Dissertations / Theses on the topic 'Séquençage en cellule unique'
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Foulon, Sophie. "Développement du séquençage ARN ciblé sur cellules uniques en microfluidique de gouttes et applications." Thesis, Paris Sciences et Lettres (ComUE), 2019. http://www.theses.fr/2019PSLET037.
Full textSingle cells technologies were introduced a few years ago and have been dramatically evolving ever since. These technologies have revolutionized biology, making it possible to better understand how heterogeneous cell systems works. For example, they permit to discover and follow cell subtypes, with applications in oncology or neurobiology. We have developed a technology to study the expression profile of genes of interest at the level of a single cell, using droplet-based microfluidics. By limiting the number of genes studied compared to commercial whole-transcriptome technologies, the targeted approach has several potential benefits: gaining deeper sequencing, increasing the number of cells studied, optimizing detection for low levels of expression, while reducing the complexity of data and costs. Targeting is sometimes essential, especially when the RNAs do not carry a generic primer sequence, as in the case of viral RNAs. Two applications are presented: the analysis of inflammation of the immune cells of the brain in the early stages of development, as well as the study of genetic recombination in the virus
Deprez, Marie. "Étude de l’hétérogénéité cellulaire et des dynamiques de régénération de l’épithélium respiratoire sain par analyses des signatures transcriptionnelles sur cellules uniques." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2019. http://www.theses.fr/2019AZUR6022.
Full textImprovements made in nucleic acid sequencing and cell handling technologies now offer the opportunity to analyze simultaneously the content of numerous single cells (RNA, DNA, ...) by global and unbiased approaches. This single-cell ‘omics’ revolution provides a new framework to revisit the “Cell Theory”, elaborated over several centuries, and essentially based on morphological and functional features. The many cell modalities now accessible at single- cell level, such as their transcriptome, spatial localization, developmental trajectories, enrich considerably this definition, and set a renewed context to precisely reassess the definition of ‘cell types’, ‘cell states’ as well as their different interactions and fates.My thesis work initially set up ad hoc approaches and statistical framework to analyze appropriately these single-cell data, which deeply differ from standard bulk RNA-seq. High variance, presence of a huge percentage of null values, large volume of data are among the specific characteristics of these datasets. My work was centered on the main experimental model of my host laboratory, e.g. the human airway epithelium. Human airways are lined by a pseudostratified epithelium mainly composed of basal, secretory, goblet and multiciliated cells. Airways also constitute a true cellular ecosystem, in which the epithelial layer interacts closely with immune and mesenchymal cells. This coordination between cells ensures proper defense of the respiratory system and its correct regeneration in case of external aggression and injuries. A better understanding of the operating sequences in normal and physiopathological situations is relevant in pathologies such as chronic obstructive pulmonary disease, asthma or cystic fibrosis.First, I characterized at a single cell level the precise and cell-specific sequence of events leading to functional regeneration of the epithelium, using a 3D model of human cells. I then built a single-cell atlas of the different cell types that are lining healthy human airways from the nose to the 12th generation of bronchi.By applying computational and statistical approaches, I have identified cell lineage hierarchies and was able to reconstruct a comprehensive cell trajectory roadmap in human airways. I not only confirmed previously described cell lineages, but I have also discovered a novel trajectory that links goblet cells to multiciliated cells, identifying novel cell populations and molecular interactors involved in the process of healthy human airway epithelium regeneration. The profiling of 12 healthy volunteers then generated a dataset of 77,969 cells, derived from 35 distinct locations. The resulting atlas is composed of more than 26 epithelial, immune and stromal cell types demonstrating the cellular heterogeneity present in the airways. Its analysis has revealed a strong proximo-distal gradient of expression in suprabasal, secretory, or multiciliated cells between the nose and lung airways. My work has also improved the characterization of rare cells, including “hillock” cells that have been previously described in mice.In conclusion, this work probably represents one of the first single-cell investigations in human airways. It brings original contributions to our understanding of differentiation’s dynamics and cellular heterogeneity in healthy human airways. The resulting resource will be extremely useful for any future single-cell investigators and also for establishing a very useful joint between clinical and biological works. As such, it will constitute a reference in any future project aiming to precisely analyze specific disease conditions
Saviano, Antonio. "Physiopathologie du foie à l'échelle de la cellule unique : caractérisation de l'hétérogénéité cellulaire et identification de nouvelles cibles thérapeutiques dans les maladies hépatiques chroniques et le cancer hépatocellulaire." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ093.
Full textHepatocellular carcinoma (HCC) is a leading cause of death worldwide and the current treatments are unsatisfactory. One reason is the limited knowledge on the complexity and microenvironment of healthy and diseased liver. To address this gap, we have developed a single cell RNA sequencing (scRNA-seq) pipeline for primary human liver tissues. We have assembled an atlas of human liver cells and compared the scRNA-seq profile of normal liver and HCC. The atlas revealed an unknown heterogeneity within the main populations of liver cells, the transcriptomic zonation of endothelial cells and the existence of an epithelial progenitor in the adult liver capable of differentiating into both cholangiocytes and hepatocytes. ScRNA-seq analysis uncovered the marked cell heterogeneity of HCC, its microenvironment changes at single-cell level and the interactions between tumor cells and hepatitis B virus discovering previously unknown pathways and drivers of hepatocarcinogenesis
Ozier-Lafontaine, Anthony. "Kernel-based testing and their application to single-cell data." Electronic Thesis or Diss., Ecole centrale de Nantes, 2023. http://www.theses.fr/2023ECDN0025.
Full textSingle-cell technologies generate data at the single-cell level. They are coumposed of hundreds to thousands of observations (i.e. cells) and tens of thousands of variables (i.e. genes). New methodological challenges arose to fully exploit the potentialities of these complex data. A major statistical challenge is to distinguish biological informationfrom technical noise in order to compare conditions or tissues. This thesis explores the application of kernel testing on single-cell datasets in order to detect and describe the potential differences between compared conditions.To overcome the limitations of existing kernel two-sample tests, we propose a kernel test inspired from the Hotelling-Lawley test that can apply to any experimental design. We implemented these tests in a R and Python package called ktest that is their first useroriented implementation. We demonstrate the performances of kernel testing on simulateddatasets and on various experimental singlecell datasets. The geometrical interpretations of these methods allows to identify the observations leading a detected difference. Finally, we propose a Nyström-based efficient implementationof these kernel tests as well as a range of diagnostic and interpretation tools
Marcy, Guillaume. "Etude des spécificités transcriptionnelles et de la compétence des progéniteurs neuraux postnataux du cerveau antérieur chez la souris." Thesis, Paris Sciences et Lettres (ComUE), 2018. http://www.theses.fr/2018PSLEP070/document.
