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  • 1.
    Alberti, Esteban
    et al.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Los, Marek Jan
    Interfaculty Institute for Biochemistry, University of Tübingen, Germany; BioApplications Enterprises, Winnipeg, MB, Canada.
    Garcia, Rocio
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Fraga, JL
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Serrano, T.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Hernandez, E.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Klonisch, Thomas
    Department of Human Anatomy and Cell Sciences, and Manitoba Institute of Child Health, Winnipeg, Canada.
    Macías, R.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Martinez, L.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    Castillo, L.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
    de la Cuétara, K.
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.
    Prolonged Survival and expression of neural markers by bone marrow-derived stem cells transplanted into brain lesions2009In: Medical Science Monitor, ISSN 1234-1010, E-ISSN 1643-3750, Vol. 15, no 2, p. BR47-BR54Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Bone marrow-derived stem cell transplantation is a potentially viable therapeutic option for the treatment of neurodegenerative disease. MATERIAL/METHODS: We have isolated bone marrow stem cells by standard method. We then evaluated the survival of rats' bone marrow mononuclear cells implanted in rats' brain. The cells were extracted from rats' femurs, and marked for monitoring purposes by adenoviral transduction with Green Fluorescent Protein (GFP). Labeled cells were implanted within the area of rats' striatum lesions that were induced a month earlier employing quinolinic acid-based method. The implants were phenotyped by monitoring CD34; CD38; CD45 and CD90 expression. Bone marrow stromal cells were extracted from rats' femurs and cultivated until monolayer bone marrow stromal cells were obtained. The ability of bone marrow stromal cells to express NGF and GDNF was evaluated by RT-PCR. RESULTS: Implanted cells survived for at least one month after transplantation and dispersed from the area of injection towards corpus callosum and brain cortex. Interestingly, passaged rat bone marrow stromal cells expressed NGF and GDNF mRNA. CONCLUSIONS: The bone marrow cells could be successfully transplanted to the brain either for the purpose of trans-differentiation, or for the expression of desired growth factors.

  • 2.
    Alexander, Helen K.
    et al.
    Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba.
    Booy, Evan P.
    Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada .
    Xiao, Wenyan
    Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba.
    Ezzati, Peyman
    Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba.
    Baust, Heinrich
    Department of Radiooncology, University of Erlangen, Erlangen, Germany .
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Selected technologies to control genes and their products for experimental and clinical purposes2007In: Archivum Immunologiae et Therapiae Experimentalis, ISSN 0004-069X, E-ISSN 1661-4917, Vol. 55, no 3, p. 139-149Article in journal (Refereed)
    Abstract [en]

    "On-demand" regulation of gene expression is a powerful tool to elucidate the functions of proteins and biologically-active RNAs. We describe here three different approaches to the regulation of expression or activity of genes or proteins. Promoter-based regulation of gene expression was among the most rapidly developing techniques in the 1980s and 1990s. Here we provide basic information and also some characteristics of the metallothionein-promoter-based system, the tet-off system, Muristerone-A-regulated expression through the ecdysone response element, RheoSwitch (R), coumermycin/novobiocin-regulated gene expression, chemical dimerizer-based promoter activation systems, the "Dual Drug Control" system, "constitutive androstane receptor"-based regulation of gene expression, and RU486/mifepristone-driven regulation of promoter activity. A large part of the review concentrates on the principles and usage of various RNA interference techniques (RNAi: siRNA, shRNA, and miRNA-based methods). Finally, the last part of the review deals with historically the oldest, but still widely used, methods of temperature-dependent regulation of enzymatic activity or protein stability (temperature-sensitive mutants). Due to space limitations we do not describe in detail but just mention the tet-regulated systems and also fusion-protein-based regulation of protein activity, such as estrogen-receptor fusion proteins. The information provided below is aimed to assist researchers in choosing the most appropriate method for the planned development of experimental systems with regulated expression or activity of studied proteins.

  • 3.
    Aman, Malin
    et al.
    Swedish Sch Sport and Hlth Sci, Sweden.
    Larsen, Karin
    Swedish Sch Sport and Hlth Sci, Sweden; Umea Univ, Sweden.
    Forssblad, Magnus
    Swedish Sch Sport and Hlth Sci, Sweden; Karolinska Inst, Sweden.
    Näsmark, Annica
    Swedish Sch Sport and Hlth Sci, Sweden; Capio Artro Clin, Sweden.
    Waldén, Markus
    Linköping University, Department of Medical and Health Sciences, Division of Community Medicine. Linköping University, Faculty of Medicine and Health Sciences. Swedish Sch Sport and Hlth Sci, Sweden.
    Hägglund, Martin
    Linköping University, Department of Medical and Health Sciences, Division of Physiotherapy. Linköping University, Faculty of Medicine and Health Sciences. Swedish Sch Sport and Hlth Sci, Sweden.
    A Nationwide Follow-up Survey on the Effectiveness of an Implemented Neuromuscular Training Program to Reduce Acute Knee Injuries in Soccer Players2018In: The Orthopaedic Journal of Sports Medicine, ISSN 2325-9671, Vol. 6, no 12, article id 2325967118813841Article in journal (Refereed)
    Abstract [en]

    Background: A cruciate ligament (CL) injury is a severe injury in soccer. Neuromuscular training programs have a well-documented preventive effect, but there are few studies on the effectiveness of such a program at a national level. The Swedish Knee Control Program (KCP) was found to be effective in preventing CL injuries in youth female soccer players. The KCP was implemented nationwide in Sweden in 2010. Purpose: To evaluate the effectiveness of the Swedish KCP in reducing acute knee injuries in soccer players at a nationwide level. Study Design: Descriptive epidemiology study. Methods: All licensed soccer players in Sweden are covered by the same insurance company. Using this insurance database, around 17,500 acute knee injuries that were reported to the insurance company between 2006 and 2015 were included in the study. By matching the number of licensed soccer players with the number of reported injuries each year, the annual incidence of knee and CL injuries was able to be calculated. To evaluate the spread of the KCP nationally, a questionnaire was sent to all 24 Swedish district football associations (FAs) with questions regarding KCP education. The number of downloads of the KCP mobile application (app) was obtained. Results: The incidence of CL injuries decreased during the study period for both male (from 2.9 to 2.4 per 1000 player-years) and female players (from 4.9 to 3.9 per 1000 player-years). The overall incidence of knee injuries decreased in both male (from 5.6 to 4.6 per 1000 player-years) and female players (from 8.7 to 6.4 per 1000 player-years). Comparing before and after the nationwide implementation of the KCP, there was a decrease in the incidence of CL injuries by 6% (rate ratio [RR], 0.94 [95% CI, 0.89-0.98]) in male players and 13% (RR, 0.87 [95% CI, 0.81-0.92]) in female players and a decrease in the incidence of knee injuries by 8% (RR, 0.92 [95% CI, 0.89-0.96]) and 21% (RR, 0.79 [95% CI, 0.75-0.83]), respectively (P amp;lt; .01 for all). This trend corresponded to a reduction of approximately 100 CL injuries each year in Sweden. A total of 21 of 24 district FAs held organized KCP educational courses during the study period. The percentage of district FAs holding KCP courses was between 46% and 79% each year. There were 101,236 downloads of the KCP app. Conclusion: The KCP can be considered partially implemented nationwide, and the incidence of knee and CL injuries has decreased in both sexes at a nationwide level.

  • 4.
    Anderson, Judy E.
    et al.
    Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada; Manitoba Institute of Child's Health (MICH), University of Manitoba, Winnipeg, Canada.
    Hansen, Lise Lotte
    Institute of Human Genetics, University of Aarhus, Denmark.
    Mooren, Frank C.
    Department of Sports Medicine, Institute of Sport Sciences, University Giessen, Germany.
    Post, Markus
    Department of Sports Medicine, Institute of Sport Sciences, University Giessen, Germany.
    Hug, Hubert
    DSM Nutritional Products Ltd, Research & Development, Kaiseraugst, Switzerland.
    Zuse, Anne
    Manitoba Institute of Cell Biology (MICB), CancerCare Manitoba, 675 McDermot Ave. Rm. ON6010, Winnipeg, Man. R3E 0V9, Canada.
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Methods and biomarkers for the diagnosis and prognosis of cancer and other diseases: Towards personalized medicine2006In: Drug resistance updates, ISSN 1368-7646, E-ISSN 1532-2084, Vol. 9, no 4-5, p. 198-210Article in journal (Refereed)
    Abstract [en]

    The rapid development of new diagnostic procedures, the mapping of the human genome, progress in mapping genetic polymorphisms, and recent advances in nucleic acid- and protein chip technologies are driving the development of personalized therapies. This breakthrough in medicine is expected to be achieved largely due to the implementation of "lab-on-the-chip" technology capable of performing hundreds, even thousands of biochemical, cellular and genetic tests on a single sample of blood or other body fluid. Focusing on a few disease-specific examples, this review discusses selected technologies and their combinations likely to be incorporated in the "lab-on-the-chip" and to provide rapid and versatile information about specific diseases entities. Focusing on breast cancer and after an overview of single-nucleofide polymorphism (SNP)-screening methodologies, we discuss the diagnostic and prognostic importance of SNPs. Next, using Duchenne muscular dystrophy (DMD) as an example, we provide a brief overview of powerful and innovative integration of traditional immuno-histochemistry techniques with advanced biophysical methods such as NMR-spectroscopy or Fourier-transformed infrared (FT-IR) spectroscopy. A brief overview of the challenges and opportunities provided by protein and aptamer microarrays follows. We conclude by highlighting novel and promising biochemical markers for the development of personalized treatment of cancer and other diseases: serum cytochrome c, cytokeratin-18 and -19 and their proteolytic fragments for the detection and quantitation of malignant tumor mass, tumor cell turn-over, inflammatory processes during hepatitis and Epstein-Barr virus (EBV)-induced hemophagocytic lymphohistiocytosis and apoptotic/necrotic cancer cell death. (c) 2006 Elsevier Ltd. All rights reserved.

  • 5.
    Atikuzzaman, Mohammad
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Sanz, Libia
    Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
    Pla, Davinia
    Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Rubér, Marie
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Wright, Dominic
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Calvete, Juan J.
    Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Selection for higher fertility reflects in the seminal fluid proteome of modern domestic chicken2017In: Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics, ISSN 1744-117X, E-ISSN 1878-0407, Vol. 21, p. 27-40Article in journal (Refereed)
    Abstract [en]

    The high egg-laying capacity of the modern domestic chicken (i.e. White Leghorn, WL) has arisen from the low egg-laying ancestor Red Junglefowl (RJF) via continuous trait selection and breeding. To investigate whether this long-term selection impacted the seminal fluid (SF)-proteome, 2DE electrophoresis-based proteomic analyses and immunoassays were conducted to map SF-proteins/cytokines in RJF, WL and a 9th generation Advanced Intercross Line (AIL) of RJF/WL-L13, including individual SF (n = 4, from each RJF, WL and AIL groups) and pools of the SF from 15 males of each group, analyzed by 2DE to determine their degree of intra-group (AIL, WL, and RJF) variability using Principal Component Analysis (PCA); respectively an inter-breed comparative analysis of intergroup fold change of specific SF protein spots intensity between breeds. The PCA clearly highlighted a clear intra-group similarity among individual roosters as well as a clear inter-group variability (e.g. between RJF, WL and AIL) validating the use of pools to minimize confounding individual variation. Protein expression varied considerably for processes related to sperm motility, nutrition, transport and survival in the female, including signaling towards immunomodulation. The major conserved SF-proteins were serum albumin and ovotransferrin. Aspartate aminotransferase, annexin A5, arginosuccinate synthase, glutathione S-transferase 2 and l-lactate dehydrogenase-A were RJF-specific. Glyceraldehyde-3-phosphate dehydrogenase appeared specific to the WL-SF while angiotensin-converting enzyme, γ-enolase, coagulation factor IX, fibrinogen α-chain, hemoglobin subunit α-D, lysozyme C, phosphoglycerate kinase, Src-substrate protein p85, tubulins and thioredoxin were AIL-specific. The RJF-SF contained fewer immune system process proteins and lower amounts of the anti-inflammatory/immunomodulatory TGF-β2 compared to WL and AIL, which had low levels- or lacked pro-inflammatory CXCL10 compared to RJF. The seminal fluid proteome differs between ancestor and modern chicken, with a clear enrichment of proteins and peptides related to immune-modulation for sperm survival in the female and fertility.

  • 6.
    Baeza, Gabriela
    Linköping University, Department of Physics, Chemistry and Biology.
    X-ray Crystallographic Structure of theMurine Norovirus protease at 1.66 Å Resolutionand Functional Studies of the β-ribbon2011Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    In humans, noroviruses (NVs) cause acute epidemic and viral gastroenteritis. NVs do not only infect humans; viruseshave also been found in pigs, cows, sheep, mice and dogs. The focus in this project has been on the murine norovirus(MNV). MNV is a member of the viral family Caliciviridae and it consists of a single-stranded, positive sense RNAgenome. The genome includes three open reading frames (ORFs), ORF1 encodes for a polyprotein that consists of theprecursor to the 6-7 non-structural (NS) proteins. The polyprotein is cleaved by the NS6 protease. The NS6 isresponsible for all the cleaving in ORF1 and that makes it an attractive target for antiviral drugs. The NS6 proteinstructure has been determined at 1.66 Å resolution using X-ray diffraction techniques. Surprisingly, the electrondensity map revealed density for a peptide bound in the active site. The peptide had a length of 7 residues andoriginated from the C-terminus of another chain in an adjacent asymmetric unit. The active site triad was composed ofthe conserved residues; histidine 30, aspargine 54 and cysteine 139, however in the structure the cysteine 139 ismutated to an alanine to inactivate the protease. Activity assays were performed to probe the importance of the residuein position 109 in the β-ribbon located close to the active site. The three full-length constructs with the mutations;I109A, I109S and I109T were found to have less activity than the full-length wt (1-183). A truncated protease, lacking9 residues in the C-terminus, also had less activity. This indicates that the terminal residues are also important foractivity.

  • 7.
    Bahrampour, Shahrzad
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Genetic mechanisms regulating proliferation and cell specification in the Drosophila embryonic CNS2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The central nervous system (CNS) consists of an enormous number of cells, and large cellular variance, integrated into an elaborate network. The CNS is the most complex animal organ, and therefore its establishment must be controlled by many different genetic programs. Considering the high level of complexity in the human CNS, addressing issues related to human neurodevelopment represents a major challenge. Since comparative studies have revealed that neurodevelopmental programs are well conserved through evolution, on both the genetic and functional levels, studies on invertebrate neurodevelopmental programs are often translatable to vertebrates. Indeed, the basis of our current knowledge about vertebrate CNS development has been greatly aided by studies on invertebrates, and in particular on the Drosophila melanogaster (fruit fly) model system.

    This thesis attempted to identify novel genes regulating neural cell specification and proliferation in the CNS, using the Drosophila model system. Moreover, I aimed to address how those genes govern neural progenitor cells (neuroblasts; NBs) to obtain/maintain their stemness identity and proliferation capacity, and how they drive NBs through temporal windows and series of programmed asymmetric division, which gradually reduces their stemness identity in favor of neural differentiation, resulting in appropriate lineage progression. In the first project, we conducted a forward genetic screen in Drosophila embryos, aimed at isolating genes involved in regulation of neural proliferation and specification, at the single cell resolution. By taking advantage of the restricted expression of the neuropeptide FMRFa in the last-born cell of the NB lineage 5-6T, the Ap4 neuron, we could monitor the entire lineage progression. This screen succeeded in identifying 43 novel genes controlling different aspects of CNS development. One of the genes isolated, Ctr9, displayed extra Ap4/FMRFa neurons. Ctr9 encodes a component of the RNA polymerase II complex Paf1, which is involved in a number of transcriptional processes. The Paf1C, including Ctr9, is highly conserved from yeast to human, and in the past couple of years, its importance for transcription has become increasingly appreciated. However, studies in the Drosophila system have been limited. In the screen, we isolated the first mutant of Drosophila Ctr9 and conducted the first detailed phenotypic study on its function in the Drosophila embryonic CNS. Loss of function of Ctr9 leads to extra NB numbers, higher proliferation ratio and lower expression of neuropeptides. Gene expression analysis identified several other genes regulated by Ctr9, which may explain the Ctr9 mutant phenotypes. In summary, we identified Ctr9 as an essential gene for proper CNS development in Drosophila, and this provides a platform for future study on the Drosophila Paf1C. Another interesting gene isolated in the screen was worniou (wor), a member of the Snail family of transcription factors. In contrast to Ctr9, whichdisplayed additional Ap4/FMRFa neurons, wor mutants displayed a loss of these neurons. Previous studies in our group have identified many genes acting to stop NB lineage progression, but how NBs are pushed to proliferate and generate their lineages was not well known. Since wor may constitute a “driver” of proliferation, we decided to study it further. Also, we identified five other transcription factors acting together with Wor as pro-proliferative in both NBs and their daughter cells. These “drivers” are gradually replaced by the previously identified late-acting “stoppers.” Early and late factors regulate each other and the cell cycle, and thereby orchestrate proper neural lineage progression.

    List of papers
    1. Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
    Open this publication in new window or tab >>Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
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    2015 (English)In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 200, no 4, p. 1229-1244Article in journal (Refereed) Published
    Abstract [en]

    The expression of neuropeptides is often extremely restricted in the nervous system, making them powerful markers for addressing cell specification . In the developing Drosophila ventral nerve cord, only six cells, the Ap4 neurons, of some 10,000 neurons, express the neuropeptide FMRFamide (FMRFa). Each Ap4/FMRFa neuron is the last-born cell generated by an identifiable and well-studied progenitor cell, neuroblast 5-6 (NB5-6T). The restricted expression of FMRFa and the wealth of information regarding its gene regulation and Ap4 neuron specification makes FMRFa a valuable readout for addressing many aspects of neural development, i.e., spatial and temporal patterning cues, cell cycle control, cell specification, axon transport, and retrograde signaling. To this end, we have conducted a forward genetic screen utilizing an Ap4-specific FMRFa-eGFP transgenic reporter as our readout. A total of 9781 EMS-mutated chromosomes were screened for perturbations in FMRFa-eGFP expression, and 611 mutants were identified. Seventy-nine of the strongest mutants were mapped down to the affected gene by deficiency mapping or whole-genome sequencing. We isolated novel alleles for previously known FMRFa regulators, confirming the validity of the screen. In addition, we identified novel essential genes, including several with previously undefined functions in neural development. Our identification of genes affecting most major steps required for successful terminal differentiation of Ap4 neurons provides a comprehensive view of the genetic flow controlling the generation of highly unique neuronal cell types in the developing nervous system.

