Stem and progenitor cell fate (self-renewal, proliferation, survival, differentiation) is tightly controlled by niche factors and the interplay of these factors is particularly important to comprehend for the development of stem cell therapies. During erythropoiesis, erythroid progenitors at the colony forming unit-erythroid (CFU-E) stage are responsive to both stem cell factor (SCF) and erythropoietin (EPO); however, the joint action of SCF and EPO in these cells and the underlying mechanisms remain to be defined. In this study, quantitative data on the activation of signaling pathways and gene expression profiles provided definitive evidence for two parallel but complementary mechanisms that resulted in enhanced generation of red blood cells from mouse bone marrow-derived CFU-E culture in the presence of SCF and EPO. First, SCF and EPO signaling intersected within the extracellular signal-regulated kinase (ERK) pathway and the sustained ERK activation was required for the maximal changes in the expression levels of genes that are involved in the proliferation and survival of CFU-Es. Second, the apparent competition between SCF and EPO in regulating c-Kit expression was found to have a dramatic impact on the terminal differentiation of CFU-Es. The latter mechanism was, for the first time, reported in a primary cell system. In addition, a fetal liver-derived conditioned medium further enhanced the survival and proliferation of bone marrow CFU-Es in the presence of SCF and EPO by not only increasing the ERK signaling duration but also, the amplitude. The agents present in the conditioned media possess significant clinical potential to stimulate erythropoiesis both in vivo and in vitro. In conclusion, our study has provided novel insights into the mechanisms by which combinations of niche factors control the fate of erythroid progenitors at a unique transitional stage and highlighted the important role of the ERK signaling dynamics in adult erythropoiesis.

Stem and progenitor cell fate (self-renewal, proliferation, survival, differentiation) is tightly controlled by niche factors and the interplay of these factors is particularly important to comprehend for the development of stem cell therapies. During erythropoiesis, erythroid progenitors at the colony forming unit-erythroid (CFU-E) stage are responsive to both stem cell factor (SCF) and erythropoietin (EPO); however, the joint action of SCF and EPO in these cells and the underlying mechanisms remain to be defined. In this study, quantitative data on the activation of signaling pathways and gene expression profiles provided definitive evidence for two parallel but complementary mechanisms that resulted in enhanced generation of red blood cells from mouse bone marrow-derived CFU-E culture in the presence of SCF and EPO. First, SCF and EPO signaling intersected within the extracellular signal-regulated kinase (ERK) pathway and the sustained ERK activation was required for the maximal changes in the expression levels of genes that are involved in the proliferation and survival of CFU-Es. Second, the apparent competition between SCF and EPO in regulating c-Kit expression was found to have a dramatic impact on the terminal differentiation of CFU-Es. The latter mechanism was, for the first time, reported in a primary cell system. In addition, a fetal liver-derived conditioned medium further enhanced the survival and proliferation of bone marrow CFU-Es in the presence of SCF and EPO by not only increasing the ERK signaling duration but also, the amplitude. The agents present in the conditioned media possess significant clinical potential to stimulate erythropoiesis both in vivo and in vitro. In conclusion, our study has provided novel insights into the mechanisms by which combinations of niche factors control the fate of erythroid progenitors at a unique transitional stage and highlighted the important role of the ERK signaling dynamics in adult erythropoiesis.

Bio-pharmaceutical industry is a vast growing market and recent recommendations of the Food and Drug Administration have put a large emphasis on the characterization of biological processes and models. As a consequence, there is a high incentive on developing modern sensors in order to more accurately monitor and control processes. In that way, Digital Holographic Microscopy (DHM) presents unique features thanks to the refocusing and quantitative phase contrast imaging capabilities. In this thesis we investigate the usage of DHM to monitor yeast cultures that are often used in both the bio-pharmaceutical and bread industries and lay the basis of a methodological framework for the study of in-line cell cultures in the context of process control. We begin with a description of Digital Holography and the microscopy setup used in the thesis as well as a detailed explanation of the image processing required to extract the holographic data and its implementation on GPU with some speed execution figures given for three popular programming paradigms. We then describe the flow setup used and infer the limitations on the dynamic range of the technique due to both Poisson statistics and overlapping phenomena. Finally, we describe an algorithm that extracts the cells position, count and morphological information such as the size, aspect ratio, circularity and refraction index. Some experimental results are presented for yeasts before drawing a general overview of the technology and its dependencies. We further end with some conclusions concerning the technology and a brief comparison with existing competitors. / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19759号 / 農博第2155号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4975(農学部図書室) / 32795 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 宮川 恒, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

