Der Tumorsuppressor p53 spielt eine wichtige Rolle bei der Aufrechterhaltung der zellulären Homöostase und verhindert die Bildung und Entwicklung von Krebs. Frühere Studien auf Einzelzellebene haben gezeigt, der p53 nach dem Auftreten von DNS Schäden in einer Serie gleichförmiger Pulse im Kern akkumuliert. Um den Einfluss von Temperatur, dem Zustand des NFκB Weges oder dem Vorhandensein von Wachstumsfaktoren auf die p53 Dynamiken nach DNS-Schäden zu untersuchen, wurden in dieser Arbeit A549 und MCF10A p53 Reporter-Zelllinien verwendet. Um Daten mit hoher zeitlicher und räumlicher Auflösung auf Einzelzellebene zu erhalten, wurde Zeitraffer-Fluoreszenzmikroskopie verwendet. Überraschenderweise zeigten A549 Zellen, die mit γ-Strahlung behandelt wurden, eine höhere p53 Akkumulation und ein verlängertes Zeitintervall zwischen den p53 Pulsen, wenn sie bei 30°C inkubiert wurden im Vergleich zu Zellen unter physiologischen Bedingungen. Hingegen zeigten Zellen bei 40°C eine höhere p53 Pulsfrequenz. Außerdem wurde die p53 Zielgen-Expression durch die Änderungen in der Dynamik beeinflusst. In den beiden Zelllinien, A549 und MCF10A, wurde die p53 Dynamik durch Inhibierung des NFκB Signalweges verändert, nicht aber durch dessen Aktivierung mittels TNF. So verlängert die Inhibierung des NFkB Signalweges das Zeitintervall zwischen p53 Pulsen, was sich auch in der Expression von p53 Zielgenen wiederspiegelt. Weiterhin konnte in MCF10A Zellen durch Experimente mit verschiedenen Medien-Bedingungen gezeigt werden, dass p53 Dynamiken vor allem durch die Anwesenheit von EGF und Hydrocortison geprägt werden. So führt EGF zu einem pulsierenden p53 Verhalten, während Hydrocortison alleine die p53 Antwort vollständig aufhebt.. Diese Studie macht deutlich, wie wichtig es ist, das Verhältnis von zellulärem Zustand und p53 Antwort systematisch zu untersuchen, um Krebstherapien wirksamer zu machen / The tumor suppressor p53 plays important roles in maintaining cellular homeostasis and in preventing the formation and development of cancer. Previous studies on p53 activation after DNA damage have reported that it shows a series of regular discrete pulses of protein accumulation over time at the single cell level. In this work A549 and MCF10A p53 reporters cell line were used to investigate how p53 dynamics after DNA damage were affected by changes in temperature, by changes in the state of the NFκB pathway and changes in the provided growth factors. Time-lapse florescent microscopy was used to obtain single cell data with high temporal and special resolution at the single cell level. Surprisingly A549 cells treated with γ-irradiation showed higher level of p53 accumulation and increased time between p53 pulses when imaged at 30°C than cells imaged under physiological conditions. Cells imaged at 40°C showed instead higher p53 pulse frequency. P53 target gene expression was also affected by these changes in dynamics. In both A549 and MCF10A cells, p53 dynamics were changed by NFκB pathways inhibition but not activation via TNFα. Upon inhibition of the NFκB pathway the timing between p53 pulses was increased leading to changes also in p53 target genes expression. In MCF10A cells experiments done under different medium conditions proved that p53 dynamic in this cell line was shaped mainly by the presence of EGF and hydrocortisone that are usual components of the media. EGF leads to a more pulsatile p53 behavior while hydrocortisone completely abrogates the p53 response. These discoveries pointed out the need to study more systematically the relationship between the p53 response to a given stress and the cellular state in order to make cancer therapies more effective.

