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  • 301.
    Pelve, Erik A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Unique Solutions to Universal Problems: Studies of the Archaeal Cell2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Archaea is one of the three domains of life and studies of archaeal biology are important for understanding of life in extreme environments, fundamental biogeochemical processes, the origin of life, the eukaryotic cell and their own, unique biology. This thesis presents four studies of the archaeal cell, using the extremophilic Sulfolobus and ocean living Nitrosopumilus as model systems.

    Cell division in crenarchaea is shown to be carried out by a previously unknown system named Cdv (cell division). The system shares homology with the eukaryotic ESCRT-III system which is used for membrane reorganization during vesicle formation, viral release and cytokinesis. Organisms of the phylum Thaumarchaeota also use the Cdv system, despite also carrying genes for the euryarchaeal and bacterial cell division system FtsZ.

    The thaumarchaeal cell cycle is demonstrated to be dominated by the prereplicative and replicative stage, in contrasts to the crenarchaeal cell cycle where the cell at the majority of the time resides in the postreplicative stage. The replication rate is remarkably low and closer to what is measured for eukaryotes than other archaea.

    The gene organization of Sulfolobus is significantly associated with the three origins of replication. The surrounding regions are dense with genes of high importance for the organisms such as highly transcribed genes, genes with known function in fundamental cellular processes and conserved archaeal genes. The overall gene density is elevated and transposons are underrepresented.

    The archaeal virus SIRV2 displays a lytic life style where the host cell at the final stage of infection is disrupted for release of new virus particles. The remarkable pyramid-like structure VAP (virus associated pyramids), that is formed independently of the virus particle, is used for cell lysis.

    The research presented in this thesis describes unique features of the archaeal cell and influences our understanding of the entire tree of life.

    List of papers
    1. A unique cell division machinery in the Archaea
    Open this publication in new window or tab >>A unique cell division machinery in the Archaea
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    2008 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 48, p. 18942-18946Article in journal (Refereed) Published
    Abstract [en]

    In contrast to the cell division machineries of bacteria, euryarchaea, and eukaryotes, no division components have been identified in the second main archaeal phylum, Crenarchaeota. Here, we demonstrate that a three-gene operon, cdv, in the crenarchaeon Sulfolobus acidocaldarius, forms part of a unique cell division machinery. The operon is induced at the onset of genome segregation and division, and the Cdv proteins then polymerize between segregating nucleoids and persist throughout cell division, forming a successively smaller structure during constriction. The cdv operon is dramatically down-regulated after UV irradiation, indicating division inhibition in response to DNA damage, reminiscent of eukaryotic checkpoint systems. The cdv genes exhibit a complementary phylogenetic range relative to FtsZ-based archaeal division systems such that, in most archaeal lineages, either one or the other system is present. Two of the Cdv proteins, CdvB and CdvC, display homology to components of the eukaryotic ESCRT-III sorting complex involved in budding of luminal vesicles and HIV-1 virion release, suggesting mechanistic similarities and a common evolutionary origin.

    Keywords
    cdv, Crenarchaeota, cytokinesis, ftsZ, Sulfolobus
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-106988 (URN)10.1073/pnas.0809467105 (DOI)000261489100060 ()
    Available from: 2009-07-15 Created: 2009-07-15 Last updated: 2022-01-28Bibliographically approved
    2. A unique virus release mechanism in the Archaea
    Open this publication in new window or tab >>A unique virus release mechanism in the Archaea
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    2009 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 27, p. 11306-11311Article in journal (Refereed) Published
    Abstract [en]

    Little is known about the infection cycles of viruses infecting cells from Archaea, the third domain of life. Here, we demonstrate that the virions of the archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) are released from the host cell through a mechanism, involving the formation of specific cellular structures. Large pyramidal virus-induced protrusions transect the cell envelope at several positions, rupturing the S-layer; they eventually open out, thus creating large apertures through which virions escape the cell. We also demonstrate that massive degradation of the host chromosomes occurs because of virus infection, and that virion assembly occurs in the cytoplasm. Furthermore, intracellular viral DNA is visualized by flow cytometry. The results show that SIRV2 is a lytic virus, and that the host cell dies as a consequence of elaborated mechanisms orchestrated by the virus. The generation of specific cellular structures for a distinct step of virus life cycle is known in eukaryal virus-host systems but is unprecedented in cells from other domains.

    Keywords
    lysis, virus factory, hyperthermophile, infection cycle
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-128369 (URN)10.1073/pnas.0901238106 (DOI)000267796100079 ()
    Available from: 2010-07-22 Created: 2010-07-20 Last updated: 2017-12-12Bibliographically approved
    3. Cdv-based cell division and cell cycle organization in the thaumarchaeon Nitrosopumilus maritimus
    Open this publication in new window or tab >>Cdv-based cell division and cell cycle organization in the thaumarchaeon Nitrosopumilus maritimus
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    2011 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 82, no 3, p. 555-566Article in journal (Refereed) Published
    Abstract [en]

    Cell division is mediated by different mechanisms in different evolutionary lineages. While bacteria and euryarchaea utilize an FtsZ-based mechanism, most crenarchaea divide using the Cdv system, related to the eukaryotic ESCRT-III machinery. Intriguingly, thaumarchaeal genomes encode both FtsZ and Cdv protein homologues, raising the question of their division mode. Here, we provide evidence indicating that Cdv is the primary division system in the thaumarchaeon Nitrosopumilus maritimus. We also show that the cell cycle is differently organized as compared to hyperthermophilic crenarchaea, with a longer pre-replication phase and a shorter post-replication stage. In particular, the time required for chromosome replication is remarkably extensive, 15-18 h, indicating a low replication rate. Further, replication did not continue to termination in a significant fraction of N. maritimus cell populations following substrate depletion. Both the low replication speed and the propensity for replication arrest are likely to represent adaptations to extremely oligotrophic environments. The results demonstrate that thaumarchaea, crenarchaea and euryarchaea display differences not only regarding phylogenetic affiliations and gene content, but also in fundamental cellular and physiological characteristics. The findings also have implications for evolutionary issues concerning the last archaeal common ancestor and the relationship between archaea and eukaryotes.

    National Category
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-162884 (URN)10.1111/j.1365-2958.2011.07834.x (DOI)000297282200004 ()21923770 (PubMedID)
    Available from: 2011-12-05 Created: 2011-12-05 Last updated: 2017-12-08Bibliographically approved
    4. Replication-biased genome organisation in the crenarchaeon Sulfolobus
    Open this publication in new window or tab >>Replication-biased genome organisation in the crenarchaeon Sulfolobus
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    2010 (English)In: BMC Genomics, E-ISSN 1471-2164, Vol. 11, p. 454-Article in journal (Refereed) Published
    Abstract [en]

    Background: Species of the crenarchaeon Sulfolobus harbour three replication origins in their single circular chromosome that are synchronously initiated during replication. Results: We demonstrate that global gene expression in two Sulfolobus species is highly biased, such that early replicating genome regions are more highly expressed at all three origins. The bias by far exceeds what would be anticipated by gene dosage effects alone. In addition, early replicating regions are denser in archaeal core genes (enriched in essential functions), display lower intergenic distances, and are devoid of mobile genetic elements. Conclusion: The strong replication-biased structuring of the Sulfolobus chromosome implies that the multiple replication origins serve purposes other than simply shortening the time required for replication. The higher-level chromosomal organisation could be of importance for minimizing the impact of DNA damage, and may also be linked to transcriptional regulation.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-134173 (URN)10.1186/1471-2164-11-454 (DOI)000282787800003 ()
    Available from: 2010-11-22 Created: 2010-11-22 Last updated: 2024-01-17Bibliographically approved
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  • 302.
    Perisynaki, Emmanouela
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Role of ribosomal biogenesis in angiogenesis2022Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
  • 303.
    Persson, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    The Role of ATF4 in Adult Neural Stem Cells During Inflammation2022Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Download full text (pdf)
    Master Thesis Report
  • 304.
    Petroutsos, Dimitris
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Physiology and Environmental Toxicology.
    Amiar, Souad
    Abida, Heni
    Dolch, Lina-Juana
    Bastien, Olivier
    Rébeillé, Fabrice
    Jouhet, Juliette
    Falconet, Denis
    Block, Maryse A
    McFadden, Geoffrey I
    Bowler, Chris
    Botté, Cyrille
    Maréchal, Eric
    Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids.2014In: Progress in lipid research, ISSN 0163-7827, E-ISSN 1873-2194, Vol. 54, p. 68-85, article id S0163-7827(14)00014-9Article in journal (Refereed)
    Abstract [en]

    Photosynthetic membranes have a unique lipid composition that has been remarkably well conserved from cyanobacteria to chloroplasts. These membranes are characterized by a very high content in galactoglycerolipids, i.e., mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively). Galactoglycerolipids make up the bulk of the lipid matrix in which photosynthetic complexes are embedded. They are also known to fulfill specific functions, such as stabilizing photosystems, being a source of polyunsaturated fatty acids for various purposes and, in some eukaryotes, being exported to other subcellular compartments. The conservation of MGDG and DGDG suggests that selection pressures might have conserved the enzymes involved in their biosynthesis, but this does not appear to be the case. Important evolutionary transitions comprise primary endosymbiosis (from a symbiotic cyanobacterium to a primary chloroplast) and secondary endosymbiosis (from a symbiotic unicellular algal eukaryote to a secondary plastid). In this review, we compare biosynthetic pathways based on available molecular and biochemical data, highlighting enzymatic reactions that have been conserved and others that have diverged or been lost, as well as the emergence of parallel and alternative biosynthetic systems originating from other metabolic pathways. Questions for future research are highlighted.

  • 305.
    Peuckert, Christiane
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Aresh, Bejan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Holenya, Pavol
    Adams, Derek
    Sreedharan, Smitha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Porthin, Annika
    Andersson, Louise
    Pettersson, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Wölfl, Stefan
    Klein, Rüdiger
    Oxburgh, Leif
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Multimodal Eph/Ephrin signaling controls several phases of urogenital development2016In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 90, no 2, p. 373-388Article in journal (Refereed)
    Abstract [en]

    A substantial portion of the human population is affected by urogenital birth defects resulting from a failure in ureter development. Although recent research suggests roles for several genes in facilitating the ureter/bladder connection, the underlying molecular mechanisms remain poorly understood. Signaling via Eph receptor tyrosine kinases is involved in several developmental processes. Here we report that impaired Eph/Ephrin signaling in genetically modified mice results in severe hydronephrosis caused by defective ureteric bud induction, ureter maturation, and translocation. Our data imply that ureter translocation requires apoptosis in the urogenital sinus and inhibition of proliferation in the common nephric duct. These processes were disturbed in EphA4/EphB2 compound knockout mice and were accompanied by decreased ERK-2 phosphorylation. Using a set of Eph, Ephrin, and signaling-deficient mutants, we found that during urogenital development, different modes of Eph/Ephrin signaling occur at several sites with EphrinB2 and EphrinA5 acting in concert. Thus, Eph/Ephrin signaling should be considered in the etiology of congenital kidney and urinary tract anomalies.

  • 306.
    Pietilä, Ilkka
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    van Mourik, Djenolan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tamelander, Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kriz, Vitezslav
    Institute of Molecular Genetics of the CAS, 14220 Prague, Czech Republic.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Temporal Dynamics of VEGFA-Induced VEGFR2/FAK Co-Localization Depend on SHB2019In: Cells, E-ISSN 2073-4409, Vol. 8, no 12, article id 1645Article in journal (Refereed)
    Abstract [en]

    Focal adhesion kinase (FAK) is essential for vascular endothelial growth factor-A (VEGFA)/VEGF receptor-2 (VEGFR2)-stimulated angiogenesis and vascular permeability. We have previously noted that presence of the Src homology-2 domain adapter protein B (SHB) is of relevance for VEGFA-stimulated angiogenesis in a FAK-dependent manner. The current study was conducted in order address the temporal dynamics of co-localization between these components in HEK293 and primary lung endothelial cells (EC) by total internal reflection fluorescence microscopy (TIRF). An early (<2.5 min) VEGFA-induced increase in VEGFR2 co-localization with SHB was dependent on tyrosine 1175 in VEGFR2. VEGFA also enhanced SHB co-localization with FAK. FAK co-localization with VEGFR2 was dependent on SHB since it was significantly lower in SHB deficient EC after VEGFA addition. Absence of SHB also resulted in a gradual decline of VEGFR2 co-localization with FAK under basal (prior to VEGFA addition) conditions. A similar basal response was observed with expression of the Y1175F-VEGFR2 mutant in wild type EC. The distribution of focal adhesions in SHB-deficient EC was altered with a primarily perinuclear location. These live cell data implicate SHB as a key component regulating FAK activity in response to VEGFA/VEGFR2.

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  • 307.
    Pinheiro, Tiago
    et al.
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.
    Otrocka, Magdalena
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Chem Biol Consortium Sweden, Stockholm, Sweden.
    Seashore-Ludlow, Brinton
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Chem Biol Consortium Sweden, Stockholm, Sweden.
    Rraklli, Vilma
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden; Karolinska Inst, Ludwig Inst Canc Res, Stockholm, Sweden.
    Holmberg, Johan
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden; Karolinska Inst, Ludwig Inst Canc Res, Stockholm, Sweden.
    Forsberg Nilsson, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Simon, Andras
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.
    Kirkham, Matthew
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.
    Reprint of: A chemical screen identifies trifluoperazine as an inhibitor of glioblastoma growth2018In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 499, no 2, p. 136-142Article in journal (Refereed)
    Abstract [en]

    Glioblastoma (GBM) is regarded as the most common malignant brain tumor but treatment options are limited. Thus, there is an unmet clinical need for compounds and corresponding targets that could inhibit GBM growth. We screened a library of 80 dopaminergic ligands with the aim of identifying compounds capable of inhibiting GBM cell line proliferation and survival. Out of 45 active compounds, 8 were further validated. We found that the dopamine receptor D2 antagonist trifluoperazine 2HCl inhibits growth and proliferation of GBM cells in a dose dependent manner. Trifluoperazine’s inhibition of GBM cells is cell line dependent and correlates with variations in dopamine receptor expression profile. We conclude that components of the dopamine receptor signaling pathways are potential targets for pharmacological interventions of GBM growth.

  • 308.
    Plashkevych, Oleksandr
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Molecular Structure of the Core-Modified siRNA Duplexes Containing Diastereomeric Pair of [C6′(R)-OH]- versus [C6′(S)-OH]-carba-LNAs Suggests a Model for RNAi Action2011In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 30, no 11, p. 815-825Article in journal (Refereed)
    Abstract [en]

    Molecular structures of native and a pair of modified small interfering RNA-RNA duplexes containing carbocyclic [6'-(R)-OH/7'-(S)-methyl]- and [6'-(S)-OH/7'-(S)-methyl]-carba-LNA-thymine nucleotides, which are two diastereomeric analogs of the native T nucleotide, incorporated at position 13 in the antisense (AS) strand of siRNA, have been simulated using molecular mechanics/dynamics techniques. The main aim of the project has been to find a plausible structural explanation of why modification of siRNA at T(13) position by the [6'(R)-O-(p-Toluoyl)-7' (S)-methyl]-carba-LNA-Thymine [IC(50) of 3.32 +/- 0.17 nM] is ca 24 times more active as an RNA silencing agent against the target HIV-1 TAR RNA than the [6' (S)-O-(p-Toluoyl)-7' (S)-methyl]-counterpart [IC(50) of 79.8 +/- 17 nM] [1]. The simulations reveal that introduction of both C6' (R)-OH and C6' (S)-OH stereoisomers does not lead even to local perturbation of the siRNA-RNA duplex structures compared to the native, and the only significant difference between 6' (S)- and 6' (R)-diastereomers found is the exposure of the 6'-OH group of the 6' (R)-diastereoisomer toward the edge of the duplex while the 6'-hydroxyl group of the 6' (S)-diastereoisomer is somewhat buried in the minor groove of the duplex. This rules out a hypothesis about any possible local distortion by the nature of chemical modification of the siRNA-target the RNA duplex, which might have influenced the formation of the effective RNA silencing complex (RISC) and puts some weight on the hypothesis about the 6'-hydroxy group being directly involved with most probably Ago protein, since it is known from exhaustive X-ray studies [2, 3] that the core residues are indeed involved with hydrogen bonding with the internucleotidyl phosphates.

