The subject of this thesis is the generation of spatial temporal structures in living cells. Specifically, we studied the Min-system in the bacterium Escherichia coli. It consists of the MinC, the MinD, and the MinE proteins, which play an important role in the correct selection of the cell division site. The Min-proteins oscillate between the two cell poles and thereby prevent division at these locations. In this way, E. coli divides at the center, producing two daughter cells of equal size, providing them with the complete genetic patrimony. Our goal is to perform a quantitative study, both theoretical and experimental, in order to reveal the mechanism underlying the Min-oscillations. Experimentally, we characterize theMin-system, measuring the temporal period of the oscillations as a function of the cell length, the time-averaged protein distributions, and the in vivo Min-protein mobility by means of different fluorescence microscopy techniques. Theoretically, we discuss a deterministic description based on the exchange of Minproteins between the cytoplasm and the cytoplasmic membrane and on the aggregation current induced by the interaction between membrane-bound proteins. Oscillatory solutions appear via a dynamic instability of the homogenous protein distributions. Moreover, we perform stochastic simulations based on a microscopic description, whereby the probability for each event is calculated according to the corresponding probability in the master equation. Starting from this microscopic description, we derive Langevin equations for the fluctuating protein densities which correspond to the deterministic equations in the limit of vanishing noise. Stochastic simulations justify this deterministic model, showing that oscillations are resistant to the perturbations induced by the stochastic reactions and diffusion. Predictions and assumptions of our theoretical model are compatible with our experimental findings. Altogether, these results enable us to propose further experiments in order to quantitatively compare the different models proposed so far and to test our model with even higher precision. They also point to the necessity of performing such an analysis through single cell measurements.

No description available.

No description available.

No description available.

La toxine bactérienne de Shiga se lie au glycosphingolipide (GSL) globotriaosylcéramide (Gb3) afin d’entrer par endocytose dans les cellules en utilisant une voie dépendante et indépendante de la clathrine. Dans la voie indépendante de la clathrine, la toxine de Shiga réorganise les lipides de la membrane de façon à imposer une contrainte mécanique sur la bicouche, conduisant ainsi à la formation de pic d’invagination d'endocytose profonds et étroits. Mécaniquement ce phénomène n’est pas encore compris, notamment il reste énigmatique, comment se traduisent les propriétés géométriques de l’agrégation des glycosphingolipides GSLS et de la toxine. Dans mon travail de thèse, via l’utilisation de la sous-unité B de la toxine de Shiga (STxB) comme un modèle, différentes espèces moléculaires de son récepteur Gb3 ont été synthétisés avec des structures délibérément choisis. Les études réalisées par imagerie de haute résolution et par la modélisation informatique ont permis d’élucider les contraintes mécano-chimique sous-jacente conduisant à une réorganisation efficace qui a pour résultat l’agrégation de la toxine et la réorganisation des lipides. En combinant des expériences de simulation sur ordinateur de dynamique des particules dissipatives (DPD) et des expériences sur des modèles de membranes cellulaires, nous avons fourni la preuve de l’induction d’une force de fluctuation-membrane, de type « force de Casimir », conduisant à l'agrégation des molécules de toxines associées à la membrane à des échelles de longueur mésoscoiques. Nous avons observé et mesuré, en outre la condensation lipidique induite par la toxine, quantitativement sur des monocouches de Langmuir en utilisant la réflectivité des rayons X (XR) et par la mesure de la diffraction des rayons X par incidence rasante (GIXD), fournissant ainsi une preuve directe de l'hypothèse que la toxine a le potentiel de réduire de façon asymétrique la surface moléculaire sur la partie membranaire exoplasmique, ce qui conduit à une déformation locale de la membrane. Durant ma thèse, nos efforts ont été consacrés à la réalisation de nouveaux glycosphinolipides (GSL) comme outils chimiques à visée biologique. Par ailleurs, une nouvelle stratégie de reconstitution de GSL fonctionnels sur la membrane cellulaire, basée sur une réaction de ligation de type « click » entre un glycosyl-cyclooctyne et un azido-sphingosine a été étudiée. Les résultats obtenus sur les cellules se sont avérés beaucoup moins efficace que ceux in vitro. Une poursuite de l'optimisation de cette méthodologie est actuellement en cours. Une sonde fluorescente du glycosphinolipide Gb3, marquée à l’Alexa Fluor 568 lui-même lié par l'intermédiaire d'un bras PEG-α à la position de la chaîne acyle, a été synthétisée. Cette sonde se lie à la STxB sur couche mince de TLC, mais pas sur des membranes modèles. D'autres améliorations sont discutées. / Bacterial Shiga toxins bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3) to enter cells by clathrin-dependent and independent endocytosis. In the clathrin-independent pathway, Shiga toxin reorganizes membrane lipids in a way such as to impose mechanical strain onto the bilayer, thus leading to the formation of deep and narrow endocytic pits. Mechanistically how this occurs is not yet understood, and notably how the geometric properties of toxin-GSLs complexes translate into function has remained enigmatic. In my thesis work, using the B-subunit of Shiga toxin (STxB) as a model, different molecular species of its receptor Gb3 have been synthesized with deliberately chosen structures, coupled with high resolution imaging and computational modeling, to understand the underlying mechano-chemical constraints leading to efficient toxin clustering and lipids reorganization. By combining dissipative particle dynamics (DPD) computer simulation and experiments on cell and model membranes, we provided evidence that a membrane fluctuation-induced force, termed Casimir-like force, drives the aggregation of tightly membrane-associated toxin molecules at mesoscopic length scales. Furthermore, toxin-induced lipid condensation was observed and measured quantitatively on Langmuir monolayers using X-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), thereby providing direct evidence for the hypothesis that the toxin has the potential to asymmetrically reduce the molecular area of the exoplasmic membrane leaflet, leading to local membrane deformation. During my PhD, effort was also invested to develop new GSL tools applied to the biological setting. A novel strategy based on the Cu-free click reaction between glycosyl-cyclooctyne and azido-sphingosine was designed with the goal to functionally incorporate GSLs into cellular membranes. Following the synthesis work, click reactions have been performed in solution and on cells. Compared to the former, results on cells were far less efficient. Further optimization is currently ongoing. A fluorescently labeled Gb3 probe with Alexa Fluor 568 coupled via a PEG linker to the α-position of the acyl chain, was synthesized, to which STxB bound on TLCs, but not on model membranes. Further improvements are discussed.

