| 1. |
- Dolatabadi, Soheila, et al.
(författare)
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Cell Cycle and Cell Size Dependent Gene Expression Reveals Distinct Subpopulations at Single-Cell Level
- 2017
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Ingår i: Frontiers in Genetics. - : Frontiers Media SA. - 1664-8021. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Cell proliferation includes a series of events that is tightly regulated by several checkpoints and layers of control mechanisms. Most studies have been performed on large cell populations, but detailed understanding of cell dynamics and heterogeneity requires single-cell analysis. Here, we used quantitative real-time PCR, profiling the expression of 93 genes in single-cells from three different cell lines. Individual unsynchronized cells from three different cell lines were collected in different cell cycle phases (GO/G1 - S - G2/M) with variable cell sizes. We found that the total transcript level per cell and the expression of most individual genes correlated with progression through the cell cycle, but not with cell size. By applying the random forests algorithm, a supervised machine learning approach, we show how a multi-gene signature that classifies individual cells into their correct cell cycle phase and cell size can be generated. To identify the most predictive genes we used a variable selection strategy. Detailed analysis of cell cycle predictive genes allowed us to define subpopulations with distinct gene expression profiles and to calculate a cell cycle index that illustrates the transition of cells between cell cycle phases. In conclusion, we provide useful experimental approaches and bioinformatics to identify informative and predictive genes at the single-cell level, which opens up new means to describe and understand cell proliferation and subpopulation dynamics.
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| 2. |
- Dolatabadi, Soheila
(författare)
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The role of fusion oncogenes and cancer stem cells in myxoid liposarcoma
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Myxoid liposarcoma (MLS) is characterised by the FUS-DDIT3, or the less common EWSR1-DDIT3 fusion oncogene and is the second most common type of liposarcoma. The fusion oncogenes encode chimeric transcription factors that are causal factors in tumourigenesis however, their functions are poorly known. Notwithstanding continuous progress in treating MLS patients, existing therapies suffer from a major flaw as they do not target the cancer stem cells (CSCs). Unique features of CSCs include self-renewal, tumour initiating capacity and increased resistance to radiotherapy- and chemotherapy-induced cell death. Thus, CSCs are crucial targets for successful therapy. The aims of this project were to define the role of fusion oncogenes in tumourigenesis and to define signalling pathways controlling CSC features in MLS. Here, we demonstrated that MLS has an intact TP53 system that may explain why this tumour entity is genetically stable. We investigated the regulatory mechanisms, expression levels and effects of FUS-DDIT3 in detail, and showed that FUS-DDIT3 was uniquely regulated at both transcriptional and post-translational level. We also screened 70 well-characterised kinase inhibitors and determined their effects on cell proliferation and FUS-DDIT3 expression at mRNA and protein levels. To facilitate these studies, we developed a novel direct lysis approach that enables us to quantify, cell proliferation, mRNA and protein expression in the same sample. This method allowed us to identify a number of previously unknown signalling pathways that regulated the expression of FUS-DDIT3. To study cell division and growth in detail, we applied single-cell analysis on unsynchronized cells at different cell cycle phases and cell sizes. We found that the total transcript level per cell and the expression of most individual genes correlated with progression of the cell cycle, but not with cell size. Detailed studies of cell cycle predictive genes revealed a previously unknown G1 subpopulation. Finally, we showed that MLS contains cells with CSC features and that JAK-STAT signalling controls their numbers. Leukaemia inhibitory factor stimuli increased the number of CSCs, while JAK inhibition depleted the CSC pool. Inhibition of JAK-STAT also showed synergistic effects when combined with chemotherapy in vitro. Our findings concerning FUS-DDIT3 function and CSCs have increased our molecular understanding of tumour development and therapy resistance in MLS that will facilitate development of specific treatment strategies.
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| 3. |
- Kroneis, Thomas, et al.
(författare)
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Global preamplification simplifies targeted mRNA quantification
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The need to perform gene expression profiling using next generation sequencing and quantitative real-time PCR (qPCR) on small sample sizes and single cells is rapidly expanding. However, to analyse few molecules, preamplification is required. Here, we studied global and target-specific preamplification using 96 optimised qPCR assays. To evaluate the preamplification strategies, we monitored the reactions in real-time using SYBR Green I detection chemistry followed by melting curve analysis. Next, we compared yield and reproducibility of global preamplification to that of target-specific preamplification by qPCR using the same amount of total RNA. Global preamplification generated 9.3-fold lower yield and 1.6-fold lower reproducibility than target-specific preamplification. However, the performance of global preamplification is sufficient for most downstream applications and offers several advantages over target-specific preamplification. To demonstrate the potential of global preamplification we analysed the expression of 15 genes in 60 single cells. In conclusion, we show that global preamplification simplifies targeted gene expression profiling of small sample sizes by a flexible workflow. We outline the pros and cons for global preamplification compared to target-specific preamplification.
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