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  • 1. Abraham, Edit
    et al.
    Miskolczi, Pal
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Ayaydin, Ferhan
    Yu, Ping
    Kotogany, Edit
    Bako, Laszlo
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Oetvoes, Krisztina
    Horvath, Gabor V.
    Dudits, Denes
    Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells2011Ingår i: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 62, nr 6, s. 2155-2168Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G(1)/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G(2)/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G(1) cells. Its amount increased upon entry into the S phase and remained high during the G(2)/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G(2)/M phase regulation in plant cells is discussed.

  • 2.
    Adhikari, Deepak
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Signaling pathways in the development of female germ cells2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Primordial follicles are the first small follicles to appear in the mammalian ovary. Women are born with a fixed number of primordial follicles in the ovaries. Once formed, the pool of primordial follicles serves as a source of developing follicles and oocytes. The first aim of this thesis was to investigate the functional role of the intra-oocyte signaling pathways, especially the phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) pathways in the regulation of primordial follicle activation and survival. We found that a primordial follicle remains dormant when the PI3K and mTORC1 signaling in its oocyte is activated to an appropriate level, which is just sufficient to maintain its survival, but not sufficient for its growth initiation. Hyperactivation of either of these signaling pathways causes global activation of the entire pool of primordial follicles leading to the exhaustion of all the follicles in young adulthood in mice. Mammalian oocytes, while growing within the follicles, remain arrested at prophase I of meiosis. Oocytes within the fully-grown antral follicles resume meiosis upon a preovulatory surge of leutinizing hormone (LH), which indicates that LH mediates the resumption of meiosis. The prophase I arrest in the follicle-enclosed oocyte is the result of low maturation promoting factor (MPF) activity, and resumption of meiosis upon the arrival of hormonal signals is mediated by activation of MPF. MPF is a complex of cyclin dependent kinase 1 (Cdk1) and cyclin B1, which is essential and sufficient for entry into mitosis. Although much of the mitotic cell cycle machinery is shared during meiosis, lack of Cdk2  in mice leads to a postnatal loss of all oocytes, indicating that Cdk2 is important for oocyte survival, and probably oocyte meiosis also. There have been conflicting results earlier about the role of Cdk2 in metaphase II arrest of Xenopus  oocytes. Thus the second aim of the thesis was to identify the specific Cdk that is essential for mouse oocyte meiotic maturation. We generated mouse models with oocytespecific deletion of Cdk1  or Cdk2  and studied the specific requirements of Cdk1 and Cdk2 during resumption of oocyte meiosis. We found that only Cdk1 is essential and sufficient for the oocyte meiotic maturation. Cdk1 does not only phosphorylate the meiotic phosphoproteins during meiosis resumption but also phosphorylates and suppresses the downstream protein phosphatase 1, which is essential for protecting the Cdk1 substrates from dephosphorylation.

  • 3.
    Aguilar, Ximena
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Folding and interaction studies of subunits in protein complexes2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Proteins function as worker molecules in the cell and their natural environment is crowded. How they fold in a cell-like environment and how they recognize their interacting partners in such conditions, are questions that underlie the work of this thesis.

    Two distinct subjects were investigated using a combination of biochemical- and biophysical methods. First, the unfolding/dissociation of a heptameric protein (cpn10) in the presence of the crowding agent Ficoll 70. Ficoll 70 was used to mimic the crowded environment in the cell and it has been used previously to study macromolecular crowding effects, or excluded volume effects, in protein folding studies. Second, the conformational changes upon interaction between the Mediator subunit Med25 and the transcription factor Dreb2a from Arabidopsis thaliana. Mediator is a transcriptional co-regulator complex which is conserved from yeast to humans. The molecular mechanisms of its action are however not entirely understood. It has been proposed that the Mediator complex conveys regulatory signals from promoter-bound transcription factors (activators/repressors) to the RNA polymerase II machinery through conformational rearrangements.

    The results from the folding study showed that cpn10 was stabilized in the presence of Ficoll 70 during thermal- and chemical induced unfolding (GuHCl). The thermal transition midpoint increased by 4°C, and the chemical midpoint by 0.5 M GuHCl as compared to buffer conditions. Also the heptamer-monomer dissociation was affected in the presence of Ficoll 70, the transition midpoint was lower in Ficoll 70 (3.1 μM) compared to in buffer (8.1 μM) thus indicating tighter binding in crowded conditions. The coupled unfolding/dissociation free energy for the heptamer increased by about 36 kJ/mol in Ficoll. Altogether, the results revealed that the stability effect on cpn10 due to macromolecular crowding was larger in the individual monomers (33%) than at the monomer-monomer interfaces (8%).

    The results from the interaction study indicated conformational changes upon interaction between the A. thaliana Med25 ACtivator Interaction Domain (ACID) and Dreb2a. Structural changes were probed to originate from unstructured Dreb2a and not from the Med25-ACID. Human Med25-ACID was also found to interact with the plant-specific Dreb2a, even though the ACIDs from human and A. thaliana share low sequence homology. Moreover, the human Med25-interacting transcription factor VP16 was found to interact with A. thaliana Med25. Finally, NMR, ITC and pull-down experiments showed that the unrelated transcription factors Dreb2a and

    VP16 interact with overlapping regions in the ACIDs of A. thaliana and human Med25.

    The results presented in this thesis contribute to previous reports in two different aspects. Firstly, they lend support to the findings that the intracellular environment affects the biophysical properties of proteins. It will therefore be important to continue comparing results between in vitro and cell-like conditions to measure the magnitude of such effects and to improve the understanding of protein folding and thereby misfolding of proteins in cells. Better knowledge of protein misfolding mechanisms is critical since they are associated to several neurodegenerative diseases such as Alzheimer’s and Parkinson's. Secondly, our results substantiate the notion that transcription factors are able to bind multiple targets and that they gain structure upon binding. They also show that subunits of the conserved Mediator complex, despite low sequence homologies, retain a conserved structure and function when comparing evolutionary diverged species.

  • 4.
    Aguilar, Ximena
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Schleucher, Jurgen
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Interaction Studies of the Human and Arabidopsis thaliana Med25-ACID Proteins with the Herpes Simplex Virus VP16-and Plant-Specific Dreb2a Transcription Factors2014Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 5, s. e98575-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional coregulator by conveying signals from activators and repressors to the RNA polymerase II transcription machinery. The Arabidopsis thaliana Med25 (aMed25) ACtivation Interaction Domain (ACID) interacts with the Dreb2a activator which is involved in plant stress response pathways, while Human Med25-ACID (hMed25) interacts with the herpes simplex virus VP16 activator. Despite low sequence similarity, hMed25-ACID also interacts with the plant-specific Dreb2a transcriptional activator protein. We have used GST pull-down-, surface plasmon resonance-, isothermal titration calorimetry and NMR chemical shift experiments to characterize interactions between Dreb2a and VP16, with the hMed25 and aMed25-ACIDs. We found that VP16 interacts with aMed25-ACID with similar affinity as with hMed25-ACID and that the binding surface on aMed25-ACID overlaps with the binding site for Dreb2a. We also show that the Dreb2a interaction region in hMed25-ACID overlaps with the earlier reported VP16 binding site. In addition, we show that hMed25-ACID/Dreb2a and aMed25-ACID/Dreb2a display similar binding affinities but different binding energetics. Our results therefore indicate that interaction between transcriptional regulators and their target proteins in Mediator are less dependent on the primary sequences in the interaction domains but that these domains fold into similar structures upon interaction.