Full textDuring development, a remarkable coordination of molecular and cellular events leads to the generation of the cortex, which orchestrates most sensorimotor and cognitive functions. Cortex development occurs in a stepwise manner: radial glia cells (RGs) - the neural stem cells (NSCs) of the developing brain - and progenitor cells from the ventricular zone (VZ) and the subventricular zone (SVZ) sequentially give rise to distinct waves of nascent neurons that form cortical layers in an inside-out manner. Around birth, RGs switch fate to produce glial cells. A fraction of neurogenic RGs that lose their radial morphology however persists throughout postnatal life in the subventricular zone that lines the lateral ventricles. These NSCs give rise to different subtypes of olfactory bulb interneurons and glial cells, according to their spatial origin and location within the postnatal SVZ. These observations raise important unresolved questions on 1) the transcriptional coding of postnatal SVZ regionalization, 2) the potential of postnatal NSCs for cellular regeneration and forebrain repair, and 3) the lineage relationship and transcriptional specificities of postnatal NSCs and of their progenies. My PhD work built upon a previously published comparative transcriptional study of defined microdomains of the postnatal SVZ. This study highlighted a high degree of transcriptional heterogeneity within NSCs and progenitors and revealed transcriptional regulators as major hallmarks sustaining postnatal SVZ regionalization. I developed bioinformatics approaches to explore these datasets further and relate expression of defined transcription factors (TFs) to the regional generation of distinct neural lineages. I then developed a model of targeted ablation that can be used to investigate the regenerative potential of postnatal progenitors in various contexts. Finally, I participated to the development of a pipeline for exploring and comparing select populations of pre- and postnatal progenitors at the single cell level. Objective 1: Transcriptomic as well as fate mapping were used to investigate the relationship between regional expression of TFs by NSCs and their acquisition of distinct neural lineage fates. Our results supported an early priming of NSCs to produce defined cell types depending of their spatial location in the SVZ and identified HOPX as a marker of a subpopulation biased to generate astrocytes. Objective 2: I established a cortical lesion model, which allowed the targeted ablation of neurons of defined cortical layers to investigate the regenerative capacity and appropriate specification of postnatal cortical progenitors. Quantitative assessment of surrounding brain regions, including the dorsal SVZ, revealed a transient response of defined progenitor populations. Objective 3: We developed a transgenic mouse line, i.e. Neurog2CreERT2Ai14, which allowed the conditional labeling of birth-dated cohorts of glutamatergic progenitors and their progeny. We used fate-mapping approaches to show that a large fraction of Glu progenitors persist in the postnatal forebrain after closure of the cortical neurogenesis period. Postnatal Glu progenitors do not accumulate during embryonal development but are produced by embryonal RGs that persist after birth in the dorsal SVZ and continue to give rise to cortical neurons, although with low efficiency. Single-cell RNA sequencing revealed a dysregulation of transcriptional programs, which correlates with the gradual decline in cortical neurogenesis observed in vivo. Altogether, these data highlight the potential of transcriptomic studies to unravel but also to approach fundamental questions such as transcriptional changes occurring in a population of progenitors over time and participating to changes in their fate potential. This knowledge will be key in developing innovative approaches to recruit and promote the generation of selected cell types, including neuronal subtypes in pathologies
Benavente, Diaz Maria. "Investigation of the molecular diversity defining muscle stem cell heterogeneity." Electronic Thesis or Diss., Sorbonne université, 2020. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2020SORUS072.pdf.
Full textAdult skeletal muscle has a remarkable regenerative capacity, being able to recover after repeated trauma. This property depends on the presence of muscle stem cells (MuSCs), which are mostly quiescent in homeostatic conditions, re-enter the cell cycle after injury and proliferate to give rise to committed myoblasts that will eventually fuse to restore the damaged fibres. Numerous studies have investigated the cell state transitions that MuSCs undergo from cell cycle entry to differentiation. Although several genetically modified reporter mice have been generated to study these events, detailed studies on the initiation of differentiation, which is generally defined by expression of the myogenic regulatory factor Myogenin, have been hampered by the lack of a reliable reporter mouse. Therefore, we developed a fluorescent reporter line where differentiating myogenic cells expressing Myogenin are marked by the expression of a tdTomato fluorescent protein. This novel knock-in mouse line allowed us to monitor the kinetics of Myogenin expression during cell differentiation in vitro, and perform preliminary experiments on the behaviour of myogenic cells in vivo by intravital imaging. Although all mouse MuSCs are characterised by the expression of the transcription factor Pax7 and they share several properties, some studies have reported differences in proliferation, engraftment ability, and sensitivity to disease of MuSCs from cranial and limb muscles. To investigate the gene regulatory networks that govern this functional heterogeneity, we have integrated single-cell transcriptomic analyses with cell biology approaches using mouse reporter lines to identify key regulators that confer distinct properties to high performing (extraocular muscles) and lower performing (limb, Tibialis anterior muscle) MuSCs in quiescence and activated states. We identified a delayed lineage progression of extraocular MuSCs in culture that was accompanied with the expression of distinct extracellular matrix remodelling factors and membrane receptors, and we validated the expression of some of these candidates at the protein level. Advanced computational analyses highlighted the dynamics underlying the maintenance of a stem-like progenitor population in extraocular MuSCs, controlled by a singular network of transcription factors acting as a co-regulating module. Taken together, these studies provide novel insights into the mechanisms underlying the differential properties of muscle stem cells in distinct anatomical locations
Cussat-Blanc, Sylvain. "Créatures Artificielles : Développement d'Organismes à partir d'une Cellule Unique." Phd thesis, Université des Sciences Sociales - Toulouse I, 2009. http://tel.archives-ouvertes.fr/tel-00449673.
Full textCaccianini, Laura. "Imagerie de l'architecture dynamique de la chromatine dans la cellule unique." Thesis, Paris Sciences et Lettres (ComUE), 2019. https://tel.archives-ouvertes.fr/tel-02896692.
Full textChromatin structure and cellular function are tightly linked in the nucleus of mammalian cells. Disruption of chromatin spatial organisation dramatically affects the life of a cell and eventually leads to severe pathologies in entire organisms. Two nuclear factors, CTCF and Cohesin, have been found to play a crucial role in the regulation and maintenance of DNA architecture. Huge advancements have been made in the understanding of the mechanisms behind chromatin arrangement but the field is still lacking a dynamic picture at the single cell and single molecule level. This study provide this study provides insight into the dynamics of CTCF and Cohesin through single particle tracking of CTCF and Cohesin dynamics achieved with single molecule tracking in living mouse embryonic stem cells. The interplay between these two factors was studied by looking at Cohesin’s behaviour in the absence of CTCF and in the context of other biological alterations
Bontoux, Nathalie. "Analyse du transcriptome d'une cellule unique à l'aide d'une puce microfluidique." Paris 6, 2006. http://www.theses.fr/2006PA066600.
Full textFarina, Francesca. "Transport de l'ADN dans le cytoplasme d'une cellule eucaryote." Paris 6, 2011. http://www.theses.fr/2011PA066283.
Full textSenecal, Adrien. "Régulation transcriptionnelle du proto-oncogène c-Fos à l’échelle de la cellule unique." Paris 6, 2013. http://www.theses.fr/2013PA066786.
