| 1. |
- Lemmens, Raf, et al.
(författare)
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Ca2+-induced Ca2+ release from the endoplasmic reticulum amplifies the Ca2+ signal mediated by activation of voltage-gated L-type Ca2+ channels in pancreatic beta-cells
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:13, s. 9971-9977
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Tidskriftsartikel (refereegranskat)abstract
- Stimulus-secretion coupling in pancreatic beta-cells involves membrane depolarization and Ca(2+) entry through voltage-gated L-type Ca(2+) channels, which is one determinant of increases in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). We investigated how the endoplasmic reticulum (ER)-associated Ca(2+) apparatus further modifies this Ca(2+) signal. When fura-2-loaded mouse beta-cells were depolarized by KCl in the presence of 3 mm glucose, [Ca(2+)](i) increased to a peak in two phases. The second phase of the [Ca(2+)](i) increase was abolished when ER Ca(2+) stores were depleted by thapsigargin. The steady-state [Ca(2+)](i) measured at 300 s of depolarization was higher in control cells compared with cells in which the ER Ca(2+) pools were depleted. The amount of Ca(2+) presented to the cytoplasm during depolarization as estimated from the integral of the increment in [Ca(2+)](i) over time (integralDelta[Ca(2+)](i).dt) was approximately 30% higher compared with that in the Ca(2+) pool-depleted cells. neo-thapsigargin, an inactive analog, did not affect [Ca(2+)](i) response. Using Sr(2+) in the extracellular medium and exploiting the differences in the fluorescence properties of Ca(2+)- and Sr(2+)-bound fluo-3, we found that the incoming Sr(2+) triggered Ca(2+) release from the ER. Depolarization-induced [Ca(2+)](i) response was not altered by, an inhibitor of phosphatidylinositol-specific phospholipase C, suggesting that stimulation of the enzyme by Ca(2+) is not essential for amplification of Ca(2+) signaling. [Ca(2+)](i) response was enhanced when cells were depolarized in the presence of 3 mm glucose, forskolin, and caffeine, suggesting involvement of ryanodine receptors in the amplification process. Pretreatment with ryanodine (100 microm) diminished the second phase of the depolarization-induced increase in [Ca(2+)](i). We conclude that Ca(2+) entry through L-type voltage-gated Ca(2+) channels triggers Ca(2+) release from the ER and that such a process amplifies depolarization-induced Ca(2+) signaling in beta-cells.
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| 2. |
- Lagae, Cis Raf, et al.
(författare)
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Raising the observed metallicity floor with a 3D non-LTE analysis of SDSS J102915.14+172927.9
- 2023
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Ingår i: Astronomy and Astrophysics. - : EDP Sciences. - 0004-6361 .- 1432-0746. ; 672, s. A90-A90
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Tidskriftsartikel (refereegranskat)abstract
- Context. The first stars marked the end of the cosmic dark ages, produced the first heavy elements, and set the stage for the formation of the first galaxies. Accurate chemical abundances of ultra metal-poor stars ([Fe/H] < −4) can be used to infer the properties of the first stars and thus the formation mechanism for low-mass second-generation stars in the early Universe. Spectroscopic studies have shown that most second-generation stars are carbon enhanced. A notable exception is SDSS J102915.14+172927.9, which is the most metal-poor star known to date, largely by virtue of the low upper limits of the carbon abundance reported in earlier studies.Aims. We re-analysed the composition of SDSS J102915.14+172927.9 with the aim of providing improved observational constraints on the lowest metallicity possible for low-mass star formation and constraining the properties of its Population III progenitor star.Methods. We developed a tailored three-dimensional model atmosphere for SDSS J102915.14+172927.9 with the Stagger code, making use of an improved surface gravity estimate based on the Gaia DR3 parallax. Snapshots from the model were used as input in the radiative transfer code Balder to compute 3D non-local thermodynamic equilibrium (non-LTE) synthetic spectra. These spectra were then used to infer abundances for Mg, Si, Ca, Fe, and Ni as well as upper limits on Li, Na, and Al. Synthetic 3D LTE spectra were computed with Scate to infer the abundance of Ti and upper limits on C and N.Results. In contrast to earlier works based on 1D non-LTE corrections applied to 3D LTE results, we are able to achieve ionisation balance for Ca I and Ca II when employing our consistent 3D non-LTE treatment. The elemental abundances are systematically higher than those found in earlier works. In particular, [Fe/H] is increased by 0.57 dex, and the upper limits of C and N are larger by 0.90 dex and 1.82 dex, respectively.Conclusions. We find that Population III progenitors with masses 10–20 M⊙ exploding with energy E ⪅ 3 × 1051 erg can reproduce our 3D non-LTE abundance pattern. Our 3D non-LTE abundances are able to better constrain the progenitor mass and explosion energy as compared to our 1D LTE abundances. Contrary to previous work, we obtain higher upper limits on the carbon abundance that are ‘marginally consistent’ with star formation through atomic line cooling, and consequently, these results prevent us from drawing strong conclusions about the formation mechanism of this low-mass star.