  • 5. Ahad, Abdul
    et al.
    Keech, Olivier
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Sjödin, Andreas
    Lindén, Pernilla
    Brouwer, Bastiaan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Stenlund, Hans
    Moritz, Thomas
    Jansson, Stefan
    Gardeström, Per
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Comparison between leaves from darkened plants and individually-darkened leaves reveals differential metabolic strategies in response to darknessManuskript (preprint) (Övrigt vetenskapligt)
  • 6. Ahlstrand, Tuuli
    et al.
    Tuominen, Heidi
    Beklen, Arzu
    Torittu, Annamari
    Oscarsson, Jan
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Sormunen, Raija
    Pöllänen, Marja T.
    Permi, Perttu
    Ihalin, Riikka
    A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-82017Ingår i: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 8, nr 2, s. 115-134Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI− mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

  • 7.
    Alanentalo, Tomas
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Optical projection tomography based 3D-spatial and quantitative assessments of the diabetic pancreas2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The gastrointestinal tract comprises a number of digestive organs including the stomach and pancreas. The stomach is involved in the digestion and short term storage of food while the pancreas is a mixed endocrine and exocrine gland which provides the body with hormones and enzymes essential for nutritional utilisation. The pancreas consists of three different cell lineages, acinar, ductal and endocrine cells. The endocrine cells, organised in the islets of Langerhans, are scattered throughout the exocrine parenchyma and regulate blood glucose levels by production of hormones such as glucagon and insulin.

    The Nkx family of homeodomain proteins controls numerous processes during development. Previous studies have identified two members belonging to the Nkx6 subfamily of Nkx proteins, Nkx6.1 and Nkx6.2. We have described the cloning and embryonic expression pattern of Nkx6.3. All three members of the Nkx6 gene family were shown to be expressed in partially overlapping domains during the development of the gastrointestinal tract and the central nervous system. Nkx6.2 was also identified as a transient marker for pancreatic exocrine cells.

    Analysing gene expression patterns and morphological features in tissues and organs is often performed by stereologic sampling which is a labour-intensive two dimensional approach that rely on certain assumptions when calculating e.g. β-cell mass and islet number in the pancreas. By combined improvements in immunohistochemical protocols, computational processing and tomographic scanning, we have developed a methodology based on optical projection tomography (OPT) allowing for 3D visualisation and quantification of specifically labelled objects within intact adult mouse organs. In the pancreas, this technique allows for spatial and quantitative measurements of total islet number and β-cell mass. We have further developed a protocol allowing for high resolution regional analyses based on global OPT assessments of the pancreatic constitution. This methodology is likely to facilitate detailed cellular and molecular analysis of user defined regions of interest in the pancreas, at the same time providing information on the overall disease state of the gland.

    Type 1 diabetes mellitus (T1D) can occur at any age and is characterized by the marked inability of the pancreas to secrete insulin due to an autoimmune destruction of the insulin producing β-cells. Information on the key cellular and molecular events underlying the recruitment of lymphocytes, their infiltration of the islets of Langerhans and consequent β-cell destruction is essential for understanding the pathogenesis of T1D. Using the developed methodology we have recorded the spatial and quantitative distribution of islet β-cells and infiltrating lymphocytes in the non obese diabetic (NOD) mouse model for T1D. This study shows that the smaller islets, which are predominantly organised in the periphery of the organ, are the first to disappear during the progression of T1D. The larger islets appear more resistant and our data suggest that a compensatory proliferative process is going on side by side with the autoimmune-induced β-cell destruction. Further, the formation of structures resembling tertiary lymphoid organs (TLOs) in areas apparently unaffected by insulitis suggests that local factors may provide cues for the homing of these lymphocytes back to the pancreas.

  • 8. Alekeyenko, Artyom A.
    et al.
    Ho, Joshua W. K.
    Peng, Shouyong
    Gelbart, Marnie
    Tolstorukov, Michael Y.
    Plachetka, Annette
    Kharchenko, Peter V.
    Jung, Youngsook L.
    Gorchakov, Andrey A.
    Larschan, Erica
    Gu, Tingting
    Minoda, Aki
    Riddle, Nicole C.
    Schwartz, Yuri B.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Elgin, Sarah C. R.
    Karpen, Gary H.
    Pirrotta, Vincenzo
    Kuroda, Mitzi I.
    Park, Peter J.
    Sequence-Specific Targeting of Dosage Compensation in Drosophila Favors an Active Chromatin Context2012Ingår i: PLoS Genetics, ISSN 1553-7390, Vol. 8, nr 4, s. e1002646-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE). However, this motif is only similar to 2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

  • 9.
    Al-Furoukh, Natalie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ianni, Alessandro
    Nolte, Hendrik
    Hölper, Soraya
    Krüger, Marcus
    Wanrooij, Sjoerd
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Braun, Thomas
    ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells2015Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1853, nr 10, s. 2580-2591Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Proteostasis is crucial for life and maintained by cellular chaperones and proteases. One major mitochondrial protease is the ClpXP complex, which is comprised of a catalytic ClpX subunit and a proteolytic ClpP subunit. Based on two separate observations, we hypothesized that ClpX may play a leading role in the cellular function of ClpXP. Therefore, we analyzed the effect of ClpX overexpression on a myoblast proteome by quantitative proteomics. ClpX overexpression results in the upregulation of markers of the mitochondria( proteostasis pathway, known as the "mitochondrial unfolded protein response" (UPRmt). Although this pathway is described in detail in Caenorhabditis elegans, it is not clear whether it is conserved in mammals. Therefore, we compared features of the classical nematode UPRmt with our mammalian ClpX-triggered UPRmt dataset. We show that they share the same retrograde mitochondria-to-nucleus signaling pathway that involves the key UPRmt transcription factor CHOP (also known as Ddit3, CEBPZ or GADD153). In conclusion, our data confirm the existence of a mammalian UPRmt that has great similarity to the C elegans pathway. Furthermore, our results illustrate that ClpX overexpression is a good and simple model to study the underlying mechanisms of the UPRmt in mammalian cells.

  • 10. Allas, Ular
    et al.
    Toom, Lauri
    Selyutina, Anastasia
    Maeorg, Uno
    Medina, Ricardo
    Merits, Andres
    Rinken, Ago
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). University of Tartu, Institute of Technology, Nooruse 1, Tartu 50411, Estonia.
    Kaldalu, Niilo
    Tenson, Tanel
    Antibacterial activity of the nitrovinylfuran G1 (Furvina) and its conversion products2016Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikel-id 36844Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    2-Bromo-5-(2-bromo-2-nitrovinyl) furan (G1 or Furvina) is an antimicrobial with a direct reactivity against thiol groups. It is active against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi. By reacting with thiol groups it causes direct damage to proteins but, as a result, is very short-living and interconverts into an array of reaction products. Our aim was to characterize thiol reactivity of G1 and its conversion products and establish how much of antimicrobial and cytotoxic effects are due to the primary activity of G1 and how much can be attributed to its reaction products. Stability of G1 in growth media as well as its conversion in the presence of thiols was characterized. The structures of G1 decomposition products were determined using NMR and mass-spectroscopy. Concentration-and time-dependent killing curves showed that G1 is bacteriostatic for Escherichia coli at the concentration of 16 mu g/ml and bactericidal at 32 mu g/ml. However, G1 is inefficient against non-growing E. coli. Addition of cysteine to medium reduces the antimicrobial potency of G1. Nevertheless, the reaction products of G1 and cysteine enabled prolonged antimicrobial action of the drug. Therefore, the activity of 2-bromo-5-(2-bromo-2-nitrovinyl) furan is a sum of its immediate reactivity and the antibacterial effects of the conversion products.