Despite extensive international acceptance of the critical role of mammalian post-dispersal seed predation in many plant communities, in New Zealand we have limited knowledge of these predators’ influence on plant recruitment in our forests. The principle objective of my thesis was to determine the importance of exotic mammals as post-dispersal seed predators in a New Zealand conifer-broadleaf forest remnant. To address this goal, I used a series of field-based experiments where the actions of different post-dispersal seed predators were separated by wire-mesh exclosures. My study was conducted at Mount Peel Forest Park Scenic Reserve, South Canterbury, New Zealand. Being a human modified conifer forest currently dominated by broadleaf species, it is typical of forest remnants in New Zealand. This presented an opportunity to study a wide range of both potential post-dispersal seed predators and broadleaf tree species. My findings indicate that exotic mammals are not only post-dispersal seed predators at Peel Forest, but are responsible for the majority of post-dispersal predation events observed. Ship rats (Rattus rattus) were the dominant post-dispersal seed predators, while brushtail possums (Trichosurus vulpecula), house mice (Mus musculus) and native invertebrates were also important post-dispersal seed predators for several tree species. Through use of time-lapse video and cafeteria experiments I found that exotic mammalian seed predators, when compared to native invertebrate seed predators, preyed upon larger-seeded plant species and were responsible for considerable seed losses of several tree species. However, exotic mammalian seed predators do share several foraging characteristics with native invertebrate seed predators, as predators foraged in similar habitats and responded in a similar way to changes in seed density. In investigating if post-dispersal seed predation by mammals had a flow-on effect to plant recruitment, I observed natural seedling densities at Peel Forest were significantly higher in the absence of mammalian seed predators, but I found no evidence that the presence of mammals significantly altered the overall species richness. At the community level, I did not find an interaction between habitat and exotic mammals, however I present evidence that for individual plant species a significant mammal : habitat interaction occurred. Consequently, even though my cafeteria experiment implied there was no significant difference in the overall amount of seed preyed upon within different habitats, the less favourable microsite conditions for germination under an intact continuous canopy allows mammals to exacerbate habitat-related patterns of seed mortality and have a noticeable effect on seedling establishment. In an effort to validate the use of manipulative experiments to predict the long-term effect of post-dispersal seed predation on plant dynamics, I attempted to link results of my cafeteria experiment with observed seedling abundance at Peel Forest. Seven tree species were used in this comparison and a strong correlation was observed. This result shows that the level of post-dispersal seed predation determined in the cafeteria experiment provided a good predictor of the effect of mammalian post-dispersal seed predation on seedling establishment. To fully gauge the impact of mammalian post-dispersal seed predators on seedling establishment, the relationship between these seed predators and the type of recruitment limitation experienced by a plant species was also investigated. By using a combination of seed addition, plot manipulations and seed predator exclusion I was able to investigate this relationship. I found evidence that seed limitation at Peel Forest is positively correlated with seed size, and that while mammalian post-dispersal seed predators can further reduce plant recruitment of plant species experiencing seed limitation, the influence of mammals in determining plant recruitment was limited for plant species experiencing microsite limitation. My study has proven that exotic mammals are now the dominant post-dispersal seed predators at Peel Forest, the amount of seed preyed upon varies among plant species, and post-dispersal seed predation by mammalian species can lead to differences in seedling richness and abundance. I proved that the influence of exotic mammals on seedling establishment is also linked to habitat structure and recruitment limitations. When combined these observations suggest that exotic mammalian post-dispersal seed predators may play an important role in determining landscape abundance and distribution of plants at Peel Forest.

Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques. In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level. To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs. The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary. / <p>QC 20130927</p>