  • 309.
    Popp, David
    et al.
    ASTAR, Inst Mol & Cell Biol, Biopolis, Singapore 138673, Singapore..
    Loh, N. Duane
    Natl Univ Singapore, Dept Phys, Singapore 117557, Singapore.;Natl Univ Singapore, Ctr BioImaging Sci, Singapore 117546, Singapore..
    Zorgati, Habiba
    ASTAR, Inst Mol & Cell Biol, Biopolis, Singapore 138673, Singapore.;Natl Univ Singapore, Dept Biochem, Singapore 117597, Singapore..
    Ghoshdastider, Umesh
    ASTAR, Inst Mol & Cell Biol, Biopolis, Singapore 138673, Singapore..
    Liow, Lu Ting
    Natl Univ Singapore, Dept Med, Singapore 119074, Singapore..
    Ivanova, Magdalena I.
    Univ Michigan, Dept Neurol, 109 Zina Pitcher Pl, Ann Arbor, MI 48109 USA..
    Larsson, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ASTAR, Inst Mol & Cell Biol, Biopolis, Singapore 138673, Singapore.
    DePonte, Daniel P.
    SLAC Natl Accelerator Lab, Linac Coherent Light Source, 2575 Sand Hill Rd, Menlo Pk, CA 94025 USA..
    Bean, Richard
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany.;European XFEL GmbH, D-22761 Hamburg, Germany..
    Beyerlein, Kenneth R.
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Gati, Cornelius
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Oberthuer, Dominik
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany.;Univ Hamburg, Inst Biochem & Mol Biol, D-22607 Hamburg, Germany..
    Arnlund, David
    Univ Gothenburg, Dept Chem & Mol Biol, S-40530 Gothenburg, Sweden..
    Branden, Gisela
    Univ Gothenburg, Dept Chem & Mol Biol, S-40530 Gothenburg, Sweden..
    Berntsen, Peter
    Univ Gothenburg, Dept Chem & Mol Biol, S-40530 Gothenburg, Sweden..
    Cascio, Duilio
    Univ Calif Los Angeles, Howard Hughes Med Inst, Los Angeles, CA 90095 USA..
    Chavas, Leonard M. G.
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Chen, Joe P. J.
    Univ Canterbury, Computat Imaging Grp, Dept Elect & Comp Engn, Christchurch, New Zealand.;Arizona State Univ, Dept Phys, Tempe, AZ 85287 USA..
    Ding, Ke
    ASTAR, Inst Mol & Cell Biol, Biopolis, Singapore 138673, Singapore..
    Fleckenstein, Holger
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Gumprecht, Lars
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Harimoorthy, Rajiv
    Univ Gothenburg, Dept Chem & Mol Biol, S-40530 Gothenburg, Sweden..
    Mossou, Estelle
    Inst Laue Langevin, F-38000 Grenoble, France.;Keele Univ, EPSAM ISTM, Keele ST5 5BG, Staffs, England..
    Sawaya, Michael R.
    Univ Calif Los Angeles, Howard Hughes Med Inst, Los Angeles, CA 90095 USA..
    Brewster, Aaron S.
    Lawrence Berkeley Natl Lab, Phys Biosci Div, Berkeley, CA 94720 USA..
    Hattne, Johan
    Lawrence Berkeley Natl Lab, Phys Biosci Div, Berkeley, CA 94720 USA.;Howard Hughes Med Inst, Janelia Res Campus,19700 Helix Dr, Ashburn, VA 20147 USA..
    Sauter, Nicholas K.
    Lawrence Berkeley Natl Lab, Phys Biosci Div, Berkeley, CA 94720 USA..
    Seibert, Marvin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Seuring, Carolin
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Stellato, Francesco
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Tilp, Thomas
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Eisenberg, David S.
    Univ Calif Los Angeles, Howard Hughes Med Inst, Los Angeles, CA 90095 USA..
    Messerschmidt, Marc
    SLAC Natl Accelerator Lab, Linac Coherent Light Source, 2575 Sand Hill Rd, Menlo Pk, CA 94025 USA..
    Williams, Garth J.
    SLAC Natl Accelerator Lab, Linac Coherent Light Source, 2575 Sand Hill Rd, Menlo Pk, CA 94025 USA..
    Koglin, Jason E.
    SLAC Natl Accelerator Lab, Linac Coherent Light Source, 2575 Sand Hill Rd, Menlo Pk, CA 94025 USA..
    Makowski, Lee
    Northeastern Univ, Dept Bioengn, 360 Huntington Ave, Boston, MA 02115 USA..
    Millane, Rick P.
    Univ Canterbury, Computat Imaging Grp, Dept Elect & Comp Engn, Christchurch, New Zealand..
    Forsyth, Trevor
    Inst Laue Langevin, F-38000 Grenoble, France.;Keele Univ, EPSAM ISTM, Keele ST5 5BG, Staffs, England..
    Boutet, Sebastien
    SLAC Natl Accelerator Lab, Linac Coherent Light Source, 2575 Sand Hill Rd, Menlo Pk, CA 94025 USA..
    White, Thomas A.
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Barty, Anton
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Chapman, Henry
    DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany.;Univ Hamburg, Dept Phys, Luruper Chaussee 149, D-22607 Hamburg, Germany..
    Chen, Swaine L.
    Natl Univ Singapore, Dept Med, Singapore 119074, Singapore.;ASTAR, Biopolis, Genome Inst Singapore, Singapore 138672, Singapore..
    Liang, Mengning
    SLAC Natl Accelerator Lab, Linac Coherent Light Source, 2575 Sand Hill Rd, Menlo Pk, CA 94025 USA.;DESY, Ctr Free Electron Laser Sci, Notkestr 85, D-22607 Hamburg, Germany..
    Neutze, Richard
    Univ Gothenburg, Dept Chem & Mol Biol, S-40530 Gothenburg, Sweden..
    Robinson, Robert C.
    ASTAR, Inst Mol & Cell Biol, Biopolis, Singapore 138673, Singapore.;Natl Univ Singapore, Dept Biochem, Singapore 117597, Singapore.;Okayama Univ, Res Inst Interdisciplinary Sci, Okayama 7008530, Japan..
    Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser2017In: CYTOSKELETON, ISSN 1949-3584, Vol. 74, no 12, p. 472-481Article in journal (Refereed)
    Abstract [en]

    A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked -strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual -synuclein amyloids.

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  • 310. Popp, David
    et al.
    Narita, Akihiro
    Lee, Lin Jie
    Larsson, Mårten
    Robinson, Robert C
    Microtubule-like properties of the bacterial actin homolog ParM-R1.2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 44, p. 37078-88Article in journal (Refereed)
    Abstract [en]

    In preparation for mammalian cell division, microtubules repeatedly probe the cytoplasm to capture chromosomes and assemble the mitotic spindle. Critical features of this microtubule system are the formation of radial arrays centered at the centrosomes and dynamic instability, leading to persistent cycles of polymerization and depolymerization. Here, we show that actin homolog, ParM-R1 that drives segregation of the R1 multidrug resistance plasmid from Escherichia coli, can also self-organize in vitro into asters, which resemble astral microtubules. ParM-R1 asters grow from centrosome-like structures consisting of interconnected nodes related by a pseudo 8-fold symmetry. In addition, we show that ParM-R1 is able to perform persistent microtubule-like oscillations of assembly and disassembly. In vitro, a whole population of ParM-R1 filaments is synchronized between phases of growth and shrinkage, leading to prolonged synchronous oscillations even at physiological ParM-R1 concentrations. These results imply that the selection pressure to reliably segregate DNA during cell division has led to common mechanisms within diverse segregation machineries.

  • 311.
    Porras Hernández, Ana Maria
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering.
    Barbe, Laurent
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering.
    Pohlit, Hannah
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering.
    Tenje, Maria
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering.
    Antfolk, Maria
    Lund Univ, Dept Biomed Engn, Lund, Sweden.;Univ Copenhagen, Biotech Res & Innovat Ctr, Copenhagen, Denmark..
    Confocal imaging dataset to assess endothelial cell orientation during extreme glucose conditions2022In: Scientific Data, E-ISSN 2052-4463, Vol. 9, no 1, article id 26Article in journal (Refereed)
    Abstract [en]

    Confocal microscopy offers a mean to extract quantitative data on spatially confined subcellular structures. Here, we provide an imaging dataset of confocal z-stacks on endothelial cells spatially confined on lines with different widths, visualizing the nucleus, F-actin, and zonula occludens-1 (ZO-1), as well as the lines. This dataset also includes confocal images of spatially confined endothelial cells challenged with different glucose conditions. We have validated the image quality by established analytical means using the MeasureImageQuality module of the CellProfilerTM software. We envision that this dataset could be used to extract data on both a population and a single cell level, as well as a learning set for the development of new image analysis tools.

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  • 312.
    Potente, Michael
    et al.
    Max Planck Inst Heart & Lung Res, Angiogenesis & Metab Lab, Ludwigstr 43, D-61231 Bad Nauheim, Germany; Int Inst Mol & Cell Biol, PL-02109 Warsaw, Poland; DZHK German Ctr Cardiovasc Res, Partner Site Frankfurt, D-13347 Berlin, Germany.
    Mäkinen, Taija
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Vascular heterogeneity and specialization in development and disease2017In: Nature reviews. Molecular cell biology, ISSN 1471-0072, E-ISSN 1471-0080, Vol. 18, no 8, p. 477-494Article, review/survey (Refereed)
    Abstract [en]

    Blood and lymphatic vessels pervade almost all body tissues and have numerous essential roles in physiology and disease. The inner lining of these networks is formed by a single layer of endothelial cells, which is specialized according to the needs of the tissue that it supplies. Whereas the general mechanisms of blood and lymphatic vessel development are being defined with increasing molecular precision, studies of the processes of endothelial specialization remain mostly descriptive. Recent insights from genetic animal models illuminate how endothelial cells interact with each other and with their tissue environment, providing paradigms for vessel type- and organ-specific endothelial differentiation. Delineating these governing principles will be crucial for understanding how tissues develop and maintain, and how their function becomes abnormal in disease.

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  • 313.
    Pähn, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The IL-5 Receptor – Expression by Mouse Mast Cells and Mast Cell Progenitors2021Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    Mast cells contribute to the symptoms of atopic asthma. They sense the microenvironment through their receptors and consequently release immunomodulators, such as cytokines. The interleukin 5 cytokine (IL-5) is elevated in asthma and produced by e.g., type 2 T-helper cells and mast cells. IL-5 binds the IL-5 receptor (IL-5R) on e.g., eosinophils. Since eosinophils are involved in asthma, monoclonal antibodies have been developed to block the IL-5/IL-5R interaction, reducing eosinophils and relieving symptoms. However, some studies have demonstrated the IL-5R expression by human in vitro-derived mast cells.

    Here, we hypothesized that mouse mast cells and their progenitors express the IL-5R and thus may be targeted by anti-IL-5 treatment in mice with airway inflammation. Using in vitro and in vivo studies, we demonstrated the IL-5R expression by mast cells and mast cell progenitors. In a mouse model of allergic airway inflammation, the IL-5R expression was significantly increased by mast cells and progenitors, suggesting an upregulation during inflammation. Also, anti-IL-5 treatment seemed to reduce the mast cell and progenitor accumulation when this treatment successfully reduced eosinophils. Our data suggest that mast cells and their progenitors may be reduced in the inflamed lung when IL-5 is neutralized. If so, whether this is a direct or indirect effect needs further investigation. Furthermore, the dosing of anti-IL-5 needs to be increased and experiments repeated to verify our preliminary results. These findings contribute to the understanding of mast cells, the IL-5/IL-5R axis and asthma, which potentiates the development of effective and safe treatments in asthmatic patients.

    The full text will be freely available from 2025-06-30 17:46
  • 314.
    Põlajeva, Jelena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Glioma as an Ecosystem: Studies of Invasion, Onco-miR Addiction and Mast Cell Infiltration2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Despite recent advances in oncology and extensive research efforts, gliomas remain essentially incurable. Glioblastoma multiforme (GBM, WHO grade IV) is the most common glioma and may arise de novo or progress from a lower-grade lesion. GBM is characterized by invasive growth, aberrant angiogenesis and necrosis. The heterogeneity of GBM is further complicated by the contribution of the inflammation that is facilitated by immune cells that reside in and infiltrate this immuno-privileged organ.

    One of the cells types present in the tumor microenvironment are mast cells (MC) that accumulate in the tumor in a grade-dependent manner. GBM cells secrete a plethora of cytokines acting as chemoattractants in MC recruitment and to a lesser degree induce MC proliferation in situ. Expression of one of the cytokines secreted by GBM cells - macrophage migration inhibitory factor (MIF) - correlates with MC accumulation in vivo.

    GBM cells invade the surrounding parenchyma making complete resection impossible. Here, migration was studied with the focus on RAP1 and its negative regulator RAP1GAP. Activation of RAP1 signaling by lentiviral silencing of RAP1GAP lead to decrease in cell migration and a shift in expression of SOX2 and GFAP, presumably enhancing stem cell phenotype.

    MicroRNAs are small non-coding RNAs known to regulate the mRNA network. miR-21 is highly overexpressed in the majority of cancers including GBM. Its expression is strictly regulated during embryonic development of the brain. SOX2 is co-regulated with miR-21 demarcating a cell population with neural/glial progenitor/stem cell properties. In an experimental mouse model, expression of miR-21 can be sustained by forced expression of PDGF-BB leading to gliomagenesis. GBM cells seem to be addicted to oncogenic properties of miR-21 as its knockdown leads to extensive apoptosis. This observation combined with the fact that miR-21 is absent in the normal adult mammalian brain suggest miR-21 to be an excellent therapeutic target.

    Effects of conventional therapy (surgery combined with radiochemotherapy) on prolonging patient survival have reached a plateau. New effective personalized therapeutic modalities need to be designed and implemented. Targeting the tumor microenvironment as well as cell intrinsic properties like invasive potential, stemness and onco-miR addiction studied in this thesis will hopefully lead to efficient disruption of GBM’s aberrant ecosystem.

    List of papers
    1. Mast Cell Accumulation in Glioblastoma with a Potential Role for Stem Cell Factor and Chemokine CXCL12
    Open this publication in new window or tab >>Mast Cell Accumulation in Glioblastoma with a Potential Role for Stem Cell Factor and Chemokine CXCL12
    Show others...
    2011 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 6, no 9, article id e25222Article in journal (Refereed) Published
    Abstract [en]

    Glioblastoma multiforme (GBM) is the most common and malignant form of glioma with high mortality and no cure. Many human cancers maintain a complex inflammatory program triggering rapid recruitment of inflammatory cells, including mast cells (MCs), to the tumor site. However, the potential contribution of MCs in glioma has not been addressed previously. Here we report for the first time that MCs infiltrate KRas+Akt-induced gliomas, using the RCAS/TV-a system, where KRas and Akt are transduced by RCAS into the brains of neonatal Gtv-a- or Ntv-a transgenic mice lacking Ink4a or Arf. The most abundant MC infiltration was observed in high-grade gliomas of Arf-/- mice. MC accumulation could be localized to the vicinity of glioma-associated vessels but also within the tumor mass. Importantly, proliferating MCs were detected, suggesting that the MC accumulation was caused by local expansion of the MC population. In line with these findings, strong expression of stem cell factor (SCF), i.e. the main MC growth factor, was detected, in particular around tumor blood vessels. Further, glioma cells expressed the MC chemotaxin CXCL12 and MCs expressed the corresponding receptor, i.e. CXCR4, suggesting that MCs could be attracted to the tumor through the CXCL12/CXCR4 axis. Supporting a role for MCs in glioma, strong MC infiltration was detected in human glioma, where GBMs contained significantly higher MC numbers than grade II tumors did. Moreover, human GBMs were positive for CXCL12 and the infiltrating MCs were positive for CXCR4. In conclusion, we provide the first evidence for a role for MCs in glioma.

