| 1. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Initiation of the transcriptional response to hyperosmotic shock correlates with the potential for volume recovery.
- 2013
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Ingår i: The FEBS journal. - : Wiley. - 1742-4658 .- 1742-464X. ; 280:16, s. 3854-67
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Tidskriftsartikel (refereegranskat)abstract
- The control of activity and localization of transcription factors is critical for appropriate transcriptional responses. In eukaryotes, signal transduction components such as mitogen-activated protein kinase (MAPK) shuttle into the nucleus to activate transcription. It is not known in detail how different amounts of nuclear MAPK over time affect the transcriptional response. In the present study, we aimed to address this issue by studying the high osmolarity glycerol (HOG) system in Saccharomyces cerevisiae. We employed a conditional osmotic system, which changes the period of the MAPK Hog1 signal independent of the initial stress level. We determined the dynamics of the Hog1 nuclear localization and cell volume by single-cell analysis in well-controlled microfluidics systems and compared the responses with the global transcriptional output of cell populations. We discovered that the onset of the initial transcriptional response correlates with the potential of cells for rapid adaptation; cells that are capable of recovering quickly initiate the transcriptional responses immediately, whereas cells that require longer time to adapt also respond later. This is reflected by Hog1 nuclear localization, Hog1 promoter association and the transcriptional response, but not Hog1 phosphorylation, suggesting that a presently uncharacterized rapid adaptive mechanism precedes the Hog1 nuclear response. Furthermore, we found that the period of Hog1 nuclear residence affects the amplitude of the transcriptional response rather than the spectrum of responsive genes.
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| 2. |
- Kantere, Despoina, et al.
(författare)
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Anti-Stokes fluorescence from endogenously formed protoporphyrin IX - Implications for clinical multiphoton diagnostics.
- 2013
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Ingår i: Journal of biophotonics. - : Wiley. - 1864-0648 .- 1864-063X. ; 6:5, s. 409-415
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Tidskriftsartikel (refereegranskat)abstract
- Multiphoton imaging based on two-photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce. No report shows conclusive evidence that endogenously formed PpIX increases tumor contrast when performing multiphoton imaging in the clinical situation. We here demonstrate by spectral analysis that two-photon excitation of endogenously formed PpIX does not provide additional contrast in superficial basal cell carcinomas. In fact, the PpIX signal is overshadowed by the autofluorescent background. The results show that PpIX should be excited at a wavelength giving rise to one-photon anti-Stokes fluorescence, to overcome the autofluorescent background. Thus, this study reports on a plausible method, which can be implemented for clinical investigations on endogenously formed PpIX using multiphoton microscopy (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
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| 3. |
- Wadskog, Ingrid, et al.
(författare)
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The yeast tumor suppressor homologue Sro7p is required for targeting of the sodium pumping ATPase to the cell surface.
- 2006
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Ingår i: Molecular biology of the cell. - 1059-1524 .- 1939-4586. ; 17:12, s. 4988-5003
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Tidskriftsartikel (refereegranskat)abstract
- The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7Delta mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7Delta mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7Delta mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7Delta mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.
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| 4. |
- Welkenhuysen, Niek, 1988, et al.
(författare)
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Single-cell study links metabolism with nutrient signaling and reveals sources of variability
- 2017
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Ingår i: Bmc Systems Biology. - : Springer Science and Business Media LLC. - 1752-0509. ; 11:59
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Tidskriftsartikel (refereegranskat)abstract
- Background: The yeast AMPK/SNF1 pathway is best known for its role in glucose de/repression. When glucose becomes limited, the Snf1 kinase is activated and phosphorylates the transcriptional repressor Mig1, which is then exported from the nucleus. The exact mechanism how the Snf1-Mig1 pathway is regulated is not entirely elucidated. Results: Glucose uptake through the low affinity transporter Hxt1 results in nuclear accumulation of Mig1 in response to all glucose concentrations upshift, however with increasing glucose concentration the nuclear localization of Mig1 is more intense. Strains expressing Hxt7 display a constant response to all glucose concentration upshifts. We show that differences in amount of hexose transporter molecules in the cell could cause cell-to-cell variability in the Mig1-Snf1 system. We further apply mathematical modelling to our data, both general deterministic and a nonlinear mixed effect model. Our model suggests a presently unrecognized regulatory step of the Snf1-Mig1 pathway at the level of Mig1 dephosphorylation. Model predictions point to parameters involved in the transport of Mig1 in and out of the nucleus as a majorsource of cell to cell variability. Conclusions: With this modelling approach we have been able to suggest steps that contribute to the cell-to-cell variability. Our data indicate a close link between the glucose uptake rate, which determines the glycolytic rate, and the activity of the Snf1/Mig1 system. This study hence establishes a close relation between metabolism and signalling.