The subject of this thesis is the generation of spatial temporal structures in living cells. Specifically, we studied the Min-system in the bacterium Escherichia coli. It consists of the MinC, the MinD, and the MinE proteins, which play an important role in the correct selection of the cell division site. The Min-proteins oscillate between the two cell poles and thereby prevent division at these locations. In this way, E. coli divides at the center, producing two daughter cells of equal size, providing them with the complete genetic patrimony. Our goal is to perform a quantitative study, both theoretical and experimental, in order to reveal the mechanism underlying the Min-oscillations. Experimentally, we characterize theMin-system, measuring the temporal period of the oscillations as a function of the cell length, the time-averaged protein distributions, and the in vivo Min-protein mobility by means of different fluorescence microscopy techniques. Theoretically, we discuss a deterministic description based on the exchange of Minproteins between the cytoplasm and the cytoplasmic membrane and on the aggregation current induced by the interaction between membrane-bound proteins. Oscillatory solutions appear via a dynamic instability of the homogenous protein distributions. Moreover, we perform stochastic simulations based on a microscopic description, whereby the probability for each event is calculated according to the corresponding probability in the master equation. Starting from this microscopic description, we derive Langevin equations for the fluctuating protein densities which correspond to the deterministic equations in the limit of vanishing noise. Stochastic simulations justify this deterministic model, showing that oscillations are resistant to the perturbations induced by the stochastic reactions and diffusion. Predictions and assumptions of our theoretical model are compatible with our experimental findings. Altogether, these results enable us to propose further experiments in order to quantitatively compare the different models proposed so far and to test our model with even higher precision. They also point to the necessity of performing such an analysis through single cell measurements.

