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  • 251.
    Söderberg, Emilia
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hessle, Viktoria
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    von Euler, Anne
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Profilin is associated with transcriptionally active genes2012In: Nucleus, ISSN 1949-1034, E-ISSN 1949-1042, Vol. 3, no 3, p. 290-299Article in journal (Refereed)
    Abstract [en]

    We have raised antibodies against the profilin of Chironomus tentans to study the location of profilin relative to chromatin and to active genes in salivary gland polytene chromosomes. We show that a fraction of profilin is located in the nucleus, where profilin is highly concentrated in the nucleoplasm and at the nuclear periphery. Moreover, profilin is associated with multiple bands in the polytene chromosomes. By staining salivary glands with propidium iodide, we show that profilin does not co-localize with dense chromatin. profilin associates instead with protein-coding genes that are transcriptionally active, as revealed by co-localization with hnRNP and snRNP proteins. We have performed experiments of transcription inhibition with actinomycin D and we show that the association of profilin with the chromosomes requires ongoing transcription. however, the interaction of profilin with the gene loci does not depend on RNA. Our results are compatible with profilin regulating actin polymerization in the cell nucleus. however, the association of actin with the polytene chromosomes of C. tentans is sensitive to RNase, whereas the association of profilin is not, and we propose therefore that the chromosomal location of profilin is independent of actin.

  • 252. Takeuchi, Nobuto
    et al.
    Salazar, Laura
    Poole, Anthony M
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hogeweg, Paulien
    The evolution of strand preference in simulated RNA replicators with strand displacement: implications for the origin of transcription2008In: Biology Direct, Vol. 3, no 33Article in journal (Refereed)
  • 253. Tamas, Ivica
    et al.
    Wernegreen, Jennifer J
    Nystedt, Björn
    Kauppinen, Seth N
    Darby, Alistair C
    Gomaz-Valero, Laura
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poole, Anthony M
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Andersson, Siv G E
    Endosymbiont gene functions impaired and rescued by polymerase infidelity at poly(A) tracts2008In: PNAS, Vol. 105, no 39, p. 14934-14939Article in journal (Refereed)
  • 254.
    Theopold, U
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Li, D
    Fabbri, M
    Scherfer, C
    Schmidt, O
    The coagulation of insect hemolymph2002In: CMLS, Vol. 59, p. 363-372Article in journal (Refereed)
  • 255.
    Theopold, U
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Schmidt, O
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Söderhäll, K
    Dushay, M S
    Coagulation in arthropods: defence, wound closure and healing2004In: Trends in Immunology, Vol. 25, no 6, p. 289-294Article in journal (Refereed)
  • 256.
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    A bad boy comes good2009In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 461, p. 486-487Article, review/survey (Other (popular science, discussion, etc.))
  • 257.
    Theopold, Ulrich
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Dorian, Camilla
    Schmidt, Otto
    Changes in glycosylation during Drosophila development.: The influence of ecdysone on hemomucin isoforms2001In: Insect Biochemistry and Molecular Biology, Vol. 31, p. 189-197Article in journal (Refereed)
  • 258.
    Theopold, Ulrich
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Dushay, Mitchell S
    Mechanisms of Drosophila Immunity: An Innate Immune System at Work2008In: Current Immunology Rewiews, ISSN 1573-3955, Vol. 3, no 4, p. 276-288Article, review/survey (Refereed)
    Abstract [en]

    Insect immune systems which lack the type of adaptive immunity known in vertebrates rely on several mechanisms including solid barriers against the environment, rapid coagulation of hemolymph after wounding, the formation of aggregates that immobilize and kill foreign invaders, phagocytosis, and the production of antimicrobial peptides. The mode of action and the regulation of the expression of antimicrobial peptides have been studied intensively for more than three decades and are now increasingly well understood. In addition, the characterization of several key molecules involved in other branches of insect immunity has led to a deeper and much more comprehensive understanding of innate immunity in insects. Here we focus on the current status of our view of immunity in the vinegar fly Drosophila melanogaster, the best characterized insect model. We also discuss how evolutionary and ecological forces may have shaped immune responses in Drosophila as compared to other insect species. Finally, several infection models reveal finely-tuned and pathogen-dependent interactions between Drosophila immunity and fly physiology.

  • 259.
    Theopold, Ulrich
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Schneider, David
    Stanford, California.
    The Tinkerer at Work2009In: Journal of Innate Immunity, Vol. 1, p. 281-Article, book review (Other academic)
  • 260.
    Thureborn, Petter
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. Södertorn University College, Sweden.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Södertorn University College, Sweden.
    Plathan, Josefin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poole, Anthony M.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. University of Canterbury, New Zealand.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöling, Sara
    A Metagenomics Transect into the Deepest Point of the Baltic Sea Reveals Clear Stratification of Microbial Functional Capacities2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, p. e74983-Article in journal (Refereed)
    Abstract [en]

    The Baltic Sea is characterized by hyposaline surface waters, hypoxic and anoxic deep waters and sediments. These conditions, which in turn lead to a steep oxygen gradient, are particularly evident at Landsort Deep in the Baltic Proper. Given these substantial differences in environmental parameters at Landsort Deep, we performed a metagenomic census spanning surface to sediment to establish whether the microbial communities at this site are as stratified as the physical environment. We report strong stratification across a depth transect for both functional capacity and taxonomic affiliation, with functional capacity corresponding most closely to key environmental parameters of oxygen, salinity and temperature. We report similarities in functional capacity between the hypoxic community and hadal zone communities, underscoring the substantial degree of eutrophication in the Baltic Proper. Reconstruction of the nitrogen cycle at Landsort deep shows potential for syntrophy between archaeal ammonium oxidizers and bacterial denitrification at anoxic depths, while anaerobic ammonium oxidation genes are absent, despite substantial ammonium levels below the chemocline. Our census also reveals enrichment in genetic prerequisites for a copiotrophic lifestyle and resistance mechanisms reflecting adaptation to prevalent eutrophic conditions and the accumulation of environmental pollutants resulting from ongoing anthropogenic pressures in the Baltic Sea.