    Place, publisher, year, edition, pages
    Genetics Society of America, 2015
    Keywords
    Drosophila; CNS development; neural cell fate specification; forward genetic screening; FMRFamide
    National Category
    Clinical Medicine
    Identifiers
    urn:nbn:se:liu:diva-121318 (URN)10.1534/genetics.115.178483 (DOI)000359917000020 ()26092715 (PubMedID)
    Available from: 2015-09-16 Created: 2015-09-14 Last updated: 2019-03-13Bibliographically approved
    2. Ctr9, a Key Component of the Paf1 Complex, Affects Proliferation and Terminal Differentiation in the Developing Drosophila Nervous System
    Open this publication in new window or tab >>Ctr9, a Key Component of the Paf1 Complex, Affects Proliferation and Terminal Differentiation in the Developing Drosophila Nervous System
    2016 (English)In: G3: Genes, Genomes, Genetics, ISSN 2160-1836, E-ISSN 2160-1836, Vol. 6, no 10, p. 3229-3239Article in journal (Refereed) Published
    Abstract [en]

    The Paf1 protein complex (Paf1C) is increasingly recognized as a highly conserved and broadly utilized regulator of a variety of transcriptional processes. These include the promotion of H3K4 and H3K36 trimethylation, H2BK123 ubiquitination, RNA Pol II transcriptional termination, and also RNA-mediated gene silencing. Paf1C contains five canonical protein components, including Paf1 and Ctr9, which are critical for overall complex integrity, as well as Rtf1, Leo1, and Cdc73/Parafibromin(Hrpt2)/Hyrax. In spite of a growing appreciation for the importance of Paf1C from yeast and mammalian studies, there has only been limited work in Drosophila. Here, we provide the first detailed phenotypic study of Ctr9 function in Drosophila. We found that Ctr9 mutants die at late embryogenesis or early larval life, but can be partly rescued by nervous system reexpression of Ctr9. We observed a number of phenotypes in Ctr9 mutants, including increased neuroblast numbers, increased nervous system proliferation, as well as downregulation of many neuropeptide genes. Analysis of cell cycle and regulatory gene expression revealed upregulation of the E2f1 cell cycle factor, as well as changes in Antennapedia and Grainy head expression. We also found reduction of H3K4me3 modification in the embryonic nervous system. Genome-wide transcriptome analysis points to additional downstream genes that may underlie these Ctr9 phenotypes, revealing gene expression changes in Notch pathway target genes, cell cycle genes, and neuropeptide genes. In addition, we find significant effects on the gene expression of metabolic genes. These findings reveal that Ctr9 is an essential gene that is necessary at multiple stages of nervous system development, and provides a starting point for future studies of the Paf1C in Drosophila.

    Place, publisher, year, edition, pages
    Genetics Society of America, 2016
    Keywords
    neuroblast, lineage tree, cell cycle, epigenetics, terminal differentiation, FlyBook
    National Category
    Genetics
    Identifiers
    urn:nbn:se:liu:diva-132856 (URN)10.1534/g3.116.034231 (DOI)000386581200018 ()27520958 (PubMedID)
    Note

    Funding Agencies|Swedish Research Council [621-2013-5258]; Knut and Alice Wallenberg Foundation [KAW2011.0165]; Swedish Cancer Foundation [120531]; Swedish Royal Academy of Sciences

    Available from: 2016-12-06 Created: 2016-11-30 Last updated: 2017-11-29
    3. Neural Lineage Progression Controlled by a Temporal Proliferation Program.
    Open this publication in new window or tab >>Neural Lineage Progression Controlled by a Temporal Proliferation Program.
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    2017 (English)In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 43, no 3, p. 332-348Article in journal (Refereed) Published
    Abstract [en]

    Great progress has been made in identifying transcriptional programs that establish stem cell identity. In contrast, we have limited insight into how these programs are down-graded in a timely manner to halt proliferation and allow for cellular differentiation. Drosophila embryonic neuroblasts undergo such a temporal progression, initially dividing to bud off daughters that divide once (type I), then switching to generating non-dividing daughters (type 0), and finally exiting the cell cycle. We identify six early transcription factors that drive neuroblast and type I daughter proliferation. Early factors are gradually replaced by three late factors, acting to trigger the type I→0 daughter proliferation switch and eventually to stop neuroblasts. Early and late factors regulate each other and four key cell-cycle genes, providing a logical genetic pathway for these transitions. The identification of this extensive driver-stopper temporal program controlling neuroblast lineage progression may have implications for studies in many other systems.less thanbr /greater than (Copyright © 2017 Elsevier Inc. All rights reserved.)

    Place, publisher, year, edition, pages
    Cell Press, 2017
    National Category
    Developmental Biology
    Identifiers
    urn:nbn:se:liu:diva-143117 (URN)10.1016/j.devcel.2017.10.004 (DOI)000414584300011 ()29112852 (PubMedID)
    Note

    Funding agencies: Swedish Research Council [621-2013-5258]; Knut and Alice Wallenberg Foundation [KAW2011.0165, KAW2012.0101]; Swedish Cancer Foundation [140780, 150633]

    Available from: 2017-11-20 Created: 2017-11-20 Last updated: 2017-11-20Bibliographically approved
  • 8.
    Banerji, Shantanu
    et al.
    Manitoba Institute of Cell Biology, Winnipeg, Manitoba, Canada .
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada.
    Important differences between topoisomerase-I and -II targeting agents2006In: Cancer Biology & Therapy, ISSN 1538-4047, E-ISSN 1555-8576, Vol. 5, no 8, p. 965-966Article in journal (Other academic)
    Abstract [en]

    Commentary to: Activation of ATM and Histone H2AX Phosphorylation Induced by Mitoxantrone But Not by Topotecan is Prevented by the Antioxidant N-acetyl-L-Cysteine Xuan Huang, Akira Kurose, Toshiki Tanaka, Frank Traganos, Wei Dai and Zbigniew Darzynkiewicz

     

  • 9.
    Bergkvist, Liza
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Amyloid-β and lysozyme proteotoxicity in Drosophila: Beneficial effects of lysozyme and serum amyloid P component in models of Alzheimer’s disease and lysozyme amyloidosis2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

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

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

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

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

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

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

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

    Place, publisher, year, edition, pages
    John Wiley & Sons, 2016
    Keywords
    Alzheimer’s disease, amyloid-β, Drosophila, lysozyme
    National Category
    Genetics Medical Genetics Developmental Biology Bioinformatics and Systems Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:liu:diva-131796 (URN)10.1111/febs.13830 (DOI)000386033700001 ()27562772 (PubMedID)
    Available from: 2016-10-07 Created: 2016-10-07 Last updated: 2018-03-20Bibliographically approved
    3. Serum Amyloid P Component Ameliorates Neurological Damage Caused by Expressing a Lysozyme Variant in the Central Nervous System of Drosophila melanogaster
    Open this publication in new window or tab >>Serum Amyloid P Component Ameliorates Neurological Damage Caused by Expressing a Lysozyme Variant in the Central Nervous System of Drosophila melanogaster
    2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 7, p. e0159294-Article in journal (Refereed) Published
    Abstract [en]

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

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

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

    Available from: 2016-09-19 Created: 2016-09-12 Last updated: 2017-11-21
  • 10.
    Bergkvist, Liza
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Sandin, Linnea
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Kågedal, Katarina
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Brorsson, Ann-Christin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    A beta PP processing results in greater toxicity per amount of A beta(1-42) than individually expressed and secreted A beta(1-42) in Drosophila melanogaster2016In: BIOLOGY OPEN, ISSN 2046-6390, Vol. 5, no 8, p. 1030-1039Article in journal (Refereed)
    Abstract [en]

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

  • 11.
    Bivik Stadler, Caroline
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Genetic pathways controlling CNS development: The role of Notch signaling in regulating daughter cell proliferation in Drosophila2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The human central nervous system (CNS) displays the greatest cellular diversity of any organ system, consisting of billions of neurons, of numerous cell sub-types, interconnected in a vast network. Given this enormous complexity, decoding the genetic programs controlling the multistep process of CNS development remains a major challenge. While great progress has been made with respect to understanding sub-type specification, considerably less is known regarding how the generation of the precise number of each sub-type is controlled.

    The aim of this thesis was to gain deeper knowledge into the regulatory programs controlling cell specification and proliferation. To address these questions I have studied the Drosophila embryonic CNS as a model system, to thereby be able to investigate the genetic mechanisms at high resolution. Despite the different size and morphology between the Drosophila and the mammalian CNS, the lineages of their progenitors share similarity. Importantly for this thesis, both species progenitors show elaborate variations in their proliferation modes, either giving rise to daughters that can directly differentiate into neurons or glia (type 0), divide once (type I), or multiple times (type II).

    The studies launched off with a comprehensive chemical forward genetic screen, for the very last born cell in the well-studied lineage of progenitor NB5-6T: the Ap4 neuron, which expresses the neuropeptide FMRFa. NB5-6T is a powerful model to use, because it undergoes a programmed type I>0 daughter cell proliferation switch. An FMRF-eGFP transgenic reporter was utilized as readout for successful terminal differentiation of Ap4/FMRFa and thereby proper lineage progression of the ∼20 cells generated. The strongest mutants were mapped to genes with both known and novel essential functions e.g., spatial and temporal patterning, cell cycle control, cell specification and chromatin modification. Subsequently, we focused on some of the genes that showed a loss of function phenotype with an excess of lineage cells. We found that Notch is critical for the type I>0 daughter cell proliferation switch in the NB5-6T lineage and globally as well. When addressing the broader relevance of these findings, and to further decipher the Notch pathway, we discovered that selective groups of E(spl) genes is controlling the switch in a close interplay with four key cell cycle factors: Cyclin E, String, E2F and Dacapo, in most if not all embryonic progenitors. The Notch mediation of the switch is likely to be by direct transcriptional regulation. Furthermore, another gene identified in the screen, sequoia, was investigated. The analysis revealed that sequoia is also controlling the daughter cell switch in the CNS, and this partly through context dependent interactions with the Notch pathway.

    Taken together, the findings presented in this thesis demonstrate that daughter cell proliferation switches in Drosophila neural lineages are genetically programmed, and that Notch contributes to the triggering of these events. Given that early embryonic processes is frequently shown to be evolutionary conserved, you can speculate that changeable daughter proliferation programs could be applied to mammals, and contribute to a broader understanding of proliferation processes in humans as well.

     

    List of papers
    1. Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
    Open this publication in new window or tab >>Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
    Show others...
    2015 (English)In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 200, no 4, p. 1229-1244Article in journal (Refereed) Published
    Abstract [en]

    The expression of neuropeptides is often extremely restricted in the nervous system, making them powerful markers for addressing cell specification . In the developing Drosophila ventral nerve cord, only six cells, the Ap4 neurons, of some 10,000 neurons, express the neuropeptide FMRFamide (FMRFa). Each Ap4/FMRFa neuron is the last-born cell generated by an identifiable and well-studied progenitor cell, neuroblast 5-6 (NB5-6T). The restricted expression of FMRFa and the wealth of information regarding its gene regulation and Ap4 neuron specification makes FMRFa a valuable readout for addressing many aspects of neural development, i.e., spatial and temporal patterning cues, cell cycle control, cell specification, axon transport, and retrograde signaling. To this end, we have conducted a forward genetic screen utilizing an Ap4-specific FMRFa-eGFP transgenic reporter as our readout. A total of 9781 EMS-mutated chromosomes were screened for perturbations in FMRFa-eGFP expression, and 611 mutants were identified. Seventy-nine of the strongest mutants were mapped down to the affected gene by deficiency mapping or whole-genome sequencing. We isolated novel alleles for previously known FMRFa regulators, confirming the validity of the screen. In addition, we identified novel essential genes, including several with previously undefined functions in neural development. Our identification of genes affecting most major steps required for successful terminal differentiation of Ap4 neurons provides a comprehensive view of the genetic flow controlling the generation of highly unique neuronal cell types in the developing nervous system.

    Place, publisher, year, edition, pages
    Genetics Society of America, 2015
    Keywords
    Drosophila; CNS development; neural cell fate specification; forward genetic screening; FMRFamide
    National Category
    Clinical Medicine
    Identifiers
    urn:nbn:se:liu:diva-121318 (URN)10.1534/genetics.115.178483 (DOI)000359917000020 ()26092715 (PubMedID)
    Available from: 2015-09-16 Created: 2015-09-14 Last updated: 2019-03-13Bibliographically approved
    2. Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression
    Open this publication in new window or tab >>Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression
    Show others...
    2012 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 139, no 4, p. 678-689Article in journal (Refereed) Published
    Abstract [en]

    During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. Here, we identify two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers.

    Place, publisher, year, edition, pages
    Company of Biologists, 2012
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-74790 (URN)10.1242/dev.074500 (DOI)000300259800005 ()
    Note

    funding agencies|Swedish Research Council||Knut and Alice Wallenberg foundation||Swedish Cancer Foundation||

    Available from: 2012-02-08 Created: 2012-02-08 Last updated: 2019-03-13
    3. Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling
    Open this publication in new window or tab >>Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling
    Show others...
    2016 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 4, article id e1005984Article in journal (Refereed) Published
    Abstract [en]

    The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21(CIP1)/p27(KIP1)/p57(Kip2)). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems.

    Place, publisher, year, edition, pages
    PUBLIC LIBRARY SCIENCE, 2016
    National Category
    Clinical Medicine
    Identifiers
    urn:nbn:se:liu:diva-128759 (URN)10.1371/journal.pgen.1005984 (DOI)000375231900032 ()27070787 (PubMedID)
    Note

    Funding Agencies|Knut and Alice Wallenberg Foundation [KAW2012.0101]; Swedish Research Council [621-2010-5214]; Swedish Cancer Foundation [120531]

    Available from: 2016-05-31 Created: 2016-05-30 Last updated: 2019-03-13
    4. sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system
    Open this publication in new window or tab >>sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system
    2016 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 143, no 20, p. 3774-3784Article in journal (Refereed) Published
    Abstract [en]

    Neural progenitors typically divide asymmetrically to renew themselves, while producing daughters with more limited potential. In the Drosophila embryonic ventral nerve cord, neuroblasts initially produce daughters that divide once to generate two neurons/glia (type I proliferation mode). Subsequently, many neuroblasts switch to generating daughters that differentiate directly (type 0). This programmed type I>0 switch is controlled by Notch signaling, triggered at a distinct point of lineage progression in each neuroblast. However, how Notch signaling onset is gated was unclear. We recently identified Sequoia (Seq), a C2H2 zinc-finger transcription factor with homology to Drosophila Tramtrack (Ttk) and the positive regulatory domain (PRDM) family, as important for lineage progression. Here, we find that seq mutants fail to execute the type I>0 daughter proliferation switch and also display increased neuroblast proliferation. Genetic interaction studies reveal that seq interacts with the Notch pathway, and seq furthermore affects expression of a Notch pathway reporter. These findings suggest that seq may act as a context-dependent regulator of Notch signaling, and underscore the growing connection between Seq, Ttk, the PRDM family and Notch signaling.

    Place, publisher, year, edition, pages
    The Company of Biologists Ltd, 2016
    Keywords
    Lineage tree, Cell cycle, Asymmetric division, Combinatorial control, Notch
    National Category
    Cell and Molecular Biology Biochemistry and Molecular Biology Cell Biology Medical Biotechnology
    Identifiers
    urn:nbn:se:liu:diva-132739 (URN)10.1242/dev.139998 (DOI)000393452500013 ()27578794 (PubMedID)
    Note

    Funding agencies: Swedish Research Council (Vetenskapsradet); Knut and Alice Wallenberg Foundation (Knut och Alice Wallenbergs Stiftelse); Swedish Cancer Foundation (Cancerfonden)

    Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2019-03-13Bibliographically approved
  • 12.
    Blissing, Annica
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Thiopurine S-methyltransferase - characterization of variants and ligand binding2017Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

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

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

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

  • 13.
    Boberg, Andreas
    et al.
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.
    Stålnacke, Alexandra
    Etvax AB, Solna, Sweden.
    Bråve, Andreas
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.
    Hinkula, Jorma
    Linköping University, Department of Clinical and Experimental Medicine, Molecular Virology. Linköping University, Faculty of Health Sciences.
    Wahren, Britta
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.
    Carlin, Nils
    Etvax AB, Solna, Sweden.
    Receptor binding by cholera toxin B-subunit and amino acid modification improves minimal peptide immunogenecity2012In: ISRN Molecular Biology, ISSN 2090-7907, Vol. 2012, article id 170676Article in journal (Refereed)
    Abstract [en]

    We increase our understanding of augmenting a cellular immune response, by using an HIV-1 protease-derived epitope (PR7584), and variants thereof, coupled to the C-terminal, of the B subunit of cholera toxin (CTB). Fusion proteins were used for immunizations of HLA-A0201 transgenic C57BL/6 mice. We observed different capacities to elicit a cellular immune response by peptides with additions of five to ten amino acids to the PR epitope. There was a positive correlation between the magnitude of the elicited cellular immune response and the capacity of the fusion protein to bind GM-1. This binding capacity is affected by its ability to form natural pentamers of CTB. Our results suggest that functional CTB pentamers containing a foreign amino acid-modified epitope is a novel way to overcome the limited cellular immunogenicity of minimal peptide antigens. This way of using a functional assay as readout for improved cellular immunogenicity might become highly valuable for difficult immunogens such as short peptides (epitopes).

