. Includes bibliographical references.

Thesis (Ph.D.)--University of Alberta, 2009.
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences-Laboratory Medicine and Pathology, Department of Laboratory Medicine and Pathology. Title from pdf file main screen (viewed on October 23, 2009). Includes bibliographical references.