  • 261.
    Torrents, Eduard
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Grinberg, Inna
    Gorovitz-Harris, Baita
    Lundström, Hanna
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Borovok, Ilya
    Aharonowitz, Yair
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Cohen, Gerald
    NrdR Controls Differential Expression of the Escherichia coli Ribonucleotide Reductase Genes2007In: Journal of Bacteriology, ISSN 0021-9193, Vol. 189, no 14, p. 5012-5021Article in journal (Refereed)
  • 262.
    Torrents, Eduard
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poplawski, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Two proteins mediate class II ribonucleotide reductase activity in Pseudomonas aeruginosa: expression and transcriptional analysis of the aerobic enzymes.2005In: J Biol Chem, ISSN 0021-9258, Vol. 280, no 17, p. 16571-8Article in journal (Other academic)
    Abstract [en]

    The opportunistic human pathogen Pseudomonas aeruginosa is one of a few microorganisms that code for three different classes (I, II, and III) of the enzyme ribonucleotide reductase (RNR). Class II RNR of P. aeruginosa differs from all hitherto known class II enzymes by being encoded by two consecutive open reading frames denoted nrdJa and nrdJb and separated by 16 bp. Split nrdJ genes were also found in the few other gamma-proteobacteria that code for a class II RNR. Interestingly, the two genes encoding the split nrdJ in P. aeruginosa were co-transcribed, and both proteins were expressed. Exponentially growing aerobic cultures were predominantly expressing the class I RNR (encoded by the nrdAB operon) compared with the class II RNR (encoded by the nrdJab operon). Upon entry to stationary phase, the relative amount of nrdJa transcript increased about 6-7-fold concomitant with a 6-fold decrease in the relative amount of nrdA transcript. Hydroxyurea treatment known to knock out the activity of class I RNR caused strict growth inhibition of P. aeruginosa unless 5'-deoxyadenosylcobalamin, a cofactor specifically required for activity of class II RNRs, was added to the rich medium. Rescue of the hydroxyurea-treated cells in the presence of the vitamin B12 cofactor strongly implies that P. aeruginosa produces a functionally active NrdJ protein. Biochemical studies showed for the first time that presence of both NrdJa and NrdJb subunits were absolutely essential for enzyme activity. Based on combined genetic and biochemical results, we suggest that the two-component class II RNR in P. aeruginosa is primarily used for DNA repair and/or possibly DNA replication at low oxygen tension.

  • 263.
    Torrents, Eduard
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Biglino, Daniele
    Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Department of Biochemistry and Biophysics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Efficient growth inhibition of Bacillus anthracis by knocking out the ribonucleotide reductase tyrosyl radical.2005In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 102, no 50, p. 17946-51Article in journal (Other academic)
    Abstract [en]

    Bacillus anthracis, the causative agent of anthrax, is a worldwide problem because of the need for effective treatment of respiratory infections shortly after exposure. One potential key enzyme of B. anthracis to be targeted by antiproliferative drugs is ribonucleotide reductase. It provides deoxyribonucleotides for DNA synthesis needed for spore germination and growth of the pathogen. We have cloned, purified, and characterized the tyrosyl radical-carrying NrdF component of B. anthracis class Ib ribonucleotide reductase. Its EPR spectrum points to a hitherto unknown three-dimensional geometry of the radical side chain with a 60 degrees rotational angle of C(alpha)-(C(beta)-C(1))-plane of the aromatic ring. The unusual relaxation behavior of the radical signal and its apparent lack of line broadening at room temperature suggest a weak interaction with the nearby diiron site and the presence of a water molecule plausibly bridging the phenolic oxygen of the radical to a ligand of the diiron site. We show that B. anthracis cells are surprisingly resistant to the radical scavenger hydroxyurea in current use as an antiproliferative drug, even though its NrdF radical is efficiently scavenged in vitro. Importantly, the antioxidants hydroxylamine and N-methyl hydroxylamine scavenge the radical several orders of magnitude faster and prevent B. anthracis growth at several hundred-fold lower concentrations compared with hydroxyurea. Phylogenetically, the B. anthracis NrdF protein clusters together with NrdFs from the pathogens Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, and Staphylococcus epidermidis. We suggest the potential use of N-hydroxylamines in combination therapies against infections by B. anthracis and closely related pathogens.

  • 264. Torrents, Eduard
    et al.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The Ribonucleotide Reductase Family: Genetics and Genomics2008In: Ribonucleotide Reductase, Nova Science Publishers Inc , 2008Chapter in book (Other (popular science, discussion, etc.))
  • 265. Torrents, Eduard
    et al.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Antibacterial activity of radical scavengers against reductase from Bacillus anthracis2010In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 391, no 2/3, p. 229-234Article in journal (Refereed)
    Abstract [en]

    Bacillus anthracis is a severe mammalian pathogen. The deoxyribonucleotides necessary for DNA replication and repair are provided via the ribonucleotide reductase (RNR) enzyme. RNR is also important for spore germination and cell proliferation upon infection. We show that the expression of B. anthracis class Ib RNR responds to the environment that the pathogen encounters upon infection. We also show that several anti-proliferative agents (radical scavengers) specifically inhibit the B. anthracis RNR. Owing to the importance of RNR in the pathogenic infection process, our results highlight a promising potential to inhibit the growth of B. anthracis early during infection.