  • 14.
    Bojmar, Linda
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Metastatic Mechanisms in Malignant Tumors2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The ultimate cause of cancer related deaths is metastasis. This thesis is about three of the main human cancers; breast, colorectal and pancreatic cancer, that together account for more than 25% of the cancer-related deaths worldwide. The focus of the thesis is the spread of cancer, metastasis, and the aim was to investigate mechanisms that can be of importance for this process. We analyzed patient samples to validate the role of epithelialto-mesenchymal transition in vivo and found regulations of many related factors. However, these changes tend to fluctuate along the metastatic process, something which makes targeting complicated. We, moreover, focused on the influence of the tumor microenvironment for metastatic spread. In pancreatic cancer, the stroma constitutes the main part of many tumors. We analyzed the crosstalk between tumor and stromal cell and focused on the mediating inflammatory factor interleukin-1 (IL-1) and regulation of microRNAs. The results showed that the most commonly mutated factor in pancreatic cancer, KRAS, associates with the expression of IL-1 and subsequent activation of stromal cells. Blocking KRAS signaling together with IL-1 blockage give a more pronounced effect on in vitro proliferation and migration of cancer cells and suggests the use of a combination therapy. The cancer-associated activation of the stroma was found to be related to changes in microRNA expression. microRNA was analyzed separately in epithelial cells and stromal cells after microdissection of matched samples of primary and secondary tumors of breast and colorectal cancers. miR-214 and miR-199a were upregulated in stroma associated with progressive tumors and in pancreatic cancer stroma we could show that their expression alters the activation of stromal cells and thereby the growth and migratory ability of associated pancreatic tumor cells. In  breast and colorectal cancers we found several common microRNAs to be up- or downregulated in line with progression. We could show that one of these candidates, miR-18a, had a prognostic value in metastatic breast cancer. To further develop these studies we analyzed this microRNA in circulating microvesicles, i.e. exosomes, and investigated their role in the preparation of a pre-metastatic niche. MicroRNAs are stable biomarkers in the circulation, especially protected in exosomes, which can moreover specifically deliver their message to recipient cells. These studies facilitate the understanding of metastatic behavior and suggest new targets to stop cancer metastasis.

    List of papers
    1. The Role of MicroRNA-200 in Progression of Human Colorectal and Breast Cancer
    Open this publication in new window or tab >>The Role of MicroRNA-200 in Progression of Human Colorectal and Breast Cancer
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    2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 12, p. 84815-Article in journal (Refereed) Published
    Abstract [en]

    The role of the epithelial-mesenchymal transition (EMT) in cancer has been studied extensively in vitro, but involvement of the EMT in tumorigenesis in vivo is largely unknown. We investigated the potential of microRNAs as clinical markers and analyzed participation of the EMT-associated microRNA-200 ZEB E-cadherin pathway in cancer progression. Expression of the microRNA-200 family was quantified by real-time RT-PCR analysis of fresh-frozen and microdissected formalin-fixed paraffin-embedded primary colorectal tumors, normal colon mucosa, and matched liver metastases. MicroRNA expression was validated by in situ hybridization and after in vitro culture of the malignant cells. To assess EMT as a predictive marker, factors considered relevant in colorectal cancer were investigated in 98 primary breast tumors from a treatment-randomized study. Associations between the studied EMTmarkers were found in primary breast tumors and in colorectal liver metastases. MicroRNA-200 expression in epithelial cells was lower in malignant mucosa than in normal mucosa, and was also decreased in metastatic compared to non-metastatic colorectal cancer. Low microRNA-200 expression in colorectal liver metastases was associated with bad prognosis. In breast cancer, low levels of microRNA-200 were related to reduced survival and high expression of microRNA-200 was predictive of benefit from radiotheraphy. MicroRNA-200 was associated with ER positive status, and inversely correlated to HER2 and overactivation of the PI3K/AKT pathway, that was associated with high ZEB1 mRNA expression. Our findings suggest that the stability of microRNAs makes them suitable as clinical markers and that the EMT-related microRNA-200 - ZEB - E-cadherin signaling pathway is connected to established clinical characteristics and can give useful prognostic and treatment-predictive information in progressive breast and colorectal cancers.

    Place, publisher, year, edition, pages
    Public Library of Science, 2013
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-103717 (URN)10.1371/journal.pone.0084815 (DOI)000328745100188 ()
    Available from: 2014-01-24 Created: 2014-01-24 Last updated: 2019-02-11
    2. IL-1α Expression in Pancreatic Ductal Adenocarcinoma Affects the Tumor Cell Migration and Is Regulated by the p38MAPK Signaling Pathway
    Open this publication in new window or tab >>IL-1α Expression in Pancreatic Ductal Adenocarcinoma Affects the Tumor Cell Migration and Is Regulated by the p38MAPK Signaling Pathway
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    2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 8Article in journal (Refereed) Published
    Abstract [en]

    The interplay between the tumor cells and the surrounding stroma creates inflammation, which promotes tumor growth and spread. The inflammation is a hallmark for pancreatic adenocarcinoma (PDAC) and is to high extent driven by IL-1α. IL-1α is expressed and secreted by the tumor cells and exerting its effect on the stroma, i.e. cancer associated fibroblasts (CAF), which in turn produce massive amount of inflammatory and immune regulatory factors. IL-1 induces activation of transcription factors such as nuclear factor-κβ (NF-κβ), but also activator protein 1 (AP-1) via the small G-protein Ras. Dysregulation of Ras pathways are common in cancer as this oncogene is the most frequently mutated in many cancers. In contrast, the signaling events leading up to the expression of IL-1α by tumor cells are not well elucidated. Our aim was to examine the signaling cascade involved in the induction of IL-1α expression in PDAC. We found p38MAPK, activated by the K-Ras signaling pathway, to be involved in the expression of IL-1α by PDAC as blocking this pathway decreased both the gene and protein expression of IL-1α. Blockage of the P38MAPK signaling in PDAC also dampened the ability of the tumor cell to induce inflammation in CAFs. In addition, the IL-1α autocrine signaling regulated the migratory capacity of PDAC cells. Taken together, the blockage of signaling pathways leading to IL-1α expression and/or neutralization of IL-1α in the PDAC microenvironment should be taken into consideration as possible treatment or complement to existing treatment of this cancer.

    Place, publisher, year, edition, pages
    Public Library of Science, 2013
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-97445 (URN)10.1371/journal.pone.0070874 (DOI)000323097300061 ()
    Note

    Funding Agencies|Swedish Research Council|AI52731|VINNMER (Vinnova)||Medical Research Council of Southeast Sweden||Swedish Society of Medicine||

    Available from: 2013-09-12 Created: 2013-09-12 Last updated: 2017-12-06
    3. MicroRNA-199a and -214 as potential therapeutic targets in pancreatic stellate cells in pancreatic tumor
    Open this publication in new window or tab >>MicroRNA-199a and -214 as potential therapeutic targets in pancreatic stellate cells in pancreatic tumor
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    2016 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 13, p. 16396-16408Article in journal (Refereed) Published
    Abstract [en]

    Pancreatic stellate cells (PSCs) are the key precursor cells for cancer-associated fibroblasts (CAFs) in pancreatic tumor stroma. Although depletion of tumor stroma is debatable, attenuation of PSC activity is still an interesting strategy to treat pancreatic cancer. In this study, we explored miRNA as therapeutic targets in tumor stroma and found miR-199a-3p and miR-214-3p induced in patient-derived pancreatic CAFs as well as in TGF-β-activated human PSCs (hPSCs). Inhibition of miR-199a or miR-214 using their hairpin inhibitors in hPSCs significantly inhibited their TGFβ-induced differentiation (gene and protein levels of α-SMA, Collagen, PDGFβR), migration and proliferation. Furthermore, heterospheroids of Panc-1 and hPSCs were prepared, which attained smaller size when hPSCs were transfected with anti-miR-199a or -214 than those transfected with control anti-miR. The conditioned medium obtained from TGFβ-activated hPSCs induced tumor cell proliferation and endothelial cell tube formation, but these effects were abrogated when hPSCs were transfected with anti-miR-199a or miR-214. Moreover, IPA analyses revealed signaling pathways related to miR-199a (TP53, mTOR, Smad1) and miR-214 (PTEN, Bax, ING4). Taken together, this study reveals miR-199a-3p and miR-214-3p as major regulators of PSC activation and PSC-induced pro-tumoral effects, representing them as key therapeutic targets in PSCs in pancreatic cancer.

    Place, publisher, year, edition, pages
    Impact press, 2016
    National Category
    Cancer and Oncology Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:liu:diva-122828 (URN)10.18632/oncotarget.7651 (DOI)000375692900085 ()
    Note

    Funding agencies: Swedish Research Council, Stockholm, Sweden [K7/60501283]

    Vid tiden för disputationen förelåg publikationen endast som manuskript

    Available from: 2015-11-26 Created: 2015-11-26 Last updated: 2018-01-10
    4. miR-18a is regulated between progressive compartments of cancers, and incorporated in exosomes with the potential of creating premetastatic niches and predict cancer outcome
    Open this publication in new window or tab >>miR-18a is regulated between progressive compartments of cancers, and incorporated in exosomes with the potential of creating premetastatic niches and predict cancer outcome
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    2015 (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    The ultimate cause of death for many cancer patients is the spread of the cancer via metastasis. Even so, there are still a lack of knowledge regarding the metastasis process. This study was performed to investigate the role of metastamirs in exosomes and their metastatic patterns. We used the well-established isogeneic murine cancer model of low metastatic 67NR cells, mimicking luminal/basal breast tumors, and highly metastatic 4T1 cells with characteristics of basal breast  tumors. We studied the exosomal properties and pre-metastatic effects in this metastasis model and compared human materials and exosomes of several other tumor types. Our data clearly demonstrated that exosomes from the highly metastatic cells home to the metastatic organs of their parental cells whereas exosomes from cells with low metastatic potential mostly located to lymph nodes. The exosome protein cargos also resembled their parental cells and potentially affects their target organs, and cells, differently. Furthermore, the exosomes from the highly metastatic cells had a more pronounced effect on tumor growth and pre-metastatic changes than the low metastatic exosomes. The microRNA-18a, a predictor of metastasis, was present to a higher extent in metastatic exosomes as compared to low metastatic exosomes, and altered the tumor progressive properties. Our findings support the role of exomirs as important players in the metastatic process, the value as biomarkers and potential therapeutic targets.

    National Category
    Cancer and Oncology Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:liu:diva-122829 (URN)
    Available from: 2015-11-26 Created: 2015-11-26 Last updated: 2018-01-10Bibliographically approved
  • 15.
    Bojmar, Linda
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Zhang, Haiying
    Children’s Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children’s Health, Meyer Cancer Center, Weill Cornell Medical College, New York, USA.
    Costa da Silva, Bruno
    Children’s Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children’s Health, Meyer Cancer Center, Weill Cornell Medical College, New York, USA.
    Karlsson, Elin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Olsson, Hans
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    Vincent, Theresa
    Departments of Physiology and Biophysics and Cell and Developmental Biology, Weill Cornell Medical College, New York, USA / Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.
    Larsson, Marie
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Stål, Olle
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Lyden, David
    Children’s Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children’s Health, Meyer Cancer Center, Weill Cornell Medical College, New York, USA.
    Sandström, Per
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Surgery in Linköping.
    miR-18a is regulated between progressive compartments of cancers, and incorporated in exosomes with the potential of creating premetastatic niches and predict cancer outcome2015Manuscript (preprint) (Other academic)
    Abstract [en]

    The ultimate cause of death for many cancer patients is the spread of the cancer via metastasis. Even so, there are still a lack of knowledge regarding the metastasis process. This study was performed to investigate the role of metastamirs in exosomes and their metastatic patterns. We used the well-established isogeneic murine cancer model of low metastatic 67NR cells, mimicking luminal/basal breast tumors, and highly metastatic 4T1 cells with characteristics of basal breast  tumors. We studied the exosomal properties and pre-metastatic effects in this metastasis model and compared human materials and exosomes of several other tumor types. Our data clearly demonstrated that exosomes from the highly metastatic cells home to the metastatic organs of their parental cells whereas exosomes from cells with low metastatic potential mostly located to lymph nodes. The exosome protein cargos also resembled their parental cells and potentially affects their target organs, and cells, differently. Furthermore, the exosomes from the highly metastatic cells had a more pronounced effect on tumor growth and pre-metastatic changes than the low metastatic exosomes. The microRNA-18a, a predictor of metastasis, was present to a higher extent in metastatic exosomes as compared to low metastatic exosomes, and altered the tumor progressive properties. Our findings support the role of exomirs as important players in the metastatic process, the value as biomarkers and potential therapeutic targets.

  • 16.
    Bolshakova, A.V.
    et al.
    Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.
    Petukhova, O.A.
    Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.
    Pinaev, G.P.
    Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.
    Magnusson, Karl-Erik
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Comparative analysis of subcellular fractionation methods for revealing a-actinin 1 and a-actinin 4 in A431 cells2009In: Cell and Tissue Biology, ISSN 1990-519X, Vol. 3, no 2, p. 188-197Article in journal (Refereed)
    Abstract [en]

    a-Actinin 1 and a-actinin 4 are actin-binding proteins with shared structural functions that are responsible for the regulation of several processes in the cell. Based on previous data on the different distribution of these proteins in the nucleus and cytoplasm, we have studied in detail the presence of a-actinin 1 and a-actinin 4 in subcellular fractions in the A431 cells spread on fibronectin. The detection of a-actinins in some particular fractions has been shown to depend on the method of lysis, as well as whether the preliminary low-temperature freezing of cells was used. The application of various fractionation methods has allowed us to conclude that a-actinin 4 is present in all cytoplasmic and nuclear subfractions, whereas, in addition to in the cytoplasm, a-actinin 1 can also be revealed in the nuclear envelope fraction.

  • 17.
    Booy, Evan P.
    et al.
    Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, Univ. Manitoba, Winnipeg, Canada.
    Johar, Dina
    Manitoba Institute of Cell Biology,CancerCare Manitoba, University of Manitoba, ON6010-675 McDermot Ave., Winnipeg, MB, R3E 0V9, Canada; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada .
    Maddika, Srilekha
    Manitoba Institute of Cell Biology,CancerCare Manitoba, University of Manitoba, ON6010-675 McDermot Ave., Winnipeg, MB, R3E 0V9, Canada; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada .
    Pirzada, Hasan
    Manitoba Institute of Cell Biology,CancerCare Manitoba, University of Manitoba, ON6010-675 McDermot Ave., Winnipeg, MB, R3E 0V9, Canada.
    Sahib, Mickey M.
    Department of Oral Biology, University of Manitoba, Winnipeg, Canada .
    Gehrke, Iris
    Manitoba Institute of Cell Biology,CancerCare Manitoba, University of Manitoba, ON6010-675 McDermot Ave., Winnipeg, MB, R3E 0V9, Canada.
    Loewen, Shauna
    Manitoba Institute of Cell Biology,CancerCare Manitoba, University of Manitoba, ON6010-675 McDermot Ave., Winnipeg, MB, R3E 0V9, Canada; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada .
    Louis, Sherif F.
    Manitoba Institute of Cell Biology,CancerCare Manitoba, University of Manitoba, ON6010-675 McDermot Ave., Winnipeg, MB, R3E 0V9, Canada.
    Kadkhoda, Kamran
    Manitoba Institute of Cell Biology,CancerCare Manitoba, University of Manitoba, ON6010-675 McDermot Ave., Winnipeg, MB, R3E 0V9, Canada; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada .
    Mowat, Michael
    Manitoba Institute of Cell Biology,CancerCare Manitoba, University of Manitoba, ON6010-675 McDermot Ave., Winnipeg, MB, R3E 0V9, Canada; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada .
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Monoclonal and bispecific antibodies as novel therapeutics2006In: Archivum Immunologiae et Therapiae Experimentalis, ISSN 0004-069X, E-ISSN 1661-4917, Vol. 54, no 2, p. 85-101Article in journal (Refereed)
    Abstract [en]

    Gene amplification, over-expression, and mutation of growth factors, or the receptors themselves, causes increased signaling through receptor kinases, which has been implicated in many human cancers and is associated with poor prognosis. Tumor growth has been shown to be decreased by interrupting this process of extensive growth factor-mediated signaling by directly targeting either the surface receptor or the ligand and thereby preventing cell survival and promoting apoptosis. Monoclonal antibodies have long been eyed as a potential new class of therapeutics targeting cancer and other diseases. Antibody-based therapy initially entered clinical practice when trastuzumab/Herceptin became the first clinically approved drug against an oncogene product as a well-established blocking reagent for tumors with hyperactivity of epidermal growth factor signaling pathways. In the first part of this review we explain basic terms related to the development of antibody-based drugs, give a brief historic perspective of the field, and also touch on topics such as the "humanization of antibodies" or creation of hybrid antibodies. The second part of the review gives an overview of the clinical usage of bispecific antibodies and antibodies "armed" with cytotoxic agents or enzymes. Further within this section, cancer-specific, site-specific, or signaling pathway-specific therapies are discussed in detail. Among other antibody-based therapeutic products, we discuss: Avastin (bevacizumab), CG76030, Theragyn (pemtumomab), daclizumab (Zenapax), TriAb, MDX-210, Herceptin (trastuzumab), panitumumab (ABX-EGF), mastuzimab (EMD-72000), Erbitux (certuximab, IMC225), Panorex (edrecolomab), STI571, CeaVac, Campath (alemtuizumab), Mylotarg (gemtuzumab, ozogamicin), and many others. The end of the review deliberates upon potential problems associated with cancer immunotherapy.