Full textThe expression level of the 21,000 genes present in a human cell must be precisely controlled according to several extra- and intracellular signals. Failures in the control of gene expression are often involved in diseases such as cancer. The choice of genes, as well as their expression level, are the result of the regulation of RNA polymerase II by a combination of transcription factors. Usually, these events are studied over large cell populations, thus masking variations between cells of the same population. In my work, I particularly focused on the transcriptional regulation of the c-Fos proto-oncogene at the single cell level. To this end, we developed a tool for quantifying single mRNA and nascent RNA on transcription site from Fluorescence in situ Hybridization data. With this program, we discovered a remarkably simple regulation of c-Fos transcription. Multiple transcripts are produced during short and infrequent transcriptional bursts. We have shown that while the burst size is not regulated, their frequency is modulated by the level of activation of intracellular signaling pathways. We also observed a dynamics clustering of RNA polymerase II on genes. This clustering may provide an explanation for the molecular origin of these transcriptional bursts as well as providing a framework to decipher their regulation
Pierrat, Sébastien. "Etude de l'adhésion cellulaire à différentes échelles de la molécule unique à la cellule." Paris 6, 2004. http://www.theses.fr/2004PA066485.
Full textMeistermann, Dimitri. "Modélisation du développement préimplantatoire humain à partir de données de transcriptome de cellule unique." Thesis, Nantes, 2020. http://www.theses.fr/2020NANT1019.
Full textCe travail de thèse est consacré à l’étude de nouveaux mélanges gazeux pour la gravure plasma du CdHgTe, à savoir : CH₃NO₂/H₂/Ar, CH₃OH/H₂/Ar et CH₄/NO₂/H₂/Ar. L’objectif est de graver sans polarisation du substrat pour limiter l’énergie déposée sur les surfaces gravées. Une première partie portant sur l’analyse de ces plasmas par sondes électrostatiques et spectroscopie d’émission optique a permis de montrer que la substitution de nitrométhane ou méthanol au méthane a un effet sur la composante chimique de la gravure. Pour ces nouveaux mélanges hydrocarbonés, l’apparition de molécules telles que CO et CN est corrélée à l’annihilation du dépôt spontané de polymère. La seconde partie, consacrée à la gravure du CdHgTe avec ces nouveaux précurseurs a prouvé la faculté de graver sans polarisation du substrat avec les mélanges CH₃NO₂/H₂/Ar et CH₄/N₂O/H₂/Ar et ainsi réduire les dommages engendrés au matériau, notamment la rugosité en surface. Une étude plus poussée de la gravure en mélange CH₄/N₂O/H₂/Ar montre notamment une augmentation de la vitesse de gravure pour les faibles polarisations jusqu’à un certain seuil, avant qu’elle ne stagne, correspondant au passage d’une gravure à dominance chimique à une gravure à dominance physique. De plus, la rugosité est indépendante de la puissance d’excitation du plasma, de la température du substrat ainsi que de la durée de gravure. Enfin, la gravure de tranchées a permis de mettre en évidence la gravure chimique et isotrope à faible polarisation avec les mélanges CH₄/N₂O/H₂/Ar et CH₃NO₂/H₂/Ar mais qui, à plus forte polarisation présente une meilleure passivation latérale que les gravures en plasma CH₄/H₂/Ar
Moussy, Alice. "Caractérisation des premières étapes de différenciation des cellules hématopoïétiques à l'échelle de la cellule unique." Thesis, Paris Sciences et Lettres (ComUE), 2017. http://www.theses.fr/2017PSLEP029/document.
Full textDespite intensively studies, the fundamental mechanisms of cell fate decision during cellular differentiation still remain unclear. The deterministic mechanisms, often based on studies of large cell populations, cannot explain the difference between individual cell fates choices placed in the same environment. The aim of my thesis work is to study the first steps of hematopoietic cell differentiation at the single cell level thanks to transcriptomic, proteomic and morphological analyses. Two differentiation models have been used: T regulatory lymphocytes and human cord blood-derived CD34+ cells. The behavior of individual cells following stimulation has been analyzed. Using time-lapse microscopy coupled to single cell molecular analyses, we could demonstrate that the cell fate choice is not a unique, programmed event. First, the cell reaches a metastable “multi-primed” state, which is characterized by a mixed lineage gene expression pattern. After transition through an “uncertain”, unstable state, characterized by fluctuations between two phenotypes, the cell reaches a stable state. Our observations are coherent with a stochastic model of cell fate decision. The differentiation is likely to be a spontaneous, dynamic, fluctuating and not a deterministic process. The cell fate decisions are taken by individual cells
Defoort, Jean-Philippe. "Étude de structures peptidiques minimales susceptibles d'intérferer avec l'infection de la cellule hôte par Trypanosoma Cruzi." Lille 1, 1990. http://www.theses.fr/1990LIL10008.
Full textMeunier, Anne. "Méthodes analytiques pour la détection de phénomènes biologiques de sécrétion à l'échelle de la cellule unique." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2011. http://tel.archives-ouvertes.fr/tel-00858915.
Full textBois, Nadège. "Développement d'un système de gouttes en microfluidique pour la récupération et l'étude de la cellule unique." Paris 6, 2013. http://www.theses.fr/2013PA066776.
Full textStudies of cell populations are necessary to better understand normal and pathological biological mechanisms. However, one must take into account the specificity of each cell within a population. The study of their heterogeneity can provide meaningful information and it is therefore essential to investigate the gene expression levels of individual cell. This is a major challenge given the small amount of material in a single cell. The aim of our project was to develop innovative and integrated tools based on microfluidics to perform gene expression analyzes at the single cell level. For that purpose we used droplet microfluidics that enables the generation of small droplets of water in oil at high throughput. We developed a platform enabling single cell encapsulation and providing an easy handling of individual droplets. This system was then used for two different applications: first, we investigated the capabilities of this platform to make monoclonal cultures for the creation of stable cell lines or the production of monoclonal antibodies. Second, we targeted an application devoted to transcriptom analysis: once encapsulated in droplets, cells can be lysed and their RNA content can then be converted to complementary DNA, amplified by RT PCR, recovered and analyzed on microarrays. This strategy provides a gene expression profile in individual cells
Jacquey, Frédéric. "Développement d'une technique de microspectrophotométrie rapide destinée à l'étude des signaux ioniques cytoplasmiques sur cellule unique." Lyon 1, 1994. http://www.theses.fr/1994LYO10092.
Full textSeeleuthner, Yoann. "Rôle des protistes hétérotrophes marins dans le cycle du carbone océanique par génomique en cellule unique." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLE002/document.
