| 1. |
- Bendrioua, Loubna, et al.
(author)
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Yeast AMP-activated protein kinase monitors glucose concentration changes and absolute glucose levels
- 2014
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 289:18, s. 12863-12875
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Journal article (peer-reviewed)abstract
- Background: Little is known about the signaling dynamics of AMP-activated protein kinase. Results: We define the dynamics of yeast AMPK signaling under different glucose concentrations. Conclusion: The Snf1-Mig1 signaling system monitors glucose concentration changes and absolute glucose levels to adjust the metabolism to a wide range of conditions. Significance: This description of AMPK signaling dynamics will stimulate studies defining the integration of signaling and metabolism. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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| 2. |
- Dyrager, Christine, 1975, et al.
(author)
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2,6,8-Trisubstituted 3-hydroxychromone derivatives as fluorophores for live-cell imaging.
- 2009
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In: Chemistry (Weinheim an der Bergstrasse, Germany). - : Wiley. - 1521-3765 .- 0947-6539. ; 15:37, s. 9417-23
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Journal article (peer-reviewed)abstract
- We present the synthesis and photophysical characterisation of a series of structurally diverse, fluorescent 2,6,8-trisubstituted 3-hydroxychromone derivatives with high fluorescence quantum yields and molar extinction coefficients. Two of these derivatives (9 and 10 a) have been studied as fluorophores for cellular imaging in HeLa cells and show excellent permeability and promising fluorescence properties in a cellular environment. In addition, we have demonstrated by photophysical characterisation of 3-isobutyroxychromone derivatives that esterification of the 3-hydroxyl group results in acceptable and useful fluorescence properties.
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| 3. |
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| 4. |
- Babazadeh, Roja, et al.
(author)
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Osmostress-induced cell volume loss delays yeast hog1 signaling by limiting diffusion processes and by hog1-specific effects.
- 2013
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In: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 8:11
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Journal article (peer-reviewed)abstract
- Signal transmission progresses via a series of transient protein-protein interactions and protein movements, which require diffusion within a cell packed with different molecules. Yeast Hog1, the effector protein kinase of the High Osmolarity Glycerol pathway, translocates transiently from the cytosol to the nucleus during adaptation to high external osmolarity. We followed the dynamics of osmostress-induced cell volume loss and Hog1 nuclear accumulation upon exposure of cells to different NaCl concentrations. While Hog1 nuclear accumulation peaked within five minutes following mild osmotic shock it was delayed up to six-fold under severe stress. The timing of Hog1 nuclear accumulation correlated with the degree of cell volume loss and the cells capacity to recover. Also the nuclear translocation of Msn2, the transcription factor of the general stress response pathway, is delayed upon severe osmotic stress suggesting a general phenomenon. We show by direct measurements that the general diffusion rate of Hog1 in the cytoplasm as well as its rate of nuclear transport are dramatically reduced following severe volume reduction. However, neither Hog1 phosphorylation nor Msn2 nuclear translocation were as much delayed as Hog1 nuclear translocation. Our data provide direct evidence that signaling slows down during cell volume compression, probably as a consequence of molecular crowding. Hence one purpose of osmotic adaptation is to restore optimal diffusion rates for biochemical and cell biological processes. In addition, there may be mechanisms slowing down especially Hog1 nuclear translocation under severe stress in order to prioritize Hog1 cytosolic targets.
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| 5. |
- Bender, Johanna, 1975, et al.
(author)
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Lipid cubic phases in topical drug delivery: Visualization of skin distribution using two-photon microscopy
- 2008
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In: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 129:3, s. 163-169
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Journal article (peer-reviewed)abstract
- The distribution of sulphorhodamine B (SRB), a fluorescent hydrophilic model drug, was investigated in human skin after passive diffusion using four different topical delivery systems. The delivery vehicles applied were two bicontinuous lipid cubic systems, a commercial ointment and water. The lipid cubic systems consisted of either monoolein (MO) or phytantriol (PT) and water. The formulations were applied on full-thickness human skin during 24 h. Thereafter the samples were investigated using two-photon microscopy (TPM). The TPM system consisted of an inverted microscope with a 40× water-immersion objective, laser scan-box, and a pulsed femtosecond titanium:sapphire laser operating at 780 nm. The fluorescence was detected using a 560 nm long-pass filter. Sequential optical sectioning was performed, resulting in images obtained at different tissue depths. TPM revealed that SRB mainly penetrates the skin via the intercellular lipid matrix. Samples exposed to the cubic phases showed a higher accumulation of SRB in micro-fissures, from which a fluorescent network of threadlike structures spread laterally in the tissue. These structures were also detected in some of the ointment samples, but not as frequent. The penetration of SRB into the stratum granulosum was deduced from the fluorescence of SRB present inside polygonal keratinocytes with cell nuclei. Higher SRB fluorescence was obtained in the outermost layer of the epidermis using the bicontinuous cubic phases, compared to when using the reference formulations. Thus, our results suggest that the dominating delivery route using the cubic phases is via micro-fissures caused by microscopic clustering of the keratinocytes in the skin. From these micro--fissures hydrophilic compounds, here modeled by SRB, can diffuse into the surrounding intercellular lipid matrix acting like a source for sustained release.
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| 6. |
- Ericson, Marica B, 1974, et al.