  • 11. Alriksson, Björn
    et al.
    Horváth, Ilona Sárvári
    Jönsson, Leif J
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Department of Chemistry and Biomedical Sciences, Karlstad University.
    Overexpression of Saccharomyces cerevisiae transcription factor and multidrug resistance genes conveys enhanced resistance to lignocellulose-derived fermentation inhibitors2010Ingår i: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 45, nr 2, s. 264-271Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The presence of fermentation inhibitors in lignocellulose hydrolysates is an obstacle for achieving efficient fermentation of lignocellulose hydrolysates to ethanol and other commodities. In this investigation, the possibility of generating more inhibitor-resistant Saccharomyces cerevisiae by genetic engineering was explored. Based on previous results from studies of deletion mutants, three S. cerevisiae genes (ATR1, FLR1, YAP1) involved in multidrug resistance and stress response of yeast were selected for overexpression in three S. cerevisiae strains. The resistance of the transformed strains to lignocellulose-derived fermentation inhibitors and a dilute-acid spruce hydrolysate was evaluated in fermentation experiments. Overexpression of FLR1 resulted in enhanced resistance to the phenolic inhibitor coniferyl aldehyde and the furan aldehyde HMF (5-hydroxymethyl-2-furaldehyde). Overexpression of ATR1 conferred increased resistance to coniferyl aldehyde. Strains overexpressing YAP1, which encodes a transcription factor, displayed increased resistance to coniferyl aldehyde, HMF, and the spruce hydrolysate. An ethanol productivity of 0.17 g ethanol × l−1 × h−1 was achieved for a YAP1-overexpressing transformant cultivated in spruce hydrolysate, whereas a control transformant, which did not overexpress YAP1, only reached a productivity of 0.05 g ethanol × l−1 × h−1

  • 12.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Costa, Tiago
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Farag, Salah
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Avican, Ummehan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis2013Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 10, artikel-id e77767Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.

  • 13.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Gurung, Jyoti
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Costa, Tiago
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ruuth, Kristina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Zavialov, Anton
    Joint Biotechnology Laboratory, Department of Chemistry, University of Turku, Turku, Finland.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis2016Ingår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, artikel-id 66Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopNW279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.

  • 14.
    Andersson, Charlotta
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Klinisk kemi.
    Li, Xingru
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Klinisk kemi.
    Lorenz, Fryderyk
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
    Golovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Wahlin, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
    Li, Aihong
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Klinisk kemi.
    Reduction in WT1 Gene Expression During Early Treatment Predicts the Outcome in Patients With Acute Myeloid Leukemia2012Ingår i: Diagnostic molecular pathology (Print), ISSN 1052-9551, E-ISSN 1533-4066, Vol. 21, nr 4, s. 225-233Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Wilms tumor gene 1 (WT1) expression has been suggested as an applicable minimal residual disease marker in acute myeloid leukemia (AML). We evaluated the use of this marker in 43 adult AML patients. Quantitative assessment of WT1 gene transcripts was performed using real-time quantitative-polymerase chain reaction assay. Samples from both the peripheral blood and the bone marrow were analyzed at diagnosis and during follow-up. A strong correlation was observed between WT1 normalized with 2 different control genes (beta-actin and ABL1, P < 0.001). WT1 mRNA level at diagnosis was of no prognostic relevance (P > 0.05). A >= 1-log reduction in WT1 expression in bone marrow samples taken < 1 month after diagnosis significantly correlated with an improved overall survival (P = 0.004) and freedom from relapse (P = 0.010) when beta-actin was used as control gene. Furthermore, a reduction in WT1 expression by >= 2 logs in peripheral blood samples taken at a later time point significantly correlated with a better outcome for overall survival (P = 0.004) and freedom from relapse (P = 0.012). This result was achieved when normalizing against both b-actin and ABL1. These results therefore suggest that WT1 gene expression can provide useful information for minimal residual disease detection in adult AML patients and that combined use of control genes can give more informative results.

  • 15.
    Andersson, David C.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Martinez, N.
    Zeller, D.
    Rondahl, S. H.
    Koza, M. M.
    Frick, B.
    Ekstrom, F.
    Peters, J.
    Linusson, Anna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Changes in dynamics of alpha-chymotrypsin due to covalent inhibitors investigated by elastic incoherent neutron scattering2017Ingår i: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 19, nr 37, s. 25369-25379Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An essential role of enzymes is to catalyze various chemical reactions in the human body and inhibition of the enzymatic activity by small molecules is the mechanism of action of many drugs or tool compounds used to study biological processes. Here, we investigate the effect on the dynamics of the serine protease alpha-chymotrypsin when in complex with two different covalently bound inhibitors using elastic incoherent neutron scattering. The results show that the inhibited enzyme displays enhanced dynamics compared to the free form. The difference was prominent at higher temperatures (240-310 K) and the type of motions that differ include both small amplitude motions, such as hydrogen atom rotations around a methyl group, and large amplitude motions, such as amino acid side chain movements. The measurements were analyzed with multivariate methods in addition to the standard univariate methods, allowing for a more in-depth analysis of the types of motions that differ between the two forms. The binding strength of an inhibitor is linked to the changes in dynamics occurring during the inhibitor-enzyme binding event and thus these results may aid in the deconvolution of this fundamental event and in the design of new inhibitors.

  • 16.
    Andersson, Karin
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Prefibrillar oligomeric Transthyretin mutants - amyloid conformation, toxicity and association with Serum amyloid P component2005Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Amyloidoses represent a heterogeneous group of diseases characterized by abnormal protein metabolism leading to extracellular deposition of fibrillar, proteinaceous amyloid in various tissues and organs of the body. To date more than 20 different proteins have been linked to diseases with amyloid depositions, of which Alzheimer’s disease and the prion-associated diseases are the most well known. Despite the origin of protein in the amyloid, the fibrils share some common biochemical and biophysical properties such as a diameter of 8-13 nm, a β-pleated sheet secondary structure packed in an ordered crystal-like way, Congo red and thioflavin binding with characteristic spectroscopic patterns and decoration of the fibrils with Serum amyloid P component and glycoseaminoglycans.

    The plasma protein transthyretin (TTR) is associated with familial amyloidosis with polyneuropathy (FAP) and senile systemic amyloidosis (SSA). FAP is a lethal, autosomal inherited disorder caused by point mutations in the TTR-gene. More than 80 different mutations have been associated with amyloid formation and linked to FAP. The interpretation is that amino acid replacements at different sites of the polypeptide lead to reduced stability. Mutant TTR were constructed that have a strong tendency to self-aggregate under physiological conditions. The precipitates were shown to be amyloid by staining with thioflavin T and Congo red. As the mutants were sensitive to trypsin cleavage compared to plasma TTR, we suggest that the mutants represent amyloid precursors or that they may share structural properties with intermediates on a pathway leading to amyloid deposition. Monoclonal antibodies were generated that exclusively recognize the amyloidogenic folding of TTR providing direct biochemical evidence for a structural change in amyloidogenic intermediates. Two cryptic epitopes were mapped to a domain of TTR, where most mutations associated with amyloidosis occur and is proposed to be displaced at the initial phase of amyloid formation. Amyloidogenic intermediates of TTR were shown to induce a toxic, free radical dependent, response in cultured neuroblastoma cells. Morphological studies revealed a correlation between toxicity (apoptosis) and the presence of immature amyloid suggesting that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.