    National Category
    Clinical Medicine
    Identifiers
    urn:nbn:se:uu:diva-159545 (URN)10.1371/journal.pone.0025222 (DOI)000295257900060 ()21949886 (PubMedID)
    Available from: 2011-10-04 Created: 2011-10-04 Last updated: 2021-06-14Bibliographically approved
    2. Mast Cells Are Recruited to Glioma and Orchestrate Cancer Cell Invasion
    Open this publication in new window or tab >>Mast Cells Are Recruited to Glioma and Orchestrate Cancer Cell Invasion
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Immunology Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-180248 (URN)
    Available from: 2012-09-01 Created: 2012-09-01 Last updated: 2013-01-23
    3. RAP1GAP Suppression Promotes Stemness and Inhibits Glioblastoma Cell Migration
    Open this publication in new window or tab >>RAP1GAP Suppression Promotes Stemness and Inhibits Glioblastoma Cell Migration
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    RAP1GAP, RAP1, glioblastoma, invasion
    National Category
    Biochemistry and Molecular Biology Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-180247 (URN)
    Available from: 2012-09-01 Created: 2012-09-01 Last updated: 2015-07-01
    4. miRNA-21 is developmentally regulated in mouse brain and is co-expressed with SOX2 in glioma
    Open this publication in new window or tab >>miRNA-21 is developmentally regulated in mouse brain and is co-expressed with SOX2 in glioma
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    2012 (English)In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 12, p. 378-Article in journal (Refereed) Published
    Abstract [en]

    Background

    MicroRNAs (miRNAs) and their role during tumor development have been studied in greatdetail during the last decade, albeit their expression pattern and regulation during normaldevelopment are however not so well established. Previous studies have shown that miRNAsare differentially expressed in solid human tumors. Platelet-derived growth factor (PDGF)signaling is known to be involved in normal development of the brain as well as in malignantprimary brain tumors, gliomas, but the complete mechanism is still lacking. We decided toinvestigate the expression of the oncogenic miR-21 during normal mouse development andglioma, focusing on PDGF signaling as a potential regulator of miR-21.

    Methods

    We generated mouse glioma using the RCAS/tv-a system for driving PDGF-BB expression ina cell-specific manner. Expression of miR-21 in mouse cell cultures and mouse brain wereassessed using Northern blot analysis and in situ hybridization. Immunohistochemistry andWestern blot analysis were used to investigate SOX2 expression. LNA-modified siRNA wasused for irreversible depletion of miR-21. For inhibition of PDGF signaling Gleevec(imatinib mesylate), Rapamycin and U0126, as well as siRNA were used. Statisticalsignificance was calculated using double-sided unpaired Student´s t-test.

    Results

    We identified miR-21 to be highly expressed during embryonic and newborn braindevelopment followed by a gradual decrease until undetectable at postnatal day 7 (P7), thiscorrelated with SOX2 expression. Furthermore, miR-21 and SOX2 showed up-regulation andoverlapping expression pattern in RCAS/tv-a generated mouse brain tumor specimens. Uponirreversible depletion of miR-21 the expression of SOX2 was strongly diminished in bothmouse primary glioma cultures and human glioma cell lines. Interestingly, in normalfibroblasts the expression of miR-21 was induced by PDGF-BB, and inhibition of PDGFsignaling in mouse glioma primary cultures resulted in suppression of miR-21 suggesting thatmiR-21 is indeed regulated by PDGF signaling.

    Conclusions

    Our data show that miR-21 and SOX2 are tightly regulated already during embryogenesisand define a distinct population with putative tumor cell of origin characteristics. We believethat miR-21 is a mediator of PDGF-driven brain tumors, which suggests miR-21 as apromising target for treatment of glioma.

    Keywords
    miRNA, miR-21, Glioma, PDGF-BB, SOX2, Imatinib (Gleevec), RCAS/tv-a
    National Category
    Biochemistry and Molecular Biology Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-180243 (URN)10.1186/1471-2407-12-378 (DOI)000312098700001 ()
    Available from: 2012-09-01 Created: 2012-09-01 Last updated: 2017-12-07Bibliographically approved
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  • 315.
    Põlajeva, Jelena
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Kastemar, Marianne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Tchougounova, Elena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Zeller, Kathrin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Swartling, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Westermark, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    RAP1GAP Suppression Promotes Stemness and Inhibits Glioblastoma Cell MigrationManuscript (preprint) (Other academic)
  • 316.
    Põlajeva, Jelena
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Swartling, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Jiang, Yiwen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Singh, Umashankar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Pietras, Kristian
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet.
    Uhrbom, Lene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Westermark, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Roswall, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    miRNA-21 is developmentally regulated in mouse brain and is co-expressed with SOX2 in glioma2012In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 12, p. 378-Article in journal (Refereed)
    Abstract [en]

    Background

    MicroRNAs (miRNAs) and their role during tumor development have been studied in greatdetail during the last decade, albeit their expression pattern and regulation during normaldevelopment are however not so well established. Previous studies have shown that miRNAsare differentially expressed in solid human tumors. Platelet-derived growth factor (PDGF)signaling is known to be involved in normal development of the brain as well as in malignantprimary brain tumors, gliomas, but the complete mechanism is still lacking. We decided toinvestigate the expression of the oncogenic miR-21 during normal mouse development andglioma, focusing on PDGF signaling as a potential regulator of miR-21.

    Methods

    We generated mouse glioma using the RCAS/tv-a system for driving PDGF-BB expression ina cell-specific manner. Expression of miR-21 in mouse cell cultures and mouse brain wereassessed using Northern blot analysis and in situ hybridization. Immunohistochemistry andWestern blot analysis were used to investigate SOX2 expression. LNA-modified siRNA wasused for irreversible depletion of miR-21. For inhibition of PDGF signaling Gleevec(imatinib mesylate), Rapamycin and U0126, as well as siRNA were used. Statisticalsignificance was calculated using double-sided unpaired Student´s t-test.

    Results

    We identified miR-21 to be highly expressed during embryonic and newborn braindevelopment followed by a gradual decrease until undetectable at postnatal day 7 (P7), thiscorrelated with SOX2 expression. Furthermore, miR-21 and SOX2 showed up-regulation andoverlapping expression pattern in RCAS/tv-a generated mouse brain tumor specimens. Uponirreversible depletion of miR-21 the expression of SOX2 was strongly diminished in bothmouse primary glioma cultures and human glioma cell lines. Interestingly, in normalfibroblasts the expression of miR-21 was induced by PDGF-BB, and inhibition of PDGFsignaling in mouse glioma primary cultures resulted in suppression of miR-21 suggesting thatmiR-21 is indeed regulated by PDGF signaling.

    Conclusions

    Our data show that miR-21 and SOX2 are tightly regulated already during embryogenesisand define a distinct population with putative tumor cell of origin characteristics. We believethat miR-21 is a mediator of PDGF-driven brain tumors, which suggests miR-21 as apromising target for treatment of glioma.

    Download full text (pdf)
    fulltext
  • 317.
    Qi, Xiaoying
    et al.
    Binzhou Med Univ, Med & Pharm Res Ctr, Yantai, Peoples R China.
    Zhang, Yunyun
    Yantai Zestern Biotech Co LTD, Yantai, Peoples R China.
    Zhang, Yuan
    Binzhou Med Univ, Med & Pharm Res Ctr, Yantai, Peoples R China.
    Ni, Tianhui
    Binzhou Med Univ, Precis Med Res Ctr, Yantai, Shandong, Peoples R China.
    Zhang, Wenfeng
    Yantai Zestern Biotech Co LTD, Yantai, Peoples R China.
    Yang, Chunhua
    Binzhou Med Univ, Med & Pharm Res Ctr, Yantai, Peoples R China.
    Mi, Jia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Binzhou Med Univ, Med & Pharm Res Ctr, Yantai, Peoples R China.
    Zhang, Jiandi
    Yantai Zestern Biotech Co LTD, Yantai, Peoples R China;Binzhou Med Univ, Precis Med Res Ctr, Yantai, Shandong, Peoples R China.
    Tian, Geng
    Binzhou Med Univ, Med & Pharm Res Ctr, Yantai, Peoples R China.
    High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)2018In: Journal of Visualized Experiments, E-ISSN 1940-087X, no 138, article id e56885Article in journal (Refereed)
    Abstract [en]

    Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

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  • 318.
    Ragunathrao, Vijay Avin Balaji
    et al.
    Univ Illinois, Coll Med, Dept Pharmacol, Chicago, IL 60612 USA;Univ Illinois, Coll Med, Ctr Lung & Vasc Biol, Chicago, IL 60612 USA.
    Anwar, Mumtaz
    Univ Illinois, Coll Med, Dept Pharmacol, Chicago, IL 60612 USA;Univ Illinois, Coll Med, Ctr Lung & Vasc Biol, Chicago, IL 60612 USA.
    Akhter, Md Zahid
    Univ Illinois, Coll Med, Dept Pharmacol, Chicago, IL 60612 USA;Univ Illinois, Coll Med, Ctr Lung & Vasc Biol, Chicago, IL 60612 USA.
    Chavez, Alejandra
    Univ Illinois, Coll Med, Dept Pharmacol, Chicago, IL 60612 USA;Univ Illinois, Coll Med, Ctr Lung & Vasc Biol, Chicago, IL 60612 USA.
    Mao, De Yu
    Univ Illinois, Coll Med, Dept Physiol, Chicago, IL 60612 USA.
    Natarajan, Viswanathan
    Univ Illinois, Coll Med, Dept Pharmacol, Chicago, IL 60612 USA;Univ Illinois, Coll Med, Ctr Lung & Vasc Biol, Chicago, IL 60612 USA;Univ Illinois, Coll Med, Dept Med, Chicago, IL 60612 USA.
    Lakshmikanthan, Sribalaji
    Blood Ctr Wisconsin, Blood Res Inst, Milwaukee, WI 53226 USA.
    Chrzanowska-Wodnicka, Magdalena
    Blood Ctr Wisconsin, Blood Res Inst, Milwaukee, WI 53226 USA.
    Dudek, Arkadiusz Z.
    Univ Illinois, Coll Med, Dept Med, Chicago, IL 60612 USA.
    Claesson-Welsh, Lena
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Kitajewski, Jan K.
    Univ Illinois, Coll Med, Dept Physiol, Chicago, IL 60612 USA.
    Wary, Kishore K.
    Univ Illinois, Coll Med, Dept Pharmacol, Chicago, IL 60612 USA;Univ Illinois, Coll Med, Ctr Lung & Vasc Biol, Chicago, IL 60612 USA.
    Malik, Asrar B.
    Univ Illinois, Coll Med, Dept Pharmacol, Chicago, IL 60612 USA;Univ Illinois, Coll Med, Ctr Lung & Vasc Biol, Chicago, IL 60612 USA.
    Mehta, Dolly
    Univ Illinois, Coll Med, Dept Pharmacol, Chicago, IL 60612 USA;Univ Illinois, Coll Med, Ctr Lung & Vasc Biol, Chicago, IL 60612 USA.
    Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling2019In: Cell Reports, E-ISSN 2211-1247, Vol. 29, no 11, p. 3472-3487Article in journal (Refereed)
    Abstract [en]

    The vascular endothelial growth factor-A (VEGF-A)-VEGFR2 pathway drives tumor vascularization by activating proangiogenic signaling in endothelial cells (ECs). Here, we show that EC-sphingosine-1-phosphate receptor 1 (S1PR1) amplifies VEGFR2-mediated angiogenic signaling to enhance tumor growth. We show that cancer cells induce S1 PR1 activity in ECs, and thereby, conditional deletion of Si PR1 in ECs (EC-Slpr1(-/-) mice) impairs tumor vascularization and growth. Mechanistically, we show that S1 PR1 engages the heterotrimeric G-protein Gi, which amplifies VEGF-VEGFR2 signaling due to an increase in the activity of the tyrosine kinase c-Abl1. c-Abl1, by phosphorylating VEGFR2 at tyrosine-951, prolongs VEGFR2 retention on the plasmalemma to sustain Rac1 activity and EC migration. Thus, S1 PR1 or VEGFR2 antagonists, alone or in combination, reverse the tumor growth in control mice to the level seen in EC-Slpr1(-/-) mice. Our findings suggest that blocking S1 PR1 activity in ECs has the potential to suppress tumor growth by preventing amplification of VEGF-VEGFR2 signaling.

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    FULLTEXT01
  • 319.
    Raja, Erna
    Ludwig Institute for Cancer Research, Faculty of Medicine, Uppsala University.
    Cross-regulation between TGFβ/BMP Signalling and the metabolic LKB1 pathway2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cell signalling determines physiological responses to many cellular stimuli and environmental changes. The transforming growth factor-beta (TGFβ)/bone morphogenetic protein (BMP) signalling pathways begin by binding of ligand to the heterodimeric receptor complex, followed by activation of Smads that translocate to the nucleus to regulate transcription of genes that further mediate cellular physiology. The TGFβ/BMP pathways are very important for proper tissue development and homeostasis, thus precise spatial and temporal regulation of the signalling pathway is required and achieved by many positive and negative signalling regulators.

    This thesis work identified the liver kinase B1 (LKB1) pathway as a negative regulator of TGFβ/BMP signalling pathways. In the first paper, we established LKB1 as a negative regulator of TGFβ signalling and TGFβ-induced epithelial to mesenchymal transition (EMT). LKB1 impairs Smad4 binding capacity to DNA leading to suppressed TGFβ-activated gene transcription. The second paper describes further the mechanism of LKB1 negative regulation on BMP signalling, by mediating BMP type I receptor degradation resulting in inhibition of BMP-induced cell differentiation.

    Downstream of LKB1, salt inducible kinase 1 (SIK1) is a TGFβ target gene and its expression is up-regulated by Smad2/3/4-mediated gene transcription. The third paper elucidates the mechanism of SIK1 transcriptional induction via an enhancer element located 3’ of the gene and SIK1-mediated type I TGFβ receptor degradation, which requires the activity of Smad7 and of the Smurf2 ubiquitin ligase.

    The fourth manuscript finds sucrose non-fermenting (SNF) 1-like kinase 2 (NUAK2) as another TGFβ target gene and its up-regulation results in modification of the mammalian target of rapamycin (mTOR) pathway that controls protein synthesis. NUAK2 cooperates with LKB1 leading to Raptor phosphorylation and inhibition of mTOR-mediated protein synthesis. Collectively, this thesis work has provided a functional link between two important signalling pathways, the metabolic LKB1 pathway and TGFβ/BMP pathway.

    List of papers
    1. Negative regulation of TGFβ signaling by the kinase LKB1 and the scaffolding protein LIP1
    Open this publication in new window or tab >>Negative regulation of TGFβ signaling by the kinase LKB1 and the scaffolding protein LIP1
    2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 1, p. 341-353Article in journal (Refereed) Published
    Abstract [en]

    Signal transduction by the Smad pathway elicits critical biological responses to many extracellular polypeptide factors, including TGFβ and bone morphogenetic protein. Regulation of Smad signaling imparts several cytoplasmic and nuclear mechanisms, some of which entail protein phosphorylation. Previous work established a protein complex between Smad4 and the scaffolding protein LKB1-interacting protein 1 (LIP1). LKB1 is a well studied tumor suppressor kinase that regulates cell growth and polarity. Here, we analyzed the LKB1-LIP1 and the Smad4-LIP1 protein complexes and found that LIP1 can self-oligomerize. We further demonstrate that LKB1 is capable of phosphorylating Smad4 on Thr(77) of its DNA-binding domain. LKB1 inhibits Smad4 from binding to either TGFβ- or bone morphogenetic protein-specific promoter sequences, which correlates with the negative regulatory effect LKB1 exerts on Smad4-dependent transcription. Accordingly, LKB1 negatively regulates TGFβ gene responses and epithelial-mesenchymal transition. Thus, LKB1 and LIP1 provide negative control of TGFβ signaling.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-145014 (URN)10.1074/jbc.M110.190660 (DOI)000285782800037 ()20974850 (PubMedID)
    Available from: 2011-02-04 Created: 2011-02-04 Last updated: 2022-01-28Bibliographically approved
    2. Transcriptional induction of salt-inducible kinase 1 by transforming growth factor β leads to negative regulation of type I receptor signaling in cooperation with the Smurf2 ubiquitin ligase
    Open this publication in new window or tab >>Transcriptional induction of salt-inducible kinase 1 by transforming growth factor β leads to negative regulation of type I receptor signaling in cooperation with the Smurf2 ubiquitin ligase
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    2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 16, p. 12867-12878Article in journal (Refereed) Published
    Abstract [en]

    Transforming growth factor β (TGFβ)1 regulates many physiological processes and requires control mechanisms to safeguard proper and timely action. We have previously described how negative regulation of TGFβ signaling is controlled by the serine/threonine kinase salt-inducible kinase (SIK) 1. SIK1 forms complexes with the TGFβ type I receptor and with the inhibitory Smad7 and downregulates the type I receptor. We now demonstrate that TGFβ induces SIK1 levels via a direct transcriptional mechanism that implicates the Smad proteins and we have mapped a putative enhancer element on the SIK1 gene. We provide evidence that the ubiquitin ligase Smurf2 forms complexes and functionally cooperates with SIK1. Both the kinase activity of SIK1 and the ubiquitin ligase activity of Smurf2 are important for proper type I receptor turnover. We also show that knockdown of endogenous SIK1 and Smurf2 enhances physiological signaling by TGFβ that leads to epithelial growth arrest. In conclusion, TGFβ induces expression of Smad7, Smurf2 and SIK1, the products of which physically and functionally interlink to control the activity of this pathway.