Full textUnicellular eukaryotes (protists) have important roles in the biogeochemical cycles of the ocean. First, although on average less abundant than cyanobacteria, photosynthetic protists account for a large proportion of net primary production. Since they are relatively easier to culture, photosynthetic organisms are relatively more studied and their genomes represent a large fraction of the genomes of marine protists available in databases. On the other hand, heterotrophic organisms require much more work for cultivation and are therefore much less well known. Moreover, the majority of genomes of heterotrophic protists sequenced to date concern organisms of interest to humans (plant pests, pathogenic organisms), but the choice of these organisms as study models does not reflect their ecological interest.The objective of this thesis is to study, using a single-cell genomic approach, several heterotrophic picoeucaryotes, that are highly abundant in the open oceans and have not yet been cultivated. For this purpose, a protocol for sequencing, assembling and annotation of genomes from single cells has been developed.The genomes of seven stramenopile lineages have been partially reconstructed and annotated, making it possible to confirm in a robust way the phylogeny of stramenopiles obtained by ribosomal markers, and, more important, to formulate hypotheses regarding their specialization in terms of mobility or trophic mode.Particularly, the comparison of gene repertoires of carbohydrate degradation indicates likely different food spectra in the organisms studied.In addition, the combined use of these genomes and metagenomic sequences from the Tara Oceans expedition made it possible to describe the geographical distribution of these organisms as well as the genetic distance between environmental populations and our reference genomes. The correlation of these distributions with the environmental parameters measured at the sampling points shows that temperature is a key factor in the distribution of these microorganisms. In addition, the use of metatranscriptomic data from the expedition allowed us to distinguish different expression profiles - potentially corresponding to different physiological states - in the most cosmopolitan lineage studied.In conclusion, this thesis shows that there is a strong genomic diversity to be explored in heterotrophic marine protists, allowing us to make hypotheses about their trophic modes. It also demonstrates the value of single-cell genomics, in particular its complementarity with metagenomic and metatranscriptomic approaches for the comprehensive understanding of marine ecosystems
Mary, Pascaline. "Génération de gouttes en microfluidique pour l'étude de la cellule unique, l'extraction liquide-liquide et la vectorisation." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2009. http://pastel.archives-ouvertes.fr/pastel-00005739.
Full textLu, Cong. "Analyse microélectrochimique du stress oxydant à l'échelle de la cellule unique : application aux cellules cancéreuses du sein." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2012. http://tel.archives-ouvertes.fr/tel-00828217.
Full textCrozatier, Cécile. "Contrôle et analyse électrochimique de la réactivité biologique à l'échelle de la cellule unique dans un dispositif microfluidique." Phd thesis, Université Paris-Diderot - Paris VII, 2007. http://tel.archives-ouvertes.fr/tel-00178656.
Full textGrâce au développement d'outils modulables de culture cellulaire et de manipulation de cellules vivantes dans des dispositifs microfluidiques, nous avons mis en place le contrôle dynamique stable de stimulations chimiques sur une population de cellules souches mésenchymateuses (CSM) en culture et poursuivons cette étude dans le but d'induire la réactivité cellulaire des CSM vers la voie de différenciation neuronale.
Le développement d'un microsystème intégré de détection électrochimique du stress oxydant sur cellules uniques est mis en oeuvre à travers la réalisation d'un dispositif microfluidique intégré consistant en un réseau de chambres de mesures, contenant des microélectrodes fonctionnelles, et permettant d'isoler des macrophages uniques et de les maintenir en survie pendant plusieurs dizaines de minutes, durée suffisante pour réaliser nos mesures électrochimiques. En faisant varier les conditions de mesure, comme le nombre de cellules sondées dans le même micro-environnement, la nature du stimulus ou la présence ou non de communication cellulaire avec une population voisine, nous posons les bases d'une analyse originale jamais réalisée jusqu'à présent.
Guille, Manon. "Détection par microélectrodes de flux atto-à femtomolaires de neuromédiateurs sur cellule unique et dans un tissu vivant." Paris 6, 2005. http://www.theses.fr/2005PA066207.
Full textChen, Tong. "Développement de biocapteurs hyperfréquences microfluidiques pour la spectroscopie diélectrique non-invasive de la cellule unique : applications en cancérologie." Toulouse 3, 2012. http://thesesups.ups-tlse.fr/2172/.
Full textDevelopment of microwave microfluidic bio-sensors for non-invasive dielectric spectroscopy of single cell: Applications in Cancerology. Biological analysis at the level of the single cell, allowing the understanding of cellular mechanisms, is of great importance in the fields of biology, medicine and especially in oncology. Microtechnology has opened up the prospect of such devices and many researches are underway on the development of analysis systems which are non-invasive, rapid, label-free or has no cell damage. The convergence of bio-microwave sensors with microfluidics can meet these challenges. We developed jointly (1) micro-devices for microwave dielectric spectroscopy of biological fluids and (2) microfluidic systems for manipulation of cell populations and single cell in the culture medium. The electromagnetic fields engineering was conducted to optimize the fluid volume analysis and the detection sensitivity. Microfabrication performed at LAAS-CNRS allowed controlled positioning of single cell in the analysis area. We finally demonstrated experimentally significant dielectric contrast between cancer cells alive and RL lymphoma apoptosis with more tracking capability longitudinal of apoptosis. These researches work on non-invasive analysis of single cell, the discriminatory capacity of different biological states, the possibility of temporal tracking of biological mechanisms open new perspectives for the cell analysis in cancerology
Trouchet, Amandine. "PCR digitale pour la détection et la caractérisation de micro-organismes pathogènes au niveau de la cellule unique." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC170/document.
Full textWe aim to develop a prototype of droplet-based microfluidic system capable of detecting and colocalizing multiple genetic markers at the single cell/bacteria level, using the Polymerase Chain Reaction (PCR) in a digital multiplexed version. This cannot be achieved using current commercial digital PCR systems, and should increase the sensitivity and reliability of the detection of pathogens. Importantly, the system will guarantee the presence of multiple markers within the same genome and enable accurate identification, and bring the false positive rate close to zero. As a first application, we will demonstrate the possibility to co-localize 3 virulence genes in the E.coli strain O157:H7, a major foodborne pathogen, which has to be detected in clinical feces samples or food samples, which may also contain non pathogenic E. coli carrying only a subset of these virulence genes. E. Coli will be encapsulated in micrometric droplets, lysed by heating in situ prior performing a multicolour end-point Taqman assay. Our objective is to demonstrate that this test can be successfully applied to real clinical or food samples
Eid, Joelle. "Etude du relargage du VIH-1 en temps réel à l'échelle de la cellule unique par la Viro-fluidique." Thesis, Université de Montpellier (2022-….), 2022. http://www.theses.fr/2022UMONT007.
Full textUpon its entry, HIV-1 replicates and produces new viral particles that are released into the extracellular environment. The crucial step of virus release remains poorly understood because it requires the study at the single cell level. Indeed, quantification of viral production from cell populations with very heterogeneous HIV-1 replication kinetics would give approximate results. This is why we have developed a microfluidic approach that allows the study of HIV-1 release dynamics in real-time at the single cell level. In this study, continuous microfluidics was combined to the virology in order to develop a sensitive and reliable technology to visualize and quantify virus production by a single cell. Three types of chips were fabricated: the trapping chip allowed us to determine the physical and biological parameters that ensure single cell trapping (~10 µm) producing VLPs-GFP. The detection chip, whose performance was compared with the Nanoparticle Tracking Analysis technique, proved to be a valuable tool for accurate and reproducible quantification of fluorescent VLPs (~140 nm) at the single particle scale in cell culture supernatants. The multiplex chip, which combines the two previous chips, allowed us to study in real-time the virus release kinetics at the single cell scale. VLPs-GFP producing HeLa and HEK 293 cell lines were used as study models. For the first time, viral production kinetics could be measured with an average of 50 VLPs / cell / h that was validated by the measurement of viruses produced by the same cells grown in culture dish, confirming the reliability and sensitivity of our approach. Interestingly, the release kinetics profile shows a periodic process (period ~4min) that could be explained by the presence of one or more limiting steps in the virion biogenesis mechanism. The new tools developed here provide novel information on the kinetics of HIV-1 salting-out. They can be used or easily adapted for the study of other pathogens or extracellular vesicles
Martineau, Eugénie. "Linking single cell directionality to dynamic multicellular transitions in Myxococcus xanthus : a multiscale analysis." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0089.
