L'analyse de l'organisation à grande échelle des chromosomes, par des approches d'imagerie et de biologie moléculaire, constitue un enjeu important de la biologie. Il est maintenant établi que l'organisation structurelle du génome est un facteur déterminant dans tous les aspects des " transactions " génomiques: transcription, recombinaison, réplication et réparation de l'ADN. Bien que plusieurs modèles aient été proposés pour décrire l'arrangement spatial des chromosomes, les principes physiques qui sous-tendent l'organisation et la dynamique de la chromatine dans le noyau sont encore largement débattus. Le noyau est le compartiment de la cellule dans lequel l'ADN chromosomique est confiné. Cependant, la mesure quantitative de l'influence de la structure nucléaire sur l'organisation du génome est délicate, principalement du fait d'un manque d'outils pour déterminer précisément la taille et la forme du noyau. Cette thèse est organisée en deux parties: le premier axe de mon projet était d'étudier la dynamique et les propriétés physiques de la chromatine dans le noyau de la levure S. cerevisiae. Le deuxième axe visait à développer des techniques pour détecter et quantifier la forme et la taille du noyau avec une grande précision. Dans les cellules de levure en croissance exponentielle, j'ai étudié la dynamique et les propriétés physiques de la chromatine de deux régions génomiques distinctes: les régions codant les ARN ribosomiques regroupés au sein d'un domaine nucléaire, le nucléole, et la chromatine du nucléoplasme. Le mouvement de la chromatine nucléoplasmique peut être modélisé par une dynamique dite de " Rouse ". La dynamique de la chromatine nucléolaire est très différente et son déplacement caractérisé par une loi de puissance d'exposant ~ 0,7. En outre, nous avons comparé le changement de la dynamique de la chromatine nucléoplasmique dans une souche sauvage et une souche porteuse d'un allèle sensible à la température (ts) permettant une inactivation conditionnelle de la transcription par l'ARN polymérase II. Les mouvements chromatiniens sont beaucoup plus importants après inactivation transcriptionnelle que dans la souche témoin. Cependant, les mouvements de la chromatine restent caractérisés par une dynamique dite de " Rouse ". Nous proposons donc un modèle biophysique prenant en compte ces résultats : le modèle de polymère dit "branched-Rouse". Dans la deuxième partie, j'ai développé "NucQuant", une méthode d'analyse d'image permettant la localisation automatique de la position de l'enveloppe nucléaire du noyau de levures. Cet algorithme comprend une correction post-acquisition de l'erreur de mesure due à l'aberration sphérique le long de l'axe Z. "NucQuant" peut être utilisée pour déterminer la géométrie nucléaire dans de grandes populations cellulaires. En combinant " NucQuant " à la technologie microfluidique, nous avons pu estimer avec précision la forme et la taille des noyaux en trois dimensions (3D) au cours du cycle cellulaire. "NucQuant" a également été utilisé pour détecter la distribution des regroupements locaux de complexes de pore nucléaire (NPCs) dans des conditions différentes, et a révélé leur répartition non homogène le long de l'enveloppe nucléaire. En particulier, nous avons pu montrer une distribution particulière sur la région de l'enveloppe en contact avec le nucléole. En conclusion, nous avons étudié les propriétés biophysiques de la chromatine, et proposé un modèle dit "branched Rouse-polymer" pour rendre compte de ces propriétés. De plus, nous avons développé "NucQuant", un algorithme d'analyse d'image permettant de faciliter l'étude de la forme et la taille nucléaire. Ces deux travaux combinés vont permettre l'étude des liens entre la géométrie du noyau et la dynamique de la chromatine. / Chromosome high-order architecture has been increasingly studied over the last decade thanks to technological breakthroughs in imaging and in molecular biology. It is now established that structural organization of the genome is a key determinant in all aspects of genomic transactions. Although several models have been proposed to describe the folding of chromosomes, the physical principles governing their organization are still largely debated. Nucleus is the cell’s compartment in which chromosomal DNA is confined. Geometrical constrains imposed by nuclear confinement are expected to affect high-order chromatin structure. However, the quantitative measurement of the influence of the nuclear structure on the genome organization is unknown, mostly because accurate nuclear shape and size determination is technically challenging. This thesis was organized along two axes: the first aim of my project was to study the dynamics and physical properties of chromatin in