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  • 51. Baker-Austin, Craig
    et al.
    Potrykus, Joanna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wexler, Margaret
    Bond, Philip L
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). School of Biological Sciences, University of East Anglia, Norwich, UK .
    Biofilm development in the extremely acidophilic archaeon 'Ferroplasma acidarmanus' Fer12010In: Extremophiles, ISSN 1431-0651, E-ISSN 1433-4909, Vol. 14, no 6, p. 485-491Article in journal (Refereed)
    Abstract [en]

    'Ferroplasma acidarmanus' Fer1 is an ironoxidizing extreme acidophile isolated from the Iron Mountain mine, California, USA This archaeon is predominantly found in biofilm-associated structures in the environment, and produces two distinct biofilm morphologies Bioinformatic analysis of the acidarmanus' Fer1 genome Identified genes annotated as involved in attachment and biofilm formation No putative quorum sensing signaling genes were identified and no N-acyl homoserine lactone-like compounds were found in acidarmanus' Fer1 biofilm supernatant Scanning confocal microscopy analysis of biofilm development on the surface of pyrite demonstrated the temporal and spatial development of biofilm growth Furthermore, two-dimensional polyacrylamide gel electrophoresis was used to examine differential protein expression patterns between biofilm and planktonic populations Ten up-regulated proteins were identified that included six enzymes associated with anaerobic growth, suggesting that the dominating phenotype in the mature biofilm was associated with anaerobic modes of growth This report increases our knowledge of the genetic and proteomic basis of biofilm formation in an extreme acidophilic archaeon.

  • 52.
    Balagopal, Vidya
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fluch, Lydia
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nissan, Tracy
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ways and means of eukaryotic mRNA decay2012In: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, ISSN 1874-9399, Vol. 1819, no 6, p. 593-603Article, review/survey (Refereed)
    Abstract [en]

    Messenger RNA degradation is an important point of control for gene expression. Genome-wide studies on mRNA stability have demonstrated its importance in adaptation and stress response. Most of the key players in mRNA decay appear to have been identified. The study of these proteins brings insight into the mechanism of general and specific targeting of transcripts for degradation. Recruitment and assembly of mRNP complexes enhance and bring specificity to mRNA decay. mRNP complexes can form larger structures that have been found to be ubiquitous in nature. Discovery of P-Bodies, an archetype of this sort of aggregates, has generated interest in the question of where mRNA degrades. This is currently an open question under extensive investigation. This review will discuss in detail the recent developments in the regulation of mRNA decay focusing on yeast as a model system. 

  • 53. Baldassarre, Maurizio
    et al.
    Baronio, Cesare M.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Barth, Andreas
    Amyloid beta-peptides 1-40 and 1-42 form oligomers with mixed beta-sheets2017In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 8, no 12, p. 8247-8254Article in journal (Refereed)
    Abstract [en]

    Two main amyloid-beta peptides of different length (A beta(40) and A beta(42)) are involved in Alzheimer's disease. Their relative abundance is decisive for the severity of the disease and mixed oligomers may contribute to the toxic species. However, little is know about the extent of mixing. To study whether A beta(40) and A beta(42) co-aggregate, we used Fourier transform infrared spectroscopy in combination with C-13-labeling and spectrum calculation and focused on the amide I vibration, which is sensitive to backbone structure. Mixtures of monomeric labeled A beta(40) and unlabeled A beta(42) (and vice versa) were co-incubated for similar to 20 min and their infrared spectrum recorded. The position of the main C-13-amide I' band shifted to higher wavenumbers with increasing admixture of C-12-peptide due to the presence of C-12-amides in the vicinity of C-13-amides. The results indicate that A beta(40) and A beta(42) form mixed oligomers with a largely random distribution of A beta(40) and A beta(42) strands in their beta-sheets. The structures of the mixed oligomers are intermediate between those of the pure oligomers. There is no indication that one of the peptides forces the backbone structure of its oligomers on the other peptide when they are mixed as monomers. We also demonstrate that isotope-edited infrared spectroscopy can distinguish aggregation modulators that integrate into the backbone structure of their interaction partner from those that do not. As an example for the latter case, the pro-inflammatory calcium binding protein S100A9 is shown not to incorporate into the b-sheets of A beta(42).

  • 54.
    Bamyaci, Sarp
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Multiple functions of YopN in the Yersinia pseudotuberculosis type III secretion system: from regulation to in vivo infection2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The type 3 secretion systems (T3SSs) are virulence mechanisms used by various Gram-negative bacteria to overcome the host immunity. They are often target-cell contact induced and activated. Activation results in targeting of virulence effector substrates into host cells. One class of secreted substrates, translocators, are required for the intracellular targeting of the second class, the virulence effectors, into host target cells. T3SSs are mainly regulated at 2 levels; a shift from environmental to host temperature results in low level induction of the system whereas target cell contact further induces and activates the system. In the Yersinia T3SS, YopN, one of the secreted substrates, is involved in the latter level of activation. Under non-inducing conditions, YopN complexes with TyeA, SycN and YscB and this complex suppresses the T3SS via an unknown mechanism. When the system is induced, the complex is believed to dissociate and YopN is secreted resulting in the activation of the system. Earlier studies indicated that YopN is not only secreted but also translocated into target cells in a T3SS dependent manner. TyeA, SycN and YscB bind to the C-terminal and N-terminal YopN respectively but so far the central region (CR) of YopN has not been characterized. In this study we have focused on the function of the YopN central region.

    We therefore generated in-frame deletion mutants within the CR of YopN. One of these deletion mutants, aa 76-181, showed decreased early translocation of both YopE and YopH into infected host cells and also failed to efficiently block phagocytosis by macrophages. However, the YopNΔ76-181 protein was expressed at lower levels compared to wt YopN and also showed a slightly deregulated phenotype when expressed from its native promoter and were as a consequence not possible to use in in vivo infection studies.

    Therefore, we generated mutants that disrupted a putative coiled coil domain located at the very N-terminal of CR. Similar to YopNΔ76-181, these substitution mutants were affected in the early translocation of effector proteins. Importantly, they were as stable as wt YopN when expressed from the native promoter. One of these mutants was unable to cause systemic infection in mice indicating that YopN indeed also has a direct role in virulence and is required for establishment of systemic infection in vivo.

  • 55.
    Barros-Rios, Jaime
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Malvar, Rosa A.
    Jung, Hans-Joachim G.
    Bunzel, Mirko
    Santiago, Rogelio
    Divergent selection for ester-linked diferulates in maize pith stalk tissues. Effects on cell wall composition and degradability2012In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 83, p. 43-50Article in journal (Refereed)
    Abstract [en]

    Cross-linking of grass cell wall components through diferulates (DFAs) has a marked impact on cell wall properties. However, results of genetic selection for DFA concentration have not been reported for any grass species. We report here the results of direct selection for ester-linked DFA concentration in maize stalk pith tissues and the associated changes in cell wall composition and biodegradability. After two cycles of divergent selection, maize populations selected for higher total DFA (DFAT) content (CHs) had 16% higher DFAT concentrations than populations selected for lower DFAT content (as). These significant DFA concentration gains suggest that DFA deposition in maize pith parenchyma cell walls is a highly heritable trait that is genetically regulated and can be modified trough conventional breeding. Maize populations selected for higher DFAT had 13% less glucose and 10% lower total cell wall concentration than CLs, suggesting that increased cross-linking of feruloylated arabinoxylans results in repacking of the matrix and possibly in thinner and firmer cell walls. Divergent selection affected esterified DFAT and monomeric ferulate ether cross link concentrations differently, supporting the hypothesis that the biosynthesis of these cell wall components are separately regulated. As expected, a more higher DFA ester cross-coupled arabinoxylan network had an effect on rumen cell wall degradability (CLs showed 12% higher 24-h total polysaccharide degradability than CHs). Interestingly, 8-8-coupled DFAs, previously associated with cell wall strength, were the best predictors of pith cell wall degradability (negative impact). Thus, further research on the involvement of these specific DFA regioisomers in limiting cell wall biodegradability is encouraged. (C) 2012 Elsevier Ltd. All rights reserved.