    Keywords
    Signal transduction, SIK1, Smad, Smurf, SNF1LK, TGFβ, Ubiquitin
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-169792 (URN)10.1074/jbc.M111.307249 (DOI)000302903700026 ()22378783 (PubMedID)
    Available from: 2012-03-06 Created: 2012-03-06 Last updated: 2022-01-28Bibliographically approved
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  • 320.
    Rath, Matthias
    et al.
    Univ Greifswald, Dept Human Genet, Univ Med Greifswald, Fleischmannstr 43, D-17475 Greifswald, Germany.;Univ Greifswald, Interfac Inst Genet & Funct Genom, Fleischmannstr 43, D-17475 Greifswald, Germany..
    Schwefel, Konrad
    Univ Greifswald, Dept Human Genet, Univ Med Greifswald, Fleischmannstr 43, D-17475 Greifswald, Germany.;Univ Greifswald, Interfac Inst Genet & Funct Genom, Fleischmannstr 43, D-17475 Greifswald, Germany..
    Malinverno, Matteo
    FIRC Inst Mol Oncol Fdn IFOM, Vasc Biol Unit, Milan, Italy..
    Skowronek, Dariush
    Univ Greifswald, Dept Human Genet, Univ Med Greifswald, Fleischmannstr 43, D-17475 Greifswald, Germany.;Univ Greifswald, Interfac Inst Genet & Funct Genom, Fleischmannstr 43, D-17475 Greifswald, Germany..
    Leopoldi, Alexandra
    Austrian Acad Sci, Inst Mol Biotechnol, Vienna, Austria..
    Pilz, Robin A.
    Univ Greifswald, Dept Human Genet, Univ Med Greifswald, Fleischmannstr 43, D-17475 Greifswald, Germany.;Univ Greifswald, Interfac Inst Genet & Funct Genom, Fleischmannstr 43, D-17475 Greifswald, Germany..
    Biedenweg, Doreen
    Univ Greifswald, Ctr Innovat Competence Humoral Immune React Cardi, Greifswald, Germany..
    Bekeschus, Sander
    Leibniz Inst Plasma Sci & Technol INP, ZIK Plasmatis, Greifswald, Germany..
    Penninger, Josef M.
    Austrian Acad Sci, Inst Mol Biotechnol, Vienna, Austria.;Univ British Columbia, Life Sci Inst, Dept Med Genet, Vancouver, BC, Canada..
    Dejana, Elisabetta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. FIRC Inst Mol Oncol Fdn IFOM, Vasc Biol Unit, Milan, Italy..
    Felbor, Ute
    Univ Greifswald, Dept Human Genet, Univ Med Greifswald, Fleischmannstr 43, D-17475 Greifswald, Germany.;Univ Greifswald, Interfac Inst Genet & Funct Genom, Fleischmannstr 43, D-17475 Greifswald, Germany..
    Contact-dependent signaling triggers tumor-like proliferation of CCM3 knockout endothelial cells in co-culture with wild-type cells2022In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 79, no 6, article id 340Article in journal (Refereed)
    Abstract [en]

    Cerebral cavernous malformations (CCM) are low-flow vascular lesions prone to cause severe hemorrhage-associated neurological complications. Pathogenic germline variants in CCM1, CCM2, or CCM3 can be identified in nearly 100% of CCM patients with a positive family history. In line with the concept that tumor-like mechanisms are involved in CCM formation and growth, we here demonstrate an abnormally increased proliferation rate of CCM3-deficient endothelial cells in co-culture with wild-type cells and in mosaic human iPSC-derived vascular organoids. The observation that NSC59984, an anticancer drug, blocked the abnormal proliferation of mutant endothelial cells further supports this intriguing concept. Fluorescence-activated cell sorting and RNA sequencing revealed that co-culture induces upregulation of proangiogenic chemokine genes in wild-type endothelial cells. Furthermore, genes known to be significantly downregulated in CCM3(-/-) endothelial cell mono-cultures were upregulated back to normal levels in co-culture with wild-type cells. These results support the hypothesis that wild-type ECs facilitate the formation of a niche that promotes abnormal proliferation of mutant ECs. Thus, targeting the cancer-like features of CCMs is a promising new direction for drug development.

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  • 321.
    Raykova, Doroteya
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Genetics of Two Mendelian Traits and Validation of Induced Pluripotent Stem Cell (iPSC) Technology for Disease Modeling2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Novel technologies for genome analysis have provided almost unlimited opportunities to uncover structural gene variants behind human disorders. Whole exome sequencing (WES) is especially useful for understanding rare Mendelian conditions, because it reduces the requirements for a priori clinical data, and can be applied on a small number of patients. However, supporting functional data on the effect of specific gene variants are often required to power these findings. A variety of methods and biological model systems exists for this purpose. Among those, induced pluripotent stem cells (iPSCs), which are capable of self-renewal and differentiation, stand out as an alternative to animal models.

    In papers I and II we took advantage of WES to identify gene variants underlying autosomal recessive pure hair and nail ectodermal dysplasia (AR PHNED) as well as autosomal dominant familial visceral myopathy (FVM). We identified a homozygous variant c.821T>C (p.Phe274Ser) in the KRT74 gene as the causative mutation in AR PHNED, supported by the fact that Keratin-74 was undetectable in hair follicles of an affected family member. In a family segregating FVM we found a heterozygous tandem base substitution c.806_807delinsAA (p.(Gly269Glu)) in the ACTG2 gene in the affected members. This novel variant is associated with a broad range of visceral symptoms and a variable age of onset.

    In Paper III we explored the similarity between clonally derived iPSC lines originating from a single parental fibroblast line and we highlighted the necessity to use lines originating from various donors in disease modeling because of biological variation. Paper IV focused on how the genomic integrity of iPSCs is affected by the choice of reprogramming methods. We described several novel cytogenetic rearrangements in iPSCs and we identified a chromosome 5q duplication as a candidate aberration for growth advantage.

    In summary, this doctoral thesis brings novel findings on unreported disease-causing variants, as supported by extensive genetic analysis and functional data. A novel molecular mechanism behind AR PHNED is presented and the phenotypic spectrum associated with FVM is expanded. In addition, the thesis brings novel understanding of benefits and limitations of the iPSC technology to be considered for disease modeling.

    List of papers
    1. Autosomal Recessive Transmission of a Rare KRT74 Variant Causes Hair and Nail Ectodermal Dysplasia: Allelism with Dominant Woolly Hair/Hypotrichosis
    Open this publication in new window or tab >>Autosomal Recessive Transmission of a Rare KRT74 Variant Causes Hair and Nail Ectodermal Dysplasia: Allelism with Dominant Woolly Hair/Hypotrichosis
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    2014 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 9, no 4, p. e93607-Article in journal (Refereed) Published
    Abstract [en]

    Pure hair and nail ectodermal dysplasia (PHNED) comprises a heterogeneous group of rare heritable disorders characterized by brittle hair, hypotrichosis, onychodystrophy and micronychia. Autosomal recessive (AR) PHNED has previously been associated with mutations in either KRT85 or HOXC13 on chromosome 12p11.1-q14.3. We investigated a consanguineous Pakistani family with AR PHNED linked to the keratin gene cluster on 12p11.1 but without detectable mutations in KRT85 and HOXC13. Whole exome sequencing of affected individuals revealed homozygosity for a rare c.821T> C variant (p.Phe274Ser) in the KRT74 gene that segregates AR PHNED in the family. The transition alters the highly conserved Phe274 residue in the coil 1B domain required for long-range dimerization of keratins, suggesting that the mutation compromises the stability of intermediate filaments. Immunohistochemical (IHC) analyses confirmed a strong keratin-74 expression in the nail matrix, the nail bed and the hyponychium of mouse distal digits, as well as in normal human hair follicles. Furthermore, hair follicles and epidermis of an affected family member stained negative for Keratin-74 suggesting a loss of function mechanism mediated by the Phe274Ser substitution. Our observations show for the first time that homozygosity for a KRT74 missense variant may be associated with AR PHNED. Heterozygous KRT74 mutations have previously been associated with autosomal dominant woolly hair/ hypotrichosis simplex (ADWH). Thus, our findings expand the phenotypic spectrum associated with KRT74 mutations and imply that a subtype of AR PHNED is allelic with ADWH.

    National Category
    Medical Genetics
    Identifiers
    urn:nbn:se:uu:diva-225058 (URN)10.1371/journal.pone.0093607 (DOI)000334160900039 ()
    Available from: 2014-06-24 Created: 2014-05-27 Last updated: 2021-06-14Bibliographically approved
    2. Phenotypic expansion of visceral myopathy associated with ACTG2 tandem base substitution
    Open this publication in new window or tab >>Phenotypic expansion of visceral myopathy associated with ACTG2 tandem base substitution
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    2015 (English)In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 23, no 12, p. 1679-1683Article in journal (Refereed) Published
    Abstract [en]

    Familial visceral myopathy (FVM) is a rare heritable and heterogeneous condition due to impaired smooth muscle function. We identified a family segregating 11 individuals with a spectrum of visceral symptoms involving the small intestine, colon, biliary tract, urinary tract and uterus. Whole-exome sequencing revealed a novel heterozygous tandem base substitution c.806_807delinsAA (p.(Gly269Glu)) in ACTG2, encoding smooth muscle actin γ-2, in affected family members. Variants in ACTG2 were recently identified in FVM with intestinal pseudo-obstruction as well as with the congenital megacystics-microcolon-intestinal hypoperistalsis syndrome. In our family, eight affected members presented with severe complications from the biliary and/or the urinary tracts in addition to gastrointestinal pseudo-obstructions. Furthermore, all affected mothers had a history of assisted deliveries owing to poor progress during labor and weak uterine contractions. The variable involvement of multiple smooth muscle-dependent organs in our family, including the biliary tract and the uterus, add to the phenotypic spectrum associated with ACTG2 missense variants.

    National Category
    Genetics
    Identifiers
    urn:nbn:se:uu:diva-244419 (URN)10.1038/ejhg.2015.49 (DOI)000365129700015 ()25782675 (PubMedID)
    Funder
    Swedish Research Council, K2013-66X-10829-20-3
    Note

    De två första författarna delar förstaförfattarskapet.

    Available from: 2015-03-03 Created: 2015-02-16 Last updated: 2017-12-04Bibliographically approved
    3. Transcriptome Profiling Reveals Degree of Variability in Induced Pluripotent Stem Cell Lines: Impact for Human Disease Modeling
    Open this publication in new window or tab >>Transcriptome Profiling Reveals Degree of Variability in Induced Pluripotent Stem Cell Lines: Impact for Human Disease Modeling
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    2015 (English)In: Cellular Reprogramming, ISSN 2152-4971, E-ISSN 2152-4998, Vol. 17, no 5, p. 327-337Article in journal (Refereed) Published
    Abstract [en]

    Induced pluripotent stem cell (iPSC) technology has become an important tool for disease modeling. Insufficient data on the variability among iPSC lines derived from a single somatic parental cell line have in practice led to generation and analysis of several, usually three, iPSC sister lines from each parental cell line. We established iPSC lines from a human fibroblast line (HDF-K1) and used transcriptome sequencing to investigate the variation among three sister lines (iPSC-K1A, B, and C). For comparison, we analyzed the transcriptome of an iPSC line (iPSC-K5B) derived from a different fibroblast line (HDF-K5), a human embryonic stem cell (ESC) line (ESC-HS181), as well as the two parental fibroblast lines. All iPSC lines fulfilled stringent criteria for pluripotency. In an unbiased cluster analysis, all stem cell lines (four iPSCs and one ESC) clustered together as opposed to the parental fibroblasts. The transcriptome profiles of the three iPSC sister lines were indistinguishable from each other, and functional pathway analysis did not reveal any significant hits. In contrast, the expression profiles of the ESC line and the iPSC-K5B line were distinct from that of the sister lines iPSC-K1A, B, and C. Differentiation to embryoid bodies and subsequent analysis of germ layer markers in the five stem cell clones confirmed that the distribution of their expression profiles was retained. Taken together, our observations stress the importance of using iPSCs of different parental origin rather than several sister iPSC lines to distinguish disease-associated mechanisms from genetic background effects in disease modeling.

    National Category
    Other Biological Topics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-244422 (URN)10.1089/cell.2015.0009 (DOI)000361523600002 ()26348590 (PubMedID)
    Funder
    Swedish Research Council, K2013-66X-10829-20-3 621-2009-4629EU, European Research Council, 282330AstraZenecaScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish National Infrastructure for Computing (SNIC), b2013214
    Available from: 2015-03-03 Created: 2015-02-16 Last updated: 2017-12-04Bibliographically approved
    4. Methods of Reprogramming to Induced Pluripotent Stem Cell Associated with Chromosomal Integrity and Delineation of a Chromosome 5q Candidate Region for Growth Advantage
    Open this publication in new window or tab >>Methods of Reprogramming to Induced Pluripotent Stem Cell Associated with Chromosomal Integrity and Delineation of a Chromosome 5q Candidate Region for Growth Advantage
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    2015 (English)In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 24, no 17, p. 2032-2040Article in journal (Refereed) Published
    Abstract [en]

    Induced pluripotent stem cells (iPSCs) have brought great promises for disease modeling and cell-based therapies. One concern related to the use of reprogrammed somatic cells is the loss of genomic integrity and chromosome stability, a hallmark for cancer and many other human disorders. We investigated 16 human iPSC lines reprogrammed by nonintegrative Sendai virus (SeV) and another 16 iPSC lines generated by integrative lentivirus for genetic changes. At early passages we detected cytogenetic rearrangements in 44% (7/16) of iPSC lines generated by lentiviral integration whereas the corresponding figure was 6% (1/16) using SeV-based delivery. The rearrangements were numerical and/or structural with chromosomes 5 and 12 as the most frequently involved chromosomes. Three iPSC lines with chromosome 5 aberrations were derived from one and the same donor. We present in this study the aberrant karyotypes including a duplication of chromosome 5q13q33 that restricts a candidate region for growth advantage. Our results suggest that the use of integrative lentivirus confers a higher risk for cytogenetic abnormalities at early passages when compared to SeV-based reprogramming. In combination, our findings expand the knowledge on acquired cytogenetic aberrations in iPSC after reprogramming and during culture.

    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Research subject
    Biology with specialization in Molecular Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-246225 (URN)10.1089/scd.2015.0061 (DOI)000359606100007 ()
    Available from: 2015-03-03 Created: 2015-03-03 Last updated: 2017-12-04Bibliographically approved
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  • 322.
    Razmara, Masoud
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Tumor Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Monazzam, Azita
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Tumor Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Skogseid, Britt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Tumor Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Reduced menin expression impairs rapamycin effects as evidenced by an increase in mTORC2 signaling and cell migration2018In: Cell Communication and Signaling, E-ISSN 1478-811X, Vol. 16, article id 64Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Mammalian target of rapamycin (mTOR) is a master regulator of various cellular responses by forming two functional complexes, mTORC1 and mTORC2. mTOR signaling is frequently dysregulated in pancreatic neuroendocrine tumors (PNETs). mTOR inhibitors have been used in attempts to treat these lesions, and prolonged progression free survival has been recorded. If this holds true also for the multiple endocrine neoplasia type 1 (MEN1) associated PNETs is yet unclear. We investigated the relationship between expression of the MEN1 protein menin and mTOR signaling in the presence or absence of the mTOR inhibitor rapamycin.

    METHODS: In addition to use of menin wild type and menin-null mouse embryonic fibroblasts (MEFs), menin was silenced by siRNA in pancreatic neuroendocrine tumor cell line BON-1. Panels of protein phosphorylation, as activation markers downstream of PI3k-mTOR-Akt pathways, as well as menin expression were evaluated by immunoblotting. The impact of menin expression in the presence and absence of rapamycin was determinate upon Wound healing, migration and proliferation in MEFs and BON1 cells.