    Serum amyloid P component (SAP) is a highly conserved plasma glycoprotein universally found associated with amyloid depositions independently of protein origin. SAP’s role in amyloid formation is contradictory since both inhibition and promotion of aggregation have been shown in the case of fibril formation from the Aβ peptide of Alzheimer’s disease. Amyloidogenic prefibrils of TTR were shown to bind SAP and no interference with aggregation was detected. SAP co-localize in patches with mutant TTR on the surface of neuroblastoma cells and prevent apoptosis induced by mutant TTR and Aβ peptide, while several other molecules known to decorate amyloid fibrils were without effect.

  • 17.
    Andersson, Marie
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Immunopathogenesis of relapsing fever borreliosis2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Relapsing fever (RF) is caused by different species of Borrelia transmitted by soft ticks or by the human body louse. Illness is characterized by reappearing peaks of high concentrations of spirochetes in blood, concordant with fever peaks separated by asymptomatic periods. Neuroborreliosis is one of the most severe manifestations of RF borreliosis. To understand the immune response during early RF, we analyzed immune cells in brain and kidney of mice infected with B. crocidurae during the acute infection. Our results indicate that brain defense is comprised primarily of innate immune cells. Despite the infiltration of innate immune cells, Borrelia was not completely eradicated. A failure of the host brain to clear the bacteria may give the pathogen a niche where it can persist. Using our mouse model, we revealed that Borrelia duttonii could persist in the mouse brain for up to 270 days, without being present in the circulation. The infection was silent with no change in host gene expression, and the spirochetes could re-enter the circulation after immunosuppression. We propose that the brain is used by the pathogen to evade host immunity and serves as a possible natural reservoir for B. duttonii, a spirochete that has rarely been found in any mammalian host other than man. Borrelia-induced complications during pregnancy have been reported, and are especially common in RF. In our established mouse model of gestational RF, we could show that the fetuses suffered from severe pathology and growth retardation, probably as a consequence of placental destruction. We could also show trans-placental transmission of the bacteria leading to neonatal RF. Surprisingly, pregnant dams had a lower bacterial load and less severe disease, showing that pregnancy has a protective effect during RF. We have used the gestational RF model to investigate host factors favoring disease resolution. Because the spleen is the primary organ responsible for trapping and removing blood-borne pathogens, we have compared temporal changes in spleen immune cell populations and cytokine/chemokine induction during the infection. Spleens of pregnant mice had earlier neutrophil infiltration, as well as faster and higher production of pro-inflammatory mediators. This rapid, robust response suggests a more effective host defense. Thus, an enhanced pro-inflammatory response during pregnancy imparts a distinct advantage in controlling the severity of relapsing fever infection.

  • 18.
    Andersson, Niki
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och geovetenskap.
    Biology and biodiversity of tardigrades in the world and in Sweden: Current status and future visions2017Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Tardigrades are small water-dwelling invertebrates that can live almost anywhere in the world. Even though they are well-known our knowledge about them is still scarce. The aim of this study was therefore to explore our current knowledge about tardigrades by: (1) explore their global phylogeny and biogeography based on bioinformatics (2) screen for tardigrades in select locations of northern Sweden and compare with other Swedish locations, and (3) identify at least one tardigrade from northern Sweden and explore the published biomarkers for further identification. The bulk of this thesis was based on evaluation of the Silva database for analyzing SSU (small subunit) and LSU (large subunit) tardigrade sequences and create phylogenetic trees. Some initial lab work was performed using samples of moss and lichen from Piteå, Vindeln and Öland. Results show that only few countries have been explored with regard to tardigrades, and in Sweden more research have been performed in the south compared to the north. The phylogenetic trees give a rough overview of tardigrade relatedness but many of the sequences need to be improved and more sequence work from additional environments is needed. In the lab tardigrades were only found from the Piteå samples, and one of those was identified as Macrobiotus hufelandi, for which a new biomarker was created. Overall, tardigrade research need to continue and expand to other regions in order to understand how these organisms differ between different environments, and more work is needed to ensure higher quality of sequences added to databases.

  • 19. Andres Valderrama, J
    et al.
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Carmona, Manuel
    Diaz, Eduardo
    AccR is a master regulator involved in carbon catabolite repression of the anaerobic catabolism of aromatic compounds in Azoarcus sp CIB2014Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, nr 4, s. 1892-1904Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Here we characterized the first known transcriptional regulator that accounts for carbon catabolite repression (CCR) control of the anaerobic catabolism of aromatic compounds in bacteria. The AccR response regulator of Azoarcus sp. CIB controls succinate-responsive CCR of the central pathways for the anaerobic catabolism of aromatics by this strain. Phosphorylation of AccR to AccR-P triggers a monomer-to-dimer transition as well as the ability to bind to the target promoter and causes repression both in vivo and in vitro. Substitution of the Asp(60) phosphorylation target residue of the N-terminal receiver motif of AccR to a phosphomimic Glu residue generates a constitutively active derivative that behaves as a superrepressor of the target genes. AccR-P binds in vitro to a conserved inverted repeat (ATGCA-N-6-TGCAT) present at two different locations within the P-N promoter of the bzd genes for anaerobic benzoate degradation. Because the DNA binding-proficient C-terminal domain of AccR is monomeric, we propose an activation mechanism in which phosphorylation of Asp(60) of AccR alleviates interdomain repression mediated by the N-terminal domain. The presence of AccR-like proteins encoded in the genomes of other -proteobacteria of the Azoarcus/Thauera group further suggests that AccR constitutes a master regulator that controls anaerobic CCR in these bacteria.

  • 20.
    Andresen, Liis
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Tenson, Tanel
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia.
    Cationic bactericidal peptide 1018 does not specifically target the stringent response alarmone (p)ppGpp2016Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikel-id 36549Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial stringent response is a key regulator of bacterial virulence, biofilm formation and antibiotic tolerance, and is a promising target for the development of new antibacterial compounds. The intracellular nucleotide (p)ppGpp acts as a messenger orchestrating the stringent response. A synthetic peptide 1018 was recently proposed to specifically disrupt biofilms by inhibiting the stringent response via direct interaction with (p) ppGpp (de la Fuente-Nunez et al. (2014) PLoS Pathogens). We have interrogated the specificity of the proposed molecular mechanism. When inhibition of Pseudomonas aeruginosa planktonic and biofilm growth is tested simultaneously in the same assay, peptides 1018 and the control peptide 8101 generated by an inversion of the amino acid sequence of 1018 are equally potent, and, importantly, do not display a preferential activity against biofilm. 1018 inhibits planktonic growth of Escherichia coli equally efficiently either when the alleged target, (p) ppGpp, is essential (MOPS media lacking amino acid L-valine), or dispensable for growth (MOPS media supplemented with L-valine). Genetic disruption of the genes relA and spoT responsible for (p) ppGpp synthesis moderately sensitizes-rather than protects-E. coli to 1018. We suggest that the antimicrobial activity of 1018 does not rely on specific recognition of the stringent response messenger (p) ppGpp.

  • 21.
    André, Domenique
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    The role of the Populus FT genes in the regulation of tree growth.2014Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    Abstract [en]

    In annual plants, flowering related genes were initially thought to have the only function to coordinate and execute reproductive development. In perennial species like poplar, however, it became clear that these genes seem to have acquired additional functions in the regulation of the yearly growth cycle. The two poplar orthologs of the Arabidopsis “florigen” FLOWERING LOCUS T (FT), PtFT1 and PtFT2, are associated with flowering as well as other aspects of phenology. After duplication they have diverged in expression pattern, and maybe also in function and their gene regulatory networks. In this study I have characterized the role of PtFT1 and PtFT2 during the yearly growth cyle, as well as the interaction between, FT-like genes, poplar homologs of the Arabidopsis flowering time repressor TFL1 and PHYTOCHROME A in the regulation of tree flowering. The preliminary data show new unexpected functional relations between these genes and new previously undescribed effects on plant growth and development.