  • 56.
    Bashar Shafiul, Shamrat
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Characterization of cell wall in transgenic aspen with modified xylan acetylation2015Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    ABSTRACT

    Mature plant cells are enclosed by inflexible wall made up of cellulose microfibrils, pectins, hemicelluloses and in some cases lignin. This cell wall provides the structure and the defense for plant cells. In secondary cell walls of dicotyledons, major hemicellulose is xylan consisting of β-(1, 4)-linked xylose units. Xylan is synthesized in Golgi apparatus by several enzymes activities. REDUCED WALL ACETYLATION (RWA) genes are involved in xylan acetylation. These genes were downregulated in hybrid aspen in order to reduce xylan acetylation activity during its biosynthesis. In addition, acetyl xylan esterase (FC2) from the fungus Aspergillus niger was expressed in hybrid aspen to modify xylan acetylation post-synthetically. In this work, I have studied effects of these modifications on wood cell wall composition.

    The cell wall components were sequentially extracted by using the small scale method and the weight of extractives, lignin, hemicelluloses and celluloses per weight of dry wood were determined. In addition, the Updegraff cellulose, Klason lignin contents per weight of extractive free wood were determined and monosugar compositions of non-cellulosic components were analyzed by methanolysis and Trimethylsilyl derivatisation (TMS). 

    I have found that content of cellulose determined by sequential extraction method was significantly increased in all constructs as compared to the wild type. Reduction of lignin (as determined by sequential extraction) was found in DFC2 construct and RWA RNAi 35S-AB and CD constructs. Furthermore, RWA RNAi 35S-CD and RWA RNAi WP-ABCD constructs showed decreased hemicellulose as compared to the wild type. Moreover, DFC2 constructs exhibited decrease in non-cellulosic sugars hydrolyzed during TMS. FC2 expressing lines showed a reduction in xylose which is the main building block of xylan. In contrast, glucose and galactose contents were increased. Inhibition of expression of all RWA genes (WP-ABCD) caused similar changes.

    Considering all the data I conclude that, reduced acetylation of xylan can affect extractability, biosynthesis or modification of polysaccharides and lignin in cell wall.

    Keywords: Cellulose microfibrils; pectins; hemicellulose; lignin, xylan; secondary wall; aspen; xylan biosynthesis; biosynthesis of polysaccharides. 

  • 57.
    Bañares de Dios, Guillermo
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Elucidating the role of CSK during early light response and chloroplast development2014Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Elucidating the role of CSK during early light response and chloroplast development

    Chloroplasts of higher plants have evolved from endosymbiotic, ancestor of modern prokaryotic cyanobacteria. During evolution most of the genes from the genome of the endosymbiont have moved to the nucleus of it host. As a consequence the components of the photosynthetic machinery are encoded both in the chloroplastic and in the nuclear genomes. Therefore, expression of both genomes must be tightly coordinated to ensure a simultaneous and stoichiometric biosynthesis of the chloroplast components at different developmental stages and under environmental or metabolic changes. This is achieved by a mechanism referred to as retrograde signalling. During retrograde signalling, signals are emitted from the chloroplast consisting on intracellular pathways emitted by the chloroplast communicating the status of the chloroplast and regulating the expression of nuclear genes encoding plastid components. The aim of this project was to elucidate the role of CSK (Chloroplast Sensor Kinase) in relation to previously described retrograde signalling components PRIN2 (Plastid Redox INsensitive 2) and GUN1 (Genomes UNcoupled 1). CSK is a plastid kinase involved in the long- term, acclimation response to balance the ratio between PSII and PSI by regulating the expression of psaA. The activity of CSK is regulated by the redox state of the plastoquinone pool. My work revealed that CSK is up- regulated upon light exposure. In addition, similarly to the prin2 and gun1 mutants, the csk mutant exhibited lower chlorophyll content, a striking yellow cotyledon tip phenotype, impaired chloroplast development and a down- regulation of PEP dependent genes psaA and psbA during a de- etiolation development and for the establishment of PEP activity in light. Furthermore, the similarity in the mutant phenotypes suggests that CSK is involved in the same signalling pathway as PRIN2 and GUN1.

  • 58. Beck, Carole
    et al.
    Rodriguez-Vargas, José Manuel
    Boehler, Christian
    Robert, Isabelle
    Heyer, Vincent
    Hanini, Najat
    Gauthier, Laurent R.
    Tissier, Agnès
    Schreiber, Valérie
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    San Martin, Bernardo Reina
    Dantzer, Françoise
    PARP3, a new therapeutic target to alter Rictor/mTORC2 signaling and tumor progression in BRCA1-associated cancers2019In: Cell Death and Differentiation, ISSN 1350-9047, E-ISSN 1476-5403, Vol. 26, no 9, p. 1615-1630Article in journal (Refereed)
    Abstract [en]

    PARP3 has been shown to be a key driver of TGF beta-induced epithelial-to-mesenchymal transition (EMT) and sternness in breast cancer cells, emerging as an attractive therapeutic target. Nevertheless, the therapeutic value of PARP3 inhibition has not yet been assessed. Here we investigated the impact of the absence of PARP3 or its inhibition on the tumorigenicity of BRCA1-proficient versus BRCA1-deficient breast cancer cell lines, focusing on the triple-negative breast cancer subtype (TNBC). We show that PARP3 knockdown exacerbates centrosome amplification and genome instability and reduces survival of BRCA1-deficient TNBC cells. Furthermore, we engineered PARP3(-/- )BRCA1-deficient or BRCA1-proficient TNBC cell lines using the CRISPR/nCas9(D10A) gene editing technology and demonstrate that the absence of PARP3 selectively suppresses the growth, survival and in vivo tumorigenicity of BRCA1-deficient TNBC cells, mechanistically via effects associated with an altered Rictor/mTORC2 signaling complex resulting from enhanced ubiquitination of Rictor. Accordingly, PARP3 interacts with and ADP-ribosylates GSK3 beta, a positive regulator of Rictor ubiquitination and degradation. Importantly, these phenotypes were rescued by re-expression of a wild-type PARP3 but not by a catalytic mutant, demonstrating the importance of PARP3's catalytic activity. Accordingly, reduced survival and compromised Rictor/mTORC2 signaling were also observed using a cell-permeable PARP3-specific inhibitor. We conclude that PARP3 and BRCA1 are synthetic lethal and that targeting PARP3's catalytic activity is a promising therapeutic strategy for BRCA1-associated cancers via the Rictor/mTORC2 signaling pathway.

  • 59.
    Behren, Sandra
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Westerlind, Ulrika
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Glycopeptides and -Mimetics to Detect, Monitor and Inhibit Bacterial and Viral Infections: Recent Advances and Perspectives2019In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 24, no 6, article id 1004Article, review/survey (Refereed)
    Abstract [en]

    The initial contact of pathogens with host cells is usually mediated by their adhesion to glycan structures present on the cell surface in order to enable infection. Furthermore, glycans play important roles in the modulation of the host immune responses to infection. Understanding the carbohydrate-pathogen interactions are of importance for the development of novel and efficient strategies to either prevent, or interfere with pathogenic infection. Synthetic glycopeptides and mimetics thereof are capable of imitating the multivalent display of carbohydrates at the cell surface, which have become an important objective of research over the last decade. Glycopeptide based constructs may function as vaccines or anti-adhesive agents that interfere with the ability of pathogens to adhere to the host cell glycans and thus possess the potential to improve or replace treatments that suffer from resistance. Additionally, synthetic glycopeptides are used as tools for epitope mapping of antibodies directed against structures present on various pathogens and have become important to improve serodiagnostic methods and to develop novel epitope-based vaccines. This review will provide an overview of the most recent advances in the synthesis and application of glycopeptides and glycopeptide mimetics exhibiting a peptide-like backbone in glycobiology.