    RESULTS: PDGF-BB markedly increased phosphorylation of mTORC2 substrate Akt, at serine 473 (S473) and threonine 450 (T450) in menin-/- MEFs but did not alter phosphorylation of mTORC1 substrates ribosomal protein S6 or eIF4B. Acute rapamycin treatment by mTORC1-S6 inhibition caused a greater enhancement of Akt phosphorylation on S473 in menin-/- cells as compared to menin+/+ MEFs (116% vs 38%). Chronic rapamycin treatment, which inhibits both mTORC1and 2, reduced Akt phosphorylation of S473 to a lesser extent in menin-/- MEFs than menin+/+ MEFs (25% vs 75%). Silencing of menin expression in human PNET cell line (BON1) also enhanced Akt phosphorylation at S473, but not activation of mTORC1. Interestingly, silencing menin in BON1 cells elevated S473 phosphorylation of Akt in both acute and chronic treatments with rapamycin. Finally, we show that the inhibitory effect of rapamycin on serum mediated wound healing and cell migration is impaired in menin-/- MEFs, as well as in menin-silenced BON1 cells.

    CONCLUSIONS: Menin is involved in regulatory mechanism between the two mTOR complexes, and its reduced expression is accompanied with increased mTORC2-Akt signaling, which consequently impairs anti-migratory effect of rapamycin.

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  • 323. Reddy, B. K. Kishore
    et al.
    Landge, Sudhir
    Ravishankar, Sudha
    Patil, Vikas
    Shinde, Vikas
    Tantry, Subramanyam
    Kale, Manoj
    Raichurkar, Anandkumar
    Menasinakai, Sreenivasaiah
    Mudugal, Naina Vinay
    Ambady, Anisha
    Ghosh, Anirban
    Tunduguru, Ragadeepthi
    Kaur, Parvinder
    Singh, Ragini
    Kumar, Naveen
    Bharath, Sowmya
    Sundaram, Aishwarya
    Bhat, Jyothi
    Sambandamurthy, Vasan K.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Das, Kaveri
    Bandodkar, Balachandra
    Malolanarasimhan, Krishnan
    Mukherjee, Kakoli
    Ramachandran, Vasanthi
    Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors2014In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 58, no 6, p. 3312-3326Article in journal (Refereed)
    Abstract [en]

    Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis.

  • 324. Reu, Pedro
    et al.
    Khosravi, Azadeh
    Bernard, Samuel
    Mold, Jeff E.
    Salehpour, Mehran
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Applied Nuclear Physics.
    Alkass, Kanar
    Perl, Shira
    Tisdale, John
    Possnert, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Applied Nuclear Physics.
    Druid, Henrik
    Frisen, Jonas
    The Lifespan and Turnover of Microglia in the Human Brain2017In: Cell Reports, E-ISSN 2211-1247, Vol. 20, no 4, p. 779-784Article in journal (Refereed)
    Abstract [en]

    The hematopoietic system seeds the CNS with microglial progenitor cells during the fetal period, but the subsequent cell generation dynamics and maintenance of this population have been poorly understood. We report that microglia, unlike most other hematopoietic lineages, renew slowly at a median rate of 28% per year, and some microglia last for more than two decades. Furthermore, we find no evidence for the existence of a substantial population of quiescent long-lived cells, meaning that the microglia population in the human brain is sustained by continuous slow turnover throughout adult life.

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  • 325.
    Richards, Mark
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Pal, Sagnik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Sjöberg, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Martinsson, Pernilla
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Venkatraman, Lakshmi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Claesson-Welsh, Lena
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Intra-vessel heterogeneity establishes enhanced sites of macromolecular leakage downstream of laminin alpha 52021In: Cell Reports, E-ISSN 2211-1247, Vol. 35, no 12, article id 109268Article in journal (Refereed)
    Abstract [en]

    Endothelial cells display heterogeneous properties based on location and function. How this heterogeneity influences endothelial barrier stability both between and within vessel subtypes is unexplored. In this study, we find that endothelial cells exhibit heterogeneous barrier properties on inter-organ and intra-vessel levels. Using intravital microscopy and sequential stimulation of the ear dermis with vascular endothelial growth factor-A (VEGFA) and/or histamine, we observe distinct, reappearing sites, common for both agonists, where leakage preferentially takes place. Through repetitive stimulation of the diaphragm and trachea, we find inter-organ conservation of such predetermined leakage sites. Qualitatively, predetermined sites display distinct leakage properties and enhanced barrier breakdown compared to less susceptible regions. Mechanistically, laminin alpha 5 is reduced at predetermined sites, which is linked to reduced junctional vascular endothelial (VE)-cadherin and enhanced VEGFA-induced VE-cadherin phosphorylation. These data highlight functional intra-vessel heterogeneity that defines predetermined sites with distinct leakage properties and that may disproportionately impact pathological vascular leakage.

  • 326.
    Richter, Franziska Sophie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Physiology and Environmental Toxicology.
    Impact of BPA, BPF and Mixture N1 on DNA-Methylation of GRIN2B and NR3C1 during human neuroprogenitor cell differentiation2023Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
    Abstract [en]

    Endocrine disrupting chemicals (EDCs) are ubiquitous and their adverse impact on nature, wildlife and humans is extensively researched. We are constantly exposed to EDCs, such as the widespread and extensively researched Bisphenol A, as well as its substitute Bisphenol F, which is coming into wider use, even though it is much less is researched and limited information is available about its endocrine effects. Realistically, we are exposed to mixtures rather than single substances. In the Swedish Environmental Longitudinal, Mother and Child, Asthma and allergy (SELMA) study, the co-exposure of EDCs was assessed. Based on the SELMA cohort data, a study identified a mixture of EDCs, Mixture N1, which is associated with delayed language development. In recent years, it has been hypothesized that epigenetic alterations are one of the underlying mechanisms for the effect of EDC exposures. For example, EDC induced changes in DNA Methylation of the promoter region of a gene might lead to altered gene expression, which can result in adverse health effects. Several studies already indicate an impact of the formerly introduced chemicals/mixtures on the DNA methylation on genes such as NR3C1 and GRIN2B in animals. However, limited research is available on the impact on NR3C1 and GRIN2B in the early human brain, which is of interest since both genes are crucial for the development of the brain and altered gene expression often leads to adverse effects. This study aimed to investigate the impact of BPA, BPF and Mixture N1 on NR3C1 and GRIN2B in the developing human brain as well as establish a protocol for differentiation of human stem cells into neuroprogenitor cells that express GRIN2B and NR3C1. In the end stem cells were differentiated in vitro into neural progenitor cells (NPCs) using the protocol of Hosseini et al. (2020). During the differentiation, the cells were exposed to different concentrations of the former mentioned chemicals. Afterwards, RNA and DNA were extracted, followed by a qPCR and bisulfite-pyrosequencing to investigate the changes in gene expression and DNA methylation of NR3C1 and GRIN2B. This study established the differentiation protocol but revealed no significant results regarding the chemical exposure. However, some chemical exposures showed a clear tendency towards an impact of the chemicals on the gene expression and the DNA methylation. Furthermore, a negative correlation between DNA methylation at 2 CpG sites and gene expression in NR3C1 could be observed. In conclusion, the DNA methylation at promoter region in NR3C1 is important for the gene expression.

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  • 327. Ricordi, C.
    et al.
    Hering, B.
    Bridges, N.
    Eggerman, T.
    Naji, A.
    Posseit, A.
    Stock, P.
    Kaufman, D.
    Larsen, C. P.
    Turgeon, N.
    Oberholzer, J.
    Barbaro, B.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Markmann, J.
    Alejandro, R.
    Rickels, M. R.
    Senior, P. A.
    Luo, X.
    Zhang, X.
    Bellin, M.
    Lei, J.
    Clarke, W.
    Hunsicker, L.
    Goldstein, J.
    Czarniecki, C.
    Priore, A.
    Green, N.
    Shapiro, A.
    Completion of the First FDA Phase 3 Multicenter Trial of Islet Transplantation in Type 1 Diabetes by the NIH CIT Consortium2014In: Cytotherapy, ISSN 1465-3249, E-ISSN 1477-2566, Vol. 16, no 4, p. S14-S14Article in journal (Other academic)
  • 328.
    Roberts, Christina Joy
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Physiological Botany.
    Cell-to-Cell Signalling in Arabidopsis Root Development2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Development in multicellular organisms requires a strict balance between cell division and differentiation. The simple architecture of the Arabidopsis thaliana root makes it an ideal model for studying molecular mechanisms controlling both the transition from cell division to cell differentiation and cell fate determination. The class III Homeodomain-Leucine Zipper (HD-ZIP III) transcription factors (TFs) are well known developmental regulators, controlling important aspects of embryogenesis, shoot meristem activity, leaf polarity and vascular patterning. The HD-ZIP III TFs are under post-transcriptional control of microRNA165 (miR165) and miR166. In this thesis, I present a cell-to-cell signalling pathway underlying root vascular patterning and describe signaling pathways downstream of the HD-ZIP III TFs in their control of root development. The TF SHORTROOT (SHR), moves from the vascular stele cells to the surrounding endodermal cell layer. We show that SHR acts here to transcriptionally activate MIR165A and MIR166B, and the miR165/6 produced in the endodermis act non-cell autonomously to post-transcriptionally restrict HD-ZIP III mRNA levels in the peripheral stele. The resulting graded HD-ZIP III activity domain in the radial stele dose-dependently determines vascular cell type; high levels of HD-ZIP III in the central stele result in metaxylem formation while lower levels in the peripheral stele result in protoxylem. We provide evidence that the HD-ZIP III factors act as de novo xylem specifiers, because the quintuple mutant lacking all five HD-ZIP III genes forms no xylem. Furthermore, reducing the plasmodesmatal aperture through callose accumulation inhibits the bi-directional mobility of both signalling molecules, providing evidence that both SHR and miR165/6 move cell-to-cell via plasmodesmata to control root development.

    I present downstream components of the miR165/HD-ZIP III TFs in the root meristem, identified through a time-course induction of miR165 coupled to transcriptome analyses. This experiment revealed novel roles for HD-ZIP III TFs in vascular patterning and meristem size control. I show that HD-ZIP III directed repression of auxin hormone signalling in the xylem axis is essential for proper xylem differentiation. Furthermore, I provide data to show that they also control the balance of reactive oxygen species in the root meristem, thereby directing meristem size and ultimately controlling root growth.

    List of papers
    1. Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate
    Open this publication in new window or tab >>Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate
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    2010 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 465, no 7296, p. 316-321Article in journal (Refereed) Published
    Abstract [en]

    A key question in developmental biology is how cells exchange positional information for proper patterning during organ development. In plant roots the radial tissue organization is highly conserved with a central vascular cylinder in which two water conducting cell types, protoxylem and metaxylem, are patterned centripetally. We show that this patterning occurs through crosstalk between the vascular cylinder and the surrounding endodermis mediated by cell-to-cell movement of a transcription factor in one direction and microRNAs in the other. SHORT ROOT, produced in the vascular cylinder, moves into the endodermis to activate SCARECROW. Together these transcription factors activate MIR165a and MIR166b. Endodermally produced microRNA165/6 then acts to degrade its target mRNAs encoding class III homeodomain-leucine zipper transcription factors in the endodermis and stele periphery. The resulting differential distribution of target mRNA in the vascular cylinder determines xylem cell types in a dosage-dependent manner.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-136559 (URN)10.1038/nature08977 (DOI)000277829200033 ()20410882 (PubMedID)
    Available from: 2010-12-14 Created: 2010-12-13 Last updated: 2017-12-11Bibliographically approved
    2. Callose Biosynthesis Regulates Symplastic Trafficking during Root Development
    Open this publication in new window or tab >>Callose Biosynthesis Regulates Symplastic Trafficking during Root Development
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    2011 (English)In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 21, no 6, p. 1144-1155Article in journal (Refereed) Published
    Abstract [en]

    Plant cells are connected through plasmodesmata (PD), membrane-lined channels that allow symplastic movement of molecules between cells. However, little is known about the role of PD-mediated signaling during plant morphogenesis. Here, we describe an Arabidopsis gene, CALS3/GSL12. Gain-of-function mutations in CALS3 result in increased accumulation of callose (beta-1,3-glucan) at the PD, a decrease in PD aperture, defects in root development, and reduced intercellular trafficking. Enhancement of CALS3 expression during phloem development suppressed loss-of-function mutations in the phloem abundant callose synthase, CALS7 indicating that CALS3 is a bona fide callose synthase. CALS3 alleles allowed us to spatially and temporally control the PD aperture between plant tissues. Using this tool, we are able to show that movement of the transcription factor SHORT-ROOT and microRNA1 65 between the stele and the endodermis is PD dependent. Taken together, we conclude that regulated callose biosynthesis at PD is essential for cell signaling.

    National Category
    Natural Sciences Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-167203 (URN)10.1016/j.devcel.2011.10.006 (DOI)000298215200018 ()
    Available from: 2012-01-23 Created: 2012-01-23 Last updated: 2017-12-08Bibliographically approved
    3. Class III HD-ZIP Transcription Factors Determine Root Meristem Size by Controlling Reactive Oxygen Species Levels
    Open this publication in new window or tab >>Class III HD-ZIP Transcription Factors Determine Root Meristem Size by Controlling Reactive Oxygen Species Levels
    (English)Manuscript (preprint) (Other academic)
    National Category
    Botany
    Identifiers
    urn:nbn:se:uu:diva-181188 (URN)
    Available from: 2012-10-03 Created: 2012-09-18 Last updated: 2016-04-25
    4. Class III HD-ZIP Transcription Factors Regulate Auxin Signalling to Pattern Arabidopsis Root Vasculature
    Open this publication in new window or tab >>Class III HD-ZIP Transcription Factors Regulate Auxin Signalling to Pattern Arabidopsis Root Vasculature
    (English)Manuscript (preprint) (Other academic)
    National Category
    Botany
    Identifiers
    urn:nbn:se:uu:diva-181189 (URN)
    Available from: 2012-10-03 Created: 2012-09-18 Last updated: 2016-04-25
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  • 329.
    Rodrigues, João S.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Kovács, László
    Lukeš, Martin
    Höper, Rune
    Steuer, Ralf
    Červený, Jan
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Zavřel, Tomáš
    Characterizing isoprene production in cyanobacteria-Insights into the effects of light, temperature, and isoprene on Synechocystis sp. PCC 68032023In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 380, article id 129068Article in journal (Refereed)
    Abstract [en]

    Engineering cyanobacteria for the production of isoprene and other terpenoids has gained increasing attention in the field of biotechnology. Several studies have addressed optimization of isoprene synthesis in cyanobacteria via enzyme and pathway engineering. However, only little attention has been paid to the optimization of cultivation conditions. In this study, an isoprene-producing strain of Synechocystis sp. PCC 6803 and two control strains were grown under a variety of cultivation conditions. Isoprene production, as quantified by modified membrane inlet mass spectrometer (MIMS) and interpreted using Flux Balance Analysis (FBA), increased under violet light and at elevated temperature. Increase of thermotolerance in the isoprene producer was attributed to the physical presence of isoprene, similar to plants. The results demonstrate a beneficial effect of isoprene on cell survival at higher temperatures. This increased thermotolerance opens new possibilities for sustainable bio-production of isoprene and other products.

  • 330.
    Rojas-López, Laura
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Regulation of differentiation during the Giardia life cycle: An insight into the molecular control2022Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Giardia intestinalis (syn. G. lamblia, G. duodenalis) is an intestinal protozoan parasite that causes diarrheal infections worldwide. A critical step in maintaining its chain of transmission is the formation of infectious cysts from the disease causing trophozoites during a cell differentiation process called encystation. Giardia establishes noninvasive infection in the epithelial cells (IECs) of the upper small intestine of mammals, including humans, causing a diarrheal disease known as giardiasis. Giardiasis is a multifactorial disease in which some of the factors involved in pathogenesis and virulence have been identified. However, several questions of the effects of host-parasite interaction during cell differentiation remain unclear.

    This thesis is divided into three sections. The first part focused on producing a high-resolution gene expression map of encystation in Giardia (Paper I). For this purpose, we used deep RNAseq combined with a rich range of timepoints during encystation that showed a gradual change in gene expression along the time course of differentiation. Genes encoding proteins involved in forming the resistant cyst wall are induced early in encystation, and most genes are regulated by the transcription factor Myb2. However, the most extensive gene expression changes were observed during the late phase of encystation, mainly among cysteine-rich surface proteins (VSPs and HCMPs) and genes involved in regulation of chromatin structure.

    Secondly, we investigated how epigenetic regulation is related to processes controlling parasite virulence, host immune evasion, transmission, and cell differentiation. As a result, we produced the first molecular map of post-translational modifications in core histones (methylation, acetylation, and phosphorylation) in Giardia using mass spectrometry (Paper II).

