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  • 151.
    Hildebrand, Sebastian
    et al.
    Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institutet and Division of Obstetrics and Gynecology, Karolinska University Hospital, Huddinge, Sweden.;Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden.
    Hultin, Sara
    Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden.
    Subramani, Aravindh
    Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden.
    Petropoulos, Sophie
    Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institutet and Division of Obstetrics and Gynecology, Karolinska University Hospital, Huddinge, Sweden.
    Zhang, Yuanyuan
    Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden.
    Cao, Xiaofang
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mpindi, John
    Medical Biotechnology, VTT Technical Research Centre of Finland, Turku, Finland.;Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland.
    Kalloniemi, Olli
    Medical Biotechnology, VTT Technical Research Centre of Finland, Turku, Finland.;Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland.
    Johansson, Staffan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Majumdar, Arindam
    Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden.
    Lanner, Fredrik
    Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institutet and Division of Obstetrics and Gynecology, Karolinska University Hospital, Huddinge, Sweden.
    Holmgren, Lars
    Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden.
    The E-cadherin/AmotL2 complex organizes actin filaments required for epithelial hexagonal packing and blastocyst hatching2017In: Scientific Reports, E-ISSN 2045-2322, Vol. 7, no 1, article id 9540Article in journal (Refereed)
    Abstract [en]

    Epithelial cells connect via cell-cell junctions to form sheets of cells with separate cellular compartments. These cellular connections are essential for the generation of cellular forms and shapes consistent with organ function. Tissue modulation is dependent on the fine-tuning of mechanical forces that are transmitted in part through the actin connection to E-cadherin as well as other components in the adherens junctions. In this report we show that p100 amotL2 forms a complex with E-cadherin that associates with radial actin filaments connecting cells over multiple layers. Genetic inactivation or depletion of amotL2 in epithelial cells in vitro or zebrafish and mouse in vivo, resulted in the loss of contractile actin filaments and perturbed epithelial packing geometry. We further showed that AMOTL2 mRNA and protein was expressed in the trophectoderm of human and mouse blastocysts. Genetic inactivation of amotL2 did not affect cellular differentiation but blocked hatching of the blastocysts from the zona pellucida. These results were mimicked by treatment with the myosin II inhibitor blebbistatin. We propose that the tension generated by the E-cadherin/AmotL2/actin filaments plays a crucial role in developmental processes such as epithelial geometrical packing as well as generation of forces required for blastocyst hatching.

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  • 152.
    Hilow, Zeinalabedin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    The regulatory effect of CsrA on cstA, glgCAP and flhDC in an RNase P ts mutant Escherichia coli2017Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    CsrA is a global post-transcriptional regulatory protein that has a great impact on many physiological pathways in a cell. It regulates central carbon metabolism, motility and biofilm formation as well as virulence, pathogenesis, quorum sensing and the oxidative stress response. However, CsrA is in turn also regulated by CsrB and CsrC sRNAs, among other factors. In this study, we explore its regulatory effect on three different genes/operons, cstA, glgCAP and flhDC. We performed beta-galactosidase assays on wild type (wt) and RNase P temperature sensitive (ts) cells to determine the dependence on RNaseP for the CsrA, CsrB and CsrC regulatory cascade on these three genes. Our results showed a clear decrease in cstA and glgCAP activity during CsrA activation, suggesting a regulatory cascade in which inhibition of RNase P leads to an inactivation of CsrB and CsrC. This in turn leads to an activation of CsrA and an inhibition of cstA and glgCAP. The effect on flhDC, however, was not as clear and needs further investigation.

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  • 153.
    Hirschi, Karen K.
    et al.
    Yale Univ, Sch Med, Yale Cardiovasc Res Ctr, Dept Med, 333 Cedar St, New Haven, CT 06520 USA;Yale Univ, Sch Med, Yale Cardiovasc Res Ctr, Dept Genet, 333 Cedar St, New Haven, CT 06520 USA;Yale Univ, Sch Med, Yale Cardiovasc Res Ctr, Dept Biomed Engn, 333 Cedar St, New Haven, CT 06520 USA;Yale Univ, Sch Med, Yale Stem Cell Ctr, 333 Cedar St, New Haven, CT 06520 USA.
    Dejana, Elisabetta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. FIRC Inst Mol Oncol, Milan, Italy.
    Resident Endothelial Progenitors Make Themselves at Home2018In: Cell Stem Cell, ISSN 1934-5909, E-ISSN 1875-9777, Vol. 23, no 2, p. 153-155Article in journal (Other academic)
    Abstract [en]

    Vascular endothelial cells adapt to their microenvironment and physiological demands to perform many essential functions. Recent studies (McDonald et al., 2018; Wakabayashi et al., 2018) suggest that quiescent endothelial stem/progenitor cells reside within blood vessels and are activated in response to injury, suggesting they can be harnessed for therapeutic applications.

  • 154.
    Huang, Miller
    et al.
    Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA;Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA.
    Tailor, Jignesh
    Univ Cambridge, MRC Stem Cell Inst, Wellcome Trust, Tennis Court Rd, Cambridge CB2 1QR, England;Inst Canc Res, London SM2 5NG, England;Hosp Sick Children, Dev & Stem Cell Biol Program, Toronto, ON, Canada;Hosp Sick Children, Div Neurosurg, Toronto, ON, Canada.
    Zhen, Qiqi
    Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA;Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA.
    Gillmor, Aaron H.
    Univ Calgary, Dept Biochem & Mol Biol, Calgary, AB, Canada;Univ Calgary, Charbonneau Canc Inst, Calgary, AB, Canada;Alberta Childrens Prov Gen Hosp, Res Inst, Calgary, AB, Canada.
    Miller, Matthew L.
    Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA;Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA.
    Weishaupt, Holger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Chen, Justin
    Stanford Univ, Dept Genet, Sch Med, Stanford, CA 94305 USA.
    Zheng, Tina
    Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA;Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA.
    Nash, Emily K.
    Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA;Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA.
    McHenry, Lauren K.
    Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA;Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA.
    An, Zhenyi
    Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA;Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA.
    Ye, Fubaiyang
    Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA;Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA.
    Takashima, Yasuhiro
    Univ Cambridge, MRC Stem Cell Inst, Wellcome Trust, Tennis Court Rd, Cambridge CB2 1QR, England.
    Clarke, James
    Univ Cambridge, MRC Stem Cell Inst, Wellcome Trust, Tennis Court Rd, Cambridge CB2 1QR, England.
    Ayetey, Harold
    Univ Cambridge, MRC Stem Cell Inst, Wellcome Trust, Tennis Court Rd, Cambridge CB2 1QR, England.
    Cavalli, Florence Mg
    Hosp Sick Children, Dev & Stem Cell Biol Program, Toronto, ON, Canada;Hosp Sick Children, Arthur & Sonia Labatt Brain Tumour Res Ctr, Toronto, ON, Canada.
    Luu, Betty
    Hosp Sick Children, Dev & Stem Cell Biol Program, Toronto, ON, Canada;Hosp Sick Children, Arthur & Sonia Labatt Brain Tumour Res Ctr, Toronto, ON, Canada.
    Moriarity, Branden S.
    Univ Minnesota, Dept Pediat, Minneapolis, MN 55455 USA;Univ Minnesota, Ctr Genome Engn, Minneapolis, MN 55455 USA;Univ Minnesota, Masonic Canc Ctr, Minneapolis, MN 55455 USA.
    Ilkhanizadeh, Shirin
    Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA;Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA.
    Chavez, Lukas
    Hopp Childrens Canc Ctr KiTZ, Heidelberg, Germany;German Canc Consortium DKTK, Div Pediat Neurooncol, German Canc Res Ctr DKFZ, Heidelberg, Germany.
    Yu, Chunying
    Hosp Sick Children, Dev & Stem Cell Biol Program, Toronto, ON, Canada.
    Kurian, Kathreena M.
    Univ Bristol, Southmead Hosp, Inst Clin Neurosci, Level 1,Learning & Res Bldg, Bristol BS10 5NB, Avon, England.
    Magnaldo, Thierry
    Nice UMR CNRS 7284 INSERM U1081 UNS UCA, Inst Res Canc & Aging, Nice, France.
    Sevenet, Nicolas
    Univ Bordeaux, Inst Bergonie, 229 Cours Argonne, F-33076 Bordeaux, France;Univ Bordeaux, INSERM U1218, 229 Cours Argonne, F-33076 Bordeaux, France.
    Koch, Philipp
    Heidelberg Univ, Cent Inst Mental Hlth, Med Fac Mannheim, Mannheim, Germany;Hector Inst Translat Brain Res HITBR gGmbH, Mannheim, Germany;German Canc Res Ctr, Heidelberg, Germany.
    Pollard, Steven M.
    Univ Edinburgh, MRC Ctr Regenerat Med, Edinburgh, Midlothian, Scotland;Univ Edinburgh, Canc Res UK Edinburgh Ctr, Edinburgh, Midlothian, Scotland.
    Dirks, Peter
    Hosp Sick Children, Dev & Stem Cell Biol Program, Toronto, ON, Canada;Hosp Sick Children, Div Neurosurg, Toronto, ON, Canada;Hosp Sick Children, Arthur & Sonia Labatt Brain Tumour Res Ctr, Toronto, ON, Canada.
    Snyder, Michael P.
    Stanford Univ, Dept Genet, Sch Med, Stanford, CA 94305 USA.
    Largaespada, David A.
    Univ Minnesota, Dept Pediat, Minneapolis, MN 55455 USA;Univ Minnesota, Ctr Genome Engn, Minneapolis, MN 55455 USA;Univ Minnesota, Masonic Canc Ctr, Minneapolis, MN 55455 USA.
    Cho, Yoon Jae
    Oregon Hlth & Sci Univ, Dept Pediat, Div Pediat Neurol, 3181 Sw Sam Jackson Pk Rd, Portland, OR 97201 USA;Oregon Hlth & Sci Univ, Dept Pediat, Pape Family Pediat Res Inst, 3181 Sw Sam Jackson Pk Rd, Portland, OR 97201 USA;Oregon Hlth & Sci Univ, Knight Canc Inst, Portland, OR 97201 USA.
    Phillips, Joanna J.
    Univ Calif San Francisco, Dept Neurol Surg, San Francisco, CA 94158 USA;Univ Calif San Francisco, Dept Pathol, San Francisco, CA 94158 USA.
    Johansson Swartling, Fredrik
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Morrissy, A. Sorana
    Hosp Sick Children, Dev & Stem Cell Biol Program, Toronto, ON, Canada;Univ Calgary, Dept Biochem & Mol Biol, Calgary, AB, Canada;Univ Calgary, Charbonneau Canc Inst, Calgary, AB, Canada;Alberta Childrens Prov Gen Hosp, Res Inst, Calgary, AB, Canada;Hosp Sick Children, Arthur & Sonia Labatt Brain Tumour Res Ctr, Toronto, ON, Canada.
    Kool, Marcel
    Hopp Childrens Canc Ctr KiTZ, Heidelberg, Germany;German Canc Consortium DKTK, Div Pediat Neurooncol, German Canc Res Ctr DKFZ, Heidelberg, Germany.
    Pfister, Stefan M.
    Hopp Childrens Canc Ctr KiTZ, Heidelberg, Germany;German Canc Consortium DKTK, Div Pediat Neurooncol, German Canc Res Ctr DKFZ, Heidelberg, Germany;Heidelberg Univ Hosp, Dept Pediat Hematol & Oncol, Heidelberg, Germany.
    Taylor, Michael D.
    Hosp Sick Children, Dev & Stem Cell Biol Program, Toronto, ON, Canada;Hosp Sick Children, Div Neurosurg, Toronto, ON, Canada;Hosp Sick Children, Arthur & Sonia Labatt Brain Tumour Res Ctr, Toronto, ON, Canada;Univ Toronto, Dept Lab Med & Pathobiol, Toronto, ON, Canada.
    Smith, Austin
    Univ Cambridge, MRC Stem Cell Inst, Wellcome Trust, Tennis Court Rd, Cambridge CB2 1QR, England.
    Weiss, William A.
    Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA;Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA;Univ Calif San Francisco, Res Ctr, Dept Pediat, San Francisco, CA 94158 USA;Univ Calif San Francisco, Res Ctr, Dept Neurosurg, San Francisco, CA 94158 USA;Univ Calif San Francisco, Res Ctr, Dept Brain Tumor, San Francisco, CA 94158 USA.
    Engineering Genetic Predisposition in Human Neuroepithelial Stem Cells Recapitulates Medulloblastoma Tumorigenesis2019In: Cell Stem Cell, ISSN 1934-5909, E-ISSN 1875-9777, Vol. 25, no 3, p. 433-+Article in journal (Refereed)
    Abstract [en]

    Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, representative of cerebellar progenitors. We transduced NES cells with MYCN, observing medulloblastoma upon orthotopic implantation in mice. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome patients, who are predis- posed to medulloblastoma due to germline-mutated PTCH1, also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3X or loss of GSE1 both accelerating tumorigenesis. These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma.