the S. cerevisiae yeast nucleus. The second objective I had was to develop techniques to detect and analyze the nuclear 3D geomtry with high accuracy. Ribosomal DNA (rDNA) is the repetitive sequences which clustered in the nucleolus in budding yeast cells. First, I studied the dynamics of non-rDNA and rDNA in exponentially growing yeast cells. The motion of the non-rDNA could be modeled as a two-regime Rouse model. The dynamics of rDNA was very different and could be fitted well with a power law of scaling exponent ~0.7. Furthermore, we compared the dynamics change of non-rDNA in WT strains and temperature sensitive (TS) strains before and after global transcription was actived. The fluctuations of non-rDNA genes after transcriptional inactivation were much higher than in the control strain. The motion of the chromatin was still consistent with the Rouse model. We propose that the chromatin in living cells is best modeled using an alternative Rouse model: the “branched Rouse polymer”. Second, we developed “NucQuant”, an automated fluorescent localization method which accurately interpolates the nuclear envelope (NE) position in a large cell population. This algorithm includes a post-acquisition correction of the measurement bias due to spherical aberration along Z-axis. “NucQuant” can be used to determine the nuclear geometry under different conditions. Combined with microfluidic technology, I could accurately estimate the shape and size of the nuclei in 3D along entire cell cycle. “NucQuant” was also utilized to detect the distribution of nuclear pore complexes (NPCs) clusters under different conditions, and revealed their non-homogeneous distribution. Upon reduction of the nucleolar volume, NPCs are concentrated in the NE flanking the nucleolus, suggesting a physical link between NPCs and the nucleolar content. In conclusion, we have further explored the biophysical properties of the chromatin, and proposed that chromatin in the nucleoplasm can be modeled as "branched Rouse polymers". Moreover, we have developed “NucQuant”, a set of computational tools to facilitate the study of the nuclear shape and size. Further analysis will be required to reveal the links between the nucleus geometry and the chromatin dynamics.

The perfusion chamber for long term observing of live cells by the means the Coherence-Controlled Holographic Microscope (CCHM) was designed. CCHM was built and designed at the Laboratory of the optical microscopy at the Institute of Physical Engineering, Brno University of Technology. CCHM can quantitatively evaluate dynamical changes inside live cells thanks to the quantitative information about phase shift in each pixel of the image. In order to demonstrate advantages of CCHM experimentally, it is important to keep the live cells in the good conditions. This is made by adding the fresh cultivation medium for studied cells directly in the microscope. In contrast to the stationary chamber the perfusion chamber allows both the cultivation medium exchange and the application of biological reagents without the necessity of removing the chamber from the microscope. Therefore we can study the vital signs of cells before and after the application of reagents. An original perfusion system with accessories compatible with CCHM was designed. The design is based on the previously published perfusion system solutions that are referred to in this thesis. The flow characteristics and medium exchange process was discussed and a modification of the internal geometry, based on numerical simulations, was introduced. The applicability of this perfusion chamber has been proven for the CCHM and even for different types of microscopes. The reactions of tumor and epithelial cells during the change of the environment from the cultivation medium to the physiologically solution were studied.

Mitochondria are the power plant of most non-green eukaryotic cells. In order to understand mitochondrial functions and their regulation, knowledge of the spatiotemporal organization of their proteins is important. Mitochondrial membrane proteins can diffuse within membranes. They are involved in diverse functions e.g. protein import, cell respiration, metabolism, metabolite transport, fusion, fission or formation of the mitochondrial architecture. Furthermore, mitochondria compose of different subcompartments with different tasks. Especially, the inner mitochondrial membrane (IM), where the oxidative phosphorylation (OXPHOS) takes places, has a complex architecture with cristae extending into the matrix. The present work revealed the restricted localization of some mitochondrial proteins to specific membrane sections and linked it to their function or bioenergetic circumstances in the living cell. 