(author)
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Two-photon laser-scanning fluorescence microscopy applied for studies of human skin
- 2008
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In: Journal of Biophotonics. - : Wiley. - 1864-0648 .- 1864-063X. ; 1:4, s. 320-330
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Journal article (peer-reviewed)abstract
- Two-photon laser scanning fluorescence microscopy (TPM) has been shown to be advantageous for imaging optically turbid media such as human skin. The ability of performing three-dimensional imaging without presectioning of the samples makes the technique not only suitable for noninvasive diagnostics but also for studies of topical delivery of xenobiotics. Here, TPM is used as a method to visualize both autofluorescent and exogenous fluorophores in skin. Samples exposed to sulforhodamine B have been scanned from two directions to investigate attenuation effects. It is shown that optical effects play a major role. Thus, TPM is excellent for visualizing the localization and distribution of fluorophores in human skin, although quantification might be difficult. Furthermore, an image-analysis algorithm has been implemented to facilitate interpretation of TPM images of autofluorescent features of nonmelanoma skin cancer obtained ex vivo. The algorithm was designed to detect cell nuclei and currently has a sensitivity and specificity of 82% and 78% to single cell nuclei. However, in order to detect multinucleated cells, the algorithm needs further development. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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| 7. |
- Ericson, Marica B, 1974, et al.
(author)
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Two-photon microscopy of non-melanoma skin cancer: initial experience and diagnostic criteria ex vivo
- 2007
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In: Proc. SPIE. - : SPIE. ; 6630
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Conference paper (peer-reviewed)abstract
- Multiphoton microscopy is an interesting optical technique, which allows for non-invasive imaging of highly light scattering media such as human skin. Recent reports have showed the potential of applying this technique for 3D visualisation of cell structures of biological tissue without previous sectioning of the tissue samples. In this study, we have applied two-photon microscopy on excised lesions of human non-melanoma skin cancer ex vivo in order to find diagnostic criteria using this technique. The skin samples have been investigated by a multiphoton microscopy system based on a fs-pulsed Ti:sapphire laser connected to a confocal microscope. The autofluorescence of the skin was detected using excitation at 780 nm. The cell nuclei distribution turned out to be one important parameter, which can be used for discriminating between tumour and normal tissue. We are now developing a technique for automatic detection and characterisation of tissue, based on an image analysis algorithm. The detection of cell nuclei has been found crucial for this purpose. The goal is to develop a fast characterisation algorithm that can be used on line in connection to in vivo investigations. This would allow for a true non-invasive biopsy technique in the future.
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| 8. |
- Frey, S, et al.
(author)
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A mathematical analysis of nuclear intensity dynamics for Mig1-GFP under consideration of bleaching effects and background noise in Saccharomyces cerevisiae
- 2011
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In: MOLECULAR BIOSYSTEMS. - : Royal Society of Chemistry (RSC). - 1742-206X .- 1742-2051. ; 7:1, s. 215-223
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Journal article (peer-reviewed)abstract
- Abstract: Fluorescence microscopy is an imaging technique that provides insights into signal transduction pathways through the generation of quantitative data, such as the spatiotemporal distribution of GFP-tagged proteins in signaling pathways. The data acquired are, however, usually a composition of both the GFP-tagged proteins of interest and of an autofluorescent background, which both undergo photobleaching during imaging. We here present a mathematical model based on ordinary differential equations that successfully describes the shuttling of intracellular Mig1-GFP under changing environmental conditions regarding glucose concentration. Our analysis separates the different bleaching rates of Mig1-GFP and background, and the background-to-Mig1-GFP ratio. By applying our model to experimental data, we can thus extract the Mig1-GFP signal from the overall acquired signal and investigate the influence of kinase and phosphatase on Mig1. We found a stronger regulation of Mig1 through its kinase than through its phosphatase when controlled by the glucose concentration, with a constant (de)phosphorylation rate independent of the glucose concentration. By replacing the term for decreasing excited Mig1-GFP concentration with a constant, we were able to reconstruct the dynamics of Mig1-GFP, as it would occur without bleaching and background noise. Our model effectively demonstrates how data, acquired with an optical microscope, can be processed and used for a systems biology analysis of signal transduction pathways.
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| 9. |
- Geijer, Cecilia, 1980, et al.
(author)
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Initiation of the transcriptional response to hyperosmotic shock correlates with the potential for volume recovery.
- 2013
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In: The FEBS journal. - : Wiley. - 1742-4658 .- 1742-464X. ; 280:16, s. 3854-67
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Journal article (peer-reviewed)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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| 10. |
- Guldbrand, Stina, 1970, et al.
(author)
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Measuring the diffusion of fluorophores in human skin by two-photon fluorescence correlation spectroscopy combined with measurements of point spread function
- 2011
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In: MULTIPHOTON MICROSCOPY scigloo.IN THE BIOMEDICAL SCIENCES XI Book Series: Proceedings of SPIE. ; 7903, s. 7903291-7903296
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Journal article (peer-reviewed)abstract
- Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been used in combination with measurements of the point spread function (PSF), for quantitative analysis of fluorophores in excised human skin. Measurements have been performed at depths between 0 and 40 μm. The PSF, measured as full width at half maximum, was found not to depend on the depth. Measurements revealed difference in diffusion coefficient depending on extra- or intracellular location of fluorophore. The number of molecules was accumulating close to the surface and then decreased by the depth. The results from our study show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.
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