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  • 155.
    Hulsart-Billstrom, Gry
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Estrada, Sergio
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Lubberink, Mark
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Larsson, Sune
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    BMP-2 Induced bone regeneration visualized by PET and SPECT2014In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, E-ISSN 1932-7005, Vol. 8, p. 513-513Article in journal (Other academic)
  • 156.
    Häggblad Sahlberg, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Colorectal cancer and radiation response: The role of EGFR, AKT and cancer stem cell markers2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The primary treatment for colorectal cancer is surgery. Radiotherapy and chemotherapy, sometimes combined, are also frequently used to diminish recurrence risk. In response to radiation exposure, several cellular signaling cascades are activated to repair DNA breaks, prevent apoptosis and to keep the cells proliferating. Several proteins in the radiation response and cell survival pathways are potential targets to enhance the effects of radiation. The epidermal growth factor receptor (EGFR), which is frequently upregulated in colorectal cancer and exhibits a radiation protective function, is an attractive target for treatment. EGFR is activated by radiation which in turn activates numerous signaling pathways such as the PI3 kinase/AKT cascade, the RAS/RAF/ERK pathway and STAT leading to tumor cell proliferation. EGFR is also believed to interact with proteins in the DNA repair process, such as DNA-PKcs and MRE11. The cytotoxic effect of an affibody molecule (ZEGFR:1907)2, with high affinity to EGFR,  in combination with radiation produced a small, but significant, reduction in survival in a KRAS mutated cell line. However, not in the BRAF mutated cell line. The next step was therefore to target proteins downstream of EGFR such as AKT. There was an interaction between AKT and the DNA repair proteins DNA-PKcs and MRE11 and both AKT1 and AKT2 were involved in the radiation response. The knockout of both AKT isoforms impaired the DNA double strand break rejoining after radiation and suppression of DNA-PKcs increased the radiations sensitivity and decreased the DNA repair further. The AKT isoforms also affected the expression of cancer stem cell markers CD133 and CD44 which are associated with the formation of metastasis as well as radiation and drug resistance. The CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. AKT was also involved in cell migration, cell-adhesion and metabolism. Overall, these results illustrate the complexity in response to radiation and drugs in cells with different mutations and the need for combining inhibitors against several targets such as EGFR, AKT, DNA-PKcs, CD133 or CD44. 

    List of papers
    1. The effect of a dimeric Affibody molecule (ZEGFR:1907)2 targeting EGFR in combination with radiation in colon cancer cell lines
    Open this publication in new window or tab >>The effect of a dimeric Affibody molecule (ZEGFR:1907)2 targeting EGFR in combination with radiation in colon cancer cell lines
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    2012 (English)In: International Journal of Oncology, ISSN 1019-6439, E-ISSN 1791-2423, Vol. 40, no 1, p. 176-184Article in journal (Refereed) Published
    Abstract [en]

    The epidermal growth factor receptor (EGFR) is frequently overexpressed in colorectal cancer and is therefore an attractive target for treatment. (ZEGFR:1907)2 is a newly developed dimeric affibody molecule with high affinity to the extracellular part of EGFR. In this study, we evaluated the cytotoxic effects of (ZEGFR:1907)2 in combination with external radiation and the possible inhibitory effects in the EGFR signalling pathways in the colon cancer cell lines HT-29 and HCT116. The effects were compared with an EGFR antibody (cetuximab) and the tyrosine kinase inhibitors (erlotinib and sunitinib). These cell lines are genotypically different with respect to e.g. KRAS and BRAF mutational status, recently shown to be of clinical significance for therapeutic effects. Both cell lines express approximately 100,000-150,000 EGFRs per cell but differ in the radiation response (HCT116, SF2=0.28 and HT-29, SF2=0.70). Exposure to (ZEGFR:1907)2 produced a small, but significant, reduction in survival in HCT116 but did not affect HT-29 cells. Similar results were obtained after exposure to EGF and the EGFR antibody cetuximab. The EGFR tyrosine kinase targeting inhibitor erlotinib and the multi-tyrosine kinase inhibitor sunitinib reduced survival in both cell lines. However, none of the drugs had any significant radiosensitizing effects in combination with radiation. Akt and Erk are central proteins in the EGFR downstream signalling and in the cellular response to ionizing radiation. The activation of Akt (Ser 473) and Erk (Thr202/Tyr204) by radiation was both dose- and time-dependent. However the activation of EGFR was not clearly affected by radiation. Neither (ZEGFR:1907)2 nor any of the other drugs were able to completely inactivate Akt or Erk. On the contrary, erlotinib stimulated Akt phosphorylation in both cell lines and in HCT116 cells Erk was activated. Overall the results illustrate the complexity in response to radiation and drugs in cells with differential phenotypic status.

    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Research subject
    Biology with specialization in Molecular Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-165752 (URN)10.3892/ijo.2011.1177 (DOI)000297403800022 ()21879255 (PubMedID)
    Note

    Online ISSN:1791-2423

    Available from: 2012-01-09 Created: 2012-01-09 Last updated: 2020-12-17Bibliographically approved
    2. The influence of AKT isoforms on radiation sensitivity and DNA repair in colon cancer cell lines
    Open this publication in new window or tab >>The influence of AKT isoforms on radiation sensitivity and DNA repair in colon cancer cell lines
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    2014 (English)In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 35, no 4, p. 3525-3534Article in journal (Refereed) Published
    Abstract [en]

    In response to ionizing radiation, several signaling cascades in the cell are activated to repair the DNA breaks, prevent apoptosis, and keep the cells proliferating. AKT is important for survival and proliferation and may also be an activating factor for DNA-PKcs and MRE11, which are essential proteins in the DNA repair process. AKT (PKB) is hyperactivated in several cancers and is associated with resistance to radiotherapy and chemotherapy. There are three AKT isoforms (AKT1, AKT2, and AKT3) with different expression patterns and functions in several cancer tumors. The role of AKT isoforms has been investigated in relation to radiation response and their effects on DNA repair proteins (DNA-PKcs and MRE11) in colon cancer cell lines. The knockout of AKT1 and/or AKT2 affected the radiation sensitivity, and a deficiency of both isoforms impaired the rejoining of radiation-induced DNA double strand breaks. Importantly, the active/phosphorylated forms of AKT and DNA-PKcs associate and exposure to ionizing radiation causes an increase in this interaction. Moreover, an increased expression of both DNA-PKcs and MRE11 was observed when AKT expression was ablated, yet only DNA-PKcs expression influenced AKT phosphorylation. Taken together, these results demonstrate a role for both AKT1 and AKT2 in radiotherapy response in colon cancer cells involving DNA repair capacity through the nonhomologous end joining pathway, thus suggesting that AKT in combination with DNA-PKcs inhibition may be used for radiotherapy sensitizing strategies in colon cancer.

    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-221446 (URN)10.1007/s13277-013-1465-9 (DOI)000334495900084 ()
    Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2017-12-05Bibliographically approved
    3. Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells.
    Open this publication in new window or tab >>Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells.
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    2014 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 9, no 4, p. e94621-Article in journal (Refereed) Published
    Abstract [en]

    The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms) expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1) expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133(high)/CD44(high)) were more resistant to gamma radiation than the ten percent with lowest expression (CD133(low)/CD44(low)). The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2) did not differ. Our results demonstrate that CD133(high)/CD44(high) expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.

    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-221449 (URN)10.1371/journal.pone.0094621 (DOI)000335298200022 ()
    Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2021-06-14Bibliographically approved
    4. Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells
    Open this publication in new window or tab >>Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells
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    2017 (English)In: International Journal of Oncology, ISSN 1019-6439, E-ISSN 1791-2423, Vol. 50, no 1, p. 5-14Article in journal (Refereed) Published
    Abstract [en]

    AKT is a central protein in many cellular pathways such as cell survival, proliferation, glucose uptake, metabolism, angiogenesis, as well as radiation and drug response. The three isoforms of AKT (AKT1, AKT2 and AKT3) are proposed to have different physiological functions, properties and expression patterns in a cell type-dependent manner. As of yet, not much is known about the influence of the different AKT isoforms in the genome and their effects in the metabolism of colorectal cancer cells. In the present study, DLD-1 isogenic AKT1, AKT2 and AKT'/2 knockout colon cancer cell lines were used as a model system in conjunction with the parental cell line in order to further elucidate the differences between the AKT isoforms and how they are involved in various cellular pathways. This was done using genome wide expression analyses, metabolic profiling and cell migration assays. In conclusion, downregulation of genes in the cell adhesion, extracellular matrix and Notch-pathways and upregulation of apoptosis and metastasis inhibitory genes in the p53-pathway, confirm that the knockout of both AKT1 and AKT2 will attenuate metastasis and tumor cell growth. This was verified with a reduction in migration rate in the AKT1 KO and AKT2 KO and most explicitly in the AKT1/2 KO. Furthermore, the knockout of AKT1, AKT2 or both, resulted in a reduction in lactate and alanine, suggesting that the metabolism of carbohydrates and glutathione was impaired. This was further verified in gene expression analyses, showing downregulation of genes involved in glucose metabolism. Additionally, both AKT1 KO and AKT2 KO demonstrated an impaired fatty acid metabolism. However, genes were upregulated in the Wnt and cell proliferation pathways, which could oppose this effect. AKT inhibition should therefore be combined with other effectors to attain the best effect.