Full textThe δ-proteobacteria Myxococcus xanthus has been a model of study for decades for its self-organized behavior as a response of environmental stimuli. It colonizes favorable ecological niches by using surface motility. In particular, this motility allows M.xanthus to predate collectively over prey microorganisms, while under starvation they start a developmental process to form macroscopic fruiting bodies, filled with environmental resistant myxospores. All these multicellular behaviors require a dynamic control of the cell polarity established by the polarity proteins MglA, MglB and RomR. Together, they define the direction of movement of the cell, which can be rapidly inverted by the Frz chemosensory system (reversion). In this thesis work, through combined computational/experimental approaches, we highlight that the regulation system forms a new type of biochemical oscillator, controlled by two proteins RomR and FrzX, which act together through complementary action to trigger the reversion at the lagging pole. The unique architecture of this system allows a wide response to various stimuli, which could be very beneficial for collective cell behaviors. To understand the importance of these transitions, we have developed a new high-resolution single cell assay linking single cMARTINEAU EUGENIE 2018AIXM0089/016ED62 2018/03/21 62 SCES SCHell behaviors to multicellular structures at unprecedented spatial and temporal resolutions. This way, we have investigated the role of the newly identified biochemical oscillator in the multicellular model of predation
Bonnaffoux, Arnaud. "Inférence de réseaux de régulation de gènes à partir de données dynamiques multi-échelles." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSEN054/document.
Full textInference of gene regulatory networks from gene expression data has been a long-standing and notoriously difficult task in systems biology. Recently, single-cell transcriptomic data have been massively used for gene regulatory network inference, with both successes and limitations.In the present work we propose an iterative algorithm called WASABI, dedicated to inferring a causal dynamical network from timestamped single-cell data, which tackles some of the limitations associated with current approaches. We first introduce the concept of waves, which posits that the information provided by an external stimulus will affect genes one-byone through a cascade, like waves spreading through a network. This concept allows us to infer the network one gene at a time, after genes have been ordered regarding their time of regulation. We then demonstrate the ability of WASABI to correctly infer small networks, which have been simulated in-silico using a mechanistic model consisting of coupled piecewise-deterministic Markov processes for the proper description of gene expression at the single-cell level. We finally apply WASABI on in-vitro generated data on an avian model of erythroid differentiation. The structure of the resulting gene regulatory network sheds a fascinating new light on the molecular mechanisms controlling this process. In particular, we find no evidence for hub genes and a much more distributed network structure than expected. Interestingly, we find that a majority of genes are under the direct control of the differentiation-inducing stimulus. Together, these results demonstrate WASABI versatility and ability to tackle some general gene regulatory networks inference issues. It is our hope that WASABI will prove useful in helping biologists to fully exploit the power of time-stamped single-cell data
Fiszman, Nicolas. "Etude de cinétique de la traduction eucaryote à l'échelle de la molécule unique." Phd thesis, Palaiseau, Institut d'optique théorique et appliquée, 2013. http://pastel.archives-ouvertes.fr/pastel-00939858.
Full textRodrigues, Peixoto Antonio José. "Stratégies de différenciation des lymphocytes T CD8 intervenant au cours d'une réponse immunitaire in vivo : étude au niveau d'une cellule unique." Paris 5, 2005. http://www.theses.fr/2005PA05N20S.
Full textThis work consisted in the study of the CD8 T cell differentiation after immunization in vivo. Due to the absence of appropriate methods to pursue this study, we have developed the necessary tools : the single cell quantitative RT-PCR. The use of this methodology has allowed us to gain a completely new insight into the differentiation of CD8 T cells during immune responses in vivo. First, cytotoxicity is only acquired once the necessary functions are co-expresseded by individual T cells after immunization. Moreover, this process is not synchronous and individual T cells engage differently in the acquisition of effector functions. Second, the generation of memory T cells is not followed by a terminal differntiation. Re-immunisation induced the accumulation and diversification of effector functions expressed by individual memory T cells. In summary, our results impose a redefinition of the stages of immune responses and challenge the current definition of memory T cells
Alberge, Jean-Baptiste. "Étude des mécanismes de l'initiation et de la progression du myélome multiple par transcriptomique en cellule-unique et cartographie de l'hydroxyméthylome." Thesis, Nantes, 2021. http://www.theses.fr/2021NANT1006.
Full textMultiple Myeloma (MM) is a hematological malignancy caused by the uncontrolled proliferation of long-lived plasma cells. Genomic heterogeneity of MM is characterized by a significant diversity of somatic genetic alterations, epigenetic modifications, and of transcriptional programs whose integration remains poorly understood, and that we discuss in three parts. First, compared to normal plasma cells, we found global DNA 5-hydroxymethylcytosine (5hmC) levels to be lower in MM. Higher 5hmC levels correlated with a less severe disease. Local 5hmC remained at active and transcribed regions of the chromatin where it mirrored the MM molecular subgroups (CCND1, MMSET, etc.) as well as their gene expression programs. Second, we described unique genomic abnormalities that were associated with clinical progression and acquired resistance to venetoclax, an anti-BCL2 targeted therapy. The BCL2 family of genes displayed numerous alterations that were clonally or sub-clonally selected. Functional tests and gene expression profiling underpinned these results. Third, transcriptional programs of MM cells were associated with biomedical imaging parameters to define new prognostic markers that mirrored the expansion and severity of the disease. Together, this work contributes to our understanding of MM oncogenesis and progression while it also unravels novel clinical biomarkers
Ruiz, Garcia Sandra. "Appréhender l'hétérogénéité cellulaire et la dynamique de différenciation des épithéliums des voies aériennes au moyen de signatures transcriptionnelles sur cellule unique." Thesis, Université Côte d'Azur (ComUE), 2018. http://www.theses.fr/2018AZUR4204/document.
Full textHuman airways are lined by a pseudostratified epithelium mainly composed of basal and columnar cells, among these cells we can find multiciliated, secretory cells and goblet cells. All these cells work together in the mucociliary clearance of the airways. This epithelium regenerates slowly under homeostatic conditions but is able to recover quickly after aggressions through proliferation, migration, polarization and differentiation processes. However, in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, asthma or cystic fibrosis, epithelial repair is defective, tissue remodeling occurs, leading to loss of multiciliated cells and goblet cell hyperplasia, impairing correct mucociliary clearance. The sequence of cellular events leading to a functional or remodelled tissue are still poorly described. Hence, we aim at identifying the successive cell types appearing during tissue regeneration and the molecular events that are responsible for healthy or pathological regeneration. We have analysed airway epithelial cell composition at several stages of differentiation using an in vitro 3D culture model which reproduces in vivo epithelial cell composition. Applying single cell transcriptomics and computational methods, we have identified cell lineage hierarchies and thus constructed a comprehensive cell trajectory roadmap in human airways. We have confirmed the cell lineages that have been previously described and have discovered a novel trajectory linking goblet cells to multiciliated cells. We have also discovered novel cell populations and molecular interactors involved in the process of healthy human airway epithelium regeneration. Using these approaches, we have finally shed light on cell-type specific responses involved in pathological goblet cell hyperplasia. Our data, by bringing significant contributions to the understanding of differentiation’s dynamics in the context of healthy and pathological human airway epithelium, may lead to the identification of novel therapeutic targets
Geisler, Hubert. "Structuration d'hydrogels thermoactivables pour l'analyse de cellules uniques." Electronic Thesis or Diss., Université Paris sciences et lettres, 2020. http://www.theses.fr/2020UPSLS001.