La grotte de Lascaux (inscrite au patrimoine mondial de l’UNESCO et l’une des plus connues au monde) nécessite, dans le cadre de sa conservation et suite aux aléas subis depuis sa découverte, une attention particulière tant pour elle-même que pour son environnement. L’utilisation d’une méthode géophysique, la Tomographie de Résistivité électrique (TRE) qui est une méthode non intrusive par excellence, est donc proposée pour la reconnaissance du milieu karstique l’environnant.Un suivi temporel par TRE a été mise en oeuvre pour caractériser l’amont hydraulique de la grotte et surveiller la variation temporelle de la résistivité des terrains. A l’aide d’analyses statistiques, et en couplant les données microclimatiques aux résultats d’un suivi temporel de vingt-deux mois, nous avons montré la capacité de ce type de mesures à caractériser l’environnement épikarstique de la grotte, particulièrement les zones d’alimentation et celles permettant l’infiltration des eaux pluviales. Une modélisation du débit d’un écoulement situé à l’entrée de la grotte est proposée à partir des données de résistivité dans la zone d’alimentation identifiée au cours du suivi. Ce modèle a la capacité de prédire les arrêts et les reprises des écoulements dans la grotte, éventuellement des évènements journaliers.A partir des données de résistivité issues d’une prospection 3D du site, des modélisations géostatistiques par krigeage ordinaire et par indicatrice ont été effectuées permettant des représentations spatiales en fonction de la résistivité des terrains. Ces modèles se sont révélés extrêmement instructifs par l’imagerie de l’environnement karstique de la grotte de Lascauxqui en a résulté. Les limites des formations détritiques et des calcaires sont identifiées à l’est et l’ouest du site. A l’intérieur des calcaires, on retrouve, bien sûr, les anomalies conductrices déjà identifiées au cours du suivi temporel mais aussi leur extension spatiale. Ainsi, on a pu mettre en évidence la continuité spatiale de certaines anomalies.Le suivi temporel par TRE a permis la compréhension de la structure et du fonctionnement de l’alimentation de l’épikarst. Les modèles géostatistiques 3D ont montré leur efficacité pour la caractérisation de l’environnement de la grotte. Les résultats aideraient à proposer des conseils pour la protection du milieu environnant la grotte et ainsi pour la préservation de cette dernière. / The Lascaux cave, one of the most important prehistoric caves in the world, located in Dordogne (24, France) needs particular attention both for itself and for the environment interms of conservation and vulnerability since its discovery. Geophysical methods in particular Electrical Resistivity Tomography (ERT) enable us, in a non-invasive way, to monitor the karsticenvironment.A Time-Lapse monitoring by ERT was carried out next to the cave. Together with analysis of the local effective rainfall (ground water recharge) and the flow in the cave, the monitoringhelped us to identify an area where upstream underground water is probably stored e.g. arecharge zone. There is a large electrical contrast between the surrounding limestone and theprobable recharge zone. Then, a multivariate analysis through the resistivity values allowed usto characterize the model blocks, showing a specific behavior over time, especially the blockswith the lowest electrical resistivity. A prediction model of the flow in relation with the recharge zone succeeded to predict the beginning and the end of flow, even the daily event withextremely high value of flow.In order to visualize the environment in 3D condition, a geostatistical modelling was then applied to the resistivity values. The geostatistical models can emphasize the limit betweenthe limestone promontory and the clayey/sandy formations to the east/west part of the site. In the limestone promontory, the models also showed the possible connection between theanomalous conductive areas that may have a special consequence in this karstic environment.The Time-Lapse monitoring by ERT allows us to understand the karstic structures andrecharge phenomena. The 3D geostatistical modeling showed efficiency for the characterization of the cave environment. Those results can help to provide advices for the cave preservation.

Future sea level rise associated to global warming is one of the greatest societal and environmental challenges of tomorrow. A large part of the contribution comes from glaciers and ice sheets discharging ice and meltwater into the ocean and the recent worldwide increase is worrying. Future predictions of sea level rise try to encompass the complex processes of ice dynamics through glacier modelling but there are still large uncertainties due to the lack of observations or too coarse parameterisation, particularly for processes occurring at the glacier interfaces with the bed (sliding) and with the ocean (calving). This thesis focuses on modelling these processes from two marine-terminating glaciers in Svalbard, Kronebreen and Tunabreen. By inverting three years of high temporal resolution time-series of surface velocities on Kronebreen, basal properties are retrieved with the ice flow model Elmer/Ice in Paper I. Results suggest that surface melt during the summer greatly influences the dynamics of the following season and that sliding laws for such glaciers should be adapted to local and global processes changing in space and time. The subglacial drainage system, fed by the surface melt, is modelled in Paper II during two melting seasons. Results show different configurations of efficient and inefficient drainage systems between years and the importance of using a sliding law dependent on spatio-temporal changes in effective pressure. The interaction with the ocean is incorporated in Paper III by combining a series of models, including an ice flow model, a plume model and a particle model for discrete calving and compares the output with observations. Results show the importance of glacier geometry, sliding and undercutting on calving rate and location. However, more observations and analytic methods are needed. Time-lapse imagery placed in front of Tunabreen have been deployed and a method of automatic detection for iceberg calving is presented in Paper IV. Results show the influence of the rising plume in calving and the front destabilisation of the local neighbourhood.