  • 22.
    Antonsson, Åsa
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak).
    Regulation of NF-κB by Calmodulin2003Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Cells experience numerous external signals which they must respond to. Such signals arriving at the cell surface are transduced via various signal transduction pathways and often ultimately result in regulation of transcription. NF-κB is a family of transcription factors involved in the regulation of genes important for processes such as immune and inflammatory responses, cell growth, development and cell survival. NF-κB proteins are normally kept inactive in the cytoplasm due to masking of their nuclear localisation signal (NLS) by inhibitory IκB proteins. A large number of stimuli lead to the activation of IκB-kinase (IKK). Active IKK phosphorylates IκB and thereby labels it for ubiquitination and, subsequently, degradation by the proteasome. Liberated NF-κB enters the nucleus, where it takes part in the regulation of its target genes.

    Calmodulin (CaM) is a ubiquitous Ca2+-binding protein which is considered to be the predominant intracellular Ca2+ sensor. CaM plays a major role in the Ca2+-dependent regulation of a wide variety of cellular processes, including transcription. CaM regulates transcription both indirectly through CaM-dependent kinases and phosphatases and directly through interaction with transcription factors.

    CaM was found to bind directly and in a Ca2+-dependent fashion to the two NF-κB family members c-Rel and RelA. The CaM-NF-κB interactions were strongly enhanced by NF-κB activating stimuli and this enhancement was blocked by the addition of IκB, suggesting that c-Rel and RelA can bind CaM after their signal-induced release from IκB. Compared to wild-type c-Rel, CaM binding-deficient mutants were shown to exhibit an increased nuclear accumulation and transcriptional activity on Ca2+-regulated cytokine promoters. The results suggest that CaM can inhibit transport of c-Rel, but not of RelA, to the nucleus and thereby differentially regulate the activation of NF-κB proteins following cell stimulation. CaM was also found to affect NF-κB activity indirectly through the action of a CaM-dependent kinase (CaMK). Studies of the events leading to IκBα phosphorylation revealed that CaM and CaMKII inhibitors blocked phorbol ester induced activation of IKK. Furthermore, CaM and CaMKII inhibitors also blocked T cell receptor/CD3 induced IκBα degradation, and expression of an inhibitor-resistant derivative of the γ isoform of CaMKII caused the inhibitors lose their effect on phorbol ester induced IκBα degradation. Finally, expression of a constitutively active CaMKII resulted in the activation of NF-κB. These results identify CaMKII as a mediator of IKK activation, specifically in response to T cell receptor/CD3 and phorbol ester stimulation.

    In conclusion, this thesis describes the identification of CaM as a dual regulator of NF-κB proteins, acting both directly and indirectly to affect the activity of this family of transcription factors.

  • 23. Arend, Andres
    et al.
    Masso, Raivo
    Masso, Marika
    Selstam, Gunnar
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Electron microscope immunocytochemical localization of cyclooxygenase-1 and-2 in pseudopregnant rat corpus luteum during luteolysis2004Ingår i: Prostaglandins & other lipid mediators, ISSN 1098-8823, E-ISSN 2212-196X, Vol. 74, nr 1-4, s. 1-10Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Prostaglandins converted from arachidonic acid by cyclooxygenases play an important regulatory role in regression of the corpus luteum. To reveal luteal distribution of cyclooxygenase isoforms during luteolysis, an electron microscope immunocytochemical study was performed. Cyclooxygenase-1 and -2 were found both in luteal steroid-producing and interstitial cells on days 13, 15 and 18 of the adult pseudopregnant rat. Cyclooxygenase-2 immunolabelling was predominantly seen in non-luteal cells. The two enzymes were localized in similar fashion to the plasma membrane, rough and smooth endoplasmic reticulum, lipid bodies and mitochondria, but differently in the nuclear compartment. Cyclooxygenase-1 labelling was found only in the perinuclear region, while cyclooxygenase-2 was localized to the nuclear envelope, region of condensed heterochromatin as well as at the perimeter of the heterochromatin. Nuclear residence may indicate additional roles for cyclooxygenase-2 in regulating gene expression. Identification of both enzymes on lipid bodies suggests that these inclusions may be involved in luteal prostanoid production.

  • 24. Arioz, Candan
    et al.
    Li, Yaozong
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wittung-Stafshede, Pernilla
    The six metal binding domains in human copper transporter, ATP7B: molecular biophysics and disease-causing mutations2017Ingår i: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 30, nr 6, s. 823-840Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Wilson Disease (WD) is a hereditary genetic disorder, which coincides with a dysfunctional copper (Cu) metabolism caused by mutations in ATP7B, a membrane-bound P-1B-type ATPase responsible for Cu export from hepatic cells. The N-terminal part (similar to 600 residues) of the multi-domain 1400-residue ATP7B constitutes six metal binding domains (MBDs), each of which can bind a copper ion, interact with other ATP7B domains as well as with different proteins. Although the ATP7B's MBDs have been investigated in vitro and in vivo intensively, it remains unclear how these domains modulate overall structure, dynamics, stability and function of ATP7B. The presence of six MBDs is unique to mammalian ATP7B homologs, and many WD causing missense mutations are found in these domains. Here, we have summarized previously reported in vitro biophysical data on the MBDs of ATP7B and WD point mutations located in these domains. Besides the demonstration of where the research field stands today, this review showcasts the need for further biophysical investigation about the roles of MBDs in ATP7B function. Molecular mechanisms of ATP7B are important not only in the development of new WD treatment but also for other aspects of human physiology where Cu transport plays a role.

  • 25. Arshadi, Mehrdad
    et al.
    Nilsson, Calle
    Magnusson, Bengt
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gas chromatography-mass spectrometry determination of the pentafluorobenzoyl derivative of methylhydrazine in false morel (Gyromitra esculenta) as a monitor for the content of the toxin gyromitrin2006Ingår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1125, nr 2, s. 229-233Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The main toxic compound found in false morel (Gyromitra esculenta) is acetaldehyde-N-methyl-N-formylhydrazone (gyromitrin). This paper describes a method of determining the total hydrazones content based on acid hydrolysis of gyromitrin and other related hydrazones in air-dried false morel followed by derivatisation of methylhydrazine with pentafluorobenzoyl chloride. The derivative, tris-pentafluorobenzoyl methylhydrazine (tris-PFB-MH) is analyzed by gas chromatography–mass spectrometry. The overall precision of the method is better than 10% (relative standard deviation) for 0.5 ng/μl methylhydrazine in solution. The minimum detectable concentration of methylhydrazine (tris-PFB-MH) by this method is estimated to be approximately 12 pg/μl, which is equal to 0.3 μg/g dry matter (DM) of false morel.