  • 266.
    Torrents, Eduard
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Westman, MariAnn
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Ribonucleotide reductase modularity: Atypical duplication of the ATP-cone domain in Pseudomonas aeruginosa.2006In: J Biol Chem, ISSN 0021-9258, Vol. 281, no 35, p. 25287-96Article in journal (Other academic)
    Abstract [en]

    The opportunistic pathogen Pseudomonas aeruginosa, which causes serious nosocomial infections, is a gamma-proteobacterium that can live in many different environments. Interestingly P. aeruginosa encodes three ribonucleotide reductases (RNRs) that all differ from other well known RNRs. The RNR enzymes are central for de novo synthesis of deoxyribonucleotides and essential to all living cells. The RNR of this study (class Ia) is a complex of the NrdA protein harboring the active site and the allosteric sites and the NrdB protein harboring a tyrosyl radical necessary to initiate catalysis. P. aeruginosa NrdA contains an atypical duplication of the N-terminal ATP-cone, an allosteric domain that can bind either ATP or dATP and regulates the overall enzyme activity. Here we characterized the wild type NrdA and two truncated NrdA variants with precise N-terminal deletions. The N-terminal ATP-cone (ATP-c1) is allosterically functional, whereas the internal ATP-cone lacks allosteric activity. The P. aeruginosa NrdB is also atypical with an unusually short lived tyrosyl radical, which is efficiently regenerated in presence of oxygen as the iron ions remain tightly bound to the protein. The P. aeruginosa wild type NrdA and NrdB proteins form an extraordinarily tight complex with a suggested alpha4beta4 composition. An alpha2beta2 composition is suggested for the complex of truncated NrdA (lacking ATP-c1) and wild type NrdB. Duplication or triplication of the ATP-cone is found in some other bacterial class Ia RNRs. We suggest that protein modularity built on the common catalytic core of all RNRs plays an important role in class diversification within the RNR family.

  • 267.
    Tryselius, Ylva
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    A molecular characterization of selected cecropins and cysteine proteinases in Drosophila1997Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Insects that have been infected with bacteria produce antibacterial peptides. Among the most potent antibacterial peptides in Drosophila are the cecropins. This work describes the cloning and expression studies of the CecC gene, which completes the picture of the cecropin locus in Drosophila melanogaster. Inducible CecC gene expression has been detected in the fat body and potential hemocytes, by RNase protection and in situ hybridizations. The CecC gene reaches its highest levels in early pupae, where gene expression can also be detected in the hindgut. A conserved region in the promoter regions of the Drosophila cecropin genes has been identified, consisting of three elements. A suitable plasmid for P element-mediated transformation in Drosophila has been constructed, pWimm. By cloning any gene into a pWimm restriction site, it can be put under the tightly regulated CecA2 gene control. CecA2 mediated pWimm expression has been shown to be induced by bacterial infection of transgenic flies. In addition to the previously described expression in the fat body, we detected inducible expression in the female reproductive tract, and constitutive expression in the male reproductive tract.

    This thesis also describes two new, blood cell expressed, cysteine proteinases in Drosophila. Calpains are specific cytosolic endopeptidases, with a putative regulatory function. This thesis presents the cloning of a new calpain, that is expressed in hemocytes, the central nervous system and the midgut. Immunostaining experiments show that the protein is only detected in a subset of the cells that express the mRNA. Differential splicing of CalpA gene transcripts results in two different calpains, of which one lacks a calcium-binding domain. We also present the cloning of a cathepsin-like cyteine proteinase gene, CP1, from Drosophila. We show that hemocytic Drosophila mbn-2 cells express CP1 in high amounts, and the protein is localized to small granules by immunofluorescence experiments. These data indicates that the CP1 might have a role in the degradation of phagocytosed material in Drosophila.

  • 268.
    Tyagi, Anu
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Ryme, Jessica
    Brodin, David
    Östlund Farrants, Ann Krristin
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    SWI/SNF Associates with Nascent Pre-mRNPs and Regulates Alternative Pre mRNA Processing2009In: PLOS Genetics, Vol. 5, no 5Article in journal (Refereed)
    Abstract [en]

    The SWI/SNF chromatin remodeling complexes regulate the transcription of many genes by remodeling nucleosomes at promoter regions. In Drosophila, SWI/SNF plays an important role in ecdysone-dependent transcription regulation. Studies in human cells suggest that Brahma (Brm), the ATPase subunit of SWI/SNF, regulates alternative pre-mRNA splicing by modulating transcription elongation rates. We describe, here, experiments that study the association of Brm with transcribed genes in Chironomus tentans and Drosophila melanogaster, the purpose of which was to further elucidate the mechanisms by which Brm regulates pre-mRNA processing. We show that Brm becomes incorporated into nascent Balbiani ring pre-mRNPs co-transcriptionally and that the human Brm and Brg1 proteins are associated with RNPs. We have analyzed the expression profiles of D. melanogaster S2 cells in which the levels of individual SWI/SNF subunits have been reduced by RNA interference, and we show that depletion of SWI/SNF core subunits changes the relative abundance of alternative transcripts from a subset of genes. This observation, and the fact that a fraction of Brm is not associated with chromatin but with nascent pre-mRNPs, suggest that SWI/SNF affects pre-mRNA processing by acting at the RNA level. Ontology enrichment tests indicate that the genes that are regulated post-transcriptionally by SWI/SNF are mostly enzymes and transcription factors that regulate postembryonic developmental processes. In summary, the data suggest that SWI/SNF becomes incorporated into nascent pre-mRNPs and acts post-transcriptionally to regulate not only the amount of mRNA synthesized from a given promoter but also the type of alternative transcript produced.