  • 18.
    Borg, Mathias
    Linköping University, Department of Physics, Chemistry and Biology.
    Study of the insulin-like peptide 3 in human platelets2009Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The insulin-like 3 peptide is autocrine/paracrine insulin-related hormone with a size of approximately 6kDa [1]. It mediates through a leucine richG-coupled receptor named LGR8. INSL3 is mainly expressed in human Leydig cells and is directly responsible for migration of the testis during the pre-natal period in maledevelopment. [2]

    INSL3 mRNA has recently been verified in human platelets whereas no mRNA has been detected for LGR8 (by Sanofi-Aventis GmbH in Frankfurt,Germany), indicating that INSL3 might be released through paracrine functions at sites of platelet adhesion and aggregation upon a vascular injury.Furthermore, has activated platelets been shown to translate essential proteins upon activation, in a term called “signal-dependent protein synthesis”.The B-Cell lymphoma-3 protein (BCL-3) is an example of such a protein [3], and there is a possibility that INSL3 might be also.

    In this thesis we wanted to detect the relaxin- like peptide 3 hormone (INSL3). (Its function, location and the timeframe of its release, when/if it issecreated in stimulated platelets).The source of platelet-derived INSL3 can be found with Western blotting and Enzyme immunoassay.

    Detection of the insulin-like 3 peptide in human platelets turned out to be a difficult challenge due to the small amount of INSL3 secretion uponplatelet activation; hence the total amount of INSL3 produced might be below detection limit.

  • 19.
    Burek, M.
    et al.
    Department of Immunology and Cell Biology, University of Münster, Münster, Germany.
    Maddika, Subbareddy
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Department of Biochemistry and Medical Genetics,University of Manitoba, Winnipeg, Canada .
    Burek, C. J.
    Department of Immunology and Cell Biology, University of Münster, Münster, Germany.
    Daniel, P. T.
    Department of Hematology, Oncology and Tumor Immunology, Charité, Berlin, Germany.
    Schulze-Osthoff, Klaus
    nstitute of Molecular Medicine, University of Düsseldorf, Düsseldorf, Germany .
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Apoptin-induced cell death is modulated by Bcl-2 family members and is Apaf-1dependent2006In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 25, no 15, p. 2213-2222Article in journal (Refereed)
    Abstract [en]

    Apoptin, a chicken anemia virus-derived protein, selectively induces apoptosis in transformed but not in normal cells, thus making it a promising candidate as a novel anticancer therapeutic. The mechanism of apoptin-induced apoptosis is largely unknown. Here, we report that contrary to previous assumptions, Bcl-2 and Bcl-x(L) inhibit apoptin-induced cell death in several tumor cell lines. In contrast, deficiency of Bax conferred resistance, whereas Bax expression sensitized cells to apoptin-induced death. Cell death induction by apoptin was associated with cytochrome c release from mitochondria as well as with caspase-3 and -7 activation. Benzyloxy-carbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad spectrum caspase inhibitor, was highly protective against apoptin-induced cell death. Apoptosis induced by apoptin required Apaf-1, as immortalized Apaf-1-deficient fibroblasts as well as tumor cells devoid of Apaf-1 were strongly protected. Thus, our data indicate that apoptin-induced apoptosis is not only Bcl-2- and caspase dependent, but also engages an Apaf-1 apoptosome-mediated mitochondrial death pathway.

  • 20.
    Carlsson, Uno
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Magnusson, Thomas
    Linköping University, Department of Management and Engineering, Project management, Innovations and Entrepreneurship . Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Svensson, Magdalena
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Science and Technology. Linköping University, The Institute of Technology.
    To design a novel protein: A CDIO experience in Molecular Biotechnology at Linköping University2006Conference paper (Other academic)
  • 21.
    Cholujová, Dana
    et al.
    Laboratory of Molecular Oncology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, Bratislava, Slovakia.
    Jakubíková, Jana
    Laboratory of Tumor Immunology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, Bratislava, Slovakia.
    Kubeš, Miroslav
    Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia.
    Arendacká, Barbora
    Institute of Measurement Science, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia.
    Sapák, Michal
    Institute of Immunology, Medical Faculty of Comenius University, Sasinkova 4, Bratislava, Slovakia.
    Ihnatko, Robert
    Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia.
    Sedlák, Ján
    Laboratory of Tumor Immunology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, Bratislava, Slovakia.
    Comparative study of four fluorescent probes for evaluation of natural killer cell cytotoxicity assays2008In: Immunobiology, ISSN 0171-2985, E-ISSN 1878-3279, Vol. 213, no 8, p. 629-640Article in journal (Refereed)
    Abstract [en]

    Cytotoxicity is one of the major defence mechanisms against both virus-infected and tumor cells. Radioactive 51chromium (51Cr) release assay is a “gold standard” for assessment of natural killer (NK) cytolytic activity in vitro. Several disadvantages of this assay led us to design alternative tools based on flow cytometry analysis. Four different fluorescent dyes, calcein acetoxymethyl ester (CAM), carboxyfluorescein succinimidyl ester (CFSE), Vybrant DiO (DiO) and MitoTracker Green (MTG) were tested for labeling of NK target K-562 cells. Target staining stability, spontaneous release of fluorochromes and subsequent accumulation in bystander unstained cells were measured using fluorimetry and flow cytometry. Healthy donor peripheral blood mononuclear cells and affinity column purified NK cells were used as effectors coincubated with target K-562 cells at different E:T ratios for 3h and 90min, respectively. Fluorescent probe 7-amino-actinomycin D was used for live and dead cell discrimination. Bland–Altman statistical method was applied to measure true agreement for all CAM–51Cr, CFSE–51Cr, DiO–51Cr and MTG–51Cr pairs analyzed. Based on the data, none of the four proposed methods can be stated equivalent to the standard 51Cr release assay. Considering linear relationships between data obtained with four fluorochromes and 51Cr release assay as well as linear regression analysis with R2=0.9393 value for CAM–51Cr pair, we found the CAM assay to be the most closely related to the 51Cr assay.

  • 22.
    Christoffersson, Jonas
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    Aronsson, Christopher
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Jury, Michael
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Selegård, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Mandenius, Carl-Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    Fabrication of modular hyaluronan-PEG hydrogels to support 3D cultures of hepatocytes in a perfused liver-on-a-chip device2018In: Biofabrication, ISSN 1758-5082, E-ISSN 1758-5090, Vol. 11, no 1, p. 1-13, article id 015013Article in journal (Refereed)
    Abstract [en]

    Liver cell culture models are attractive in both tissue engineering and for development of assays for drug toxicology research. To retain liver specific cell functions, the use of adequate cell types and culture conditions, such as a 3D orientation of the cells and a proper supply of nutrients and oxygen, are critical. In this article, we show how extracellular matrix mimetic hydrogels can support hepatocyte viability and functionality in a perfused liver-on-a-chip device. A modular hydrogel system based on hyaluronan and poly(ethylene glycol) (HA-PEG), modified with cyclooctyne moieties for bioorthogonal strain-promoted alkyne-azide 1, 3-dipolar cycloaddition (SPAAC), was developed, characterized, and compared for cell compatibility to hydrogels based on agarose and alginate. Hepatoma cells (HepG2) formed spheroids with viable cells in all hydrogels with the highest expression of albumin and urea in alginate hydrogels. By including an excess of cyclooctyne in the HA backbone, azide-modified cell adhesion motifs (linear and cyclic RGD peptides) could be introduced in order to enhance viability and functionality of human induced pluripotent stem cell derived hepatocytes (hiPS-HEPs). In the HA-PEG hydrogels modified with cyclic RGD peptides hiPS-HEPs migrated and grew in 3D and showed an increased viability and higher albumin production compared to when cultured in the other hydrogels. This flexible SPAAC crosslinked hydrogel system enabled fabrication of perfused 3D cell culture of hiPS-HEPs and is a promising material for further development and optimization of liver-on-a-chip devices.

  • 23.
    Christoffersson, Jonas
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    Meier, Florian
    Boehringer Ingelheim Pharma GmbH and Co. KG, Nonclinical Drug Safety Germany, D-88397 Biberach an der Riss, Germany.
    Kempf, Henning
    Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
    Schwanke, Kristin
    Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
    Coffee, Michelle
    Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
    Beilmann, Mario
    Boehringer Ingelheim Pharma GmbH and Co. KG, Nonclinical Drug Safety Germany, D-88397 Biberach an der Riss, Germany.
    Zweigerdt, Robert
    Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
    Mandenius, Carl-Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    A Cardiac Cell Outgrowth Assay for Evaluating Drug Compounds Using a Cardiac Spheroid-on-a-Chip Device2018In: Bioengineering, E-ISSN 2306-5354, Vol. 5, no 2, p. 1-13, article id 36Article in journal (Refereed)
    Abstract [en]

    Three-dimensional (3D) models with cells arranged in clusters or spheroids have emerged as valuable tools to improve physiological relevance in drug screening. One of the challenges with cells cultured in 3D, especially for high-throughput applications, is to quickly and non-invasively assess the cellular state in vitro. In this article, we show that the number of cells growing out from human induced pluripotent stem cell (hiPSC)-derived cardiac spheroids can be quantified to serve as an indicator of a drug’s effect on spheroids captured in a microfluidic device. Combining this spheroid-on-a-chip with confocal high content imaging reveals easily accessible, quantitative outgrowth data. We found that effects on outgrowing cell numbers correlate to the concentrations of relevant pharmacological compounds and could thus serve as a practical readout to monitor drug effects. Here, we demonstrate the potential of this semi-high-throughput “cardiac cell outgrowth assay” with six compounds at three concentrations applied to spheroids for 48 h. The image-based readout complements end-point assays or may be used as a non-invasive assay for quality control during long-term culture.

  • 24.
    Cros, Olivier
    Linköping University, Department of Biomedical Engineering. Linköping University, Faculty of Science & Engineering.
    Structural properties of the mastoid using image analysis and visualization2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The mastoid, located in the temporal bone, houses an air cell system whose cells have a variation in size that can go far below current conventional clinical CT scanner resolution. Therefore, the mastoid air cell system is only partially represented in a CT scan. Where the conventional clinical CT scanner lacks level of minute details, micro-CT scanning provides an overwhelming amount of ne details. The temporal bone being one of the most complex in the human body, visualization of micro-CT scanning of this boneawakens the curiosity of the experimenter, especially with the correct visualization settings.

    This thesis first presents a statistical analysis determining the surface area to volume ratio of the mastoid air cell system of human temporal bone, from micro-CT scanning using methods previously applied for conventional clinical CT scans. The study compared current results with previous studies, with successive downsampling the data down to a resolution found in conventional clinical CT scanning. The results from the statistical analysis showed that all the small mastoid air cells, that cannot be detected in conventional clinical CT scans, do heavily contribute to the estimation of the surface area, and in consequence to the estimation of the surface area to volume ratio by a factor of about 2.6. Such a result further strengthens the idea of the mastoid to play an active role in pressure regulation and gas exchange.

    Discovery of micro-channels through specific use of a non-traditional transfer function was then reported, where a qualitative and a quantitative pre-analysis were performed and reported. To gain more knowledge about these micro-channels, a local structure tensor analysis was applied where structures are described in terms of planar, tubular, or isotropic structures. The results from this structural tensor analysis suggest these microchannels to potentially be part of a more complex framework, which hypothetically would provide a separate blood supply for the mucosa lining the mastoid air cell system.

    The knowledge gained from analysing the micro-channels as locally providing blood to the mucosa, led to the consideration of how inflammation of the mucosa could impact the pneumatization of the mastoid air cell system. Though very primitive, a 3D shape analysis of the mastoid air cell system was carried out. The mastoid air cell system was first represented in a compact form through a medial axis, from which medial balls could be used. The medial balls, representative of how large the mastoid air cells can be locally, were used in two complementary clustering methods, one based on the size diameter of the medial balls and one based on their location within the mastoid air cell system. From both quantitative and qualitative statistics, it was possible to map the clusters based on pre-defined regions already described in the literature, which opened the door for new hypotheses concerning the effect of mucosal inflammation on the mastoid pneumatization.

    Last but not least, discovery of other structures, previously unreported in the literature, were also visually observed and briefly discussed in this thesis. Further analysis of these unknown structures is needed.

    List of papers
    1. Determination of the mastoid surface area and volume based on micro-CT scanning of human temporal bone: Geometrical parameters dependence on scanning resolutions
    Open this publication in new window or tab >>Determination of the mastoid surface area and volume based on micro-CT scanning of human temporal bone: Geometrical parameters dependence on scanning resolutions
    Show others...
    2016 (English)In: Hearing Research, ISSN 0378-5955, E-ISSN 1878-5891, Vol. 340, p. 127-134Article in journal (Other academic) Published
    Abstract [en]

    The mastoid air cell system (MACS) with its large complex of interconnected air cells reflects an enhanced surface area (SA) relative to its volume (V), which may indicate that the MACS is adapted to gas exchange and has a potential role in middle ear pressure regulation. Thus, these geometric parameters of the MACS have been studied by high resolution clinical CT scanning. However, the resolution of these scans is limited to a voxel size of around 0.6 mm in all dimensions, and so, the geometrical parameters are also limited. Small air cells may appear below the resolution and cannot be detected. Such air cells may contribute to a much higher SA than the V, and thus, also the SA/V ratio. More accurate parameters are important for analysis of the function of the MACS including physiological modeling.

    Our aim was to determine the SA, V, and SA/V ratio in MACS in human temporal bones at highest resolution by using micro-CT-scanning. Further, the influence of the resolution on these parameters was investigated by downsampling the data. Eight normally aerated temporal bones were scanned at the highest possible resolution (30-60 μm). The SA was determined using a triangular mesh fitted onto the segmented MACS. The V was determined by summing all the voxels containing air. Downsampling of the original data was applied four times by a factor of 2.

    The mean SA was 194 cm2, the mean V was 9 cm3, and the mean SA/V amounted to 22 cm-1. Decreasing the resolution resulted in a non-linear decrement of SA and SA/V, whereas V was mainly independent of the resolution.

    The current study found significantly higher SA and SA/V compared with previous studies using clinical CT scanning at lower resolutions. These findings indicate a separate role of the MACS compared with the tympanum, and the results are important for a more accurate modeling of the middle ear physiology.

    Keywords
    Mastoid air cells; medical imaging; micro-CT; surface area; volume
    National Category
    Radiology, Nuclear Medicine and Medical Imaging
    Identifiers
    urn:nbn:se:liu:diva-122176 (URN)10.1016/j.heares.2015.12.005 (DOI)000386417900016 ()
    Available from: 2015-10-23 Created: 2015-10-23 Last updated: 2017-12-01Bibliographically approved
    2. Micro-channels in the mastoid anatomy. Indications of a separate blood supply of the air cell system mucosa by micro-CT scanning
    Open this publication in new window or tab >>Micro-channels in the mastoid anatomy. Indications of a separate blood supply of the air cell system mucosa by micro-CT scanning
    Show others...
    2013 (English)In: Hearing Research, ISSN 0378-5955, E-ISSN 1878-5891, Vol. 301, p. 60-65Article in journal (Refereed) Published
    Abstract [en]

    The mastoid air cell system has traditionally been considered to have a passive role in gas exchange and pressure regulation of the middle ear possibly with some acoustic function. However, more evidence has focused on the mucosa of the mastoid, which may play a more active role in regulation of middle ear pressure.

    In this study we have applied micro-CT scanning on a series of three human temporal bones. This approach greatly enhances the resolution (40–60 μm), so that we have discovered anatomical details, which has not been reported earlier. Thus, qualitative analysis using volume rendering has demonstrated notable micro-channels connecting the surface of the compact bone directly to the mastoid air cells as well as forming a network of connections between the air cells. Quantitative analysis on 2D slices was employed to determine the average diameter of these micro-channels (158 μm; range = 40–440 μm) as well as their density at a localized area (average = 75 cm−2; range = 64–97 cm−2).

    These channels are hypothesized to contain a separate vascular supply for the mastoid mucosa. However, future studies of the histological structure of the micro-channels are warranted to confirm the hypothesis. Studies on the mastoid mucosa and its blood supply may improve our knowledge of its physiological properties, which may have important implications for our understanding of the pressure regulation of the middle ear.

    Place, publisher, year, edition, pages
    Elsevier, 2013
    Keywords
    mastoid, micro CT, middle ear
    National Category
    Otorhinolaryngology Radiology, Nuclear Medicine and Medical Imaging Medical Image Processing
    Identifiers
    urn:nbn:se:liu:diva-92813 (URN)10.1016/j.heares.2013.03.002 (DOI)000320478100009 ()23518400 (PubMedID)
    Available from: 2013-05-22 Created: 2013-05-22 Last updated: 2017-12-06Bibliographically approved
    3. Structural Analysis of Micro-channels in Human Temporal Bone
    Open this publication in new window or tab >>Structural Analysis of Micro-channels in Human Temporal Bone
    2015 (English)In: IEEE 12th International Symposium on Biomedical Imaging (ISBI), 2015 IEEE 12th International Symposium on, Institute of Electrical and Electronics Engineers (IEEE), 2015, p. 9-12Conference paper, Published paper (Refereed)
    Abstract [en]

    Recently, numerous micro-channels have been discovered in the human temporal bone by micro-CT-scanning. Preliminary structure of these channels has suggested they contain a new separate blood supply for the mucosa of the mastoid air cells, which may have important functional implications. This paper proposes a structural analysis of the microchannels to corroborate this role. A local structure tensor is first estimated. The eigenvalues obtained from the estimated local structure tensor were then used to build probability maps representing planar, tubular, and isotropic tensor types. Each tensor type was assigned a respective RGB color and the full structure tensor was rendered along with the original data. Such structural analysis provides new and relevant information about the micro-channels but also their connections to mastoid air cells. Before carrying a future statistical analysis, a more accurate representation of the micro-channels in terms of local structure tensor analysis using adaptive filtering is needed.