Philosophiae Doctor - PhD / The aim of this thesis is first, to evaluate various culture conditions to improve embryo development, and secondly, to analyze spent culture media for any biomarkers that may be predictive of embryo health. Single-step and sequential culture media were compared in both Planer and EmbryoScope™ incubators. Single-step media resulted in better blastocyst development compared to sequential media and the EmbryoScope™ incubation system showed slight improvements in embryo development than the Planer system. The benefits of supplementing the culture medium with either insulin or insulin-like growth factor 1 (IGF-1) or culturing in a 2% O2 environment, using two different strains of mice (hybrid and C57), as well as the suitability of these strains for quality control were compared. In insulin, hybrid embryos were slower to blastulate and had a lower blastocyst rate, whereas C57 embryos were slower to the morula and faster to blastocyst stages, and lower blastocyst rate than the controls. IGF-1 showed no difference in time-lapse morphokinetics (TLM) or blastocyst rates compared to controls in both hybrid and C57 embryos. Under 2% O2, hybrid embryos showed no significant difference in TLM up to the 8-cell stage, but slowed down afterwards, resulting in blastocysts with significantly lower cell counts than the 6% O2 group. The C57 embryos were slower to reach morula and expanded blastocyst, and had lower blastocyst rates in 2%O2 vs 6%O2. The C57 strain had significant slower overall embryo development for all time points than hybrid embryos in insulin, IGF-1 and ultra-low O2, as well as lower blastocyst rates. Measurement of growth differentiation factor 9 (GDF-9) and oxidation-reduction potential (ORP) in spent media as markers for embryo health were evaluated. Day 5 human blastocysts yielded higher pregnancy rates and GDF-9 levels in spent media compared to Day 6 blastocysts, but TLM parameters showed no impact on pregnancy outcome. In Day 6 blastocysts, the non-pregnant group showed significantly faster embryo development compared to the clinically pregnant group up to the 8-cell stage and start of blastulation. GDF-9 did not show any significant differences between non-pregnant and pregnant groups of Day 5 or Day 6 embryo transfers. ORP in spent media from good quality Day 3 embryos that developed into blastocysts were significantly higher than from those that did not, with no difference in control medium ORP. Spent media from arrested embryos showed lower ORP than their corresponding controls. Arrested embryos had slower development at syngamy, morula, blastulation and blastocyst stages. The single step medium in the EmbryoScope™ is the preferred choice for embryo culture. Insulin or IGF-1 media supplementation or 2% O2 culture did not provide any benefit to embryo development. The C57 mouse strain is more sensitive and may be better to detect changes in culture conditions, and therefore better model for quality control assays. GDF-9 values decrease from Day 5 to Day 6 which gives new insight to understanding the role of GDF-9 during embryogenesis. ORP in spent media indicate that embryos that developed into blastocysts did not contribute to ROS, but maintained ORP balance.

Infertility affects 15% of reproductive-aged couples. The milieu surrounding the growing embryo is of outmost importance, and should be optimised during in vitro fertilisation (IVF). Many biological processes, such as angiogenesis, coagulation, and immune processes need to be well regulated for a pregnancy to occur and progress normally. Histidine-rich glycoprotein (HRG) is a plasma protein that regulates components of these systems by building complexes with various ligands. A single nucleotide polymorphism (SNP) in HRG, denoted HRG C633T, seem to be of importance for IVF treatment outcomes. The aim of this thesis was to further investigate the proposed human fertility effects of the HRG C633T SNP. According to the findings of this thesis, the HRG C633T genotype is associated with primary recurrent miscarriage. Male HRG C633T genotype is associated with semen characteristics in infertile men, and pregnancy rates following IVF. However, the distribution of the HRG C633T SNP does not differ between infertile and fertile couples. We further examined the role of the region surrounding the HRG C633T SNP for regulation of endometrial angiogenesis and human embryo development. The region affects primary endometrial endothelial cell migration, proliferation and tube-formation in vitro but does not appear to affect human embryo development. No effect of the HRG peptide was noted on the secretome of human embryos. However, early embryos secrete proteins into the surrounding culture media and the level of secretion of VEGF-A, IL-6, EMMPRIN and PlGF is greater in embryos of higher developmental stages. In conclusion, the HRG C633T genotype appears to play a role only if infertility is established. The region surrounding HRG C633T SNP is of relevance in vitro for regulation of human endometrial endothelial cell angiogenesis. To predict which embryos to transfer in IVF, we have highlighted a number of proteins of interest for further investigation.

No description available.