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| 5. |
- Abbaszadehbanaeiyan, Amin, et al.
(författare)
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Design and fabrication of high-throughput application-specific microfluidic devices for studying single-cell responses to extracellular perturbations
- 2013
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Ingår i: Proc. SPIE 8765, Bio-MEMS and Medical Microdevices, 87650K. ; 8765
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Konferensbidrag (refereegranskat)abstract
- Single cell analysis techniques provide a unique opportunity of determining the intercellular heterogeneity in a cell population, which due to genotype variations and different physiological states of the cells i.e. size, shape and age, cannot be retrieved from averaged cell population values. In order to obtain high-value quantitative data from single-cell experiments it is important to have experimental platforms enabling high-throughput studies. Here, we present a microfluidic chip, which is capable of capturing individual cells in suspension inside separate traps. The device consists of three adjacent microchannels with separate inlets and outlets, laterally connected through the V-shaped traps. Vshaped traps, with openings smaller than the size of a single cell, are fabricated in the middle (main) channel perpendicular to the flow direction. Cells are guided into the wells by streamlines of the flows and are kept still at the bottom of the traps. Cells can then be exposed to extracellular stimuli either in the main or the side channels. Microchannels and traps of different sizes can be fabricated in polydimethylsiloxane (PDMS), offering the possibility of independent studies on cellular responses with different cell types and different extracellular environmental changes. We believe that this versatile high-throughput cell trapping approach will contribute to further development of the current knowledge and information acquired from single-cell studies and provide valuable statistical experimental data required for systems biology. © (2013) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
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| 6. |
- Abbaszadehbanaeiyan, Amin, 1983, et al.
(författare)
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Hydrodynamic Cell Trapping for High Throughput Single-Cell Applications
- 2013
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Ingår i: Micromachines. - : MDPI AG. - 2072-666X. ; 4:4, s. 414-430
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Tidskriftsartikel (refereegranskat)abstract
- The possibility to conduct complete cell assays under a precisely controlled environment while consuming minor amounts of chemicals and precious drugs have made microfluidics an interesting candidate for quantitative single-cell studies. Here, we present an application-specific microfluidic device, cellcomb, capable of conducting high-throughput single-cell experiments. The system employs pure hydrodynamic forces for easy cell trapping and is readily fabricated in polydimethylsiloxane (PDMS) using soft lithography techniques. The cell-trapping array consists of V-shaped pockets designed to accommodate up to six Saccharomyces cerevisiae (yeast cells) with the average diameter of 4 μm. We used this platform to monitor the impact of flow rate modulation on the arsenite (As(III)) uptake in yeast. Redistribution of a green fluorescent protein (GFP)-tagged version of the heat shock protein Hsp104 was followed over time as read out. Results showed a clear reverse correlation between the arsenite uptake and three different adjusted low = 25 nL min−1, moderate = 50 nL min−1, and high = 100 nL min−1 flow rates. We consider the presented device as the first building block of a future integrated application-specific cell-trapping array that can be used to conduct complete single cell experiments on different cell types.
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| 7. |
- Abbaszadehbanaeiyan, Amin, et al.