    Place, publisher, year, edition, pages
    Institute of Electrical and Electronics Engineers (IEEE), 2015
    Series
    IEEE International Symposium on Biomedical Imaging, ISSN 1945-7928
    Keywords
    Human temporal bone, mastoid, microchannels, quadrature filters, structure tensor, visualization
    National Category
    Radiology, Nuclear Medicine and Medical Imaging
    Identifiers
    urn:nbn:se:liu:diva-122177 (URN)10.1109/ISBI.2015.7163804 (DOI)000380546000003 ()978-1-4799-2374-8 (ISBN)
    Conference
    IEEE 12th International Symposium on Biomedical Imaging (ISBI), 2015 IEEE 12th International Symposium on, 16-19 April, New York, USA
    Available from: 2015-10-23 Created: 2015-10-23 Last updated: 2017-05-10Bibliographically approved
    4. Enhancement of micro-channels within the human mastoid bone based on local structure tensor analysis
    Open this publication in new window or tab >>Enhancement of micro-channels within the human mastoid bone based on local structure tensor analysis
    2016 (English)In: Image Proceessing Theory, Tools and Apllications, IEEE, 2016Conference paper, Published paper (Refereed)
    Abstract [en]

    Numerous micro-channels have recently been discovered in the human temporal bone by x-ray micro-CT-scanning. After a preliminary study suggesting that these micro-channels form a separate blood supply for the mucosa of the mastoid air cells, a structural analysis of the micro-channels using a local structure tensor was carried out. Despite the high-resolution of the micro-CT scan, presence of noise within the air cells along with missing information in some micro-channels suggested the need of image enhancement. This paper proposes an adaptive enhancement of the micro-channels based on a local structure analysis while minimizing the impact of noise on the overall data. Comparison with an anisotropic diffusion PDE based scheme was also performed.

    Place, publisher, year, edition, pages
    IEEE, 2016
    Series
    International Conference on Image Processing Theory Tools and Applications (IPTA), E-ISSN 2154-512X
    Keywords
    Micro-channels, Structure tensor analysis, Image enhancement, Adaptive filtering, Human temporal bone, Mastoid bone
    National Category
    Medical Engineering
    Identifiers
    urn:nbn:se:liu:diva-134434 (URN)10.1109/IPTA.2016.7821019 (DOI)000393589800071 ()9781467389105 (ISBN)9781467389112 (ISBN)
    Conference
    6th International Conference on Image Processing Theory Tools and Applications (IPTA), Oulu, Finland, 12-15 December 2016
    Note

    Funding agencies: Obel Family Foundation (Denmark)

    Available from: 2017-02-13 Created: 2017-02-13 Last updated: 2017-06-21
  • 25.
    Dassie, Justin P.
    et al.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Hernandez, Luiza I.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Thomas, Gregory S.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Long, Matthew E.
    Molecular and Cellular Biology Program, University of of Iowa, Iowa City, IA, United States; Inflammation Program, University of of Iowa, Iowa City, IA, United States.
    Rockey, William M.
    Department of Radiation Oncology, University of of Iowa, Iowa City, IA, United States.
    Howell, Craig A.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Chen, Yani
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Hernandez, Frank J
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Liu, Xiu Y.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Wilson, Mary E.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States; Department of Microbiology, University of of Iowa, Iowa City, IA, United States; Veterans Affairs Medical Center, University of of Iowa, Iowa City, IA, United States.
    Allen, Lee-Ann
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States; Molecular and Cellular Biology Program, University of of Iowa, Iowa City, IA, United States; Inflammation Program, University of of Iowa, Iowa City, IA, United States; Department of Microbiology, University of of Iowa, Iowa City, IA, United States; Veterans Affairs Medical Center, University of of Iowa, Iowa City, IA, United States.
    Vaena, Daniel A.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Meyerholz, David K.
    Department of Pathology, University of of Iowa, Iowa City, IA, United States.
    Giangrande, Paloma H.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States; Molecular and Cellular Biology Program, University of of Iowa, Iowa City, IA, United States; Department of Radiation Oncology, University of of Iowa, Iowa City, IA, United States.
    Targeted inhibition of prostate cancer metastases with an RNA aptamer to prostate-specific membrane antigen2014In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 22, no 11, p. 1910-1922Article in journal (Refereed)
    Abstract [en]

    Cell-targeted therapies (smart drugs), which selectively control cancer cell progression with limited toxicity to normal cells, have been developed to effectively treat some cancers. However, many cancers such as metastatic prostate cancer (PC) have yet to be treated with current smart drug technology. Here, we describe the thorough preclinical characterization of an RNA aptamer (A9g) that functions as a smart drug for PC by inhibiting the enzymatic activity of prostate-specific membrane antigen (PSMA). Treatment of PC cells with A9g results in reduced cell migration/invasion in culture and metastatic disease in vivo. Importantly, A9g is safe in vivo and is not immunogenic in human cells. Pharmacokinetic and biodistribution studies in mice confirm target specificity and absence of non-specific on/off-target effects. In conclusion, these studies provide new and important insights into the role of PSMA in driving carcinogenesis and demonstrate critical endpoints for the translation of a novel RNA smart drug for advanced stage PC. © The American Society of Gene amp; Cell Therapy.

  • 26.
    Diczfalusy, Elin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology.
    Broberg, Sarah
    Linköping University, Department of Physics, Chemistry and Biology.
    Non-Invasive Methods for Detecting Drug and Alcohol Impaired Drivers: - a Study of Alcohol and Drug Biomarkers and Optical Detection Techniques2009Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    In recent years, the use of alcohol and psychoactive drugs in combination withdriving has recieved increased attention. The lack of in-vehicle devices capable ofdetecting recent drug consumption and the difficulties associated with the breathbasedalcolocks in use today makes it interesting to investigate methods that areable to non-invasivelly measure analytes directly in the blood.

    The assignment of this project, commissioned by Volvo Technology Corporationand Volvo Car Corporation, is to map substances that constitute a possible threatto traffic safety, identify suitable detection markers as a proof of administrationof these substances, and study possible non-invasive techniques to detect thesemarkers. The objective is to present for Volvo if and how to continue evaluatingand developing a non-invasive detection device.

    The project has been carried out by performing an extensive literature study and averification experiment. From the literature review, a number of substances affectingdriving performance could be identified, and a metabolic study was performedfor each drug to map suitable biomarkers. Furthermore, two potential techniquesfor non-invasive detection, near-infrared Raman spectroscopy and near-infraredspectroscopy, were found and evaluated. The experiment was conducted usingnear-infrared Raman spectroscopy, with the aim of investigating the sensitivityand linearity of the method for ethanol detection.

    Based on the theoretical evaluation, both near-infrared Raman spectroscopy andnear-infrared spectroscopy are expected to have potential for non-invasive detectionof ethanol. The experiment further proved the theoretical conclusionsmade for near-infrared Raman spectroscopy. However, neither of the techniquesis thought to have potential for drug detection.Altogether, we believe that non-invasive ethanol detection is possible, but suggestfurther experiments in order to determine which technique to be preferred.

  • 27.
    Duran Rosich, David
    et al.
    ViRVIG Group, UPC Barcelona, Barcelona, Spain.
    Hermosilla, Pedro
    Visual Computing Group, U. Ulm, Ulm, Germany.
    Ropinski, Timo
    Visual Computing Group, U. Ulm, Ulm, Germany.
    Kozlikova, Barbora
    Masaryk University, Brno, Czech Republic.
    Vinacua, Àlvar
    ViRVIG Group, UPC Barcelona, Barcelona, Spain.
    Vazquez, Pere-Pau
    ViRVIG Group, UPC Barcelona, Barcelona, Spain.
    Visualization of Large Molecular Trajectories2018In: IEEE Transactions on Visualization and Computer Graphics, ISSN 1077-2626, E-ISSN 1941-0506Article in journal (Refereed)
    Abstract [en]

    The analysis of protein-ligand interactions is a time-intensive task. Researchers have to analyze multiple physico-chemical properties of the protein at once and combine them to derive conclusions about the protein-ligand interplay. Typically, several charts are inspected, and 3D animations can be played side-by-side to obtain a deeper understanding of the data. With the advances in simulation techniques, larger and larger datasets are available, with up to hundreds of thousands of steps. Unfortunately, such large trajectories are very difficult to investigate with traditional approaches. Therefore, the need for special tools that facilitate inspection of these large trajectories becomes substantial. In this paper, we present a novel system for visual exploration of very large trajectories in an interactive and user-friendly way. Several visualization motifs are automatically derived from the data to give the user the information about interactions between protein and ligand. Our system offers specialized widgets to ease and accelerate data inspection and navigation to interesting parts of the simulation. The system is suitable also for simulations where multiple ligands are involved. We have tested the usefulness of our tool on a set of datasets obtained from protein engineers, and we describe the expert feedback.

  • 28.
    Erdtman, Edvin
    et al.
    Institutionen för naturvetenskap and Modelling and Simulation Research Center, Örebro University, Sweden.
    dos Santos, Daniel J. V. A.
    i. Med. UL/Institute for Medicine and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon. Av. Prof. Gama Pinto, Lisbon, Portugal.
    Löfgren, Lennart
    Head- and Neck Oncology Center, Örebro University Hospital, Örebro, Sweden.
    Eriksson, Leif A.
    Örebro universitet, Institutionen för naturvetenskap.
    Modelling the behavior of 5-aminolevulinic acid and its alkyl esters in a lipid bilayer2008In: Chemical Physics Letters, ISSN 0009-2614, E-ISSN 1873-4448, Vol. 463, no 1-3, p. 178-182Article in journal (Refereed)
    Abstract [en]

    5-Aminolevulinic acid (5ALA) and ester derivates thereof are used as prodrugs in photodynamic therapy (PDT). The behavior of 5ALA and three esters of 5ALA in a DPPC lipid bilayer is investigated. In particular, the methyl ester displays a very different free energy profile, where the highest barrier is located in the region with highest lipid density, while the others have their peak in the middle of the membrane, and also displays a considerably lower permeability coefficient than neutral 5ALA and the ethyl ester. The zwitterion of 5ALA has the highest permeability constant, but a significant free energy minimum in the polar head-group region renders an accumulation in this region.

  • 29.
    Erdtman, Edvin
    et al.
    Örebro universitet, Institutionen för naturvetenskap.
    Eriksson, Leif A.
    Örebro universitet, Institutionen för naturvetenskap.
    Theoretical study of 5-aminolevulinic acid tautomerization: a novel self-catalyzed mechanism2008In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 112, no 18, p. 4367-4374Article in journal (Refereed)
    Abstract [en]

    5-Aminolevulinic acid (5ALA) is the key synthetic building block in protoporphyrin IX (PpIX), the heme chromophore in mitochondria. In this study density functional theory calculations were performed on the tautomers of 5ALA and the tautomerization reaction mechanism from its enolic forms (5-amino-4-hydroxypent-3-enoic acid and 5-amino-4-hydroxypent-4-enoic acid) to the more stable 5ALA. The hydrated form 5-amino-4,4-dihydroxypentanoic acid was also studied. The lowest energy pathway of 5ALA tautomerization is by means of autocatalysis, in that an oxygen of the carboxylic group transfers the hydrogen atom as a "crane", with an activation energy of similar to 15 kcal/mol. This should be compared to the barriers of about 35 kcal/mol for water assisted tautomerization, and 60 kcal/mol for direct hydrogen transfer. For hydration of 5ALA, the water catalyzed activation barrier is found to be similar to 35 kcal/mol, approximately 5 kcal/mol lower than direct hydration.

  • 30.
    Fernius, Josefin
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Starkenberg, Annika
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Thor, Stefan
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Bar-coding neurodegeneration: identifying subcellular effects of human neurodegenerative disease proteins using Drosophila leg neurons2017In: Disease Models and Mechanisms, ISSN 1754-8403, E-ISSN 1754-8411, Vol. 10, no 8, p. 1027-1038Article in journal (Refereed)
    Abstract [en]

    Genetic, biochemical and histological studies have identified a number of different proteins as key drivers of human neurodegenerative diseases. Although different proteins are typically involved in different diseases, there is also considerable overlap. Addressing disease protein dysfunction in an in vivo neuronal context is often time consuming and requires labor-intensive analysis of transgenic models. To facilitate the rapid, cellular analysis of disease protein dysfunction, we have developed a fruit fly (Drosophila melanogaster) adult leg neuron assay. We tested the robustness of 41 transgenic fluorescent reporters and identified a number that were readily detected in the legs and could report on different cellular events. To test these reporters, we expressed a number of human proteins involved in neurodegenerative disease, in both their mutated and wild-type versions, to address the effects on reporter expression and localization. We observed strikingly different effects of the different disease proteins upon the various reporters with, for example, A beta(1-42) being highly neurotoxic, tau, parkin and HTT128Q affecting mitochondrial distribution, integrity or both, and A beta(1-42), tau, HTT128Q and ATX1(82Q) affecting the F-actin network. This study provides proof of concept for using the Drosophila adult leg for inexpensive and rapid analysis of cellular effects of neurodegenerative disease proteins in mature neurons.

  • 31.
    Folkesson, Maggie
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Sadowska, Natalia
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Karlsson, Matts
    Linköping University, Department of Management and Engineering, Applied Thermodynamics and Fluid Mechanics. Linköping University, Faculty of Science & Engineering. Linköping University, Center for Medical Image Science and Visualization (CMIV).
    Carlhäll, Carl-Johan
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Heart and Medicine Center, Department of Clinical Physiology in Linköping. Linköping University, Center for Medical Image Science and Visualization (CMIV).
    Länne, Toste
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Heart and Medicine Center, Department of Thoracic and Vascular Surgery.
    Wågsäter, Dick
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Jensen, Lasse
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pharmacology.
    Differences in cardiovascular toxicities associated with cigarette smoking and snuff use revealed using novel zebrafish models2016In: Biology Open, ISSN 2046-6390, Vol. 5, no 7, p. 970-978Article in journal (Refereed)
    Abstract [en]

    Tobacco use is strongly associated with cardiovascular disease and the only avoidable risk factor associated with development of aortic aneurysm. While smoking is the most common form of tobacco use, snuff and other oral tobacco products are gaining popularity, but research on potentially toxic effects of oral tobacco use has not kept pace with the increase in its use. Here, we demonstrate that cigarette smoke and snuff extracts are highly toxic to developing zebrafish embryos. Exposure to such extracts led to a palette of toxic effects including early embryonic mortality, developmental delay, cerebral hemorrhages, defects in lymphatics development and ventricular function, and aneurysm development. Both cigarette smoke and snuff were more toxic than pure nicotine, indicating that other compounds in these products are also associated with toxicity. While some toxicities were found following exposure to both types of tobacco product, other toxicities, including developmental delay and aneurysm development, were specifically observed in the snuff extract group, whereas cerebral hemorrhages were only found in the group exposed to cigarette smoke extract. These findings deepen our understanding of the pathogenic effects of cigarette smoking and snuff use on the cardiovascular system and illustrate the benefits of using zebrafish to study mechanisms involved in aneurysm development.

  • 32.
    Franz, F
    et al.
    Clinic of Endocrinology and Nephrology, Department of Internal Medicine, Neurology and Dermatology, University of Leipzig, Leipzig, Germany.
    Weidinger, C
    Clinic of Endocrinology and Nephrology, Department of Internal Medicine, Neurology and Dermatology, University of Leipzig, Leipzig, Germany.
    Krause, K
    Clinic of Endocrinology and Nephrology, Department of Internal Medicine, Neurology and Dermatology, University of Leipzig, Leipzig, Germany.
    Gimm, Oliver
    Department of General, Visceral, and Vascular Surgery, Martin-Luther-University of Halle-Wittenberg, Halle/Saale, Germany.
    Dralle, H
    Department of General, Visceral, and Vascular Surgery, Martin-Luther-University of Halle-Wittenberg, Halle/Saale, Germany.
    Führer, D
    Department of Endocrinology & Metabolism and Division of Laboratory Research, University Hospital Essen, Essen, Germany.
    The Transcriptional Regulation of FOXO Genes in Thyrocytes.2016In: Hormone and Metabolic Research, ISSN 0018-5043, E-ISSN 1439-4286, Vol. 48, no 9, p. 601-6Article in journal (Refereed)
    Abstract [en]

    FOXO transcription factors are key regulators of DNA damage repair, proliferation and apoptosis in thyrocytes. Thyroid malignancies show impaired FOXO function. In this study, we investigated the transcriptional regulation of FOXO isoforms in thyroid epithelial cells. mRNA expression of FOXO isoforms (FOXO1, 3 and 4) was determined in FRTL-5 cells stimulated with different growth factors and H2O2. Furthermore, the impact of PI3K/AKT signalling on FOXO transcription was investigated in PI3K p110α mutant FRTL-5 cells and regulatory dependence of FOXO transcription on FOXO was studied in FRTL-5 cells with hFOXO3 overexpression. Finally, mRNA expression levels of FOXO isoforms were determined in human epithelial thyroid tumours. Growth factor deprivation induced transcription of FOXO1, 3 and 4, whereas insulin stimulation decreased FOXO1 and FOXO4 transcription in FRTL-5 cells. Inhibition of the PI3K/AKT cascade amplified FOXO1 and FOXO4 expression. In contrast, H2O2 and TSH did not influence FOXO transcription in thyrocytes. Overexpression of PI3K p110α inhibited FOXO3 and induced FOXO4 transcription. In human thyroid tumours, FOXO1 and FOXO3 mRNA levels were significantly downregulated in papillary thyroid carcinoma when compared to normal tissues. In contrast, follicular thyroid carcinomas showed significant upregulation of FOXO4 mRNA.In this paper, we demonstrate an influence of PI3K signalling on FOXO transcription in thyrocytes. Moreover, we show that thyroid cancers exhibit alterations in FOXO transcription besides the previously reported alterations in posttranslational FOXO3 regulation. These findings may add to the concept of targeting the PI3K pathway in advanced thyroid cancers.