Full textWe present in this work a new microfluidic technology aiming at isolating single cells by the use of thermoactuable polymers. One of the polymers we use is polyNIPAM, a polymer that can expand its volume by 400% in water when the temperature is set under 32°C and can shrink down when it is set over 34°C. We use this reversible swelling capability to open and close compartments embedded in a microfluidic chip.Grafting and structuring these hydrogel features relies on thiol-en click chemistry, initiated thermally or by UV irradiation. We have developed methods and microfabrication protocols in order to diversify the substrate materials (from glass to PDMS, COC, PMMA, etc), to expand the structures thickness range (from few microns to a tenth of microns) and to strengthen our knowledge regarding the fabrication impact on the hydrogel’s behavior. A robust protocol of photolithography has finally been worked on allowing the design of any type of 2D features on a large choice of substrates.One of the realistic applications detailed here is the development of microfluidic chips aiming at isolating single cells in hydrogel compartments. (confidential)
Ben, Meriem Zacchari. "Memory of stress response in the budding yeast Saccharomyces cerevisiae." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC311.
Full textCellular memory is a critical ability displayed by micro-organisms in order to adapt to potentially detrimental environmental fluctuations. In the unicellular eukaryote S. cerevisiae, it has been shown at the population level that cellular memory can take the form of a faster or a decreased response following repeated stresses. We here present a study on how yeasts respond to short, pulsed hyperosmotic stresses at the single-cell level. We analyzed the dynamical behavior of the stress responsive STL1 promoter fused to a fluorescent reporter using microfluidics and fluorescence time-lapse microscopy. We established that pSTL1 displays a dynamical variability in its successive activations following two short and repeated stresses. Despite this variability, most cells displayed a memory of past stresses through a decreased activity of pSTL1 upon repeated stresses. We showed that this memory does not require do novo protein synthesis. Rather, the genomic location is important for the memory since promoter displacement to a pericentromeric chromatin domain leads to its decreased transcriptional strength and to the loss of the memory. Interestingly, our results points towards an unreported involvement of the SIR complex on the activity of pSTL1 only when displaced to the pericentromeric domain in our experimental conditions. This study provides a quantitative description of a cellular memory that includes single-cell variability and points towards the contribution of the chromatin structure in stress memory. Our work could serve as a basis to broader studies on the positioning of stress response genes at subtelomeric positions in the budding yeast, from a genetic point of view as well as an evolutionary one
Ducousso, Mathieu. "Acoustique picoseconde dans une cellule biologique individuelle." Phd thesis, Université Sciences et Technologies - Bordeaux I, 2010. http://tel.archives-ouvertes.fr/tel-00537030.
Full textBrisset, Sophie. "Apport de l'hybridation génomique comparative (CGH) sur puce en cytogénétique constitutionnelle : applications à l'étude de remaniements télomériques et à l'analyse chromosomique d'une cellule unique." Paris 5, 2006. http://www.theses.fr/2006PA05N03S.
Full textComparative genomic hybridisation (CGH) on metaphase spreads and microarray CGH are genome-wide analyses of DNA copy number imbalances. First of all, we studied telomeric rearrangements by microarray CGH. Fetuses with increased nuchal translucency have been analysed with conventional and microarray CGH. Precise characterisation of a de novo unbalanced chromosomal abnormality has been achieved using microarray CGH. Secondly, we performed CGH analysis of a single cell. A reliable DOP-PCR protocol have been developed to amplify single lymphocytes DNA. Trisomy 21 has been detected on a single lymphocyte by microarray CGH. Chromosomal analysis of a single cell or few cells is promising and should be applied in prenatal diagnosis or cancer
Tondeur, Sylvie. "Cellule souches hématopoïétiques et cellules souches embryonnaires humaines : analyse du transcriptome et mise en ligne des données par la création d'un atlas d'expression." Montpellier 1, 2009. http://www.theses.fr/2009MON1T012.
Full textDNA-microarray based transcriptome study can monitor the expression of a whole genome in one experiment and has completely changed our manner to conceive biological questions. We applied this technology to the study of stem cells. The transcriptome analysis of human embryonic stem cells (hESC), a main model of pluripotent stem cells, led us to better define molecular mechanisms of pluripotency. Of note we compared hESC to human oocytes in order to identify intinsic determinants of pluripotency. Affymetrix Exon ST 1. 0 microarrays are high resolution platforms that can measure the expression of all the exons of a genome. We used this new microarray to map exonic expression profile (exome) of hematopoietic stem cells and mature blood cells. Our work showed hematopoietic specific alternative splicing events (NEDD9, CD74) and an alternative switch for some transcripts during maturation (INPP4B, PTPLA, CXCL3, COMMD6). Alternatively spliced genes are notably involved in cell motility and immune response. Finally, we created a web atlas, Amazonia! (http://amazonia. Transcriptome. Eu/), for an easy access to public transciptome data. Gene expression profiles can be visualised as histograms or colored matrixes in more than 5,000 samples regrouped in thematic pages such as «stem cell», «hematology» or «exon»
Nissant, Antoine. "Caractérisation des canaux chlorures de la membrane basolatérale du néphron distal de souris." Paris 6, 2005. http://www.theses.fr/2005PA066450.
Full textSantamaria, Kathleen. "Etude de l’hétérogénéité des centrocytes humains à travers l’expression du CD23 : différenciation en plasmablastes et expression d’une signature minimale transcriptionnelle au niveau cellule-unique comportant DEC2." Thesis, Rennes 1, 2020. http://www.theses.fr/2020REN1B038.
Full textTerminal B cell differentiation through the germinal center leads to the production of antibody-secreting cells: plasma cells (PC) with a long lifespan and high affinity for the antigen. This reaction involves the formation of an anatomical microstructure that includes a dark zone: site of intense proliferation and affinity maturation of the BCR of the centroblasts, and a light zone in which the class switch recombination of the BCR and centrocyte (CC) selection take place. This differentiation is finely controlled by follicular helper T cells (Tfh) that produce molecules such as IL-4, CD40L and IL-21. This phD work focus on the CD23 expression on the surface of CC during their metamorphosis into PC. Firstly, I showed that the expression of the low affinity IgE receptor is regulated by Tfh derived IL-4 and CD40L. Moreover, I showed that CC exposed to Tfh signals and which do not express the CD23 were the ones that have the ability to differentiate into PC. In this context, I identified at the single cell level a transcriptional signature expressed specifically by these cells which contains a gene never described in PC, encoding the transcription factor DEC2 whose function remains to be determined. In addition, I studied CD23 expression in follicular lymphoma and showed the existence of differences between these two populations, suggesting a preferential survival and differentiation of CD23neg cells compared to CD23pos cells
Sakakini, Nathalie. "Rôle du facteur de transcription EGR1 dans le contrôle de l' autorenouvellement des cellules souches de glioblastomes." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4071.