    Keywords
    Microarray, metabolism, cell migration AKT1, AKT2, AKT, PKB, gene expression, colon-cancer, DLD-1, metabolomics, CD44, CD133
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biomedical Radiation Science; Biology with specialization in Molecular Cell Biology; Biology with specialization in Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-222834 (URN)10.3892/ijo.2016.3771 (DOI)000391419200001 ()
    Available from: 2014-04-14 Created: 2014-04-14 Last updated: 2020-12-17Bibliographically approved
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  • 157.
    Ibrahim, Nader
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University.
    Avian Malaria infection and its effect on cellular metabolic rate in Ficedula Flycatchers2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Chronic avian malaria and low-intensity parasite infections can have a negative influence on reproduction and survival which can reduce the fitness of the host. Therefore, the effect on host due to avian malaria infection can affect a bird’s lifespan. Consequently, avian parasites can influence evolution, physiology, and host morphology. Among birds, there are several blood parasites used as model organisms for the study of parasite interactions with hosts, of which three genera of a haemosporidian parasite called avian malaria parasite. It is possible nowadays, to investigate and detect if the organism is infected with a blood parasite using fast and accurate molecular techniques.    

    In this study, I investigated how cellular respiration, an important trait regulating energy demands in animals, is affected by the infection of malaria in pied and collared flycatchers and if sex and age have a cross-relation influence on the cellular respiration rate. I used recently collected respiratory data and molecular techniques to evaluate the infection’s influence on cellular respiration. 138 samples were extracted and the nested-Polymerase chain reaction (PCR) was used to assess the presence of the parasite. Data was collected and interpreted and statistical analyses were performed.    

    I found that, females and juvenile flycatchers have higher cellular respiration rates and that there is a significant difference in respiration rate between the sexes. Also, I found that Leucocytozoon, but not Haemoproteus and/or Plasmodium infection show significant effect on flycatcher cellular respiration rate. However, parasite infection shows no significant effect on the total number of red blood cells count. Therefore, our study supports the idea that parasites can influence the organism’s metabolic activity. The study also shows how the identification of factors underlying the effect on cellular respiration rate can get us closer to understanding the important links between infection, age, sex and metabolic activity and performance

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  • 158.
    Ibrahim, Yasir H.
    et al.
    Araucaria Labs Inc, New York, NY 10065 USA..
    Pantelios, Spyridon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala Univ, Dept Immunol Pathol & Genet, Uppsala, Sweden..
    Mutvei, Anders P.
    Karolinska Inst, Dept Lab Med, Div Pathol, Stockholm, Sweden..
    An affinity tool for the isolation of endogenous active mTORC1 from various cellular sources2023In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 299, no 5, article id 104644Article in journal (Refereed)
    Abstract [en]

    The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of mammalian cell growth that is dysregulated in a number of human diseases, including metabolic syndromes, aging, and cancer. Structural, biochemical, and pharmacological studies that have increased our understanding of how mTORC1 executes growth control often relied upon purified mTORC1 protein. However, current immunoaffinitybased purification methods are expensive, inefficient, and do not necessarily isolate endogenous mTORC1, hampering their overall utility in research. Here we present a simple tool to isolate endogenous mTORC1 from various cellular sources. By GTPases from Escherichia coli and using them as affinity probes, we demonstrate that mTORC1 can be isolated from mouse, bovine, and human sources. Our results indicate that mTORC1 isolated by this relatively inexpensive method is catalytically active and amenable to scaling. Collectively, this tool may be utilized to isolate mTORC1 from various cellular sources, organs, and disease contexts, aiding mTORC1-related research.

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  • 159.
    Idevall Hagren, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gylfe, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 30, p. 23005-23016Article in journal (Refereed)
    Abstract [en]

    Pulsatile insulin release from glucose-stimulated beta-cells is driven by oscillations of the Ca2+ and cAMP concentrations in the subplasma membrane space ([Ca2+](pm) and [cAMP](pm)). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP](pm), [Ca2+](pm), and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 beta-cells. Elevation of the glucose concentration from 3 to 11 mM induced, after a 2.7-min delay, coordinated oscillations of [Ca2+](pm), [cAMP](pm), and PIP3. Inhibitors of protein kinase A (PKA) markedly diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The reduced PIP3 response could be attributed to accelerated depolarization causing early rise of [Ca2+](pm) that preceded the elevation of [cAMP](pm). However, the amplitude of the PIP3 response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP3 oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP3 oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca2+](pm) and amplifying [cAMP](pm) signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.

  • 160.
    Idevall Hagren, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Dynamic control of Epac2 localization by cAMP and Ca2+-mediated activation of RasManuscript (preprint) (Other academic)
    Abstract [en]

    Epac2, a cAMP-regulated guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2, is an important mediator of a variety of cAMP-regulated cellular processes, including insulin secretion from pancreatic β-cells. Epac2 has been suggested to associate with the plasma membrane by interacting with active Ras (Ras-GTP), but the dynamics and regulation of membrane binding is unknown. Using real-time confocal and total internal reflection fluorescence microscopy we demonstrate that cAMP-elevating agents cause rapid translocation of GFP-tagged Epac2 from the cytoplasm to the plasma membrane in insulin-secreting MIN6 β-cells. Glucose concentrations that stimulate insulin secretion often triggered oscillatory translocation of GFP-Epac2 following oscillations of the sub-membrane concentrations of cAMP and Ca2+ ([cAMP]pm and [Ca2+]pm). The translocation was suppressed after inhibition of adenylyl cyclases or removal of extracellular Ca2+. GFP-Epac2 translocation by rise of [Ca2+]pm required concomitant elevation of [cAMP]pm and cAMP-induced translocation was enhanced by moderate [Ca2+]pm elevations. However the effect of Ca2+ was dual since translocation was inhibited by high [Ca2+]pm spikes. Epac2 mutants lacking the cAMP-binding or Ras-association domains were unable to translocate and localized constitutively to the plasma membrane and cytoplasm, respectively. Ras activity monitored with a fluorescent Ras-GTP binding reporter was tightly correlated with the translocation of Epac2. It is concluded that Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras, and that reversible translocation of Epac2 between the cytoplasm and plasma membrane requires both Ras-association and cAMP-binding domains. Spatio-temporal control of Epac2 in β-cells has implications for the understanding of its involvement in insulin secretion kinetics by Rap GTPases and other downstream effectors at the plasma membrane.

  • 161.
    Idevall Hagren, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Metabolic regulation of calcium signaling in beta cells2020In: Seminars in Cell and Developmental Biology, ISSN 1084-9521, E-ISSN 1096-3634, Vol. 103, p. 20-30Article, review/survey (Refereed)
    Abstract [en]

    The cytoplasmic Ca2+ concentration ([Ca2+](cyt)) regulates a vast number of cellular functions, including insulin secretion from beta cells. The major physiological insulin secretagogue, glucose, triggers [Ca2+](cyt) oscillations in beta cells. Synchronization of the oscillations among the beta cells within an islet underlies the generation of pulsatile insulin secretion. This review describes the mechanisms generating [Ca2+](cyt) oscillations, the interactions between [Ca2+](cyt) and cell metabolism, as well as the contribution of various organelles to the shaping of [Ca2+](cyt) signals and insulin secretion. It also discusses how Ca2+ signals are coordinated and spread throughout the islets and data indicating that altered Ca2+ signaling is associated with beta cell dysfunction and development of type 2 diabetes.

  • 162.
    Imsland, Freyja
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    McGowan, Kelly
    HudsonAlpha Institute for Biotechnology / Department of Genetics, Stanford University School of Medicine.
    Rubin, Carl-Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Henegar, Corneliu
    Department of Genetics, Stanford University School of Medicine.
    Sundström, Elisabeth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Berglund, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Schwochow, Doreen
    Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences / INRA - AgroParisTech.
    Gustafson, Ulla
    Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences.
    Imsland, Páll
    Menntaskólinn við Hamrahlíð.
    Lindblad-Toh, Kerstin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Broad Institute of Harvard and MIT.
    Lindgren, Gabriella
    Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences.
    Mikko, Sofia
    Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences.
    Millon, Lee
    Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis.
    Wade, Claire
    Broad Institute of Harvard and MIT.
    Schubert, Mikkel
    Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen.
    Orlando, Ludovic
    Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen.
    Penedo, Maria Cecilia T.
    Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis.
    Barsh, Gregory S.
    HudsonAlpha Institute for Biotechnology / Department of Genetics, Stanford University School of Medicine.
    Andersson, Leif
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Texas A&M University.
    Regulatory mutations in TBX3 disrupt asymmetric hair pigmentation underlying Dun camouflage colour in horses2016In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 48, no 2, p. 152-158Article in journal (Refereed)
    Abstract [en]

    Dun is a wild-type coat color in horses characterized by pigment dilution with a striking pattern of dark areas termed primitive markings. Here we show that pigment dilution in Dun horses is due to radially asymmetric deposition of pigment in the growing hair caused by localized expression of the T-box 3 (TBX3) transcription factor in hair follicles, which in turn determines the distribution of hair follicle melanocytes. Most domestic horses are non-dun, a more intensely pigmented phenotype caused by regulatory mutations impairing TBX3 expression in the hair follicle, resulting in a more circumferential distribution of melanocytes and pigment granules in individual hairs. We identified two different alleles (non-dun1 and non-dun2) causing non-dun color. non-dun2 is a recently derived allele, whereas the Dun and non-dun1 alleles are found in ancient horse DNA, demonstrating that this polymorphism predates horse domestication. These findings uncover a new developmental role for T-box genes and new aspects of hair follicle biology and pigmentation.

  • 163.
    Janouskovec, Jan
    et al.
    UCL, Dept Genet Evolut & Environm, London, England.;San Diego State Univ, Dept Biol, San Diego, CA 92182 USA.;Univ British Columbia, Bot Dept, Vancouver, BC, Canada..
    Tikhonenkov, Denis V.
    Univ British Columbia, Bot Dept, Vancouver, BC, Canada.;Russian Acad Sci, Inst Biol Inland Waters, Borok, Russia..
    Burki, Fabien
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Univ British Columbia, Bot Dept, Vancouver, BC, Canada.
    Howe, Alexis T.
    Univ British Columbia, Bot Dept, Vancouver, BC, Canada..
    Rohwer, Forest L.
    San Diego State Univ, Dept Biol, San Diego, CA 92182 USA..
    Mylnikov, Alexander P.
    Russian Acad Sci, Inst Biol Inland Waters, Borok, Russia..
    Keeling, Patrick J.
    Univ British Columbia, Bot Dept, Vancouver, BC, Canada..
    A New Lineage of Eukaryotes Illuminates Early Mitochondrial Genome Reduction2017In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 27, no 23, p. 3717-3724.e5Article in journal (Refereed)
    Abstract [en]

    The origin of eukaryotic cells represents a key transition in cellular evolution and is closely tied to outstanding questions about mitochondrial endosymbiosis [1, 2]. For example, gene-rich mitochondrial genomes are thought to be indicative of an ancient divergence, but this relies on unexamined assumptions about endosymbiont-to-host gene transfer [3-5]. Here, we characterize Ancoracysta twista, a new predatory flagellate that is not closely related to any known lineage in 201-protein phylogenomic trees and has a unique morphology, including a novel type of extrusome (ancoracyst). The Ancoracysta mitochondrion has a gene-rich genome with a coding capacity exceeding that of all other eukaryotes except the distantly related jakobids and Diphylleia, and it uniquely possesses heterologous, nucleus-, and mitochondrion-encoded cytochrome c maturase systems. To comprehensively examine mitochondrial genome reduction, we also assembled mitochondrial genomes from picozoans and colponemids and re-annotated existing mitochondrial genomes using hidden Markov model gene profiles. This revealed over a dozen previously overlooked mitochondrial genes at the level of eukaryotic supergroups. Analysis of trends over evolutionary time demonstrates that gene transfer to the nucleus was non-linear, that it occurred in waves of exponential decrease, and that much of it took place comparatively early, massively independently, and with lineage-specific rates. This process has led to differential gene retention, suggesting that gene-rich mitochondrial genomes are not a product of their early divergence. Parallel transfer of mitochondrial genes and their functional replacement by new nuclear factors are important in models for the origin of eukaryotes, especially as major gaps in our knowl-edge of eukaryotic diversity at the deepest level remain unfilled.