    Finally, to study how cell differentiation affects the host and vice versa, we used a host-parasite interaction model combining IECs (differentiated Caco-2 cells) with different cell stages of Giardia (trophozoites, early encysting cells (7 h), and cysts). We analyzed these interactions using Dual RNA sequencing (Dual RNAseq) to identify differentially expressed genes (DEGs) in Giardia and the host cells. Our results showed that different life cycle stages of Giardia induce different gene expression responses in the host cells. Furthermore, the IECs affect the gene expression in trophozoites and early encysting cells (Paper III). This life cycle stage-specific host-parasite crosstalk stands as a crucial step to consider for future studies in the molecular pathogenesis of Giardia. 

    In summary, this thesis has provided a deeper understanding of cell differentiation of Giardia intestinalis and its interaction with host cells. It shows that specific gene expression changes, regulated via transcription factors and epigenetic regulation, are involved in the host-parasite crosstalk during Giardia infections.

    List of papers
    1. A Detailed Gene Expression Map of Giardia Encystation
    Open this publication in new window or tab >>A Detailed Gene Expression Map of Giardia Encystation
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    2021 (English)In: Genes, ISSN 2073-4425, E-ISSN 2073-4425, Vol. 12, no 12, p. 1932-, article id 1932Article in journal (Refereed) Published
    Abstract [en]

    Giardia intestinalis is an intestinal protozoan parasite that causes diarrheal infections worldwide. A key process to sustain its chain of transmission is the formation of infectious cysts in the encystation process. We combined deep RNAseq of a broad range of encystation timepoints to produce a high-resolution gene expression map of Giardia encystation. This detailed transcriptomic map of encystation confirmed a gradual change of gene expression along the time course of encystation, showing the most significant gene expression changes during late encystation. Few genes are differentially expressed early in encystation, but the major cyst wall proteins CWP-1 and -2 are highly up-regulated already after 3.5 h encystation. Several transcription factors are sequentially up-regulated throughout the process, but many up-regulated genes at 7, 10, and 14 h post-induction of encystation have binding sites in the upstream regions for the Myb2 transcription factor, suggesting that Myb2 is a master regulator of encystation. We observed major changes in gene expression of several meiotic-related genes from 10.5 h of encystation to the cyst stage, and at 17.5 h encystation, there are changes in many different metabolic pathways and protein synthesis. Late encystation, 21 h to cysts, show extensive gene expression changes, most of all in VSP and HCMP genes, which are involved in antigenic variation, and genes involved in chromatin modifications. This high-resolution gene expression map of Giardia encystation will be an important tool in further studies of this important differentiation process.

    Place, publisher, year, edition, pages
    MDPIMDPI AG, 2021
    Keywords
    diarrhea, RNAseq, small intestine, protozoa, differentiation
    National Category
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-465062 (URN)10.3390/genes12121932 (DOI)000737881000001 ()34946882 (PubMedID)
    Available from: 2022-01-21 Created: 2022-01-21 Last updated: 2024-01-15Bibliographically approved
    2. Eukaryote-conserved histone post-translational modification landscape in Giardia duodenalis revealed by mass spectrometry
    Open this publication in new window or tab >>Eukaryote-conserved histone post-translational modification landscape in Giardia duodenalis revealed by mass spectrometry
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    2021 (English)In: International Journal of Parasitology, ISSN 0020-7519, E-ISSN 1879-0135, Vol. 51, no 4, p. 225-239Article in journal (Refereed) Published
    Abstract [en]

    Diarrheal disease caused by Giardia duodenalis is highly prevalent, causing over 200 million cases globally each year. The processes that drive parasite virulence, host immune evasion and transmission involve coordinated gene expression and have been linked to epigenetic regulation. Epigenetic regulatory systems are eukaryote-conserved, including in deep branching excavates such as Giardia, with several studies already implicating histone post-translational modifications in regulation of its pathogenesis and life cycle. However, further insights into Giardia chromatin dynamics have been hindered by a lack of site-specific knowledge of histone modifications. Using mass spectrometry, we have provided the first known molecular map of histone methylation, acetylation and phosphorylation modifications in Giardia core histones. We have identified over 50 previously unreported histone modifications including sites with established roles in epigenetic regulation, and co-occurring modifications indicative of post-translational modification crosstalk. These demonstrate conserved histone modifications in Giardia which are equivalent to many other eukaryotes, and suggest that similar epigenetic mechanisms are in place in this parasite. Further, we used sequence, domain and structural homology to annotate putative histone enzyme networks in Giardia, highlighting representative chromatin modifiers which appear sufficient for identified sites, particularly those from H3 and H4 variants. This study is to our knowledge the first and most comprehensive, complete and accurate view of Giardia histone post-translational modifications to date, and a substantial step towards understanding their associations in parasite development and virulence.

    Place, publisher, year, edition, pages
    Elsevier, 2021
    Keywords
    Giardia, Histone, Chromatin, Phosphorylation, Methylation, Acetylation
    National Category
    Infectious Medicine Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-441715 (URN)10.1016/j.ijpara.2020.09.006 (DOI)000631819400001 ()33275945 (PubMedID)
    Available from: 2021-05-04 Created: 2021-05-04 Last updated: 2024-01-15Bibliographically approved
    3. Dual RNA sequencing reveals key events when different Giardia life-cycle stages interact with human intestinal epithelial cells in vitro
    Open this publication in new window or tab >>Dual RNA sequencing reveals key events when different Giardia life-cycle stages interact with human intestinal epithelial cells in vitro
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Giardia intestinalis is a protozoan parasite causing diarrheal disease, giardiasis, after extracellular infection of humans and other mammals’ intestinal epithelial cells (IECs) of the upper small intestine. The parasite has two main life cycle stages: replicative trophozoites and transmissive cysts. Differentiating parasites (encysting cells) and trophozoites have recently been shown to be present in the same regions of the upper small intestine, whereas most mature cysts are found further down in the intestinal system. To learn more about host-parasite interactions during Giardia infections, we used an in vitro model of the parasite’s interaction with host IECs (differentiated Caco-2 cells) and Giardia WB trophozoites, early encysting cells (7 h), and cysts. Dual RNA sequencing (Dual RNAseq) was used to identify differentially expressed genes (DEGs) in both Giardia and the IECs, which might relate to establishing infection and disease induction. In the human cells, the largest gene expression changes were found in immune and MAPK signaling, transcriptional regulation, apoptosis, cholesterol metabolism and oxidative stress. The different life cycle stages of Giardia induced a core of similar DEGs but at different levels and there are many life cycle stage-specific DEGs. The metabolic protein PCK1, the transcription factors HES7, HEY1 and JUN, the peptide hormone CCK and the mucins MUC2 and MUC5A are up-regulated in the IECs by trophozoites but not cysts. Cysts specifically induce the chemokines CCL4L2, CCL5 and CXCL5, the signaling protein TRKA and the anti-bacterial protein WFDC12. The parasite, in turn, up-regulated a large number of hypothetical genes, high cysteine membrane proteins (HCMPs) and oxidative stress response genes. Early encysting cells have unique DEGs compared to trophozoites and interaction of these cells with IECs negatively affected the encystation process. Our data show that different life cycle stages of Giardia induce different gene expression responses in the host cells and that the IECs in turn differentially affect the gene expression in trophozoites and early encysting cells. This life cycle stage-specific host-parasite cross-talk is an important aspect to consider during further studies of Giardia’s molecular pathogenesis. 

    Keywords
    diarrhea, dual RNAseq, small intestinal epithelial cells, protozoa, differentiation
    National Category
    Microbiology Cell Biology Immunology
    Identifiers
    urn:nbn:se:uu:diva-466616 (URN)
    Funder
    Swedish Research Council, 2020-02918
    Available from: 2022-01-31 Created: 2022-01-31 Last updated: 2022-01-31
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  • 331.
    Rojas-López, Laura
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Grüttner, Jana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Ma’ayeh, Showgy
    Xu, Feifei
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Dual RNA sequencing reveals key events when different Giardia life-cycle stages interact with human intestinal epithelial cells in vitroManuscript (preprint) (Other academic)
    Abstract [en]

    Giardia intestinalis is a protozoan parasite causing diarrheal disease, giardiasis, after extracellular infection of humans and other mammals’ intestinal epithelial cells (IECs) of the upper small intestine. The parasite has two main life cycle stages: replicative trophozoites and transmissive cysts. Differentiating parasites (encysting cells) and trophozoites have recently been shown to be present in the same regions of the upper small intestine, whereas most mature cysts are found further down in the intestinal system. To learn more about host-parasite interactions during Giardia infections, we used an in vitro model of the parasite’s interaction with host IECs (differentiated Caco-2 cells) and Giardia WB trophozoites, early encysting cells (7 h), and cysts. Dual RNA sequencing (Dual RNAseq) was used to identify differentially expressed genes (DEGs) in both Giardia and the IECs, which might relate to establishing infection and disease induction. In the human cells, the largest gene expression changes were found in immune and MAPK signaling, transcriptional regulation, apoptosis, cholesterol metabolism and oxidative stress. The different life cycle stages of Giardia induced a core of similar DEGs but at different levels and there are many life cycle stage-specific DEGs. The metabolic protein PCK1, the transcription factors HES7, HEY1 and JUN, the peptide hormone CCK and the mucins MUC2 and MUC5A are up-regulated in the IECs by trophozoites but not cysts. Cysts specifically induce the chemokines CCL4L2, CCL5 and CXCL5, the signaling protein TRKA and the anti-bacterial protein WFDC12. The parasite, in turn, up-regulated a large number of hypothetical genes, high cysteine membrane proteins (HCMPs) and oxidative stress response genes. Early encysting cells have unique DEGs compared to trophozoites and interaction of these cells with IECs negatively affected the encystation process. Our data show that different life cycle stages of Giardia induce different gene expression responses in the host cells and that the IECs in turn differentially affect the gene expression in trophozoites and early encysting cells. This life cycle stage-specific host-parasite cross-talk is an important aspect to consider during further studies of Giardia’s molecular pathogenesis. 

  • 332.
    Rossi, Fiorella
    et al.
    Nova Southeastern Univ, NSU Cell Therapy Inst, 3301 Coll Ave,3200 South Univ Dr, Ft Lauderdale, FL 33328 USA..
    Noren, Hunter
    Nova Southeastern Univ, NSU Cell Therapy Inst, 3301 Coll Ave,3200 South Univ Dr, Ft Lauderdale, FL 33328 USA..
    Jove, Richard
    Nova Southeastern Univ, NSU Cell Therapy Inst, 3301 Coll Ave,3200 South Univ Dr, Ft Lauderdale, FL 33328 USA..
    Beljanski, Vladimir
    Nova Southeastern Univ, NSU Cell Therapy Inst, 3301 Coll Ave,3200 South Univ Dr, Ft Lauderdale, FL 33328 USA..
    Grinnemo, Karl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Thoracic Surgery. Nova Southeastern Univ, NSU Cell Therapy Inst, 3301 Coll Ave,3200 South Univ Dr, Ft Lauderdale, FL 33328 USA.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Differences and similarities between cancer and somatic stem cells: therapeutic implications2020In: Stem Cell Research & Therapy, E-ISSN 1757-6512, Vol. 11, no 1, article id 489Article, review/survey (Refereed)
    Abstract [en]

    Over the last decades, the cancer survival rate has increased due to personalized therapies, the discovery of targeted therapeutics and novel biological agents, and the application of palliative treatments. Despite these advances, tumor resistance to chemotherapy and radiation and rapid progression to metastatic disease are still seen in many patients. Evidence has shown that cancer stem cells (CSCs), a sub-population of cells that share many common characteristics with somatic stem cells (SSCs), contribute to this therapeutic failure. The most critical properties of CSCs are their self-renewal ability and their capacity for differentiation into heterogeneous populations of cancer cells. Although CSCs only constitute a low percentage of the total tumor mass, these cells can regrow the tumor mass on their own. Initially identified in leukemia, CSCs have subsequently been found in cancers of the breast, the colon, the pancreas, and the brain. Common genetic and phenotypic features found in both SSCs and CSCs, including upregulated signaling pathways such as Notch, Wnt, Hedgehog, and TGF-beta. These pathways play fundamental roles in the development as well as in the control of cell survival and cell fate and are relevant to therapeutic targeting of CSCs. The differences in the expression of membrane proteins and exosome-delivered microRNAs between SSCs and CSCs are also important to specifically target the stem cells of the cancer. Further research efforts should be directed toward elucidation of the fundamental differences between SSCs and CSCs to improve existing therapies and generate new clinically relevant cancer treatments.

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  • 333.
    Roy, J.
    et al.
    Stanford Univ, Biol, Stanford, CA USA.
    Wigington, C. P.
    Stanford Univ, Biol, Stanford, CA USA.
    Damle, N. P.
    Stanford Univ, Biol, Stanford, CA USA.
    Ulengin-Talkish, I.
    Stanford Univ, Biol, Stanford, CA USA.
    Cho, S. Ei
    Stanford Univ, Biol, Stanford, CA USA.
    Davey, N. E.
    Univ Coll Dublin, Conway Inst Biomol & Biomed Res, Dublin, North Ireland.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Wong, C. J.
    Univ Toronto, Mt Sinai Hosp, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada.
    Gingras, A.
    Univ Toronto, Mt Sinai Hosp, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada.
    Cyert, M. S.
    Stanford Univ, Biol, Stanford, CA USA.
    Mapping the human calcineurin phosphatase signaling network through global identification of short linear motifs that mediate substrate recognition2017In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal (Other academic)
  • 334.
    Sainero-Alcolado, Lourdes
    et al.
    Department of Microbiology, Tumor and Cell Biology, Biomedicum B7, Karolinska Institutet, Stockholm 17165, Sweden.
    Garde-Lapido, Elisa
    Department of Microbiology, Tumor and Cell Biology, Biomedicum B7, Karolinska Institutet, Stockholm 17165, Sweden.
    Snaebjörnsson, Marteinn Thor
    Division of Tumor Metabolism and Microenvironment, German Cancer Research Center, Heidelberg 69120, Germany.
    Schoch, Sarah
    Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund 22100, Sweden.
    Stevens, Irene
    Science for Life Laboratory, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm 17165, Sweden.
    Ruiz-Pérez, María Victoria
    Department of Microbiology, Tumor and Cell Biology, Biomedicum B7, Karolinska Institutet, Stockholm 17165, Sweden.
    Dyrager, Christine
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Pelechano, Vicent
    Science for Life Laboratory, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm 17165, Sweden.
    Axelson, Håkan
    Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund 22100, Sweden.
    Schulze, Almut
    Division of Tumor Metabolism and Microenvironment, German Cancer Research Center, Heidelberg 69120, Germany.
    Arsenian-Henriksson, Marie
    Department of Microbiology, Tumor and Cell Biology, Biomedicum B7, Karolinska Institutet, Stockholm 17165, Sweden.
    Targeting MYC induces lipid droplet accumulation by upregulation of HILPDA in clear cell renal cell carcinoma2024In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 121, no 7, article id e2310479121Article in journal (Refereed)
    Abstract [en]

    Metabolic reprogramming is critical during clear cell renal cell carcinoma (ccRCC) tumorigenesis, manifested by accumulation of lipid droplets (LDs), organelles that have emerged as new hallmarks of cancer. Yet, regulation of their biogenesis is still poorly understood. Here, we demonstrate that MYC inhibition in ccRCC cells lacking the von Hippel Lindau (VHL) gene leads to increased triglyceride content potentiating LD formation in a glutamine-dependent manner. Importantly, the concurrent inhibition of MYC signaling and glutamine metabolism prevented LD accumulation and reduced tumor burden in vivo. Furthermore, we identified the hypoxia-inducible lipid droplet–associated protein (HILPDA) as the key driver for induction of MYC-driven LD accumulation and demonstrated that conversely, proliferation, LD formation, and tumor growth are impaired upon its downregulation. Finally, analysis of ccRCC tissue as well as healthy renal control samples postulated HILPDA as a specific ccRCC biomarker. Together, these results provide an attractive approach for development of alternative therapeutic interventions for the treatment of this type of renal cancer.