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| 3. |
- Podowski, Raf M
(författare)
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Applied bioinformatics for gene characterization
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- As the vast majority of human protein-encoding genes are now identified, the new challenge placed before life scientists is the determination of the functions of the proteins. Traditionally, intense, directed efforts are applied to decipher the function of a novel protein using laboratory techniques. Currently, increasing efforts are directed at the generation of high-throughput results for large numbers of genes using new technologies and the application of robotics to established methods. These efforts can generate large, complex, often noisy datasets, which are difficult to interpret. The extraction of information from genomics data that is relevant for specific scientific research efforts is required to accelerate functional characterization and annotation of genes by the scientific community. The research presented in this thesis highlights and addresses deficiencies in gene/protein function annotation. The bioinformatics tools and methodologies presented share the common theme of facilitating research scientists with means to understand and to interpret gene-specific data. The work, which addresses both diverse types of genomics data and a broad set of computational approaches, is united by the hypothesis that computational approaches to genomics data analysis can assist in the characterization of human protein-encoding genes. The initial sections of the thesis describe the identification of human protein-encoding genes for which there is little or no functional annotation. Chapter I presents the Gene Characterization Index, a bioinformatics method for quantifying the level of annotation of individual genes and monitoring progress. The Gene Characterization Index serves both as a tool for assessing the novelty of individual genes, and for the assessment of short-term annotation progress on a genome scale. In chapter 2, computational approaches are used to identify specific protein families that share evolutionarilyconserved domains for which the biochemical function is unknown. For the identified domains, a genecentric data centre, NovelFam3000, is created to facilitate shared annotation of protein function. Knowledge of a domain´s function, or the protein within which it arises, can facilitate the analysis of the entire set of proteins. The subsequent sections of the thesis focus on the creation of bioinformatics methods to assist human interpretation of gene function. Interpretation of large-scale biological data can be aided by visualization - humans can perform complex interpretation of data through visual assessment. In order to enable rapid identification of biological relationships within high-throughput genomics data, the Parallel HeatMap viewer was developed for four-dimensional data display and pattern observation. Researchers seeking to understand gene function often turn initially to the scientific literature. Hindered by a historic lack of standard gene and protein-naming conventions, they endure long, sometimes fruitless literature searches. The final chapter of the thesis focuses on the computational identification of abstracts which may be relevant to gene function - the essential and difficult challenge that must be overcome for computational assisted literature review. The SureGene system is based on supervised machine learning, resulting in a distinct model for the identification of relevant abstracts for each gene. The system was able to achieve high quality gene disambiguation using scalable automated techniques. This thesis explores the hypothesis that computational methods can facilitate the identification and characterization of poorly annotated genes. The bioinformatics approaches to this problem assist researchers in advancing our understanding of the functional of human protein encoding genes.
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| 4. |
- Arbajian, Elsa, et al.
