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See detailThe Effect of ATP INDUCED CALCIUM DYNAMICS ON EPITHELIAL TO MESENCHYMAL TRANSITIONS
Grzyb, Kamil UL

Doctoral thesis (2019)

Cells respond to a multitude of external triggers by a limited number of signaling pathways activated by receptors on plasma membrane, such as receptor tyrosine kinases (RTKs) or G protein-coupled ... [more ▼]

Cells respond to a multitude of external triggers by a limited number of signaling pathways activated by receptors on plasma membrane, such as receptor tyrosine kinases (RTKs) or G protein-coupled receptors (GPCRs). These pathways do not simply convey the downstream signal, but instead the signal is very often processed by encoding and integrated with the current state of the cell. A traditional transcriptional analysis tends to provide an averaged output measured in a population, what often masks the behavior of individual cells. However, with recent single cell techniques developments, it is possible to investigate transcription in individual living cells. This contributed tremendously to the understanding of development and progression of many diseases including cancer. The more we understand about this high complexity of signaling mechanisms and multitude of cellular safety countermeasures, the more we see cancer as a microevolution state of “rebellious cells” (cells entering the fate opposite to the one intended) following a patch through a discreet system. This thesis specifically focused on the temporal aspect of signaling in the context of the epithelia-to-mensenchymal transition (EMT) by combining single cell experiments and bioinformatics analysis. We investigated cellular signaling changes in response to different dynamical profiles of the stimuli. In particular, we used the HMLER cell line, which is a metastatic breast cancer model for the epithelial to mesenchymal transition. By applying stochastic or oscillatory pulses of extracellular ATP-induced Ca2+ signals with different interspike intervals, we were investigating different transcription states from those evoked by constant ATP-induced Ca2+ dose responses. In order to precisely apply those stimulation profiles, we have developed and established a perfusion system. This device allows to treat population of cells simultaneously with the exact same dynamical profiles. Cells treated by these well controlled signals were subsequently processed by the single cell RNA-seq technique Drop-seq for transcriptional analysis. The resulting high dimensional digital gene expression matrices were analyzed by a developed high-throughput computational analysis pipeline. This analysis includes the identification of differentially expressed genes and cellular clusters (states) by dimensionality reduction methods (PCA, t-SNE) and pathway analysis. We evaluated changes and trends of genes from difference dynamical profiles by investigating their involvement in stress, stemness and regulation of motility. First, we confirmed that oscillatory stimulation with extracellular ATP (eATP) tends to lower the burden of cellular stress and apoptosis related pathways while maintaining its other effector functions compared to constant eATP stimulation. Interestingly, stochastic spiking of extracellular ATP in our setup led to a massive (~80%) increase in overall differential gene expression compared to deterministic oscillatory stimulation with the same period. Consequentially, stochastic signaling seems to activate a much wider range of biological pathways, which indicates the much higher complexity in information processing capability of producing rebellious cells during cancer progression and metastasis. On the other hand, our findings suggests that oscillatory eATP stimulation could contribute to EMT by lowering ID3 expression compared to stochastic stimulation where we observed a stronger upregulation of IRS2. Finally, we integrated the DEGs into biological processes involved in each conditions and put these new insights into the context of the eATP-induced Ca2+ induced epithelial to mesenchymal transition. Overall, this thesis has applied recent single cell technologies to characterize underlying principles of cellular heterogeneity induced by cell signaling and specifically investigated the complex mechanisms of cell fate in the context of EMT [less ▲]

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See detailSingle-cell transcriptomics reveals multiple neuronal cell types in human midbrain-specific organoids
Smith, Lisa M.; Magni, Stefano UL; Grzyb, Kamil UL et al

E-print/Working paper (2019)

Human stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulate in vitro the organisation and function of a respective organ or part of an ... [more ▼]

Human stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulate in vitro the organisation and function of a respective organ or part of an organ. Human midbrain organoids (hMOs) have been described to contain midbrain-specific dopaminergic neurons that release the neurotransmitter dopamine. However, the human midbrain contains also additional neuronal cell types, which are functionally interacting with each other. Here, we analysed hMOs at high-resolution by means of single-cell RNA-sequencing (scRNA-seq), imaging and electrophysiology to unravel cell heterogeneity. Our findings demonstrate that hMOs show essential neuronal functional properties as spontaneous electrophysiological activity of different neuronal subtypes, including dopaminergic, GABAergic, and glutamatergic neurons. Recapitulating these in vivo features makes hMOs an excellent tool for in vitro disease phenotyping and drug discovery. [less ▲]

Detailed reference viewed: 113 (21 UL)
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See detailCaSiAn: a Calcium Signaling Analyzer tool
Moein, Mahsa UL; Grzyb, Kamil UL; Gonçalves Martins, Teresa et al

in Bioinformatics (2018), 1

Ca2þ is a central second messenger in eukaryotic cells that regulates many cellular proc- esses. Recently, we have indicated that typical Ca2þ signals are not purely oscillatory as widely assumed, but ... [more ▼]

Ca2þ is a central second messenger in eukaryotic cells that regulates many cellular proc- esses. Recently, we have indicated that typical Ca2þ signals are not purely oscillatory as widely assumed, but exhibit stochastic spiking with cell type and pathway specific characteristics. Here, we present the Calcium Signaling Analyzer (CaSiAn), an open source software tool that allows for quantifying these signal characteristics including individual spike properties and time course statis- tics in a semi-automated manner. CaSiAn provides an intuitive graphical user interface allowing experimentalists to easily process a large amount of Ca2þ signals, interactively tune peak detection, revise statistical measures and access the quantified signal properties as excel or text files. [less ▲]

Detailed reference viewed: 46 (4 UL)