| 1. |
- Alm, Kersti, et al.
(författare)
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Cells and holograms : holograms and digital holographic microscopy as a tool to study the morphology of living cells
- 2013
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Ingår i: Holography. - : INTECH. - 9789535111177 ; , s. 335-351
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- We present a method to study the morphology of living, dividing and dying cells using DHM. DHM is a non-invasive, non-destructive and non-phototoxic method which allows the user to perform both qualitative and quantitative measurements of living cells over time. We show here our results on cell division and cell death in single cells. The morphological analyses performed here show changes caused by cell death and cell division, and indicate the possibilities to discriminate between different types of cell death. Cells dying in an apoptosis-like manner display different cell area and cell thickness profiles over time compared to cells dying in a necrosis-like manner, although their volume profiles are very similar. Dividing cells show a characteristic dip in the volume profile, which makes them easily distinguishable. Also, several previous studies show the versatile abilities of DHM. Different cell types have been studied and the morphology has been used to determine cell functionality as well as changes in morphology related to the environment. Cell morphology parameters can be very useful when following the effects of different treatments, the process of differentiation as well as cell growth and cell death. Cell morphology studied by DHM can be useful in toxicology, stem cell and cancer research.
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| 2. |
- Alm, Kersti, et al.
(författare)
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Cells and polyamines do it cyclically
- 2009
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Ingår i: Essays in Biochemistry. - : Portland Press Ltd.. - 0071-1365 .- 1744-1358. ; 46, s. 63-76
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Tidskriftsartikel (refereegranskat)abstract
- Cell-cycle progression is a one-way journey where the cell grows in size to be able to divide into two equally sized daughter cells. The cell cycle is divided into distinct consecutive phases defined as G(1) (first gap), S (synthesis), G(2) (second gap) and M (mitosis). A non-proliferating cell, which has retained the ability to enter the cell cycle when it receives appropriate signals, is in G(0) phase, and cycling cells that do not receive proper signals leave the cell cycle from G(1) into G(0). One of the major events of the cell cycle is the duplication of DNA during S-phase. A group of molecules that are important for proper cell-cycle progression is the polyamines. Polyamine biosynthesis occurs cyclically during the cell cycle with peaks in activity in conjunction with the G(1)/S transition and at the end of S-phase and during G(2)-phase. The negative regulator of polyamine biosynthesis, antizyme, shows an inverse activity compared with the polyamine biosynthetic activity. The levels of the polyamines, putrescine, spermidine and spermine, double during the cell cycle and show a certain degree of cyclic variation in accordance with the biosynthetic activity. When cells in G(0)/G(1) -phase are seeded in the presence of compounds that prevent the cell-cycle-related increases in the polyamine pools, the S-phase of the first cell cycle is prolonged, whereas the other phases are initially unaffected. The results point to an important role for polyamines with regard to the ability of the cell to attain optimal rates of DNA replication.
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| 3. |
- Alm, Kersti, et al.
(författare)
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Digital holography and cell studies
- 2011
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Ingår i: Holography, Research and Technologies. - : DKV - Deutscher Kälte- und Klimatechnischer Verein. - 9789533072272 ; , s. 237-252
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Digital holography microscopy (DHM) has developed into a broad field, and one of all the interesting applications is to study cells without staining, labeling or in any other way affecting them. Both fixed and living, dying or dead cells can be studied. The first DHM images showing living cells were published in 2004 and 2005 (Carl et al. 2004, Marquet et al. 2005), making this field of research rather new. Digital holography makes it possible to easily measure cell properties that previously have been very difficult to study, such as cell thickness and volume (Marquet et al. 2005, Mölder et al. 2008). Two of the major advantages of DHM is the 3-D imaging possibility and measurements over time. Digital holography has ben used to study several types of cells, such as nerve cells, red blood cells, stem cells and cancer cells (Emery et al. 2007, Kemper et al. 2006, Langehanenberg et al. 2009) . It has also been applied for studies of cell proliferation, cell movement, sub-cellular structures and cell morphology (Kemper et al. 2009, Yu et al. 2009). Both 2-D and 3-D cell movement can be determined ( Langehanenberg et al. 2009). Even cell viability status can be determined using DHM. Interestingly, it is possible to study both single cells and entire populations simultaneously, allowing for very nuanced studies. Older, well known techniques often require some degree of cell disturbance such as the fluorescent antibody labeling required for fluorescense or confocal microscopy studies. In this paper we will present some of the studies made possible by DHM. We will compare DHM with previously used techniques and discuss the benefits and drawbacks of digital holography cell measurements.