Ce mémoire par article est une étude des différences hémodynamiques entre la dextro-transposition des gros vaisseaux (TGV) avec communication interventriculaire (CIV) et la TGV avec septum intact (SI) pendant la période fœtale et périopératoire. Il est à noter que SI fait référence au septum inter ventriculaire et non au septum inter auriculaire. La présence d’une communication inter auriculaire étant, comme nous le verrons dans ce travail, un élément important de la physiologie des fœtus/nouveau-nés porteur de TGV. Le document est divisé en deux parties importantes. La première partie est composée du chapitre 1 qui présente une revue de littérature détaillant les notions importantes à la compréhension de la problématique et du chapitre 2 qui décrit la méthodologie utilisée pour répondre à la question de recherche. On détaille d’abord les méthodes d’acquisition des échocardiographies fœtales ainsi que les principales mesures effectuées à partir de celles-ci. Ensuite, on y décrit les technologies de la spectroscopie proche infrarouge avancée et de la spectroscopie à corrélation diffuse (NIRS-DCS) permettant de recueillir les données hémodynamiques sur la microvascularisation cérébrale des nouveau-nés. La seconde partie est constituée du chapitre 3 qui est le manuscrit accepté au journal Ultrasound in Obstetric & Gynecology pour publication. Celui-ci décrit les différences hémodynamiques entre les patients ayant une TGV&CIV et les TGV&SI et présente les différences retrouvées en échocardiographie fœtale et en hémodynamie cérébrale périopératoire étudiée à l’aide de la NIRS avancée. Ensuite, nous présentons dans le chapitre 4 une discussion sur les principaux impacts cliniques et sur d’éventuelles améliorations. / This master’sthesis is composed of an article and a study. They present hemodynamic differences between patients with transposition of the great arteries (TGA) and ventricular septum defect (VSD), and TGA with intact ventricular septum (IVS) during fetal and perioperative periods. This document is divided into two principal sections. The first section includes Chapter 1 that presents a review of literature detailing important notions to understand the problematic, and Chapter 2, that describes the methodology used to answer our research question. First, we detailed acquisition data and measured parameters of the fetal echocardiography exams. Then, we describe advanced near infrared spectroscopy (NIRS-DCS) technology that allowed hemodynamic data acquisition on the cerebral microvascularization of neonates. The second section is composed of Chapter 3, the manuscript accepted in Ultrasound in Obstetric & Gynecology journal for publication. The aim of this article is to describe hemodynamic differences between patients with TGA&VSD and TGA&IVS. It describes fetal echocardiography and cerebral perioperative hemodynamic differences studied with advanced NIRS. Next, we present in Chapter 4 a more detailed discussion with principal impacts on the clinical field and future improvements.

A new method, probability distribution of diffusivities (time scaled square displacements between succeeding video frames), was developed to analyze single molecule tracking (SMT) experiments. This method was then applied to SMT experiments on ultrathin liquid tetrakis(2-ethylhexoxy)silane (TEHOS) films on Si wafer with 100 nm thermally grown oxide, and on thin semectic liquid crystal films. Spatial maps of diffusivities from SMT experiments on 220 nm thick semectic liquid crystal films reveal structure related dynamics. The SMT experiments on ultrathin TEHOS films were complemented by fluorescence correlation spectroscopy (FCS). The observed strongly heterogeneous single molecule dynamics within those films can be explained by a three-layer model consisting of (i) dye molecules adsorbed to the substrate, (ii) slowly diffusing molecules in the laterally heterogeneous near-surface region of 1 - 2 molecular diameters, and (iii) freely diffusing dye molecules in the upper region of the film. FCS and SMT experiments reveal a strong influence of substrate heterogeneity on SM dynamics. Thereby chemisorption to substrate surface silanols plays an important role. Vertical mean first passage times (mfpt) in those films are below 1 µs. This appears as fast component in FCS autocorrelation curves, which further contain a contribution from lateral diffusion and from adsorption events. Therefore, the FCS curves are approximated by a tri-component function, which contains an exponential term related to the mfpt, the correlation function for translational diffusion and a stretched exponential term for the broad distribution of adsorption events. Lateral diffusion coefficients obtained by FCS on 10 nm thick TEHOS films, thereby, are effective diffusion coefficients from dye transients in the focal area. They strongly depend on the substrate heterogeneity. Variation of the frame times for the acquisition of SMT experiments in steps of 20 ms from 20 ms to 200 ms revealed a strong dependence of the corresponding probability distributions of diffusivities on time, in particular in the range between 20 ms and 100 ms. This points to average dwell times of the dye molecules in at least one type of the heterogeneous regions (e.g. on and above silanol clusters) in the range of few tens of milliseconds. Furthermore, time series of SM spectra from Nile Red in 25 nm thick poly-n-alkyl-methacrylate (PnAMA) films were studied. In analogy to translational diffusion, spectral diffusion (shifts in energetic positions of SM spectra) can be studied by probability distributions of spectral diffusivities, i.e. time scaled square energetic displacements. Simulations were run and analyzed to study contributions from noise and fitting uncertainty to spectral diffusion. Furthermore the effect of spectral jumps during acquisition of a SM spectrum was investigated. Probability distributions of spectral diffusivites of Nile Red probing vitreous PnAMA films reveal a two-level system. In contrast, such probability distributions obtained from Nile Red within a 25 nm thick poly-n-butylmethacrylate film around glass transition and in the melt state, display larger spectral jumps. Moreover, for longer alkyl side chains a solvent shift to higher energies is observed, which supports the idea of nanophase separation within those polymers.