Single particle tracking (SPT) techniques like tracking and localization microscopy (TALM) allow to localize proteins with a precision below 20 nm. Additionally, tracking single proteins provides information about their mobility, dynamic and their spatiotemporal organization. TALM uses proteins, which were genetically tagged either with the HaloTag® (HaloTag) or the fSnapTag® (fSnapTag). These tags can be orthogonally and posttranslationally stained with specific and self-marking dyes. If the dyes are conjugated to the respective substrate of the tag. Single molecule labeling of mitochondrial proteins was performed substoichiometrically using membrane permeable rhodamine dyes, either tetramethylrhodamine (TMR) or silicon rhodamine (SiR). TALM allowed to localize proteins in different mitochondrial subcompartments. The gained trajectories and trajectory maps of mitochondrial proteins revealed their spatiotemporal organization. In the case of IM proteins like F1FO ATP synthase (Complex V - CV) a restricted diffusion in the CM, which is part of the continuous IM, was determined. The unimpeded diffusion of mitochondrial proteins in the outer mitochondrial membrane (OM) was compared with the mobility of IM proteins. The diffusion of mitochondrial IM proteins was restricted by the IM architecture and their diffusion coefficients were lower. Furthermore, significant differences of different mitochondrial IM proteins were compared, showing different localizations in the IM often coupled to their function, accompanied by different spatiotemporal organization and diffusion coefficients. Furthermore, a distinction was made between diffusion of proteins in the inner boundary membrane (IBM) and proteins that preferentially diffuse in the cristae membrane (CM). Evaluating trajectory maps, the different subcompartments in the IM were revealed by trajectories and the trajectory directionality, allowing the identification of mitochondrial proteins, which mark these subcompartments.
The morphology of mitochondria / mitochondrial networks and their bioenergetic parameters are linked to the metabolic states of the cell. In this work, the connection of the spatiotemporal protein organization of CV and the IM architecture was uncovered on the micro- and nanoscopic level and linked to the metabolic state of the cell. It was determined that the spatiotemporal organization of the CV was altered, when CV was inhibited. In addition, the bioenergetic influence of cells on the spatiotemporal behavior of CV and the reorganization of the IM architecture was investigated by TALM and compared with results of electron microscopy images. It was shown that starvation of cells led to a loss of cristae and thus to an increased mobility and spatiotemporal reorganization of CV. Taken together, the results presented in this work showed that a correctly functioning and active CV helps to maintain the IM architecture and both, the spatiotemporal organization of CV and the IM architecture were coupled to the metabolic state.. 
In order to investigate putative protein-protein interactions by colocalization and co-locomotion studies on single molecule level, dual color SPT is needed. Therefore, posttranslational and substoichimetric labeling as performed in TALM was tested for its potential of protein-protein interaction studies of mitochondrial membrane proteins. Here, a genetically double tagged translocase of the outer membrane subunit-20 (Tom20) (Tom20:HaloTag:fSnapTag) acted as a positive control. It turned out that substoichimetric, posttranslational labeling of mitochondrial proteins was not suitable for protein-protein interaction studies on mitochondrial proteins, because it was restricted by the low labeling degrees needed for TALM. However, dual-color TALM still allowed to study effects of proteins influencing the IM architecture and to study their influence on the spatiotemporal organization of CV. The co-transfection of Mic10, as the central protein of the mitochondrial inner membrane organizing system / mitochondrial contact site complex / mitochondrial organizing structure (MINOS / MICOS / MitOS (MINOS/MICOS)), altered the regular and aligned organization of the cristae. This was measured by a changed spatiotemporal organization of the CV, such as the loss of the perpendicular oriented of CV subunit-γ (CV-SUγ) cristae trajectories. In contrast to this, co-transfection of CV subunit-e (CV-SUe), important for dimerization of CV, increased the number of cristae trajectories. 
Mitochondria are three-dimensional (3D) cell organelles. Consequently, subcompartments like the IBM and CM are a 3D space in which CV is localized and diffuses. Thus, the diffusion of mitochondrial proteins is underestimated by two-dimensional SPT e.g. lateral confined diffusion can result from mitochondrial proteins diffusing along the z-axis of the microscope. In order to reveal the 3D spatiotemporal organization of CV, the potential of TALM to be extended to a 3D-SPT technique was investigated. Therto a cylindrical lens was installed in the emission path of a total internal reflection fluorescence (TIRF) microscope. This leads to an astigmatically distorted point spread function (PSF) of the fluorescent single molecule signals. This distortion allowed the reconstruction of single molecule localizations of CV to a superresolved image of the IM, in living cells. In addition, 3D-TALM enabled to display the 3D architecture of the IM by 3D trajectories of CV. 3D-TALM was able to detect whether CV diffuses in the IBM or in the CM, and extended the information about its mobility in the CM that it takes place in a disc-like manner. In this way it could be shown that CV is mobile within the cristae in all directions. Finally, 3D-TALM revealed an altered IM architecture caused by the metabolic state of the cell. As performed in two-dimensional TALM, the cells were kept under starving conditions. Here the now tubular IM architecture was revealed by 3D-TALM. The reversed metabolic state under improved respiratory conditions unexpectedly led to a more diverse IM architecture. These ultrastructural changes were also revealed by electron microscopy. Consequently, 3D-TALM enabled the study of IM architecture by tracking CV under different metabolic conditions, allowing an ultrastructural analysis of mitochondria in living cells. In addition, 3D TALM provided the spatiotemporal organization of CV under different metabolic conditions, so that the diffusion coefficients of CV could be related to changes in IM architecture caused by the metabolic condition.