  • 164.
    Jauhiainen, Suvi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Univ Eastern Finland, AI Virtanen Inst Mol Sci, Kuopio, Finland.
    Laakkonen, Johanna P.
    Univ Eastern Finland, AI Virtanen Inst Mol Sci, Kuopio, Finland.
    Ketola, Kirsi
    Univ Eastern Finland, Inst Biomed, Kuopio, Finland.
    Toivanen, Pyry, I
    Univ Eastern Finland, AI Virtanen Inst Mol Sci, Kuopio, Finland.
    Nieminen, Tina
    Univ Eastern Finland, AI Virtanen Inst Mol Sci, Kuopio, Finland.
    Ninchoji, Takeshi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Levonen, Anna-Liisa
    Univ Eastern Finland, AI Virtanen Inst Mol Sci, Kuopio, Finland.
    Kaikkonen, Minna U.
    Univ Eastern Finland, AI Virtanen Inst Mol Sci, Kuopio, Finland.
    Ylä-Herttuala, Seppo
    Univ Eastern Finland, AI Virtanen Inst Mol Sci, Kuopio, Finland;Kuopio Univ Hosp, Heart Ctr & Gene Therapy Unit, Kuopio, Finland.
    Axon Guidance-Related Factor FLRT3 Regulates VEGF-Signaling and Endothelial Cell Function2019In: Frontiers in Physiology, E-ISSN 1664-042X, Vol. 10, article id 224Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth factors (VEGFs) are key mediators of endothelial cell (EC) function in angiogenesis. Emerging knowledge also supports the involvement of axon guidance-related factors in the regulation of angiogenesis and vascular patterning. In the current study, we demonstrate that fibronectin and leucine-rich transmembrane protein-3 (FLRT3), an axon guidance-related factor connected to the regulation of neuronal cell outgrowth and morphogenesis but not to VEGF-signaling, was upregulated in ECs after VEGF binding to VEGFR2. We found that FLRT3 exhibited a transcriptionally paused phenotype in non-stimulated human umbilical vein ECs. After VEGF-stimulation its nascent RNA and mRNA-levels were rapidly upregulated suggesting that the regulation of FLRT3 expression is mainly occurring at the level of transcriptional elongation. Blockage of FLRT3 by siRNA decreased survival of ECs and their arrangement into capillary-like structures but enhanced cell migration and wound closure in wound healing assay. Bifunctional role of FLRT3 in repulsive vs. adhesive cell signaling has been already detected during embryogenesis and neuronal growth, and depends on its interactions either with UNC5B or another FLRT3 expressed by adjacent cells. In conclusion, our findings demonstrate that besides regulating neuronal cell outgrowth and morphogenesis, FLRT3 has a novel role in ECs via regulating VEGF-stimulated EC-survival, migration, and tube formation. Thus, FLRT3 becomes a new member of the axon guidance-related factors which participate in the VEGF-signaling and regulation of the EC functions.

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  • 165.
    Jerlström-Hultqvist, Jon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Einarsson, Elin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Stable transfection of the diplomonad parasite Spironucleus salmonicida2012In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 11, no 11, p. 1353-1361Article in journal (Refereed)
    Abstract [en]

    Eukaryotic microbes are highly diverse and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has mainly been studied due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad casuing systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3xHA, 2xOLLAS, 3xMYC) and purification of proteins (2xStrepII-FLAG or SBP-GST) under the control of native or constitutive promoters. Three selectable markers, puromycin acetyltransferase (pac), blasticidin S-deaminase (bsr) or neomycin phosphotransferase (nptII) were successfully applied for generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope-tagged six proteins and confirmed their expression by Western Blot. Next, we demonstrated the utility of these vectors by recording the sub-cellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we describe the creation of a S. salmonicida double transfectant suitable for co-localization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific as well as general diplomonad traits, benefiting research on both Giardia and Spironucleus.