  • 60.
    Beier, Frank
    et al.
    Institute for Experimental Medicine, University of Erlangen-Nürnberg, Erlangen, Germany.
    Eerola, Iiro
    Department of Medical Biochemistry and Molecular Biology, University of Turku, Turku, Finland.
    Vuorio, Eero
    Department of Medical Biochemistry and Molecular Biology, University of Turku, Turku, Finland.
    Luvalle, Phyllis
    Department of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada.
    Reichenberger, Ernest
    Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
    Bertling, Wolf
    Institute for Genetics, University of Bayreuth, Bayreuth, Germany.
    von der Mark, Klaus
    Institute for Experimental Medicine, University of Erlangen-Nürnberg, Erlangen, Germany.
    Lammi, Mikko
    Variability in the upstream promoter and intron sequences of the human, mouse and chick type X collagen genes.1996In: Matrix Biology, ISSN 0945-053X, E-ISSN 1569-1802, Vol. 15, no 6, p. 415-422, article id 9049979Article in journal (Refereed)
    Abstract [en]

    The type X collagen gene is specifically expressed in hypertrophic chondrocytes during endochondral ossification. Transcription of the type X collagen gene by these differentiated cells is turned on at the same time as transcription of several other cartilage specific genes is switched off and before mineralization of the matrix begins. Analysis of type X collagen promoters for regulatory regions in different cell culture systems and in transgenic mice has given contradictory results suggesting major differences among species. To approach this problem, we have determined the nucleotide sequences of the two introns and upstream promoter sequences of the human and mouse type X collagen genes and compared them with those of bovine and chick. Within the promoter regions, we found three boxes of homology which are nearly continuous in the human gene but have interruptions in the murine gene. One of these interruptions was identified as a complex 1.9 kb repetitive element with homology to LINE, B1, B2 and long terminal repeat sequences. Regulatory elements of the human type X collagen gene are located upstream of the region where the repetitive element is inserted in the mouse gene, making it likely that the repetitive element is inserted between the coding region and regulatory sequences of the murine gene without interfering with its expression pattern. We also compared the sequences of the introns of both genes and found strong conservation. Comparisons of the mammalian sequences with promoter and first intron sequences of the chicken type X collagen gene revealed that only the proximal 120 nucleotides of the promoter were conserved, whereas all other sequences displayed no obvious homology to the murine and human sequences.

  • 61.
    Beier, Frank
    et al.
    Institute Experimental Medicine, University of Erlangen-Nürnberg, Erlangen, Germany.
    Lammi, Mikko
    Institute Experimental Medicine, University of Erlangen-Nürnberg, Erlangen, Germany.
    Bertling, Wolf
    Institute for Genetics, University of Bayreuth, Bayreuth, Germany.
    von der Mark, Klaus
    Institute Experimental Medicine, University of Erlangen-Nürnberg, Erlangen, Germany.
    Transcriptional regulation of the human type X collagen gene expression.1996In: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 785, p. 209-211, article id 8702131Article in journal (Refereed)
  • 62.
    Beier, Frank
    et al.
    Institut für Experimentelle Medizin, Universität Erlangen-Nürnberg, Erlangen, Germany; Department of Medical Biochemistry, University of Calgary, Calgary, Canada.
    Vornehm, Silvia
    Institut für Experimentelle Medizin, Universität Erlangen-Nürnberg, Erlangen, Germany.
    Pöschl, Ernst
    Institut für Experimentelle Medizin, Universität Erlangen-Nürnberg, Erlangen, Germany.
    von der Mark, Klaus
    Institut für Experimentelle Medizin, Universität Erlangen-Nürnberg, Erlangen, Germany.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Localization of silencer and enhancer elements in the human type X collagen gene.1997In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 66, no 2, p. 210-218, article id 9213222Article in journal (Refereed)
    Abstract [en]

    Collagen type X is a short, network-forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5'-flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non-tissue-specific positive regulatory element was present in the first intron. Third, a cell-type-specific enhancer element active only in hypertrophic chondrocytes was located between -2.4 and -0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291-296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements.

  • 63. Beljantseva, Jelena
    et al.
    Kudrin, Pavel
    Andresen, Liis
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Shingler, Vicky
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Atkinson, Gemma C.
    Tenson, Tanel
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Institute of Technology, University of Tartu, 50411 Tartu, Estonia.
    Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 14, p. 3726-3731Article in journal (Refereed)
    Abstract [en]

    The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ: RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5 alpha. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes.

  • 64. Bengtsson, Anders A.
    et al.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wuttge, Dirk M.
    Sturfelt, Gunnar
    Theander, Elke
    Donten, Magdalena
    Moritz, Thomas
    Sennbro, Carl-Johan
    Torell, Frida
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lood, Christian
    Surowiec, Izabella
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rännar, Stefan
    Lundstedt, Torbjörn
    Metabolic Profiling of Systemic Lupus Erythematosus and Comparison with Primary Sjögren’s Syndrome and Systemic Sclerosis2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 7, article id e0159384Article in journal (Refereed)
    Abstract [en]

    Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease which can affect most organ systems including skin, joints and the kidney. Clinically, SLE is a heterogeneous disease and shares features of several other rheumatic diseases, in particular primary Sjögrens syndrome (pSS) and systemic sclerosis (SSc), why it is difficult to diag- nose The pathogenesis of SLE is not completely understood, partly due to the heterogeneity of the disease. This study demonstrates that metabolomics can be used as a tool for improved diagnosis of SLE compared to other similar autoimmune diseases. We observed differences in metabolic profiles with a classification specificity above 67% in the comparison of SLE with pSS, SSc and a matched group of healthy individuals. Selected metabolites were also significantly different between studied diseases. Biochemical pathway analysis was conducted to gain understanding of underlying pathways involved in the SLE pathogenesis. We found an increased oxidative activity in SLE, supported by increased xanthine oxidase activity and an increased turnover in the urea cycle. The most discriminatory metabolite observed was tryptophan, with decreased levels in SLE patients compared to control groups. Changes of tryptophan levels were related to changes in the activity of the aromatic amino acid decarboxylase (AADC) and/or to activation of the kynurenine pathway. 

  • 65.
    Benlloch, Reyes
    et al.
    Department of Forest Genetics and Plant Physiology, SLU.
    Shevela, Dmitriy
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hainzl, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Shutova, Tatyana
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Messinger, Johannes
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Samuelsson, Göran
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Crystal structure and functional characterization of Photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii2015In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 167, no 3, p. 950-962Article in journal (Refereed)
    Abstract [en]

    In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates.The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place inphotosystem II (PSII). Recent studies show that in higher plants, HCO3– increases PSII activity by acting as a mobile acceptor ofthe protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested toimprove proton removal from PSII, possibly by rapid reformation of HCO3– from CO2. In this study, we investigated the interplaybetween PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometrymeasurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen underillumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature notpreviously observed in a-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 functionwith dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3– on PSIIactivity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSIIpreparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at lowpH and CO2 concentration.

  • 66.
    Berglund, Anders
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brorsson, Ann-Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jonsson, Bengt-Harald
    Sethson, Ingmar
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The equilibrium unfolding of MerP characterized by multivariate analysis of 2D NMR data2005In: Journal of magnetic resonance, ISSN 1090-7807, E-ISSN 1096-0856, Vol. 172, no 1, p. 24-30Article in journal (Refereed)
    Abstract [en]

    A general problem when analysing NMR spectra that reflect variations in the environment of target molecules is that different resonances are affected to various extents. Often a few resonances that display the largest frequency changes are selected as probes to reflect the examined variation, especially in the case, where the NMR spectra contain numerous resonances. Such a selection is dependent on more or less intuitive judgements and relying on the observed spectral variation being primarily caused by changes in the NMR sample. Second, recording changes observed for a few (albeit significant) resonances is inevitably accompanied by not using all available information in the analysis. Likewise, the commonly used chemical shift mapping (CSM) [Biochemistry 39 (2000) 26, Biochemistry 39 (2000) 12595] constitutes a loss of information since the total variation in the data is not retained in the projection into this single variable. Here, we describe a method for subjecting 2D NMR time-domain data to multivariate analysis and illustrate it with an analysis of multiple NNIR experiments recorded at various folding conditions for the protein MerP. The calculated principal components provide an unbiased model of variations in the NNIR spectra and they can consequently be processed as NMR data, and all the changes as reflected in the principal components are thereby made available for visual inspection in one single NMR spectrum. This approach is much less laborious than consideration of large numbers of individual spectra, and it greatly increases the interpretative power of the analysis.

  • 67.
    Bernardo, Lisandro
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    On the role of ppGpp and DksA mediated control of σ54-dependent transcription2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to promoter affinity for σ54-holoenzyme, ii) that σ54 is under significant competition with other σ-factors in the cell, and iii) that mutants of σ70, and the beta- and beta prime-subunits of RNA polymerase that can bypass the need for ppGpp and DksA in vivo have defects that would favour the formation of σ54-RNA holoenzyme over that with σ70, and that mimic the effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. A purely passive model for ppGpp/DksA regulation of σ54-dependent transcription that functions through their potent negative effects on transcription from powerful σ70-stringent promoters is presented.

  • 68. Bernardo, Lisandro
    et al.
    Johansson, Linda U M
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    σ54-promoter discrimination and regulation by ppGpp and DksA2009In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 2, p. 828-838Article in journal (Refereed)
    Abstract [en]

    The sigma(54)-factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of sigma(54)-dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of sigma(54)-dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of sigma(54)-dependent transcription in combination with a series of sigma(54)-promoters with graded affinities for sigma(54)-RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted sigma(54)-transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous sigma(54)-RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive sigma(54)-transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity sigma(54)-promoters that is conferred by the strong DNA binding properties of the P. putida sigma(54)-RNA polymerase holoenzyme.