Tous les sites étudiés lors de cette thèse, qui se situent tous dans des régions tabulaires ou monoclinales, ont permis d'observer et d'analyser d'un point de vue géophysique une diversité de morphologies associées aux karsts de plateaux et aux karsts sous couverture : tertres émissifs et dolines de suffusion dans l'Est de la France, dolines de dissolution et dolines masquées dans les Causses du Quercy, épikarst et altération sur le Causse du Larzac. Les tomographies de résistivité électrique et de sismique réfraction ont en effet été très utiles pour caractériser la géométrie de ces objets. L'apport de la sismique réfraction 3D s'est révélée considérable pour l'imagerie de la fracturation et de l'altération dans l'épikarst. Un suivi temporel électrique et sismique a également été mis en place sur un aquifère karstique en Corrèze et sur deux secteurs épikarstiques sur le Causse du Larzac. Si le suivi par tomographie de résistivité ne s'est pas avéré très efficace, le suivi par tomographie de sismique réfraction montre un potentiel conséquent pour identifier les zones de stockage et les zones d'infiltration, particulièrement dans l'épikarst. Des résultats nouveaux ont été obtenus dans : (1) la compréhension des morphologies karstiques par l'analyse de leur structure, (2) la structure et le fonctionnement de l'épikarst, (3) l'efficacité des méthodes électriques et sismiques pour l'imagerie structurale et le suivi temporel en milieu karstique.

Le suivi temporel est un processus d'acquisition et d'analyse d'acquisitions multiples répétées au même endroit sur la même cible à différentes périodes de temps. Cela s'applique bien à l'exploration sismique quand les propriétés de la cible varient au cours du temps comme pour les réservoirs pétroliers. Cette technique de sismique, dite 4D en raison de l'intégration du temps dans la construction des images, permet une détection et une estimation des variations du sous-sol survenues lors de l'évolution en temps du milieu. En particulier, dans l'industrie, le suivi et la surveillance peuvent améliorer notre compréhension d'un réservoir de pétrole/gaz ou d'un site de stockage de CO2. Analyser la sismique 4D peut aider à mieux gérer les programmes de production des réservoirs. Ainsi, des acquisitions répétées permettent de suivre l'évolutiondes fronts de fluide injectés: on peut optimiser les programmes d'injection de fluides pour une récupération améliorée des hydrocarbures (enhanced oil recovery). Plusieurs méthodes ont été développées pour l'imagerie variable dans le temps en utilisant les informations des ondes sismiques. Dans ma thèse, je montre que l'inversion de forme d'onde complété (FWI) peut être utilisée pour cette imagerie. Cette m'méthode offre des images sismiques quantitatives haute résolution. Elle est une technique prometteuse pour reconstruire les petites variations de propriétés physiques macro-échelle du sous-sol. Sur une cible identifiée pour ces imageries 4D, plusieurs informations a priori sont souvent disponibles et peuvent être utilisées pour augmenter la résolution de l'image. J'ai introduit ces informations grâce à la définition d'un modèle a priori dans une approche classique FWI en l'accompagnant de la construction d'un modèle d'incertitudes a priori. On peut réaliser deux reconstructions indépendantes et faire la différence les reconstruits: on parle de différence parallèle. On peut aussi effectuer une différence séquentielle o'u l'inversion de l'ensemble de données de la second acquisition, dite moniteur, se fait 'a partir du modèle de base et non plus à partir du modèle utilisé initialement. Enfin, l'approche double-différence conduit à l'inversion des différences entre les deux jeux de données que l'on rajoute aux données synthétiques du modèle de base reconstruit. J'étudie quelle stratégie est à adopter pour obtenir des changements vitesse plus précis et plus robustes. En plus, je propose une imagerie 4D ciblée en construisant un modèle d'incertitude a priori grâce 'a une information (si elle existe) sur la localisation potentielle des variations attendues. Il est démontré que l'inversion 4D ciblée empêche l'apparition d'artéfacts en dehors des zones cibles: on évite la contamination des zones extérieures qui pourrait compromettre la reconstruction des changements 4D réels. Une étude de sensibilité, concernant l'échantillonnage en fréquence pour cette imagerie 4D, montre qu'il est nécessaire de faire agir simultanément un grand nombre de fréquences au cours d'un cycle d'inversion. Ce faisant, l'inversion fournit un modèle de base plus précis que l'approche temporelle, ainsi qu'un modèle des variations 4D plus robuste avec moins d'artéfacts. Toutefois, la FWI effectuée dans le domaine temporel semble être une approche plus intéressante pour l'imagerie 4D. Enfin, l'approche d'inversion 4D régularisée avec un modèle a priori est appliquée sur des ensembles de données réelles d'acquisitions sismiques répétées fournis par TOTAL. Cette reconstruction des variations locales s'inscrit dans un projet d'injection de vapeur pour améliorer la récupération des hydro-carbures: Il est possible de reconstituer des variations de vitesse fines causées par la vapeur injectée.