  • 166.
    Jiao, Xiang
    et al.
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Aravidis, Christos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Marikkannu, Rajeshwari
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Rantala, Johanna
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Picelli, Simone
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Adamovic, Tatjana
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Liu, Tao
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Maguire, Paula
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Kremeyer, Barbara
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Luo, Liping
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Von Holst, Susanna
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Kontham, Vinaykumar
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Thutkawkorapin, Jessada
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Margolin, Sara
    Karolinska Inst, Dept Oncol Pathol, Stockholm, Sweden..
    Du, Quan
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Lundin, Johanna
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Michailidou, Kyriaki
    Univ Cambridge, Dept Publ Hlth & Primary Care, Ctr Canc Genet Epidemiol, Cambridge, England.;Cyprus Inst Neurol & Genet, Dept Elect Microscopy Mol Pathol, Nicosia, Cyprus..
    Bolla, Manjeet K.
    Univ Cambridge, Dept Publ Hlth & Primary Care, Ctr Canc Genet Epidemiol, Cambridge, England..
    Wang, Qin
    Univ Cambridge, Dept Publ Hlth & Primary Care, Ctr Canc Genet Epidemiol, Cambridge, England..
    Dennis, Joe
    Univ Cambridge, Dept Publ Hlth & Primary Care, Ctr Canc Genet Epidemiol, Cambridge, England..
    Lush, Michael
    Univ Cambridge, Dept Publ Hlth & Primary Care, Ctr Canc Genet Epidemiol, Cambridge, England..
    Ambrosone, Christine B.
    Roswell Pk Canc Inst, Buffalo, NY 14263 USA..
    Andrulis, Irene L.
    Mt Sinai Hosp, Lunenfeld Tanenbaum Res Inst, Fred A Litwin Ctr Canc Genet, Toronto, ON, Canada.;Univ Toronto, Dept Mol Genet, Toronto, ON, Canada..
    Anton-Culver, Hoda
    Univ Calif Irvine, Dept Epidemiol, Irvine, CA USA..
    Antonenkova, Natalia N.
    NN Alexandrov Res Inst Oncol & Med Radiol, Minsk, Byelarus..
    Arndt, Volker
    German Canc Res Ctr, Div Clin Epidemiol & Aging Res, Heidelberg, Germany..
    Beckmann, Matthias W.
    Friedrich Alexander Univ Erlangen Nuremberg, Univ Hosp Erlangen, Comprehens Canc Ctr Erlangen EMN, Dept Gynaecol & Obstet, Erlangen, Germany..
    Blomqvist, Carl
    Univ Helsinki, Helsinki Univ Hosp, Dept Oncol, Helsinki, Finland..
    Blot, William
    Vanderbilt Univ, Sch Med, Div Epidemiol,Dept Med, Vanderbilt Ingram Canc Ctr,Vanderbilt Epidemiol C, Nashville, TN 37212 USA.;Int Epidemiol Inst, Rockville, MD USA..
    Boeckx, Bram
    VIB, VIB Ctr Canc Biol, Leuven, Belgium.;Univ Leuven, Dept Human Genet, Lab Translat Genet, Leuven, Belgium..
    Bojesen, Stig E.
    Copenhagen Univ Hosp, Herlev & Gentofte Hosp, Copenhagen Gen Populat Study, Herlev, Denmark.;Copenhagen Univ Hosp, Herlev & Gentofte Hosp, Dept Clin Biochem, Herlev, Denmark.;Univ Copenhagen, Fac Hlth & Med Sci, Copenhagen, Denmark..
    Bonanni, Bernardo
    Ist Europeo Oncol, Div Canc Prevent & Genet, Milan, Italy..
    Brand, Judith S.
    Karolinska Inst, Dept Med Epidemiol & Biostat, Stockholm, Sweden..
    Brauch, Hiltrud
    Dr Margarete Fischer Bosch Inst Clin Pharmacol, Stuttgart, Germany.;Univ Tubingen, Tubingen, Germany.;German Canc Res Ctr, German Canc Consortium DKTK, Heidelberg, Germany..
    Brenner, Hermann
    German Canc Res Ctr, Div Clin Epidemiol & Aging Res, Heidelberg, Germany.;German Canc Res Ctr, German Canc Consortium DKTK, Heidelberg, Germany.;German Canc Res Ctr, Div Prevent Oncol, Heidelberg, Germany.;Natl Ctr Tumor Dis NCT, Heidelberg, Germany..
    Broeks, Annegien
    Antoni Leeuwenhoek Hosp, Netherlands Canc Inst, Div Mol Pathol, Amsterdam, Netherlands..
    Bruning, Thomas
    Ruhr Univ Bochum, Inst Prevent & Occupat Med, German Social Accid Insurance, Bochum, Germany..
    Burwinkel, Barbara
    Heidelberg Univ, Dept Obstet & Gynecol, Heidelberg, Germany.;German Canc Res Ctr, Mol Epidemiol Grp, C080, Heidelberg, Germany..
    Cai, Qiuyin
    Vanderbilt Univ, Sch Med, Div Epidemiol,Dept Med, Vanderbilt Ingram Canc Ctr,Vanderbilt Epidemiol C, Nashville, TN 37212 USA..
    Chang-Claude, Jenny
    German Canc Res Ctr, Div Canc Epidemiol, Heidelberg, Germany.;Univ Canc Ctr Hamburg, Univ Med Ctr Hamburg Eppendorf, Res Grp Genet Canc Epidemiol, Hamburg, Germany. Haukeland Hosp, Dept Oncol, Bergen, Norway. Univ Bergen, Inst Med, Sect Oncol, Bergen, Norway. Akershus Univ Hosp, Dept Pathol, Lorenskog, Norway. Akershus Univ Hosp, Dept Breast Endocrine Surg, Lorenskog, Norway..
    Couch, Fergus J.
    Mayo Clin, Dept Lab Med & Pathol, Rochester, MN USA..
    Cox, Angela
    Univ Sheffield, Dept Oncol & Metab, Sheffield Inst Nucle Acids SInFoNiA, Sheffield, S Yorkshire, England..
    Cross, Simon S.
    Univ Sheffield, Dept Neurosci, Acad Unit Pathol, Sheffield, S Yorkshire, England..
    Deming-Halverson, Sandra L.
    Vanderbilt Univ, Sch Med, Div Epidemiol,Dept Med, Vanderbilt Ingram Canc Ctr,Vanderbilt Epidemiol C, Nashville, TN 37212 USA..
    Devilee, Peter
    Leiden Univ, Med Ctr, Dept Pathol, Leiden, Netherlands.;Leiden Univ, Med Ctr, Dept Human Genet, Leiden, Netherlands..
    dos-Santos-Silva, Isabel
    London Sch Hyg & Trop Med, Dept Noncommun Dis Epidemiol, London, England..
    Dork, Thilo
    Hannover Med Sch, Gynaecol Res Unit, Hannover, Germany..
    Eriksson, Mikael
    Karolinska Inst, Dept Med Epidemiol & Biostat, Stockholm, Sweden..
    Fasching, Peter A.
    Friedrich Alexander Univ Erlangen Nuremberg, Univ Hosp Erlangen, Comprehens Canc Ctr Erlangen EMN, Dept Gynaecol & Obstet, Erlangen, Germany.;Univ Calif Los Angeles, Dept Med, David Geffen Sch Med, Los Angeles, CA 90024 USA.;Univ Calif Los Angeles, Div Hematol & Oncol, Los Angeles, CA USA..
    Figueroa, Jonine
    Univ Edinburgh, Med Sch, Usher Inst Populat Hlth Sci & Informat, Edinburgh, Midlothian, Scotland.;NCI, Div Canc Epidemiol & Genet, Rockville, MD USA..
    Flesch-Janys, Dieter
    Univ Med Ctr Hamburg Eppendorf, Inst Med Biometr & Epidemiol, Hamburg, Germany.;Univ Med Ctr Hamburg Eppendorf, Clin Canc Registry, Dept Canc Epidemiol, Hamburg, Germany..
    Flyger, Henrik
    Copenhagen Univ Hosp, Herlev & Gentofte Hosp, Dept Breast Surg, Herlev, Denmark..
    Gabrielson, Marike
    Karolinska Inst, Dept Med Epidemiol & Biostat, Stockholm, Sweden..
    Garcia-Closas, Montserrat
    NCI, Div Canc Epidemiol & Genet, Rockville, MD USA..
    Giles, Graham G.
    Canc Council Victoria, Canc Epidemiol & Intelligence Div, Melbourne, Vic, Australia.;Univ Melbourne, Melbourne Sch Populat & Global Hlth, Ctr Epidemiol & Biostat, Melbourne, Vic, Australia..
    Gonzalez-Neira, Anna
    Spanish Natl Canc Res Ctr, Human Canc Genet Program, Madrid, Spain..
    Guenel, Pascal
    Univ Paris Saclay, Univ Paris Sud, INSERM, Ctr Res Epidemiol & Populat Hlth CESP,Canc & Envi, Villejuif, France..
    Guo, Qi
    Univ Cambridge, Dept Publ Hlth & Primary Care, Cardiovasc Epidemiol Unit, Cambridge, England..
    Gundert, Melanie
    Heidelberg Univ, Dept Obstet & Gynecol, Heidelberg, Germany.;German Canc Res Ctr, Mol Epidemiol Grp, C080, Heidelberg, Germany..
    Haiman, Christopher A.
    Univ Southern Calif, Keck Sch Med, Dept Prevent Med, Los Angeles, CA 90033 USA..
    Hallberg, Emily
    Mayo Clin, Dept Hlth Sci Res, Rochester, MN USA..
    Hamann, Ute
    German Canc Res Ctr, Mol Genet Breast Canc, Heidelberg, Germany..
    Harrington, Patricia
    Univ Cambridge, Dept Oncol, Ctr Canc Genet Epidemiol, Cambridge, England..
    Hooning, Maartje J.
    Erasmus MC Canc Inst, Family Canc Clin, Dept Med Oncol, Rotterdam, Netherlands..
    Hopper, John L.
    Univ Melbourne, Melbourne Sch Populat & Global Hlth, Ctr Epidemiol & Biostat, Melbourne, Vic, Australia..
    Huang, Guanmengqian
    German Canc Res Ctr, Mol Genet Breast Canc, Heidelberg, Germany..
    Jakubowska, Anna
    Pomeranian Med Univ, Dept Genet & Pathol, Szczecin, Poland..
    Jones, Michael E.
    Inst Canc Res, Div Genet & Epidemiol, London, England..
    Kerin, Michael J.
    Natl Univ Ireland, Sch Med, Galway, Ireland..
    Kosma, Veli-Matti
    Univ Eastern Finland, Translat Canc Res Area, Kuopio, Finland.;Univ Eastern Finland, Inst Clin Med Pathol & Forens Med, Kuopio, Finland.;Kuopio Univ Hosp, Dept Clin Pathol, Imaging Ctr, Kuopio, Finland..
    Kristensen, Vessela N.
    Univ Oslo, Hosp Radiumhosp, Inst Canc Res, Dept Canc Genet, Oslo, Norway. Oslo Univ Hosp, Dept Breast & Endocrine Surg, Oslo, Norway. Vestre Viken Hosp, Dept Res, Drammen, Norway. Univ Oslo, Hosp Radiumhosp, Inst Canc Res, Dept Tumor Biol, Oslo, Norway.;Univ Oslo, Fac Med, Inst Clin Med, Oslo, Norway.;Univ Oslo, Oslo Univ Hosp, Dept Clin Mol Biol, Oslo, Norway. Univ Oslo, Hosp Radiumhosp, Natl Advisory Unit Late Effects Canc Treatment, Oslo, Norway. Univ Oslo, Hosp Radiumhosp, Dept Oncol, Oslo, Norway. Univ Oslo, Hosp Radiumhosp, Dept Radiol & Nucl Med, Oslo, Norway. Oslo Univ Hosp, Oslo, Norway..
    Lambrechts, Diether
    VIB, VIB Ctr Canc Biol, Leuven, Belgium.;Univ Leuven, Dept Human Genet, Lab Translat Genet, Leuven, Belgium..
    Le Marchand, Loic
    Univ Hawaii, Ctr Canc, Program Epidemiol, Honolulu, HI 96822 USA..
    Lubinski, Jan
    Pomeranian Med Univ, Dept Genet & Pathol, Szczecin, Poland..
    Mannermaa, Arto
    Univ Eastern Finland, Translat Canc Res Area, Kuopio, Finland.;Univ Eastern Finland, Inst Clin Med Pathol & Forens Med, Kuopio, Finland.;Kuopio Univ Hosp, Dept Clin Pathol, Imaging Ctr, Kuopio, Finland..
    Martens, John W. M.
    Erasmus MC Canc Inst, Family Canc Clin, Dept Med Oncol, Rotterdam, Netherlands..
    Meindl, Alfons
    Tech Univ Munich, Div Gynaecol & Obstet, Munich, Germany..
    Milne, Roger L.
    Canc Council Victoria, Canc Epidemiol & Intelligence Div, Melbourne, Vic, Australia.;Univ Melbourne, Melbourne Sch Populat & Global Hlth, Ctr Epidemiol & Biostat, Melbourne, Vic, Australia..
    Mulligan, Anna Marie
    Univ Toronto, Dept Lab Med & Pathobiol, Toronto, ON, Canada.;Univ Hlth Network, Lab Med Program, Toronto, ON, Canada..
    Neuhausen, Susan L.
    Beckman Res Inst City Hope, Dept Populat Sci, Duarte, CA USA..
    Nevanlinna, Heli
    Univ Helsinki, Helsinki Univ Hosp, Dept Obstet & Gynecol, Helsinki, Finland..
    Peto, Julian
    London Sch Hyg & Trop Med, Dept Noncommun Dis Epidemiol, London, England..
    Pylkaes, Katri
    Univ Oulu, Bioctr Oulu, Canc & Translat Med Res Unit, Lab Canc Genet & Tumor Biol, Oulu, Finland.;Northern Finland Lab Ctr Oulu, Lab Canc Genet & Tumor Biol, Oulu, Finland..
    Radice, Paolo
    INT, Fdn IRCCS, Ist Ricovero Cura Carattere Sci, Dept Res, Milan, Italy..
    Rhenius, Valerie
    Univ Cambridge, Dept Oncol, Ctr Canc Genet Epidemiol, Cambridge, England..
    Sawyer, Elinor J.
    Kings Coll London, Guys Hosp, Res Oncol, London, England..
    Schmidt, Marjanka K.
    Antoni Leeuwenhoek Hosp, Netherlands Canc Inst, Div Mol Pathol, Amsterdam, Netherlands.;Antoni Leeuwenhoek Hosp, Netherlands Canc Inst, Div Psychosocial Res & Epidemiol, Amsterdam, Netherlands..
    Schmutzler, Rita K.
    Univ Hosp Cologne, Ctr Hereditary Breast & Ovarian Canc, Cologne, Germany.;Univ Hosp Cologne, Ctr Integrated Oncol, Cologne, Germany.;Univ Cologne, Ctr Mol Med Cologne, Cologne, Germany..
    Seynaeve, Caroline
    Erasmus MC Canc Inst, Family Canc Clin, Dept Med Oncol, Rotterdam, Netherlands..
    Shah, Mitul
    Univ Cambridge, Dept Oncol, Ctr Canc Genet Epidemiol, Cambridge, England..
    Simard, Jacques
    Laval Univ, Univ Quebec, Res Ctr, Ctr Hosp,Gen Ctr, Quebec City, PQ, Canada..
    Southey, Melissa C.
    Univ Melbourne, Dept Pathol, Melbourne, Vic, Australia..
    Swerdlow, Anthony J.
    Inst Canc Res, Div Genet & Epidemiol, London, England.;Inst Canc Res, Div Breast Canc Res, London, England. Peter MacCallum Canc Ctr, Melbourne, Vic, Australia..
    Truong, Therese
    Univ Paris Saclay, Univ Paris Sud, INSERM, Ctr Res Epidemiol & Populat Hlth CESP,Canc & Envi, Villejuif, France..
    Wendt, Camilla
    Karolinska Inst, Dept Oncol Pathol, Stockholm, Sweden..
    Winqvist, Robert
    Univ Oulu, Bioctr Oulu, Canc & Translat Med Res Unit, Lab Canc Genet & Tumor Biol, Oulu, Finland.;Northern Finland Lab Ctr Oulu, Lab Canc Genet & Tumor Biol, Oulu, Finland..
    Zheng, Wei
    Vanderbilt Univ, Sch Med, Div Epidemiol,Dept Med, Vanderbilt Ingram Canc Ctr,Vanderbilt Epidemiol C, Nashville, TN 37212 USA..
    Benitez, Javier
    Spanish Natl Canc Res Ctr, Human Canc Genet Program, Madrid, Spain.;Ctr Invest Red Enfermedades Raras CIBERER, Valencia, Spain..
    Dunning, Alison M.
    Univ Cambridge, Dept Oncol, Ctr Canc Genet Epidemiol, Cambridge, England..
    Pharoah, Paul D. P.
    Univ Cambridge, Dept Publ Hlth & Primary Care, Ctr Canc Genet Epidemiol, Cambridge, England.;Univ Cambridge, Dept Oncol, Ctr Canc Genet Epidemiol, Cambridge, England..
    Easton, Douglas F.
    Univ Cambridge, Dept Publ Hlth & Primary Care, Ctr Canc Genet Epidemiol, Cambridge, England.;Univ Cambridge, Dept Oncol, Ctr Canc Genet Epidemiol, Cambridge, England..
    Czene, Kamila
    Karolinska Inst, Dept Med Epidemiol & Biostat, Stockholm, Sweden..
    Hall, Per
    Karolinska Inst, Dept Med Epidemiol & Biostat, Stockholm, Sweden..
    Lindblom, Annika
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    PHIP - a novel candidate breast cancer susceptibility locus on 6q14.12017In: Oncotarget, E-ISSN 1949-2553, Vol. 8, no 61, p. 102769-102782Article in journal (Refereed)
    Abstract [en]

    Most non-BRCA1/2 breast cancer families have no identified genetic cause. We used linkage and haplotype analyses in familial and sporadic breast cancer cases to identify a susceptibility locus on chromosome 6q. Two independent genome-wide linkage analysis studies suggested a 3 Mb locus on chromosome 6q and two unrelated Swedish families with a LOD > 2 together seemed to share a haplotype in 6q14.1. We hypothesized that this region harbored a rare high-risk founder allele contributing to breast cancer in these two families. Sequencing of DNA and RNA from the two families did not detect any pathogenic mutations. Finally, 29 SNPs in the region were analyzed in 44,214 cases and 43,532 controls from BCAC, and the original haplotypes in the two families were suggested as low-risk alleles for European and Swedish women specifically. There was also some support for one additional independent moderate-risk allele in Swedish familial samples. The results were consistent with our previous findings in familial breast cancer and supported a breast cancer susceptibility locus at 6q14.1 around the PHIP gene.