Connaître en temps réel la réponse et le comportement des cellules et organismes modèles suite à des changements de leur environnement, ou à des modulations de leurs fonctions biologiques est devenu essentiel dans les sciences du vivant. Ces réponses nous permettent ensuite de comprendre les mécanismes qui régissent le fonctionnement des cellules vivantes, avec des implications en recherche fondamentale, appliquée et biomédicale. Un des plus gros défis technologiques reste le contrôle des paramètres environnementaux en microscopie haute résolution. De nos jours, aucun système ne permet de réguler un ensemble complexe de paramètres de manière précise, dynamique et simultanée tout en observant les cellules dans leur environnement. L’objectif de ma thèse est de mettre au point un tel dispositif permettant a minima une régulation fine de la température, de la composition du milieu, et notamment de la concentration de divers drogues. Ce système doit être compatible avec les applications les plus poussées en microscopie photonique. Mon approche au cours de ma thèse pour élaborer un tel système est l’utilisation de la microfluidique. En effet, c’est la seule technologie qui puisse de réaliser un tel multiplexage. Elle permet de manipuler des petites quantités de fluide à travers un système contenant des canaux de dimensions allant du micromètre au centimètre. Cet ordre de grandeur des canaux constitue un atout majeur (réduction de la consommation des réactifs, réduction des couts, cinétiques des réactions chimiques et biologiques élevées, temps de diffusion court, etc.) et permet d’allier les expériences biologiques à la microscopie. Mon objectif est de concevoir une puce microfluidique qui représentera un pas technologique majeur et ouvrira de nouvelles possibilités de recherche. / Monitoring in real-time the response of cells and model organisms to the changes in their environment or to modulations of their biological functions has become essential in life sciences. One of the main technical challenges for biologists is the precise and dynamic control of various environmental parameters while doing high-resolution microscopy. My thesis consists of building a robust and versatile system, dedicated to live-cell imaging that will be compatible with adherent and non adherent models, that could provide a precise and simultaneous control of 1) the temperature, 2) the media exchanges and 3) the drug concentration while doing photonic microscopy. My approach is to use microfluidics, which is the best candidate in order to achieve this system and provides all the necessary controls of micro-scaled volumes for culturing, maintaining or analyzing cells. It produces miniaturized systems used as tools for biological experiments, in which channels of a micro-scaled dimension are used for the fluid circulation. The laminar flow in these chips allows fast molecule diffusion as well as fast temperature diffusion. Because of the high surface to volume ratio, the consumption of reagents is reduced, and media switches can be fast. This system will represent a major technical and beneficial step and will open new possibilities of research in biology.

Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

Most biological experiments are performed on an ensemble of cells under the assumption that all cells are identical. However, recent evidence from single cells studies reveals that this assumption is incorrect. Individual cells within the same generation may differ dramatically, and these differences have important consequences for the health and function of the entire living body. I have used Scanning Ion Conductance Microscopy (SICM) for imaging and analysis of topographical change of single cell membrane, which is difficult to be revealed by optical microscopes. Morphological change in the fixed and live HeLa cell membrane during endocytosis of conjugated polymer nanoparticles was studied. Results demonstrated SICM is a powerful tool to study the interaction between nanoparticle and cell membrane during internalization of nanoparticles through the membrane. This research can improve our fundamental understanding of cellular behavior and will be helpful for drug delivery applications. Based on conventional SICM, we have developed a novel method to simultaneous map the topography and potential distributions of the single living cells membranes. At the first step, multifunctional nanopipettes (nanopore/nanoelectrode) have been fabricated and characterized. To demonstrate the potential sensing capability and understand the mechanism, I measured the ionic current and local electric potential change during translocation of 40 nm charged gold nanoparticles. Our results reveal the capability of the multifunctional probe for the highly sensitive detection of the ionic current and local electrical potential changes during the translocation of the charged entity through the nanopore. From the potential change, we revealed the dynamic assembly of GNPs before entering the nanopore. The experimental results are also nicely explained by the finite element method based numerical simulation results. At the second step, I have measured the surface potential of living cell membrane at selected locations. Very recently, I have obtained results to show that we can map the extracellular membrane potential distribution of the complicated living cell membrane with sub-micron spatial resolution.This new imaging technique can help biologist to explore the extracellular potential distribution of varieties of cells quantitatively.These studies will have impacts on several biomedical applications such as regenerative repair and cancer treatment.

