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  • 1.
    Aguilar-Calvo, Patricia
    et al.
    University of Calif San Diego, CA 92093 USA; University of Calif San Diego, CA 92093 USA.
    Xiao, Xiangzhu
    Case Western Reserve University, OH 44116 USA.
    Bett, Cyrus
    University of Calif San Diego, CA 92093 USA; University of Calif San Diego, CA 92093 USA; US FDA, MD USA.
    Erana, Hasier
    CIC bioGUNE, Spain.
    Soldau, Katrin
    University of Calif San Diego, CA 92093 USA.
    Castilla, Joaquin
    University of Calif San Diego, CA 92093 USA; CIC bioGUNE, Spain; Ikerbasque, Spain.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Surewicz, Witold K.
    Case Western Reserve University, OH 44116 USA.
    Sigurdson, Christina J.
    University of Calif San Diego, CA 92093 USA; University of Calif San Diego, CA 92093 USA; University of Calif Davis, CA 95616 USA.
    Post-translational modifications in PrP expand the conformational diversity of prions in vivo2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, 43295Article in journal (Refereed)
    Abstract [en]

    Misfolded prion protein aggregates (PrPSc) show remarkable structural diversity and are associated with highly variable disease phenotypes. Similarly, other proteins, including amyloid-beta, tau, alpha-synuclein, and serum amyloid A, misfold into distinct conformers linked to different clinical diseases through poorly understood mechanisms. Here we use mice expressing glycophosphatidylinositol (GPI)anchorless prion protein, PrPC, together with hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and a battery of biochemical and biophysical tools to investigate how posttranslational modifications impact the aggregated prion protein properties and disease phenotype. Four GPI-anchorless prion strains caused a nearly identical clinical and pathological disease phenotype, yet maintained their structural diversity in the anchorless state. HXMS studies revealed that GPIanchorless PrPSc is characterized by substantially higher protection against hydrogen/deuterium exchange in the C-terminal region near the N-glycan sites, suggesting this region had become more ordered in the anchorless state. For one strain, passage of GPI-anchorless prions into wild type mice led to the emergence of a novel strain with a unique biochemical and phenotypic signature. For the new strain, histidine hydrogen-deuterium mass spectrometry revealed altered packing arrangements of beta-sheets that encompass residues 139 and 186 of PrPSc. These findings show how variation in posttranslational modifications may explain the emergence of new protein conformations in vivo and also provide a basis for understanding how the misfolded protein structure impacts the disease.

  • 2.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins are essential for all known forms of life and in many lethal diseases protein failure is the cause of the disease. To understand proteins and the processes they are involved in, it is valuable to know their structures as well as their dynamics and interactions. The structures may not be directly inspected because proteins are too small to be visible in a light microscope, which is why indirect methods such as nuclear magnetic resonance (NMR) spectroscopy have to be utilized. This method provides atomic information about the protein and, in contrast to other methods with similar resolution, the measurements are performed in solution resulting in more physiological conditions, enabling analysis of dynamics. Important dynamical processes are the ones on the millisecond timeframe, which may contribute to interactions of proteins and their catalysis of chemical reactions, both of significant value for the function of the proteins.

    To better understand proteins, not only do we need to study them, but also develop the methods we are using. This thesis presents four papers about improved NMR techniques as well as a fifth where the kinase domain of ephrinB receptor 2 (EphB2) has been studied regarding the importance of millisecond dynamics and interactions for the activation process. The first paper presents the software COMPASS, which combines statistics and the calculation power of a computer with the flexibility and experience of the user to facilitate and speed up the process of assigning NMR signals to the atoms in the protein. The computer program PINT has been developed for easier and faster evaluation of NMR experiments, such as those that evaluate protein dynamics. It is especially helpful for NMR signals that are difficult to distinguish, so called overlapped peaks, and the soft- ware also converts the detected signals to the indirectly measured physical quantities, such as relaxation rate constants, principal for dynamics. Next are two new versions of the Carr-Purcell-Maiboom-Gill (CPMG) dispersion pulse sequences, designed to measure millisecond dynamics in a way so that the signals are more separated than in standard experiments, to reduce problems with overlaps. To speed up the collection time of the data set, a subset is collected and the entire data set is then reconstructed, by multi-dimensional decomposition co-processing. Described in the thesis is also a way to produce suitably labeled proteins, to detect millisecond dynamics at Cα positions in proteins, using the CPMG dispersion relaxation experiment at lower protein concentrations. Lastly, the kinase domain of EphB2 is shown to be more dynamic on the millisecond time scale as well as more prone to interact with itself in the active form than in the inactive one. This is important for the receptor function of the protein, when and how it mediates signals.

    To conclude, this work has extended the possibilities to study protein dynamics by NMR spectroscopy and contributed to increased understanding of the activation process of EphB2 and its signaling mechanism. 

    List of papers
    1. Fast and Accurate Resonance Assignment of Small-to-Large Proteins by Combining Automated and Manual Approaches
    Open this publication in new window or tab >>Fast and Accurate Resonance Assignment of Small-to-Large Proteins by Combining Automated and Manual Approaches
    Show others...
    2015 (English)In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 11, no 1, e1004022- p.Article in journal (Refereed) Published
    Abstract [en]

    The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. Source code and binaries for Linux, Mac OS X and Microsoft Windows are available.

    Place, publisher, year, edition, pages
    Public Library of Science, 2015
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-115010 (URN)10.1371/journal.pcbi.1004022 (DOI)000349309400013 ()25569628 (PubMedID)
    Note

    Funding Agencies|Swedish Research Council [Dnr. 2012-5136]

    Available from: 2015-03-09 Created: 2015-03-06 Last updated: 2015-04-15
    2. PINT: a software for integration of peak volumes and extraction of relaxation rates
    Open this publication in new window or tab >>PINT: a software for integration of peak volumes and extraction of relaxation rates
    2013 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 56, no 3, 191-202 p.Article in journal (Refereed) Published
    Abstract [en]

    We present the software Peak INTegration (PINT), designed to perform integration of peaks in NMR spectra. The program is very simple to run, yet powerful enough to handle complicated spectra. Peaks are integrated by fitting predefined line shapes to experimental data and the fitting can be customized to deal with, for instance, heavily overlapped peaks. The results can be inspected visually, which facilitates systematic optimization of the line shape fitting. Finally, integrated peak volumes can be used to extract parameters such as relaxation rates and information about low populated states. The utility of PINT is demonstrated by applications to the 59 residue SH3 domain of the yeast protein Abp1p and the 289 residue kinase domain of murine EphB2.

    Place, publisher, year, edition, pages
    Springer Verlag (Germany), 2013
    Keyword
    Peak integration, Overlapped peaks, Relaxation rates, Protein dynamics
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:liu:diva-95951 (URN)10.1007/s10858-013-9737-7 (DOI)000321544600001 ()
    Note

    Funding Agencies|Swedish Research Council||

    Available from: 2013-08-19 Created: 2013-08-12 Last updated: 2015-04-23Bibliographically approved
    3. Measurement of Protein Backbone 13CO and 15N Relaxation Dispersion at High Resolution
    Open this publication in new window or tab >>Measurement of Protein Backbone 13CO and 15N Relaxation Dispersion at High Resolution
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Three-dimensional pulse sequences for the measurement of Carr-Purcell-Meiboom-Gill relaxation dispersions and new methods for co-processing non-uniformly sampled data are presented. The new methodology was validated for the disordered protein IgA and for an SH3 domain from Abp1p in exchange between its free form and bound to a peptide from the protein Ark1p. We show that the results are similar to ones obtained using traditional experiments and that accurate excited state chemical shifts can be determined. Furthermore, we show that jackknife analysis of down sampled spectra yields robust estimates of peak intensities errors, eliminating the need for recording duplicate data points. The methodology should be useful for characterization of millisecond dynamics in small to medium-sized proteins with poorly dispersed spectra.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-117070 (URN)
    Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2015-04-15Bibliographically approved
    4. Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity
    Open this publication in new window or tab >>Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity
    Show others...
    2015 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 62, no 3, 341-351 p.Article in journal (Refereed) Published
    Abstract [en]

    A selective isotope labeling scheme based on the utilization of [2-13C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13Cα chemical shifts using Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-13C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state 13Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s−1, despite the small fraction of 13Cα–13Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using 13Cα spin probes.

    Place, publisher, year, edition, pages
    Springer Netherlands, 2015
    Keyword
    CPMG, 13Cα labeling, [2-13C]-Glycerol, Excited states
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-117073 (URN)10.1007/s10858-015-9948-1 (DOI)000357489200010 ()
    Note

    At the time for thesis presentation publication was in status: Manuscript

    At the time for thesis presentation name of publication was: Fractional enrichment using [2-13C]-glycerol as the carbon source facilitates measurements of excited state 13Cα chemical shifts with improved sensitivity

    Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2016-12-28Bibliographically approved
    5. Conformational Dynamics and Multimerization of Active Forms of the EphrinB Receptor 2 Kinase Domain
    Open this publication in new window or tab >>Conformational Dynamics and Multimerization of Active Forms of the EphrinB Receptor 2 Kinase Domain
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Active and autoinhibited forms of the ephrinB receptor 2 (EphB2) kinase domain have been studied using NMR spectroscopy. The project was initiated because of the finding that the crystal structures of active forms of the kinase domain and previous NMR studies suggested that a change in inter-lobe flexibility and the sampling of catalytically competent excited states conformations are responsible for activity. Using Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, we have measured millisecond dynamics to identify such states. We have also performed concentration dependent relaxation experiments and analytical ultracentrifugation experiments that report on the effective protein size to look for possible differences in self-association for active and autoinhibited forms of the EphB2 kinase domain. We show that the active but not autoinhibited forms exchange between a ground state and an excited state at a rate of 1900 s-1. Similar results were found for the S677/680A mutant of the protein. The nature and importance of the excited state is still unknown. Our most important finding is that active forms of the kinase domain self-associate in a concentration dependent manner and form tetramers and possibly larger oligomers. Multimerization of the kinase domain may enable the assembly of complexes of downstream proteins and could be important for Eph signaling.

    Keyword
    Kinase activation | Eph receptors | chemical exchange | nmr spectroscopy | protein dynamics | self-association
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-117071 (URN)
    Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2015-04-15Bibliographically approved
  • 3.
    Ahlner, Alexandra
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Andresen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Khan, Shahid N.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Kay, Lewis E.
    Departments of Medical Genetics, Biochemistry and Chemistry, The University of Toronto, Canada.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity2015In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 62, no 3, 341-351 p.Article in journal (Refereed)
    Abstract [en]

    A selective isotope labeling scheme based on the utilization of [2-13C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13Cα chemical shifts using Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-13C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state 13Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s−1, despite the small fraction of 13Cα–13Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using 13Cα spin probes.

  • 4.
    Ahlner, Alexandra
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Khan, Shahid N.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Forman-Kay, Julie D.
    Molecular Structure and Function Program, Hospital for Sick Children; Department and Biochemistry, University of Toronto, Canada.
    Sicheri, Frank
    cDepartment and Biochemistry, University of Toronto; Department of Molecular Genetics, University of Toronto; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Conformational Dynamics and Multimerization of Active Forms of the EphrinB Receptor 2 Kinase DomainManuscript (preprint) (Other academic)
    Abstract [en]

    Active and autoinhibited forms of the ephrinB receptor 2 (EphB2) kinase domain have been studied using NMR spectroscopy. The project was initiated because of the finding that the crystal structures of active forms of the kinase domain and previous NMR studies suggested that a change in inter-lobe flexibility and the sampling of catalytically competent excited states conformations are responsible for activity. Using Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, we have measured millisecond dynamics to identify such states. We have also performed concentration dependent relaxation experiments and analytical ultracentrifugation experiments that report on the effective protein size to look for possible differences in self-association for active and autoinhibited forms of the EphB2 kinase domain. We show that the active but not autoinhibited forms exchange between a ground state and an excited state at a rate of 1900 s-1. Similar results were found for the S677/680A mutant of the protein. The nature and importance of the excited state is still unknown. Our most important finding is that active forms of the kinase domain self-associate in a concentration dependent manner and form tetramers and possibly larger oligomers. Multimerization of the kinase domain may enable the assembly of complexes of downstream proteins and could be important for Eph signaling.

  • 5.
    Ahlner, Alexandra
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Mayzel, Maxim
    The Swedish NMR Centre, University of Gothenburg, Sweden.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Orekhov, Vladislav Y.
    The Swedish NMR Centre, University of Gothenburg, Sweden.
    Measurement of Protein Backbone 13CO and 15N Relaxation Dispersion at High ResolutionManuscript (preprint) (Other academic)
    Abstract [en]

    Three-dimensional pulse sequences for the measurement of Carr-Purcell-Meiboom-Gill relaxation dispersions and new methods for co-processing non-uniformly sampled data are presented. The new methodology was validated for the disordered protein IgA and for an SH3 domain from Abp1p in exchange between its free form and bound to a peptide from the protein Ark1p. We show that the results are similar to ones obtained using traditional experiments and that accurate excited state chemical shifts can be determined. Furthermore, we show that jackknife analysis of down sampled spectra yields robust estimates of peak intensities errors, eliminating the need for recording duplicate data points. The methodology should be useful for characterization of millisecond dynamics in small to medium-sized proteins with poorly dispersed spectra.