  • 269. Umek, Tea
    et al.
    Sollander, Karin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Bergquist, Helen
    Wengel, Jesper
    Lundin, Karin E.
    Smith, C. I. Edvard
    Zain, Rula
    Oligonucleotide Binding to Non-B-DNA in MYC2019In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 24, no 5, article id 1000Article in journal (Refereed)
    Abstract [en]

    MYC, originally named c-myc, is an oncogene deregulated in many different forms of cancer. Translocation of the MYC gene to an immunoglobulin gene leads to an overexpression and the development of Burkitt's lymphoma (BL). Sporadic BL constitutes one subgroup where one of the translocation sites is located at the 5'-vicinity of the two major MYC promoters P-1 and P-2. A non-B-DNA forming sequence within this region has been reported with the ability to form an intramolecular triplex (H-DNA) or a G-quadruplex. We have examined triplex formation at this site first by using a 17 bp triplex-forming oligonucleotide (TFO) and a double strand DNA (dsDNA) target corresponding to the MYC sequence. An antiparallel purine-motif triplex was detected using electrophoretic mobility shift assay. Furthermore, we probed for H-DNA formation using the BQQ-OP based triplex-specific cleavage assay, which indicated the formation of the structure in the supercoiled plasmid containing the corresponding region of the MYC promoter. Targeting non-B-DNA structures has therapeutic potential; therefore, we investigated their influence on strand-invasion of anti-gene oligonucleotides (ON)s. We show that in vitro, non-B-DNA formation at the vicinity of the ON target site facilitates dsDNA strand-invasion of the anti-gene ONs.

  • 270. Uvell, H
    et al.
    Engström, Y
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Functional Characterization of a Novel Promoter Element Required for an Innate Immune Response in Drosophila2003In: Molecular and Cellular Biology, p. 8272-8281Article in journal (Refereed)
  • 271.
    Uvell, Hanna
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Signaling and transcriptional regulation of antimicrobial peptide genes in Drosophila melanogaster2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Insects rely solely on innate immune reactions for protection against infect-ing microbes in their environment. In Drosophila, one major defense mechanism is the production of a battery of antimicrobial peptides (AMPs). The expression of AMPs is primarily regulated at the level of transcription and constitutes both constitutive expression in a tissue-specific manner and inducible systemic expression in response to infection. The aim of my thesis has been to investigate the regulation of AMP gene expression at different levels. I have studied a novel cis-regulatory element, Region 1 (R1) found in the proximal promoter of all Cecropin genes in Drosophila melanogaster, as well as in other species of Drosophila. We found that the R1 element was important for the expression of CecropinA1 (CecA1) both in vitro and in vivo. A signaling-dependent R1-binding activity (RBA) was identified in nuclear extracts from Drosophila cells and flies. The molecular nature of the RBA, has despite considerable effort, not yet been identified. I also have studied the role of the JNK pathway in transcriptional regulation of AMP genes. The role of the JNK pathway in the regulation of AMP genes has long been elusive, however, in this study we showed that the pathway is directly involved in the expression of AMP genes. Analysis of cells mutant for JNK pathway components showed severely reduced AMP gene expression. Fur-thermore, over-expression of a JNK pathway-inhibitor also inhibited AMP gene expression. Lastly, I have studied transcription factors that have not previously been implicated in transcriptional regulation of AMP genes. In a yeast screen, three members of the POU family of transcription factors were identified as regulators of CecA1. Two of them, Drifter (Dfr) and POU do-main protein 1 (Pdm1) were further characterized. We showed that Dfr was able to promote AMP gene expression in the absence of infection, suggest-ing it to play a role in constitutive expression of AMP genes. Indeed, down-regulation of Dfr expression using RNAi severely reduced the constitutive expression of AMP genes in the male ejaculatory duct. We also identified an enhancer element important for Dfr-mediated expression of CecA1. Pdm1, on the other hand, was shown to be important for the systemic expression of AMP genes. In Pdm1 mutant flies, several AMP genes are systemically expressed even in the absence of infection, suggesting that Pdm1 works as a repressor of those genes. However, at least on AMP gene, AttacinA (AttA) requires Pdm1 for its expression, suggesting that Pdm1 works as an activator for this gene. Upon infection, Pdm1 was rapidly degraded, but, regenerated shortly after infection. We propose that the degradation of Pdm1 is important for the activation of the Pdm1-repressed genes and that regeneration sup-ports the expression of AttA.

  • 272.
    Uvell, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Engström, Ylva
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    A multilayered defense against infection: combinatorial control of insect immune genes2007In: Trends in Genetics, ISSN 0168-9525, Vol. 23, no 7, p. 342-349Article in journal (Refereed)
  • 273.
    Uvell, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Engström, Ylva
    Functional characterization of a novel promoter element required for an innate immune response in Drosophila2003In: Molecular and Cellular Biology, ISSN 0270-7306, Vol. 23, no 22, p. 8272-8281Article in journal (Refereed)
  • 274.
    Uvell, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Junell, Anna
    Antonsson, Åsa
    Cantera, Rafael
    Engström, Ylva
    The Drosophila transcription factor Pdm1 inhibits antimicrobial peptide gene expressionManuscript (Other academic)
  • 275. Valanne, Susanna
    et al.
    Myllymäki, Henna
    Kallio, Jenni
    Schmid, Martin Rudolf
    Kleino, Anni
    Murumägi, Astrid
    Airaksinen, Laura
    Kotipelto, Tapio
    Kaustio, Meri
    Ulvila, Johanna
    Esfahani, Shiva Seyedoleslami
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Engström, Ylva
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Silvennoinen, Olli
    Hultmark, Dan
    Parikka, Mataleena
    Rämet, Mika
    Genome-Wide RNA Interference in Drosophila Cells Identifies G Protein-Coupled Receptor Kinase 2 as a Conserved Regulator of NF-kappa B Signaling2010In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 184, no 11, p. 6188-6198Article in journal (Refereed)
    Abstract [en]

