Vitiligo is an autoimmune disease in which CD8+ T cells selectively destroy melanocytes, leading to a patchy, disfiguring depigmentation of the skin. Our group and others have highlighted the central role of IFN-γ-dependent chemokines in the progression of disease; however, IFN-γ is also reported to have pleiotropic effects on melanocyte biology. We examined whether IFN-γ has a direct role in melanocyte killing. We tested the T-cell effector functions IFN-γ, Fas ligand and perforin by deleting them from autoreactive T cells used to induce vitiligo in mice. We found that disease incidence, disease severity and T cell accumulation in the skin was reduced in mice receiving adoptive transfer of either IFN-γ deficient or Fas ligand deficient gp100-specific T cells; however, perforin was dispensable and led to increased disease scores and T cell accumulation. To determine how melanocytes are affected by IFN-γ signaling during vitiligo, we performed single-cell RNA-sequencing on suction blister biopsies obtained from vitiligo and healthy subjects. We discovered that integrin expression and TGFb2 signaling was decreased only in lesional melanocyte transcriptomes. Moreover, melanocytes appear to participate in their own demise by increasing HLA expression and recruiting effector cells through the chemotactic ligand CCL18. The loss of melanocyte retention factors may explain their clean disappearance from the skin during keratinocyte turnover. Taken together, we believe IFN-γ production by autoreactive T cells in the skin leads to clean loss of melanocytes by downregulation of melanocyte retention factors and by increasing their potential to be detected by effector cells during vitiligo.

Panarthropoda is a monophyletic group comprised of arthropods and lobopods, molting animals with a segmented body, paired appendages, dorsal brain, and ventral nerve cords. Evolutionary Developmental Biology (EvoDevo) is an interdisciplinary field that seeks to understand how changes in development form the basis for variations in morphology and phenotypic evolution, including the genetic network underlying these processes. To study the evolution of panarthropods from such an EvoDevo perspective, one typically uses standard molecular techniques. A first step here is to investigate the expression of a gene of interest in order to find out where and when it is transcribed during development. A hallmark of EvoDevo studies is its comparative character, often with respect to model organisms such as the fruit fly Drosophila melanogaster. Recently developed single-cell RNA sequencing technologies allow the profiling of a plethora of gene expression on the level of individual cells, and thus provide a much more detailed insight into gene expression. In Paper I, I applied standard molecular techniques used in EvoDevo research such as PCR, gene cloning, probe synthesis and whole mount in situ hybridization, to investigate the embryonic expression patterns of the tiptop/teashirt (tio/tsh) and spalt (sal) genes in a range of arthropods representing all main groups of this phylum, and an onychophoran. In the arthropod model Drosophila, these genes act as trunk-specifiers, and the objective of my work was to find out if this is conserved in Arthropoda or even Panarthropoda as a whole. I provide comprehensive data on arthropod tio/tsh and sal expression, including the first data from an onychophoran. The results support the idea that tio/tsh genes are involved in the development of ‘trunk’ segments by regulating limb development. In addition, my data suggest that the function of Sal is unlikely to be conserved in trunk vs head development. Early expression of sal, however, is in line with a potential homeotic function of this gene, at least in Arthropoda. In Paper II, I provide an embryonic tissue dissociation protocol for embryos of the common house spider Parasteatoda tepidariorum that I developed and that I successfully applied for single-cell RNA sequencing. In addition, I report on the progress of this experiment, and provide and discuss preliminary results.

No description available.