Technological advancement allowed for the advent of single-cell technologies capable of measuring a large number of cellular features simultaneously. These technologies have been subsequently used to shed light on the heterogeneity of cellular systems previously considered homogeneous, identifying the exclusive features of individual cells within cellular niches. Today, single-cell technologies represent an essential tool for studying the underlying immunological mechanisms correlating with disease. In this context, cytometry is one of the diverse high-throughput methods capable of examining more than 50 features per cell. However, utilising cytometry at its full potential requires the development of optimized assays. Additionally, the resulting high-dimensional data represent a challenge for existing computational techniques. This thesis attempts to address these challenges. The first part of the thesis is focused on developing a non-linear embedding algorithm for rapid analysis of cytometry datasets called EmbedSOM. The comparison of EmbedSOM with other state-of-the-art algorithms suggested the superiority of EmbedSOM with faster runtime. This is critical for the analysis of large datasets with millions of cells. Furthermore, EmbedSOM has additional functionality such as landmark guided...

Droplet-based microfluidics is a powerful tool permitting massive-scale single-cell analysis in pico-/nano-liter water-in-oil droplets. It has been integrated into various library preparation techniques to accomplish high-throughput scRNA-seq, scDNA-seq, scATAC-seq, scChIP-seq, as well as scMulti-omics-seq. These advanced technologies have been providing unique and novel insights into both normal differentiation and disease development at single-cell level. In this thesis, we develop four new droplet-based tools for single-cell omics profiling. First, the developed Drop-BS is the first droplet-based platform to construct single-cell bisulfite sequencing libraries for DNA methylome profiling and allows production of BS library of 2,000-10,000 single cells within 2 d. We applied the technology to separately profile mixed cell lines, mouse brain tissues, and human brain tissues to reveal cell type heterogeneity. Second, the new Drop-ChIP platform only requires two steps of droplet generation to achieve multiple steps of reactions in droplets such as single-cell lysis, chromatin fragmentation, ChIP, and barcoding. Third, we aim to establish a droplet-based platform to accomplish high-throughput full-length RNA-seq (Drop-full-seq), which both current tube-based and droplet-based methods cannot realize. Last, we constructed an in-house droplet-based tool to assist single-cell ATAC-seq library preparation (Drop-ATAC), which provided a low-cost and facile protocol to conduct scATAC-seq in laboratories without the expensive instrument. / Doctor of Philosophy / Microfluidics is a collection of techniques to manipulate fluids in the micrometer scale. One of microfluidic techniques is called "droplet-based microfluidics". It can manipulate (i.e., generate, merge, sort, split, etc) pico-/nano-liter of water-in-oil droplets. First, since the water phase is separated by the continuous oil phase, these droplets are discrete and individual reactors. Second, droplet-based microfluidics can achieve highly parallel manipulation of thousands to millions of droplets. These two advantages make droplet-based microfluidics an ideal tool to perform single-cell assays. Over the past 10 years, various droplet-based platforms have been developed to study single-cell transcriptome, genome, epigenome, as well as multi-ome. To expand droplet-based tools for single-cell analysis, we aim to develop four novel platforms in this thesis. First, Drop-BS, by integrating droplet generation and droplet fusion techniques, can achieve high-throughput single-cell bisulfite sequencing library preparation. It can generate 10,000 single-cell BS libraries within 2 days which is difficult to achieve for conventional library preparation in tubes/microwells. Second, we developed a novel and facile Drop-ChIP platform to prepare single-cell ChIP-seq library. It is easy to operate since it only requires two steps of droplet generation. It also generates higher quality of data compared to previous work. In addition, we are working on the development and characterization of the other two droplet-based tools to achieve full-length single-cell RNA-seq and single-cell ATAC-seq.