    Because NF-kappa B signaling pathways are highly conserved in evolution, the fruit fly Drosophila melanogaster provides a good model to study these cascades. We carried out an RNA interference (RNAi)-based genome-wide in vitro reporter assay screen in Drosophila for components of NF-kappa B pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-kappa B regulators. Of these, nine dsRNA-treatments affect primarily the Toll pathway. G protein-coupled receptor kinase (Gprk) 2, CG15737/Toll pathway activation mediating protein, and u-shaped were required for normal Drosomycin response in vivo. Interaction studies revealed that Gprk2 interacts with the Drosophila I kappa B homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-kappa B regulation. Morpholino silencing of the zebrafish ortholog of Gprk2 in fish embryos caused impaired cytokine expression after Escherichia coli infection, indicating a conserved role in NF-kappa B signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-kappa B reporter activity. Gprk2 RNAi flies are susceptible to infection with Enterococcus faecalis and Gprk2 RNAi rescues Toll(10b)-induced blood cell activation in Drosophila larvae in vivo. We conclude that Gprk2/GRK5 has an evolutionarily conserved role in regulating NF-kappa B signaling. The Journal of Immunology, 2010, 184: 6188-6198.

  • 276. Veno, Morten T.
    et al.
    Bramsen, Jesper B.
    Bendixen, Christian
    Panitz, Frank
    Holm, Ida Elisabeth
    Ohman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Kjems, Jorgen
    Spatio temporal regulation of adar editing during development in porcine neural tissues2012In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 9, no 8, p. 1054-1065Article in journal (Refereed)
    Abstract [en]

    Editing by ADAR enzymes is essential for mammalian life. Still, knowledge of the spatio-temporal editing patterns in mammals is limited. By use of 454 amplicon sequencing we examined the editing status of 12 regionally extracted mRNAs from porcine developing brain encompassing a total of 64 ADAR editing sites. In total 24 brain tissues, dissected from up to five regions from embryonic gestation day 23, 42, 60, 80, 100 and 115, were examined for editing. Generally, editing increased during embryonic development concomitantly with an increase in ADAR2 mRNA level. Notably, the Gria2 (GluR-B) Q/R site, reported to be similar to 100% edited in previous studies, is only 54% edited at embryonic day 23. Transcripts with multiple editing sites in close proximity to each other exhibit coupled editing and an extraordinary incidence of long-range coupling of editing events more than 32 kb apart is observed for the kainate glutamate receptor 2 transcript, Grik2. Our study reveals complex spatio-temporal ADAR editing patterns of coordinated editing events that may play important roles in the development of the mammalian brain.

  • 277.
    Visa, N
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Actin in transcription2005In: EMBO, Vol. 6, no 3, p. 218-219Article in journal (Other academic)
  • 278.
    Visa, Neus
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Percipalle, Piergiorgio
    Nuclear Functions of Actin2010In: COLD SPRING HARBOR PERSPECT B, ISSN 1943-0264, Vol. 2, no 4, p. a000620-Article in journal (Refereed)
    Abstract [en]

    Actin participates in several essential processes in the cell nucleus. Even though the presence of actin in the nucleus was proposed more than 30 years ago, nuclear processes that require actin have been only recently identified. Actin is part of chromatin remodeling complexes; it is associated with the transcription machineries; it becomes incorporated into newly synthesized ribonucleoproteins; and it influences long-range chromatin organization. As in the cytoplasm, nuclear actin works in conjunction with different types of actin-binding proteins that regulate actin function and bridge interactions between actin and other nuclear components.

  • 279.
    Wahlstedt, Helene
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Regulation of site-selective A-to-I RNA editing: During mammalian brain development2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Adenosine (A) to inosine (I) RNA editing is a widespread post-transcriptional mechanism in mammals that contributes to increase the protein diversity. Adenosine deaminases that act on RNA (ADARs) are the enzymes catalyzing RNA editing. ADARs are particularly active within the brain where they act on transcripts involved in neurotransmission. In this work the editing efficiency of all known site-selectively edited substrates have been analyzed during development of the mouse brain. We show that there is a global regulation of RNA editing, where editing levels of sites increase as the brain matures. This increase in editing efficiency cannot be explained by an increase in ADAR protein expression. During differentiation of primary cells from the mouse brain, editing levels increases similar to what we observe in vivo. Interestingly, the subcellular localization of the ADAR enzymes of cultured neurons show a different distribution in immature compared mature neurons. An accumulation of the ADAR enzymes in the nucleus may explain elevated A-to-I editing during brain development. Furthermore, we find that certain adenosines work as principal sites where editing of the transcript is initiated. Presumably, these sites are kinetically favored and are hypothesized to recruit the ADAR enzymes to the RNA substrate. Editing is then coupled to sites located in multiples of 12 nucleotides from each other. Interestingly, these sites reside on the same side in the 3D helix structure. The Gabra-3 transcript is site-selectively edited at a single position changing an isoleucine codon for a methionine upon editing. Gabra-3 encodes the a3 subunit of the GABAA receptor. We show that receptors assembled with edited a3 are less stable at the cell surface than the non-edited a3. We propose that the amino acid change upon editing, could affect protein interactions important for trafficking and stability of the GABAA receptors. Further, the editing event in a3 may have the function to reduce the number of a3 subunits in favor of other a subunits.