L'électroporation est un procédé physique qui consiste à appliquer des impulsions de champ électrique pour perméabiliser de manière transitoire ou permanente la membrane plasmique. Ce phénomène est d'un grand intérêt dans le domaine clinique ainsi que dans l'industrie en raison de ses diverses applications, notamment l'électrochimiothérapie qui combine les impulsions électriques à l'administration d'une molécule cytotoxique, dans le cadre du traitement des tumeurs. L'analyse de ce phénomène est traditionnellement réalisée à l'aide des méthodes optique et biochimique (microscopie, cytométrie en flux, test biochimique). Elles sont très efficaces mais nécessitent l'utilisation d'une large gamme de fluorochromes et de marqueurs dont la mise en œuvre peut être laborieuse et coûteuse tout en ayant un caractère invasif aux cellules. Durant ces dernières années, le développement de nouveaux outils biophysiques pour l'étude de l'électroporation a pris place, tels que la diélectrophorèse et la spectroscopie d'impédance (basse fréquence). Outre une facilité de mise en œuvre, ces méthodes représentent un intérêt dans l'étude des modifications membranaires de la cellule. De là vient l'intérêt d'opérer au-delà du GHz, dans la gamme des micro-ondes, pour laquelle la membrane cytoplasmique devient transparente et le contenu intracellulaire est exposé. L'extraction de la permittivité relative suite à l'interaction champ électromagnétique/cellules biologiques reflète alors l'état cellulaire. Cette technique, la spectroscopie diélectrique hyperfréquence, se présente comme une méthode pertinente pour analyser les effets de l'électroporation sur la viabilité cellulaire. De plus, elle ne nécessite aucune utilisation des molécules exogènes (non-invasivité) et les mesures sont directement réalisées dans le milieu de culture des cellules. Deux objectifs ont été définis lors de cette thèse dont les travaux se situent à l'interface entre trois domaines scientifiques : la biologie cellulaire, l'électronique hyperfréquence et les micro-technologies. Le premier objectif concerne la transposition de l'électroporation conventionnelle à l'échelle micrométrique, qui a montré une efficacité aussi performante que la première. La deuxième partie du travail concerne l'étude par spectroscopie diélectrique HyperFréquence de cellules soumises à différents traitements électriques (combinés ou non à une molécule cytotoxique). Ces travaux présentent une puissance statistique et montrent une très bonne corrélation (R2 >0 .94) avec des techniques standards utilisées en biologie, ce qui valide 'biologiquement' la méthode d'analyse HF dans le contexte d'électroporation. Ces travaux montrent en outre que la spectroscopie diélectrique hyperfréquence s'avère être une technique puissante, capable de révéler la viabilité cellulaire suite à un traitement chimique et/ou électrique. Ils ouvrent la voie à l'analyse 'non-invasive' par spectroscopie diélectrique HyperFréquence de cellules électroporées in-situ. / Electroporation 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.

Microorganisms are ubiquitously distributed on the earth and drive the fundamental element cycling in the biosphere. Their metabolic activities serve human societies in countless areas such as biotechnological engineering, food engineering, energy production, waste disposal et cetera. For human beings, and also for animals, microorganisms are imperative for health especially as colonizers of the gut system. Microbial resource management, especially when complex communities are exploited in biotechnology is a key challenge. Therefore, communities are more and more in the focus of basic research in microbiology complementary to pure cultivation technologies. Owing to their complexity, microbial communities are almost exclusively studied on the basis of bulk parameters and empirical expert knowledge. Bulk parameters are representative for an entire community performance but do not allow a segregated analysis of subpopulations or subcommunities, let alone individuals and their disparate functions within a community. This thesis aims to resolve microbial community properties and mechanisms of assembly in managed ecosystems on the individual level (i.e. single cell). For this the flow cytometric toolbox was employed and further expanded, which phenotypically classifies microbial individuals into sub-communities according to their physiological similarities. Workflows for the fast analysis and evaluation of dynamics in community structure, assembly and interaction were developed. Stability properties of communities, i.e. resistance, resilience, displacement speed and elasticity, can now quantitatively be determined based on cytometric data. For resilience behavior an on-line tool was developed. In addition, the relative proportions of neutral and deterministic forces that structure a microbial community can now be unraveled. As consequence, microbial flow cytometry has been proven to be a powerful tool for analysing complex microbial communities, and will allow huge improvements in understanding and control of microbial communities in managed and natural ecosystems.:Contents Summary ............................................................................................................. I Zusammenfassung ........................................................................................... IV 1 Introduction ..................................................................................................... 1 1.1 Microbial community and ecology ........................................................... 1 1.1.1 What is a microbial community? ...................................................... 1 1.1.2 Flow cytometry as a tool to study microbial communities ................ 2 1.2 Community structure and diversity ........................................................ 10 1.2.1 Community structure...................................................................... 10 1.2.2 Diversity metrics ............................................................................ 10 1.2.3 Evaluating structure and diversity with flow cytometry ................... 12 1.3 Community assembly and dynamics ..................................................... 13 1.3.1 Basic assembly processes ............................................................ 13 1.3.2 Evaluating assembly processes with flow cytometry ..................... 16 1.4 Community interactions ......................................................................... 18 1.4.1 Abiotic interactions of microbes and their surroundings................. 18 1.4.2 Biotic interactions of microbial partners ......................................... 18 1.4.3 Evaluating interactions with flow cytometry ................................... 20 1.5 Community functions ............................................................................. 22 1.5.1 Omics approaches to study functions in microbial communities .... 22 1.5.2 Evaluating functions with flow cytometry ....................................... 23 1.6 Aims of this study .................................................................................. 25 2 Publications .................................................................................................. 27 2.1 Overview of publications ....................................................................... 27 2.2 Published articles .................................................................................. 28 2.2.1 Publication 1 .................................................................................. 29 2.2.2 Publication 2 .................................................................................. 42 2.2.3 Publication 3 (under review) .......................................................... 60 3 Discussion .................................................................................................... 81 3.1 The importance of perceiving ecological situations ............................... 81 3.2 Stability properties of a microbial community ........................................ 84 3.3 Assembly processes in insular environments ....................................... 87 3.3.1 Niche differentiation under balanced cultivation conditions ........... 88 3.3.2 Neutral assembly under balanced cultivation conditions ............... 89 3.3.3 From intermediate disturbance to a non‐equilibrium system ......... 90 3.4 On-line analysis of reactor data ............................................................ 93 3.5 Conclusion and outlook ......................................................................... 95 4 References ................................................................................................... 97 5 Acknowledgement ...................................................................................... 105 6 Appendix .................................................................................................... 106 6.1 Declaration of independent work ......................................................... 106 6.2 Author contributions of published articles............................................ 107 6.3 Curriculum vitae .................................................................................. 111 6.4 List of Publications and conference contributions ............................... 112 6.5 Supplementary materials .................................................................... 113 6.5.1 Supplementary material for publication 1 .................................... 113 6.5.2 Supplementary material for publication 2 .................................... 140 6.5.3 Supplementary material for publication 3 .................................... 174