  • 69. Birkholtz, Lyn-Marie
    et al.
    Williams, Marni
    Niemand, Jandeli
    Louw, Abraham I.
    Persson, Lo
    Heby, Olle
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Polyamine homoeostasis as a drug target in pathogenic protozoa: peculiarities and possibilities2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 438, p. 229-244Article, review/survey (Refereed)
    Abstract [en]

    New drugs are urgently needed for the treatment of tropical and subtropical parasitic diseases, such as African sleeping sickness. Chagas' disease, leishmaniasis and malaria. Enzymes in polyamine biosynthesis and thiol metabolism, as well as polyamine transporters, are potential drug targets within these organisms. In the present review, the current knowledge of unique properties of polyamine metabolism in these parasites is outlined. These properties include prozyme regulation of AdoMetDC (S-adenosylmethionine decarboxylase) activity in trypanosomatids, co-expression of ODC (ornithine decarboxylase) and AdoMetDC activities in a single protein in plasmodia, and formation of trypanothione, a unique compound linking polyamine and thiol metabolism in trypanosomatids. Particularly interesting features within polyamine metabolism in these parasites are highlighted for their potential in selective therapeutic strategies.

  • 70.
    Björk, Glenn R
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Jacobsson, Kerstin
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Nilsson, Kristina
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J O
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Persson, Olof P
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    A primordial tRNA modification required for the evolution of life?2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 1-2, p. 231-239Article in journal (Refereed)
    Abstract [en]

    The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m(1)G37), which prevents frameshifting and is present adjacent to and 3' of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.

  • 71.
    Björnfot, Ann-Catrin
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Meticulous control of the T3SS of Yersinia is essential for full virulence2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The type III secretion system (T3SS) of pathogenic Yersinia pseudotuberculosis is involved in virulence. The syringe-like secretion system spans both bacterial membranes and is responsible for the ability of Yersinia to transfer toxic proteins (Yop proteins) into the eukaryotic target cell. The T3SS is believed to have evolved from the flagellum and regulation of the T3SS is a complex event that involves a series of regulatory proteins, whereby two are YscP and YscU. In a regulatory model, called the substrate specificity switch, both proteins act together to ensure proper T3SS structure and function by regulating a stop in YscF needle protein export with a shift to Yop effector secretion. YscU undergoes autoproteolysis at a conserved motif consisting of amino acids Asparagine-Proline-Threonine-Histidine (NPTH). Processing generates a C-terminal 10 kDa peptide, YscUCC. Processing is crucial for proper T3SS regulation and function both in vitro and in vivo. Full-length YscU does not support Yop secretion and after cleavage, YscUCC remains attached to the rest of YscU and acts as a negative block on T3S. Relief of this negative block is suggested to occur through displacement of YscUCC from the rest of YscU. Thorough control of many different cellular processes is brought by the heat shock proteins (HSPs) DnaK and DnaJ. Due to their multiple regulatory functions, mutations in the hsp-genes lead to pleiotropic effects. DnaK and DnaJ are essential for proper flagellum driven motion of bacteria, but more so; they ensure proper Yersinia T3SS function in vivo. Furthermore, DnaJ interacts with YscU and may be directly involved in T3SS regulation. Virulence of Yersinia is regulated on many levels. A previously identified virulence associated protein, VagH, is now characterized as an S-adenosyl-methionine dependent methyltransferase. The targets of the methylation activity of VagH are release factors 1 and 2 (RF1 and RF2), that are important for translation termination. The enzymatic activity of VagH is important for Yop secretion and a vagH mutant up-regulates a T3SS negative regulatory protein, YopD. Furthermore, a vagH mutant is avirulent in a mouse infection model, but is not affected in macrophage intracellular survival. The importance of VagH in vivo makes it a possible target for novel antimicrobial therapy.

  • 72.
    Björnfot, Ann-Catrin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Login, Frédéric H.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Edgren, Tomas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Involvement of the heat shock proteins DnaK/DnaJ in Yersinia T3SManuscript (preprint) (Other academic)
    Abstract [en]

    Yersinia  pseudotuberculosis  uses  a  type  III  secretion  system  (T3SS)  to  secrete  and  deliver  effectors  called Yops into target cells. These processes are highly regulated and the pathogen senses cell contact and respond accordingly by inducing Yop-effector expression.  A key component of the T3SS is the YscF needle present on the  surface of  the  pathogen. It has  been  suggested  that the  bacterium  can  switch  from  needle  export  to  Yop expression  and  secretion  and  that this  substrate  switch  is  important  for proper  regulation  during infection. YscU  is  an  essential  protein  regulating  the  substrate  switch  and  autoproteolysis  of  YscU  is  essential  for accurate  T3SS  regulation.  To  study  regulation  of  Yop  translocation  in  more  detail,  we  generated  mutants defective for expression of the heat shock proteins (HSPs) DnaJ and DnaK, since earlier studies had indicated a role of these proteins in regulation of effector translocation in Salmonella. The dnaJ mutant and the double dnaK/J  mutant  showed  significant  defects  in  Yop  translocation,  but  surprisingly  both  mutants  were  able  to secrete Yops in vitro much like the wild type.  However, both mutants showed a changed export pattern of the YscF  needle  with  a  pronounced  increased  export  of  the  YscF  needle  protein  after  incubation  in  calcium containing media. This phenotype was linked to defects in YscU autoproteolysis and in this respect the  hsp-mutants  were  identical  to  earlier  identified  autoprocessing  defective  mutants  in  YscU  (Single  amino  acid exchange mutants N263A and P264A). The hsp-mutants and the processing mutants accumulated full-length YscU,  which  surprisingly  was  associated  with  the  outer  membrane,  while  the  processed  form  of  YscU  was found  in  the  inner  membrane  fraction.  The  dnaJ  and  dnaK/J  mutants  were  strongly  affected  in  YscU autoproteolysis, which indicates a possible direct role for DnaJ in this process. Indeed a specific interaction between  YscU  and  DnaJ  could  be  found  suggesting  a  direct  role  of  the  HSPs  in  regulation  of  the  substrate switch in the T3SS.

  • 73.
    Björnham, Oscar
    et al.
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Bugaytsova, Jeanna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Dynamic force spectroscopy of the Helicobacter pylori BabA-Lewis b binding2009In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 143, no 1-2, p. 102-105Article in journal (Refereed)
    Abstract [en]

    The binding strength of the Helicobacter pylori adhesin–receptor complex BabA-ABO/Lewis b has been analyzed by means of dynamic force pectroscopy. High-resolution measurements of rupture forces were performed in situ on single bacterial cells, expressing the high-affinity binding BabA adhesin, by the use of force measuring optical tweezers. The resulting force spectra revealed the mechanical properties of a single BabA–Leb bond. It was found that the bond is dominated by one single energy barrier and that it is a slipbond. The bond length and thermal off-rate were assessed to be 0.86±0.07 nm and 0.015±0.006 s−1, respectively.

  • 74.
    Björnham, Oscar
    et al.
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Nilsson, Håkan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Physical properties of the specific PapG–galabiose binding in E. coli P pili-mediated adhesion2009In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 38, no 2, p. 245-254Article in journal (Refereed)
    Abstract [en]

    Detailed analyses of the mechanisms thatmediate binding of the uropathogenic Escherichia coli tohost cells are essential, as attachment is a prerequisite forthe subsequent infection process. We explore, by means offorce measuring optical tweezers, the interaction betweenthe galabiose receptor and the adhesin PapG expressed byP pili on single bacterial cells. Two variants of dynamicforce spectroscopy were applied based on constant andnon-linear loading force. The specific PapG–galabiosebinding showed typical slip-bond behaviour in the forceinterval (30–100 pN) set by the pilus intrinsic biomechanicalproperties. Moreover, it was found that the bondhas a thermodynamic off-rate and a bond length of2.6×10-3 s-1 and 5.0 Å , respectively. Consequently, thePapG–galabiose complex is significantly stronger thanthe internal bonds in the P pilus structure that stabilizes thehelical chain-like macromolecule. This finding suggeststhat the specific binding is strong enough to enable the Ppili rod to unfold when subjected to strong shear forces inthe urinary tract. The unfolding process of the P pili rodpromotes the formation of strong multipili interaction,which is important for the bacterium to maintain attachmentto the host cells.