  • 280.
    Wahlstedt, Helene
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The subcellular localization of ADAR determines A-to-I editing levels in developing neuronsManuscript (preprint) (Other academic)
  • 281.
    Wahlstedt, Helene
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Daniel, Chammiran
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Ensterö, Mats
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Large-scale mRNA sequencing determines global regulation of RNA editing during brain development2009In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 19, p. 978-986Article in journal (Refereed)
    Abstract [en]

    RNA editing by adenosine deamination has been shown to generate multiple isoforms of several neural receptors, often with profound effects on receptor function. However, little is known about the regulation of editing activity during development. We have developed a large-scale RNA sequencing protocol to determine adenosine-to-inosine (A-to-I) editing frequencies in the coding region of genes in the mammalian brain. Using the 454 Life Sciences (Roche) Amplicon Sequencing technology, we were able to determine even low levels of editing with high accuracy. The efficiency of editing for 28 different sites was analyzed during the development of the mouse brain from embryogenesis to adulthood. We show that, with few exceptions, the editing efficiency is low during embryogenesis, increasing gradually at different rates up to the adult mouse. The variation in editing gave receptors like HTR2C and GABAA (gamma-aminobutyric acid type A) a different set of protein isoforms during development from those in the adult animal. Furthermore, we show that this regulation of editing activity cannot be explained by an altered expression of the ADAR proteins but, rather, by the presence of a regulatory network that controls the editing activity during development.

  • 282.
    Wahlstedt, Helene
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Site-selective versus promiscuous A-to-I editing2011In: Wiley Interdiscip Reviews - RNA, ISSN 1757-7012, Vol. 2, no 6, p. 761-771Article, review/survey (Refereed)
    Abstract [en]

    RNA editing by adenosine deamination is acting on polymerase II derived transcripts in all metazoans. Adenosine-to-inosine (A-to-I) editing is mediated by the adenosine deaminase that acts on RNA (ADAR) enzymes. Two types of adenosine to inosine (A-to-I) RNA editing have been defined: site selective and hyper-editing. Typically, in site selectively edited substrates, one or a few A-to-I sites are edited in double-stranded RNA structures, frequently interrupted by single-stranded bulges and loops. Hyper-editing occurs in long stretches of duplex RNA where multiple adenosines are subjected to deamination. In this review, recent findings on editing within noncoding RNA as well as examples of site selective editing within coding regions are presented. We discuss how these two editing events have evolved and the structural differences between a site selective and hyper-edited substrate.

  • 283.
    Waldholm, Johan
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wang, Zhi
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Brodin, David
    Tyagi, Anu
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. University of Würzburg, Germany.
    Yu, Simei
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Theopold, Uli
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Östlund Farrants, Ann Kristin
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in Drosophila melanogaster2011In: BMC Molecular Biology, ISSN 1471-2199, E-ISSN 1471-2199, Vol. 12, article id 46Article in journal (Refereed)
    Abstract [en]

    Background: The SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila melanogaster is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA.

    Results: We have investigated whether the function of Brm in pre-mRNA processing in Drosophila melanogaster is mediated by Brm alone or by the SWI/SNF complex. We have analyzed the effects of depleting individual SWI/SNF subunits on pre-mRNA processing throughout the genome, and we have identified a subset of transcripts that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. We have also depleted Brm in larvae and we have shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo.

    Conclusions: We have shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. Our results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes.

  • 284. Wang, Q
    et al.
    Young, P
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Walters, K J
    Structure of S5a Bound to Monoubiqitin Provides a Model for Polyubiquitin Recognition2005In: Journal of Molecular Biology, Vol. 348, p. 727-739Article in journal (Refereed)
  • 285.
    Wang, Zhi
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Functional study of hemolymph coagulation in Drosophila larvae2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Many pathogen infections in nature are accompanied by injury and subsequent coagulation. Despite the contribution of hemolymph coagulation to wound sealing, little is known about its immune function. Based on the molecular knowledge of Drosophila innate immunity, this thesis investigated the immune function of clot both in vitro and in vivo, the immune relevant genes involved in a natural infection model, involving entomopathgenic nematodes (EPN) and the factors leading to crystal cell activation. Transglutaminase (TG) and its substrate Fondue (Fon) have been identified as bona fide clot components in Drosophila larvae. By knocking down TG or Fon via RNAi, we observed an increased susceptibility to EPN in larvae. In addition, this increased susceptibility was associated with an impaired ability of hemolymph clots to entrap bacteria. Immunostaining revealed that both clot components (Fon and TG) were able to target microbial surfaces. All these data suggest an immune function for the Drosophila hemolymph clot. Strikingly, similar results were obtained when we ran parallel experiments with human FXIIIa, an ortholog of Drosophila TG, indicating a functional conservation. We also found evidence for the regulation on both clot and immunity by eicosanoids in Drosophila larvae. The combination of EPN infection with the Drosophila model system allowed us to discover an immune function for TEP3 and Glutactin. However the molecular mechanism underlying the involvement of these two proteins in this particular host-pathogen interaction remains to be elucidated. Prophenoloxidase, the proform of enzyme involved in hardening the clot matrix, has been shown to be released by rupture of crystal cells. This cell rupture is dependent on activation of the JNK pathway, Rho GTPases and Eiger. Our work further identified the cytoskeletal component, Moesin, and the cytoskeletal regulator Rac2 as mediators of cell rupture. Despite the possible role of caspases in crystal cell activation, such cell rupture was turned out to be different from apoptosis. The implication of Rab5 in this process indicated that proper endocytosis is required for cell activation and subsequent melanization. Our findings furthered not only our understanding of the release of proPO via cell rupture but also our knowledge on different paths of immune cell activation.

  • 286.
    Wang, Zhi
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Bidla, Gawa
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Markus, Robert
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Crystal cell activation through ruptureManuscript (preprint) (Other academic)
    Abstract [en]

    Cell lysis is increasingly recognized as a mode of immune cell activation. We studied secretion by cell rupture using crystal cells, a specialized type of immune cell from the model insect

    Drosophila melanogaster. Crystal cells contain a multifunctional enzyme in a proform within crystalline inclusions, which are released during rupture and dissolve within minutes. Using targeted screens we identified cytoskeletal components and regulators as mediators of cell rupture. We also find evidence for an involvement of the endocytic pathway and for caspases. In addition we find no evidence for a release of nuclear DNA. Taken together our results set crystal cell activation apart from other activation processes that involve cell lysis such as the release of neutrophil traps.