Les anticorps thérapeutiques sont des molécules de choix pour le traitement standard de nombreuses formes de cancers. Leur application est à ce jour restreinte au compartiment extracellulaire à cause de leur taille trop importante qui les empêche de traverser la membrane cellulaire. Comme la plupart des cibles thérapeutiques du cancer semblent être situées dans le milieu intracellulaire, ce serait un plus de pouvoir exploiter les propriétés des anticorps dans les cellules pour étudier et perturber l’activité de ces cibles. Néanmoins, l’utilisation des anticorps dans le milieu intracellulaire constitue un véritable challenge, notamment à cause de la membrane cellulaire et de l’environnement réducteur du cytoplasme. L’ensemble des travaux de thèse présentés dans ce manuscrit ont permis d’établir les bases de plusieurs stratégies innovantes d’immunociblage intracellulaire et de mettre en lumière l’importance des différentes étapes de validation d’anticorps ou de fragments dérivés utilisés comme anticorps intracellulaires. La vectorisation d’anticorps complets par électroporation, le développement d’un intracorps bispécifique original anti-PCNA et la mise au point d’une méthode de mutagenèse inspirée de l’hypermutation somatique constituent les principales avancées apportées par ce travail dans le domaine de la recherche technologique en immuno biotechnologie. / Therapeutic antibodies are interesting molecules used to treat numerous pathologies such as cancer. Because of their size, their application is currently limited to the extracellular space. Indeed, antibodies cannot cross the cell membrane. Almost all therapeutic targets in cancer seem to be located inside cells, it would be beneficial to take advantage of antibodies in cells in order to neutralize the activity of these targets. The use of antibodies inside the cells is a real challenge, because of the cell membrane and the reducing environment of the cytoplasm. Several strategies of intracellular immunotargeting are presented in this thesis.

Introduction: Cancer vaccines are a therapeutic approach to initiate an antigen specific cytotoxic immune responses against tumors. Cancer vaccines are composed by an antigen (tumor peptide) and adjuvant. A peptide in combination with adjuvants effectively activate dendritic cells (DCs), the most efficient antigen presenting cells in our immune system. DCs prime and activate CD8+ cytotoxic T cells which generates an antigen specific response.Aim: Developing an assay to study the internalisation rout of an antigen-adjuvant conjugate in human dendritic cells by using live cell imaging. Method: Immobilisation of cells is necessary for the ability to perform live cell imaging for several hours. The immobilisation ability of three coatings, collagen type I, fibronectin and matrigel, at different concentrations were evaluated by using live cell imaging in a fluorescence microscope. The potential induction of activation of the cells were evaluated by using flow cytometry and ELISA. Results: Immature DCs internalise antigen-adjuvant conjugate more efficiently than mature and activated DCs. Therefore, it is important that the coating do not induce activation. Cells must also be immobilised for the possibility of long term detection. Collagen type I immobilised cells and induced activation in all investigated concentrations. Fibronectin and matrigel had concentration-dependent abilities to immobilise the cells. Matrigel did not activate the cells whilst fibronectin was concentration dependent. Conclusion: Matrigel immobilise the cells which enables long term single cell imaging without activation.

The goal of this diploma thesis was using of a coherence-controlled holographic microscope in cell’s life research. A brief history of interference microscopy and it’s applications in biology is described. Also other microscopy techniques routinely used for transparent objects imaging are mentioned and the biology of cell’s life cycle briefly explained. Characteristics describing the shape of a cell were proposed and tested with respect to identification of particular phases of its life cycle. The method of dynamic phase differences was modified in order to distinguish the internal motion of cell’s mass from the movement of the whole cell. Selected characteristics were used to evaluate observations carried out with the holographic microscope and the possibilities of their further applications were depicted. In conclusion, obtained findings were summarized and modifications of microscope construction as well as data-processing software were suggested.