  • 75. Blanchard, P. G.
    et al.
    Turcotte, V.
    Cote, M.
    Gelinas, Y.
    Nilsson, S.
    Olivecrona, Gunilla
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Deshaies, Y.
    Festuccia, W. T.
    Peroxisome proliferator-activated receptor activation favours selective subcutaneous lipid deposition by coordinately regulating lipoprotein lipase modulators, fatty acid transporters and lipogenic enzymes2016In: Acta Physiologica, ISSN 1748-1708, E-ISSN 1748-1716, Vol. 217, no 3, p. 227-239Article in journal (Refereed)
    Abstract [en]

    Aim: Peroxisome proliferator-activated receptor (PPAR) γ activation is associated with preferential lipoprotein lipase (LPL)-mediated fatty acid storage in peripheral subcutaneous fat depots. How PPARγ agonism acts upon the multi-level modulation of depot-specific lipid storage remains incompletely understood.

    Methods: We evaluated herein triglyceride-derived lipid incorporation into adipose tissue depots, LPL mass and activity, mRNA levels and content of proteins involved in the modulation of LPL activity and fatty acid transport, and the expression/activity of enzymes defining adipose tissue lipogenic potential in rats treated with the PPARγ ligand rosiglitazone (30 mg kg−1 day−1, 23 days) after either a 10-h fasting period or a 17-h fast followed by 6 h of ad libitum refeeding.

    Results: Rosiglitazone stimulated lipid accretion in subcutaneous fat (SF) ~twofold and significantly reduced that of visceral fat (VF) to nearly half. PPARγ activation selectively increased LPL mass, activity and the expression of its chaperone LMF1 in SF. In VF, rosiglitazone had no effect on LPL activity and downregulated the mRNA levels of the transendothelial transporter GPIHBP1. Overexpression of lipid uptake and fatty acid transport proteins (FAT/CD36, FATP1 and FABP4) and stimulation of lipogenic enzyme activities (GPAT, AGPAT and DGAT) upon rosiglitazone treatment were of higher magnitude in SF.

    Conclusions: Together these findings demonstrate that the depot-specific transcriptional control of LPL induced by PPARγ activation extends to its key interacting proteins and post-translational modulators to favour subcutaneous lipid storage.

  • 76.
    Blanco-Rivero, Amaya
    et al.
    Madrid, Spain.
    Shutova, Tatiana
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    José Román, María
    Madrid, Spain.
    Villarejo, Arsenio
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Department of Biology, Universidad Autónoma de Madrid, Madrid, Spain.
    Martinez, Flor
    Madrid, Spain.
    Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, article id e49063Article in journal (Refereed)
    Abstract [en]

    Background: Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO2 conditions. Results/Conclusions: In the present work we demonstrate that after transfer to low CO2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO2 conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. Significance: This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

  • 77.
    Blomberg, Jeanette
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Aguilar, Ximena
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rautio, Linn
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 13, p. 5938-5950Article in journal (Refereed)
    Abstract [en]

    Mediator is a multiprotein coregulatory complex that conveys signals from DNA-bound transcriptional regulators to the RNA polymerase II transcription machinery in eukaryotes. The molecular mechanisms for how these signals are transmitted are still elusive. By using purified transcription factor Dreb2a, mediator subunit Med25 from Arabidopsis thaliana, and a combination of biochemical and biophysical methods, we show that binding of Dreb2a to its canonical DNA sequence leads to an increase in secondary structure of the transcription factor. Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes. However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25. We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.

  • 78. Bobay, Benjamin G
    et al.
    Thompson, Richele J
    Milton, Debra L
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Southern Research Institute, Birmingham, AL, USA.
    Cavanagh, John
    Chemical shift assignments and secondary structure prediction of the phosphorelay protein VanU from Vibrio anguillarum2014In: Biomolecular NMR Assignments, ISSN 1874-2718, E-ISSN 1874-270X, Vol. 8, no 1, p. 177-179Article in journal (Refereed)
    Abstract [en]

    Vibrio anguillarum is a biofilm forming Gram-negative bacterium that survives prolonged periods in seawater and causes vibriosis in marine life. A quorum-sensing signal transduction pathway initiates biofilm formation in response to environmental stresses. The phosphotransferase protein VanU is the focal point of the quorum-sensing pathway and facilitates the regulation between independent phosphorelay systems that activate or repress biofilm formation. Here we report the (1)H, (13)C, and (15)N backbone and side chain resonance assignments and secondary structure prediction for VanU from V. anguillarum.

  • 79. Bogomolovas, Julius
    et al.
    Simon, Bernd
    Sattler, Michael
    Stier, Gunter
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP). Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
    Screening of fusion partners for high yield expression and purification of bioactive viscotoxins2009In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 64, no 1, p. 16-23Article in journal (Refereed)
    Abstract [en]

    Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6-tagged fusion protein was captured with Ni(2+) affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipitation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2 mg of visctoxin A3 from 11 of culture medium corresponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by heteronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins.

  • 80.
    Boija, Ann
    et al.
    Dept. of Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden.
    Holmqvist, Per-Henrik
    Dept. of Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden.
    Philip, Philge
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Computational Life Science Cluster (CLiC), Umeå, Sweden.
    Zare, Aman
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Computational Life Science Cluster (CLiC), Umeå, Sweden.
    Meyers, David J.
    Dept. Pharmacology and Molecular Sciences, The Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
    Cole, Philip A.
    Dept. Pharmacology and Molecular Sciences, The Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Division of CBRN Defence and Security, FOI, Swedish Defence Research Agency, Sweden.
    Mannervik, Mattias
    Dept. of Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, SwedenDept. Pharmacology and Molecular Sciences, The Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
    Drosophila CBP cooperates with GAGA factor to induce high levels of Pol II promoter-proximal pausingManuscript (preprint) (Other academic)
  • 81. Boija, Ann
    et al.
    Mahat, Dig Bijay
    Zare, Aman
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Holmqvist, Per-Henrik
    Philip, Philge
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Meyers, David J
    Cole, Philip A
    Lis, John T
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mannervik, Mattias
    CBP Regulates Recruitment and Release of Promoter-Proximal RNA Polymerase II2017In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 68, no 3, p. 491-503.e5Article in journal (Refereed)
    Abstract [en]

    Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is unclear. Here, we identify a novel activity of the histone acetyltransferase p300/CREB-binding protein (CBP) in regulating promoter-proximal paused Pol II. We find that Drosophila CBP inhibition results in "dribbling" of Pol II from the pause site to positions further downstream but impedes transcription through the +1 nucleosome genome-wide. Promoters strongly occupied by CBP and GAGA factor have high levels of paused Pol II, a unique chromatin signature, and are highly expressed regardless of cell type. Interestingly, CBP activity is rate limiting for Pol II recruitment to these highly paused promoters through an interaction with TFIIB but for transit into elongation by histone acetylation at other genes. Thus, CBP directly stimulates both Pol II recruitment and the ability to traverse the first nucleosome, thereby promoting transcription of most genes.

  • 82. Boldinova, Elizaveta O.
    et al.
    Wanrooij, Paulina H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shilkin, Evgeniy S.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Makarova, Alena V.
    DNA Damage Tolerance by Eukaryotic DNA Polymerase and Primase PrimPol2017In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 18, no 7, article id 1584Article, review/survey (Refereed)
    Abstract [en]

    PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of PrimPol, as well as in identification of new protein-protein interaction partners. Furthermore, we discuss the possible functions of PrimPol in both the nucleus and the mitochondria.

  • 83.
    Bolivar, Juan M
    et al.
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, Madrid, Spain.
    Rocha-Martin, Javier
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, Madrid, Spain.
    Mateo, Cesar
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, Madrid, Spain.
    Cava, Felipe
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Berenguer, Jose
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Fernandez-Lafuente, Roberto
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, Madrid, Spain.
    Guisan, Jose M
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, Madrid, Spain.
    Coating of soluble and immobilized enzymes with ionic polymers: full stabilization of the quaternary structure of multimeric enzymes2009In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, no 4, p. 742-747Article in journal (Refereed)
    Abstract [en]

    This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes are very unstable at acidic pH values due to the rapid dissociation of their subunits (half-life of diluted preparations is few minutes at pH 4 and 25 degrees C). GDH and FDH were incubated in the presence of PEI yielding an enzyme-PEI composite with full activity. To stabilize the enzyme-polymer composite, a treatment with glutaraldehyde was required. These enzyme-PEI composites can be crosslinked with glutaraldehyde by immobilizing previously the composite onto a weak cationic exchanger. The soluble GDH-PEI composite was much more stable than unmodified GDH at pH 4 and 30 degrees C (retaining over 90% activity after 24 h incubation) with no effect of the GDH concentration in the inactivation course. The composite could be very strongly, but reversibly, adsorbed on cationic exchangers. Similarly, FDH could be treated with PEI and glutaraldehyde after adsorption on cationic exchangers, This permitted a stabilized FDH preparation. In this way, the coating of the enzymes surfaces with PEI is used as a simple and efficient strategy to prevent enzyme dissociation of multimeric enzymes. These composites can be used as a soluble catalyst or reversibly immobilized onto a cationic exchanger (e.g., CM-agarose).