  • 287.
    Wang, Zhi
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wilhelmsson, Christine
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hyrsl, Pavel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Loof, Torsten G.
    Dobes, Pavel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Klupp, Martina
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Loseva, Olga
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Moergelin, Matthias
    Ikle, Jennifer
    Cripps, Richard M.
    Herwald, Heiko
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Pathogen Entrapment by Transglutaminase - A Conserved Early Innate Immune Mechanism2010In: PLOS pathogens, ISSN 1553-7366, Vol. 6, no 2, p. e1000763-Article in journal (Refereed)
    Abstract [en]

    Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or-as previously shown-the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.

  • 288.
    Wetterberg, Ingela
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Zhao, Jian
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wieslander, Lars
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Masich, Sergej
    Skoglund, Ulf
    In situ transcription and splicing in the Balbiani ring 3 gene2001In: The EMBO Journal, Vol. 20, no 10, p. 2564-2574Article in journal (Refereed)
  • 289.
    Wieslander, L
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The cell nucleus2004In: Experimental Cell research, Vol. 296, p. 1-3Article in journal (Refereed)
  • 290.
    Wieslander, Lars
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Björk, Petra
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Nucleocytoplasmic mRNP export is an integral part of mRNP biogenesis2011In: Chromosoma, ISSN 0009-5915, E-ISSN 1432-0886, Vol. 120, no 1, p. 23-38Article in journal (Refereed)
    Abstract [en]

    Nucleocytoplasmic export and biogenesis of mRNPs are closely coupled. At the gene, concomitant with synthesis of the pre-mRNA, the transcription machinery, hnRNP proteins, processing, quality control and export machineries cooperate to release processed and export competent mRNPs. After diffusion through the interchromatin space, the mRNPs are translocated through the nuclear pore complex and released into the cytoplasm. At the nuclear pore complex, defined compositional and conformational changes are triggered, but specific cotranscriptionally added components are retained in the mRNP and subsequently influence the cytoplasmic fate of the mRNP. Processes taking place at the gene locus and at the nuclear pore complex are crucial for integrating export as an essential part of gene expression. Spatial, temporal and structural aspects of these events have been highlighted in analyses of the Balbiani ring genes

  • 291. Wiklund, Magda-Lena
    et al.
    Steinert, Stefanie
    Junell, Anna
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hultmark, Dan
    Stoven, Svenja
    The N-terminal half of the Drosophila Rel/NF-kappa B factor Relish, REL-68, constitutively activates transcription of specific Relish target genes2009In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 33, no 5, p. 690-696Article in journal (Refereed)
    Abstract [en]

    The Rel/NF-kappa B transcription factor Relish is a major regulator of the antimicrobial response in Drosophila. Upon immune challenge, Relish is cleaved to generate two fragments, the DNA-binding transcription factor REL-68 and the I kappa B-like REL-49. Using transgenic fly strains we show here that overexpression of REL-68 separately from REL-49 is sufficient to activate strong constitutive transcription of the Diptericin gene, but little constitutive or inducible transcription of Attacin and Cecropin, two other Relish target genes. Their transcription may therefore require additional modifications of Relish. However, phosphorylation of the conserved serine residue S431 is not involved in such modifications. This is unlike p65 and Dorsal, which are modulated by phosphorylation at their homologous site. In contrast to other I kappa B proteins, overexpression of REL-49 had no inhibitory effect on Relish-dependent transcription. Instead, we propose that the C-terminal I kappa B-like domain executes a scaffolding and recruiting function for full activation of Relish.

  • 292.
    Wilhelmsson, Christine
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Proteomics of the Drosophila hemolymph clot and the function of transglutaminase2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Insects rely on a fast and effective coagulation and wound response to avoid loss of body fluids and immobilize pathogens. Arthropod coagulation is in some respect equivalent to vertebrate coagulation but most factors and the regulation of coagulation systems seem not to be phylogenetically conserved. To get a more complete picture of insect clotting we studied the molecular and functional nature of Drosophila hemolymph coagulation.

    We developed new proteomic methods to collect Drosophila clotting factors. Several candidate factors were identified, including both predicted and novel clot proteins. Five putative TG (transglutaminase) substrates were found and we could also demonstrate that the clot is involved in immobilization of bacteria. Further investigating the role of TG we found TG to be important for Drosophila coagulation and that Fondue is a major substrate of the enzyme. Using fon RNAi knockdown we showed that Fondue affects the physical properties of the clot. A fon-GFP fusion construct was generated to follow its expression. The cuticle and the clot were labelled suggesting that Fondue is incorporated into both cuticle and clot. Clot properties and composition were affected by inhibiting TG chemically (MDC) and genetically (RNAi). Moreover, interaction between Fondue and Eig71Ee was demonstrated. Previous results indicated that coagulation could have an immune function. In hemolymph preparations, containing selected microorganisms, small deposits were seen on the microbial surfaces. The contents of these were investigated, revealing the presence of procoagulants. The targeting of microbes is instant and depends on TG and its substrates. Entomopathogenic nematode infections were performed to validate the functional importance of TG. TG RNAi knockdown larvae showed increased mortality, supporting an immune function for TG. Altogether, our data provide a more comprehensive picture of Drosophila immunity, and may further improve the understanding of innate immunity in general.