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  • 167.
    Jimenez, Brenda Irene Medina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Earth Sciences, Department of Earth Sciences, Palaeobiology.
    Janssen, Ralf
    Uppsala University, Disciplinary Domain of Science and Technology, Earth Sciences, Department of Earth Sciences, Palaeobiology.
    A simple dissociation protocol for single-cell RNA sequencing of spider embryosManuscript (preprint) (Other academic)
    Abstract [en]

    The common house spider Parasteatoda tepidariorum is an emerging model organism used in Evo-devo research. However, and despite the rising demand for single cell RNA sequencing of arthropod species, dissociation protocols for arthropods are scarce. We provide the following protocol as a tool for such analysis in spider embryos. 

  • 168.
    Johansson, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Interaction Between Drosophila melanogaster mbn-2 Cells and Bacteria2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Innate immunity relies on a repertoire of germline-encoded non-rearranging pattern recognition receptors that bind to invariant microbial surface molecules. This event initiates a number of signal transduction cascades that lead to humoral and cellular defense responses like synthesis of antimicrobial peptides, phagocytosis and coagulation – mechanisms that efficiently fight infectious microorganisms and have been evolutionary conserved to exist in parallel with the antibody-based adaptive immunity found in vertebrates.

    The fruit fly, Drosophila melanogaster represents a widely used animal model for studies of a pristine innate immune system. Its immune responsive intracellular signalling pathways display a high degree of similarity with the NF-κB /Rel-signalling pathways that mediate the inflammatory response in mammals. Insects are also vectors for medically important parasitic diseases which can trigger immune responses in the vector so basal knowledge about the regulation and function of insect immune systems can contribute to our understanding of inflammation and microbial disease in higher animals and open new strategies for biological vector control.

    Drosophila hemocytes play a key role in executing and coordinating local and systemic defenses in response to infection. This thesis describes in vitro studies of Drosophila gene expression in response to bacterial infection using the larval hemocyte-like cell line – mbn-2. Our results show that immune challenge with bacterial cell wall components and intact live bacteria induces differential gene expression that gives clues to how cellular immune responses could be activated and regulated.

    List of papers
    1. Enteric bacteria counteract lipopolysaccharide induction of antimicrobial peptide genes
    Open this publication in new window or tab >>Enteric bacteria counteract lipopolysaccharide induction of antimicrobial peptide genes
    Show others...
    2001 (English)In: Journal of Immunology, ISSN 0022-1767, Vol. 167, p. 6920-6923Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-92568 (URN)
    Available from: 2005-02-11 Created: 2005-02-11 Last updated: 2009-04-05Bibliographically approved
    2. Microarray analysis of immune challenged Drosophila hemocytes
    Open this publication in new window or tab >>Microarray analysis of immune challenged Drosophila hemocytes
    2005 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 305, no 1, p. 145-155Article in journal (Refereed) Published
    Abstract [en]

    nsect hemocytes play multiple roles in immunity and carry out cellular responses like phagocytosis, encapsulation and melanization as well as producing humoral effector proteins in the first line of defense after injury and invasion of microorganisms. In this work, we used the Drosophila melanogaster hemocyte-like cell line mbn-2 and Affymetrix Drosophila GeneChips to investigate the transcriptome of a single type of immune competent tissue exposed to Gram-negative cell wall components (crude LPS) or high dose infection with live Escherichia coli. We found that gene expression profiles of both treatments overlap but show important differences in expression levels of several genes involved in immunity. In addition, cell morphology during infection was monitored and revealed distinct alterations in cell shape and adhesion. Presence of large numbers of bacteria also increased the number of cells taking on crystal cell fate. Taken together, our results indicate that hemocytes sense and respond differently to purified bacterial surface molecules and infection with live and actively growing bacteria both at the level of gene expression and in cell behavior.

    Keywords
    Animals, Cell Line, Drosophila Proteins/genetics, Drosophila melanogaster/*genetics/immunology, Escherichia coli, Gene Expression Regulation, Gram-Negative Bacteria/immunology, Hemocytes/*immunology, Lipopolysaccharides/toxicity, Microscopy; Electron; Scanning, Oligonucleotide Array Sequence Analysis/*methods, Research Support; Non-U.S. Gov't
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-92569 (URN)10.1016/j.yexcr.2004.12.018 (DOI)15777795 (PubMedID)
    Available from: 2005-02-11 Created: 2005-02-11 Last updated: 2017-12-14Bibliographically approved
    3. Diptericin expression in bacteria infected Drosophila mbn-2 cells - effect of infection dose and phagocytosis
    Open this publication in new window or tab >>Diptericin expression in bacteria infected Drosophila mbn-2 cells - effect of infection dose and phagocytosis
    (English)Manuscript (Other (popular science, discussion, etc.))
    Identifiers
    urn:nbn:se:uu:diva-92570 (URN)
    Available from: 2005-02-11 Created: 2005-02-11 Last updated: 2010-10-11Bibliographically approved
    4. Pefabloc: A sulfonyl fluoride serine protease inhibitor blocks induction of Diptericin in Drosophila l(2)mbn cells
    Open this publication in new window or tab >>Pefabloc: A sulfonyl fluoride serine protease inhibitor blocks induction of Diptericin in Drosophila l(2)mbn cells
    2012 (English)In: Insect Science, ISSN 1672-9609, E-ISSN 1744-7917, Vol. 19, no 4, p. 472-476Article in journal (Refereed) Published
    Abstract [en]

    Insects protect themselves against microbial infection by an efficient innate immune system that is activated by recognition of invariant microbial surface molecules. In the fruit fly Drosophila melanogaster the presence of bacteria is associated with expression of antimicrobial peptides in host immune-competent tissues. Host receptors detect infection and relay the signal to mount the appropriate immune response. In Drosophila hemocyte-like l(2)mbn cells pre-infection treatment with Pefabloc, a commonly used serine protease inhibitor, induced two major effects: it blocked expression of the antibacterial peptide Diptericin in response to live Gram-negative bacteria and bacterial surface molecules (crude lipopolysaccharide contaminated by peptidoglycans) and it induced morphological changes.

    Keywords
    cell morphology, diptericin, Drosophila, IMD pathway, immune response, proteinase inhibitor
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-92571 (URN)10.1111/j.1744-7917.2011.01482.x (DOI)000306786800005 ()
    Available from: 2005-02-11 Created: 2005-02-11 Last updated: 2017-12-14
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    FULLTEXT01
  • 169.
    Johansson, Patrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Krona, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kundu, Soumi
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Doroszko, Milena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Baskaran, Sathishkumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Schmidt, Linnea
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vinel, Claire
    Queen Mary Univ London, Barts & London Sch Med & Dent, Blizard Inst, London E1 2AT, England..
    Almstedt, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elgendy, Ramy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elfineh, Lioudmila
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Gallant, Caroline J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundsten, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gago, Fernando J. Ferrer
    Agcy Sci Technol & Res Star, Singapore 138648, Singapore..
    Hakkarainen, Aleksi
    Univ Turku, Res Ctr Integrat Physiol & Pharmacol, Inst Biomed, Turku 20500, Finland..
    Sipila, Petra
    Univ Turku, Res Ctr Integrat Physiol & Pharmacol, Inst Biomed, Turku 20500, Finland..
    Haggblad, Maria
    Stockholm Univ, Dept Biochem & Biophys, SciLifeLab, S-10405 Stockholm, Sweden..
    Martens, Ulf
    Stockholm Univ, Dept Biochem & Biophys, SciLifeLab, S-10405 Stockholm, Sweden..
    Lundgren, Bo
    Stockholm Univ, Dept Biochem & Biophys, SciLifeLab, S-10405 Stockholm, Sweden..
    Frigault, Melanie M.
    AstraZeneca Oncol, Waltham, MA 02451 USA..
    Lane, David P.
    Agcy Sci Technol & Res Star, Singapore 138648, Singapore.;Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Sci Life Lab, S-17177 Stockholm, Sweden..
    Swartling, Fredrik J
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Uhrbom, Lene
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Nestor, Marika
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Marino, Silvia
    Queen Mary Univ London, Barts & London Sch Med & Dent, Blizard Inst, London E1 2AT, England..
    Nelander, Sven
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    A Patient-Derived Cell Atlas Informs Precision Targeting of Glioblastoma2020In: Cell Reports, E-ISSN 2211-1247, Vol. 32, no 2, article id 107897Article in journal (Refereed)
    Abstract [en]

    Glioblastoma (GBM) is a malignant brain tumor with few therapeutic options. The disease presents with a complex spectrum of genomic aberrations, but the pharmacological consequences of these aberrations are partly unknown. Here, we report an integrated pharmacogenomic analysis of 100 patient-derived GBM cell cultures from the human glioma cell culture (HGCC) cohort. Exploring 1,544 drugs, we find that GBM has two main pharmacological subgroups, marked by differential response to proteasome inhibitors and mutually exclusive aberrations in TP53 and CDKN2A/B. We confirm this trend in cell and in xenotransplantation models, and identify both Bcl-2 family inhibitors and p53 activators as potentiators of proteasome inhibitors in GBM cells, We can further predict the responses of individual cell cultures to several existing drug classes, presenting opportunities for drug repurposing and design of stratified trials. Our functionally profiled biobank provides a valuable resource for the discovery of new treatments for GBM.

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    FULLTEXT01
  • 170.
    Johnsson, Anna-Karin
    et al.
    Karolinska Inst, Inst Environm Med, Stockholm, Sweden.;Karolinska Inst, Ctr Allergy Res, Stockholm, Sweden..
    Rönnberg, Elin
    Karolinska Inst, Immunol & Allergy Div, Dept Med, Stockholm, Sweden.;Karolinska Univ Hosp Solna, Stockholm, Sweden.;Karolinska Inst, Ctr Allergy Res, Stockholm, Sweden..
    Fuchs, David
    Karolinska Inst, Dept Med Biochem & Biophys, Div Physiol Chem 2, Stockholm, Sweden..
    Kolmert, Johan
    Karolinska Inst, Inst Environm Med, Stockholm, Sweden.;Karolinska Inst, Ctr Allergy Res, Stockholm, Sweden..
    Säfholm, Jesper
    Karolinska Inst, Inst Environm Med, Stockholm, Sweden.;Karolinska Inst, Ctr Allergy Res, Stockholm, Sweden..
    Claesson, Hans-Erik
    Karolinska Inst, Dept Internal Med, Stockholm, Sweden..
    Hamberg, Mats
    Karolinska Inst, Dept Med Biochem & Biophys, Div Physiol Chem 2, Stockholm, Sweden..
    Wheelock, Craig E.
    Karolinska Inst, Dept Med Biochem & Biophys, Div Physiol Chem 2, Stockholm, Sweden..
    Nilsson, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology. Karolinska Inst, Immunol & Allergy Div, Dept Med, Stockholm, Sweden.;Karolinska Univ Hosp Solna, Stockholm, Sweden.;Karolinska Inst, Ctr Allergy Res, Stockholm, Sweden..
    Dahlén, Sven-Erik
    Karolinska Inst, Inst Environm Med, Stockholm, Sweden.;Karolinska Inst, Ctr Allergy Res, Stockholm, Sweden..
    COX-1 dependent biosynthesis of 15-hydroxyeicosatetraenoic acid in human mast cells2021In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1866, no 5, article id 158886Article in journal (Refereed)
    Abstract [en]

    15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined.