  • 6.
    Ahrén, Maria
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, The Institute of Technology.
    Selegård, Linnéa
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, The Institute of Technology.
    Klasson, Anna
    Linköping University, Center for Medical Image Science and Visualization (CMIV). Linköping University, Department of Medical and Health Sciences, Radiology. Linköping University, Faculty of Health Sciences.
    Söderlind, Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Abrikossova, Natalia
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, The Institute of Technology.
    Skoglund, Caroline
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, Faculty of Science & Engineering.
    Bengtsson, Torbjörn
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, The Institute of Technology.
    Engström, Maria
    Linköping University, Center for Medical Image Science and Visualization (CMIV). Linköping University, Department of Medical and Health Sciences, Radiology. Linköping University, Faculty of Health Sciences.
    Käll, Per-Olov
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Uvdal, Kajsa
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, The Institute of Technology.
    Synthesis and Characterization of PEGylated Gd2O3 Nanoparticles for MRI Contrast Enhancement2010In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 26, no 8, 5753-5762 p.Article in journal (Refereed)
    Abstract [en]

    Recently, much attention has been given to the development of biofunctionalized nanoparticles with magnetic properties for novel biomedical imaging. Guided, smart, targeting nanoparticulate magnetic resonance imaging (MRI) contrast agents inducing high MRI signal will be valuable tools for future tissue specific imaging and investigation of molecular and cellular events. In this study, we report a new design of functionalized ultrasmall rare earth based nanoparticles to be used as a positive contrast agent in MRI. The relaxivity is compared to commercially available Gd based chelates. The synthesis, PEGylation, and dialysis of small (3−5 nm) gadolinium oxide (DEG-Gd2O3) nanoparticles are presented. The chemical and physical properties of the nanomaterial were investigated with Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, and dynamic light scattering. Neutrophil activation after exposure to this nanomaterial was studied by means of fluorescence microscopy. The proton relaxation times as a function of dialysis time and functionalization were measured at 1.5 T. A capping procedure introducing stabilizing properties was designed and verified, and the dialysis effects were evaluated. A higher proton relaxivity was obtained for as-synthesized diethylene glycol (DEG)-Gd2O3 nanoparticles compared to commercial Gd-DTPA. A slight decrease of the relaxivity for as-synthesized DEG-Gd2O3 nanoparticles as a function of dialysis time was observed. The results for functionalized nanoparticles showed a considerable relaxivity increase for particles dialyzed extensively with r1 and r2 values approximately 4 times the corresponding values for Gd-DTPA. The microscopy study showed that PEGylated nanoparticles do not activate neutrophils in contrast to uncapped Gd2O3. Finally, the nanoparticles are equipped with Rhodamine to show that our PEGylated nanoparticles are available for further coupling chemistry, and thus prepared for targeting purposes. The long term goal is to design a powerful, directed contrast agent for MRI examinations with specific targeting possibilities and with properties inducing local contrast, that is, an extremely high MR signal at the cellular and molecular level.

  • 7.
    Ahvenniemi, Esko
    et al.
    Aalto University, Finland.
    Akbashev, Andrew R.
    Stanford University, CA 94305 USA.
    Ali, Saima
    Aalto University, Finland.
    Bechelany, Mikhael
    University of Montpellier, France.
    Berdova, Maria
    University of Twente, Netherlands.
    Boyadjiev, Stefan
    Bulgarian Academic Science, Bulgaria.
    Cameron, David C.
    Masaryk University, Czech Republic.
    Chen, Rong
    Huazhong University of Science and Technology, Peoples R China.
    Chubarov, Mikhail
    University of Grenoble Alpes, France.
    Cremers, Veronique
    University of Ghent, Belgium.
    Devi, Anjana
    Ruhr University of Bochum, Germany.
    Drozd, Viktor
    St Petersburg State University, Russia.
    Elnikova, Liliya
    Institute Theoret and Expt Phys, Russia.
    Gottardi, Gloria
    Fdn Bruno Kessler, Italy.
    Grigoras, Kestutis
    VTT Technical Research Centre Finland, Finland.
    Hausmann, Dennis M.
    Lam Research Corp, OR 97062 USA.
    Seong Hwang, Cheol
    Seoul National University, South Korea; Seoul National University, South Korea.
    Jen, Shih-Hui
    Globalfoundries, NY 12203 USA.
    Kallio, Tanja
    Aalto University, Finland.
    Kanervo, Jaana
    Aalto University, Finland; Abo Akad University, Finland.
    Khmelnitskiy, Ivan
    St Petersburg Electrotech University of LETI, Russia.
    Han Kim, Do
    MIT, MA 02139 USA.
    Klibanov, Lev
    Techinsights, Canada.
    Koshtyal, Yury
    Ioffe Institute, Russia.
    Krause, A. Outi I.
    Aalto University, Finland.
    Kuhs, Jakob
    University of Ghent, Belgium.
    Kaerkkaenen, Irina
    Sentech Instruments GmbH, Germany.
    Kaariainen, Marja-Leena
    NovaldMedical Ltd Oy, Finland.
    Kaariainen, Tommi
    NovaldMedical Ltd Oy, Finland; University of Helsinki, Finland.
    Lamagna, Luca
    STMicroelectronics, Italy.
    Lapicki, Adam A.
    Seagate Technology Ireland, North Ireland.
    Leskela, Markku
    University of Helsinki, Finland.
    Lipsanen, Harri
    Aalto University, Finland.
    Lyytinen, Jussi
    Aalto University, Finland.
    Malkov, Anatoly
    Technical University, Russia.
    Malygin, Anatoly
    Technical University, Russia.
    Mennad, Abdelkader
    CDER, Algeria.
    Militzer, Christian
    Technical University of Chemnitz, Germany.
    Molarius, Jyrki
    Summa Semicond Oy, Finland.
    Norek, Malgorzata
    Mil University of Technology, Poland.
    Ozgit-Akgun, Cagla
    ASELSAN Inc, Turkey.
    Panov, Mikhail
    St Petersburg Electrotech University of LETI, Russia.
    Pedersen, Henrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Piallat, Fabien
    KOBUS, France.
    Popov, Georgi
    University of Helsinki, Finland.
    Puurunen, Riikka L.
    VTT Technical Research Centre Finland, Finland.
    Rampelberg, Geert
    University of Ghent, Belgium.
    Ras, Robin H. A.
    Not Found:[Ahvenniemi, Esko] Aalto Univ, Dept Chem, POB 16100, FI-00076 Espoo, Finland; [Akbashev, Andrew R.] Stanford Univ, Dept Mat Sci and Engn, Stanford, CA 94305 USA; [Ali, Saima; Krause, A. Outi I.; Lyytinen, Jussi] Aalto Univ, Sch Chem Technol, Dept Mat Sci and Engn, POB 16200, FI-00076 Aalto, Finland; [Bechelany, Mikhael] Univ Montpellier, ENSCM, CNRS, IEM,UMR 5635, Pl Eugene Bataillon, F-34095 Montpellier 5, France; [Berdova, Maria] Univ Twente, Ind Focus Grp XUV Opt, NL-7522 ND Enschede, Netherlands; [Boyadjiev, Stefan] Bulgarian Acad Sci, Inst Solid State Phys, 72 Tzarigradsko Chaussee Blvd, Sofia 1784, Bulgaria; [Cameron, David C.] Masaryk Univ, CEPLANT, Kotlarska 267-2, CS-61137 Brno, Czech Republic; [Chen, Rong] Huazhong Univ Sci and Technol, Sch Mech Sci and Engn, Sch Opt and Elect Informat, 1037 Luoyu Rd, Wuhan 430074, Hubei, Peoples R China; [Chubarov, Mikhail] Univ Grenoble Alpes, CNRS, SIMAP, F-38000 Grenoble, France; [Cremers, Veronique; Kuhs, Jakob; Rampelberg, Geert] Univ Ghent, CoCooN, Dept Solid State Sci, Krijgslaan 281-S1, B-9000 Ghent, Belgium; [Devi, Anjana] Ruhr Univ Bochum, Inorgan Mat Chem, D-44801 Bochum, Germany; [Drozd, Viktor] St Petersburg State Univ, Inst Chem, Univ Skaya Emb 7-9, St Petersburg 199034, Russia; [Elnikova, Liliya] Inst Theoret and Expt Phys, Bolshaya Cheremushkinskaya 25, Moscow 117218, Russia; [Gottardi, Gloria] Fdn Bruno Kessler, Ctr Mat and Microsyst, I-38123 Trento, Italy; [Grigoras, Kestutis; Puurunen, Riikka L.; Ylivaara, Oili M. E.] VTT Tech Res Ctr Finland, POB 1000,Tietotie 3, FI-02044 Espoo, Vtt, Finland; [Hausmann, Dennis M.] Lam Res Corp, Tualatin, OR 97062 USA; [Hwang, Cheol Seong] Seoul Natl Univ, Dept Mat Sci and Engn, Coll Engn, Seoul 08826, South Korea; [Hwang, Cheol Seong] Seoul Natl Univ, Interuniv Semicond Res Ctr, Coll Engn, Seoul 08826, South Korea; [Jen, Shih-Hui] Globalfoundries, Albany, NY 12203 USA; [Kallio, Tanja; Kanervo, Jaana] Aalto Univ, Sch Chem Engn, Dept Chem, POB 16100, FI-00076 Aalto, Finland; [Kanervo, Jaana] Abo Akad Univ, FI-20500 Turku, Finland; [Khmelnitskiy, Ivan] St Petersburg Electrotech Univ LETI, Res and Educ Ctr Nanotechnol, Ul Prof Popova 5, St Petersburg 197376, Russia; [Kim, Do Han] MIT, Dept Chem Engn, 77 Massachusetts Ave, Cambridge, MA 02139 USA; [Klibanov, Lev] Techinsights, 3000 Solandt Rd, Ottawa, ON K2K2X2, Canada; [Koshtyal, Yury] Ioffe Inst, Lab Lithium Ion Technol, 26 Politekhnicheskaya, St Petersburg 194021, Russia; [Kaerkkaenen, Irina] Sentech Instruments GmbH, Schwarzschildstr 2, D-12489 Berlin, Germany; [Kaariainen, Marja-Leena; Kaariainen, Tommi] NovaldMed Ltd Oy, Telkantie 5, FI-82500 Kitee, Finland; [Kaariainen, Tommi] Univ Helsinki, Inorgan Chem Lab, POB 55,AI Virtasen Aukio 1, FI-00014 Helsinki, Finland; [Lamagna, Luca] STMicroelectronics, Via C Olivetti 2, I-20864 Agrate Brianza, MB, Italy; [Lapicki, Adam A.] Seagate Technol Ireland, 1 Disc Dr, Derry BT48 7BD, North Ireland; [Leskela, Markku; Popov, Georgi] Univ Helsinki, Dept Chem, POB 55, FI-00014 Helsinki, Finland; [Lipsanen, Harri; Savin, Hele] Aalto Univ, Dept Micro and Nanosci, Tietotie 3, Espoo 02150, Finland; [Malkov, Anatoly; Malygin, Anatoly] Tech Univ, St Petersburg State Inst Technol, Dept Chem Nanotechnol and Mat Elect, 26 Moskovsky Prosp, St Petersburg 190013, Russia; [Mennad, Abdelkader] CDER, UDES, RN 11 BP 386 Bou Ismail, Tipasa 42415, Algeria; [Militzer, Christian] Tech Univ Chemnitz, Inst Chem, Phys Chem, Str Nationen 62, D-09111 Chemnitz, Germany; [Molarius, Jyrki] Summa Semicond Oy, PL 11, Espoo 02131, Finland; [Norek, Malgorzata] Mil Univ Technol, Fac Adv Technol and Chem, Dept Adv Mat and Technol, Str Kaliskiego 2, PL-00908 Warsaw, Poland; [Ozgit-Akgun, Cagla] ASELSAN Inc, Microelect Guidance and Electroopt Business Sect, TR-06750 Ankara, Turkey; [Panov, Mikhail] St Petersburg Electrotech Univ LETI, Ctr Microtechnol and Diagnost, Ul Prof Popova 5, St Petersburg 197376, Russia; [Pedersen, Henrik] Linkoping Univ, Dept Phys Chem and Biol, SE-58183 Linkoping, Sweden; [Piallat, Fabien] KOBUS, F-38330 Montbonnot St Martin, France; [Rauwel, Erwan] Tallinn Univ Technol, Tartu Coll, Puiestee 78, EE-51008 Tartu, Estonia; [Roozeboom, Fred] Eindhoven Univ Technol, Dept Appl Phys, Grp Plasma and Mat Proc, POB 513, NL-5600 MB Eindhoven, Netherlands; [Roozeboom, Fred] TNO, High Tech Campus 21, NL-5656 AE Eindhoven, Netherlands; [Sajavaara, Timo] Univ Jyvaskyla, Dept Phys, POB 35, Jyvaskyla 40014, Finland; [Salami, Hossein] Univ Maryland, Dept Chem and Biomol Engn, College Pk, MD 20742 USA; [Schneider, Nathanaelle] IRDEP CNRS, 6 Quai Watier, F-78401 Chatou, France; [Schneider, Nathanaelle] IPVF, 8 Rue Renaissance, F-92160 Antony, France; [Seidel, Thomas E.] Seitek50, POB 350238, Palm Coast, FL 32135 USA; [Sundqvist, Jonas] Fraunhofer Inst Ceram Technol and Syst IKTS, Syst Integrat and Technol Transfer, Winterbergstr 28, D-01277 Dresden, Germany; [Suyatin, Dmitry B.] Lund Univ, Div Solid State Phys, Box 118, SE-22100 Lund, Sweden; [Suyatin, Dmitry B.] Lund Univ, NanoLund, Box 118, SE-22100 Lund, Sweden; [Torndahl, Tobias] Uppsala Univ, Solid State Elect, POB 534, SE-75121 Uppsala, Sweden; [van Ommen, J. Ruud] Delft Univ Technol, Dept Chem Engn, Van der Maasweg 9, NL-2629 HZ Delft, Netherlands; [Wiemer, Claudia] CNR, IMM, Lab MDM, Via C Olivetti 2, I-20864 Agrate Brianza, MB, Italy; [Yurkevich, Oksana] Immanuel Kant Balt Fed Univ, Res and Educ Ctr Funct Nanomat, A Nevskogo 14, Kaliningrad 236041, Russia.
    Rauwel, Erwan
    Tallinn University of Technology, Estonia.
    Roozeboom, Fred
    Eindhoven University of Technology, Netherlands; TNO, Netherlands.
    Sajavaara, Timo
    University of Jyvaskyla, Finland.
    Salami, Hossein
    University of Maryland, MD 20742 USA.
    Savin, Hele
    Aalto University, Finland.
    Schneider, Nathanaelle
    IRDEP CNRS, France; IPVF, France.
    Seidel, Thomas E.
    Seitek50, FL 32135 USA.
    Sundqvist, Jonas
    Fraunhofer Institute Ceram Technology and Syst IKTS, Germany.
    Suyatin, Dmitry B.
    Lund University, Sweden; Lund University, Sweden.
    Torndahl, Tobias
    Uppsala University, Sweden.
    van Ommen, J. Ruud
    Delft University of Technology, Netherlands.
    Wiemer, Claudia
    CNR, Italy.
    Ylivaara, Oili M. E.
    VTT Technical Research Centre Finland, Finland.
    Yurkevich, Oksana
    Immanuel Kant Balt Federal University, Russia.
    Recommended reading list of early publications on atomic layer deposition-Outcome of the "Virtual Project on the History of ALD"2017In: Journal of Vacuum Science & Technology. A. Vacuum, Surfaces, and Films, ISSN 0734-2101, E-ISSN 1520-8559, Vol. 35, no 1, 010801Article, review/survey (Refereed)
    Abstract [en]

    Atomic layer deposition (ALD), a gas-phase thin film deposition technique based on repeated, self-terminating gas-solid reactions, has become the method of choice in semiconductor manufacturing and many other technological areas for depositing thin conformal inorganic material layers for various applications. ALD has been discovered and developed independently, at least twice, under different names: atomic layer epitaxy (ALE) and molecular layering. ALE, dating back to 1974 in Finland, has been commonly known as the origin of ALD, while work done since the 1960s in the Soviet Union under the name "molecular layering" (and sometimes other names) has remained much less known. The virtual project on the history of ALD (VPHA) is a volunteer-based effort with open participation, set up to make the early days of ALD more transparent. In VPHA, started in July 2013, the target is to list, read and comment on all early ALD academic and patent literature up to 1986. VPHA has resulted in two essays and several presentations at international conferences. This paper, based on a poster presentation at the 16th International Conference on Atomic Layer Deposition in Dublin, Ireland, 2016, presents a recommended reading list of early ALD publications, created collectively by the VPHA participants through voting. The list contains 22 publications from Finland, Japan, Soviet Union, United Kingdom, and United States. Up to now, a balanced overview regarding the early history of ALD has been missing; the current list is an attempt to remedy this deficiency. (C) 2016 Author(s).

