5 Abstract Although yeasts are unicellular microorganisms, they form complex multicellular formations such as biofilms and colonies under natural conditions. Within these structures, processes such as cell differentiation, specialization by particular cell populations and cell signalling, which are typical of multicellular organisms, take place. The literature introduction to this thesis summarizes current knowledge regarding the development of biofilms and colonies, in particular those of the model organism, Saccharomyces cerevisiae, and some selected regulations that are important for the formation of multicellular structures. In the results section, I focus on two lines of research. The first is directed towards mechanisms, involved in the formation of multicellular structures. In studying the formation of SLI biofilms (biofilms at the solid/liquid interface), we have documented the antagonistic role of the regulators CYC8 and TUP1 in their formation and have also described the effect of the presence of glucose on the development and stability of SLI biofilms of strain BR-F. During this study we[D1] have developed an imaging method that allows us to prepare and observe the internal structure (vertical cross-section) of SLI biofilms, as well as the growth of unattached cells, under physiological...

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Duchenne Muscular Dystrophy (DMD) is a disorder caused by loss-of-function mutations in dystrophin, a critical protein that maintains muscle fiber integrity. Our lab discovered that dystrophin-deficient skeletal muscle stem cells, also known as satellite cells, cannot generate enough myogenic progenitors for proper muscle regeneration. Previously, we demonstrated that WNT7A, a protein expressed during muscle regeneration, stimulates symmetric division of satellite cells, and gives rise to two daughter satellite cells. Conversely, epidermal growth factor (EGF) induces asymmetric division, which generates one daughter satellite cell and one committed precursor cell. We aimed to investigate these satellite cell division mechanisms following WNT7A or EGF treatment in a human model using healthy and DMD-patient derived hiPSCs differentiated into the myogenic lineage. The presence of satellite-like cells was confirmed in both lines by their characteristic expression of PAX7 and other myogenic markers. Intriguingly, DMD-patient hiPSCs precociously differentiated compared to healthy control human induced pluripotent stem cells (hiPSCs). More notably, WNT7A treatment had a potent effect on the DMD differentiated cells. High content analysis revealed an expansion of the satellite-like cell pool as observed by a higher number of PAX7+ cells within the total population and gene expression analysis demonstrated a significant increase in global PAX7 expression. In contrast, EGF treatment reduced the number of PAX7+ cells and increased the proportion of MYOG+ cells within the myogenic population, indicating an increase in myogenic progenitors. Taken together, WNT7A and EGF can alter the myogenic differentiation program of healthy and DMD-patient derived hiPSCs by modulating the satellite-like cell division dynamics.

Indiana University-Purdue University Indianapolis (IUPUI) / Mechanical force environment is a major factor that influences cellular homeostasis and remodeling. The prevailing wisdom in this field demonstrated that a threshold of mechanical forces or deformation was required to affect cell signaling. However, by using a fluorescence resonance energy transfer (FRET)-based approach, we found that C28/I2 chondrocytes exhibited an increase in RhoA activities in response to high shear stress (10 or 20 dyn/cm2), while they showed a decrease in their RhoA activities to intermediate shear stress at 5 dyn/cm2. No changes were observed under low shear stress (2 dyn/ cm2). The observed two-level switch of RhoA activities was closely linked to the shear stress-induced alterations in actin cytoskeleton and traction forces. In the presence of constitutively active RhoA (RhoA-V14), intermediate shear stress suppressed RhoA activities, while high shear stress failed to activate them. Collectively, these results herein suggest that intensities of shear stress are critical in differential activation and inhibition of RhoA activities in chondrocytes.