Droplet microfluidics is an emerging technology for analysis of single cells and biomolecules at high throughput. The controlled encapsulation of particles along with the surrounding microenvironment in discrete droplets, which acts as miniaturized reaction vessels, allows millions of particles to be screened in parallel. By utilizing the unit operations developed to generate, manipulate and analyze droplets, this technology platform has been used to miniaturize a wide range of complex biological assays including, but not limited to, directed evolution, rare cell detection, single cell transcriptomics, rare mutation detection and drug screening. The aim of this thesis is to develop droplet microfluidics based methods for analysis of single cells and nucleic acids. In Paper I, a method for time-series analysis of mammalian cells, using automated fluorescence microscopy and image analysis technique is presented. The cell-containing droplets were trapped on-chip and imaged continuously to assess the viability of hundreds of isolated individual cells over time. This method can be used for studying the dynamic behavior of cells. In Paper II, the influence of droplet size on cell division and viability of mammalian cell factories during cultivation in droplets is presented. The ability to achieve continuous cell division in droplets will enable development of mammalian cell factory screening assays in droplets. In Paper III, a workflow for detecting the outcome of droplet PCR assay using fluorescently color-coded beads is presented. This workflow was used to detect the presence of DNA biomarkers associated with poultry pathogens in a sample. The use of color-coded detection beads will help to improve the scalability of the detection panel, to detect multiple targets in a sample. In Paper IV, a novel unit operation for label-free enrichment of particles in droplets using acoustophoresis is presented. This technique will be useful for developing droplet-based assays that require label-free enrichment of cells/particles and removal of droplet content. In general, droplet microfluidics has proven to be a versatile tool for biological analysis. In the years to come, droplet microfluidics could potentially be used to improve clinical diagnostics and bio-based production processes. / <p>QC 20160926</p>

Cette thèse concerne le développement d’outils microfluidique pour l’ingénierie d’enzymes d’intérêt thérapeutique. La microfluidique à base de gouttelettes présente un énorme potentiel dans le domaine de la biologie quantitative. Nous développons des outils microfluidiques pour l’évolution dirigée de l’enzyme L-asparaginase, enzyme utilisée comme traitement de laleucémie lymphoblastique aiguë. Ce traitement est basée sur une enzyme d’origine bactérienne,ce qui conduit à déclencher des réactions immunitaires qui se traduit par l’interruption du traitement, souvent fatale pour le patient. Cependant, une version humaine de l’enzyme L-asparaginase, qui est moins immunogénique, n’est à l’heure actuelle pas suffisamment active pour être utilisée. L’objectif principal de cette thèse est d’alors d’analyser et de cribler des banques de mutants d’enzymes en utilisant des méthodes classiques de mutagenèse et d’analyser chaque mutant individuellement par le biais de la microfluidique. Pour cela, plusieurs systèmes microfluidiques ont été développés et optimisés afin de répondre à différents critères de sélection pour l’analyse et la sélection de l’enzyme L-asparaginase. La version bactérienne a servi de contrôle positif pour l’optimisation des systèmes microfluidiques afin de pouvoir analyser et de cribler des banques de mutants de la version humaine de l’enzyme L-asparaginase. / This thesis deals with the development of microfluidic tools for the engineering ofenzymes of therapeutic interest. Droplet microfluidics has enormous potential in the field ofquantitative biology. We are developing microfluidic tools based on the directed evolutionof the enzyme L-asparaginase, an enzyme used to treat acute lymphoblastic leukemia. Thistreatment is based on an enzyme of bacterial origin, which leads to immune reactions thatresult in the interruption of treatment, often fatal for the patient. However, a human version ofthe enzyme L-asparaginase, which is less immunogenic, is currently not sufficiently active to beused. The main objective of this thesis is to analyze and screen enzyme mutant libraries usingstandard mutagenesis methods and to analyze each mutant individually through microfluidics.For this, several microfluidic systems have been developed and optimized for different selectioncriteria for the analysis and selection of the enzyme L-asparaginase. The bacterial versionserving as a positive control for the optimization of microfluidic workflows to analyze andscreen mutant libraries of the human version of the enzyme L-asparaginase.