  • 8.
    Alfredsson, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry.
    Synthesis and Characterization of Acrylfentanyl Metabolites2017Independent thesis Basic level (degree of Bachelor), 10,5 credits / 16 HE creditsStudent thesis
    Abstract [en]

    Acrylfentanyl is a synthetic opioid that has been widely used in the last year. To help in the fight against synthetic drugs two potential metabolites of acrylfentanyl, one monohydroxy and one dihydroxy were synthesized. These metabolites will hopefully later be implemented in the analytical methods for metabolites of acrylfentanyl in urine by the Swedish National Board of Forensic Medicine.

    To have metabolites for analysis are very important as they are the main target in drug testing.

    The method used to synthesize the metabolites is a five-step synthesis with an additional 6th step for the dihydroxy metabolite. The methods used in the synthesis includes protection of amine with tert-butyloxycarbonyl, reductive amination with sodium triaceto boronhydride, alkylation and demethylation with boron tribromide. The methods used produced good results with high yields in nearly all steps.

  • 9.
    Ammenberg, Jonas
    et al.
    Linköping University, Department of Management and Engineering, Environmental Technology and Management. Linköping University, The Institute of Technology. Linköping University, Biogas Research Center.
    Svensson, Bo
    Linköping University, Department of Thematic Studies, Tema Environmental Change. Linköping University, Faculty of Arts and Sciences. Linköping University, Biogas Research Center.
    Karlsson, Magnus
    Linköping University, Department of Management and Engineering, Energy Systems. Linköping University, Biogas Research Center.
    Svensson, Niclas
    Linköping University, Department of Management and Engineering, Environmental Technology and Management. Linköping University, The Institute of Technology. Linköping University, Biogas Research Center.
    Björn, Annika
    Linköping University, Department of Thematic Studies, Tema Environmental Change. Linköping University, Faculty of Arts and Sciences. Linköping University, Biogas Research Center.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Linköping University, Biogas Research Center.
    Tonderski, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, The Institute of Technology. Linköping University, Biogas Research Center.
    Eklund, Mats
    Linköping University, Department of Management and Engineering, Environmental Technology and Management. Linköping University, The Institute of Technology. Linköping University, Biogas Research Center.
    Biogas Research Center, BRC: Slutrapport för etapp 12015Report (Other academic)
    Abstract [en]

    Biogas Research Center (BRC) is a center of excellence in biogas research funded by the Swedish Energy Agency, Linköping University and a number of external organizations with one-third each. BRC has a very broad interdisciplinary approach, bringing together biogas-related skills from several areas to create interaction on many levels:

    • between industry, academia and society,
    • between different perspectives, and
    • between different disciplines and areas of expertise.

    BRC’s vision is:

    BRC contributes to the vision by advancing knowledge and technical development, as well as by facilitating development, innovation and business. Resource efficiency is central, improving existing processes and systems as well as establishing biogas solutions in new sectors and enabling use of new substrates.

    For BRC phase 1, the first two year period from 2012-2014, the research projects were organized in accordance with the table below showing important challenges for biogas producers and other stakeholders, and how these challenges were tackled in eight research projects. Five of the projects had an exploratory nature, meaning that they were broader, more future oriented and, for example, evaluated several different technology paths (EP1-5). Three projects focused more on technology and process development (DP6-8).

    This final report briefly presents the background and contains some information about competence centers in general. Thereafter follows more detailed information about BRC, for example, regarding the establishment, relevance, organization, vision, corner stones and development. The participating organizations are presented, both the research groups within Linköping University and the partners and members. Further on, there is a more detailed introduction to and description of the challenges mentioned in the table above and a short presentation from each of the research projects, followed by some sections dealing with fulfillment of objectives and an external assessment of BRC. Detailed, listed information is commonly provided in the appendices.

    Briefly, the fulfillment of objectives is good and it is very positive that so many scientific articles have been published (or are to be published) from the research projects and also within the wider center perspective. Clearly, extensive and relevant activities are ongoing within and around BRC. In phase 2 it essential to increase the share of very satisfied partners and members, where now half of them are satisfied and the other half is very satisfied. For this purpose, improved communication, interaction and project management are central. During 2015, at least two PhD theses are expected, to a large extent based on the research from BRC phase 1.

    In the beginning of 2014 an external assessment of BRC was carried out, with the main purpose to assess how well the center has been established and to review the conditions for a future, successful competence center. Generally, the outcome was very positive and the assessors concluded that BRC within a short period of time had been able to establish a well-functioning organization engaging a large share of the participants within relevant areas, and that most of the involved actors look upon BRC as a justifiable and well working investment that they plan to continue to support. The assessment also contributed with several relevant tips of improvements and to clarify challenges to address.

    This report is written in Swedish, but for each research project there will be reports and/or scientific papers published in English.

    The work presented in this report has been financed by the Swedish Energy Agency and the participating organizations.

  • 10.
    Anandapadamanaban, Madhanagopal
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Virtually every aspect of the cellular processes in eukaryotes requires that the interactions between protein molecules are well coordinated in different regulatory pathways. Any protein dysfunction involved in these regulatory pathways might lead to various pathological conditions. Understanding the structural and functional peculiarities of these proteins molecular machineries will help in formulating structure-based drug design.

    The first regulatory process studied here is the RNA polymerase-II mediated transcription of the eukaryotic protein-coding genes to produce mRNAs. This process requires the formation of the ‘transcription initiation’ by the assembly of Pre-Initiation Complex (PIC) formation at a core promoter region. Regulation at this initiation level is a key mechanism for the control of gene expression that governs cellular growth and differentiation. The transcription Factor IID (TFIID) is a conserved multiprotein general transcription factor with an essential role in  nucleating the PIC formation, composed of TATA Binding Protein (TBP) and about 14 TBP Associated Factors (TAFs). The here presented crystal structure (1.97Å) of TBP bound to TAND1 and TAND2 domains from TAF1 reveals a detailed molecular pattern of interactions involving both transcriptionally activating and repressing regions in TBP, thereby uncovering central principles for anchoring of TBP-binding motifs. Together with NMR and cellular analysis, this work provides the structural basis of competitive binding with TFIIA to modulate TBP in promoter recognition.

    In eukaryotes, another fundamental mechanism in the regulation of cellular physiology is the posttranslational modification of substrate proteins by ubiquitin, termed ‘ubiquitination’. Important actors in this mechanism are the ubiquitin-ligases (E3s) that culminate the transfer of ubiquitin to the substrate and govern the specificity of this system. One E3 ligase in particular, TRIM21, defines a subgroup of the Tripartite Motif (TRIM) family, which belongs to the major RING-type of E3 ubiquitin ligases, and plays an important role in pathogenesis of autoimmunity by mediating ubiquitination of transcription factors. The crystal structure (2.86Å) of the RING domain from TRIM21 in complex with UBE2E1, an E2 conjugating enzyme, together with the NMR and SAXS analysis as well as biochemical functional analysis, reveals the molecular basis for the dynamic binding interfaces. The TRIM21 mode of ubiquitin recognition and activation for catalytic transfer of ubiquitin can be modeled onto the entire TRIM family.

    Finally, we explored the concepts of conformational selection in proteins as a possible key component for protein-mediated transcriptional regulation. In this framework, MexR, a bacterial repressor of the MexAB-OprM efflux pump, and its mutant Arg21Trp were studied as an example for proteins presenting different conformations. The residue Arg21Trp mutation is clinically identified to cause of Multi-Drug Resistant (MDR) by attenuated DNA binding, and leads to the overexpression of the MexAB-OprM efflux pump. With the crystal structure (2.19Å) of MexR mutant Arg21Trp, in combination with MD-simulations and SAXS for both wild-type and mutant, we could unravel the atomic details of the wild-type conformations consisting in subsets of populations of DNA bound and unbound forms. Remarkably, the mutant Arg21Trp stabilize the DNA unbound state and shifts MexR in a pre-existing equilibrium, from a repressed to a derepressed state.

    Taken together, these studies substantially broaden our knowledge at a molecular level in protein interactions that are involved in transcriptional regulation and ubiquitination, studied by a carefully selected combination of complementary structural methods spanning different resolutions and time scales.

    List of papers
    1. High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation
    Open this publication in new window or tab >>High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation
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    2013 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 20, no 8, 1008-+ p.Article in journal (Refereed) Published
    Abstract [en]

    The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 angstrom) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBPs concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

    Place, publisher, year, edition, pages
    NATURE PUBLISHING GROUP, 75 VARICK ST, 9TH FLR, NEW YORK, NY 10013-1917 USA, 2013
    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-96977 (URN)10.1038/nsmb.2611 (DOI)000322715300016 ()
    Note

    Funding Agencies|Swedish Research Council|621-2011-6028621-2012-5250621-2012-5136|VINNOVA|P32045-1|Swedish Cancer Foundation|11 0681|Swedish Child Cancer Foundation|PROJ09/092|Forum Scientium Award||Canadian Institutes for Health Research|MT-13611|Japan Society for the Promotion of Science|23370077|Knut and Alice Wallenberg foundation||Canada Research Chair||

    Available from: 2013-09-05 Created: 2013-09-02 Last updated: 2015-11-03
    2. Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression
    Open this publication in new window or tab >>Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression
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    2016 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 8, 1311-1321 p.Article in journal (Refereed) Published
    Abstract [en]

    MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 angstrom) presented here is closely similar to wildtype MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members.

    Place, publisher, year, edition, pages
    CELL PRESS, 2016
    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:liu:diva-131908 (URN)10.1016/j.str.2016.06.008 (DOI)000383244600012 ()27427478 (PubMedID)
    Note

    Funding Agencies|European Communitys Seventh Framework Program (FP7) under BioStruct-X [283570]; Swedish e-Science Research Center; Swedish Research Council; Tage Erlander Visiting Professor grant.

    The original status of this article was Manuscript and the titel was Population shift disengages DNA binding in a multidrug resistance MexR mutant.

    Available from: 2016-10-13 Created: 2016-10-11 Last updated: 2017-01-10
    3. Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface
    Open this publication in new window or tab >>Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface
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    2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 42, 36478-36491 p.Article in journal (Refereed) Published
    Abstract [en]

    Ro52 (TRIM21) is an E3 ligase of the tripartite motif family that negatively regulates proinflammatory cytokine production by ubiquitinating transcription factors of the interferon regulatory factor family. Autoantibodies to Ro52 are present in patients with lupus and Sjögren's syndrome, but it is not known if these autoantibodies affect the function of Ro52. To address this question, the requirements for Ro52 E3 ligase activity were first analyzed in detail. Scanning a panel of E2 ubiquitin-conjugating enzymes, we found that UBE2D1–4 and UBE2E1–2 supported the E3 ligase activity of Ro52 and that the E3 ligase activity of Ro52 was dependent on its RING domain. We also found that the N-terminal extensions in the class III E2 enzymes affected their interaction with Ro52. Although the N-terminal extension in UBE2E3 made this E2 enzyme unable to function together with Ro52, the N-terminal extensions in UBE2E1 and UBE2E2 allowed for a functional interaction with Ro52. Anti-Ro52-positive patient sera and affinity-purified anti-RING domain autoantibodies inhibited the E3 activity of Ro52 in ubiquitination assays. Using NMR, limited proteolysis, ELISA, and Ro52 mutants, we mapped the interactions between Ro52, UBE2E1, and anti-Ro52 autoantibodies. We found that anti-Ro52 autoantibodies inhibited the E3 ligase activity of Ro52 by sterically blocking the E2/E3 interaction between Ro52 and UBE2E1. Our data suggest that anti-Ro52 autoantibodies binding the RING domain of Ro52 may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52-mediated ubiquitination.

    Place, publisher, year, edition, pages
    American Society for Biochemistry and Molecular Biology, 2011
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-53170 (URN)10.1074/jbc.M111.241786 (DOI)000296538300033 ()
    Note

    Funding agencies|Swedish Research Council||Swedish Foundation for Strategic Research||VINNOVA||CeNano||Swedish Cancer Society||Karolinska Institutet||Linkoping University||King Gustaf Vs 80-Year Foundation||Heart-Lung Foundation||Stockholm County Council||Gustafsson Foundation||Soderberg Foundation||National Cancer Institute of Canada||Swedish Rheumatism Association||Wallenberg Foundation||

    Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2015-11-03Bibliographically approved
    4. Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGs
    Open this publication in new window or tab >>Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGs
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    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    TRIM21, a RlNG-containing E3 ubiquitin-ligase of the TRIM   protein family, is a major autoantigen in SLE and Sjögren's syndrome as well as a modifier of interferon regulatory factors, thereby regulating innate immune signalling. We herein report the 2.86 Å crystal structure ofhuman TRIM211-91 comprising the RING domain (residues 16-55), in complex with the human E2 conjugating UBE2El enzyme (also denoted UbcH6). The crystal structure, joint with analysis by NMR and SAXS as well as structure-directed mutations and functional assays provides a detailed view of the specificity-determining contacts that support specific E2 recognition in the TRIM family. A detailed comparison of our structure with known E2 bound ubiquitin complexes, supported by biochemical analyses, reveals the molecular basis for TRIM21 interactions with donor ubiquitin that activates catalytic ubiquitin transfer. Finally, our structure convincingly demonstrates the placement of the Ub-targeted Lys61 of the adjacent TRIM211- 91 close to the catalytically active UBE2El cysteine, and how the Lys61 amide is activated fora nucleophilic attack by hydrogen-bondeffected deshielding by conserved acidic residues at the E2 active site. In all, our structural findings provide molecular details ofthe selectivity involved in TRIM21 interactions with its cognate UBE2E1 enzyme and how TRIM21 positions ubiquitin in a catalytic conformation for ubiquitin transfer, and presents a snapshot of the Ub ligation step on a specific target residue of TRIM211-91 as an auto-ubiquitinated pseudo-substrate at high concentration. Increased structural and functional understanding of the TRIM mediated ubiquitination will aid development ofnovel therapeutic approaches in the entire TRIM family ofproteins.