<p>  </p> <p>IL-9-producing CD4+ T helper (Th9) cells contribute to inflammatory responses during infection, anti-cancer responses and autoimmune disease. Thus, elucidating the signals that regulate their differentiation is critical for understanding the roles of Th9 cells in protective immunity and disease. Th9 cells differentiate in response to IL-4, TGF-β and IL-2, where IL-2 signaling through STAT5 is crucial for transactivating <em>Il9</em> locus. While the roles of IL-4 and TGF- β-mediated signaling are relatively well understood, how IL-2 signaling contributes to Th9 cell differentiation outside of directly inducing the <em>Il9</em> locus remains less clear. I found that human allergen-induced Th9 cells exhibited a strong signature of STAT5-mediated gene repression that was associated with inhibition of a Th17-like transcriptional signature. Likewise, blockade of IL-2/STAT5 signaling increased IL-17 and RORγt expression in murine Th9 cells <em>in vitro</em>. Interestingly, development of this Th17-like phenotype was independent of STAT3. While STAT3 was not required for IL-17 expression, it was required for their long-term persistence. These results suggest that IL-2/STAT5 signaling controls the balance between Th9 and Th17-like cell differentiation in vitro and during allergy. Additionally, I found that murine Th9 cells cultured in a low IL-2 environment had reduced IL-9 production and a diminished NF-kB-associated transcriptional signature, suggesting that IL-2 signaling is associated with NF-kB activation in Th9 cells. Interestingly, NF-kB activation via IL-1β stimulation enhanced Th9 differentiation under IL-2 limiting conditions and promoted their inflammatory potential in a mouse model of Lung inflammation. Mechanistically, we found that IL-2- limiting conditions enhanced IL-1β receptor expression and that IL-1β/NF-kB signaling increased the sensitivity to IL-2 and silenced the expression of the anti-Th9 transcription factor BCL6. Together, these findings indicate that IL-1β /NF-kB signaling can promote Th9 cell differentiation in IL-2-limiting conditions and that this pathway may be targeted to enhance Th9 differentiation and their inflammatory function.  Collectively, these data revealed two novel roles for the IL-2/STAT5 axis in Th9 cells.</p> <p>The Thymocyte associated High Mobility Group (HMG) box, known as TOX has been previously described to have paramount functions in the development of all the lineages of CD4+ T cells during thymic selection, during CD8+ T cell exhaustion and in Tfh cell differentiation and function. However, the role of TOX in non-Tfh CD4+ T cells in the periphery has not been addressed. In these studies, I found that CD4+ T cells express TOX in the steady state in secondary lymphoid organs like spleen, lymph nodes, and Peyer’s patches. Specifically, TOX was expressed remarkably in Tfh, Th1, Treg cells, and other non-Tfh unidentified Th cells, as well as Th2 cells in the lungs. Transcriptomics analyses using bulk RNA-seq revealed that TOX minimally alters s gene expression, however it revealed for the first time, that TOX induced genes associated with cell migration i.e., <em>Xcl1</em> <em>Ccl3</em>, <em>Ccl4</em> and also the inhibitory cytokine <em>Il10</em>. The induction of IL-10 and CCL3 was validated at the protein levels, and mechanistic studies revealed that the induction of these molecules required the transcription factor BATF, indicating for the first time a mechanism of TOX-mediated functions. Together, these data shed light in novel roles of TOX in CD4+ T cell function and opens the door for future functional and mechanistic studies that may be relevant during health and disease.</p>

RNA-bindende Proteine sind zentrale Regulatoren der Genexpression, aber ihre Funktionen bei der Koordinierung von Zellschicksalsentscheidungen sind unzureichend verstanden. In dieser Studie haben wir RNA interactome capture angewandt, um die globalen Dynamiken des RNA-gebundenen Proteoms während der Auflösung der Pluripotenz und neuronaler Differenzierung zu bestimmen. Wir haben entdeckt, dass 30-40% der RNA-bindenden Proteine sehr dynamisch während der Zellschicksalentscheidungen sind, die Abundanzdynamiken dieser Proteine aber nicht hauptursächlich dafür zu sein scheinen. Basierend auf unseren Daten haben wir ZAP (ZC3HAV1) als einen Faktor identifiziert, der mit Pluripotenz assoziiert ist. Um die Rolle von ZAP in der Stammzellbiologie zu analysieren, haben wir PAR-CLIP, SLAMseq und einen Differenzierungsassay angewandt. Unsere Daten haben gezeigt, dass ZAP mehr als 2,000 mRNA-Transkripte innerhalb des murinen Stammzelltranskriptoms in Abhängigkeit von CG-Dinukleotiden bindet. Zieltranskripte sind angereichert mit Genfunktionen in Zell-Zell-Interaktionen, Gewebemorphogenese und Pluripotenzregulation und werden in Abwesenheit von ZAP stabilisiert. Auβerdem haben wir herausgefunden, dass Depletion von ZAP zu flacherer und breiterer Koloniemorphologie von Stammzellen bei gleichzeitiger Fehlexpression von hunderten von Genen inklusive Lineage-Faktoren führt. Desweiteren führt Abwesenheit von ZAP zu erhöhter Geschwindigkeit bei der Auflösung der Pluripotenz. Zusammengefasst stellen wir die These auf, dass ZAP ein multi-modaler Regulator der Pluripotenz ist. ZAP agiert als positiver Regulator während Aufrechterhaltung der Pluripotenz, während es am Anfang der Pluripotenzauflösung pluripotenz-fördernde Faktoren herunterreguliert. Schlussendlich demonstriert diese Studie, wie die Erforschung von Dynamiken des RNA-gebundenen Proteoms während Zellschicksalsentscheidungen neue Wege öffnet, um die Funktion von RNA-bindenden Proteinen im entwicklungsbiologischen Kontext zu analysieren. / RNA-binding proteins are key regulators of gene expression, but their functions in coordinating cell fate transitions are poorly understood. In this study, we applied RNA interactome capture to determine the global dynamics of the RNA-bound proteome during dissolution of pluripotency and neuronal differentiation. We discovered that 30-40% of RNA-binding proteins are highly dynamic during cell fate transitions and that these dynamics do not appear to be predominantly governed by alterations in their abundance. Based on our data, we identified ZAP (ZC3HAV1) as a factor highly associated with pluripotency. In order to dissect the role of ZAP in mESC biology, we applied a variety of approaches including PAR-CLIP, SLAMseq and pluripotency exit reporter assays. We found that ZAP binds more than 2,000 mRNAs in the mESC transcriptome in a CG dinucleotide-dependent manner. Targets are enriched for transcripts encoding cell-cell adhesion, tissue morphogenesis and pro-pluripotency regulators and stabilized in absence of ZAP. Furthermore, we found that ZAP depletion leads to flattened and spreading stem cell colony morphology, concomitant misexpression of hundreds of transcripts including lineage factors and accelerated early dissolution of pluripotency. In conclusion, we propose that ZAP is a multi-modal stem cell RNA-binding protein acting as a positive regulator in maintenance of pluripotency while aiding downregulation of pro-pluripotent factors at the onset of differentiation. Ultimately, this study demonstrates how exploration of RNA-bound proteome dynamics during cell fate transitions can open paths to dissecting functions of RNA-binding proteins in a developmental context.