  • 26.
    Artursson, Elisabet
    et al.
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Andersson, Per Ola
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Akfur, Christine
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Linusson, Anna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Börjegren, Susanne
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Ekström, Fredrik
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Catalytic-site conformational equilibrium in nerve-agent adducts of acetylcholinesterase: Possible implications for the HI-6 antidote substrate specificity2013Ingår i: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 85, nr 9, s. 1389-1397Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Nerve agents such as tabun, cyclosarin and Russian VX inhibit the essential enzyme acetylcholinesterase (AChE) by organophosphorylating the catalytic serine residue. Nucleophiles, such as oximes, are used as antidotes as they can reactivate and restore the function of the inhibited enzyme. The oxime HI-6 shows a notably low activity on tabun adducts but can effectively reactivate adducts of cyclosarin and Russian VX. To examine the structural basis for the pronounced substrate specificity of HI-6, we determined the binary crystal structures of Mus musculus AChE (mAChE) conjugated by cyclosarin and Russian VX and found a conformational mobility of the side chains of Phe338 and His447. The interaction between HI-6 and tabun-adducts of AChE were subsequently investigated using a combination of time resolved fluorescence spectroscopy and X-ray crystallography. Our findings show that HI-6 binds to tabun inhibited Homo sapiens AChE (hAChE) with an IC50 value of 300 μM and suggest that the reactive nucleophilic moiety of HI-6 is excluded from the phosphorus atom of tabun. We propose that a conformational mobility of the side-chains of Phe338 and His447 is a common feature in nerve-agent adducts of AChE. We also suggest that the conformational mobility allow HI-6 to reactivate conjugates of cyclosarin and Russian VX while a reduced mobility in tabun conjugated AChE results in steric hindrance that prevents efficient reactivation.

  • 27. Artursson, Tom
    et al.
    Hagman, Anders
    Björk, Seth
    Trygg, Johan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wold, Svante
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Jacobsson, Sven P
    Study of Preprocessing Methods for the Determination of Crystalline Phases in Binary Mixtures of Drug Substances by X-ray Powder Diffraction and Multivariate Calibration2000Ingår i: Applied Spectroscopy, ISSN 0003-7028, E-ISSN 1943-3530, Vol. 54, nr 8, s. 272A-301AArtikel i tidskrift (Refereegranskat)
    Abstract [en]

    In this paper, various preprocessing methods were tested on data generated by X-ray powder diffraction (XRPD) in order to enhance the partial least-squares (PLS) regression modeling performance. The preprocessing methods examined were 22 different discrete wavelet transforms, Fourier transform, Savitzky-Golay, orthogonal signal correction (OSC), and combinations of wavelet transform and OSC, and Fourier transform and OSC. Root mean square error of prediction (RMSEP) of an independent test set was used to measure the performance of the various preprocessing methods. The best PLS model was obtained with a wavelet transform (Symmlet 8), which at the same time compressed the data set by a factor of 9.5. With the use of wavelet and X-ray powder diffraction, concentrations of less than 10% of one crystal from could be detected in a binary mixture. The linear range was found to be in the range 10-70% of the crystalline form of phenacetin, although semiquantitative work could be carried out down to a level of approximately 2%. Furthermore, the wavelet-pretreated models were able to handle admixtures and deliberately added noise.

  • 28.
    Ashelford, Kevin
    et al.
    School of Biological Sciences, University of Liverpool, Liverpool, UK.
    Eriksson, Maria E
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Allen, Christopher M
    Applied Biosystems, part of Life Technologies, Warrington, UK.
    D’Amore, Linda
    School of Biological Sciences, University of Liverpool, Liverpool, UK.
    Johansson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Gould, Peter
    School of Biological Sciences, University of Liverpool, Liverpool, UK.
    Kay, Susanne
    School of Biological Sciences, University of Liverpool, Liverpool, UK.
    Millar, Andrew J.
    Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, Edinburgh, UK.
    Hall, Neil
    School of Biological Sciences, University of Liverpool, Liverpool, UK.
    Hall, Anthony
    School of Biological Sciences, University of Liverpool, Liverpool, UK.
    Full genome re-sequencing reveals a novel circadian clock mutationin Arabidopsis2011Ingår i: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 12, s. R28-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Map based cloning in Arabidopsis thaliana can be a difficult and time-consuming process,specifically if the phenotype is subtle and scoring labour intensive. An alternative to map basedcloning would be to directly sequence the whole genome of a mutant to uncover the mutationresponsible for the phenotype.

    Results: Here, we have re-sequenced the 120 Mb genome of a novel Arabidopsis clock mutant earlybird (ebi-1), using massively parallel sequencing by ligation. This process was further complicated by the fact that ebi-1 is in Wassilewskija (Ws-2), not the reference accession ofArabidopsis. The approach reveals evidence of DNA strand bias in the ethyl methanesulfonate(EMS) mutation process. We have demonstrated the utility of sequencing a backcrossed line andusing gene expression data to limit the number of SNP considered. Using new SNP informationwe have excluded a previously identified clock gene, PRR7. Finally, we have identified a SNPin the gene AtNFXL-2 as the likely cause of the ebi-1 phenotype and validated this bycharacterising a further allele.

    Conclusion: In Arabidopsis, as in other organisms, the (EMS) mutation load can be high. Here wedescribe how sequencing a backcrossed line, using functional genomics and analysing new SNPinformation can be used to reduce the number EMS mutations for consideration. Moreover, theapproach we describe here does not require out-crossing and scoring F2 mapping populations, anapproach which can be compromised by background effects. The strategy has broad utility andwill be an extremely useful tool to identify causative SNP in other organisms.

  • 29.
    Atkinson, Gemma C.
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia .
    Kuzmenko, Anton
    University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia & Department of Molecular Biology, Faculty of Biology, Moscow State University, Moscow, Russia .
    Chicherin, Ivan
    Department of Molecular Biology, Faculty of Biology, Moscow State University, Moscow, Russia.
    Soosaar, Axel
    University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia .
    Tenson, Tanel
    University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia .
    Carr, Martin
    School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield, UK.
    Kamenski, Piotr
    Department of Molecular Biology, Faculty of Biology, Moscow State University, Moscow, Russia .
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia .
    An evolutionary ratchet leading to loss of elongation factors in eukaryotes2014Ingår i: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 14, s. 35-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The GTPase eEF1A is the eukaryotic factor responsible for the essential, universal function of aminoacyl-tRNA delivery to the ribosome. Surprisingly, eEF1A is not universally present in eukaryotes, being replaced by the paralog EFL independently in multiple lineages. The driving force behind this unusually frequent replacement is poorly understood. Results: Through sequence searching of genomic and EST databases, we find a striking association of eEF1A replacement by EFL and loss of eEF1A's guanine exchange factor, eEF1Ba, suggesting that EFL is able to spontaneously recharge with GTP. Sequence conservation and homology modeling analyses indicate several sequence regions that may be responsible for EFL's lack of requirement for eEF1Ba. Conclusions: We propose that the unusual pattern of eEF1A, eEF1Ba and EFL presence and absence can be explained by a ratchet-like process: if either eEF1A or eEF1Ba diverges beyond functionality in the presence of EFL, the system is unable to return to the ancestral, eEF1A:eEFBa-driven state.

  • 30.
    Atkinson, Gemma Catherine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Institute of Technology, University of Tartu, Nooruse 1, Tartu, 50411, Estonia.
    The evolutionary and functional diversity of classical and lesser-known cytoplasmic and organellar translational GTPases across the tree of life2015Ingår i: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 16, artikel-id 78Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The ribosome translates mRNA to protein with the aid of a number of accessory protein factors. Translational GTPases (trGTPases) are an integral part of the 'core set' of essential translational factors, and are some of the most conserved proteins across life. This study takes advantage of the wealth of available genomic data, along with novel functional information that has come to light for a number of trGTPases to address the full evolutionary and functional diversity of this superfamily across all domains of life. 

    Results: Through sensitive sequence searching combined with phylogenetic analysis, 57 distinct subfamilies of trGTPases are identified: 14 bacterial, 7 archaeal and 35 eukaryotic (of which 21 are known or predicted to be organellar). The results uncover the functional evolution of trGTPases from before the last common ancestor of life on earth to the current day. 