Recent breakthroughs in the RNA quantification of single cells are rapidly transforming the view on biology and medicine. Flexibility and sensitivity of reverse transcription quantitative PCR (RT-qPCR) make it an ideal method for quantification of single-cell material, but its limits had not been yet fully explored. In this thesis, various factors influencing RT-qPCR performance in single-cell application have been assessed, including conditions of sample collection and processing, importance of quality control, performance of reverse transcription, preamplification and role of qPCR assays. We showed that prolonged time for single cell collection as well as repeated freeze-thaw cycles had negligible effect on RT-qPCR data quality. Direct lysis routinely applied for RNA extraction from single cells may be scaled up to 256 cells. The comprehensive comparison of 11 reverse transcriptases in low RNA input conditions identified 2 best-performing enzymes. Decrease in preamplification volume as well as poor primer design resulted in the loss of sensitivity. Finally, the established workflow has been applied to profile gene expression of astrocytes in mouse model of amyotrophic lateral sclerosis (ALS) identifying important components of ALS-induced changes to astrocyte transcriptome. Altogether, the thesis...

Despite spectacular progress in biophysics, molecular biology and biochemistry our ability to predict the dynamic behavior of multicellular systems under different conditions is very limited. An important reason for this is that still not enough is known about how cells change their physical and biological properties by genetic or metabolic regulation, and which of these changes affect the cell behavior. For this reason, it is difficult to predict the system behavior of multicellular systems in case the cell behavior changes, for example, as a consequence of regulation or differentiation. The rules that underlie the regulation processes have been determined on the time scale of evolution, by selection on the phenotypic level of cells or cell populations. We illustrate by detailed computer simulations in a multi-scale approach how cell behavior controlled by regulatory networks may emerge as a consequence of an evolutionary process, if either the cells, or populations of cells are subject to selection on particular features. We consider two examples, migration strategies of single cells searching a signal source, or aggregation of two or more cells within minimal multiscale models of biological evolution. Both can be found for example in the life cycle of the slime mold Dictyostelium discoideum. However, phenotypic changes that can lead to completely different modes of migration have also been observed in cells of multi-cellular organisms, for example, as a consequence of a specialization in stem cells or the de-differentiation in tumor cells. The regulatory networks are represented by Boolean networks and encoded by binary strings. The latter may be considered as encoding the genetic information (the genotype) and are subject to mutations and crossovers. The cell behavior reflects the phenotype. We find that cells adopt naturally observed migration strategies, controlled by networks that show robustness and redundancy. The model simplicity allow us to unambiguously analyze the regulatory networks and the resulting phenotypes by different measures and by knockouts of regulatory elements. We illustrate that in order to maintain a cells' phenotype in case of a knockout, the cell may have to be able to deal with contradictory information. In summary, both the cell phenotype as well as the emerged regulatory network behave as their biological counterparts observed in nature.