  • 84.
    Bonde, Mari
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Östberg, Yngve
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bunikis, Ignas
    Uppsala University.
    Nyunt Wai, Sun
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Effects of osmotic stress in P13 and P66 deficient Borrelia burgdorferi mutantsManuscript (preprint) (Other (popular science, discussion, etc.))
  • 85.
    Bos, Antoine
    Umeå University, Faculty of Science and Technology, Ecology and Environmental Science.
    Natural variation in cold adaptation and freezing tolerance in Arabidopsis thaliana2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Plants have spread to almost everywhere in the world. As they disperse, they meet many different environments to which they may be able to adapt. For a plant species to adapt to a new environment, genetic variation is needed. The individuals differ from each other in their genetic composition, which often means differences in phenotypes. Those individuals that manage to reproduce will form the next generation. With different conditions in different environments, it will not be the same phenotypes that reproduce everywhere. In that way, plant species will form into a mosaic of locally adapted populations varying genetically as the species disperses.

    After the last ice age plants have started to disperse away from the equators. With increasing latitudes come increasing challenges to migrating plants. As plant species disperse northwards along this gradient of varying conditions individuals are selected for cold adaptive traits like flowering time and freezing tolerance, acquired by cold acclimation. In this way, genetic variation from the original populations for these traits becomes sorted out along a latitudinal cline.

    The aim of this thesis was to understand how selection along a latitudinal gradient has shaped natural variation in cold adaptive traits in plants dispersing northwards, and specifically, to investigate what variation can be observed in phenotypes for these traits and how these traits correlate with genetic variation in genes known to be involved in cold acclimation.

    In this study significant variation was found in a sample of the model plan Arabidopsis thaliana accessions in cold adaptive traits flowering time and freezing tolerance. A clear latitudinal cline in the cold adaptive traits freezing tolerance for A. thaliana was observed.

    Analysis of nucleotide polymorphism for the cold responsive ICE1 (inducer of CBF expression 1) transcription factor revealed a haplotype structure with two allelic clades as well as unusually high levels of synonymous polymorphism.

    Nucleotide polymorphism analysis for the transcription factors CBF1, CBF2 and CBF3 (C-repeat binding factors) that play a key role in regulating the expression of a group of target genes known as the “CBF regulon” showed a distinct geographical haplotype structure. One haplotype was dominant in southern accessions while in the other northern accessions overrepresented. There was a significant effect of CBF haplotype on both freezing tolerance and flowering time even after correcting for latitude.

    Significant differences in CBF expression levels were found between the different CBF genes as well as between different accessions. Sequence variation at CBF was shown to have a significant effect on expression levels of CBF2. No clear correlations were found between CBF gene expression and freezing tolerance or temperature sensitivity for any of the accessions used in the study. This highlights the complex relationship between sequence variation in candidate genes and gene expression, and the problems associated with unraveling the genetic basis of ecologically important traits.

  • 86. Bosley, Katrine S
    et al.
    Botchan, Michael
    Bredenoord, Annelien L
    Carroll, Dana
    Charo, R Alta
    Charpentier, Emmanuelle
    Cohen, Ron
    Corn, Jacob
    Doudna, Jennifer
    Feng, Guoping
    Greely, Henry T
    Isasi, Rosario
    Ji, Weihzi
    Kim, Jin-Soo
    Knoppers, Bartha
    Lanphier, Edward
    Li, Jinsong
    Lovell-Badge, Robin
    Martin, G Steven
    Moreno, Jonathan
    Naldini, Luigi
    Pera, Martin
    Perry, Anthony C F
    Venter, J Craig
    Zhang, Feng
    Zhou, Qi
    CRISPR germline engineering: the community speaks2015In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 33, no 5, p. 478-486Article in journal (Refereed)
  • 87.
    Botelho, Hugo M.
    et al.
    Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
    Leal, Sonia S.
    Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
    Cardoso, Isabel
    Molecular Neurobiology Unit, Instituto de Biologia Molecular e Celular, Porto, Portugal.
    Yanamandra, Kiran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Fritz, Günter
    Department of Neuropathology, University of Freiburg, Germany.
    Gomes, Claudio M.
    Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
    S100A6 Amyloid Fibril formation is Calcium-modulated and enhances Superoxide Dismutase-1 (SOD1) aggregation2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 50, p. 42233-42242Article in journal (Refereed)
    Abstract [en]

    S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel beta-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metalmodulated aggregation propensity may be a key aspect in their physiology and function.

  • 88. Brandsma, Joost
    et al.
    Goss, Victoria M.
    Yang, Xian
    Bakke, Per S.
    Caruso, Massimo
    Chanez, Pascal
    Dahlén, Sven-Erik
    Fowler, Stephen J.
    Horvath, Ildiko
    Krug, Norbert
    Montuschi, Paolo
    Sanak, Marek
    Sandström, Thomas
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Section of Medicine.
    Shaw, Dominick E.
    Chung, Kian Fan
    Singer, Florian
    Fleming, Louise J.
    Sousa, Ana R.
    Pandis, Ioannis
    Bansal, Aruna T.
    Sterk, Peter J.
    Djukanovic, Ratko
    Postle, Anthony D.
    Lipid phenotyping of lung epithelial lining fluid in healthy human volunteers2018In: Metabolomics, ISSN 1573-3882, E-ISSN 1573-3890, Vol. 14, no 10, article id 123Article in journal (Refereed)
    Abstract [en]

    Background: Lung epithelial lining fluid (ELF)-sampled through sputum induction-is a medium rich in cells, proteins and lipids. However, despite its key role in maintaining lung function, homeostasis and defences, the composition and biology of ELF, especially in respect of lipids, remain incompletely understood.

    Objectives: To characterise the induced sputum lipidome of healthy adult individuals, and to examine associations between different ELF lipid phenotypes and the demographic characteristics within the study cohort.

    Methods: Induced sputum samples were obtained from 41 healthy non-smoking adults, and their lipid compositions analysed using a combination of untargeted shotgun and liquid chromatography mass spectrometry methods. Topological data analysis (TDA) was used to group subjects with comparable sputum lipidomes in order to identify distinct ELF phenotypes.

    Results: The induced sputum lipidome was diverse, comprising a range of different molecular classes, including at least 75 glycerophospholipids, 13 sphingolipids, 5 sterol lipids and 12 neutral glycerolipids. TDA identified two distinct phenotypes differentiated by a higher total lipid content and specific enrichments of diacyl-glycerophosphocholines, -inositols and -glycerols in one group, with enrichments of sterols, glycolipids and sphingolipids in the other. Subjects presenting the lipid-rich ELF phenotype also had significantly higher BMI, but did not differ in respect of other demographic characteristics such as age or gender.

    Conclusions: We provide the first evidence that the ELF lipidome varies significantly between healthy individuals and propose that such differences are related to weight status, highlighting the potential impact of (over)nutrition on lung lipid metabolism.