    Conclusions: While some trGTPases are universal, others are limited to certain taxa, suggesting lineage-specific translational control mechanisms that exist on a base of core factors. These lineage-specific features may give organisms the ability to tune their translation machinery to respond to their environment. Only a fraction of the diversity of the trGTPase superfamily has been subjected to experimental analyses; this comprehensive classification brings to light novel and overlooked translation factors that are worthy of further investigation.

  • 31. Aureliano, Manuel
    et al.
    Ohlin, C. André
    Vieira, Michele O.
    Marques, M. Paula M.
    Casey, William H.
    Batista de Carvalho, Luis A. E.
    Characterization of decavanadate and decaniobate solutions by Raman spectroscopy2016Ingår i: Dalton Transactions, ISSN 1477-9226, E-ISSN 1477-9234, Vol. 45, nr 17, s. 7391-7399Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The decaniobate ion, (Nb-10 = [Nb10O28](6-)) being isoelectronic and isostructural with the decavanadate ion (V-10 = [V10O28](6-)), but chemically and electrochemically more inert, has been useful in advancing the understanding of V-10 toxicology and pharmacological activities. In the present study, the solution chemistry of Nb-10 and V-10 between pH 4 and 12 is studied by Raman spectroscopy. The Raman spectra of V-10 show that this vanadate species dominates up to pH 6.45 whereas it remains detectable until pH 8.59, which is an important range for biochemistry. Similarly, Nb-10 is present between pH 5.49 and 9.90 and this species remains detectable in solution up to pH 10.80. V-10 dissociates at most pH values into smaller tetrahedral vanadate oligomers such as V-1 and V-2, whereas Nb-10 dissociates into Nb-6 under mildly (10 > pH > 7.6) or highly alkaline conditions. Solutions of V-10 and Nb-10 are both kinetically stable under basic pH conditions for at least two weeks and at moderate temperature. The Raman method provides a means of establishing speciation in the difficult niobate system and these findings have important consequences for toxicology activities and pharmacological applications of vanadate and niobate polyoxometalates.

  • 32. Avagyan, Rozanna
    et al.
    Nyström, Robin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik.
    Boman, Christoffer
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik.
    Westerholm, Roger
    Determination of hydroxylated polycyclic aromatic hydrocarbons by HPLC-photoionization tandem mass spectrometry in wood smoke particles and soil samples2015Ingår i: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 407, nr 16, s. 4523-4534Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A simple and fast method for analysis of hydroxylated polycyclic aromatic hydrocarbons using pressurized liquid extraction and high performance liquid chromatography utilizing photoionization tandem mass spectrometry was developed. Simultaneous separation and determination of nine hydroxylated polycyclic aromatic hydrocarbons and two hydroxy biphenyls could be performed in negative mode with a run time of 12 min, including equilibration in 5 min. The calibration curves were in two concentration ranges; 1-50 ng/mL and 0.01-50 mu g/mL, with coefficients of correlation R (2) > 0.997. The limits of detection and method quantification limits were in the range of 9-56 pg and 5-38 ng/g, respectively. A two-level full factorial experimental design was used for screening of conditions with the highest impact on the extraction. The extraction procedure was automated and suitable for a large number of samples. The extraction recoveries ranged from 70 to 102 % and the matrix effects were between 92 and 104 %. The overall method was demonstrated on wood smoke particles and soil samples with good analytical performance, and five OH-PAHs were determined in the concentration range of 0.19-210 mu g/g. As far as we know, hydroxylated polycyclic aromatic hydrocarbons were determined in wood smoke and soil samples using photoionization mass spectrometry for the first time in this present study. Accordingly, this study shows that high performance liquid chromatography photoionization tandem mass spectrometry can be a good option for the determination of hydroxylated polycyclic aromatic hydrocarbons in complex environmental samples.

  • 33.
    Avican, Kemal
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Persistent infection by Yersinia pseudotuberculosis2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Enteropathogenic Yersinia species can infect many mammalian organs such as the small intestine, cecum, Peyer’s patches, liver, spleen, and lung and cause diseases that resemble a typhoid-like syndrome, as seen for other enteropathogens. We found that sublethal infection doses of Y. pseudotuberculosis gave rise to asymptomatic persistent infection in mice and identified the cecal lymphoid follicles as the primary site for colonization during persistence. Persistent Y. pseudotuberculosis is localized in the dome area, often in inflammatory lesions, as foci or as single cells, and also in neutrophil exudates in the cecal lumen. This new mouse model for bacterial persistence in cecum has potential as an investigative tool for deeper understanding of bacterial adaptation and host immune defense mechanisms during persistent infection. Here, we investigated the nature of the persistent infection established by Y. pseudotuberculosis in mouse cecal tissue using in vivo RNA-seq of bacteria during early and persistent stages of infection. Comparative analysis of the bacterial transcriptomes revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence in the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26°C. Genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, we show that ArcA, Fnr, FrdA, WrbA, RovA, and RfaH play critical roles in persistence. An extended investigation of the transcriptional regulator rfaH employing mouse infection studies, phenotypic characterizations, and RNA-seq transcriptomics analyses indicated that this gene product contributes to establishment of infection and confirmed that it regulates O-antigen biosynthesis genes in Y. pseudotuberculosis. The RNA-seq results also suggest that rfaH has a relatively global effect. Furthermore, we also found that the dynamics of the cecal tissue organization and microbial composition shows changes during different stages of the infection. Taken together, based on our findings, we speculate that this enteropathogen initiates infection by using its virulence factors in meeting the innate immune response in the cecal tissue. Later on, these factors lead to dysbiosis in the local microbiota and altered tissue organization. At later stages of the infection, the pathogen adapts to the environment in the cecum by reprogramming its transcriptome from a highly virulent mode to a more environmentally adaptable mode for survival and shedding. The in vivo transcriptomic analyses for essential genes during infections present strong candidates for novel targets for antimicrobials.

  • 34.
    Avican, Kemal
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Huss, Mikael
    Heroven, Ann Kathrin
    Beckstette, Michael
    Dersch, Petra
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Reprogramming of Yersinia from Virulent to Persistent Mode Revealed by Complex In Vivo RNA-seq Analysis2015Ingår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 11, nr 1, artikel-id e1004600Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding.

  • 35.
    Avican, Ummehan
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Twin-arginine translocation in Yersinia: the substrates and their role in virulence2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Pathogenic Yersinia cause a manifold of diseases in humans ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to pneumonic and bubonic plague (Y. pestis), while all three have a common virulence strategy that relies on a well-studied type III secretion system and its effector proteins to colonize the host and evade immune responses. However, the role of other protein secretion and/or translocation systems in virulence of Yersinia species is not well known. In this thesis, we sought to investigate the contribution of twin-arginine translocation (Tat) pathway and its secreted substrates to the physiology and virulence of Y. pseudotuberculosis. Tat pathway uniquely exports folded proteins including virulence factors across the cytoplasmic membranes of bacteria. The proteins exported by Tat pathway contain a highly conserved twin-arginine motif in the N-terminal signal peptide. We found that the loss of Tat pathway causes a drastic change of the transcriptome of Y. pseudotuberculosis in stationary phase at environmental temperature with differential regulation of genes involved in virulence, carbon metabolism and stress responses. Phenotypic analysis revealed novel phenotypes of the Tat-deficient strain with defects in iron acquisition, acid resistance, copper oxidation and envelope integrity, which we were partly able to associate with the related Tat substrates. Moreover, increased glucose consumption and accumulation of intracellular fumarate were observed in response to inactivation of Tat pathway implicating a generic effect in cellular physiology. We evaluated the direct role of 22 in silico predicted Tat substrate mutants in the mouse infection model and found only one strain, ΔsufI, exhibited a similar degree of attenuation as Tat-deficient strain. Comparative in vivo characterization studies demonstrated a minor defect for ΔsufI in colonization of intestinal tissues compared to the Tat-deficient strain during early infection, whereas both SufI and TatC were required for dissemination from mesenteric lymph nodes and further systemic spread during late infection. This verifies that SufI has a major role in attenuation seen for the Tat deficient strain both during late infection and initial colonization. It is possible that other Tat substrates such as those involved in iron acquisition and copper resistance also has a role in establishing infection. Further phenotypic analysis indicated that SufI function is required for cell division and stress-survival. Transcriptomic analysis revealed that the highest number of differentially regulated genes in response to loss of Tat and SufI were involved in metabolism and transport. Taken together, this thesis presents a thorough analysis of the involvement of Tat pathway in the overall physiology and virulence strategies of Y. pseudotuberculosis. Finally, we propose that strong effects in virulence render TatC and SufI as potential targets for development of novel antimicrobial compounds