    National Category
    Chemical Sciences Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:liu:diva-122466 (URN)
    Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2015-11-13Bibliographically approved
  • 11.
    Anandapadamanaban, Madhanagopal
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Kyriakidis, Nikolaos
    Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden.
    Espinosa, Alexander
    Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden.
    Round, Adam R.
    European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France.
    Trewhella, Jill
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. School of Molecular Bioscience, The University of Sydney, New South Wales, Australia.
    Wahren-Herlenius, Marie
    Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden.
    Moche, Martin
    Department of Medical Biochemistry and Biophysics, Protein Science Facility, Karolinska Institutet, Stockholm, Sweden.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGsManuscript (preprint) (Other academic)
    Abstract [en]

    TRIM21, a RlNG-containing E3 ubiquitin-ligase of the TRIM   protein family, is a major autoantigen in SLE and Sjögren's syndrome as well as a modifier of interferon regulatory factors, thereby regulating innate immune signalling. We herein report the 2.86 Å crystal structure ofhuman TRIM211-91 comprising the RING domain (residues 16-55), in complex with the human E2 conjugating UBE2El enzyme (also denoted UbcH6). The crystal structure, joint with analysis by NMR and SAXS as well as structure-directed mutations and functional assays provides a detailed view of the specificity-determining contacts that support specific E2 recognition in the TRIM family. A detailed comparison of our structure with known E2 bound ubiquitin complexes, supported by biochemical analyses, reveals the molecular basis for TRIM21 interactions with donor ubiquitin that activates catalytic ubiquitin transfer. Finally, our structure convincingly demonstrates the placement of the Ub-targeted Lys61 of the adjacent TRIM211- 91 close to the catalytically active UBE2El cysteine, and how the Lys61 amide is activated fora nucleophilic attack by hydrogen-bondeffected deshielding by conserved acidic residues at the E2 active site. In all, our structural findings provide molecular details ofthe selectivity involved in TRIM21 interactions with its cognate UBE2E1 enzyme and how TRIM21 positions ubiquitin in a catalytic conformation for ubiquitin transfer, and presents a snapshot of the Ub ligation step on a specific target residue of TRIM211-91 as an auto-ubiquitinated pseudo-substrate at high concentration. Increased structural and functional understanding of the TRIM mediated ubiquitination will aid development ofnovel therapeutic approaches in the entire TRIM family ofproteins.

  • 12.
    Anandapadmanaban, Madhanagopal
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Helander, Sara
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Ohyama, Yoshifumi
    Yokohama City University, Japan .
    Siponen, Marina I.
    Karolinska Institute, Sweden .
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Kokubo, Tetsuro
    Yokohama City University, Japan .
    Ikura, Mitsuhiko
    University of Toronto, Canada .
    Moche, Martin
    Karolinska Institute, Sweden .
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation2013In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 20, no 8, 1008-+ p.Article in journal (Refereed)
    Abstract [en]

    The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 angstrom) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBPs concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

  • 13.
    Anandapadmanaban, Madhanagopal
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
    Pilstål, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Trewhella, Jill
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. University of Sydney, Australia.
    Moche, Martin
    Karolinska Institute, Sweden.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression2016In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 8, 1311-1321 p.Article in journal (Refereed)
    Abstract [en]

    MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 angstrom) presented here is closely similar to wildtype MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members.

  • 14.
    Andrasko, Jan
    et al.
    GC UV Centre, Kobergsgränd 2, SE-58731 Linkoping, Sweden.
    Lagesson-Andrasko, Ludmila
    GC UV Centre, Kobergsgränd 2, SE-58731 Linkoping, Sweden.
    Dahlén, Johan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Analysis of Explosives by GC-UV2017In: Journal of Forensic Sciences, ISSN 0022-1198, E-ISSN 1556-4029, Vol. 62, no 4, 1022-1027 p.Article in journal (Refereed)
    Abstract [en]

    A mixture of explosives was analyzed by gas chromatography (GC) linked to ultraviolet (UV) spectrophotometry that enabled detection in the range of 178-330 nm. The gas-phase UV spectra of 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), ethylene glycol dinitrate (EGDN), glycerine trinitrate (NG, nitroglycerine), triacetone triperoxide (TATP), and pentaerythritol tetranitrate (PETN) were successfully recorded. The most interesting aspect of the current application is that it enabled simultaneous detection of both the target analyte and its decomposition products. At suitable elevated temperatures of the transfer line between the GC instrument and the UV detector, a partial decomposition was accomplished. Detection was made in real time and resulted in overlaid spectra of the mother compound and its decomposition product. Hence, the presented approach added another level to the qualitative identification of the explosives in comparison with traditional methods that relies only on the detection of the target analyte. As expected, the decomposition product of EGDN, NG, and PETN was NO, while TATP degraded to acetone. DNT and TNT did not exhibit any decomposition at the temperatures used.

    The full text will be freely available from 2018-01-10 16:23
  • 15.
    Andres Cisneros, Gerardo
    et al.
    Wayne State University, MI 48202 USA.
    Thor Wikfeldt, Kjartan
    University of Iceland, Iceland; Stockholm University, Sweden.
    Ojamäe, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lu, Jibao
    University of Utah, USA.
    Xu, Yao
    Ruhr University of Bochum, Germany.
    Torabifard, Hedieh
    Wayne State University, USA.
    Bartok, Albert P.
    University of Cambridge, England.
    Csanyi, Gabor
    University of Cambridge, England.
    Molinero, Valeria
    University of Utah, USA.
    Paesani, Francesco
    University of Calif San Diego, USA.
    Modeling Molecular Interactions in Water: From Pairwise to Many Body Potential Energy Functions2016In: Chemical Reviews, ISSN 0009-2665, E-ISSN 1520-6890, Vol. 116, no 13, 7501-7528 p.Article, review/survey (Refereed)
    Abstract [en]

    Almost 50 years have passed from the first computer simulations of water, and a large number of molecular models have been proposed since then to elucidate the unique behavior of water across different phases. In this article, we review the recent progress in the development of analytical potential energy functions that aim at correctly representing many-body effects. Starting from the many-body expansion of the interaction energy, specific focus is on different classes of potential energy functions built upon a hierarchy of approximations and on their ability to accurately reproduce reference data obtained from state-of-the-art electronic structure calculations and experimental measurements. We show that most recent potential energy functions, which include explicit short-range representations of two-body and three-body effects along with a physically correct description of many-body effects at all distances, predict the properties of water from the gas to the condensed phase with unprecedented accuracy, thus opening the door to the long-sought "universal model" capable of describing the behavior of water under different conditions and in different environments.

  • 16.
    Andrésen, Cecilia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Helander, Sara
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lemak, Alexander
    University of Toronto, Canada .
    Fares, Christophe
    University of Toronto, Canada .
    Csizmok, Veronika
    Hospital for Sick Children, Canada .
    Carlsson, Jonas
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, The Institute of Technology.
    Penn, Linda Z
    University of Toronto, Canada .
    Forman-Kay, Julie D
    Hospital Sick Children, Canada University of Toronto, Canada .
    Arrowsmith, Cheryl H
    University of Toronto, Canada.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 13, 6353-6366 p.Article in journal (Refereed)
    Abstract [en]

    The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions in residues 22-33 and in the Myc homology box I (MBI; residues 45-65); both these regions are conserved in other members of the Myc family. Binding of Bin1 to Myc-1-88 as assayed by NMR and surface plasmon resonance (SPR) revealed primary binding to the S62 region in a dynamically disordered and multivalent complex, accompanied by population shifts leading to altered intramolecular conformational dynamics. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.

  • 17.
    Andrésen, Cecilia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Niklasson, Markus
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Cassman Eklöf, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Biophysical characterization of the calmodulin-like domain of Plasmodium falciparum calcium dependent protein kinase 32017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 7, e0181721Article in journal (Refereed)
    Abstract [en]

    Calcium dependent protein kinases are unique to plants and certain parasites and comprise an N-terminal segment and a kinase domain that is regulated by a C-terminal calcium binding domain. Since the proteins are not found in man they are potential drug targets. We have characterized the calcium binding lobes of the regulatory domain of calcium dependent protein kinase 3 from the malaria parasite Plasmodium falciparum. Despite being structurally similar, the two lobes differ in several other regards. While the monomeric N-terminal lobe changes its structure in response to calcium binding and shows global dynamics on the sub-millisecond time-scale both in its apo and calcium bound states, the C-terminal lobe could not be prepared calcium-free and forms dimers in solution. If our results can be generalized to the full-length protein, they suggest that the C-terminal lobe is calcium bound even at basal levels and that activation is caused by the structural reorganization associated with binding of a single calcium ion to the N-terminal lobe.

  • 18.
    Andrésen, Cecilia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Wang, Yi
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Jarl, Anngelica
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Jalal, Shah
    Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Stockholm, Sweden.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Wretlind, Bengt
    Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Stockholm, Sweden.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Molecular causes for deficient repression in multidrug resistant mutants in the Pseudomonas aeruginosa efflux gene regulator MexRManuscript (preprint) (Other academic)
    Abstract [en]

    n/a

  • 19.
    Appelqvist, Hanna
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Stranius, Kati
    University of Gothenburg, Sweden.
    Börjesson, Karl
    University of Gothenburg, Sweden.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Dyrager, Christine
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Specific Imaging of Intracellular Lipid Droplets Using a Benzothiadiazole Derivative with Solvatochromic Properties2017In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 28, no 5, 1363-1370 p.Article in journal (Refereed)
    Abstract [en]

    Altered lipid metabolism and extensive lipid storage in cells have been associated with various medical disorders, including cancer. The development of fluorescent probes that specifically accumulate in lipid deposits is therefore of great interest in order to study pathological processes that are linked to dysregulated lipogenesis. In the present study, we present a small fluorescent benzothiadiazole dye that specifically stains lipid droplets in living and fixated cells. The photophysical characterization of the probe revealed strong solvatochromic behavior, large Stokes shifts, and high fluorescent quantum yields in hydrophobic solvents. In addition, the fluorophore exhibits a nontoxic profile and a high signal-to-noise ratio in cells (i.e., lipid droplets vs cytosol), which make it an excellent candidate for studying lipid biology using confocal fluorescent microscopy.

    The full text will be freely available from 2018-04-12 14:34
  • 20.
    Appelqvist, Hanna
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Wäster, Petra
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Eriksson, Ida
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Rosdahl, Inger
    Linköping University, Department of Clinical and Experimental Medicine, Division of Inflammation Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Dermatology and Venerology.
    Öllinger, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    Lysosomal exocytosis and caspase-8-mediated apoptosis in UVA-irradiated keratinocytes2013In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 126, no 24, 5578-5584 p.Article in journal (Refereed)
    Abstract [en]

    Ultraviolet (UV) irradiation is a major environmental carcinogen involved in the development of skin cancer. To elucidate the initial signaling during UV-induced damage in human keratinocytes, we investigated lysosomal exocytosis and apoptosis induction. UVA, but not UVB, induced plasma membrane damage, which was repaired by Ca2+-dependent lysosomal exocytosis. The lysosomal exocytosis resulted in extracellular release of cathepsin D and acid sphingomyelinase (aSMase). Two hours after UVA irradiation, we detected activation of caspase-8, which was reduced by addition of anti-aSMAse. Furthermore, caspase-8 activation and apoptosis was reduced by prevention of endocytosis and by the use of cathepsin inhibitors. We conclude that lysosomal exocytosis is part of the keratinocyte response to UVA and is followed by cathepsin-dependent activation of caspase-8. The findings have implications for the understanding of UV-induced skin damage and emphasize that UVA and UVB initiate apoptosis through different signaling pathways in keratinocytes.

  • 21.
    Arhammar, C.
    et al.
    Sandvik Coromant, Sweden .
    Silvearv, F.
    Luleå University of Technology, Sweden .
    Bergman, A.
    Uppsala University, Sweden .
    Norgren, S.
    Sandvik Coromant, Sweden Uppsala University, Sweden .
    Pedersen, Henrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Ahuja, R.
    Uppsala University, Sweden .
    A theoretical study of possible point defects incorporated into alpha-alumina deposited by chemical vapor deposition2013In: Theoretical Chemistry accounts, ISSN 1432-881X, E-ISSN 1432-2234, Vol. 133, no 2, 1433- p.Article in journal (Refereed)
    Abstract [en]

    The energetics and electronic structure of carbon, chlorine, hydrogen, and sulfur in alpha-Al2O3 was investigated by first principles and thermodynamical calculations. These species are present in the gas phase during the synthesis of alpha-Al2O3 by chemical vapor deposition (CVD) but little is known of their solubility in this compound. The heat of formation from standard reference states of the elements varying the chemical potential of each element was calculated. An attempt to model the actual conditions in the CVD process was made, using the species and solid compounds present in a common CVD process as reference states. Our calculations suggest that sulfur from the catalyzing agent H2S will not solve in alpha-Al2O3 during deposition by CVD. It is found that the neutral chlorine and hydrogen interstitial defects display the lowest heat of formation, 281 and 280 kJ/mol, respectively, at the modeled CVD conditions. This energy is too high in order for neutral defects to form during CVD of alpha-Al2O3 at any significant amounts. The charged defects and their compensation were studied. Carbon substituting oxygen is found to be energetically favored under the modeled CVD conditions, considering carbon dioxide as competing species to solid solubility in alpha-Al2O3 at an energy of -128 kJ/mol. However, care needs to be taken when choosing the possible competing carbon-containing phases. Compensation of carbon substituting for oxygen by oxygen vacancies takes place at 110 kJ/mol from standard reference states, graphite, fcc-Al and O-2. The carbon solubility in Al2O3 is difficult to measure with standard analysis techniques such as X-ray diffraction and energy dispersive X-ray spectroscopy, but several stable compounds in the Al-C-O are available in the literature.