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| 4. |
- Alm, Kersti
(författare)
-
Polyamines are involved in the regulation of S phase and DNA synthesis
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Our research group has previously shown that treatment with the polyamine biosynthesis inhibitors a-difluoromethylornithine (DFMO) or amidinoindan-1-one 2´-amidinohydrazone (CGP 48664) inhibited S phase progression before any other cell cycle phase was affected. This study was undertaken to further investigate the role of polyamines in the regulation of S phase progression and DNA synthesis. I have found that treatment with the polyamine analog N1,N11-diethylnorspermine (DENSPM) also caused a prolongation of the S phase. The common denominator for DFMO, CGP 48664, or DENSPM treatment is a depletion of the cellular spermidine pool. CGP 48664 and DENSPM in addition deplete the spermine pool. CGP 48664 or DENSPM treatment prolonged the S phase more than did DFMO treatment. Thus, mainly spermine but also spermidine may have a function in S phase progression. Using the single cell gel electrophoresis assay, I have shown that polyamine deficiency resulted in DNA strand breaks. I have also shown that the topoisomerase II that is present in polyamine deficient cells is not functional. The results imply that there might be a change in chromatin structure rendering topoisomerase II non-functional in polyamine deficient cells. The number and organization of replicon clusters was not affected by polyamine deficiency. However, replicon clusters were less fluorescent in polyamine deficient cells compared to control cells, pointing to a lower rate of DNA elongation in the former cells. Polyamine deficiency resulted in an aberrant regulation of cell cycle progression in Chinese hamster ovary cells (CHO). The results may be related to the fact that CHO cells have a mutated p53 gene. MCF-7 cells, which have a wild type p53 gene, reacted somewhat differently to polyamine deficiency than did CHO cells. As a general conclusion of my results I suggest that normal levels of spermidine and spermine are required for an optimal rate of S phase progression and that the first cell cycle phase affected by polyamine biosynthesis inhibition is the S phase. I do believe that many of the effects observed after more than 1-2 days of polyamine biosynthesis inhibitor treatment are secondary to the initial perturbances that have taken place.
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| 5. |
- Chen, Y, et al.
(författare)
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The role of mitogen-activated protein kinase activation in determining cellular outcomes in polyamine analogue-treated human melanoma cells
- 2003
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Ingår i: Cancer Research. - 1538-7445. ; 63:13, s. 3619-3625
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Tidskriftsartikel (refereegranskat)abstract
- The clinically relevant polyamine analogue N-1,N-11-diethylnorspermine (DENSPM) inhibits cell growth by down-regulating polyamine biosynthesis, up-regulating polyamine catabolism at the level of spermidine/spermine N-1-acetyltransferase (SSAT), and depleting intracellular polyamine pools. Among human melanoma cell lines, the analogue causes rapid apoptosis in SK-MEL-28 cells and a sharp G(1) arrest in MALME-3M cells. This study reveals that DENSPM potently activates the mitogen-activated protein kinase (MAPK) pathways in melanoma cells and investigates the role of this response in determining cellular outcomes. Onset of apoptosis was preceded by an intense phosphorylation of the MAPKs, including extracellular signal-regulated kinase 1/2, c-jun NH2-terminal kinase, and p38 in both SK-MEL-28 and MALME-3M cells. A panel of DENSPM analogues differing only in their ability to induce SSAT was used to show that MAPK activation was causally linked to induction of SSAT. activity and related oxidative events. The latter was confirmed with the polyamine oxidase inhibitor MDL-75275 and the antioxidant N-acetyl-L-cysteine, which when used in combination with DENSPM, decreased MAPK activation and as previously shown, reduced apoptosis. The MAP/extracellular signal-regulated kinase-1 inhibitor PD 98059 reduced activation of all three kinases but failed to alter apoptosis in DENSPM-treated SK-MEL-28 cells. By contrast, the inhibitor prevented p21(waf1/cip1) induction and enhanced apoptosis in MALME-3M cells as indicated by accelerated caspase-3 activation and positive annexin V staining. The generality of this effect was demonstrated in DENSPM-treated A375 and LOX human melanoma cells. Taken together, the importance of the MAPK pathways in determining the biological response to DENSPM treatment is dependent on the genetic environment of the cell.
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| 6. |
- El-Schich, Zahra, et al.
(författare)
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Digital holographic microscopy : innovative and non-destructive analysis of living cells
- 2010
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Ingår i: Microscopy: Science, Technology, Applications and Education. - : Formatex Research Center.
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Digital holography is a novel technique that has been developed recently to study living cells. The technique is an innovative, non-destructive method with possibilities to study living cells over time. We are investigating cell number, growth, viability and death of adherent cells using digital holography, which is a novel, label-free, imaging technique for biological applications. We have recently demonstrated that digital holography is highly comparable to the conventional manual cell counting method using a hemocytometer (Mölder et al., 2008). Digital holography is a method that gives us information about the refractive index of cells, which can change under different circumstances. The technique is cheap, fast and simple to use. The unique measurable parameters are the cell number, cell area, thickness and volume, which can be transformed to proliferation, migration, viability and cell death. The digital holographic images produced can provide both quantitative and qualitative phase information from a single hologram. Future applications can include real-time cell monitoring of various parameters of cells of different diseases in response to clinically relevant compounds.
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| 7. |
- El-Schich, Zahra, et al.