  • 293.
    Witman, Nevin
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Murtuza, Bari
    Davis, Ben
    Arner, Anders
    Morrison, Jamie Ian
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Recapitulation of developmental cardiogenesis governs the morphological and functional regeneration of adult newt hearts following injury2011In: Developmental Biology, ISSN 0012-1606, E-ISSN 1095-564X, Vol. 354, no 1, p. 67-76Article in journal (Refereed)
    Abstract [en]

    Urodele amphibians, like the newt, are the champions of regeneration as they are able to regenerate many body parts and tissues. Previous experiments, however, have suggested that the newt heart has only a limited regeneration capacity, similar to the human heart. Using a novel, reproducible ventricular resection model, we show for the first time that adult newt hearts can fully regenerate without any evidence of scarring. This process is governed by increased proliferation and the up-regulation of cardiac transcription factors normally expressed during developmental cardiogenesis. Furthermore, we are able to identify cells within the newly regenerated regions of the myocardium that express the LIM-homeodomain protein Istet1 and GATA4, transcription factors found in cardiac progenitors. Information acquired from using the newt as a model organism may help to shed light on the regeneration deficits demonstrated in damaged human hearts.

  • 294. Xin, Sun
    et al.
    Zhao, Jian
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Jin, Shao-Bo
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Palka, Kevin
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Aissouni, Youssef
    Daneholt, Bertil
    Alzhanova-Ericsson, Alla T.
    A novel protein localized to the fibrillar compartment of the nucleolus and to the brush border of a secretory cell2002In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 81, no 3, p. 125-137Article in journal (Refereed)
    Abstract [en]

    We report the identification and molecular characterization of a novel abundant nucleolar protein of the dipteran Chironomus tentans. As shown by Western blot analysis, this protein is present in nuclear extracts in a phosphorylated form with a mobility corresponding to 100 kDa. Therefore, the protein has been termed Chironomus tentans p100, or p100 for short. Analysis of the cDNA-derived primary structure of p100 indicates a protein that contains a combination of structural domains which could be involved in interactions with proteins and nucleic acids: twelve alternating acidic and basic repeats, a glycine-arginine-rich domain and a region with two zinc fingers of the C4-type. Acidic and basic repeats are typical for a group of nonribosomal nucleolar proteins. The best-studied representatives of this group are Nopp140 and nucleolin, proteins with structural and regulatory functions in rDNA transcription. Immunocytology and immunoelectron microscopy of Chironomus tentans salivary gland cells have shown that the p100 protein is located in the fibrillar compartment of the nucleolus, while it is almost absent from the granular compartment and from the nucleoplasm. The p100 protein remains in the nucleolus after removal of RNA and DNA by digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. The presence of p100 both in the nucleolus and at the apical plasma membrane suggests that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus.

  • 295. Zaid, A
    et al.
    Sun, J-S
    Nguyen, C-H
    Bisagni, E
    Garestier, T
    Grierson, D S
    Zain, R
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Triple-Helix Directed Cleavage of Double-Stranded DNA by Benzoquinoquionxaline-1, 10-phenanthroline Conjugates2004In: ChemBioChem, Vol. 5, p. 1550-1557Article in journal (Refereed)
  • 296.
    Zain, R
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Polverari, D
    Nguyen, C-H
    Blouquit, Y
    Bisagni, E
    Garestier, T
    Grierson, D S
    Sun, J-S
    Optimization of Triple-Helix-Directed DNA Cleavage by Benzoquinoquinoxaline - Ethylene - diaminetetraacetic Acid Conjugates2003In: ChemBioChem, Vol. 4, p. 856-862Article in journal (Refereed)
  • 297.
    Zain, R
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sun, J-S
    Do natural DNA tripple-helical structures occur and function in vivo?2003In: CMLS, Vol. 60, p. 862-870Article in journal (Other academic)
  • 298. Zhao, J
    et al.
    Jin, S
    Stockholm University, Faculty of Science, Wenner-Gren Institute for Experimental Biology.
    Björkroth, B
    Wieslander, L
    Department of Molecular Biology and Functional Genomics.
    Daneholt, B
    The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from gene to cytoplasm2002In: The EMBO Journal, Vol. 21, no 5, p. 1177-1187Article in journal (Refereed)
  • 299. Zisoulis, Dimitrios G.
    et al.
    Kai, Zoya S.
    Chang, Roger K.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Pasquinelli, Amy E.
    Autoregulation of microRNA biogenesis by let-7 and Argonaute2012In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 486, no 7404, p. 541-U140Article in journal (Refereed)
    Abstract [en]

    MicroRNAs (miRNAs) comprise a large family of small RNA molecules that post-transcriptionally regulate gene expression in many biological pathways(1). Most miRNAs are derived from long primary transcripts that undergo processing by Drosha to produce similar to 65-nucleotide precursors that are then cleaved by Dicer, resulting in the mature 22-nucleotide forms(2,3). Serving as guides in Argonaute protein complexes, mature miRNAs use imperfect base pairing to recognize sequences in messenger RNA transcripts, leading to translational repression and destabilization of the target messenger RNAs4,5. Here we show that the miRNA complex also targets and regulates non-coding RNAs that serve as substrates for the miRNA-processing pathway. We found that the Argonaute protein in Caenorhabditis elegans, ALG-1, binds to a specific site at the 3' end of let-7 miRNA primary transcripts and promotes downstream processing events. This interaction is mediated by mature let-7 miRNA through a conserved complementary site in its own primary transcript, thus creating a positive-feedback loop. We further show that ALG-1 associates with let-7 primary transcripts in nuclear fractions. Argonaute also binds let-7 primary transcripts in human cells, demonstrating that the miRNA pathway targets non-coding RNAs in addition to protein-coding messenger RNAs across species. Moreover, our studies in C. elegans reveal a novel role for Argonaute in promoting biogenesis of a targeted transcript, expanding the functions of the miRNA pathway in gene regulation. This discovery of autoregulation of let-7 biogenesis establishes a new mechanism for controlling miRNA expression.

  • 300. Öhman, M
    et al.
    Källman, A.M.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Bass, B.L.
    In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site2000In: RNA, ISSN 1355-8382, Vol. 6, p. 687-697Article in journal (Refereed)
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