  • 22.
    Armstrong, Andrea
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Mattsson, Niklas
    Sahlgrens University Hospital, Sweden University of Calif San Francisco, CA 94143 USA .
    Appelqvist, Hanna
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Janefjord, Camilla
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Sandin, Linnea
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Agholme, Lotta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Olsson, Bob
    Sahlgrens University Hospital, Sweden .
    Svensson, Samuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. AlzeCure Fdn.
    Blennow, Kaj
    Sahlgrens University Hospital, Sweden .
    Zetterberg, Henrik
    Sahlgrens University Hospital, Sweden UCL Institute Neurol, England .
    Kågedal, Katarina
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Lysosomal Network Proteins as Potential Novel CSF Biomarkers for Alzheimers Disease2014In: Neuromolecular medicine, ISSN 1535-1084, E-ISSN 1559-1174, Vol. 16, no 1, 150-160 p.Article in journal (Refereed)
    Abstract [en]

    The success of future intervention strategies for Alzheimers disease (AD) will likely rely on the development of treatments starting early in the disease course, before irreversible brain damage occurs. The pre-symptomatic stage of AD occurs at least one decade before the clinical onset, highlighting the need for validated biomarkers that reflect this early period. Reliable biomarkers for AD are also needed in research and clinics for diagnosis, patient stratification, clinical trials, monitoring of disease progression and the development of new treatments. Changes in the lysosomal network, i.e., the endosomal, lysosomal and autophagy systems, are among the first alterations observed in an AD brain. In this study, we performed a targeted search for lysosomal network proteins in human cerebrospinal fluid (CSF). Thirty-four proteins were investigated, and six of them, early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were significantly increased in the CSF from AD patients compared with neurological controls. These results were confirmed in a validation cohort of CSF samples, and patients with no neurochemical evidence of AD, apart from increased total-tau, were found to have EEA1 levels corresponding to the increased total-tau levels. These findings indicate that increased levels of LAMP-1, LAMP-2, LC3, Rab3 and Rab7 in the CSF might be specific for AD, and increased EEA1 levels may be a sign of general neurodegeneration. These six lysosomal network proteins are potential AD biomarkers and may be used to investigate lysosomal involvement in AD pathogenesis.

  • 23.
    Asplund, Gunilla
    et al.
    Linköping University, The Tema Institute, Department of Water and Environmental Studies. Linköping University, Faculty of Arts and Sciences.
    Borén, Hans
    Linköping University, The Tema Institute, Department of Water and Environmental Studies. Linköping University, Faculty of Arts and Sciences.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Grimvall, Anders
    Linköping University, The Tema Institute, Department of Water and Environmental Studies. Linköping University, Faculty of Arts and Sciences.
    Soil Peroxidase-Mediated Chlorination of Fulvic Acid1991In: Humic substances in the aquatic and terrestrial environment : proceedings of an international symposium, Linköping, Sweden, August 21-23, 1989 / [ed] B. Allard, H. Borén and A. Grimvall, Berlin Heidelberg New York: Springer, 1991, 474-483 p.Chapter in book (Refereed)
    Abstract [en]

    Humic matter has recently been shown to contain considerable quantities of naturally produced organohalogens. The present study investigated the possibility of a non-specific, enzymatically mediated halogenation of organic matter in soil. The results showed that, in the presence of chloride and hydrogen peroxide, the enzyme chloroperox1dase (CPO) from the fungus Caldariomyces fumago catalyzes chlorination of fulvic acid. At pH 2.5 - 6.0, the chlorine to fulvic acid ratio in the tested sample was elevated from 12 mg/g to approximately 40-50 mg/g. It was also shown that this reaction can take place at chloride and hydrogen peroxide concentrations found in the environment. An extract from spruce forest soil was shown to have a measurable chlorinating capacity. The activity of an extract of 0.5 kg soil corresponded to approximately 0.3 enzyme units, measured as CPO activity. Enzymatically mediated halogenation of humic substances may be one of the mechanisms explaining the w1despread occurrence of adsorbable organic halogens (AOX) in soil and water.

  • 24.
    Atakan, Aylin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Nanostructured Materials. Linköping University, Faculty of Science & Engineering.
    Mäkie, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Nanostructured Materials. Linköping University, Faculty of Science & Engineering.
    Söderlind, Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Keraudy, Julien
    Linköping University, Department of Physics, Chemistry and Biology, Plasma and Coating Physics. Linköping University, Faculty of Science & Engineering.
    Björk, Emma
    Linköping University, Department of Physics, Chemistry and Biology, Nanostructured Materials. Linköping University, Faculty of Science & Engineering.
    Odén, Magnus
    Linköping University, Department of Physics, Chemistry and Biology, Nanostructured Materials. Linköping University, Faculty of Science & Engineering.
    Synthesis of a Cu-infiltrated Zr-doped SBA-15 catalyst for CO2 hydrogenation into methanol and dimethyl ethert2017In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 19, no 29, 19139-19149 p.Article in journal (Refereed)
    Abstract [en]

    A catalytically active nanoassembly comprising Cu-nanoparticles grown on integrated and active supports (large pore Zr-doped mesoporous SBA-15 silica) has been synthesized and used to promote CO2 hydrogenation. The doped mesoporous material was synthesized using a sal-gel method, in which the pore size was tuned between 11 and 15 nm while maintaining a specific surface area of about 700 m(2) g (1). The subsequent Cu nanoparticle growth was achieved by an infiltration process involving attachment of different functional groups on the external and internal surfaces of the mesoporous structure such that 7-10 nm sized Cu nanoparticles grew preferentially inside the pores. Chemisorption showed improved absorption of both CO2 and H-2 for the assembly compared to pure SBA-15 and 15% of the total CO2 was converted to methanol and dimethyl ether at 250 degrees C and 33 bar.

  • 25.
    Babu Moparthi, Satish
    et al.
    Aix Marseille University, France.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Vincentelli, Renaud
    University of Aix Marseille, France.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wenger, Jerome
    Aix Marseille University, France.
    Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, no 28386Article in journal (Refereed)
    Abstract [en]

    Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of beta-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes.

  • 26.
    Babu Moparthi, Satish
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Institut Fresnel, CNRS UMR 7249, Aix-Marseille Université, Marseille, France.
    Sjölander, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Villebeck, Laila
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Transient conformational remodeling of folding proteins by GroES - Individually and in concert with GroEL2014In: Journal of chemical biology, ISSN 1864-6158, E-ISSN 1864-6166, Vol. 7, no 1, 1-15 p.Article, review/survey (Refereed)
    Abstract [en]

    The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.

  • 27.
    Bagheri, Maryam
    et al.
    Ilam University of Medical Science, Iran .
    Rezakhani, Arjang
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Nyström, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Turkina, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Roghani, Mehrdad
    Shahed University, Iran .
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Mohseni, Simin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Amyloid Beta1-40-Induced Astrogliosis and the Effect of Genistein Treatment in Rat: A Three-Dimensional Confocal Morphometric and Proteomic Study2013In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 10Article in journal (Refereed)
    Abstract [en]

    Astrocytes are highly involved in regulation and homeostasis of the extracellular environment in the healthy brain. In pathological conditions, these cells play a major role in the inflammatory response seen in CNS tissues, which is called reactive astrogliosis and includes hypertrophy and proliferation of astrocytes. Here, we performed 3D confocal microscopy to evaluate the morphological response of reactive astrocytes positive for glial fibrillary acidic protein (GFAP) in rats, to the presence of Aβ1–40 in the rat brain before and after treatment with genistein. In 50 astrocytes per animal, we measured the volume and surface area for the nucleus, cell body, the entire cell, the tissue covered by single astrocytes and quantified the number and length of branches, the density of the astrocytes and the intensity of GFAP immunoreactivity. Injecting Aβ1–40 into the brain of rats caused astrogliosis indicated by increased values for all measured parameters. Mass spectrometric analysis of hippocampal tissue in Aβ1–40-injected brain showed decreased amounts of tubulins, enolases and myelin basic protein, and increased amounts of dihydropyrimidinase-related protein 2. In Aβ1–40-injected rats pretreated with genistein, GFAP intensity was decreased to the sham-operated group level, and Aβ1–40-induced astrogliosis was significantly ameliorated.

  • 28.
    Ballem, Mohamed Ali
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Nanostructured Materials. Linköping University, The Institute of Technology.
    Söderlind, Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Nanostructured Materials. Linköping University, The Institute of Technology.
    Nordblad, Per
    Uppsala Unversity, Sweden.
    Käll, Per-Olov
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Odén, Magnus
    Linköping University, Department of Physics, Chemistry and Biology, Nanostructured Materials. Linköping University, The Institute of Technology.
    Growth of Gd2O3 nanoparticles inside mesoporous silica frameworks2013In: Microporous and Mesoporous Materials, ISSN 1387-1811, Vol. 168, 221-224 p.Article in journal (Refereed)
    Abstract [en]

    Gadolinium oxide (Gd2O3) nanoparticles with very small size, and narrow size distribution were synthesized by infiltration of Gd(NO3)3.6H2O as an oxide precursor into the pores of SBA-15 mesoporous silica using a wet-impregnation technique. High resolution transmission electron microscopy and X-ray diffraction show that during the hydrothermal treatment of the precursor at 550 °C, gadolinium oxide nanoparticles inside the silica pores are formed. Subsequent dissolution of the silica template by NaOH resulted in well dispersed nanoparticles with an average diameter of 3.6 ± 0.9 nm.

  • 29.
    Becker, Richard
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Söderlind, Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Käll, Per-Olov
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Synthesis of silver nanowires in aqueous solutions2010In: Materials letters (General ed.), ISSN 0167-577X, E-ISSN 1873-4979, Vol. 64, no 8, 956-958 p.Article in journal (Refereed)
    Abstract [en]

    Silver nanowires with a diameter of 30 nm and typical lengths of 5–10 μm have been synthesized in an aqueous medium. To initiate the reaction, citrate ions were used, and during the reaction the aromatic organicmolecules polymerize forming “straight” chain surfactants which support the formation of nanowires. Characterization by TEM and HRETM revealed the nanowires to be highly crystalline with a growth along the [110] direction.

  • 30.
    Bednarska, Natalia G.
    et al.
    KULeuven, Belgium; VIB, Belgium.
    van Eldere, Johan
    KULeuven, Belgium.
    Gallardo, Rodrigo
    VIB, Belgium; KULeuven, Belgium.
    Ganesan, Ashok
    VIB, Belgium; KULeuven, Belgium.
    Ramakers, Meine
    VIB, Belgium; KULeuven, Belgium.
    Vogel, Isabel
    KULeuven, Belgium.
    Baatsen, Pieter
    VIB11, Belgium; KULeuven, Belgium.
    Staes, An
    VIB, Belgium; University of Ghent, Belgium.
    Goethals, Marc
    VIB, Belgium; University of Ghent, Belgium.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Nilsson, K. Peter R.
    VIB, Belgium.
    Gevaert, Kris
    VIB, Belgium; University of Ghent, Belgium.
    Schymkowitz, Joost
    VIB, Belgium; KULeuven, Belgium.
    Rousseau, Frederic
    VIB, Belgium; KULeuven, Belgium.
    Protein aggregation as an antibiotic design strategy2016In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 99, no 5, 849-865 p.Article in journal (Refereed)
    Abstract [en]

    Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin-resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.

  • 31.
    Berg, Ina
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Thor, Stefan
    Linköping University, Department of Clinical and Experimental Medicine, Developmental Biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Modeling Familial Amyloidotic Polyneuropathy (Transthyretin V30M) in Drosophila melanogaster2009In: NEURODEGENERATIVE DISEASES, ISSN 1660-2854, Vol. 6, no 3, 127-138 p.Article in journal (Refereed)
    Abstract [en]

    Background/Aims: Transthyretin (TTR) is a prevalent plasma and cerebrospinal fluid protein associated with sporadic and heritable amyloidosis. TTR amyloidosis is linked to a vast number of mutations with varying phenotype, tissue distribution and age of onset. The most prevalent mutation associated with familial amyloidotic polyneuropathy (FAP) is the V30M mutation. Studies of transgenic mouse models of TTR V30M FAP have been hampered by variable phenotype, low disease penetrance, and slow onset. Methods/Results: To model TTR-associated amyloid disease in the Drosophila model system, transgenic Drosophila were generated, expressing wild-type (wt) TTR or TTR V30M, associated with sporadic senile systemic amyloidosis (SSA) and inherited FAP, respectively. We found that expression of FAP-associated TTR V30M mutant in the nervous system resulted in reduced lifespan and in reduced climbing ability indicating neurological impairment, whereas expression of TTR wt showed a milder phenotype. Congo red staining of the Drosophila brain shows positive amyloid binding in the aged TTR V30M flies. Extensive brain vacuole formation was evident for the aged TTR V30M flies, whereas a milder phenotype was shown by the TTR wt flies. In addition, expression of TTR V30M in the eye leads to tissue damage, including rough eye, morphological changes and fibrous deposition. Conclusion: Our results suggest that Drosophila is a promising complementary system for studies of TTR-associated amyloid diseases.

  • 32.
    Berg, Kirsti
    et al.
    Norwegian University of Science and Technology, Trondheim, Norway.
    Ericsson, Madelene
    Umeå University, Sweden.
    Lindgren, Mikael
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Norwegian University of Science and Technology, Trondheim, Norway.
    Gustafsson, Håkan
    Linköping University, Department of Medical and Health Sciences, Division of Radiological Sciences. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Biomedical Engineering.
    A High Precision Method for Quantitative Measurements of Reactive Oxygen Species in Frozen Biopsies2014In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 3Article in journal (Refereed)
    Abstract [en]

    Objective

    An electron paramagnetic resonance (EPR) technique using the spin probe cyclic hydroxylamine 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetr​amethylpyrrolidine(CMH) was introduced as a versatile method for high precision quantification of reactive oxygen species, including the superoxide radical in frozen biological samples such as cell suspensions, blood or biopsies.

    Materials and Methods

    Loss of measurement precision and accuracy due to variations in sample size and shape were minimized by assembling the sample in a well-defined volume. Measurement was carried out at low temperature (150 K) using a nitrogen flow Dewar. The signal intensity was measured from the EPR 1st derivative amplitude, and related to a sample, 3-carboxy-proxyl (CP•) with known spin concentration.

    Results

    The absolute spin concentration could be quantified with a precision and accuracy better than ±10 µM (k = 1). The spin concentration of samples stored at −80°C could be reproduced after 6 months of storage well within the same error estimate.

    Conclusion

    The absolute spin concentration in wet biological samples such as biopsies, water solutions and cell cultures could be quantified with higher precision and accuracy than normally achievable using common techniques such as flat cells, tissue cells and various capillary tubes. In addition; biological samples could be collected and stored for future incubation with spin probe, and also further stored up to at least six months before EPR analysis, without loss of signal intensity. This opens for the possibility to store and transport incubated biological samples with known accuracy of the spin concentration over time.