  • 89.
    Brattsand, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Dermatology and Venerology.
    Egelrud, Torbjörn
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Dermatology and Venerology.
    Purification and characterization of interleukin 1 beta from human plantar stratum corneum. Evidence of interleukin 1 beta processing in vivo not involving interleukin 1 beta convertase1998In: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 10, no 7, p. 506-513Article in journal (Refereed)
    Abstract [en]

    The major interleukin 1 beta (IL-1 beta) species from human plantar stratum corneum was purified and found to have an N-terminal amino acid sequence homologous to a stretch of the human IL-1 beta precursor, starting with His115. Whereas SDS-polyacrylamide gel electrophoresis followed by immunoblotting revealed only one component in plantar stratum corneum with IL-1 beta-like immunoreactivity, and with an apparent molecular mass around 18 kDa, isoelectric focusing under non-denaturing conditions showed one major component with isoelectric point around 6.1 and two minor components isoelectric at pH 6.3 and 6.9, respectively. Digestion of recombinant human IL-1 beta precursor with chymotrypsin, producing a C-terminal fragment with N-terminal Yal114, yielded a component with IL-1 beta-like immunoreactivity isoelectric at pH 6.3. Recombinant bacterial variants of human IL-1 beta with N-terminal amino acids corresponding to Val114, His115 and Ala117 were isoelectric at pH 6.3, 6.1 and 6.9, respectively. Cloning and subsequent nucleotide sequencing of IL-1 beta precursor cDNA from a human keratinocyte line showed total identify with the sequence previously published for the human monocyte IL-1 beta precursor. The authors conclude that the IL-1 beta species present in plantar stratum corneum have isoelectric points determined by their respective amino acid sequences, and that there is a mechanism for IL-1 beta activation in human epidermis not involving interleukin 1 beta convertase.

  • 90.
    Brattsand, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine.
    Egelrud, Torbjörn
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine.
    Purification, molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 42, p. 30033-40Article in journal (Refereed)
    Abstract [en]

    A new human 33-kDa serine protease was purified from human epidermis, and its cDNA was cloned from a keratinocyte library, from mRNA from a human keratinocyte line (HaCat) and from mRNA from human skin. Polyclonal antibodies specific for the new protein detected three groups of proteins in partially purified extracts of cornified eptihelium of human plantar skin. The three components are proposed to correspond to proenzyme, active enzyme, and proteolytically modified active enzyme. After N-deglycosylation, there was a decrease in apparent molecular mass of all detected components. Expression of the cloned cDNA in a eukaryotic virus-derived system yielded a recombinant protein that could be converted to an active protease by treatment with trypsin. Polymerase chain reaction analyses of cDNA from a number of human tissues showed high expression of the new enzyme in the skin and low expression in brain, placenta, and kidney. Homology searches yielded the highest score for porcine enamel matrix protease (55% amino acid sequence homology). High scores were also obtained for human and mouse neuropsin and for human stratum corneum chymotryptic enzyme. The function of this new protease, tentatively named stratum corneum tryptic enzyme, may be related to stratum corneum turnover and desquamation in the epidermis.

  • 91. Broglia, Laura
    et al.
    Materne, Solange
    Lecrivain, Anne-Laure
    Hahnke, Karin
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Institute for Biology, Humboldt University, Berlin, Germany; Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B2018In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 15, no 10, p. 1336-1347Article in journal (Refereed)
    Abstract [en]

    Endoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5′ untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5′ UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5′ UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5′ UTR and on the role of RNase Y in speB regulation.

  • 92.
    Brorsson, Ann-Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The Folding Energy Landscape of MerP2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis is based on studies, described in four papers, in which the folding energy landscape of MerP was investigated by various techniques. MerP is a water-soluble 72 amino acid protein with a secondary structure consisting of four anti-parallel β-strands and two α-helices on one side of the sheet in the order β1α1β2β3α2β4.

    The first paper describes the use of CD and fluorescence analysis to examine the folding/unfolding process of MerP. From these experiments it was found that the protein folds according to a two-state model in which only the native and unfolded forms are populated without any visible intermediates. With a rate constant of 1.2 s-1, the folding rate was found to be unusually slow for a protein of this size.

    The studies presented in the second and third papers were based on measurements of native-state amide proton exchange at different temperatures (Paper II) and GuHCl concentrations (Paper III) in the pre-transitional region. In these studies partially unfolded forms were found for MerP which are essentially unrelated to each other. Thus, in the folding energy landscape of MerP, several intermediates seem to occur on different folding trajectories that are parallel to each other. The slow folding rate of MerP might be coupled to extensive visitation of these conformations. Hydrogen exchange in MerP did also reveal structure-dependent differences in compactness between the denatured states in GuHCl and H2O.

    In the last paper multivariate data analysis was applied to 2-dimensional NMR data to detect conformational changes in the structure of MerP induced by GuHCl. From this analysis it was suggested that regions involved in the most flexible part of the protein structure are disrupted at rather low denaturant concentrations (< 2.1 M GuHCl) while the native structures of the most stable parts are still not completely ruptured at 2.9 M GuHCl.

    Finally, the stability, kinetics, contact order and folding nuclei of six proteins with similar topology (MerP, U1A, S6, ADA2h, AcP and HPr) were compared. In this analysis it was found that their folding properties are quite diverse, despite their topological similarities, and no general rules that have been formulated yet can adequately predict their folding behaviour.

  • 93.
    Brouwer, Bastiaan
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Shedding Light on Shade- and Dark-Induced Leaf Senescence2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Leaf senescence is the final stage of leaf development, during which the leaf relocates most of itsvaluable nutrients to developing or storing parts of the plant. As this process progresses, leaves losetheir green color and their capacity to perform photosynthesis. Shade and darkness are well-knownas factors inducing leaf senescence and it has been proposed that senescence can be initiated byreductions in photosynthesis, photomorphogenesis and transpiration. However, despite the fact thatthe signaling mechanisms regulating each of these processes have been extensively described,particularly in seedlings, their contribution to the initiation of senescence in mature leaves stillremains unclear. Furthermore, the use of different experimental systems to study shade-inducedleaf senescence has yielded several divergent results, which altogether complicate the overallunderstanding of leaf senescence.

    To address this, darkened plants and individually darkened leaves, which show different rates of leafsenescence, were studied. Comparing the transcriptome and metabolome of these two darktreatmentsrevealed that they differed distinctly with regard to their metabolic strategies. Wholedarkened plants were severely carbohydrate-starved, accumulated amino acids and slowed downtheir metabolism. In contrast, individually darkened leaves showed continued active metabolismcoupled to senescence-associated degradation and relocation of amino acids.

    This knowledge was used to set up a new system to study how shade affects leaf senescence in themodel plant Arabidopsis thaliana. Use of this system revealed that different senescence-associatedhallmarks appeared in response to different intensities of shade. Some of these hallmarks werefurther shown to be part of both leaf senescence and photosynthetic acclimation to low light.

    Finally, using this system on phytochrome mutants revealed that loss of phytochrome A increasedthe loss of chlorophyll under shade, without increasing the expression of senescence-associatedgenes.

    Together, these findings suggest that shade-induced leaf senescence, which is generally perceived asa single process, is actually an intricate network of different processes that work together tomaintain an optimal distribution of nutrients within the plant.

  • 94.
    Brouwer, Bastiaan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Gardeström, Per
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Keech, Olivier
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Far-red light reduces senescence-associated chlorophyll loss under low light via a Phytochrome A-mediated Far-red High Irradiance ResponseManuscript (preprint) (Other academic)
  • 95.
    Brouwer, Bastiaan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Ziolkowska, Agnieszka
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Bagard, Matthieu
    Keech, Olivier
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Gardeström, Per
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    The impact of light intensity on shade-induced leaf senescence2012In: Plant, Cell and Environment, ISSN 0140-7791, E-ISSN 1365-3040, Vol. 35, no 6, p. 1084-1098Article in journal (Refereed)
    Abstract [en]

    Plants often have to cope with altered light conditions, which in leaves induce various physiological responses ranging from photosynthetic acclimation to leaf senescence. However, our knowledge of the regulatory pathways by which shade and darkness induce leaf senescence remains incomplete. To determine to what extent reduced light intensities regulate the induction of leaf senescence, we performed a functional comparison between Arabidopsis leaves subjected to a range of shading treatments. Individually covered leaves, which remained attached to the plant, were compared with respect to chlorophyll, protein, histology, expression of senescence-associated genes, capacity for photosynthesis and respiration, and light compensation point (LCP). Mild shading induced photosynthetic acclimation and resource partitioning, which, together with a decreased respiration, lowered the LCP. Leaf senescence was induced only under strong shade, coinciding with a negative carbon balance and independent of the red/far-red ratio. Interestingly, while senescence was significantly delayed at very low light compared with darkness, phytochrome A mutant plants showed enhanced chlorophyll degradation under all shading treatments except complete darkness. Taken together, our results suggest that the induction of leaf senescence during shading depends on the efficiency of carbon fixation, which in turn appears to be modulated via light receptors such as phytochrome A.

  • 96.
    Bruce, Stephen J
    et al.
    Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Jonsson, Pär
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Antti, Henrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cloarec, Olivier
    Technologie Servier, 45000 Orleans, France.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Marklund, Stefan L
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Moritz, Thomas
    Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis2009In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 372, no 2, p. 237-249Article in journal (Refereed)
    Abstract [en]

    The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS–DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.