(författare)
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In-depth genetic analysis of sclerosing epithelioid fibrosarcoma reveals recurrent genomic alterations and potential treatment targets
- 2017
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Ingår i: Clinical Cancer Research. - 1078-0432. ; 23:23, s. 7426-7434
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: Sclerosing epithelioid fibrosarcoma (SEF) is a highly aggressive soft tissue sarcoma closely related to low-grade fibromyxoid sarcoma (LGFMS). Some tumors display morphological characteristics of both SEF and LGFMS, so called hybrid SEF/LGFMS. Despite the overlap of gene fusion variants between these two tumor types, SEF is much more aggressive. The present study aimed to further characterize SEF and hybrid SEF/LGFMS genetically in order to better understand the role of the characteristic fusion genes and possible additional genetic alterations in tumorigenesis.EXPERIMENTAL DESIGN: We performed whole exome sequencing, single nucleotide polymorphism (SNP) array analysis, RNA-sequencing (RNA-seq), global gene expression analyses and/or IHC on a series of 13 SEFs and 6 hybrid SEF/LGFMS. We also expressed the FUS-CREB3L2 and EWSR1-CREB3L1 fusion genes conditionally in a fibroblast cell line; these cells were subsequently analyzed by RNA-seq and expression of the CD24 protein was assessed by FACS analysis.RESULTS: The SNP array analysis detected a large number of structural aberrations in SEF and SEF/LGFMS, many of which were recurrent, notably DMD microdeletions. RNA-seq identified FUS-CREM and PAX5-CREB3L1 as alternative fusion genes in one SEF each. CD24 was strongly upregulated, presumably a direct target of the fusion proteins. This was further confirmed by the gene expression analysis and FACS analysis on Tet-On 3G cells expressing EWSR1-CREB3L1.CONCLUSIONS: While gene fusions are the primary tumorigenic events in both SEF and LGFMS, additional genomic changes explain the differences in aggressiveness and clinical outcome between the two types. CD24 and DMD constitute potential therapeutic targets.
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| 5. |
- Bennemo, Mia, et al.
(författare)
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A chromatographic method for determination of supercoiled plasmid DNA concentration in complex solutions.
- 2009
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Ingår i: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 877:24, s. 2530-6
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Tidskriftsartikel (refereegranskat)abstract
- A method for determination of the plasmid DNA concentration with subsequent analysis of the ratio supercoiled to open circular form is presented. The method is suitable for samples from all steps of the manufacturing process, from fermentation to final product. The analysis consists of size exclusion chromatography, followed by analytical thiophilic aromatic chromatography. In the first step, the plasmid DNA concentration is determined by group separation, including removal of RNA and other impurities, within less than 2 min. The limit of detection and quantification was 0.28 and 0.83 microg/ml, respectively. The precision of the method is high, providing a coefficient of variation as low as below 2%. In the second step, the ratio of open circular to supercoiled plasmid DNA is determined following separation of the two plasmid DNA isoforms with a linear salt gradient. The precision of the second step was evaluated using serial injections of aliquots of a sample stock solution. In comparison with the two most commonly used methods, the developed analysis was found to be significantly more accurate than agarose gel electrophoresis and equivalent to capillary gel electrophoresis. The combined methods for quantification and control of homogeneity of plasmid DNA presented here enable reliable and precise analysis at all steps of the manufacturing process.
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| 6. |
- Blom, Hans, et al.
(författare)
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Flocculate removal after alkaline lysis in plasmid DNA production.
- 2010
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Ingår i: Vaccine. - : Elsevier BV. - 0264-410X .- 1873-2518. ; 29:1, s. 6-10
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Tidskriftsartikel (refereegranskat)abstract
- Alkaline lysis is the most commonly used method following harvest of bacterial cells for production of plasmid DNA. The method was originally developed for laboratory scale experiments and has shown to be challenging at larger scales. A major problem prior to further downstream processing is the risk of filter clogging without efficient removal of the flocculate that occurs after neutralization. For this purpose we here present a scalable method where the clarification of alkaline lysate is greatly simplified. Through a rapid procedure, involving the addition of ammonium hydrogen carbonate to the neutralized alkaline lysate, the flocculate is lifted to the surface of the solution by the released carbon dioxide and ammonium. After this step a clarified solution can be drained from the bottom of the vessel. The procedure does not impact pH, plasmid DNA concentration or the ratio of open circular to supercoiled plasmid DNA in the solution.
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| 7. |
- Bruton, Joseph D., et al.