Full textGlioblastoma is the most commun and agressive cerebral tumor. The current treatments combine surgery with chemotherapy and radiotherapy. However these treatments are poor effective. The relapse is frequent and the rate survival is less than 18 months.The relapse is in part due to the presence of glioblastoma initiating cells (GIC). The cells have stem cell properties. They can self-renew to maintain a pool of tumor cells and they can differentiate in different kind of tumor cells. They are also able to resist to the therapies by activating mechanisms of drug efflux. The commitment of GIC toward a differentiated tumor state decreases strongly their tumorigenic potential.EGR1 transcription factor is involved in many biological processes such as proliferation and differentiation. In the GIC EGR1 expression is abnormally elevated. This level decreases when cells are differentiated. EGR1 expression is strongly correlated with stem state suggesting its contribution in the proliferation regulation of GIC or in the maintenance of this state.My aim is to characterize the role of EGR1 in the regulation of proliferating state of the GIC.We have demonstrated the involvement of EGR1 in the pathway involving the mir18a* and the genes SHH and GLI1. It contributes so to the self-renewal, to the proliferation and to the maintenance of the stem state of GIC. In addition by directly regulating the gene PDGFa EGR1 maintains this system by a second molecular loop
Koh, Alaric C. W. "Analyses électrochimiques d’espèces réactives de l’oxygène et de l’azote produites par un macrophage immunostimulé." Paris 6, 2009. http://www.theses.fr/2009PA066465.
Full textGrosselin, Kevin. "Cartographie épigénétique de cellules cancéreuses résistantes rares par microfluidique en gouttelettes." Thesis, Paris Sciences et Lettres (ComUE), 2018. http://www.theses.fr/2018PSLET015.
Full textThe dynamic nature of chromatin and transcriptional features play a critical role in normal differentiation and are expected to contribute to tumor evolution. Studying the heterogeneity of chromatin alterations with single-cell resolution is mandatory to understand the contribution of epigenetic plasticity in cancer.In this thesis, I describe a droplet microfluidics approach to profile chromatin landscapes of thousands of cells at single-cell resolution, with an unprecedented coverage of 10,000 loci per cell.The system was evaluated to profile histone modifications associated with active (H3K4me3) and inactive transcription (H3K27me3) of human B cells and T cells, and revealed that >99% of the cells were correctly assigned to one cell type, defining distinct chromatin states of immune cells with high accuracy.In patient-derived xenograft (PDX) models of breast cancer with acquired drug resistance, the system enabled the detection of a rare subpopulation of cells in the untreated, drug-sensitive tumors with chromatin features characteristic of resistant cancer cells. These cells had lost chromatin marks (H3K27me3) associated with stable transcriptional repression for a number of genes known to promote resistance, potentially priming them for transcriptional activation.These results highlight the potential selection of cells with specific chromatin marks in response and in resistance to cancer therapy
Vandecasteele, Marie. "Interactions locales (synapses chimiques et électriques) entre les neurones dopaminergiques de la substance noire compacte." Paris 6, 2006. http://www.theses.fr/2006PA066323.
Full textGalindo, González Luis Javier. "Deep eukaryotic phylogenomics : the holomycota branch Combined cultivation and single-cell approaches to the phylogenomics of nucleariid amoebae, close relatives of fungi Evolutionary Genomics of Metchnikovella incurvata (Metchnikovellidae): an early Branching Microsporidium A new fungal clade helps reconstructing the tree of Fungi and the evolution of the flagellum in Holomycota Ancient Adaptive Lateral Gene Transfers in the Symbiotic Opalina–Blastocystis Stramenopile Lineage." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS050.
Full textDespite the astonishing diversity of plants, animals and macroscopic fungi, most eukaryotic diversity is actually microbial. The eukaryotic tree comprises several large monophyletic supergroups. One of these groups is the Opisthokonta, which encompasses two branches, Holozoa, including animals, and Holomycota, grouping Fungi and their unicellular relatives. While multicellular fungi are well known, knowledge on the diversity of unicellular Fungi and their phylogenetic relatives is still poor. This unicellular fraction includes several zoosporic lineages (e.g. Chytridiomycota and Blastocladiomycota) within Fungi, but also a variety of lineages related to the classical core Fungi: nucleariids, rozellids, aphelids and Microsporidia. However, the phylogenetic relationships of these lineages among them and with classical Fungi remain to be solidly established. Molecular phylogenetic trees of 18S rRNA genes retrieved from environmental studies have showed a wide diversity of unicellular holomycotans in almost all environments on Earth. However, the phylogenetic signal of this gene is limited and does not allow robustly resolving most deep phylogenetic relationships. During past years, high-throughput techniques have allowed sequencing hundreds of new genomes and transcriptomes. This has made possible to carry out multi-gene phylogenomic studies, which increase the available signal to resolve evolutionary relationships. Nevertheless, most sequenced genomes correspond to easy-to-culture fungal species, often with particular interest for humans (e.g. parasites, plant symbionts, yeast). Recently, single-cell omics has become a potential useful approach to study uncultured unicellular eukaryotes, making it possible to reconstruct robust phylogenetic analyses of a wide environmental diversity using genomic and transcriptomic data. During my PhD work, I have applied single-cell techniques to get phylogenetic information from divergent holomycotan lineages, clarify phylogenetic relationships among fungi and their close relatives and infer trait evolution. More specifically, I have used this approach to: 1) Generate genomic and transcriptomic data for nucleariids and better reconstruct inner relationships in the clade and the characters present in the nucleariid ancestor. Our results confirm that the cover-bearing unicellular genera Pompholyxophrys and Lithocolla are indeed nucleariids and branch together with Nuclearia, Parvularia and Fonticula. The reconstruction of a robust phylogeny for the group allowed us to infer the traits (e.g. no flagellum, glycocalyx, no cover) already present in their ancestor. 2) Sequence and comparatively analyze the genome of Metchnikovella incurvata, to confirm its relatively basal position within Microsporidia, and determine synapomorphies for the clade. Phylogenomic analysis of the metchnikovellid Metchnikovella incurvata confirmed that Metchnikovellidae branch at the base of Core-Microsporidia. We also confirmed their metabolic profile to be more similar to Core-microsporidia, being both similarly reduced in genes/functions. 3) Generate genomic data for Amoeboradix gromovi and Sanchytrium tribonematis, which form the newly described zoosporic fungal clade of sanchytrids, and resolve their phylogenetic position. The study of the two sanchytrid genomes clarified their placement within Fungi as a new clade sister to Blastocladiomycota. Comparative genomics showed that their metabolic composition was reduced in comparison with related lineages. This reduction was especially important in their flagellar toolkit when compared with other Holomycota, confirming 4 independent flagellum loss events in the clade
Bost, Pierre. "Decoding cellular communications and interactions between immune cells by using single-cell approaches." Electronic Thesis or Diss., Sorbonne université, 2020. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2020SORUS020.pdf.