  • 33.
    Bergkvist, Liza
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Amyloid-β and lysozyme proteotoxicity in Drosophila: Beneficial effects of lysozyme and serum amyloid P component in models of Alzheimer’s disease and lysozyme amyloidosis2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In the work presented this thesis, two different conditions that are classified as protein misfolding diseases: Alzheimer's disease and lysozyme amyloidosis and proteins that could have a beneficial effect in these diseases, have been studied using Drosophila melanogaster, commonly known as the fruit fly. The fruit fly has been used for over 100 years to study and better understand fundamental biological processes. Although the fruit fly, unlike humans, is an invertebrate, many of its central biological mechanisms are very similar to ours. The first transgenic flies were designed in the early 1980s, and since then, the fruit fly has been one of the most widely used model organisms in studies on the effects of over-expressed human proteins in a biological system; one can regard the fly as a living, biological test tube. For  most proteins, it is necessary that they fold into a three-dimensional structure to function properly. But sometimes the folding goes wrong; this may be due to mutations that make the protein unstable and subject to misfolding. A misfolded protein molecule can then aggregate with other misfolded proteins. In Alzheimer's disease, which is the most common form of dementia, protein aggregates are present in the brains of patients. These aggregates are composed of the amyloid-β (Aβ) peptide, a small peptide of around 42 amino acids which is cleaved from the larger, membrane-bound, protein AβPP by two different enzymes, BACE1 and γ-secretase. In the first part of this thesis, two different fly models for Alzheimer’s disease were used: the Aβ fly model, which directly expresses the Aβ peptide, and the AβPP-BACE1 fly model, in which all the components necessary to produce the Aβ peptide in the fly are expressed in the fly central nervous system (CNS). The two different fly models were compared and the results show that a significantly smaller amount of the Aβ peptide is needed to achieve the same, or an even greater, toxic effect in the AβPP-BACE1 model compared to the Aβ model. In the second part of the thesis, these two fly models for Alzheimer’s disease were again used, but now to investigate whether lysozyme, a protein involved in our innate immune system, can counteract the toxic effect of Aβ generated in the fly models. And indeed, lysozyme is able to save the flies from Aβ-induced toxicity. Aβ and lysozyme were found to interact with each other in vivo. The second misfolding disease studied in this thesis is lysozyme amyloidosis. It is a rare, dominantly inherited amyloid disease in which mutant variants of lysozyme give rise to aggregates, weighing up to several kilograms, that accumulate around the kidneys and liver, eventually leading to organ failure. In the third part of this thesis, a fly model for lysozyme amyloidosis was used to study the effect of co-expressing the serum amyloid P component (SAP), a protein that is part of all protein aggregates found within this disease class. SAP is able to rescue the toxicity induced by expressing the mutant variant of lysozyme, F57I, in the fly's CNS. To further investigate how SAP was able to do this, double-expressing lysozyme flies, which exhibit stronger disease phenotypes than those of the single-expressing lysozyme flies previously studied, were used in the fourth part of this thesis. SAP was observed to reduce F57I toxicity and promote F57I to form aggregates with more distinct amyloid characteristics. In conclusion, the work included in this thesis demonstrates that: i) Aβ generated from AβPP processing in the fly CNS results in higher proteotoxicity compared with direct expression of Aβ from the transgene, ii) lysozyme can prevent Aβ proteotoxicity in Drosophila and could thus be a potential therapeutic molecule to treat Alzheimer’s disease and iii) in a Drosophila model of lysozyme amyloidosis, SAP can prevent toxicity from the disease-associated lysozyme variant F57I and promote formation of aggregated lysozyme morphotypes with amyloid properties; this is important to take into account when a reduced level of SAP is considered as a treatment strategy for lysozyme amyloidosis.

    List of papers
    1. A beta PP processing results in greater toxicity per amount of A beta(1-42) than individually expressed and secreted A beta(1-42) in Drosophila melanogaster
    Open this publication in new window or tab >>A beta PP processing results in greater toxicity per amount of A beta(1-42) than individually expressed and secreted A beta(1-42) in Drosophila melanogaster
    2016 (English)In: BIOLOGY OPEN, ISSN 2046-6390, Vol. 5, no 8, 1030-1039 p.Article in journal (Refereed) Published
    Abstract [en]

    The aggregation of the amyloid-beta (A beta) peptide into fibrillar deposits has long been considered the key neuropathological hallmark of Alzheimers disease (AD). A beta peptides are generated from proteolytic processing of the transmembrane A beta precursor protein (A beta PP) via sequential proteolysis through the beta-secretase activity of beta-site A beta PP-cleaving enzyme (BACE1) and by the intramembranous enzyme gamma-secretase. For over a decade, Drosophila melanogaster has been used as a model organism to study AD, and two different approaches have been developed to investigate the toxicity caused by AD-associated gene products in vivo. In one model, the A beta peptide is directly over-expressed fused to a signal peptide, allowing secretion of the peptide into the extracellular space. In the other model, human A beta PP is co-expressed with human BACE1, resulting in production of the A beta peptide through the processing of A beta PP by BACE1 and by endogenous fly gamma-secretase. Here, we performed a parallel study of flies that expressed the A beta(1-42) peptide alone or that co-expressed A beta PP and BACE1. Toxic effects (assessed by eye phenotype, longevity and locomotor assays) and levels of the A beta(1-42), A beta(1-40) and A beta(1-38) peptides were examined. Our data reveal that the toxic effect per amount of detected A beta(1-42) peptide was higher in the flies co-expressing A beta PP and BACE1 than in the A beta(1-42)-expressing flies, and that the co-existence of A beta(1-42) and A beta(1-40) in the flies co-expressing A beta PP and BACE1 could be of significant importance to the neurotoxic effect detected in these flies. Thus, the toxicity detected in these two fly models seems to have different modes of action and is highly dependent on how and where the peptide is generated rather than on the actual level of the A beta(1-42) peptide in the flies. This is important knowledge that needs to be taken into consideration when using Drosophila models to investigate disease mechanisms or therapeutic strategies in AD research.

    Place, publisher, year, edition, pages
    COMPANY OF BIOLOGISTS LTD, 2016
    Keyword
    Alzheimers disease; Amyloid-beta (A beta); A beta PP processing; Drosophila melanogaster; Proteotoxicity
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:liu:diva-131685 (URN)10.1242/bio.017194 (DOI)000382304400003 ()27387531 (PubMedID)
    Note

    Funding Agencies|Torsten Soderbergs Stiftelse [M26/11]; Alzheimer Foundation [03-069]; Dementia Foundation; Ahlen Foundation; Gamla Tjanarinnor [2015-00187]

    Available from: 2016-10-03 Created: 2016-09-30 Last updated: 2017-05-16
    2. Beneficial effects of increased lysozyme levels in Alzheimer’s disease modelled in Drosophila melanogaster
    Open this publication in new window or tab >>Beneficial effects of increased lysozyme levels in Alzheimer’s disease modelled in Drosophila melanogaster
    Show others...
    2016 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, no 19, 3508-3522 p.Article in journal (Refereed) Published
    Abstract [en]

    Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimer’s disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome-wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aβ1-42 or AβPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aβ1-42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aβ1-42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aβ1-42 and caused a beneficial effect by binding of lysozyme to toxic species of Aβ1-42, which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD.

    Place, publisher, year, edition, pages
    John Wiley & Sons, 2016
    Keyword
    Alzheimer’s disease, amyloid-β, Drosophila, lysozyme
    National Category
    Genetics Medical Genetics Developmental Biology Bioinformatics and Systems Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:liu:diva-131796 (URN)10.1111/febs.13830 (DOI)000386033700001 ()27562772 (PubMedID)
    Available from: 2016-10-07 Created: 2016-10-07 Last updated: 2017-05-16Bibliographically approved
    3. Serum Amyloid P Component Ameliorates Neurological Damage Caused by Expressing a Lysozyme Variant in the Central Nervous System of Drosophila melanogaster
    Open this publication in new window or tab >>Serum Amyloid P Component Ameliorates Neurological Damage Caused by Expressing a Lysozyme Variant in the Central Nervous System of Drosophila melanogaster
    2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 7, e0159294- p.Article in journal (Refereed) Published
    Abstract [en]

    Lysozyme amyloidosis is a hereditary disease in which mutations in the gene coding for lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT) lysozyme and the disease-associated variant F57I in the central nervous system (CNS) of a Drosophila melanogaster model of lysozyme amyloidosis, with and without co-expression of serum amyloid p component (SAP). SAP is known to be a universal constituent of amyloid deposits and to associate with lysozyme fibrils. There are clear indications that SAP may play an important role in lysozyme amyloidosis, which requires further elucidation. We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan than flies expressing WT lysozyme. We also identified apoptotic cells in the brains of F57I flies demonstrating that the flies neurological functions are impaired when F57I is expressed in the nerve cells. However, co-expression of SAP in the CNS prevented cell death and restored the F57I flies lifespan. Thus, SAP has the apparent ability to protect nerve cells from damage caused by F57I. Furthermore, it was found that co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies. Our findings suggest that the F57I mutation affects the aggregation process of lysozyme resulting in the formation of cytotoxic species and that SAP is able to prevent cell death in the F57I flies by preventing accumulation of toxic F57I structures.

    Place, publisher, year, edition, pages
    PUBLIC LIBRARY SCIENCE, 2016
    National Category
    Developmental Biology
    Identifiers
    urn:nbn:se:liu:diva-131183 (URN)10.1371/journal.pone.0159294 (DOI)000380169300043 ()27428539 (PubMedID)
    Note

    Funding Agencies|Swedish Research Council; Soderberg foundation [M26/11]; Linkoping University Neurobiology Center

    Available from: 2016-09-19 Created: 2016-09-12 Last updated: 2017-05-16
  • 34.
    Bergkvist, Liza
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Helmfors, Linda
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Brorsson, Ann-Christin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Co-expression of a disease-associated lysozyme variant with human lysozyme in Drosophila causes accumulation of amyloid deposits and neurodegenerationManuscript (preprint) (Other academic)
    Abstract [en]

    Lysozyme amyloidosis is a dominantly inherited form of amyloid disease. Mutant variants of the protein, with increased tendencies to aggregate compared to the wild type (WT), accumulate in large amyloid deposits in multiple organs, eventually leading to organ failure. Humans affected by lysozyme amyloidosis carry one allele for the wild type protein and one allele encoding for a mutant variant of lysozyme. We have used a Drosophila melanogaster model to investigate the effect of co-expressing WT lysozyme and a mutated variant, F57I, in the central nervous system (CNS) of the fly. In this study, using activity and longevity assays, WT-F57I flies showed a lower activity and a shorter lifespan than flies expressing only WT or the F57I variant of lysozyme (median survival 16 days compared to 34 and 23 respectively). This indicates deteriorating neurological functions in WT-F57I flies; exceeding the decrease in neurological function previously observed for flies only expressing the mutated variant, F57I. In addition, accumulation of insoluble species with amyloid structure was detected for the WT-F57I flies but not for the WT or the F57I flies. Our study show that co-expression of WT lysozyme and the amyloidogenic variant F57I results in neurological damage and is required for accumulation of amyloid deposits, which is characteristic for the disease observed in humans. Our data suggest that insoluble amyloid species or intermediate species, formed on the pathway toward amyloid species, may be cytotoxic and thus contribute to the impaired neurological functions observed for the WT-F57I flies.

  • 35.
    Bergkvist, Liza
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Sandin, Linnea
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Kågedal, Katarina
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Brorsson, Ann-Christin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    A beta PP processing results in greater toxicity per amount of A beta(1-42) than individually expressed and secreted A beta(1-42) in Drosophila melanogaster2016In: BIOLOGY OPEN, ISSN 2046-6390, Vol. 5, no 8, 1030-1039 p.Article in journal (Refereed)
    Abstract [en]

    The aggregation of the amyloid-beta (A beta) peptide into fibrillar deposits has long been considered the key neuropathological hallmark of Alzheimers disease (AD). A beta peptides are generated from proteolytic processing of the transmembrane A beta precursor protein (A beta PP) via sequential proteolysis through the beta-secretase activity of beta-site A beta PP-cleaving enzyme (BACE1) and by the intramembranous enzyme gamma-secretase. For over a decade, Drosophila melanogaster has been used as a model organism to study AD, and two different approaches have been developed to investigate the toxicity caused by AD-associated gene products in vivo. In one model, the A beta peptide is directly over-expressed fused to a signal peptide, allowing secretion of the peptide into the extracellular space. In the other model, human A beta PP is co-expressed with human BACE1, resulting in production of the A beta peptide through the processing of A beta PP by BACE1 and by endogenous fly gamma-secretase. Here, we performed a parallel study of flies that expressed the A beta(1-42) peptide alone or that co-expressed A beta PP and BACE1. Toxic effects (assessed by eye phenotype, longevity and locomotor assays) and levels of the A beta(1-42), A beta(1-40) and A beta(1-38) peptides were examined. Our data reveal that the toxic effect per amount of detected A beta(1-42) peptide was higher in the flies co-expressing A beta PP and BACE1 than in the A beta(1-42)-expressing flies, and that the co-existence of A beta(1-42) and A beta(1-40) in the flies co-expressing A beta PP and BACE1 could be of significant importance to the neurotoxic effect detected in these flies. Thus, the toxicity detected in these two fly models seems to have different modes of action and is highly dependent on how and where the peptide is generated rather than on the actual level of the A beta(1-42) peptide in the flies. This is important knowledge that needs to be taken into consideration when using Drosophila models to investigate disease mechanisms or therapeutic strategies in AD research.

  • 36.
    Bergsten, Johan
    et al.
    Chalmers, Gothenburg, Sweden.
    Li, Xun
    Linköping University, Department of Physics, Chemistry and Biology, Semiconductor Materials. Linköping University, Faculty of Science & Engineering.
    Nilsson, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Semiconductor Materials. Linköping University, Faculty of Science & Engineering.
    Danielsson, Örjan
    Linköping University, Department of Physics, Chemistry and Biology, Semiconductor Materials. Linköping University, Faculty of Science & Engineering.
    Pedersen, Henrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Janzén, Erik
    Linköping University, Department of Physics, Chemistry and Biology, Semiconductor Materials. Linköping University, Faculty of Science & Engineering.
    Forsberg, Urban
    Linköping University, Department of Physics, Chemistry and Biology, Semiconductor Materials. Linköping University, Faculty of Science & Engineering.
    Rorsman, Niklas
    Chalmers, Gothenburg, Sweden.
    AlGaN/GaN high electron mobility transistors with intentionally doped GaN buffer using propane as carbon precursor2016In: Japanese Journal of Applied Physics, ISSN 0021-4922, E-ISSN 1347-4065, Vol. 55, 05FK02-1-05FK02-4 p., 05FK02Article in journal (Refereed)
    Abstract [en]

    AlGaN/GaN high electron mobility transistors (HEMTs) fabricated on a heterostructure grown by metalorganic chemical vapor deposition using analternative method of carbon (C) doping the buffer are characterized. C-doping is achieved by using propane as precursor, as compared to tuningthe growth process parameters to control C-incorporation from the gallium precursor. This approach allows for optimization of the GaN growthconditions without compromising material quality to achieve semi-insulating properties. The HEMTs are evaluated in terms of isolation anddispersion. Good isolation with OFF-state currents of 2 ' 10%6A/mm, breakdown fields of 70V/μm, and low drain induced barrier lowering of0.13mV/V are found. Dispersive effects are examined using pulsed current–voltage measurements. Current collapse and knee walkout effectslimit the maximum output power to 1.3W/mm. With further optimization of the C-doping profile and GaN material quality this method should offer aversatile approach to decrease dispersive effects in GaN HEMTs.