  • 171.
    Jones, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Unoson, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Leroy, Prune
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Curic, Vladimir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Kinetics of dCas9 Target Search in Escherichia Coli2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 314A-314AArticle in journal (Other academic)
  • 172.
    Junkunlo, Kingkamon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Regulation of hematopoiesis in the freshwater crayfish, Pacifastacus leniusculus: role of transglutaminase2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The freshwater crayfish, Pacifastacus leniusculus, has been used as a model for studying hematopoiesis or blood cell production or hematopoiesis and immunity. The work of this thesis aims to investigate the impact of factors such as ROS signaling, Ast1, and the PVF/PVR signaling pathway in controlling stem cell behavior during hematopoiesis and specifically the role of the crosslinking enzyme transglutaminase (TGase) in regulation of hematopoiesis.

    The role of ROS in crayfish hematopoiesis was characterized by using the antioxidant named NAC to inhibit ROS production. Low ROS level resulted in a prolonged decrease in hemocyte numbers and a combined injection of LPS and NAC caused a slower rate of new hemocyte production. A low ROS level in cell cultures supplemented with crude Ast1 was found to inhibit cell spreading and a high extracellular TGase activity was detected on the surfaces of APC and HPT cells. We suggest that ROS serves as a prime signal to control proliferation and differentiation of progenitor cells by affecting extracellular TGase activity. We reported an inhibitory effect of Ast1 on TGase enzyme activity and on its crosslinking activity and consequently Ast1 affects the clot formation and thus coagulation by inhibiting the crosslinking activity of the TGase enzyme. Secretion of the clot protein (CP) and the production of CP filament network between spreading cells were observed in HPT cell cultures in vitro. In the presence of CP together with Ast1 in 3D-collagen-I cultures, HPT cells were found to be more elongated and they formed chains of cells throughout the surrounding matrix. In the HPT tissue, CP was located around the HPT cells or around the lobules of HPT, and thus, CP was demonstrated to be a part of ECM and to possibly function together with collagen in generating a suitable environment for HPT progenitor cells. The inhibition of PVF/PVR downstream signaling pathway by Sunitinib malate resulted in a dramatic change of cell morphology and induction of an increase cell surface area during cell culture. The addition of crude Ast1 into the cell cultures in vitro enhanced this effect. Consequently, cell migration was stimulated and a high extracellular TGase activity on HPT cell surface was found after this inhibition. In conclusion, the work in this thesis provides new insight in understanding the role of the extracellular matrix (ECM) and extracellular TGase activity in controlling stem cell activity.

    List of papers
    1. Reactive oxygen species affect transglutaminase activity and regulate hematopoiesis in a crustacean
    Open this publication in new window or tab >>Reactive oxygen species affect transglutaminase activity and regulate hematopoiesis in a crustacean
    2016 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 34, p. 17593-17601Article in journal (Refereed) Published
    Abstract [en]

    Reactive oxygen species (ROS) serve as a prime signal in the commitment to hematopoiesis in both mammals and Drosophila. In this study, the potential function of ROS during hematopoiesis in the crayfish Pacifastacus leniusculus was examined. The antioxidant N-acetylcysteine (NAC) was used to decrease ROS in both in vivo and in vitro experiments. An increase in ROS was observed in the anterior proliferation center (APC) after LPS injection. In the absence of NAC, the LPS-induced increase in ROS levels resulted in the rapid restoration of the circulating hemocyte number. In the presence of NAC, a delay in the recovery rate of the hemocyte number was observed. NAC treatment also blocked the spread of APC and other hematopoietic tissue (HPT) cells, maintaining these cells at an undifferentiated stage. Extracellular transglutaminase (TGase) has been shown previously to play a role in maintaining HPT cells in an undifferentiated form. In this study, we show that extracellular TGase activity increased when the ROS level in HPT or APC cells was reduced after NAC treatment. In addition, collagen, a major component of the extracellular matrix and a TGase substrate were co-localized on the HPT cell surface. Taken together, the results of this study show that ROS are involved in crayfish hematopoiesis, in which a low ROS level is required to maintain hematopoietic progenitor cells in the tissue and to reduce hemocyte release. The potential roles of TGase in this process are investigated and discussed.

    Keywords
    extracellular matrix, hematopoiesis, invertebrate, reactive oxygen species (ROS), transglutaminase
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biology with specialization in Comparative Physiology
    Identifiers
    urn:nbn:se:uu:diva-305568 (URN)10.1074/jbc.M116.741348 (DOI)000383241300011 ()27339892 (PubMedID)
    Funder
    Swedish Research Council, VR 20114797, VR 621-2012-2418
    Available from: 2016-10-19 Created: 2016-10-19 Last updated: 2017-08-22
    2. Role of astakine1 in regulating transglutaminase activity
    Open this publication in new window or tab >>Role of astakine1 in regulating transglutaminase activity
    2017 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 76, p. 77-82Article in journal (Refereed) Published
    Abstract [en]

    Transglutaminase (TGase) has been implicated in maintaining the undifferentiated stage of hematopoietic stem cells (HSC) in the crayfish Pacifastacus leniusculus. TGase activity has been reported to be regulated by astakine1, an essential crayfish cytokine for inducing new hemocyte synthesis in hematopoietic tissue (HPT). Here, the role of astakine1 in TGase activity regulation and clotting protein (CP) cross-linking was characterized. A reduction in TGase activity was observed by the addition of purified astakine1 in vitro for both endogenous crayfish TGase and a commercial purified guinea pig liver TGase. As a result, we observed that astakine1 inhibits TGase enzyme activity and acts as a non-competitive inhibitor for the TGase enzyme. Additionally, the clotting reaction was impaired in the presence of astakine1. A decrease in TGase-mediated crosslinking of ε(γ-glutamyl)-lysine bonds was also observed in the presence of astakine1. In conclusion, this study shows that astakine1 acts as an inhibitor of TGase activity and that it also affects CP cross-linking during crayfish hematopoiesis.

    Place, publisher, year, edition, pages
    Elsevier, 2017
    Keywords
    Astakine1, Clotting protein, Hematopoiesis, Transglutaminase activity
    National Category
    Developmental Biology Immunology Zoology
    Research subject
    Biology with specialization in Comparative Physiology
    Identifiers
    urn:nbn:se:uu:diva-327217 (URN)10.1016/j.dci.2017.05.015 (DOI)000407985100009 ()28528959 (PubMedID)
    Funder
    Swedish Research Council, VR 621-2012-2418
    Available from: 2017-08-07 Created: 2017-08-07 Last updated: 2017-10-09Bibliographically approved
    3. PDGF/VEGF-related receptor affects transglutaminase activity to control cell migration during crustacean hematopoiesis
    Open this publication in new window or tab >>PDGF/VEGF-related receptor affects transglutaminase activity to control cell migration during crustacean hematopoiesis
    2017 (English)In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 26, no 20, p. 1449-1459Article in journal (Refereed) Published
    Abstract [en]

    The platelet-derived growth factor (PDGF) receptor, a tyrosine kinase (TK) receptor whose ligand is PDGF, is crucial in the transduction of extracellular signals into cells and mediates numerous processes, such as cell proliferation, differentiation, survival, and migration. We demonstrate the important roles of a receptor TK related to the PDGF/VEGF family protein (PVR) in controlling hematopoietic progenitor cell migration by affecting extracellular transglutaminase (TGase) activity. Pl_PVR1, GenBank accession No. KY444650, is highly expressed in hemocytes and the hematopoietic tissue (HPT). Sunitinib malate was used to block the PVF/PVR downstream pathway in HPT cell culture. The addition of Sunitinib also caused the HPT cells to increase in size and begin spreading. An increase in extracellular TGase activity on the HPT cell membrane was observed in a dose-dependent manner after treatment with Sunitinib malate. The presence of crude Ast1 provided a combinatorial beneficial effect that enhanced the number of spreading cells after inhibition of the Pl_PVR downstream signaling cascade. In addition, an increased immunoreactivity for beta-tubulin and elongation of beta-tubulin filaments were found in Pl_PVR signaling-inhibited cells. The potential roles of PVF/PVR signaling in controlling progenitor cell activity during hematopoiesis in crayfish were investigated and discussed.

    Keywords
    PDGF/VEGF, hematopoiesis, Transglutaminase, Ast1, crayfish
    National Category
    Developmental Biology Cell Biology Immunology
    Research subject
    Biology with specialization in Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-327243 (URN)10.1089/scd.2017.0086 (DOI)000412919700001 ()28805145 (PubMedID)
    Funder
    Swedish Research Council, VR 2011-4797, VR 621-2012-2418
    Available from: 2017-08-07 Created: 2017-08-07 Last updated: 2019-08-22Bibliographically approved
    4. Clotting protein - an extracellular matrix (ECM) protein involved in crustacean hematopoiesis
    Open this publication in new window or tab >>Clotting protein - an extracellular matrix (ECM) protein involved in crustacean hematopoiesis
    (English)In: ISSN 0145-305XArticle in journal (Refereed) Submitted
    Keywords
    clotting protein, ECM, hematopoiesis, crustacean
    National Category
    Immunology Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-327248 (URN)
    Available from: 2017-08-07 Created: 2017-08-07 Last updated: 2017-08-14
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  • 173.
    Junkunlo, Kingkamon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Noonin, Chadanat
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    PDGF/VEGF-related receptor affects transglutaminase activity to control cell migration during crustacean hematopoiesis2017In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 26, no 20, p. 1449-1459Article in journal (Refereed)
    Abstract [en]

    The platelet-derived growth factor (PDGF) receptor, a tyrosine kinase (TK) receptor whose ligand is PDGF, is crucial in the transduction of extracellular signals into cells and mediates numerous processes, such as cell proliferation, differentiation, survival, and migration. We demonstrate the important roles of a receptor TK related to the PDGF/VEGF family protein (PVR) in controlling hematopoietic progenitor cell migration by affecting extracellular transglutaminase (TGase) activity. Pl_PVR1, GenBank accession No. KY444650, is highly expressed in hemocytes and the hematopoietic tissue (HPT). Sunitinib malate was used to block the PVF/PVR downstream pathway in HPT cell culture. The addition of Sunitinib also caused the HPT cells to increase in size and begin spreading. An increase in extracellular TGase activity on the HPT cell membrane was observed in a dose-dependent manner after treatment with Sunitinib malate. The presence of crude Ast1 provided a combinatorial beneficial effect that enhanced the number of spreading cells after inhibition of the Pl_PVR downstream signaling cascade. In addition, an increased immunoreactivity for beta-tubulin and elongation of beta-tubulin filaments were found in Pl_PVR signaling-inhibited cells. The potential roles of PVF/PVR signaling in controlling progenitor cell activity during hematopoiesis in crayfish were investigated and discussed.