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  • 335.
    Salloum, Hanin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Actin filaments as an indicator of impaired neuronal differentiation mediated by disruption of the retinoic acid signalling pathway2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Retinoic acid (RA) is a well-known neurodevelopmental signaling molecule. It is reported to induce effects on neurite formation in differentiating neurons and to interfere with the actin cytoskeleton. Therefore, this project aimed to investigate the mechanisms behind effects of RA on the actin cytoskeleton of developing neurons using the C17.2 neural progenitor cells (NPCs) in vitro model. The goal was to evaluate the morphological effects the growth cone had upon exposure to RA agonist and antagonist, and to analyze the expression of three genes: Coronin actin-binding protein 1C(Coro1c), Cdc42 effector protein 4 gene (Cdc42), and Fibronectin (Fn1). These genes were selected because of their relation to actin dynamics and/or their regulation by the Wnt pathway, which regulates/affects actin reorganization. Since the Wnt pathway was also shown to be affected by RA, this study aimed to investigate the relationship between RA and actin through the Wnt pathway. Cdc42 and Fn1 are related to both the Wnt pathway and actin dynamics, whereas Coro1cis a known actin-related protein. The expressions showed significant increase with Coro1c, while Cdc42 and Fn1 had a similar overall trend increase with the RA agonist. The RA antagonist showed no significant effect, except a trend decrease in all the genetic expressions. All genetic expression effects subside with the increase of RA agonist and antagonist concentrations. The results suggest the changes in actin filaments are related to a low dose effect of RA. The findings indicate a possibility of a regulation mechanism that controls actin-related gene expression in response to RA. This mechanism is possibly not restricted to the Wnt pathway seeing that a non-Wnt related gene was affected as well. 

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  • 336.
    Sampson, Christopher J
    et al.
    Univ Aberdeen, Inst Biol & Environm Sci, Aberdeen, Scotland.
    Williams, Michael J.
    Univ Aberdeen, Inst Biol & Environm Sci, Aberdeen, Scotland.
    Real-time analysis of Drosophila post-embryonic haemocyte behaviour2012In: PLOS ONE, E-ISSN 1932-6203, Vol. 7, no 1, article id e28783Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The larval stage of the model organism Drosophila is frequently used to study host-pathogen interactions. During embryogenesis the cellular arm of the immune response, consisting of macrophage-like cells known as plasmatocytes, is extremely motile and functions to phagocytise pathogens and apoptotic bodies, as well as produce extracellular matrix. The cellular branch of the larval (post-embryonic) innate immune system consists of three cell types-plasmatocytes, crystal cells and lamellocytes-which are involved in the phagocytosis, encapsulation and melanisation of invading pathogens. Post-embryonic haemocyte motility is poorly understood thus further characterisation is required, for the purpose of standardisation.

    METHODOLOGY: In order to examine post-embryonic haemocyte cytoskeletal dynamics or migration, the most commonly used system is in vitro cell lines. The current study employs an ex vivo system (an adaptation of in vitro cell incubation using primary cells), in which primary larval or pre-pupal haemocytes are isolated for short term analysis, in order to discover various aspects of their behaviour during events requiring cytoskeleton dynamics.

    SIGNIFICANCE: The ex vivo method allows for real-time analysis and manipulation of primary post-embryonic haemocytes. This technique was used to characterise, and potentially standardised, larval and pre-pupal haemocyte cytoskeleton dynamics, assayed on different extracellular matrices. Using this method it was determined that, while larval haemocytes are unable to migrate, haemocytes recovered from pre-pupae are capable of migration.

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  • 337.
    Sanchez-Vera, Victoria
    et al.
    Swedish University of Agricultural Sciences, The Linnean Centre of Plant Biology in Uppsala, Department of Plant Biology.
    Kenchappa, Chandra Shekar
    Swedish University of Agricultural Sciences, The Linnean Centre of Plant Biology in Uppsala, Department of Plant Biology.
    Landberg, Katarina
    Swedish University of Agricultural Sciences, The Linnean Centre of Plant Biology in Uppsala, Department of Plant Biology.
    Bressendorff, Simon
    University of Copenhagen, Department of Biology.
    Schwarzbach, Stefan
    Swedish University of Agricultural Sciences, The Linnean Centre of Plant Biology in Uppsala, Department of Plant Biology.
    Martin, Tom
    Swedish University of Agricultural Sciences, The Linnean Centre of Plant Biology in Uppsala, Department of Plant Biology.
    Mundy, John
    University of Copenhagen, Department of Biology.
    Petersen, Morten
    University of Copenhagen, Department of Biology.
    Thelander, Mattias
    Swedish University of Agricultural Sciences, The Linnean Centre of Plant Biology in Uppsala, Department of Plant Biology.
    Sundberg, Eva
    Swedish University of Agricultural Sciences, The Linnean Centre of Plant Biology in Uppsala, Department of Plant Biology.
    Autophagy is required for gamete differentiation in the moss Physcomitrella patens2017In: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 13, no 11, p. 1939-1951Article in journal (Refereed)
    Abstract [en]

    Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

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  • 338. Sandrini, Michael Paolo Bastner
    et al.
    Söderbom, Fredrik
    Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, Box 590, SE-75124 Uppsala, Sweden.
    Mikkelsen, Nils Egil
    Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, Box 590, SE-75124 Uppsala, Sweden.
    Piskur, Jure
    Dictyostelium discoideum salvages purine deoxyribonucleosides by highly specific bacterial-like deoxyribonucleoside kinases2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 369, no 3, p. 653-664Article in journal (Refereed)
    Abstract [en]

    The salvage of deoxyribonucleosides in the social amoeba Dictyostelium discoideum, which has an extremely A+T-rich genome, was investigated. All native deoxyribonucleosides were phosphorylated by D. discoideum cell extracts and we subcloned three deoxyribonucleoside kinase (dNK) encoding genes. D. discoideum thymidine kinase was similar to the human thymidine kinase 1 and was specific for thymidine with a K(m) of 5.1 microM. The other two cloned kinases were phylogenetically closer to bacterial deoxyribonucleoside kinases than to the eukaryotic enzymes. D. discoideum deoxyadenosine kinase (DddAK) had a K(m) for deoxyadenosine of 22.7 microM and a k(cat) of 3.7 s(-1) and could not efficiently phosphorylate any other native deoxyribonucleoside. D. discoideum deoxyguanosine kinase was also a purine-specific kinase and phosphorylated significantly only deoxyguanosine, with a K(m) of 1.4 microM and a k(cat) of 3 s(-1). The two purine-specific deoxyribonucleoside kinases could represent ancient enzymes present in the common ancestor of bacteria and eukaryotes but remaining only in a few eukaryote lineages. The narrow substrate specificity of the D. discoideum dNKs reflects the biased genome composition and we attempted to explain the strict preference of DddAK for deoxyadenosine by modeling the active center with different substrates. Apart from its native substrate, deoxyadenosine, DddAK efficiently phosphorylated fludarabine. Hence, DddAK could be used in the enzymatic production of fludarabine monophosphate, a drug used in the treatment of chronic lymphocytic leukemia.

  • 339.
    Sasaki, Mari
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Terabayashi, Takeshi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Oita Univ, Dept Pharmacol, Fac Med, 1-1 Idaigaoka, Hasamamachi, Oita, Japan.
    Weiss, Stefanie M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Toplab Gesell Angew Biotechnol mbH, Martinsried, Germany.
    Ferby, Ingvar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The Tumor Suppressor MIG6 Controls Mitotic Progression and the G2/M DNA Damage Checkpoint by Stabilizing the WEE1 Kinase2018In: Cell Reports, E-ISSN 2211-1247, Vol. 24, no 5, p. 1278-1289, article id S2211-1247(18)30991-4Article in journal (Refereed)
    Abstract [en]

    MIG6 is an important tumor suppressor that binds to and negatively regulates epidermal growth factor receptor (EGFR). Here, we report an EGFR-independent function for MIG6 as an integral component of the cell cycle machinery. We found that depletion of MIG6 causes accelerated entry into and delayed exit from mitosis. This is due to premature and prolonged activation of CDK1, a key regulator of mitotic progression at the G2/M and meta- and anaphase transitions. Furthermore, MIG6 is required for inhibition of CDK1 upon DNA damage and subsequent G2/M cell cycle arrest. Mechanistically, we found that MIG6 depletion results in reduced phosphorylation of CDK1 on the inhibitory WEE1-targeted tyrosine-15 residue. MIG6 interacts with WEE1 and promotes its stability by interfering with the recruitment of the βTrCP-SCF E3 ubiquitin ligase and consequent proteasomal degradation of WEE1. Our findings uncover a critical role of MIG6 in cell cycle progression that is likely to contribute to its potent tumor-suppressive properties.

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  • 340.
    Schizas, Nikos
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    König, Niclas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Andersson, Brittmarie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Vasylovska, Svitlana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Hoeber, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Kozlova, Elena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Regenerative neurobiology.
    Hailer, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Neural crest stem cells protect spinal cord neurons from excitotoxic damage and inhibit glial activation by secretion of brain-derived neurotrophic factor2018In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, Vol. 372, no 3, p. 493-505Article in journal (Refereed)
    Abstract [en]

    The acute phase of spinal cord injury is characterized by excitotoxic and inflammatory events that mediate extensive neuronal loss in the gray matter. Neural crest stem cells (NCSCs) can exert neuroprotective and anti-inflammatory effects that may be mediated by soluble factors. We therefore hypothesize that transplantation of NCSCs to acutely injured spinal cord slice cultures (SCSCs) can prevent neuronal loss after excitotoxic injury. NCSCs were applied onto SCSCs previously subjected to N-methyl-d-aspartate (NMDA)-induced injury. Immunohistochemistry and TUNEL staining were used to quantitatively study cell populations and apoptosis. Concentrations of neurotrophic factors were measured by ELISA. Migration and differentiation properties of NCSCs on SCSCs, laminin, or hyaluronic acid hydrogel were separately studied. NCSCs counteracted the loss of NeuN-positive neurons that was otherwise observed after NMDA-induced excitotoxicity, partly by inhibiting neuronal apoptosis. They also reduced activation of both microglial cells and astrocytes. The concentration of brain-derived neurotrophic factor (BDNF) was increased in supernatants from SCSCs cultured with NCSCs compared to SCSCs alone and BDNF alone mimicked the effects of NCSC application on SCSCs. NCSCs migrated superficially across the surface of SCSCs and showed no signs of neuronal or glial differentiation but preserved their expression of SOX2 and Krox20. In conclusion, NCSCs exert neuroprotective, anti-apoptotic and glia-inhibitory effects on excitotoxically injured spinal cord tissue, some of these effects mediated by secretion of BDNF. However, the investigated NCSCs seem not to undergo neuronal or glial differentiation in the short term since markers indicative of an undifferentiated state were expressed during the entire observation period.

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  • 341.
    Schwarz, Johanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Characterization of encystation in Giardia intestinalis2023Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
    Abstract [en]

    Giardia intestinalis is a protozoan parasite causing the diarrheal disease called giardiasis that infects millions of people worldwide each year. The life cycle of Giardia intestinalis is characterized by two stages; the sturdy, infectious cyst and the vegetative, motile trophozoite. This project sought to investigate the regulation of the encystation process where trophozoites transform into cysts. Twelve genes with interesting transcriptomic profiles were chosen to study as putative transcription factors and regulators of encystation. These genes were cloned onto a plasmid with a Strep-Tag and transfected into Giardia intestinalis. The protein expression and localization was studied using immunofluorescence microscopy with antibodies against the Strep-Tag at different time points after inducing encystation. Although the project did not fully characterize these genes, protein expression was seen in all cases except two. Some proteins were seen localized to the nuclei and others had a localization pattern similar to the localization of cyst wall protein. In addition, a dramatic phenotype resembling cells going through programmed cell death was observed in one of the transfectants early in encystation and would be interesting to study further. The transfectant strains generated from this project remain interesting candidates to investigate as putative transcription factors.

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    Characterization of encystation in Giardia intestinalis
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  • 342. Scolari, Silvia
    et al.
    Engel, Stephanie
    Krebs, Nils
    Plazzo, Anna Pia
    De Almeida, Rodrigo F M
    Prieto, Manuel
    Veit, Michael
    Herrmann, Andreas
    Lateral distribution of the transmembrane domain of influenza virus hemagglutinin revealed by time-resolved fluorescence imaging.2009In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 23, p. 15708-16Article in journal (Refereed)
    Abstract [en]

    Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy to study the interaction of the cytoplasmic and transmembrane domain (TMD) of HA with agly co sylphos pha tidyl ino si tol (GPI)-anchored peptide, an established marker for rafts in the exoplasmic leaflet of living mammalian plasma membranes. Cyan fluorescent protein (CFP) was fused to GPI, whereas the HA sequence was tagged with yellow fluorescent protein (YFP) on its exoplasmic site (TMD-HA-YFP), avoiding any interference of fluorescent proteins with the proposed role of the cytoplasmic domain in lateral organization of HA. Constructs were expressed in Chinese hamster ovary cells (CHO-K1) for which cholesterol-sensitive lipid nanodomains and their dimension in the plasma membrane have been described (Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004) Cell 116, 577-589). Upon transfection in CHO-K1 cells, TMD-HA-YFP is partially expressed as a dimer. Only dimers are targeted to the plasma membrane. Clustering of TMD-HA-YFP with GPI-CFP was observed and shown to be reduced upon cholesterol depletion, a treatment known to disrupt rafts. No indication for association of TMD-HA-YFP with GPI-CFP was found when palmitoylation, an important determinant of raft targeting, was suppressed. Clustering of TMD-HA-YFP and GPI-CFP was also observed in purified plasma membrane suspensions by homoFRET. We concluded that the pal mit oy lated TMD-HA alone is sufficient to recruit HA to cholesterol-sensitive nanodomains. The corresponding construct of the spike protein E2 of Semliki Forest virus did not partition preferentially in such domains.

  • 343.
    Seignez, Cedric
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Phillipson, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Implanted biomaterials: Neutrophil-mediated vascularization2017In: Nature Biomedical Engineering, E-ISSN 2157-846X, Vol. 1, no 6, article id UNSP 0086Article in journal (Other academic)
  • 344.
    Seltenhammer, Monika H.
    et al.
    Dept Forens Sci, A-1090 Vienna, Austria;Med Univ Vienna, Ctr Physiol & Pharmacol, Vascobiolab, A-1090 Vienna, Austria; Univ Vienna, Dept Pharmacol & Toxicol, A-1090 Vienna, Austria.
    Sundström, Elisabeth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Meisslitzer-Ruppitsch, Claudia
    Med Univ Vienna, Dept Cell Biol & Ultrastruct Res, Ctr Anat & Cell Biol, A-1090 Vienna, Austria.
    Cejka, Petra
    Med Univ Vienna, Inst Immunol, A-1090 Vienna, Austria.
    Kosiuk, Jedrzej
    Med Univ Vienna, Dept Cell Biol & Ultrastruct Res, Ctr Anat & Cell Biol, A-1090 Vienna, Austria.
    Neumüller, Josef
    Med Univ Vienna, Dept Cell Biol & Ultrastruct Res, Ctr Anat & Cell Biol, A-1090 Vienna, Austria.
    Almeder, Marlene
    Med Univ Vienna, Dept Cell Biol & Ultrastruct Res, Ctr Anat & Cell Biol, A-1090 Vienna, Austria.
    Majdic, Otto
    Med Univ Vienna, Inst Immunol, A-1090 Vienna, Austria.
    Steinberger, Peter
    Med Univ Vienna, Inst Immunol, A-1090 Vienna, Austria.
    Losert, Udo M.
    Med Univ Vienna, Core Unit Biomed Res, A-1090 Vienna, Austria.
    Stöckl, Johannes
    Med Univ Vienna, Inst Immunol, A-1090 Vienna, Austria.
    Andersson, Leif
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Swedish Univ Agr Sci, Dept Anim Breeding & Genet, S-75124 Uppsala, Sweden.
    Sölkner, Johann
    Univ Nat Resources & Appl Life Sci, Dept Sustainable Agr Syst, A-1180 Vienna, Austria.
    Vetterlein, Monika
    Med Univ Vienna, Dept Cell Biol & Ultrastruct Res, Ctr Anat & Cell Biol, A-1090 Vienna, Austria.
    Golovko, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Establishment and characterization of a primary and a metastatic melanoma cell line from Grey horses2014In: In vitro Cellular & Developmental Biology-Animal, ISSN 1071-2690, E-ISSN 1543-706X, Vol. 50, no 1, p. 56-65Article in journal (Refereed)
    Abstract [en]

    The Grey horse phenotype, caused by a 4.6 kb duplication in Syntaxin 17, is strongly associated with high incidence of melanoma. In contrast to most human melanomas with an early onset of metastasis, the Grey horse melanomas have an extended period of benign growth, after which 50% or more eventually undergo progression and may metastasize. In efforts to define changes occurring during Grey horse melanoma progression, we established an in vitro model comprised of two cell lines, HoMel-L1 and HoMel-A1, representing a primary and a metastatic stage of the melanoma, respectively. The cell lines were examined for their growth and morphological characteristics, in vitro and in vivo oncogenic potential, chromosome numbers, and expression of melanocytic antigens and tumor suppressors. Both cell lines exhibited malignant characteristics; however, the metastatic HoMel-A1 showed a more aggressive phenotype characterized by higher proliferation rates, invasiveness, and a stronger tumorigenic potential both in vitro and in vivo. HoMel-A1 displayed a near-haploid karyotype, whereas HoMel-L1 was near-diploid. The cell lines expressed melanocytic lineage markers such as TYR, TRP1, MITF, PMEL, ASIP, MC1R, POMC, and KIT. The tumor suppressor p53 was strongly expressed in both cell lines, while the tumor suppressors p16 and PTEN were absent in HoMel-A1, potentially implicating significance of these pathways in the melanoma progression. This in vitro model system will not only aid in understanding of the Grey horse melanoma pathogenesis, but also in unraveling the steps during melanoma progression in general as well as being an invaluable tool for development of new therapeutic strategies.