A new method, probability distribution of diffusivities (time scaled square displacements between succeeding video frames), was developed to analyze single molecule tracking (SMT) experiments. This method was then applied to SMT experiments on ultrathin liquid tetrakis(2-ethylhexoxy)silane (TEHOS) films on Si wafer with 100 nm thermally grown oxide, and on thin semectic liquid crystal films. Spatial maps of diffusivities from SMT experiments on 220 nm thick semectic liquid crystal films reveal structure related dynamics. The SMT experiments on ultrathin TEHOS films were complemented by fluorescence correlation spectroscopy (FCS). The observed strongly heterogeneous single molecule dynamics within those films can be explained by a three-layer model consisting of (i) dye molecules adsorbed to the substrate, (ii) slowly diffusing molecules in the laterally heterogeneous near-surface region of 1 - 2 molecular diameters, and (iii) freely diffusing dye molecules in the upper region of the film. FCS and SMT experiments reveal a strong influence of substrate heterogeneity on SM dynamics. Thereby chemisorption to substrate surface silanols plays an important role. Vertical mean first passage times (mfpt) in those films are below 1 µs. This appears as fast component in FCS autocorrelation curves, which further contain a contribution from lateral diffusion and from adsorption events. Therefore, the FCS curves are approximated by a tri-component function, which contains an exponential term related to the mfpt, the correlation function for translational diffusion and a stretched exponential term for the broad distribution of adsorption events. Lateral diffusion coefficients obtained by FCS on 10 nm thick TEHOS films, thereby, are effective diffusion coefficients from dye transients in the focal area. They strongly depend on the substrate heterogeneity. Variation of the frame times for the acquisition of SMT experiments in steps of 20 ms from 20 ms to 200 ms revealed a strong dependence of the corresponding probability distributions of diffusivities on time, in particular in the range between 20 ms and 100 ms. This points to average dwell times of the dye molecules in at least one type of the heterogeneous regions (e.g. on and above silanol clusters) in the range of few tens of milliseconds. Furthermore, time series of SM spectra from Nile Red in 25 nm thick poly-n-alkyl-methacrylate (PnAMA) films were studied. In analogy to translational diffusion, spectral diffusion (shifts in energetic positions of SM spectra) can be studied by probability distributions of spectral diffusivities, i.e. time scaled square energetic displacements. Simulations were run and analyzed to study contributions from noise and fitting uncertainty to spectral diffusion. Furthermore the effect of spectral jumps during acquisition of a SM spectrum was investigated. Probability distributions of spectral diffusivites of Nile Red probing vitreous PnAMA films reveal a two-level system. In contrast, such probability distributions obtained from Nile Red within a 25 nm thick poly-n-butylmethacrylate film around glass transition and in the melt state, display larger spectral jumps. Moreover, for longer alkyl side chains a solvent shift to higher energies is observed, which supports the idea of nanophase separation within those polymers.