  • 37.
    Bett, Cyrus
    et al.
    Department of Pathology, University of California, San Diego, La Jolla, California, United States of America.
    Kurt, Tim D.
    Department of Pathology, University of California, San Diego, La Jolla, California, United States of America.
    Lucero, Melanie
    Department of Pathology, University of California, San Diego, La Jolla, California, United States of America.
    Trejo, Margarita
    Department of Neuroscience, University of California, San Diego, La Jolla, California, United States of America.
    Rozemuller, Annemieke J.
    Dutch Surveillance Centre for Prion Diseases, University Medical Centre Utrecht, Utrecht, The Netherlands.
    Kong, Qingzhong
    Department of Pathology, Case Western Reserve University, Cleveland, Ohio, United States of America.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Masliah, Eliezer
    Department of Neuroscience, University of California, San Diego, La Jolla, California, United States of America.
    Oldstone, Michael B.
    Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America.
    Sigurdson, Christina J.
    Department of Pathology, Immunology, and Microbiology, University of California, Davis, Davis, California, United States of America.
    Defining the Conformational Features of Anchorless, Poorly Neuroinvasive Prions2013In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 9, no 4Article in journal (Refereed)
    Abstract [en]

    Infectious prions cause diverse clinical signs and form an extraordinary range of structures, from amorphous aggregates to fibrils. How the conformation of a prion dictates the disease phenotype remains unclear. Mice expressing GPI-anchorless or GPI-anchored prion protein exposed to the same infectious prion develop fibrillar or nonfibrillar aggregates, respectively, and show a striking divergence in the disease pathogenesis. To better understand how a prion's physical properties govern the pathogenesis, infectious anchorless prions were passaged in mice expressing anchorless prion protein and the resulting prions were biochemically characterized. Serial passage of anchorless prions led to a significant decrease in the incubation period to terminal disease and altered the biochemical properties, consistent with a transmission barrier effect. After an intraperitoneal exposure, anchorless prions were only weakly neuroinvasive, as prion plaques rarely occurred in the brain yet were abundant in extracerebral sites such as heart and adipose tissue. Anchorless prions consistently showed very high stability in chaotropes or when heated in SDS, and were highly resistant to enzyme digestion. Consistent with the results in mice, anchorless prions from a human patient were also highly stable in chaotropes. These findings reveal that anchorless prions consist of fibrillar and highly stable conformers. The additional finding from our group and others that both anchorless and anchored prion fibrils are poorly neuroinvasive strengthens the hypothesis that a fibrillar prion structure impedes efficient CNS invasion.

  • 38.
    Blissing, Annica
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Thiopurine S-methyltransferase - characterization of variants and ligand binding2017Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects.

    Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function.

    Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.

  • 39.
    Boesze-Battaglia, Kathleen
    et al.
    University of Penn, PA 19104 USA.
    Walker, Lisa P.
    University of Penn, PA 19104 USA.
    Zekavat, Ali
    University of Penn, PA 19104 USA.
    Dlakic, Mensur
    Montana State University, MT 59717 USA.
    Damek Scuron, Monika
    University of Penn, PA 19104 USA.
    Nygren, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Shenker, Bruce J.
    University of Penn, PA 19104 USA.
    The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization2015In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 83, no 10, 4042-4055 p.Article in journal (Refereed)
    Abstract [en]

    Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1 beta [IL-1 beta] and tumor necrosis factor alpha [TNF-alpha] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.

  • 40.
    Bogoeva, Vanya
    et al.
    Bulgarian Academic Science, Bulgaria.
    Siksjö, Monica
    Norwegian University of Science and Technology, Norway.
    Säterbo, Kristin G.
    Norwegian University of Science and Technology, Norway.
    Melo, Thor Bernt
    Norwegian University of Science and Technology, Norway.
    Björköy, Astrid
    Norwegian University of Science and Technology, Norway.
    Lindgren, Mikael
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Norwegian University of Science and Technology, Norway.
    Gederaas, Odrun A.
    Norwegian University of Science and Technology, Norway.
    Ruthenium porphyrin-induced photodamage in bladder cancer cells2016In: Photodiagnosis and Photodynamic Therapy, ISSN 1572-1000, E-ISSN 1873-1597, Vol. 14Article in journal (Refereed)
    Abstract [en]

    Photodynamic therapy (PDT) is a noninvasive treatment for solid malignant and flat tumors. Light activated sensitizers catalyze photochemical reactions that produce reactive oxygen species which can cause cancer cell death. In this work we investigated the photophysical properties of the photosensitizer ruthenium(II) porphyrin (RuP), along with its PDT efficiency onto rat bladder cancer cells (AY27). Optical spectroscopy verified that RuP is capable to activate singlet oxygen via blue and red absorption bands and inter system crossing (ISC) to the triplet state. In vitro experiments on AY27 indicated increased photo-toxicity of RuP (20 mu M, 1811 incubation) after cell illumination (at 435 nm), as a function of blue light exposure. Cell survival fraction was significantly reduced to 14% after illumination of 20 mu M RuP with 15.6 J/cm(2), whereas the "dark toxicity" of 20 mu M RuP was 17%. Structural and morphological changes of cells were observed, due to RuP accumulation, as well as light-dependent cell death was recorded by confocal microscopy. Flow cytometry verified that PDT-RuP (50 mu M) triggered significant photo-induced cellular destruction with a photoxicity of (93% +/- 0.9%). Interestingly, the present investigation of RuP-PDT showed that the dominating mode of cell death is necrosis. RuP "dark toxicity" compared to the conventional chemotherapeutic drug cisplatin was higher, both evaluated by the MIT assay (24 h). In conclusion, the present investigation shows that RuP with or without photoactivation induces cell death of bladder cancer cells. (C) 2016 Elsevier B.V. All rights reserved.

  • 41.
    Bohlin, Nina
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Kemiskt försvar mot havstulpanskolonisering i marina svampdjur2013Independent thesis Basic level (degree of Bachelor), 10,5 credits / 16 HE creditsStudent thesis
    Abstract [en]

    The purpose of this project is to analyze and study the absorption of the substances ivermectin, spinosad and barettin to hydrophobic and hydrophilic surfaces, and to analyze and study if the barnacle larva are effected when they try to settle on the treated surface.

    Incubation tests with barnacle larva in Petri dishes were performed as well as absorption tests on hydrophobic and hydrophilic surfaces which were tested with ellipsometry. To determine the surface thickness.

    The barnacles were placed in hydrophobic as well as hydrophilic Petri dishes that had been incubated with ivermectin, spinosad or barettin. After four to six days the larva was counted to analyze the settling. From the results conclusions could be drawn about the adsorption abilities of the substances to the different surfaces.

    The barnacles were placed in hydrophobic as well as hydrophilic Petri dishes that had been incubated with ivermectin, spinosad or barettin. After four to six days the larva was counted to analyze the settling. From the results conclusions could be drawn about the adsorption abilities of the substances to the different surfaces.

    To analyze the adsorption abilities, pieces of silicon oxide were prepared with hydrophobic and hydrophilic poly dimethyl siloxan groups, and albumine. The pieces were then analyzed with ellipsometry.

    The larva's mortality was very high in the first tests. It can be explained with them being stored too cold the first days, which might have caused their death. It might also be because of contamination from the net used to move the larva, since it was in contact with all the substances concentrations.

    The standard deviations from the ellipsometry tests are very high, most likely due to uneven adsorption of the substances to the surface. With more tests and measurements, more accurate results could have been sustained

    The purpose of this project is to analyze and study the absorption of the substances ivermectin, spinosad and barettin to hydrophobic and hydrophilic surfaces, and to analyze and study if the barnacle larva are effected when they try to settle on the treated surface.

    Incubation tests with barnacle larva in Petri dishes were performed as well as absorption tests on hydrophobic and hydrophilic surfaces which were tested with ellipsometry. To determine the surface thickness.

    The barnacles were placed in hydrophobic as well as hydrophilic Petri dishes that had been incubated with ivermectin, spinosad or barettin. After four to six days the larva was counted to analyze the settling. From the results conclusions could be drawn about the adsorption abilities of the substances to the different surfaces.

    The barnacles were placed in hydrophobic as well as hydrophilic Petri dishes that had been incubated with ivermectin, spinosad or barettin. After four to six days the larva was counted to analyze the settling. From the results conclusions could be drawn about the adsorption abilities of the substances to the different surfaces.

    To analyze the adsorption abilities, pieces of silicon oxide were prepared with hydrophobic and hydrophilic poly dimethyl siloxan groups, and albumine. The pieces were then analyzed with ellipsometry.

    The larva's mortality was very high in the first tests. It can be explained with them being stored too cold the first days, which might have caused their death. It might also be because of contamination from the net used to move the larva, since it was in contact with all the substances concentrations.

    The standard deviations from the ellipsometry tests are very high, most likely due to uneven adsorption of the substances to the surface. With more tests and measurements, more accurate results could have been sustained

  • 42.
    Bounechada, Djamela
    et al.
    Chalmers, Dept Chem & Biol Engn, SE-41296 Gothenburg, Sweden Chalmers, Competence Ctr Catalysis, SE-41296 Gothenburg, Sweden .
    Darmastuti, Zhafira
    Linköping University, Department of Physics, Chemistry and Biology, Applied Sensor Science. Linköping University, The Institute of Technology.
    Andersson, Mike
    Linköping University, Department of Physics, Chemistry and Biology, Applied Sensor Science. Linköping University, The Institute of Technology.
    Ojamäe, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Lloyd Spetz, Anita
    Linköping University, Department of Physics, Chemistry and Biology, Applied Sensor Science. Linköping University, The Institute of Technology.
    Skoglundh, Magnus
    Chalmers, Dept Chem & Biol Engn, SE-41296 Gothenburg, Sweden Chalmers, Competence Ctr Catalysis, SE-41296 Gothenburg, Sweden.
    Carlsson, Per-Anders
    Chalmers, Dept Chem & Biol Engn, SE-41296 Gothenburg, Sweden Chalmers, Competence Ctr Catalysis, SE-41296 Gothenburg, Sweden.
    Vibrational Study of SOx Adsorption on Pt/SiO22014In: The Journal of Physical Chemistry C, ISSN 1932-7447, E-ISSN 1932-7455, Vol. 118, no 51, 29713-29723 p.Article in journal (Refereed)
    Abstract [en]

    The formation of ad-SOx species on Pt/SiO2 upon exposure to SO2 in concentrations ranging from 10 to 50 ppm at between 200 and 400 degrees C has been studied by in situ diffuse reflectance infrared Fourier transformed spectroscopy. In parallel, first-principles calculations have been carried out to consolidate the experimental interpretations. It was found that sulfate species form on the silica surface with a concomitant removal/rearrangement of silanol groups. Formation of ad-SOx species occurs only after SO2 oxidation to SO3 on the platinum surface. Thus, SO2 oxidation to SO3 is the first step in the SOx adsorption process, followed by spillover of SO3 to the oxide, and finally, the formation of sulfate species on the hydroxyl positions on the oxide. The sulfate formation is influenced by both temperature and SO2 concentration. Furthermore, exposure to hydrogen is shown to be sufficiently efficient as to remove ad-SOx species from the silica surface.

  • 43.
    Bounechada, Djamela
    et al.
    Chalmers Institute of Technology, Gothenburg.
    Darmastuti, Zhafira
    Linköping University, Department of Physics, Chemistry and Biology, Applied Sensor Science. Linköping University, The Institute of Technology.
    Ojamae, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Andersson, Mike
    Linköping University, Department of Physics, Chemistry and Biology, Applied Sensor Science. Linköping University, The Institute of Technology.
    Lloyd Spetz, Anita
    Linköping University, Department of Physics, Chemistry and Biology, Applied Sensor Science. Linköping University, The Institute of Technology.
    Skoglundh, Magnus
    Competence Centre for Catalysis / Department of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden.
    Carlsson, P-A
    Competence Centre for Catalysis / Department of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden.
    Vibrational analysis of SO2 on Pt / SiO2 systemManuscript (preprint) (Other academic)
    Abstract [en]

    In situ diffuse reflectance infrared Fourier transformed spectroscopy was used to study the interactions of SOx species with Pt/SiO2 between 200 and 400°C, and for SO2 concentrations between 10 and 50 ppm, which represents a concentration range where MISFET sensors exhibit good responses. In parallel, first-principles calculations have been carried out to support the experimental interpretations. It was found that sulfate species were formed on the silica surface, accompanied with removal/rearrangement of silanol groups upon exposure to SO2. Both experimental and theoretical calculations also suggest that the surface species were only formed after SO2 oxidation to SO3 on the metal surface. These evidences support the idea of SO2 oxidation to SO3 as the first step in the process of sulfate formation, followed by spillover of SO3 to the oxide, and finally the formation of sulfate species on the hydroxyl positions on the oxide. The results also indicate that the sulfate formation on silica depends both on the temperature and the SO2 concentration. Furthermore, hydrogen exposure was shown to be efficient for sulfur removal from the silica surface.

  • 44.
    Boyd, Robert
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Plasma and Coating Physics. Linköping University, Faculty of Science & Engineering.
    Söderlind, Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Helmersson, Ulf
    Linköping University, Department of Physics, Chemistry and Biology, Plasma and Coating Physics. Linköping University, Faculty of Science & Engineering.
    Odén, Magnus
    Linköping University, Department of Physics, Chemistry and Biology, Nanostructured Materials. Linköping University, Faculty of Science & Engineering.
    Pilch, Iris
    Linköping University, Department of Physics, Chemistry and Biology, Thin Film Physics. Linköping University, Faculty of Science & Engineering.
    Complex 3D nanocoral like structures formed by copper nanoparticle aggregation on nanostructured zinc oxide rods2016In: Materials letters (General ed.), ISSN 0167-577X, E-ISSN 1873-4979, Vol. 184, 127-130 p.Article in journal (Refereed)
    Abstract [en]

    This paper reports a new strategy for nanoparticle surface assembly so that they form anisotropic fibril like features, consisting of particles directly attached to each other, which can extend 500 nm from the surface. The particles are both formed and deposited in a single step process enabled via the use of a pulsed plasma based technique. Using this approach, we have successfully modified zinc oxide rods, up to several hundred nanometers in diameter, with 25 nm diameter copper nanoparticles for catalytic applications. The resulting structure could be modelled using a diffusion limited aggregation based approach. This gives the material the appearance of marine coral, hence the term nanocoral. (C) 2016 Elsevier B.V. All rights reserved.