  • 36.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Avican, Kemal
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Transcriptomic and phenotypic analysis of sufI and tatC mutants of Yersinia pseudotuberculosisManuskript (preprint) (Övrigt vetenskapligt)
  • 37.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Beckstette, Michael
    Heroven, Ann Kathrin
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Dersch, Petra
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis2016Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, nr 20, s. 2876-2886Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Twin-arginine translocation (Tat) system mediates secretion of folded proteins that in bacteria, plants and archaea are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and ∆tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26oC and 37oC. The most significant changes in the transcriptome of the ∆tatC mutant were seen at 26oC during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper conditions as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.

  • 38.
    Backman, Lars
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Alpha-actinin of the chlorarchiniophyte Bigelowiella natans2018Ingår i: PeerJ, ISSN 2167-8359, E-ISSN 2167-8359, Vol. 6, artikel-id e4288Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The genome of the chlorarchiniophyte Bigelowiella natans codes for a protein annotated as an α-actinin-like protein. Analysis of the primary sequence indicate that this protein has the same domain structure as other α-actinins, a N-terminal actin-binding domain and a C-terminal calmodulin-like domain. These two domains are connected by a short rod domain, albeit long enough to form a single spectrin repeat. To analyse the functional properties of this protein, the full-length protein as well as the separate domains were cloned and isolated. Characerisation showed that the protein is capable of cross-linking actin filaments into dense bundles, probably due to dimer formation. Similar to human α-actinin, calcium-binding occurs to the most N-terminal EF-hand motif in the calmodulin-like C-terminal domain. The results indicate that this Bigelowiella protein is a proper α-actinin, with all common characteristics of a typical α-actinin.

  • 39.
    Backman, Lars
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Calcium affinity of human α-actinin12015Ingår i: PeerJ, Vol. 3, artikel-id e944Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Due to alternative splicing, the human ACTN1 gene codes for three different transcripts of α-actinin; one isoform that is expressed only in the brain and two with a more general expression pattern. The sequence difference is located to the C-terminal domains and the EF-hand motifs. Therefore, any functional or structural distinction should involve this part of the protein. To investigate this further, the calcium affinities of these three isoforms of α-actinin 1 have been determined by isothermal calorimetry.

  • 40.
    Bajhaiya, Amit K.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Univ Manchester, Fac Life Sci, Manchester, Lancs, England.
    Dean, Andrew P.
    Zeef, Leo A. H.
    Webster, Rachel E.
    Pittman, Jon K.
    PSR1 Is a Global Transcriptional Regulator of Phosphorus Deficiency Responses and Carbon Storage Metabolism in Chlamydomonas reinhardtii2016Ingår i: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 170, nr 3, s. 1216-1234Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many eukaryotic microalgae modify their metabolism in response to nutrient stresses such as phosphorus (P) starvation, which substantially induces storage metabolite biosynthesis, but the genetic mechanisms regulating this response are poorly understood. Here, we show that P starvation-induced lipid and starch accumulation is inhibited in a Chlamydomonas reinhardtii mutant lacking the transcription factor Pi Starvation Response1 (PSR1). Transcriptomic analysis identified specific metabolism transcripts that are induced by P starvation but misregulated in the psr1 mutant. These include transcripts for starch and triacylglycerol synthesis but also transcripts for photosynthesis-, redox-, and stress signaling-related proteins. To further examine the role of PSR1 in regulating lipid and starch metabolism, PSR1 complementation lines in the psr1 strain and PSR1 overexpression lines in a cell wall-deficient strain were generated. PSR1 expression in the psr1 lines was shown to be functional due to rescue of the psr1 phenotype. PSR1 overexpression lines exhibited increased starch content and number of starch granules per cell, which correlated with a higher expression of specific starch metabolism genes but reduced neutral lipid content. Furthermore, this phenotype was consistent in the presence and absence of acetate. Together, these results identify a key transcriptional regulator in global metabolism and demonstrate transcriptional engineering in microalgae to modulate starch biosynthesis.

  • 41. Baker-Austin, Craig
    et al.
    Potrykus, Joanna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wexler, Margaret
    Bond, Philip L
    Dopson, Mark
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). School of Biological Sciences, University of East Anglia, Norwich, UK .
    Biofilm development in the extremely acidophilic archaeon 'Ferroplasma acidarmanus' Fer12010Ingår i: Extremophiles, ISSN 1431-0651, E-ISSN 1433-4909, Vol. 14, nr 6, s. 485-491Artikel i tidskrift (Refereegranskat)
    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.

  • 42.
    Balagopal, Vidya
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fluch, Lydia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Nissan, Tracy
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ways and means of eukaryotic mRNA decay2012Ingår i: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, ISSN 1874-9399, Vol. 1819, nr 6, s. 593-603Artikel, forskningsöversikt (Refereegranskat)
    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. 

  • 43. Baldassarre, Maurizio
    et al.
    Baronio, Cesare M.
    Morozova-Roche, Ludmilla A.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Barth, Andreas
    Amyloid beta-peptides 1-40 and 1-42 form oligomers with mixed beta-sheets2017Ingår i: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 8, nr 12, s. 8247-8254Artikel i tidskrift (Refereegranskat)
    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).

  • 44.
    Barros-Rios, Jaime
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    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 degradability2012Ingår i: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 83, s. 43-50Artikel i tidskrift (Refereegranskat)
    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.

  • 45.
    Bashar Shafiul, Shamrat
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Characterization of cell wall in transgenic aspen with modified xylan acetylation2015Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    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. 

  • 46.
    Bañares de Dios, Guillermo
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Elucidating the role of CSK during early light response and chloroplast development2014Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    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.

  • 47.
    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.1996Ingår i: Matrix Biology, ISSN 0945-053X, E-ISSN 1569-1802, Vol. 15, nr 6, s. 415-422, artikel-id 9049979Artikel i tidskrift (Refereegranskat)
    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.

  • 48.
    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.1996Ingår i: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 785, s. 209-211, artikel-id 8702131Artikel i tidskrift (Refereegranskat)
  • 49.
    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.1997Ingår i: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 66, nr 2, s. 210-218, artikel-id 9213222Artikel i tidskrift (Refereegranskat)
    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.

  • 50. Beljantseva, Jelena
    et al.
    Kudrin, Pavel
    Andresen, Liis
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Shingler, Victoria
    Atkinson, Gemma C.
    Tenson, Tanel
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Institute of Technology, University of Tartu, 50411 Tartu, Estonia.
    Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA2017Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 14, s. 3726-3731Artikel i tidskrift (Refereegranskat)
    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.

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