  • 33.
    Gallardo, Rodrigo
    et al.
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Ramakers, Meine
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    De Smet, Frederik
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Claes, Filip
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Khodaparast, Ladan
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Khodaparast, Laleh
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Couceiro, Jose R.
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Langenberg, Tobias
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Siemons, Maxime
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Nyström, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Young, Laurence J.
    University of Cambridge, England; University of Leeds, England; University of Leeds, England.
    Laine, Romain F.
    University of Cambridge, England.
    Young, Lydia
    University of Cambridge, England; University of Leeds, England; University of Leeds, England.
    Radaelli, Enrico
    VIB Centre Biol Disease, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Benilova, Iryna
    VIB Centre Biol Disease, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Kumar, Manoj
    Katholieke University of Leuven, Belgium.
    Staes, An
    VIB, Belgium; University of Ghent, Belgium.
    Desager, Matyas
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Beerens, Manu
    Katholieke University of Leuven, Belgium.
    Vandervoort, Petra
    Katholieke University of Leuven, Belgium.
    Luttun, Aernout
    Katholieke University of Leuven, Belgium.
    Gevaert, Kris
    VIB, Belgium; University of Ghent, Belgium.
    Bormans, Guy
    Katholieke University of Leuven, Belgium.
    Dewerchin, Mieke
    Katholieke University of Leuven, Belgium; VIB, Belgium.
    Van Eldere, Johan
    Katholieke University of Leuven, Belgium.
    Carmeliet, Peter
    Katholieke University of Leuven, Belgium; VIB, Belgium.
    Vande Velde, Greetje
    Katholieke University of Leuven, Belgium.
    Verfaillie, Catherine
    Katholieke University of Leuven, Belgium.
    Kaminski, Clemens F.
    University of Cambridge, England.
    De Strooper, Bart
    VIB Centre Biol Disease, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Serpell, Louise
    University of Sussex, England.
    Schymkowitz, Joost
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Rousseau, Frederic
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    De novo design of a biologically active amyloid2016In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 354, no 6313, p. 720-+Article in journal (Refereed)
    Abstract [en]

    Most human proteins possess amyloidogenic segments, but only about 30 are associated with amyloid-associated pathologies, and it remains unclear what determines amyloid toxicity. We designed vascin, a synthetic amyloid peptide, based on an amyloidogenic fragment of vascular endothelial growth factor receptor 2 (VEGFR2), a protein that is not associated to amyloidosis. Vascin recapitulates key biophysical and biochemical characteristics of natural amyloids, penetrates cells, and seeds the aggregation of VEGFR2 through direct interaction. We found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, amyloid toxicity here appears to be both protein-specific and conditional-determined by VEGFR2 loss of function in a biological context in which target protein function is essential.

  • 34.
    Gao, Feng
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, Faculty of Science & Engineering.
    A New Acceptor for Highly Efficient Organic Solar Cells2019In: JOULE, ISSN 2542-4351, Vol. 3, no 4, p. 908-909Article in journal (Other academic)
    Abstract [en]

    Research into organic solar cells has gone from pure scientific curiosity to a topic of commercial relevance in the past few years, as a result of rapid development of non-fullerene acceptors. This transition is mainly driven by the development of new materials. Recently in Joule, Zou and co-workers developed a new acceptor material and reached a record efficiency for single-junction organic solar cells.

  • 35.
    Ghavami, Saeid
    et al.
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Asoodeh, Ahmad
    Department of Biophysics and Biochemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran; Department of Chemistry Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran .
    Klonisch, Thomas
    Department of Human Anatomy and Cell Sciences, and Manitoba Institute of Child Health, Winnipeg, Canada.
    Halayko, Andrew J.
    Department of Physiology, University of Manitoba, Winnipeg, MB, Canada; Manitoba Institute of Child Health, Winnipeg, MB, Canada.
    Kadkhoda, Kamran
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Kroczak, Tadeusz J.
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Gibson, Spencer B.
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada; BioApplications Enterprises, Winnipeg, Canada.
    Booy, Evan P.
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Naderi-Manesh, Hossein
    Department of Biophysics and Biochemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran.
    Los, Marek Jan
    BioApplications Enterprises, Winnipeg, MB, Canada.
    Brevinin-2R semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway2008In: Journal of Cellular and Molecular Medicine (Print), ISSN 1582-1838, E-ISSN 1582-4934, Vol. 12, no 3, p. 1005-1022Article in journal (Refereed)
    Abstract [en]

    Brevinin-2R is a novel non-hemolytic defensin that was isolated from the skin of the frog Rana ridibunda. It exhibits preferential cytotoxicity towards malignant cells, including Jurkat (T-cell leukemia), BJAB (B-cell lymphoma), HT29/219, SW742 (colon carcinomas), L929 (fibrosarcoma), MCF-7 (breast adenocarcinoma), A549 (lung carcinoma), as compared to primary cells including peripheral blood mononuclear cells (PBMC), T cells and human lung fibroblasts. Jurkat and MCF-7 cells overexpressing Bcl2, and L929 and MCF-7 over-expressing a dominant-negative mutant of a pro-apoptotic BNIP3 (ΔTM-BNIP3) were largely resistant towards Brevinin-2R treatment. The decrease in mitochondrial membrane potential (ΔΨm), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin-2R-triggered death. Brevinin-2R interacts with both early and late endosomes. Lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin-B and cathepsin-L prevented Brevinin-2R-induced cell death. Autophagosomes have been detected upon Brevinin-2R treatment. Our results show that Brevinin-2R activates the lysosomalmitochondrial death pathway, and involves autophagy-like cell death.

  • 36.
    Ghavami, Saeid
    et al.
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
    Hashemi, M.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Kadkhoda, K.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Alavian, S. M.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Bay, G. H.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Apoptosis in liver diseases - detection and therapeutic applications2005In: Medical Science Monitor, ISSN 1234-1010, E-ISSN 1643-3750, Vol. 11, no 11, p. RA337-RA345Article, review/survey (Refereed)
    Abstract [en]

    The liver is continuously exposed to a large antigenic load that includes pathogens, toxins, tumor cells and dietary antigens. Amongst the hepatitis viruses, only hepatitis B virus (HBV) and hepatitis C virus (HCV) cause chronic hepatitis, which can progress to cirrhosis and hepatocellular carcinoma. Of the different antiviral defense systems employed by the tissue, apoptosis significantly contributes to the prevention of viral replication, dissemination, and persistence. Loss of tolerance to the liver autoantigens may result in autoimmune hepatitis (AIH). This review outlines the recent findings that highlight the role and mechanisms of apoptotic processes in the course of liver diseases. Among factors that contribute to liver pathology, we discuss the role of tumor necrosis factor (TNF)-alpha, HBx, ds-PKR, TRAIL, FasL, and IL-1 alpha. Since TNF and FasL-induced hepatocyte apoptosis is implicated in a wide range of liver diseases, including viral hepatitis, alcoholic hepatitis, ischemia/reperfusion liver injury, and fulminant hepatic failure, these items will be discussed in greater detail in this review. We also highlight some recent discoveries that pave the way for the development of new therapeutic strategies by protecting hepatocytes (for example by employing Bcl-2, Bcl-X-L or A1/Bfl-1, IAPs, or synthetic caspase inhibitors), or by the induction of apoptosis in stellate cells. The assessment of the severity of liver disease, as well as monitoring of patients with chronic liver disease, remains a major challenge in clinical hepatology practice. Therefore, a separate chapter is devoted to a novel cytochrome c - based method useful for the diagnosis and monitoring of fulminant hepatitis.

  • 37.
    Glogowska, Aleksandra
    et al.
    Department of Human Anatomy and Cell Science, Winnipeg, Manitoba, Canada.
    Pyka, Janette
    Clinics of Surgery, Medical Faculty, Martin Luther University Halle-Wittenberg, Halle, Germany.
    Kehlen, Astrid
    Probiodrug AG, Weinbergweg, Halle, Germany.
    Los, Marek Jan
    BioApplications Enterprises, Winnipeg, MB, Canada.
    Perumal, Paul
    Department of Human Anatomy and Cell Science, Winnipeg, Manitoba, Canada.
    Weber, Ekkehard
    Cheng, Sheue-yann
    Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA.
    Hoang-Vu, Cuong
    Clinics of Surgery, Medical Faculty, Martin Luther University Halle-Wittenberg, Halle, Germany.
    Klonisch, Thomas
    Department of Human Anatomy and Cell Science; Department of Medical Microbiology and Infectious Diseases, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
    The cytoplasmic domain of proEGF negatively regulates motility and elastinolytic activity in thyroid carcinoma cells2008In: Neoplasia, ISSN 1522-8002, E-ISSN 1476-5586, Vol. 10, no 10, p. 1120-1130Article in journal (Refereed)
    Abstract [en]

    The intracellular domains of the membrane-anchoring regions of some precursors of epidermal growth factor (EGF) family members have intrinsic biologic activities. We have determined the role of the human proEGF cytoplasmic domain (proEGFcyt) as part of the proEGF transmembrane-anchored region (proEGFctF) in the regulation of motility and elastinolytic invasion in human thyroid cancer cells. We found proEGFctF to act as a negative regulator of motility and elastin matrix penetration and the presence of proEGFcyt or proEGF22.23 resulted in a similar reduction in motility and elastinolytic migration. This activity was counteracted by EGF-induced activation of EGF receptor signaling. Decreased elastinolytic migratory activity in the presence of proEGFctF and proEGFcyt/proEGF22.23 coincided with decreased secretion of elastinolytic procathepsin L. The presence of proEGFctF and proEGFcyt/proEGF22.23 coincided with the specific transcriptional up-regulation of t-SNARE member SNAP25. Treatment with siRNA-SNAP25 resulted in motility and elastin migration being restored to normal levels. Epidermal growth factor treatment down-regulated SNAP25 protein by activating EGF receptor-mediated proteasomal degradation of SNAP25. These data provide first evidence for an important function of the cytoplasmic domain of the human proEGF transmembrane region as a novel suppressor of motility and cathepsin L-mediated elastinolytic invasion in human thyroid carcinoma cells and suggest important clinical implications for EGF-expressing tumors.

  • 38.
    Guan, Na N.
    et al.
    Department of Molecular Medicine and Surgery, Section of Urology, Karolinska Institutet, Stockholm, Sweden / Department of Urology, Karolinska University Hospital, Stockholm, Sweden / Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
    Sharma, Nimish
    Department of Molecular Medicine and Surgery, Section of Urology, Karolinska Institutet, Stockholm, Sweden / Department of Urology, Karolinska University Hospital, Stockholm, Sweden.
    Hallén‐Grufman, Katarina
    Department of Molecular Medicine and Surgery, Section of Urology, Karolinska Institutet, Stockholm, Sweden / Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
    Jager, Edwin W. H.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor and Actuator Systems. Linköping University, Faculty of Science & Engineering.
    Svennersten, Karl
    Department of Molecular Medicine and Surgery, Section of Urology, Karolinska Institutet, Stockholm, Sweden / Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
    The role of ATP signalling in response to mechanical stimulation studied in T24 cells using new microphysiological tools2018In: Journal of Cellular and Molecular Medicine (Print), ISSN 1582-1838, E-ISSN 1582-4934, Vol. 22, no 4, p. 2319-2328Article in journal (Refereed)
    Abstract [en]

    The capacity to store urine and initiate voiding is a valued characteristic of the human urinary bladder. To maintain this feature, it is necessary that the bladder can sense when it is full and when it is time to void. The bladder has a specialized epithelium called urothelium that is believed to be important for its sensory function. It has been suggested that autocrine ATP signalling contributes to this sensory function of the urothelium. There is well‐established evidence that ATP is released via vesicular exocytosis as well as by pannexin hemichannels upon mechanical stimulation. However, there are still many details that need elucidation and therefore there is a need for the development of new tools to further explore this fascinating field. In this work, we use new microphysiological systems to study mechanostimulation at a cellular level: a mechanostimulation microchip and a silicone‐based cell stretcher. Using these tools, we show that ATP is released upon cell stretching and that extracellular ATP contributes to a major part of Ca2+ signalling induced by stretching in T24 cells. These results contribute to the increasing body of evidence for ATP signalling as an important component for the sensory function of urothelial cells. This encourages the development of drugs targeting P2 receptors to relieve suffering from overactive bladder disorder and incontinence.

  • 39.
    Gunnar, Erika
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Regulatory programs controlling profileration during Drosophila nervous system development2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The central nervous system (CNS) is the most complex organ in the body, responsible for complex functions, including thinking, reasoning and memory. The CNS contains cells of many different types, often generated in vast numbers. Hence, CNS development requires precise genetic control of both cell fate and of cell proliferation, to generate the right number of cells, with the proper identity, and in the proper location. The cells also need to make connections with each other for correct signaling and function. This complexity evokes the question of how this is regulated. How does the stem cells, responsible for building the CNS, know how many times to divide, and how does the daughters know which identity to acquire and in which location they shall end up? During Drosophila melanogaster development, the neuroblasts (NBs) are responsible for generating the CNS. In each hemisegment, every NB is unique in identity, and generates a predetermined number of daughters with specific identities. The lineages of different NBs vary in size, but are always the same for each specific NB, and the division modes of each NBs is hence stereotyped. Most NBs start dividing by renewing themselves while generating daughters that will in turn divide once to generate two neurons and/or glia (denoted type I mode). Many, maybe all, NBs later switch to generating daughters that will differentiate directly into a neuron or glia (denoted type 0 mode). This type I>0 switch occurs at different time-points during lineage progression, and influences the total numbers of cells generated from a single NB.

    The work presented in this thesis aimed at investigating the genetic regulation of proliferation, with particular focus on the type I>0 switch. In the first project, the implication of the Notch pathway on the type I>0 switch was studied. Mutants of the Notch pathway do not switch, and the results show that the Notch pathway regulates the switch by activation of several target genes, both regulators and cell cycle genes. One of the target genes, the E(spl)-C genes, have been difficult to study due to functional redundancy. This study reveals that even though they can functionally compensate for each other, they have individual functions in different lineages. Regarding cell cycle genes, both Notch and E(spl)-C regulate several key cell cycle genes, and molecular analysis indicated that this regulation is direct. In the second project we studied the seq gene, previously identified in a genetic screen. We found that seq controls the type I>0 switch by regulating the key cell cycle genes, but also through interplay with the Notch pathway. Notch and seq stop proliferation, and in the third project we wanted to identify genes that drive proliferation. We found that there is battery of early NB genes, socalled early factors, which activate the cell cycle, and drive NB and daughter proliferation. These are gradually replaced by late regulators, and the interplay between early and late factors acts to achieve precise control of lineage progression.

    The work presented here increases our understanding of how regulatory programs act to control the development of the CNS; to generate the right number of cells of different identities. These results demonstrate the importance of correct regulation of proliferation in both stem cells and daughters. Problems in this control can result in either an underdeveloped CNS or loss of control such as in cancer. Knowledge about these regulatory programs can contribute to the development of therapeutics against these diseases.

    List of papers
    1. Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling
    Open this publication in new window or tab >>Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling
    Show others...
    2016 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 4, article id e1005984Article in journal (Refereed) Published
    Abstract [en]

    The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21(CIP1)/p27(KIP1)/p57(Kip2)). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems.

    Place, publisher, year, edition, pages
    PUBLIC LIBRARY SCIENCE, 2016
    National Category
    Clinical Medicine
    Identifiers
    urn:nbn:se:liu:diva-128759 (URN)10.1371/journal.pgen.1005984 (DOI)000375231900032 ()27070787 (PubMedID)
    Note

    Funding Agencies|Knut and Alice Wallenberg Foundation [KAW2012.0101]; Swedish Research Council [621-2010-5214]; Swedish Cancer Foundation [120531]

    Available from: 2016-05-31 Created: 2016-05-30 Last updated: 2019-03-13
    2. sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system
    Open this publication in new window or tab >>sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system
    2016 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 143, no 20, p. 3774-3784Article in journal (Refereed) Published
    Abstract [en]

    Neural progenitors typically divide asymmetrically to renew themselves, while producing daughters with more limited potential. In the Drosophila embryonic ventral nerve cord, neuroblasts initially produce daughters that divide once to generate two neurons/glia (type I proliferation mode). Subsequently, many neuroblasts switch to generating daughters that differentiate directly (type 0). This programmed type I>0 switch is controlled by Notch signaling, triggered at a distinct point of lineage progression in each neuroblast. However, how Notch signaling onset is gated was unclear. We recently identified Sequoia (Seq), a C2H2 zinc-finger transcription factor with homology to Drosophila Tramtrack (Ttk) and the positive regulatory domain (PRDM) family, as important for lineage progression. Here, we find that seq mutants fail to execute the type I>0 daughter proliferation switch and also display increased neuroblast proliferation. Genetic interaction studies reveal that seq interacts with the Notch pathway, and seq furthermore affects expression of a Notch pathway reporter. These findings suggest that seq may act as a context-dependent regulator of Notch signaling, and underscore the growing connection between Seq, Ttk, the PRDM family and Notch signaling.

    Place, publisher, year, edition, pages
    The Company of Biologists Ltd, 2016
    Keywords
    Lineage tree, Cell cycle, Asymmetric division, Combinatorial control, Notch
    National Category
    Cell and Molecular Biology Biochemistry and Molecular Biology Cell Biology Medical Biotechnology
    Identifiers
    urn:nbn:se:liu:diva-132739 (URN)10.1242/dev.139998 (DOI)000393452500013 ()27578794 (PubMedID)
    Note

    Funding agencies: Swedish Research Council (Vetenskapsradet); Knut and Alice Wallenberg Foundation (Knut och Alice Wallenbergs Stiftelse); Swedish Cancer Foundation (Cancerfonden)

    Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2019-03-13Bibliographically approved
  • 40.
    Halling Linder, Cecilia
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
    Biochemical and functional properties of mammalian bone alkaline phosphatase isoforms during osteogenesis2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The human skeleton is a living and dynamic tissue that constantly is being renewed in a process called bone remodeling. Old bone is resorbed by osteoclasts and new bone is formed by osteoblasts. Bone is a composite material made up by mineral crystals in the form of hydroxyapatite (calcium and phosphate) that provides the hardness of bone, and collagen fibrils that provides elasticity and flexibility. Alkaline phosphatase (ALP) is a family of enzymes that is present in most species and catalyzes the hydrolysis of various phosphomonoesters at alkaline pH. Despite the generalized use of ALP as a biochemical marker of bone formation, the precise function of bone ALP (BALP) is only now becoming clear. Three circulating human BALP isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue.