Interactions of cells with the extracellular matrix (ECM) have important roles in various physiological and pathological processes, including tissue morphogenesis during embryonic development, wound healing and tumor invasion. Although most of the proteins involved in cell-ECM interactions have been identified, the underlying mechanisms and involved signaling pathways are incompletely understood. Here, atomic force microscope-based imaging and single-cell force measurements were used to characterize the interactions of different cell types with ECM proteins. The interplay between cells and ECM is complex. However, two interaction types, protein-protein and protein-carbohydrate, predominate. Integrins, adhesion receptors for ECM, mediate the former, galectins, a family of animal lectins, the latter. In the second chapter of this thesis, the contributions of both receptor families to the interactions of epithelial MDCK cells with ECM proteins are presented. It was found that galectins-3 and 9 are highly expressed in MDCK cells and required for optimal long-term adhesion (90 minutes) to ECM proteins collagen-I and laminin-111. Interestingly, early adhesion (< 2 minutes) to laminin-111, was integrin-independent and instead mediated by carbohydrate interactions and galectins. In contrast, early adhesion to collagen-I was exclusively mediated by integrins. Moreover, cells frequently entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Although adhesion was mediated by integrins, adhesion enhancement was especially observed in cells depleted for galectin-3. It was proposed that galectin-3 influences integrin-mediated adhesion complex formation by altering receptor clustering. To control their attachment to ECM proteins, cells regulate integrin receptors. One regulatory process is integrin crosstalk, where the binding of one type of integrin influences the activity of another type. In the third chapter, the implementation of a single-cell force spectroscopy assay to identify such crosstalks and gain insight into their mechanisms is described. In this assay the interactions of integrin receptors being specifically attached to one ligand are characterized in dependence of another ligand-bond receptor pair. With this assay a crosstalk between collagen-binding integrin α1β1 and fibronectin-binding integrin α5β1 was identified in HeLa cells. This crosstalk was directional from integrin α1β1 to integrin α5β1 and appeared to regulate integrin α5β1 by inducing its endocytosis. In the fourth and final chapter, mechanisms of matrix-induced cell alignment were studied by imaging cells on two-dimensional matrices assembled of highly aligned collagen fibrils. Integrin α2β1 was identified as the predominant receptor mediating cell polarization. Time-lapse AFM demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization did not occur. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix and lead to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction. The work presented here adds to the understanding of cell-ECM interactions. Atomic force microscopy imaging allowed characterizing the behavior of cells on nanopatterned collagen matrices whereas single-cell force spectroscopy revealed insights into the regulation of cell adhesion by galectins. Furthermore, methodological advances in the single-cell force spectroscopy assay allowed the intracellular regulation of receptor molecules to be studied. The work demonstrates that atomic force microscopy is a versatile tool to study cell-ECM interactions.

During vertebrate development, gastrulation leads to the formation of three distinct germlayers. In zebrafish a central process is the delamination and the ingression of single cells from a common ancestor tissue - that will lead to the formation of the germlayers. Several molecules have been identified to regulate this process but the precise cellular mechanisms are poorly understood. Differential adhesiveness, a concept first introduced by Steinberg over 40 years ago, has been proposed to represent a key phenomena by which single hypoblast cells separate from the epiblast to form the mesendoderm at later stages. In this work it is shown that differential adhesion among the germlayer progenitor cells alone cannot predict germlayer formation. It is a combination of several mechanical properties such as cell cortex tension, cell adhesion and membrane mechanical properties that influence the migratory behavior of the constituent cells.