(författare)
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Ryanodine receptors of pancreatic beta-cells mediate a distinct context-dependent signal for insulin secretion
- 2003
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 17:2, s. 301-303
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Tidskriftsartikel (refereegranskat)abstract
- The ryanodine (RY) receptors in beta-cells amplify signals by Ca2+-induced Ca2+ release (CICR). The role of CICR in insulin secretion remains unclear in spite of the fact that caffeine is known to stimulate secretion. This effect of caffeine is attributed solely to the inhibition of cAMP-phosphodiesterases (cAMP-PDEs). We demonstrate that stimulation of insulin secretion by caffeine is due to a sensitization of the RY receptors. The dose-response relationship of caffeine-induced inhibition of cAMP-PDEs was not correlated with the stimulation of insulin secretion. Sensitization of the RY receptors stimulated insulin secretion in a context-dependent manner, that is, only in the presence of a high concentration of glucose. This effect of caffeine depended on an increase in [Ca2+]i. Confocal images of beta-cells demonstrated an increase in [Ca2+]i induced by caffeine but not by forskolin. 9-Methyl-7-bromoeudistomin D (MBED), which sensitizes RY receptors, did not inhibit cAMP-PDEs, but it stimulated secretion in a glucose-dependent manner. The stimulation of secretion by caffeine and MBED involved both the first and the second phases of secretion. We conclude that the RY receptors of beta-cells mediate a distinct glucose-dependent signal for insulin secretion and may be a target for developing drugs that will stimulate insulin secretion only in a glucose-dependent manner.
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| 8. |
- Currie, Thomas E., et al.
(författare)
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Integrating evolutionary theory and social–ecological systems research to address the sustainability challenges of the Anthropocene
- 2024
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Ingår i: Philosophical Transactions of the Royal Society of London. Biological Sciences. - 0962-8436 .- 1471-2970. ; 379:1893
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Forskningsöversikt (refereegranskat)abstract
- The rapid, human-induced changes in the Earth system during the Anthropocene present humanity with critical sustainability challenges. Social–ecological systems (SES) research provides multiple approaches for understanding the complex interactions between humans, social systems, and environments and how we might direct them towards healthier and more resilient futures. However, general theories of SES change have yet to be fully developed. Formal evolutionary theory has been applied as a dynamic theory of change of complex phenomena in biology and the social sciences, but rarely in SES research. In this paper, we explore the connections between both fields, hoping to foster collaboration. After sketching out the distinct intellectual traditions of SES research and evolutionary theory, we map some of their terminological and theoretical connections. We then provide examples of how evolutionary theory might be incorporated into SES research through the use of systems mapping to identify evolutionary processes in SES, the application of concepts from evolutionary developmental biology to understand the connections between systems changes and evolutionary changes, and how evolutionary thinking may help design interventions for beneficial change. Integrating evolutionary theory and SES research can lead to a better understanding of SES changes and positive interventions for a more sustainable Anthropocene.
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| 9. |
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| 10. |
- Dahlén, Anna, et al.
(författare)
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Clustering of deletions on chromosome 13 in benign and low-malignant lipomatous tumors
- 2003
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136. ; 103:5, s. 616-623
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Tidskriftsartikel (refereegranskat)abstract
- Deletions and structural rearrangements of the long arm of chromosome 13 are frequently observed in benign and low-malignant lipomatous tumors, but nothing is known about their molecular genetic consequences. We assessed the karyotypes of 40 new and 22 previously published cases (35 ordinary lipomas, 15 spindle cell/pleomorphic lipomas, 2 myxolipomas, 1 angiomyxolipoma and 9 atypical lipomatous tumors) with chromosome 13-abnormalities, and found bands 13q12-22 to be frequently affected. Twenty-seven cases with structural abnormalities within this region were selected for breakpoint and deletion mapping by metaphase fluorescence in situ hybridization (FISH), using a set of 20 probes. Deletions were found in 23 of 27 cases. The remaining 4 cases had seemingly balanced rearrangements. The breakpoints were scattered but clustered to band 13q14, and in all cases with unbalanced abnormalities, a limited region within band 13q14 was partially or completely deleted. A deletion within band 13q14 was found together with a breakpoint on the other homologue in 5 cases, 4 of which could be tested further with regard to the status of the retinoblastoma (RB1)-gene. In all 4 cases, only 1 copy of the gene was deleted. In addition to the breaks and deletions in the vicinity of the RB1-locus, several other regions of 13q were recurrently affected, e.g., in the vicinity of the hereditary breast cancer (BRCA2; 13q12)- and lipoma HMGIC fusion partner (LHFP; 13q13)- genes. Our findings strongly indicate that deletion of a limited region (approximately 2.5 Mbp) within 13q14, distal to the RB1-locus, is of importance in the development of a subset of lipomatous tumors.
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