(författare)
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Liver-lobule-on-a-chip microfluidic device for long-term maintenance of human hepatocytes
- 2017
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Ingår i: Presented at EMBEC’17 & NBC’17 (conference), 11-15 juni 2017, Tampere, Finland.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The pressing need for in vitro micro-physiological platforms for drug discovery and development has given rise to the emergence of organs-on-a-chip (OOC) microfluidic devices. The possibility of reproducing the native niche of each organ in a dynamic microenvironment offers advantages over current static 2D and 3D cell culture techniques. Constant removal of waste products and metabolites from the culture while providing a continuous flow of growth media is one of the major benefits of dynamic OOC systems. Additionally, physiological flow conditions can be introduced to the system allowing for reproduction of the vasculature parameters of organs in vitro. The liver is the main organ in the body for drug clearance and detoxification. The key role of the liver in the metabolism system of the human body makes it an interesting target organ to mimic in the dynamic OOC systems. Here we present a PDMS-based liver-lobule-on-a-chip microfluidic device designed to reproduce the geometrical as well as convection-diffusion mass transport aspects of the classic liver lobule. We cultured human induced pluripotent stem cell (hiPSC)-derived hepatocytes (CDI) in honeycomb cell culture chambers with involvement of two different extra-cellular matrix (ECM) materials. In the first approach, microfluidic devices were pre-treated with rat-tail collagen I and cell suspension was seeded in the devices afterwards. Cells were seeded in the devices with the supplemented plating medium (RPMI) and culture for 5 days. The medium was changed to the supplemented mainte-nance media (RPMI) thereafter and replaced every other day. In the second approach, we mixed the cell suspension with 20% diluted GeltrexTM (15 mg/ml) in a 1:1 ratio. Cells were seeded in the supplemented plating media (RPMI) and were kept under conditions identical to approach one during the hepatocyte maturation period. After day 5, however, the formulation of maintenance media was changed to supplemented DMEM/F12. Cultures were kept viable and functional for at least three weeks. In both scenarios cells formed 3D tissue-like structures and formation of bile canaliculi network was observed in the devices versus 2D static cultures. The compatibility of the device for drug toxicity applications and multi-cellular in vitro organotype construction is currently under exploration.
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| 8. |
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| 9. |
- Ahmadpour, Doryaneh, 1973, et al.
(författare)
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Effect of MAPK Inhibitor on the Arsenite uptake by Aquaglyceroporin in Single Yeast Cells.
- 2013
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Ingår i: Optical Molecular Probes, Imaging and Drug Delivery.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Regulating arsenic uptake is imperative due to its carcinogenicity. Combining microfluidics, optical tweezers and fluorescence microscopy, the arsenite uptake by Fps1 using a selective kinase inhibitor is investigated using a single cell analysis platform.
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| 10. |
- Ahmadpour, Doryaneh, 1973, et al.
(författare)
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Inhibition of MAPK Hog1 Results in Increased Hsp104 Aggregate Formation Probably through Elevated Arsenite Influx into the Cells, an Approach with Numerous Potential Applications
- 2014
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Ingår i: American Journal of Molecular Biology. - : Scientific Research Publishing, Inc.. - 2161-6620 .- 2161-6663. ; 4:2, s. 59-71
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Tidskriftsartikel (refereegranskat)abstract
- Arsenic is a highly toxic and carcinogenic metalloid widely dispersed in the environment, contaminating water and soil and accumulating in crops. Paradoxically, arsenic is also part of modern therapy and employed in treating numerous ailments and diseases. Hence, inventing strategies to tune cellular arsenic uptake based on purpose is striking. Here, we describe an approach in which the arsenite uptake can be increased using a MAPK inhibitor. Employing microfluidic flow chambers in combination with optical tweezers and fluorescent microscopy, we elevated the influx of arsenite into the yeast Saccharomyces cerevisiae cells following short-term treatment with a Hog1 kinase inhibitor. The increase in arsenite uptake was followed on arsenite triggered redistribution of a reporter protein, Hsp104-GFP, which was imaged over time. The effect was even more pronounced when the yeast mother and daughter cells were analyzed disjointedly, an opportunity provided owing to single-cell analysis. Our data firstly provide a strategy to increase arsenite uptake and secondly show that arsenite triggered aggregates, previously shown to be sites of damaged proteins, are distributed asymmetrically and less accumulated in daughter cells. Inventing approaches to tune arsenite uptake has a great value for its use in environmental as well as medical applications.
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