    Paper I. Three endogenous phosphocompounds, (i.e., inorganic pyrophosphate (PPi), pyridoxal 5′-phosphate (PLP) and phosphoethanolamine (PEA)), have been suggested to serve as  physiological substrates for BALP. The BALP isoforms display different catalytic properties towards PPi and PLP, which is attributed to their distinct N-linked glycosylation patterns. The catalytic activity, using PEA as substrate, was barely detectable for all BALP isoforms indicating that PEA is not a physiological substrate for BALP.

    Paper II. Mouse serum ALP is frequently measured and interpreted in mammalian bone research. However, little is known about the circulating ALPs in mice and their relation to human ALP. We characterized the circulating and tissue-derived mouse ALP isozymes and isoforms from mixed strains of wild-type and knockout mice. All four BALP isoforms (B/I, B1x, B1, and B2) were identified in mouse serum and bone tissues, in good correspondence with those found in human bones. All mouse tissues, except liver, contained significant ALP activities. This is a notable difference as human liver contains vast amounts of ALP.

    Paper III. The objective of this study was to investigate the binding properties of human collagen type I to human BALP, including the two BALP isoforms B1 and B2, together with ALP from human liver, human placenta and E. coli. A surface plasmon resonance-based analysis showed that BALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. The B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform, indicating that glycosylation differences in human ALPs are of crucial importance for protein–protein interactions with collagen type I.

    Paper IV. Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and frequently used as a marker of bone resorption. Intriguingly, recent studies show that TRAP is also expressed in osteoblasts and osteocytes. TRAP displays enzymatic activity towards the endogenous substrates for BALP, i.e., PPi and PLP. Both TRAP and BALP can alleviate the inhibitory effect of osteopontin on mineralization by dephosphorylation, which suggests a novel role for TRAP in skeletal mineralization.

    List of papers
    1. Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms.
    Open this publication in new window or tab >>Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms.
    2009 (English)In: Bone, ISSN 1873-2763, Vol. 45, no 5, p. 987-993Article in journal (Refereed) Published
    Abstract [en]

    Three circulating human bone alkaline phosphatase (BALP) isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD(10), a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia. The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PP(i)), pyridoxal 5'-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation. The k(cat)/K(M) was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD(10) was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates. In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP-flag and the sALP-FcD(10) isoforms faithfully mimic the biological properties of the human BALP isoforms in vivo validating the use of these recombinant enzymes in studies aimed at dissecting the pathophysiology and treating hypophosphatasia.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-21351 (URN)10.1016/j.bone.2009.07.009 (DOI)19631305 (PubMedID)
    Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2016-04-14
    2. Isozyme profile and tissue-origin of alkaline phosphatases in mouse serum
    Open this publication in new window or tab >>Isozyme profile and tissue-origin of alkaline phosphatases in mouse serum
    Show others...
    2013 (English)In: Bone, ISSN 8756-3282, E-ISSN 1873-2763, Vol. 53, no 2, p. 399-408Article in journal (Refereed) Published
    Abstract [en]

    Mouse serum alkaline phosphatase (ALP) is frequently measured and interpreted in mammalian bone research. However, little is known about the circulating ALPs in mice and their relation to human ALP isozymes and isoforms. Mouse ALP was extracted from liver, kidney, intestine, and bone from vertebra, femur and calvaria tissues. Serum from mixed strains of wild-type (WT) mice and from individual ALP knockout strains were investigated, i.e., Alpl(-/-) (a.k.a. Akp2 encoding tissue-nonspecific ALP or TNALP), Akp3(-/-) (encoding duodenum-specific intestinal ALP or dIALP), and Alpi(-/-) (a.k.a. Akp6 encoding global intestinal ALP or gIALP). The ALP isozymes and isoforms were identified by various techniques and quantified by high-performance liquid chromatography. Results from the WT and knockout mouse models revealed identical bone-specific ALP isoforms (B/I. B1, and B2) as found in human serum, but in addition mouse serum contains the B1x isoform only detected earlier in patients with chronic kidney disease and in human bone tissue. The two murine intestinal isozymes, dIALP and gIALP, were also identified in mouse serum. All four bone-specific ALP isoforms (B/I, B1x, B1, and B2) were identified in mouse bones, in good correspondence with those found in human bones. All mouse tissues, except liver and colon, contained significant ALP activities. This is a notable difference as human liver contains vast amounts of ALP. Histochemical staining, Northern and Western blot analyses confirmed undetectable ALP expression in liver tissue. ALP activity staining showed some positive staining in the bile canaliculi for BALB/c and FVB/N WT mice, but not in C57BI/6 and ICR mice. Taken together, while the main source of ALP in human serum originates from bone and liver, and a small fraction from intestine (andlt;5%), mouse serum consists mostly of bone ALP, including all four isoforms, B/I, B1x, B1, and B2, and two intestinal ALP isozymes dIALP and gIALR We suggest that the genetic nomenclature for the Alpl gene in mice (i.e., ALP liver) should be reconsidered since murine liver has undetectable amounts of ALP activity. These findings should pave the way for the development of user-friendly assays measuring circulating bone-specific ALP in mouse models used in bone and mineral research.

    Place, publisher, year, edition, pages
    Elsevier, 2013
    Keywords
    Alkaline phosphatase, Bone, Glycosylation, Hypophosphatasia, Knockout mice, Mineralization
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-90744 (URN)10.1016/j.bone.2012.12.048 (DOI)000315763700010 ()
    Note

    Funding Agencies|County Council of Ostergotland in Sweden||National Institutes of Health, USA|DE012889|

    Available from: 2013-04-05 Created: 2013-04-05 Last updated: 2017-12-06
    3. Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I
    Open this publication in new window or tab >>Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I
    2016 (English)In: Calcified Tissue International, ISSN 0171-967X, E-ISSN 1432-0827, Vol. 98, no 3, p. 284-293Article in journal (Refereed) Published
    Abstract [en]

    Bone is a biological composite material comprised primarily of collagen type I and mineral crystals of calcium and phosphate in the form of hydroxyapatite (HA), which together provide its mechanical properties. Bone alkaline phosphatase (ALP), produced by osteoblasts, plays a pivotal role in the mineralization process. Affinity contacts between collagen, mainly type II, and the crown domain of various ALP isozymes were reported in a few in vitro studies in the 1980s and 1990s, but have not attracted much attention since, although such interactions may have important implications for the bone mineralization process. The objective of this study was to investigate the binding properties of human collagen type I to human bone ALP, including the two bone ALP isoforms B1 and B2. ALP from human liver, human placenta and E. coli were also studied. A surface plasmon resonance-based analysis, supported by electrophoresis and blotting, showed that bone ALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. Further, the B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform. Human bone and liver ALP (with identical amino acid composition) displayed pronounced differences in binding, revealing that post-translational glycosylation properties govern these interactions to a large extent. In conclusion, this study presents the first evidence that glycosylation differences in human ALPs are of crucial importance for protein–protein interactions with collagen type I, although the presence of the ALP crown domain may also be necessary. Different binding affinities among the bone ALP isoforms may influence the mineral-collagen interface, mineralization kinetics, and degree of bone matrix mineralization, which are important factors determining the material properties of bone.

    Place, publisher, year, edition, pages
    Springer-Verlag New York, 2016
    Keywords
    Alkaline phosphatase; Bone; Collagen; Glycosylation; Mineralization; Surface plasmon resonance
    National Category
    Endocrinology and Diabetes Dentistry
    Identifiers
    urn:nbn:se:liu:diva-127099 (URN)10.1007/s00223-015-0088-0 (DOI)000373744700008 ()26645431 (PubMedID)
    Note

    Funding agencies:  Region Ostergotland, Sweden

    Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2017-11-30Bibliographically approved
  • 41.
    Halvarsson, Camilla
    Linköping University, Department of Physics, Chemistry and Biology.
    Substitution of disulphide bonds to hydrophobic amino acids in BACE12009Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The study and understanding of Alzheimer’s disease on protein level is fundamentally important in the search for its treatment and there is a demand for proteins that can be used together with candidate drugs in crystallography trials. The refolding time reaching up to three weeks for beta-site APP cleaving enzyme 1 (BACE1), the proposed disease-generating protein, is presently not optimal and new protein constructs are needed. In attempts to shorten the refolding time the six cysteins in BACE1 were substituted to hydrophobic valine or alanine residues. The proteins, both wild type and mutant BACE1, were expressed in Escherichia coli, refolded for one week and purified by ion exchange chromatography and gel filtration. The final products were characterised by measuring stability, homogeneity and enzyme activity. There was significantly lower protein yield for the mutants compared to the wild type BACE1, indicating that generation of the disulphide bonds are important for correctly folded and stable BACE1. Also, it was found that the three different disulphide bonds are not equally important during refolding, with Cys278-Cys443 being the most important and Cys216-Cys420 and Cys330-Cys380 being of less importance. The present work shows that one week of refolding is enough for a sufficient protein yield of wt BACE1 and that the current refolding time for wt BACE1 can be shortened. Furthermore the disulphide bridges in BACE1 are important for forming an active protein with correct fold.

  • 42.
    Hashemi, Mohammad
    et al.
    Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.
    Ghavami, Saeid
    Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada; Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.
    Eshraghi, Mehdi
    Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, Univ. Manitoba, Winnipeg, Canada.
    Booy, Evan P.
    Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, Univ. Manitoba, Winnipeg, Canada.
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Cytotoxic effects of intra and extracellular zinc chelation on human breast cancer cells2007In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 557, no 1, p. 9-19Article in journal (Refereed)
    Abstract [en]

    Zinc is an essential trace element with cofactor functions in a large number of proteins of intermediary metabolism, hormone secretion pathways, immune defence mechanisms, and as a cofactor of transcription factors it is also involved in the control of gene expression. Our study demonstrates that the modulation of intra and extracellular zinc alone is sufficient to induce metabolic changes or even apoptosis in two model human breast cancer cell lines MCF-7 and MDA-MB468. Treatment of breast cancer cells with different concentrations of a cell membrane permeable zinc chelator, N,N,N'N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the membrane impermeable zinc chelator, diethylenctriaminepentacetic acid, (DTPA) resulted in a significant increase of cell death. Features of apoptosis, such as chromatin condensation and nuclear fragmentation accompanied the DTPA and TPEN-induced cell death. A significant increase in the activity of caspase-9 was observed in both cell lines; whereas, caspase-3 activity was only increased in MDA-MB468 cells since caspase-3 is not expressed in MCF-7 cells. Caspase-8 activation was negligible in both cell lines. Addition of Zn2+ or Cu2+ prevented DTPA and TPEN-induced cytotoxicity, indicating that both bivalent cations can be replaced functionally to a certain extent in our experimental system. Interestingly, addition of Ca2+, or Mg2+ had no effect. The antioxidant N-Acetyl-L-Cysteine inhibited the cytotoxic effect of DTPA and TPEN, indicating that oxidative stress is the likely mediator of Zn-deficiency-related cell death. (c) 2006 Elsevier B.V. All rights reserved.

  • 43.
    Hauff, K.
    et al.
    Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada; Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Zamzow, C.
    Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada.
    Law, W. J.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    De Melo, J.
    Department of Anatomy, University of Manitoba, Winnipeg, Canada; Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Kennedy, K.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Peptide-based approaches to treat asthma, arthritis, other autoimmune diseases and pathologies of the central nervous system2005In: Archivum Immunologiae et Therapiae Experimentalis, ISSN 0004-069X, E-ISSN 1661-4917, Vol. 53, no 4, p. 308-320Article in journal (Refereed)
    Abstract [en]

    In this review we focus on peptide- and peptidomimetic-based approaches that target autoimmune diseases and some pathologies of the central nervous system. Special attention is given to asthma, allergic rhinitis, osteoarthritis, and Alzheimer's disease, but other related pathologies are also reviewed, although to a lesser degree. Among others, drugs like Diacerhein and its active form Rhein, Pralnacasan, Anakinra (Kineret), Omalizumab, an antibody "BION-1", directed against the common beta-chain of cytokine receptors, are described below as well as attempts to target beta-amyloid peptide aggregation. Parts of the review are also dedicated to targeting of pathologic conditions in the brain and in other tissues with peptides as well as methods to deliver larger molecules through the "blood-brain barrier" by exploring receptor-mediated transport, or elsewhere in the body by using peptides as carriers through cellular membranes. In addition to highlighting current developments in the field, we also propose, for future drug targets, the components of the inflammasome protein complex, which is believed to initiate the activation of caspase-1 dependent signaling events, as well as other pathways that signal inflammation. Thus we discuss the possibility of targeting inflammasome components for negative or positive modulation of an inflammatory response.

  • 44.
    Hennig, Janosch
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Structure-function studies on TRIM21/Ro52, a protein involved in autoimmune diseases2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Several members of the tripartite motif (TRIM) protein family are involvedin antiviral activity and immunity and have been linked to severaldiseases. TRIM21, the main object of this thesis, is involved in Sjögrensyndrome (SS) and systemic lupus erythematosus (SLE), where patientsoften have autoantibodies against different epitopes on TRIM21. Duringthe course of this study a role of TRIM21 in regulation of proinflammatorycytokines and autoimmunity emerged. The aim of this thesis is to providea better understanding of the structure-function relationship of TRIM21.A conformational epitope in the coiled-coil domain of TRIM21 has beencharacterized, whose autoantibodies cause congenital heart block. A widerange of biophysical methods were employed to establish a model of theprotein domain arrangement of TRIM21, and functional implications werederived. By sequence comparisons, TRIM proteins were classified into threesubgroups, sharing a conserved amphipathic helix in the region, linkingthe conserved N-terminal Zn2+-binding domains RING and B-box, calledthe RING-B-box linker (RBL). A structural dependence of this region on theRING has been observed and a model of the RING-RBL was derived frombioinformatics and proteolysis data. Anti-RING-RBL antibodies inhibit theE3 ligase activity of TRIM21 in ubiquitination. Interferon regulatory factors(IRFs), the substrate for TRIM21-dependent ubiquitination could thereforeretain their high cellular levels after stress-induced inflammation, increasingthe susceptibility to SS and SLE. According to NMR data, the antibodiesbind to the Zn2+-binding loop regions of the RING, which usually bind tothe E2 conjugating enzyme. Antibodies against the C-terminus of the RBLregion do not inhibit the E3 ligase activity.

    List of papers
    1. Structurally derived mutations define congenital heart block-related epitopes within the 200-239 amino acid stretch of the Ro52 protein
    Open this publication in new window or tab >>Structurally derived mutations define congenital heart block-related epitopes within the 200-239 amino acid stretch of the Ro52 protein
    Show others...
    2005 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 61, no 2, p. 109-118Article in journal (Refereed) Published
    Abstract [en]

    Congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with Ro/SSA autoantibodies. During pregnancy, the antibodies are transported across the placenta and affect the fetus. We have previously demonstrated that antibodies directed to the 200-239 amino acid (aa) stretch of the Ro52 component of the Ro/SSA antigen correlate with the development of congenital heart block. In this report, we investigated the antibody-antigen interaction of this target epitope in detail at a molecular and structural level. Peptides representing aa 200-239 (p200) with structurally derived mutations were synthesized to define the epitopes recognized by two Ro52 human monoclonal antibodies, S3A8 and M4H1, isolated from patient-derived phage display libraries. Analyses by ELISA, circular dichroism and MALDI-TOF-MS demonstrate that the antibody recognition is dependent on a partly a-helical fold within the putative leucine zipper of the 200-239 aa stretch and that the two human anti-p200 monoclonal antibodies, M4H1 and S3A8, recognize different epitopic structures within the p200 peptide. In addition, we investigated the representation of each fine specificity within the sera of mothers with children born with congenital heart block, and in such sera, antibodies of the S3A8 idiotype were more commonly detected and at higher levels than M4H1-like antibodies.

    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-45521 (URN)10.1111/j.0300-9475.2005.01542.x (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
    2. Structural, functional and immunologic characterization of folded subdomains in the Ro52 protein targeted in Sjögren's syndrome
    Open this publication in new window or tab >>Structural, functional and immunologic characterization of folded subdomains in the Ro52 protein targeted in Sjögren's syndrome
    Show others...
    2006 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 43, no 6, p. 588-598Article in journal (Refereed) Published
    Abstract [en]

    Ro52, one of the major autoantigens in the rheumatic disease Sjögren's syndrome (SS), belongs to the tripartite motif (TRIM) or RING-B-box-coiled-coil (RBCC) protein family, thus comprising an N-terminal RING, followed by a B-box and a coiled-coil region. Several different proteomic functions have been suggested for Ro52, including DNA binding, protein interactions and Zn 2+-binding. To analyze the presence and/or absence of these functions and, in particular, map those to different subregions, the modular composition of the Ro52 protein was experimentally characterized. Two structured parts of Ro52 were identified, corresponding to the RING-B-box and the coiled-coil regions, respectively. Secondary structure analysis by circular dichroism (CD) spectroscopy indicated that the two subregions are independently structured. The entire RING-B-box region displayed Zn2+-dependent stabilization against proteolysis in the presence of Zn2+, indicating functional Zn2+-binding sites in both the RING and the B-box. However, no stabilization with DNA was detected, irrespective of Zn2+, thus suggesting that the RING-B-box region does not bind DNA. Oligomerization of the coiled-coil was investigated by analytical ultracentrifugation and in a mammalian two-hybrid system. Both methods show weak homodimer affinity, in parity with other coiled-coil domains involved in regulatory interactions. The C-terminal B30.2 region was rapidly degraded both during cellular expression and refolding, indicating a less stable structure. Immunologic analysis of the stable protein regions with sera from patients with Sjögren's syndrome shows that immunodominant epitopes to a large extent are localized in the structurally stable parts of Ro52. The results form a basis for further Ro52 functional studies on the proteome level. © 2005 Elsevier Ltd. All rights reserved.