    The full text will be freely available from 2018-07-01 15:18
  • 45.
    Brelstaff, Jack
    et al.
    University of Cambridge, England.
    Ossola, Bernardino
    University of Cambridge, England.
    Neher, Jonas J.
    University of Tubingen, Germany.
    Klingstedt, Therese
    MRC, England.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Goedert, Michel
    MRC, England.
    Grazia Spillantini, Maria
    University of Cambridge, England.
    Tolkovsky, Aviva M.
    University of Cambridge, England.
    The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice2015In: Frontiers in Neuroscience, ISSN 1662-4548, E-ISSN 1662-453X, Vol. 9, no 184Article in journal (Refereed)
    Abstract [en]

    Identification of fluorescent dyes that label the filamentous protein aggregates characteristic of neurodegenerative disease, such as beta-amyloid and tau in Alzheimers disease, in a live cell culture system has previously been a major hurdle. Here we show that pentameric formyl thiophene acetic acid (pFTAA) fulfills this function in living neurons cultured from adult P301S tau transgenic mice. Injection of pFTAA into 5-month-old P301S tau mice detected cortical and DRG neurons immunoreactive for AT100, an antibody that identifies solely filamentous tau, or MC1, an antibody that identifies a conformational change in tau that is commensurate with neurofibrillary tangle formation in Alzheimers disease brains. In fixed cultures of dorsal root ganglion (DRG) neurons, pFTAA binding, which also identified AT100 or MC1+ve neurons, followed a single, saturable binding curve with a half saturation constant of 0.14 mu M, the first reported measurement of a binding affinity of a beta-sheet reactive dye to primary neurons harboring filamentous tau. Treatment with formic acid, which solubilizes filamentous tau, extracted pFTAA, and prevented the re-binding of pFTAA and MC1 without perturbing expression of soluble tau, detected using an anti-human tau (HT7) antibody. In live cultures, pFTAA only identified DRG neurons that, after fixation, were AT100/MC1+ve, confirming that these forms of tau pre-exist in live neurons. The utility of pFTAA to discriminate between living neurons containing filamentous tau from other neurons is demonstrated by showing that more pFTAA+ve neurons die than pFTAA-ve neurons over 25 days. Since pFTAA identifies fibrillar tau and other misfolded proteins in living neurons in culture and in animal models of several neurodegenerative diseases, as well as in human brains, it will have considerable application in sorting out disease mechanisms and in identifying diseasemodifying drugs that will ultimately help establish the mechanisms of neurodegeneration in human neurodegenerative diseases.

  • 46.
    Buchholt, Kristina
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Ieva, E.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry.
    Käll, Per-Olov
    Linköping University, Department of Physics, Chemistry and Biology, Physical Chemistry. Linköping University, The Institute of Technology.
    Ojamäe, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Physical Chemistry. Linköping University, The Institute of Technology.
    Torsi, L
    Universita degli Studi di Bari, Italy.
    Lutic, D.
    Växjö universitet.
    Strand, M
    Växjö universitet.
    Sanati, M.
    Växjö universitet.
    Lloyd Spetz, Anita
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    FET devices with gold nanoparticle gate material as nitrogen oxide gas sensors2006In: Proceedings from E-MRS 2006, Nice France, May 29- June 1, 2006, 2006, 87-92 p.Conference paper (Refereed)
    Abstract [en]

       

  • 47.
    Bäck, Marcus
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Appelqvist, Hanna
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    LeVine, Harry III
    University of Kentucky, KY 40536 USA.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Anionic Oligothiophenes Compete for Binding of X-34 but not PIB to Recombinant A beta Amyloid Fibrils and Alzheimers Disease Brain-Derived A beta2016In: CHEMISTRY-A EUROPEAN JOURNAL, ISSN 0947-6539, Vol. 22, no 51, 18335-18338 p.Article in journal (Refereed)
    Abstract [en]

    Deposits comprised of amyloid- (A) are one of the pathological hallmarks of Alzheimers disease (AD) and small hydrophobic ligands targeting these aggregated species are used clinically for the diagnosis of AD. Herein, we observed that anionic oligothiophenes efficiently displaced X-34, a Congo Red analogue, but not Pittsburgh compoundB (PIB) from recombinant A amyloid fibrils and Alzheimers disease brain-derived A. Overall, we foresee that the oligothiophene scaffold offers the possibility to develop novel high-affinity ligands for A pathology only found in human AD brain, targeting a different site than PIB.

  • 48.
    Campos Melo, Raul Ivan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Studies on molecular aspects of Transthyretin Amyloidosis2015Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins are versatile molecules that play a variety of roles in maintaining the human body, e.g. transport of nutrients. Transthyretin (TTR) is a 55 kDa homotetrameric protein found in human plasma and in the cerebrospinal fluid, responsible for the transport of retinol (vitamin A) and T4 (thyroxine). This protein is probably not essential for life, since TTR knockout mice have normal fetal development and lifespan. TTR, like 25 other human proteins, has been associated to the deposition of amyloid aggregates. Previous research has shown that mutations considerably increase the propensity of the protein to form aggregates. However, the wild type protein also exhibits this ability to aggregate, giving rise to the senile systemic amyloidosis disease that affects 20% people over 80 years of age. It is well accepted that self-association of monomeric subunits triggers the disease through tetramer dissociation, since stabilization of the quaternary structure suppresses aggregate formation.

    However, a detailed description of the self-assembly mechanism and fibril structure remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state.

    In our efforts to study both native and protofibrillar TTR, we realized the need for development of a fluorescent small molecule capable of binding native and protofibrillar TTR, providing distinguishable emission spectra. We used microwave heating for efficient synthesis and fluorescence spectral screening of compounds. We synthesized and tested 22 analogs displaying a variety of functional groups, most of them linked to a stilbene scaffold. We successfully developed two compounds that detect both TTR states at physiological concentrations. The compounds bound with nM-μM affinities and displayed very distinct emission maxima upon binding native or protofibrillar TTR (> 100 nm difference).

    We expect these new findings regarding protofibril self-assembly mechanism, together with our novel molecules serve as important tools in future studies of TTR amyloid formation.

    List of papers
    1. Considerably Unfolded Transthyretin Monomers Preceed and Exchange with Dynamically Structured Amyloid Protofibrils
    Open this publication in new window or tab >>Considerably Unfolded Transthyretin Monomers Preceed and Exchange with Dynamically Structured Amyloid Protofibrils
    Show others...
    2015 (English)In: Scientific Reports, ISSN 2045-2322, Vol. 5, no 11443Article in journal (Refereed) Published
    Abstract [en]

    Despite numerous studies, a detailed description of the transthyretin (TTR) self-assembly mechanism and fibril structure in TTR amyloidoses remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry (HXMS) analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. The protofibrils only grow to an approximate final size of 2,900 kDa and a length of 70 nm and a comparative HXMS analysis of native and aggregated samples revealed a much higher average solvent exposure of TTR upon fibrillation. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state. Together, these data reveal that the fibrillar state interchanges with the solution state. Accordingly, we suggest that TTR fibrillation proceeds via addition of considerably unfolded monomers, and the continuous presence of amyloidogenic structures near the protofibril surface offers a plausible explanation for secondary nucleation. We argue that the presence of such dynamic structural equilibria must impact future therapeutic development strategies.

    Place, publisher, year, edition, pages
    Nature Publishing Group: Open Access Journals - Option C / Nature Publishing Group, 2015
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-120227 (URN)10.1038/srep11443 (DOI)000356857900001 ()26108284 (PubMedID)
    Note

    Funding Agencies|This work was funded by the Danish Council for Independent Research, Medical Sciences; Danish Council for Independent Research, Medical Sciences, Sapere Aude programme; Drug Research Academy; DANSCATT; Swedish Research Council; Linux cluster at University of Copenhagen by the Carlsberg Research Foundation; FTIR spectrophotometer by the Apotekerfonden

    Available from: 2015-07-21 Created: 2015-07-20 Last updated: 2015-11-26
    2. Novel Trans-Stilbene-based Fluorophores as Probes for Spectral Discrimination of Native and Protofibrillar Transthyretin
    Open this publication in new window or tab >>Novel Trans-Stilbene-based Fluorophores as Probes for Spectral Discrimination of Native and Protofibrillar Transthyretin
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    2016 (English)In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 7, no 7, 924-940 p.Article in journal (Refereed) Published
    Abstract [en]

    Accumulation of misfolded transthyretin (TTR) as amyloid fibrils causes various human disorders. Native transthyretin is a neurotrophic protein and is a putative extracellular molecular chaperone. Several fluorophores have been shown in vitro to bind selectively to native TTR. Other compounds, such as thioflavin T, bind TTR amyloid fibrils. The probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to both native and fibrillar TTR, becoming highly fluorescent, but with indistinguishable emission spectra for native and fibrillar TTR. Herein we report our efforts to develop a fluorescent small molecule capable of binding both native and misfolded protofibrillar TTR, providing distinguishable emission spectra. We used microwave synthesis for efficient production of a small library of trans-stilbenes and fluorescence spectral screening of their binding properties. We synthesized and tested 22 trans-stilbenes displaying a variety of functional groups. We successfully developed two naphthyl-based trans-stilbenes probes that detect both TTR states at physiological concentrations. The compounds bound with nanomolar to micromolar affinities and displayed distinct emission maxima upon binding native or misfolded protofibrillar TTR (>100 nm difference). The probes were mainly responsive to environment polarity providing evidence for the divergent hydrophobic structure of the binding sites of these protein conformational states. Furthermore, we were able to successfully use one of these probes to quantify the relative amounts of native and protofibrillar TTR in a dynamic equilibrium. In conclusion, we identified two trans-stilbene-based fluorescent probes, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (11) and (E)-4-(2-(naphthalen-2-yl)vinyl)benzene-1,2-diol (14), that bind native and protofibrillar TTR, providing a wide difference in emission maxima allowing conformational discrimination by fluorescence spectroscopy. We expect these novel molecules to serve as important chemical biology research tools in studies of TTR folding and misfolding.

    Place, publisher, year, edition, pages
    American Chemical Society (ACS), 2016
    Keyword
    transthyretin, amyloid, stilbene, fluorescence, probe, spectrum
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-122842 (URN)10.1021/acschemneuro.6b00062 (DOI)000380297500009 ()27144293 (PubMedID)
    Note

    At the time for thesis presentation publication was in status: Manuscript

    Funding agencies:The work was supported by Goran Gustafsson's Foundation (PH), The Swedish Research Council (PH), The Linkoping center for systemic neuroscience, LiU-Neuro, (XW), and Sven and Lilly Lawski's foundation (ME).

    Available from: 2015-11-26 Created: 2015-11-26 Last updated: 2016-09-19Bibliographically approved
  • 49.
    Campos Melo, Raúl Ivan
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wu, Xiongyu
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Elgland, Mathias
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Novel Trans-Stilbene-based Fluorophores as Probes for Spectral Discrimination of Native and Protofibrillar Transthyretin2016In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 7, no 7, 924-940 p.Article in journal (Refereed)
    Abstract [en]

    Accumulation of misfolded transthyretin (TTR) as amyloid fibrils causes various human disorders. Native transthyretin is a neurotrophic protein and is a putative extracellular molecular chaperone. Several fluorophores have been shown in vitro to bind selectively to native TTR. Other compounds, such as thioflavin T, bind TTR amyloid fibrils. The probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to both native and fibrillar TTR, becoming highly fluorescent, but with indistinguishable emission spectra for native and fibrillar TTR. Herein we report our efforts to develop a fluorescent small molecule capable of binding both native and misfolded protofibrillar TTR, providing distinguishable emission spectra. We used microwave synthesis for efficient production of a small library of trans-stilbenes and fluorescence spectral screening of their binding properties. We synthesized and tested 22 trans-stilbenes displaying a variety of functional groups. We successfully developed two naphthyl-based trans-stilbenes probes that detect both TTR states at physiological concentrations. The compounds bound with nanomolar to micromolar affinities and displayed distinct emission maxima upon binding native or misfolded protofibrillar TTR (>100 nm difference). The probes were mainly responsive to environment polarity providing evidence for the divergent hydrophobic structure of the binding sites of these protein conformational states. Furthermore, we were able to successfully use one of these probes to quantify the relative amounts of native and protofibrillar TTR in a dynamic equilibrium. In conclusion, we identified two trans-stilbene-based fluorescent probes, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (11) and (E)-4-(2-(naphthalen-2-yl)vinyl)benzene-1,2-diol (14), that bind native and protofibrillar TTR, providing a wide difference in emission maxima allowing conformational discrimination by fluorescence spectroscopy. We expect these novel molecules to serve as important chemical biology research tools in studies of TTR folding and misfolding.

  • 50.
    Carlsson, Anders
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Identification of potential plasma biomarkers of inflammation in farmers with musculoskeletal disorders: A proteomic study2012Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    In this thesis we look for potential chronic inflammation biomarkers because studies have shown that farmers with musculoskeletal disorders might be affected by the environment to develop musculoskeletal disorders. Animal farmers are highly exposed to dust, aerosols, molds and other toxins in the air and environment leading to musculoskeletal disorders, respiratory disorders, airway symptoms and febrile reactions. There is reason to believe that the farmers have a constant or chronic inflammation that develops into musculoskeletal disorders. By using a proteomic approach with Two-dimensional Gel Electrophoresis and silver staining our goal was to find biomarkers by quantifying protein spots that differ significantly from farmers with musculoskeletal disorders compared to rural controls. In our study we found 8 significant proteins, two from Alpha-2-HS-glycoprotein, one from Apolipoprotein A1, three from Haptoglobin, one from Hemopexin and 1 from Antithrombin. All 5 proteins are involved in inflammation response in some way and some proteins are linked to chronic inflammation. Out of the 5 proteins Alpha-2-HS-glycoprotein, Apolipoprotein A1 and Hemopexin seem like the most likely proteins to investigate further as potential inflammation biomarkers.

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