The local microenvironment of hematopoietic stem cells (HSCs) in the bone marrow -referred to as stem cell niche- is thought to regulate the balance of stem cell maintenance and differentiation by a complex interplay of extrinsic signals including spatial constraints, extracellular matrix (ECM) components and cell-cell interactions. To dissect the role of niche ECM components, a set of well-defined matrix biomolecular coatings including fibronectin, laminin, collagen IV, tropocollagen I, heparin, heparan sulphate, hyaluronic acid and co-fibrils of collagen I with heparin or hyaluronic acid were prepared and analyzed with respect to adhesive interactions of human CD133+ HSCs in vitro. ECM molecule dependent adhesion areas as well as fractions of adherent HSCs were assessed by reflection interference contrast microscopy and differential interference contrast microscopy. HSCs, so far mostly classified as suspension cells, exhibited intense adhesive interactions with fibronectin, laminin, collagen IV, heparin, heparan sulphate, and collagen I based co-fibrils. An integrin mediated adhesion on fibronectin and a L-selectin mediated adhesion on heparin pointed to specific interactions based on different adhesion mechanisms. As a consequence of HSC adhesion to molecules of the vascular and the endosteal regions, both regions were confirmed as possible stem cell niches and adhesive signals were suggested as potential regulators of stem cell fate. Furthermore, the impact of a spatially organized ECM on the HSC behavior was analyzed by single cell tracking. These studies required the development of engineered three-dimensional, ECM coated microcavities with the option for single cell tracking. A semi-automated cell-tracking tool was established to accelerate data access from time-lapse image sequences. From this analysis it was possible to reveal the genealogy, localization, morphology and migration of single HSCs over a time period of 4 days. A decreased cycling frequency was observed depending on the HSC localization in the spatially constraining microcavities. Besides the revealed impact of spatial constraints on HSC fate, the newly engineered ECM-coated microcavity setup and the semi-automated cell tracking tool provide new options to study the cell fate in engineered microenvironments at single cell level for other cell types ex vivo. / Die lokale Mikroumgebung von Blutstammzellen (BSZ) im Knochenmark, bezeichnet als Stammzellnische, reguliert das Gleichgewicht von Stammzellerhaltung und -differenzierung durch ein komplexes Zusammenspiel von extrinsischen Signalen wie räumliche Beschränkungen, Komponenten der extrazellulären Matrix (EZM) und Zell-Zell Wechselwirkungen. Um die Rolle der EZM-Komponenten zu analysieren, wurden definierte Beschichtungen von Fibronektin, Laminin, Kollagen IV, monomerem Kollagen I, Heparin, Heparan Sulphat, Hyaluronsäure und Co-Fibrillen aus Kollagen I und Heparin oder Hyaluronsäure hergestellt und in vitro bezüglich der adhäsiven Wechselwirkungen von humanen CD133+ BSZ untersucht. Die Adhäsionsflächen und der Anteil adhärenter Zellen wurden in Abhängigkeit von der EZM-Beschichtung mittels Reflexions- Interferenz-Kontrast-Mikroskopie und Differentieller Interferenz Kontrast Mikroskopie bestimmt. BSZ, bisher als Suspensionszellen definiert, zeigten intensive adhäsive Wechselwirkungen mit Fibronektin, Laminin, Kollagen IV, Heparin, Heparan Sulphat und den Co-Fibrillen. Eine Integrin abhängige Adhäsion auf Fibronektin und eine L-Selektin abhängige Adhäsion auf Heparin, wiesen auf spezifische Wechselwirkungen hin, die auf unterschiedlichen Mechanismen basieren. Aufgrund der Adhäsion von BSZ sowohl zu Molekülen der vaskulären als auch der endostealen Knochenmarkregion, wurden beide Bereiche als mögliche Stammzellnische bestätigt. Adhäsive Signale sind potentielle Regulatoren der Stammzellentwicklung. Im Weiteren wurde der Einfluss einer räumlich beschränkenden EZM auf das Verhalten der BSZ durch Einzelzellverfolgung untersucht. Diese Studien erforderten die Entwicklung von dreidimensionalen EZM-beschichteten Mikrokavitäten, die das Verfolgen einzelner Zellen ermöglichten. Es wurde ein halbautomatischer Algorithmus für die Zellverfolgung etabliert, um die Datengenerierung von den Zeitreihenaufnahmen zu beschleunigen. Die Analysen ermöglichten Aussagen über die Genealogie, Lokalisierung, Morphologie und Migration einzelner BSZ während einer Analysenzeit von 4 Tagen. Eine verringerte Zellteilungsaktivität wurde in Abhängigkeit von der BSZ Lokalisierung innerhalb der räumlich einschränkenden Mikrokavitäten festgestellt. Neben diesen Erkenntnissen bieten die entwickelten Mikrokavitäten und die etablierte Einzelzellverfolgung neue Möglichkeiten auch andere Zelltypen auf Einzelzellniveau ex vivo zu untersuchen.