  • 345.
    Selvin, Tove
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Andersson, Claes
    Jarvius, Malin
    Berglund, Malin
    Larsson, Rolf
    Fryknäs, Mårten
    The Immuno-Oncology Hollow Fiber AssayManuscript (preprint) (Other academic)
    Abstract [en]

    In order to facilitate the translation of novel immunotherapies from bench to bedside, continued development of predictive preclinical models is essential. Herein, we developed the immuno-oncology hollow fiber assay (HFA) to bridge the gap between cell based in vitro assays and more complex mouse models for evaluation of immuno-oncological agents. The colorectal cancer (CRC) cell line HCT116-GFP and human peripheral blood mononuclear cells (PBMCs) were co-cultured inside semipermeable hollow fibers. As a proof of concept, aCD3 and IL-2 was used to induce immune cell-mediated cancer cell death. During in vitro characterization of the model system, an enhanced effect of aCD3 and IL-2 was observed in the HFA compared to conventional monolayers. Further investigation demonstrated that increased cell proximity alone is sufficient to augment immune cell activation and effector function. To assess the functionality of the assay in vivo, a pilot study was performed using nude mice. Hollow fibers were surgically implanted intraperitoneally (i.p.) and the mice received local injections of aCD3 at the time of implantation and/ or systemic IL-2 via i.p. injection once daily for 3 consecutive days. Compared to untreated mice and mice receiving IL-2 alone, the combination of aCD3 and IL-2 resulted in a significant decrease in cancer cell viability. Traditional in vivo models often necessitate lengthy observation periods to monitor tumor growth and treatment response. We have developed a simplified model system that enables initial in vivo evaluation of immunological agents on cancer and immune cells of human origin within a matter of days. 

  • 346.
    Senkowski, Wojciech
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    High-throughput screening using multicellular tumor spheroids to reveal and exploit tumor-specific vulnerabilities2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS.

    In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines.

    In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.

    List of papers
    1. Three-Dimensional Cell Culture-Based Screening Identifies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer
    Open this publication in new window or tab >>Three-Dimensional Cell Culture-Based Screening Identifies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer
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    2015 (English)In: Molecular Cancer Therapeutics, ISSN 1535-7163, E-ISSN 1538-8514, Vol. 14, no 6, p. 1504-1516Article in journal (Refereed) Published
    Abstract [en]

    Because dormant cancer cells in hypoxic and nutrient-deprived regions of solid tumors provide a major obstacle to treatment, compounds targeting those cells might have clinical benefits. Here, we describe a high-throughput drug screening approach, using glucose-deprived multicellular tumor spheroids (MCTS) with inner hypoxia, to identify compounds that specifically target this cell population. We used a concept of drug repositioning-using known molecules for new indications. This is a promising strategy to identify molecules for rapid clinical advancement. By screening 1,600 compounds with documented clinical history, we aimed to identify candidates with unforeseen potential for repositioning as anticancer drugs. Our screen identified five molecules with pronounced MCTS-selective activity: nitazoxanide, niclosamide, closantel, pyrvinium pamoate, and salinomycin. Herein, we show that all five compounds inhibit mitochondrial respiration. This suggests that cancer cells in low glucose concentrations depend on oxidative phosphorylation rather than solely glycolysis. Importantly, continuous exposure to the compounds was required to achieve effective treatment. Nitazoxanide, an FDA-approved antiprotozoal drug with excellent pharmacokinetic and safety profile, is the only molecule among the screening hits that reaches high plasma concentrations persisting for up to a few hours after single oral dose. Nitazoxanide activated the AMPK pathway and downregulated c-Myc, mTOR, and Wnt signaling at clinically achievable concentrations. Nitazoxanide combined with the cytotoxic drug irinotecan showed anticancer activity in vivo. We here report that the FDA-approved anthelmintic drug nitazoxanide could be a potential candidate for advancement into cancer clinical trials.

    National Category
    Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-260735 (URN)10.1158/1535-7163.MCT-14-0792 (DOI)000358054300025 ()25911689 (PubMedID)
    Funder
    Swedish Cancer SocietySwedish Research CouncilSwedish Childhood Cancer Foundation
    Available from: 2015-08-24 Created: 2015-08-24 Last updated: 2022-01-28Bibliographically approved
    2. Large-Scale Gene Expression Profiling Platform for Identification of Context-Dependent Drug Responses in Multicellular Tumor Spheroids
    Open this publication in new window or tab >>Large-Scale Gene Expression Profiling Platform for Identification of Context-Dependent Drug Responses in Multicellular Tumor Spheroids
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    2016 (English)In: CELL CHEMICAL BIOLOGY, ISSN 2451-9448, Vol. 23, no 11, p. 1428-1438Article in journal (Refereed) Published
    Abstract [en]

    Cancer cell lines grown as two-dimensional (2D) cultures have been an essential model for studying cancer biology and anticancer drug discovery. However, 2D cancer cell cultures have major limitations, as they do not closely mimic the heterogeneity and tissue context of in vivo tumors. Developing three-dimensional (3D) cell cultures, such as multicellular tumor spheroids, has the potential to address some of these limitations. Here, we combined a high-throughput gene expression profiling method with a tumor spheroid-based drug-screening assay to identify context-dependent treatment responses. As a proof of concept, we examined drug responses of quiescent cancer cells to oxidative phosphorylation (OXPHOS) inhibitors. Use of multicellular tumor spheroids led to discovery that the mevalonate pathway is upregulated in quiescent cells during OXPHOS inhibition, and that OXPHOS inhibitors and mevalonate pathway inhibitors were synergistically toxic to quiescent spheroids. This work illustrates how 3D cellular models yield functional and mechanistic insights not accessible via 2D cultures.

    National Category
    Cell and Molecular Biology Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-311191 (URN)10.1016/j.chembiol.2016.09.013 (DOI)000388373200015 ()27984028 (PubMedID)
    Funder
    Swedish Cancer SocietySwedish Foundation for Strategic Research
    Available from: 2016-12-22 Created: 2016-12-22 Last updated: 2019-04-29Bibliographically approved
    3. Drug combination screening in multicellular tumor spheroids identifies synthetic lethalities in quiescent cancer cells
    Open this publication in new window or tab >>Drug combination screening in multicellular tumor spheroids identifies synthetic lethalities in quiescent cancer cells
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Cancer and Oncology Medicinal Chemistry
    Research subject
    Molecular Medicine
    Identifiers
    urn:nbn:se:uu:diva-320596 (URN)
    Available from: 2017-04-22 Created: 2017-04-22 Last updated: 2020-05-13Bibliographically approved
    4. Targeting tumor cells based on PDE3A expression
    Open this publication in new window or tab >>Targeting tumor cells based on PDE3A expression
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Medicinal Chemistry Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-320597 (URN)
    Available from: 2017-04-22 Created: 2017-04-22 Last updated: 2018-01-13
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  • 347. Serra-Vinardell, Jenny
    et al.
    Diaz, Lucia
    Guitierrez-de Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Sanchez-Olle, Gessarni
    Bujons, Jordi
    Michelakakis, Helen
    Mavridou, Irene
    Aerts, Johannes M. F. G.
    Delgado, Antonio
    Grinberg, Daniel
    Vilageliu, Lluisa
    Casas, Josefina
    Selective chaperone effect of aminocyclitol derivatives on G202R and other mutant glucocerebrosidases causing Gaucher disease2014In: International Journal of Biochemistry and Cell Biology, ISSN 1357-2725, E-ISSN 1878-5875, Vol. 54, p. 245-254Article in journal (Refereed)
    Abstract [en]

    Gaucher disease is an autosomal recessive lysosomal disorder characterized by the accumulation of glucosylceramide as a result of a deficiency of the enzyme glucocerebrosidase. Several competitive glucocerebrosidase inhibitors are able to act as pharmacological chaperones for an efficient rescue of the mutated, misfolded forms of the enzyme. Along this line, we report in this work on the ability of several aminocyclitols to increase the residual glucocerebrosidase activity in patient fibroblasts with different genotypes. Some of the compounds were slightly active on fibroblasts bearing some mutations, including the highly prevalent N370S mutation. All compounds were highly active as enzyme activity enhancers on fibroblasts from Gaucher disease patients containing the G202R mutation. Moreover, using the novel tagged sphingolipid omega-azidosphingosine, a reduction in the tagged glucosylceramide accumulation was also observed for selected aminocyclitols. Attempts to explain the activity impairment observed in glucocerebrosidase bearing the G202R mutation by comparative molecular dynamic studies on wild type and the G202R mutated proteins (free and isofagomine-bound, in both cases) were unsuccessful. Under the simulation conditions used, no clear effect of the G202R mutation neither over the global structure of the protein nor on the loops that constitute the glucocerebrosidase active site was observed. Since the G202R residue is located on the protein surface, altered protein-membrane or protein-protein interactions could account for the observed differences. In conclusion, we have tested novel compounds that have shown some chaperone effect on particular glucocerebrosidase mutant enzymes, supporting the enhancement therapy as an alternative approach for Gaucher disease. (C) 2014 Elsevier Ltd. All rights reserved.

  • 348.
    Seton, Kristina
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    Eosinophil Apoptosis2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Apoptosis or programmed cell death is crucial for the resolution of inflammation, and phagocytosis of apoptotic cells initiates the release of actively anti-inflammatory responses from the phagocytes. Eosinophils are one of the most potent inflammatory cells in the body and is involved in a number of diseases, most commonly associated with parasitic infections and allergic diseases. Apoptosis in eosinophils is therefore one of the most important systems to avoid inflammation. This aim of the present investigation was to examine the mechanisms behind, and the consequences of this process in eosinophils. Apoptotic eosinophils have a unique surface receptor expression that indicates abilities to communicate with T-, B- and antigen presenting cells. They have a novel expression of CD49f, indicating an importance for binding to laminin or unknown functions of the VLA-6 receptor, possibly in the concept of phagocytosis of the apoptotic cell.

    In apoptotic eosinophils the granules are translocated to the periphery of the cell, probably through a disruption of the cytoskeleton. This translocation makes the granules easily accessible and the apoptotic eosinophil can release considerable amounts of granule proteins in response to specific stimuli. The spontaneous release however, is decreased as compared with living cells.

    Furthermore, the survival of eosinophils in response to an allergen challenge is increased in healthy subjects, but not in allergic patients. Mechanistically, this needs further investigation, but one theory is that it is due to the presence of specific IgE in patients in combination with differences in the response from the epithelial cells.

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  • 349.
    Shahidi Dadras, Mahsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Regulation of cell polarity and invasion by TGF-β and BMP signaling2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways are involved in many physiological processes during embryonic and adult life. TGF-β promotes epithelial to mesenchymal transition (EMT). We identified a gene target of TGF-β signaling, encoding the salt-inducible kinase 1 (SIK1). A potential substrate of this kinase, the polarity protein Par3, is an established regulator of tight junction assembly. SIK1 associates with Par3, can potentially phosphorylate Par3 and leads to its degradation, contributing to tight junction disassembly.

    Glioblastoma multiforme (GBM) is a common malignancy in the central nervous system, characterized by high heterogeneity, invasiveness, and resistance to therapy. One of the causes of heterogeneity and therapy-resistance is the existence of glioblastoma stem cells (GSCs). TGF-β signaling promotes self-renewal while BMP signaling induces differentiation of GSCs. Snail is a potent inducer of the EMT in carcinomas. However, in the context of GBM, Snail induces BMP signaling and represses TGF-β signaling through interaction with SMADs, the signaling mediators of TGF-β and BMP. In conclusion, Snail differentially regulates the activity of the opposing BMP and TGF-β pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.

    Although profound changes in cell polarity is a hallmark of invasive malignancies, little is known about the role of the polarity machinery in tumor suppression. Patient transcriptomic data suggested low Par3 expression, correlating with poor survival of the GBM patients. Par3 silencing decreased the GSC self-renewal capacity and enhanced their invasiveness. Transcriptomic analysis indicates that loss of Par3 leads to downregulation of genes encoding mitochondrial enzymes that generate ATP. These results support a novel role of Par3 in GBM, beyond its contribution to junctional contacts between cells.

    Another regulator of TGF-β and BMP signaling is the liver kinase B1 (LKB1). According to GBM patient mRNA analysis, high levels of LKB1 correlate with poor prognosis. Silencing of LKB1 in GSCs impairs invasion and self-renewal capacity due to downregulation of genes involved in these processes. Moreover, loss of LKB1 induces mitochondrial dysfunction, leading to decreased ATP levels. Collectively, this thesis has delivered a group of novel regulatory pathways that control critical aspects of cancer cell polarity, invasion and stemness.

    List of papers
    1. The protein kinase SIK downregulates the polarity protein Par3
    Open this publication in new window or tab >>The protein kinase SIK downregulates the polarity protein Par3
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    2018 (English)In: Oncotarget, E-ISSN 1949-2553, Vol. 9, p. 5716-5735Article in journal (Refereed) Published
    Abstract [en]

    The multifunctional cytokine transforming growth factor β (TGFβ) controls homeostasis and disease during embryonic and adult life. TGFβ alters epithelial cell differentiation by inducing epithelial-mesenchymal transition (EMT), which involves downregulation of several cell-cell junctional constituents. Little is understood about the mechanism of tight junction disassembly by TGFβ. We found that one of the newly identified gene targets of TGFβ, encoding the serine/threonine kinase salt-inducible kinase 1 (SIK), controls tight junction dynamics. We provide bioinformatic and biochemical evidence that SIK can potentially phosphorylate the polarity complex protein Par3, an established regulator of tight junction assembly. SIK associates with Par3, and induces degradation of Par3 that can be prevented by proteasomal and lysosomal inhibition or by mutation of Ser885, a putative phosphorylation site on Par3. Functionally, this mechanism impacts on tight junction downregulation. Furthermore, SIK contributes to the loss of epithelial polarity and examination of advanced and invasive human cancers of diverse origin displayed high levels of SIK expression and a corresponding low expression of Par3 protein. High SIK mRNA expression also correlates with lower chance for survival in various carcinomas. In specific human breast cancer samples, aneuploidy of tumor cells best correlated with cytoplasmic SIK distribution, and SIK expression correlated with TGFβ/Smad signaling activity and low or undetectable expression of Par3. Our model suggests that SIK can act directly on the polarity protein Par3 to regulate tight junction assembly.

    National Category
    Biochemistry and Molecular Biology Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-334429 (URN)10.18632/oncotarget.23788 (DOI)29464029 (PubMedID)
    Note

    Michael Vanlandewijck and Mahsa Shahidi Dadras contributed equally to this work.

    Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2024-01-17Bibliographically approved
    2. Snail regulates BMP and TGF-β pathways to control the differentiation status of glioma initiating cells
    Open this publication in new window or tab >>Snail regulates BMP and TGF-β pathways to control the differentiation status of glioma initiating cells
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    (English)Manuscript (preprint) (Other academic)
    Abstract
    National Category
    Biochemistry and Molecular Biology Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-334439 (URN)
    Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2018-12-03
    3. Par3 promotes glioblastoma stem cell self-renewal while inhibiting cell invasion
    Open this publication in new window or tab >>Par3 promotes glioblastoma stem cell self-renewal while inhibiting cell invasion
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-334454 (URN)
    Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2017-11-28
    4. The protein kinase LKB1 takes oncogenic actions in glioblastoma promoting self-renewal and invasiveness
    Open this publication in new window or tab >>The protein kinase LKB1 takes oncogenic actions in glioblastoma promoting self-renewal and invasiveness
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-334464 (URN)
    Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2017-12-04
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  • 350. Shahidi Dadras, Mahsa
    et al.
    Caja, Laia
    Heldin, Carl-Henrik
    Moustakas, Aristidis
    Par3 promotes glioblastoma stem cell self-renewal while inhibiting cell invasionManuscript (preprint) (Other academic)
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