Cell differentiation is the process of a cell developing from one cell type to another. It is of interest to analyse the differentiation from stem cells to different types of mature cells, and discover what genes are involved in regulating the differentiation to specific cells, for instance to get insights to what is causing certain diseases and find potential treatments.  In this project, two mathematical models are developed for analysing blood cell differentiation (haematopoiesis) with methods based on optimal transportation. Optimal transportation is about moving one mass distribution to another at minimal cost. Modelling a sample of cells as point masses placed in a space based on the cells' gene expressions, accessed by single-cell RNA sequencing, optimal transportation is used to find transitions between cells that costs the least in terms of changes in gene expression. With this, cell-to-cell trajectories, from haematopoietic stem cells to mature blood cells, are obtained. With the first model, cells are divided into groups based on their maturity, which is determined by using diffusion pseudotime, and optimal transportation is preformed between groups. The resulting trajectories suggest that haematopoietic stem cells possibly can develop into the same mature cell type in different ways, and that the cell fate for some cell types is decided late on in development. In future work, the gene regulation along the obtained trajectories can be analysed. The second model is developed to be more general than the first, and not be dependent on a group division before preforming optimal transportation. / Celldifferentiering är processen då en cell utvecklas från en celltyp till en annan. Det är av intresse att analysera differentieringen från stamcell till olika typer av mogna celler, och undersöka vilka gener som har betydelse i regleringen av differentieringen till specifika celler, bland annat för att få en inblick i vad som orsakar vissa sjukdomar och hitta potentiella botemedel. I detta projekt utvecklas två matematiska modeller för att analysera blodcellsutveckling (hematopoes) med metoder som är baserade på optimal transport. Optimal transport handlar om att förflytta en massfördelning till en annan till lägst kostnad. Genom att modellera celler som punktmassor, placerade i ett rum baserat på cellernas genuttryck som fås genom singel-cell RNA-sekvensering, används optimal transport för att hitta förflyttningar mellan celler som kostar minst i termer av förändringar i genuttryck. Från detta skapas vägar mellan celler, från hematopoetiska stamceller till mogna celler. I den första modellen delas cellerna upp i grupper baserat på deras mognadsgrad, som bestäms genom att använda pseudotid baserad på en diffusionsavbildning, och optimal transport används sedan mellan grupperna. De resulterande vägarna visar på att hematopoetiska stamceller möjligen kan utvecklas till samma typ av mogen cell på olika sätt, och att cellödet för vissa typer av celler bestäms sent i utvecklingen. I framtida arbete kan genregleringen längs de funna vägarna analyseras. Den andra modellen utvecklas för att vara mer generell än den första, och inte bero på en gruppuppdelning innan optimal transport används.

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