  • 97.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Molecular dissection of established and proposed members of the Op18/Stathmin family of tubulin binding proteins2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    My initial aim was a functional analysis of the conserved Op18/stathmin family of microtubule-regulators, which includes the ubiquitous cytosolic Op18 protein and the neural membrane-attached RB3 and SCG10 proteins. The solved X-ray structure has shown that these proteins form a complex with tubulin -heterodimers via two imperfect helical repeats, which result in two head-to-tail aligned heterodimers in a tandem-tubulin complex. We have analyzed GTP exchange and GTP hydrolysis at the two exchangeable GTP-binding sites (E-site) within the tandem-tubulin complex. A comparison of Op18, RB3 and SCG10 proteins indicates that Op18/Stathmin family proteins have evolved to maintain the two heterodimers in a configuration that restrains the otherwise potent GTPase productive interactions facilitated by the head-to-head alignment of heterodimers in protofilaments. We concluded from these studies that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis.

    To understand the significance of the large differences in tubulin affinity of Op18, RB3 and SCG10, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell-surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. We showed that, in contrast to CD2-Op18, both the CD2-SCG10 and CD2-RB3 chimeras sequester tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels. However, all three CD2-chimeras, including the tubulin sequestration-incompetent CD2-Op18, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during the interphase, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules.

    Sm16/SmSLP (Stathmin-Like Protein) has been identified as a protein released during skin penetration of the Schistosoma mansoni parasite. This protein has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. However, our studies refuted any functional similarity with stathmin/Op18 and we found instead that Sm16/SmSLP is a lipid bilayer binding protein that is taken up by cells through endocytosis.

    To study immuno-modulatory properties of Sm16/SmSLP, we designed an engineered version with decreased aggregation propensity, thus facilitating expression and purification of a soluble Sm16 /SmSLP protein from the eukaryotic organism Pichia pastoris. Determination of the hydrodynamic parameters revealed that both the recombinant and native Sm16/SmSLP is a ~9-subunits oligomer. The recombinant protein was found to have no effect on T lymphocyte activation, cell proliferation or the basal level of cytokine production of whole human blood or monocytic cells. Interestingly, however, recombinant Sm16 was found to potently inhibit the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and Poly(I:C). Since Sm16 specifically inhibits degradation of the IRAK1 signaling protein in LPS stimulated monocytes, it seems likely that inhibition is exerted proximal to the TLR-complex.

  • 98.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Anders
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Aβ peptide fibrillar architectures controlled by conformational constraints of the monomer2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, p. e25157-Article in journal (Refereed)
    Abstract [en]

    Anomalous self-assembly of the Aβ peptide into fibrillar amyloid deposits is strongly correlated with the development of Alzheimer's disease. Aβ fibril extension follows a template guided "dock and lock" mechanism where polymerisation is catalysed by the fibrillar ends. Using surface plasmon resonance (SPR) and quenched hydrogen-deuterium exchange NMR (H/D-exchange NMR), we have analysed the fibrillar structure and polymerisation properties of both the highly aggregation prone Aβ1-40 Glu22Gly (Aβ(40Arc)) and wild type Aβ1-40 (Aβ(40WT)). The solvent protection patterns from H/D exchange experiments suggest very similar structures of the fibrillar forms. However, through cross-seeding experiments monitored by SPR, we found that the monomeric form of Aβ(40WT) is significantly impaired to acquire the fibrillar architecture of Aβ(40Arc). A detailed characterisation demonstrated that Aβ(40WT) has a restricted ability to dock and isomerise with high binding affinity onto Aβ(40Arc) fibrils. These results have general implications for the process of fibril assembly, where the rate of polymerisation, and consequently the architecture of the formed fibrils, is restricted by conformational constraints of the monomers. Interestingly, we also found that the kinetic rate of fibril formation rather than the thermodynamically lowest energy state determines the overall fibrillar structure.

  • 99.
    Bröms, Jeanette E
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Type III secretion- the various functions of the translocon operon in bacterial pathogenesis2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In order to establish colonisation of a human host, pathogenic Yersinia use a type III protein secretion system to directly intoxicate host immune cells. Activation of this system requires target cell contact and is a highly regulated process. Both the intoxication and regulation events depend on the lcrGVHyopBD translocon operon, which is highly conserved in many bacterial pathogens. In this study, the role of individual operon members was analysed and functional domains identified by using the highly homologous pcrGVHpopBD operon of P. aeruginosa as a comparative tool.

    Yersinia spp. and P. aeruginosa were shown to form translocation pores of a similar size that promoted equally efficient protein delivery. A strong dependency on interactions between native translocator(s) in protein delivery was revealed, suggesting that each pathogen has delicately fine-tuned this process to suit its own infection niche. In particular, the C-terminus of YopD was shown to possess functional specificity for effector delivery in Yersinia that could not be conferred by the comparable region in homologous PopD. Moreover, a role for LcrV and PcrV in substrate recognition during the protein delivery process was excluded.

    The N-terminus of LcrH was recognized as a unique regulatory domain, mediating formation of LcrH-YscY regulatory complexes in Yersinia, while equivalent complexes with analogous proteins were not formed in P. aeruginosa. These results compliment the idea that a negative regulatory pathway involving LcrH, YopD, LcrQ and YscY is unique to Yersinia.

    Finally, PcrH was identified as a new member of the translocator class of chaperones, being essential for assembly of a functional PopB/PopD mediated translocon in P. aeruginosa. However, in contrast to the other members of this family, PcrH was dispensable for type III regulation. Moreover, both LcrH and PcrH were shown to possess tetratricopeptide repeats crucial for their chaperone function. One tetratricopeptide repeat mutant in LcrH was even isolated that failed to secrete both YopB and YopD substrates, even though stability was maintained. This demonstrates for the first time that LcrH has a role in substrate secretion in addition to its critical role in promoting substrate stability.

  • 100.
    Buckland, Philip I.
    et al.
    Umeå University, Faculty of Arts, Department of historical, philosophical and religious studies, Environmental Archaeology Lab.
    Hammarlund, Dan
    Lund University.
    Hjärthner-Holdar, Eva
    Swedish National Historical Museums.
    Lidén, Kerstin
    Stockholm University.
    Lindahl, Anders
    Lund University.
    Palm, Fredrik
    Umeå University, Faculty of Arts, Humlab.
    Possnert, Göran
    Uppsala University.
    The Strategic Environmental Archaeology Database: a resource for international, multiproxy and transdisciplinary studies of environmental and climatic change2015Conference paper (Refereed)
    Abstract [en]

    Climate and environmental change are global challenges which require global data and infrastructure to investigate. These challenges also require a multi-proxy approach, integrating evidence from Quaternary science and archaeology with information from studies on modern ecology and physical processes among other disciplines. The Strategic Environmental Archaeology Database (SEAD http://www.sead.se) is a Swedish based international research e-infrastructure for storing, managing, analysing and disseminating palaeoenvironmental data from an almost unlimited number of analysis methods. The system currently makes available raw data from over 1500 sites (>5300 datasets) and the analysis of Quaternary fossil insects, plant macrofossils, pollen, geochemistry and sediment physical properties, dendrochronology and wood anatomy, ceramic geochemistry and bones, along with numerous dating methods. This capacity will be expanded in the near future to include isotopes, multi-spectral and archaeo-metalurgical data. SEAD also includes expandable climate and environment calibration datasets, a complete bibliography and extensive metadata and services for linking these data to other resources. All data is available as Open Access through http://qsead.sead.se and downloadable software.

     

    SEAD is maintained and managed at the Environmental Archaeology Lab and HUMlab at Umea University, Sweden. Development and data ingestion is progressing in cooperation with The Laboratory for Ceramic Research and the National Laboratory for Wood Anatomy and Dendrochronology at Lund University, Sweden, the Archaeological Research Laboratory, Stockholm University, the Geoarchaeological Laboratory, Swedish National Historical Museums Agency and several international partners and research projects. Current plans include expanding its capacity to serve as a data source for any system and integration with the Swedish National Heritage Board's information systems.

     

    SEAD is partnered with the Neotoma palaeoecology database (http://www.neotomadb.org) and a new initiative for building cyberinfrastructure for transdisciplinary research and visualization of the long-term human ecodynamics of the North Atlantic funded by the National Science Foundation (NSF).

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