(författare)
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Discrimination between Breast Cancer Cells and White Blood Cells by Non-Invasive Measurements : Implications for a Novel In Vitro-Based Circulating Tumor Cell Model Using Digital Holographic Cytometry
- 2020
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Ingår i: Applied Sciences. - : MDPI. - 2076-3417. ; 10:14
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Tidskriftsartikel (refereegranskat)abstract
- Breast cancer is the second most common cancer worldwide. Metastasis is the main reason for death in breast cancer, and today, there is a lack of methods to detect and isolate circulating tumor cells (CTCs), mainly due to their heterogeneity and rarity. There are some systems that are designed to detect rare epithelial cancer cells in whole blood based on the most common marker used today, the epithelial cell adhesion molecule (EpCAM). It has been shown that aggressive breast cancer metastases are of non-epithelial origin and are therefore not always detected using EpCAM as a marker. In the present study, we used an in vitro-based circulating tumor cell model comprising a collection of six breast cancer cell lines and white blood cell lines. We used digital holographic cytometry (DHC) to characterize and distinguish between the different cell types by area, volume and thickness. Here, we present significant differences in cell size-related parameters observed when comparing white blood cells and breast cancer cells by using DHC. In conclusion, DHC can be a powerful diagnostic tool for the characterization of CTCs in the blood.
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| 8. |
- El-Schich, Zahra, et al.
(författare)
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Holography : The Usefulness of Digital Holographic Microscopy for Clinical Diagnostics
- 2017
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Ingår i: Holographic Materials and Optical Systems. - : INTECH. - 9789535130383 ; , s. 319-333
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Digital holographic (DH) microscopy is a digital high-resolution holographic imaging technique with the capacity of quantification of cellular conditions without any staining or labeling of cells. The unique measurable parameters are the cell number, cell area, thickness, and volume, which can be coupled to proliferation, migration, cell cycle analysis, viability, and cell death. The technique is cell friendly, fast and simple to use and has unique imaging capabilities for time-lapse investigations on both the single cell and the cell-population levels. The interest for analyzing specifically cell volume changes with DH microscopy, resulting from cytotoxic treatments, drug response, or apoptosis events has recently increased in popularity. We and others have used DH microscopy showing that the technique has the sensitivity to distinguish between different cells and treatments. Recently, DH microscopy has been used for cellular diagnosis in the clinic, providing support for using the concept of DH, e.g., screening of malaria infection of red blood cells (RBC), cervix cancer screening, and sperm quality. Because of its quick and label-free sample handling, DH microscopy will be an important tool in the future for personalized medicine investigations, determining the optimal therapeutic concentration for both different cancer types and individual treatments.
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| 9. |
- Fernandez, Céline, et al.
(författare)
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Omics Analyses Reveal a Potential Link between Hormone-Sensitive Lipase and Polyamine Metabolism.
- 2009
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 8, s. 5008-5019
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Tidskriftsartikel (refereegranskat)abstract
- Hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization from lipid stores, is expressed in the liver and decreased hepatic insulin sensitivity has been reported in our HSL null mouse model. Here, an integrated approach, comprising transcriptomics and proteomics together with targeted metabolite analysis, was used to investigate the liver phenotype of HSL null mice. Oligonucleotide microarray analysis revealed altered expression of genes involved in lipid and polyamine metabolism in HSL null mice compared with wild-type mice and in genes controlling the immune system in mice on high-fat diet versus mice on normal diet. Two-dimensional gel electrophoresis followed by MS and/or MS/MS allowed identification of 52 and 22 unique proteins differentially regulated according to the genotype and diet, respectively. Changes were observed mainly for proteins related to metabolism, including several proteins involved in polyamine metabolism or exhibiting methyl transferase activity. Despite the coordinated changes in mRNA and protein levels in polyamine pathways, no significant differences in levels of key polyamine metabolites were detected between the two genotypes. This study identifies a link between HSL and polyamine metabolism, which deserves further attention in view of the emerging data suggesting that disturbances in polyamine metabolism may affect insulin sensitivity. The present work also describes a limited correlation between mRNA, protein and metabolite levels, thus, underscoring the importance of integrated approaches.
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| 10. |
- Ghatnekar, Johannes, et al.
(författare)
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Construction of polyamine-modified uridine and adenosine derivatives-evaluation of DNA binding capacity and cytotoxicity in vitro
- 2007
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Ingår i: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896. ; 15:23, s. 7426-7433
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Tidskriftsartikel (refereegranskat)abstract
- We here report the synthesis of the two polyamine-based nucleoside derivatives 5-{[bis-(3-aminopropyl)amino]acetamido-1-propynyl}uridine and 2-{[bis-(3-aminopropyl)amino]-acetamido-1-propynyl}adenosine. The various polyamine derivatives have been used in thermal melting analysis using DNA from herring testes, and in cellular studies using four different cell lines. The compounds were all found to be non-toxic, thus holding good promise for future use as siRNA building blocks.
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