Full textCellular communications are essential to the proper functioning of multi-cellular organisms, particularly in order to adapt to a constantly changing environment. The cells of the immune system are no exception to this rule, but the interactions between immune cells remain little known and complicated to study. The recent emergence of 'single cell' sequencing technologies represents a unique opportunity to study these communications. In this thesis, different experimental and analytical approaches have been developed to study these communications on a single cell scale. These strategies were then applied to different disease contexts, including COVID-19, Alzheimer's disease or immunisation with inactivated pathogens, and identified previously unknown or poorly understood cellular communication pathways. However, the effectiveness of these approaches is limited by the lack of information on cell location and further work integrating such data will be essential to go further in the dissection of immune cell communications
Chen, Wenli. "Spectroscopie diélectrique hyperfréquence de cellules uniques cancéreuses : de l'optimisation du capteur en sensibilité et répétabilité jusqu'au suivi en temps réel de stimuli chimiques." Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30219/document.
Full textThe measurement of biological cells is a routine step in many biological investigations. Current techniques used by biologists are mainly based on staining or fluorescent labelings, which provide very precise and effective molecular and cellular observations. Within this context, the microwave dielectric spectroscopy for cell analysis represents a new and attractive method, due to the lack of cells preparation and manipulation, without adding chemicals that could interfere with other cellular constituents. Its compatibility with the analysis of single-cells, potentially in real-time monitoring, constitute also two major assets of the analysis technique. This PhD thesis therefore focused on the optimization of a microfluidic and microwave based biosensor, which is dedicated to the dielectric spectroscopy of individual biological cells, and the development of its metrology to assess the dielectric behavior of cells subjected to chemical stimuli. After a state of the art on the current techniques available to analyze single cells, we focused on the optimization of the microwave biosensor to improve its performances in terms of sensitivity and repeatability. These optimizations dealt with the microfabrication process, the component architecture through the investigation of single cell loading efficacy as well as an electromagnetic parametric study. These developments were validated first experimentally with the measurement of polystyrene beads, which present a simplified dielectric model compared to the complexity of a biological cell, followed then by living individual cells in their culture medium. The test bench was also optimized to allow the dielectric measurement of cells over time, and especially in response to a chemical stimulus. The reaction kinetics of a single-cell subjected to saponin was recorded automatically for different cells. This work opens the door to single-cell analysis with microwave dielectric spectroscopy of complex biological processes in real-time
Sun, Hui. "Développement d'une méthode microspectrophotométrique destinée à la mesure des concentrations ioniques cytoplasmiques sur cellule unique et étude physico-chimique des colorants microinjectés dans les myoballs de rat pour la détermination précise du pH et du magnésium libre cytoplasmiques." Lyon 1, 1994. http://www.theses.fr/1994LYO1T189.
Full textTamra, Amar. "Spectroscopie diélectrique hyperfréquence de cellules individualisées sous électroporation." Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30011/document.
Full textElectroporation is a physical process that consists in applying electric field pulses to transiently or permanently permeabilize the plasma membrane. This phenomenon is of great interest in the clinical field as well as in the industry because of its various applications, in particular electrochemotherapy which combines electrical pulses with the administration of a cytotoxic molecule in the treatment of tumors. The evaluation of this phenomenon is raditionally carried out using optical and biochemical methods (microscopy, flow cytometry, biochemical test). They are very effective but require the use of a wide range of fluorochromes and markers, which can be laborious and costly to implement, while being invasive to the cells. In recent years, the development of new biophysical tools for the study of electroporation has taken place, such as dielectrophoresis and impedance spectroscopy (low frequency). In addition to the ease of implementation, these methods are of interest in the study of membrane modifications of the cell. Hence the advantage of operating beyond the GHz, in the range of microwaves, for which the cytoplasmic membrane becomes transparent and the intracellular content is exposed. The extraction of the relative permittivity as a result of the electromagnetic field / biological cell interaction then reflects the cell state. This technique, microwave dielectric spectroscopy, is a relevant method for analyzing the effects of electroporation on cell viability. Moreover, it does not require any use of the exogenous molecules (non-invasive) and the measurements are directly carried out in the culture medium of the cells. Two objectives were defined during this thesis whose work is located at the interface between three scientific fields: cellular biology, microwave electronics and micro-technologies. The first objective concerns the transposition of conventional electroporation to the micrometric scale, which has shown an efficiency as efficient as the first. The second part of the work concerns the study by HighFrequency dielectric spectroscopy of cells subjected to different electrical treatments (combined or not with a cytotoxic molecule). This work presents a statistical power and shows a very good correlation (R2> 0.94) with standard techniques used in biology, which biologically validates the HF analysis method in the context of electroporation. This work also shows that microwave dielectric spectroscopy proves to be a powerful technique capable of revealing cell viability following chemical and / or electrical treatment. They open the way to 'non-invasive' analysis by hyper-frequency dielectric spectroscopy of electroporated cells in situ
Richard, Angélique. "Analyse de la variabilité de l’expression génique et du métabolisme glycolytique au cours du processus de différenciation érythrocytaire : de l’analyse à grande échelle aux questions mécanistiques." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1058/document.
Full textThe meaning of cell decision making consists in the capacity of every living cell to integrate environmental information and to transform it in a coherent biological response. Nowadays it is increasingly demonstrated that cell populations present a significant quantitative and qualitative heterogeneity that could be involved in living organisms functions. Thus, the first part of my thesis consisted in studying gene expression variability at the single-cell level during the differentiation process of primary avian erythroid progenitor cells. The expression of 92 genes was analyzed using RT-qPCR in cells isolated at different differentiation time-points. The main results of this study showed that gene expression variability, as measured by Shannon entropy, reached a maximal level, simultaneously to a drop in the number of correlated genes, at 8-24h of differentiation. This increase of the gene expression variability preceded the irreversible commitment of cells into differentiation, identified between 24h and 48h. This analysis also highlighted the potential importance ofLDHA(Lactate dehydrogenase A) encoding a glycolytic enzyme, in erythroid progenitors self-renewal and at the critical differentiation time-point 8-24h. Therefore the second part of my thesis consisted in analyzing the role of LDHA in erythroid progenitors self-renewal and the variations of glucose metabolism during the differentiation process. Our first results suggested that erythroid differentiation might be accompanied with a metabolic change, corresponding to a switch from anaerobic glycolysisdepending upon LDHA, toward aerobic energy production, relying upon oxidative phosphorylation
Lieber, Diana. "Functional assays for screening antibody activity in droplet-based microfluidics." Strasbourg, 2010. https://publication-theses.unistra.fr/public/theses_doctorat/2010/LIEBER_Diana_2010.pdf.
Full textHigh-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturisation of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we developed droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Furthermore, this system allowed for an automated analysis of individual compartments subsequent to an incubation period as required for high-throughput, cell-based assays. In addition, we focused on the development of functional assays for screening antibody activity, e. G. Neutralisation of HIV or the inhibition of ACE-1. Common high throughput approaches for antibody screening use phage display or hybridoma cells. Phage display is powerful but based on binding properties rather than neutralising effects. Hybridoma cells allow for direct screening of neutralising activity, but are very restricted in the number of clones that can be screened due to their generation and proliferation as required for conventional screens. The aim of this study was the development of novel screening technology with the ultimate goal of screening single antibody-releasing cells (hybridoma cells). This system should also allow for direct B-cell screening. Once established, this technology could be used for the screening/ selection of many more therapeutic antibodies