El comportamiento fagotrófico en especies fitoplanctónicas fue evaluado por medio de técnicas de análisis celular que permiten establecer patrones individuales de cada una de ellas. Primeramente, se calibró el contenido vacuolar como tasas de ingestión comparando la captación de microesferas fluorescentes de látex y el contenido de las vacuolas alimenticias por medio de la hibridación fluorescente in situ (CARD-FISH) en especies fitoplanctónicas bajo condiciones controladas de cultivo. Se obtuvo que el contenido de las vacuolas alimenticias caracterizado por la técnica del CARD-FISH refleja las tasas de ingestión obtenidas por la captura de microesferas al menos a escalas de horas. Así mismo, las tasas de digestión fueron diferentes entres protistas mixotróficos dependiendo de su tamaño celular, los individuos pequeños (<10 μm) digieren a una mayor velocidad que aquellos individuos de mayor tamaño. Posteriormente, se evaluaron los cambios a corta escala de tiempo (horas) de la actividad fagotrófica y los comportamientos selectivos en tres especies fitoplanctónicas (Cryptomonas ovata, Rhodomonas minuta y Dinobryon cylindricum) de un lago de alta montaña. Los resultados muestran diferencias temporales entre las especies, indicando cambios en las tasas de ingestión y digestión a lo largo del dia. Así mismo, se observó una clara preferencia por la ingestión de presas del dominio Archaea, la cual no puede ser explicada ni por la abundancia ni por el tamaño de las presas en el sistema. Finalmente, se comparo la actividad fagotrófica y la selección de presas en comunidades fitoplanctónicas de un conjunto de lagos de alta montaña para identificar generalidades de los patrones de ingestión observados. El patrón selectivo más general entre los diferentes protistas mixotróficos y en los diferentes sistemas estudiados es una preferencia sobre el grupo Actinobacteria, aunque esta preferencia puede ser muy variable. El tamaño celular de las presas es un parámetro importante en determinar las tasas de depredación selectiva que exhiben los mixótrofos sobre ellas. Así mismo, el tamaño celular de los protistas mixotróficos condiciona su actividad fagotrófica. Individuos con tamaños celulares grandes (>10 μm) presentan mayor contenido de presas en sus vacuolas, pero, debido a por su baja abundancia, el impacto sobre el bacterioplancton es bajo. Especies con tamaño pequeños (<10 μm) presentan un menor contenido, pero su alta abundancia y digestión más rápida hace que representen un mayor impacto sobre las comunidades bacterianas. La conclusión global de la tesis es que, incluso en condiciones óptimas de luz, la actividad fagotrófica de algunos grupos fitoplanctónicos típicos de los lagos de alta montaña (Chrysophyta y Cryptophyta) es muy importante, se han observado particularidades en el comportamiento y en la selección de presas y se ha evidenciado que el tamaño celular, tanto de los depredadores como de las presas, es el parámetro determinante para explicar las tasas de ingestión. / The feeding behaviour of phytoplankton species was evaluated through single cell analysis. Firstly, food vacuole content was calibrated as ingestion rate, comparing the intake of fluorescent microspheres and the prey vacuole content measured by Catalized Reported Deposition-Fluorescent in Situ Hybridization (CARD-FISH) on phytoplankton species in culture conditions. It has been observed that the food vacuole content properly reflects the ingestion rates at scales of hours. Likewise, digestion rates varied between species as a function of cell size: small individuals (<10μm) digest faster than bigger ones. Secondly, temporal changes of phagotrophic activity and selective behaviour were assessed in three phytoplanktonic species (Cryptomonas ovata, Rhodomonas minuta and Dinobryon cylindricum) in a high mountain lake. The results have shown that there are temporal differences between species, which indicates that changes in ingestion and digestion rates occurred along the day. A clear preference to ingest Archaea was observed in all three species. This preference could be explained neither by the abundance nor the size of the prey. Finally, the feeding behaviour of phytoplankton communities in a set of mountain lakes was compared, in order to identify general phagotrophic patterns. The most general selective behaviour in mixotrophic protists was a preference for Actinobacteria prey; however, this preference was highly variable and clearly related to the prey cell size. In addition, the cell size of mixotrophic protists determined their phagotrophic activity; small individuals (>10 μm), presented lower content of preys than bigger individuals, although, due to their higher abundance and faster digestion rates, they exerted a high grazing pressure on the bacterial communities. The main conclusion of this work is that, even under optimum light conditions, the phagotrophic activity in some typical phytoplankton groups of high mountain lakes (Chrysophyta and Cryptophyta), is significant; nevertheless, some particularities have been observed in the feeding behaviour of some species. Also, it has become clear that the size of both predator and prey are the most important parameters to explain ingestion rates.