    Keywords
    Immunologi, Ro52, Biofysik
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-38851 (URN)10.1016/j.molimm.2005.04.013 (DOI)45868 (Local ID)45868 (Archive number)45868 (OAI)
    Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
    3. Structural organization and Zn2+ -dependent subdomain interactions involving autoantigenic epitopes in the Ring-B-box-coiled-coil (RBCC) region of Ro52
    Open this publication in new window or tab >>Structural organization and Zn2+ -dependent subdomain interactions involving autoantigenic epitopes in the Ring-B-box-coiled-coil (RBCC) region of Ro52
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    2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 39, p. 33250-33261Article in journal (Refereed) Published
    Abstract [en]

    Ro52 is one of the major autoantigens targeted in the autoimmune disease Sjögren syndrome. By sequence similarity, Ro52 belongs to the RING-B-box-coiled-coil (RBCC) protein family. Disease-related antibodies bind Ro52 in a conformation-dependent way both in the coiled-coil region and in the Zn2+-binding Ring-B-box region. Primarily associated with Sjögren syndrome, Ro52 autoantibodies directed to a specific, partially structured epitope in the coiled-coil region may also induce a congenital heart block in the fetus of pregnant Ro52-positive mothers. To improve our understanding of the pathogenic effects of autoantibody binding to the Zn 2+-binding region, a multianalytical mapping of its structural, biophysical, and antigenic properties is presented. Structure content and ligand binding of subregions, dissected by peptide synthesis and subcloning, were analyzed by fluorescence and circular dichroism spectroscopy. A novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry strategy for time-resolved proteolysis experiments of large protein domains was developed to facilitate analysis and to help resolve the tertiary arrangement of the entire RBCC subregion. The linker region between the RING and B-box motifs is crucial for full folding, and Zn2+ affinity of the RING-B-box region is further protected in the entire RBCC region and appears to interact with the coiled-coil region. Murine monoclonal antibodies raised toward the RING-B-box region were primarily directed toward the linker, further supporting a highly functional role for the linker in a cellular environment. Taken together with our previous analysis of autoantigenic epitopes in the coiled-coil region, localization of autoantigenic epitopes in Ro52 appears closely related to molecular functionalities. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-50421 (URN)10.1074/jbc.M503066200 (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12
    4. The fellowship of the RING: The RING-B-box linker region (RBL) interacts with the RING in TRIM21/Ro52, contributes to an autoantigenic epitope in Sjögren's syndrome, and is an integral and conserved region in TRIM proteins
    Open this publication in new window or tab >>The fellowship of the RING: The RING-B-box linker region (RBL) interacts with the RING in TRIM21/Ro52, contributes to an autoantigenic epitope in Sjögren's syndrome, and is an integral and conserved region in TRIM proteins
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    2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 377, no 2, p. 431-449Article in journal (Refereed) Published
    Abstract [en]

    Ro52 is a major autoantigen that is targeted in the autoimmune disease Sjögren syndrome and belongs to the tripartite motif (TRIM) protein family. Disease-related antigenic epitopes are mainly found in the coiled-coil domain of Ro52, but one such epitope is located in the Zn2+-binding region, which comprises an N-terminal RING followed by a B-box, separated by a ∼40-residue linker peptide. In the present study, we extend the structural, biophysical, and immunological knowledge of this RING-B-box linker (RBL) by employing an array of methods. Our bioinformatic investigations show that the RBL sequence motif is unique to TRIM proteins and can be classified into three distinct subtypes. The RBL regions of all three subtypes are as conserved as their known flanking domains, and all are predicted to comprise an amphipathic helix. This helix formation is confirmed by circular dichroism spectroscopy and is dependent on the presence of the RING. Immunological studies show that the RBL is part of a conformation-dependent epitope, and its antigenicity is likewise dependent on a structured RING domain. Recombinant Ro52 RING-RBL exists as a monomer in vitro, and binding of two Zn2+ increases its stability. Regions stabilized by Zn2+ binding are identified by limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry. Furthermore, the residues of the RING and linker that interact with each other are identified by analysis of protection patterns, which, together with bioinformatic and biophysical data, enabled us to propose a structural model of the RING-RBL based on modeling and docking experiments. Sequence similarities and evolutionary sequence patterns suggest that the results obtained from Ro52 are extendable to the entire TRIM protein family.

    Keywords
    Ro52; TRIM21; RING; linker; zinc binding
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-12887 (URN)10.1016/j.jmb.2008.01.005 (DOI)
    Available from: 2008-01-28 Created: 2008-01-28 Last updated: 2017-12-14Bibliographically approved
    5. Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface
    Open this publication in new window or tab >>Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface
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    2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 42, p. 36478-36491Article in journal (Refereed) Published
    Abstract [en]

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

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

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

    Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2017-12-12Bibliographically approved
    6. MTMDAT: Automated analysis and visualization of mass spectrometry data for tertiary and quaternary structure probing of proteins
    Open this publication in new window or tab >>MTMDAT: Automated analysis and visualization of mass spectrometry data for tertiary and quaternary structure probing of proteins
    2008 (English)In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 24, no 10, p. 1310-1312Article in journal (Refereed) Published
    Abstract [en]

    In structural biology and -genomics, nuclear magnetic resonance (NMR) spectroscopy and crystallography are the methods of choice, but sample requirements can be hard to fulfil. Valuable structural information can also be obtained by using a combination of limited proteolysis and mass spectrometry, providing not only knowledge of how to improve sample conditions for crystallization trials or NMR spectrosopy by gaining insight into subdomain identities but also probing tertiary and quaternary structure, folding and stability, ligand binding, protein interactions and the location of post-translational modifications. For high-throughput studies and larger proteins, however, this experimentally fast and easy approach produces considerable amounts of data, which until now has made the evaluation exceedingly laborious if at all manually possible. MTMDAT, equipped with a browser-like graphical user interface, accelerates this evaluation manifold by automated peak picking, assignment, data processing and visualization. © 2008 The Author(s).

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-42790 (URN)10.1093/bioinformatics/btn116 (DOI)68779 (Local ID)68779 (Archive number)68779 (OAI)
    Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
    7. MTMDAT-HADDOCK: high-throughput, data-driven protein complex structure modeling
    Open this publication in new window or tab >>MTMDAT-HADDOCK: high-throughput, data-driven protein complex structure modeling
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Background: MTMDAT is a recently developed program facilitating the analysis of mass spectrometry data of proteins and biomolecular complexes (Hennig et al, 2008), which were structurally probed by limited proteolysis. This can provide information about stable fragments of multidomain proteins, yields tertiary and quaternary structure data, and residue-wise origins of stability changes can be determined.

    Results: A new feature allows for the direct identification of residues that are involved in complex formation bycomparing the mass spectra of bound and unbound proteins after proteolysis. If 3D structures of the unboundcomponents are available, this data can be used to restrain data-driven docking to calculate the structure ofthe complex. Here, we provide a new implementation of MTMDAT, with a pipeline to the data-driven dockingprogram HADDOCK (Dominguez et al., 2003; De Vries et al., 2007), streamlining the entire procedure. This,together with usability improvements in MTMDAT, enables direct high-throughput modelling of protein complexesfrom mass spectrometry data.

    Conclusions: MTMDAT can be downloaded from http://cms.ifm.liu.se/chemistry/molbiotech/maria¯sunnerhagens¯group/mtmdat/ (windows- or unix-based, with or without HADDOCK pipeline) together withthe manual and example files. The program is free for academic/non-commercial purposes.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-53169 (URN)
    Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2018-09-17
  • 45. Hermosilla Casajus, Pedro
    et al.
    Maisch, Sebastian
    Vázquez Alcocer, Pere Pau
    Ropinski, Timo
    Linköping University, Department of Science and Technology, Media and Information Technology. Linköping University, Faculty of Science & Engineering.
    Improving Perception of Molecular Surface Visualizations by Incorporating Translucency Effects2018In: VCBM 2018, 2018, p. 185-195Conference paper (Refereed)
  • 46.
    Hermosilla, Pedro
    et al.
    VIRVIG Group, Universitat Politècnica de Catalunya, Barcelona, Spain.
    Vazquez, Pere-Pau
    VIRVIG Group, Universitat Politècnica de Catalunya, Barcelona, Spain.
    Vinacua, Àlvar
    VIRVIG Group, Universitat Politècnica de Catalunya, Barcelona, Spain.
    Ropinski, Timo
    Visual Computing Group, Ulm University, Ulm, Germany.
    A General Illumination Model for Molecular Visualization2018In: Computer Graphics Forum (Proceedings of EuroVis 2018), Vol. 37, no 3, p. 367-378Article in journal (Refereed)
    Abstract [en]

    Several visual representations have been developed over the years to visualize molecular structures, and to enable a better understanding of their underlying chemical processes. Today, the most frequently used atom‐based representations are the Space‐filling, the Solvent Excluded Surface, the Balls‐and‐Sticks, and the Licorice models. While each of these representations has its individual benefits, when applied to large‐scale models spatial arrangements can be difficult to interpret when employing current visualization techniques. In the past it has been shown that global illumination techniques improve the perception of molecular visualizations; unfortunately existing approaches are tailored towards a single visual representation. We propose a general illumination model for molecular visualization that is valid for different representations. With our illumination model, it becomes possible, for the first time, to achieve consistent illumination among all atom‐based molecular representations. The proposed model can be further evaluated in real‐time, as it employs an analytical solution to simulate diffuse light interactions between objects. To be able to derive such a solution for the rather complicated and diverse visual representations, we propose the use of regression analysis together with adapted parameter sampling strategies as well as shape parametrization guided sampling, which are applied to the geometric building blocks of the targeted visual representations. We will discuss the proposed sampling strategies, the derived illumination model, and demonstrate its capabilities when visualizing several dynamic molecules.

  • 47.
    Hombach-Klonisch, Sabine
    et al.
    Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada .
    Panigrahi, Soumya
    Department of Physiology, University of Manitoba, Winnipeg, Canada; Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada.
    Rashedi, Iran
    Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada .
    Seifert, Anja
    Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada.
    Alberti, Esteban
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.
    Pocar, Paola
    Department of Animal Science, Faculty of Veterinary Medicine, University of Milan, Milan, Italy .
    Kurpisz, Maciej
    Institute of Human Genetics, Polish Academy of Science, Poznan, Poland .
    Schulze-Osthoff, Klaus
    Institute of Molecular Medicine, University of Duesseldorf, Duesseldorf, Germany .
    Mackiewicz, Andrzej
    Department of Cancer Immunology, Poznan University of Medical Sciences, and Great-Poland Cancer Center, Poznan, Poland.
    Los, Marek Jan
    BioApplications Enterprises, Winnipeg, MB, Canada.
    Adult stem cells and their trans-differentiation potential-perspectives and therapeutic applications2008In: Journal of Molecular Medicine, ISSN 0946-2716, E-ISSN 1432-1440, Vol. 86, no 12, p. 1301-1314Article in journal (Refereed)
    Abstract [en]

    Stem cells are self-renewing multipotent progenitors with the broadest developmental potential in a given tissue at a given time. Normal stem cells in the adult organism are responsible for renewal and repair of aged or damaged tissue. Adult stem cells are present in virtually all tissues and during most stages of development. In this review, we introduce the reader to the basic information about the field. We describe selected stem cell isolation techniques and stem cell markers for various stem cell populations. These include makers for endothelial progenitor cells (CD146/MCAM/MUC18/S-endo-1, CD34, CD133/prominin, Tie-2, Flk1/KD/VEGFR2), hematopoietic stem cells (CD34, CD117/c-Kit, Sca1), mesenchymal stem cells (CD146/MCAM/MUC18/S-endo-1, STRO-1, Thy-1), neural stem cells (CD133/prominin, nestin, NCAM), mammary stem cells (CD24, CD29, Sca1), and intestinal stem cells (NCAM, CD34, Thy-1, CD117/c-Kit, Flt-3). Separate section provides a concise summary of recent clinical trials involving stem cells directed towards improvement of a damaged myocardium. In the last part of the review, we reflect on the field and on future developments.

  • 48.
    Hombach-Klonisch, Sabine
    et al.
    Department Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada .
    Paranjothy, Ted
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada .
    Wiechec, Emilia
    Department of Human Genetics, University of Aarhus, Aarhus, Denmark.
    Pocar, Paola
    Department of Animal Science, Faculty of Veterinary Medicine, University of Milan, Milan, Italy .
    Mustafa, Tarek
    Department of Oral and Maxillo-Facial Surgery, Faculty of Medicine, University Magdeburg, Magdeburg, Germany; Experimental Surgery and Oncology Research Group, Department of General, Visceral and Vascular Surgery, University of Halle-Wittenberg, Halle, Germany .
    Seifert, Anja
    Department Anatomy and Cell Biology, University of Halle-Wittenberg, Halle, Germany .
    Zahl, Christian
    Department of Oral and Maxillo-Facial Surgery, Faculty of Medicine, University Magdeburg, Magdeburg, Germany .
    Luis Gerlach, Klaus
    Department of Oral and Maxillo-Facial Surgery, Faculty of Medicine, University Magdeburg, Magdeburg, Germany .
    Biermann, Katharina
    Institute of Pathology, University Hospital Bonn, Bonn, Germany .
    Steger, Klaus
    Department of Urology and Pediatric Urology, University of Giessen, Giessen, Germany .
    Hoang-Vu, Cuong
    Experimental Surgery and Oncology Research Group, Department of General, Visceral and Vascular Surgery, University of Halle-Wittenberg, Halle, Germany .
    Schulze-Osthoff, Klaus
    Institute of Molecular Medicine, University of Düsseldorf, Düsseldorf, Germany .
    Los, Marek Jan
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada; BioApplications Enterprises, Winnipeg, MB, Canada.
    Cancer stem cells as targets for cancer therapy: selected cancers as examples2008In: Archivum Immunologiae et Therapiae Experimentalis, ISSN 0004-069X, E-ISSN 1661-4917, Vol. 56, no 3, p. 165-180Article, review/survey (Refereed)
    Abstract [en]

    It is becoming increasingly evident that cancer constitutes a group of diseases involving altered stem-cell maturation/differentiation and the disturbance of regenerative processes. The observed malignant transformation is merely a symptom of normal differentiation processes gone astray rather than the primary event. This review focuses on the role of cancer stem cells (CSCs) in three common but also relatively under-investigated cancers: head and neck, ovarian, and testicular cancer. For didactic purpose, the physiology of stem cells is first introduced using hematopoietic and mesenchymal stem cells as examples. This is followed by a discussion of the (possible) role of CSCs in head and neck, ovarian, and testicular cancer. Aside from basic information about the pathophysiology of these cancers, current research results focused on the discovery of molecular markers specific to these cancers are also discussed. The last part of the review is largely dedicated to signaling pathways active within various normal and CSC types (e.g. Nanog, Nestin, Notch1, Notch2, Oct3 and 4, Wnt). Different elements of these pathways are also discussed in the context of therapeutic opportunities for the development of targeted therapies aimed at CSCs. Finally, alternative targeted anticancer therapies arising from recently identified molecules with cancer-(semi-)selective capabilities (e.g. apoptin, Brevinin-2R) are considered.

  • 49.
    Ihnatko, Robert
    et al.
    Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 842 45 Bratislava, Slovakia.
    Kubes, M
    Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 842 45 Bratislava, Slovakia.
    TNF signaling: early events and phosphorylation.2007In: General Physiology and Biophysics, ISSN 0231-5882, E-ISSN 1338-4325, Vol. 26, no 3, p. 159-67Article in journal (Refereed)
    Abstract [en]

    Tumor necrosis factor-alpha (TNF) is a major mediator of apoptosis as well as immunity and inflammation. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated in the pathogenesis of a wide spectrum of human diseases, including cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. TNF binds to two specific receptors, TNF-receptor type I (TNF-R1, CD120a, p55/60) and TNF-receptor type II (TNF-R2, CD120b, p75/80). Signaling through TNF-R1 is extremely complex, leading to both cell death and survival signals. Many findings suggest an important role of phosphorylation of the TNF-R1 by number of protein kinases. Role of TNF-R2 phosphorylation on its signaling properties is understood less than TNF-R1. Other cellular substrates as TRADD adaptor protein, TRAF protein family and RIP kinases are reviewed in relation to TNF receptor-mediated apoptosis or survival pathways and regulation of their actions by phosphorylation.

  • 50.
    Ihnatko, Robert
    et al.
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Kubes, Miroslav
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Takacova, Martina
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Sedlakova, Olga
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Sedlak, Jan
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Pastorek, Jaromir
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Kopacek, Juraj
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Pastorekova, Silvia
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Extracellular acidosis elevates carbonic anhydrase IX in human glioblastoma cells via transcriptional modulation that does not depend on hypoxia2006In: International Journal of Oncology, ISSN 1019-6439, Vol. 29, no 4, p. 1025-33Article in journal (Refereed)
    Abstract [en]

    Most solid tumors display extracellular acidosis, which only partially overlaps with hypoxia and induces distinct adaptive changes leading to aggressive phenotype. Although acidosis is mainly attributable to excessive production of lactic acid, it also involves carbonic anhydrase (CA) IX-mediated conversion of CO(2) to an extracellular proton and a bicarbonate ion transported to cytoplasm. CA IX is pre-dominantly expressed in tumors with poor prognosis and its transcription and activity are induced by hypoxia. Here we investigated whether low extracellular pH in absence of hypoxia can influence CA IX expression in cell lines derived from glioblastoma, a tumor type particularly linked with acidosis. Our data show that extracellular acidosis increased the level of CA IX protein, mRNA and the activity of minimal CA9 promoter that contains binding sites for HIF-1 and SP-1 transcription factors. Mutation within each of these two biding sites reduced the promoter activity, but did not eliminate the increase by acidosis. Transfection of HIF-1alpha cDNA produced additive inducing effect with acidosis. Normoxic acidosis was accompanied by HIF-1alpha protein accumulation and transiently increased phosphorylation of ERK1/2. Expression of a dominant-negative mutant of ERK2 reduced the CA9 promoter activity in both standard and acidic conditions. Similar result was obtained by inhibitors of MAPK and PI3K pathways, whose combination completely suppressed CA IX expression and abolished induction by acidosis. Altogether, our results suggest that acidosis increases the CA IX expression via a hypoxia-independent mechanism that operates through modulation of the basic CA9 transcriptional machinery.

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