    Download full text (pdf)
    Junkunlo et al 2017 SCAD
  • 174.
    Junkunlo, Kingkamon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    A transcription factor glial cell missing (Gcm) in the freshwater crayfish Pacifastacus leniusculus2020In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 113, article id 103782Article in journal (Refereed)
    Abstract [en]

    The transcription factor glial cell missing, Gcm, is known to be an important protein in the determination of glial cell fate as well as embryonic plasmatocyte differentiation in Drosophila melanogaster. So far, no function for Gcm in crustaceans has been reported. In this study, we show the cDNA sequence of a Gcm homologue in the freshwater crayfish Pacifastacus leniusculus. The P. leniusculus Gcm transcript is expressed exclusively in brain and nervous tissue, and by in situ hybridization we show that the expression is restricted to a small number of large cells with morphology similar to neurosecretory cells. Furthermore, we show that the expression of Gcm coincides with the expression of a Repo homologue, that is induced in expression by Gcm in Drosophila. Moreover, the Gcm transcript is increased shortly and transiently after injection of cystamine, a substance that inhibits transglutaminase and also strongly affects the movement behavior of crayfish. This finding of Gcm transcripts in a subpopulation of brain cells in very low numbers may enable more detailed studies about Gcm in adult crustaceans.

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    fulltext
  • 175.
    Junkunlo, Kingkamon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Clotting protein - an extracellular matrix (ECM) protein involved in crustacean hematopoiesisIn: ISSN 0145-305XArticle in journal (Refereed)
  • 176.
    Junkunlo, Kingkamon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Transglutaminase inhibition stimulates hematopoiesis and reduces aggressive behavior of crayfish, Pacifastacus leniusculus2019In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, no 2, p. 708-715Article in journal (Refereed)
    Abstract [en]

    Transglutaminase (TGase) is a Ca2+-dependent cross-linking enzyme, which has both enzymatic and nonenzymatic properties. TGase is involved in several cellular activities, including adhesion, migration, survival, apoptosis, and extracellular matrix (ECM) organization. In this study, we focused on the role of the TGase enzyme in controlling hematopoiesis in the crayfish, Pacifastacus leniusculus. We hypothesized that a high TGase activity could mediate an interaction of progenitor cells with the ECM to maintain cells in an undifferentiated stage in the hematopoietic tissue (HPT). We found here that the reversible inhibitor cystamine decreases the enzymatic activity of TGase from crayfish HPT, as well as from guinea pig, in a concentration-dependent manner. Cystamine injection decreased TGase activity in HPT without affecting production of reactive oxygen species. Moreover, the decrease in TGase activity in the HPT increased the number of circulating hemocytes. Interestingly the cystamine-mediated TGase inhibition reduced aggressive behavior and movement in crayfish. In conclusion, we show that cystamine-mediated TGase inhibition directly releases HPT progenitor cells from the HPT into the peripheral circulation in the hemolymph and strongly reduces aggressive behavior in crayfish.

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    Junkunlo et al 2019 JBC
  • 177.
    Jurjee, Zain
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala univeritet.
    Östrogen receptor och EGFR samarbete i cancerutveckling2023Independent thesis Basic level (degree of Bachelor), 180 HE creditsStudent thesis
    Abstract [sv]

    Bakgrund: Denna studie riktar sig mot att förstå samspelet mellan intracellurlära receptorer och receptortyrosinkinas som resulterar i cancerutveckling. Mer specifikt riktar denna studie sig mot Östrogena receptorer och epidermal growth factor receptor. Deras interaktion är viktig att förstå då cancer är en livshotande och komplex sjukdom som kräver djupgående förståelse. Detta är av betydelse för att förstå exakta mekanismer för uppkomst av en cancercell och detta är till god kunskap för hälsosjukvården att få en bättre förståelse som underlättar hur man ska gå till väga när det kommer till diagnostik samt vad som skulle vara bästa möjliga tillvägagångssätt för den mest optimala terapin. Det ger även information för oss som gör det möjligt att utveckla nya bättre behandlingar mot bröstcancer.  Syfte: Syftet med denna studie är att försöka förstå interaktionen mellan intracellulära receptorer och tyrosinkinasreceptor samt hur man kan tillämpa informationen bakom denna interaktion för att få bättre och mer optimala behandlingar vid bröstcancer.   Resultat: Flera studier har tyder på att man får ett ökat uttryck av signalvägarna MAPK, PI3K-akt och STAT5 genom aktiveringen av ER och EGFR. Det har visat attt EGFR har möjligheten att fosforylera ER och dess co-aktivator vilket förstärker den transkriptionella aktiviteten. Dessutom så delar de signalvägar vilket gör det möjligt för ett samspel mellan receptorerna då aktivering av en signalväg kan fosforylera proteiner som har andra signalvägar som mål, lättare sagt bidrar det även till aktivering av andra signalvägar.  Slutsats: Det har visat sig finnas ett samarbete mellan ER och EGFR genom dess signalvägar som står till grund bakom bröstcancerutvecklingen. Dessa mekanismer kommer vara av nytta till framtida forskning genom att rikta terapierna mot dessa för att stoppa utveckling och ge patienter en bättre behandling. 

  • 178.
    Kahata, Kaoru
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Ludwig Institute for Cancer Research.
    Maturi, Varun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Ludwig Institute for Cancer Research.
    Moustakas, Aristidis
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Ludwig Institute for Cancer Research.
    TGF-beta Family Signaling in Ductal Differentiation and Branching Morphogenesis2018In: Cold Spring Harbor Perspectives in Biology, E-ISSN 1943-0264, Vol. 10, no 3, article id a031997Article in journal (Refereed)
    Abstract [en]

    Epithelial cells contribute to the development of various vital organs by generating tubular and/or glandular architectures. The fully developed forms of ductal organs depend on processes of branching morphogenesis, whereby frequency, total number, and complexity of the branching tissue define the final architecture in the organ. Some ductal tissues, like the mammary gland during pregnancy and lactation, disintegrate and regenerate through periodic cycles. Differentiation of branched epithelia is driven by antagonistic actions of parallel growth factor systems that mediate epithelial-mesenchymal communication. Transforming growth factor-beta (TGF-beta) family members and their extracellular antagonists are prominently involved in both normal and disease-associated (e.g., malignant or fibrotic) ductal tissue patterning. Here, we discuss collective knowledge that permeates the roles of TGF-beta family members in the control of the ductal tissues in the vertebrate body.

  • 179.
    Kaminska, Mathilda
    et al.
    Institute of Biochemistry II, INSPIRE laboratory, Jena University Hospital, Jena, Germany.
    Maurer, Michelle
    Institute of Biochemistry II, INSPIRE laboratory, Jena University Hospital, Jena, Germany.
    Gensheimer, Tarek
    Applied Stem Cell Technologies, Twente University, Enschede, Netherlands.
    Fuchs, Stefanie
    Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Graz, Austria.
    Werr, Gabriel
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering, Microsystems Technology.
    Johansson, Sofia
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering, Microsystems Technology.
    van der Meer, Andries
    Mayr, Torsten
    Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Graz, Austria.
    Tenje, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering, Microsystems Technology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mahé, Maxime
    Inserm UMR 1235-TENS, INSERM, University of Nantes, Nantes, France.
    Mosig, Alexander
    Department of Materials Science and Engineering, Science for Life laboratory Uppsala University, Uppsala, Sweden.
    Increasing the physiological relevance of the gut-on-chip model2020Conference paper (Other academic)
  • 180.
    Kamranvar, Siamak A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gupta, Deepesh Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wasberg, Anishia
    Liu, Liangwen
    Roig, J
    Johansson, Staffan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Focal adhesion kinase (FAK) is required for centrosome separation and subsequent bipolar mitotic spindle  assemblyManuscript (preprint) (Other academic)
  • 181.
    Kamranvar, Siamak A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Rani, Bhavna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Johansson, Staffan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cell Cycle Regulation by Integrin-Mediated Adhesion2022In: Cells, E-ISSN 2073-4409, Vol. 11, no 16, article id 2521Article, review/survey (Refereed)
    Abstract [en]

    Cell cycle and cell adhesion are two interdependent cellular processes regulating each other, reciprocally, in every cell cycle phase. The cell adhesion to the extracellular matrix (ECM) via integrin receptors triggers signaling pathways required for the cell cycle progression; the passage from the G1 to S phase and the completion of cytokinesis are the best-understood events. Growing evidence, however, suggests more adhesion-dependent regulatory aspects of the cell cycle, particularly during G2 to M transition and early mitosis. Conversely, the cell cycle machinery regulates cell adhesion in manners recently shown driven mainly by cyclin-dependent kinase 1 (CDK1). This review summarizes the recent findings regarding the role of integrin-mediated cell adhesion and its downstream signaling components in regulating the cell cycle, emphasizing the cell cycle progression through the G2 and early M phases. Further investigations are required to raise our knowledge about the molecular mechanisms of crosstalk between cell adhesion and the cell cycle in detail.

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  • 182. Kaucka, Marketa
    et al.
    Ivashkin, Evgeny
    Gyllborg, Daniel
    Zikmund, Tomas
    Tesarova, Marketa
    Kaiser, Jozef
    Xie, Meng
    Petersen, Julian
    Pachnis, Vassilis
    Nicolis, Silvia K.
    Yu, Tian
    Sharpe, Paul
    Arenas, Ernest
    Brismar, Hjalmar
    Blom, Hans
    Clevers, Hans
    Suter, Ueli
    Chagin, Andrei S.
    Fried, Kaj
    Hellander, Andreas
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computational Science.
    Adameyko, Igor
    Analysis of neural crest-derived clones reveals novel aspects of facial development2016In: Science Advances, E-ISSN 2375-2548, Vol. 2, no 8, p. e1600060:1-16, article id e1600060Article in journal (Refereed)
  • 183. Kaucka, Marketa
    et al.
    Zikmund, Tomas
    Tesarova, Marketa
    Gyllborg, Daniel
    Hellander, Andreas
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computational Science.
    Jaros, Josef
    Kaiser, Jozef
    Petersen, Julian
    Szarowska, Bara
    Newton, Phillip T.
    Dyachuk, Vyacheslav
    Li, Lei
    Qian, Hong
    Johansson, Anne-Sofie
    Mishina, Yuji
    Currie, Josh
    Tanaka, Elly M.
    Erickson, Alek
    Dudley, Andrew
    Brismar, Hjalmar
    Southam, Paul
    Coen, Enrico
    Chen, Min
    Weinstein, Lee S.
    Hampl, Ales
    Arenas, Ernest
    Chagin, Andrei S.
    Fried, Kaj
    Adameyko, Igor
    Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage2017In: eLIFE, E-ISSN 2050-084X, Vol. 6, article id e25902Article in journal (Refereed)
  • 184.
    Kermpatsou, Despoina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wåhlén, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lennartsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Silencing of 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) receptor in prostate cancer cell lines PC3 and DU145: effects on cellular responsesManuscript (preprint) (Other academic)
    Abstract [en]

    1,25(OH)2-vitamin D3 has well-documented roles in a variety of cellular processes, including proliferation and differentiation. 1,25(OH)2-vitamin D3 signalling is largely mediated by vitamin D receptor (VDR), however, 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) receptor has been investigated as a potential transducer of 1,25(OH)2-vitamin D3 membrane-initiated response. In this work we aimed to study the role of 1,25D3-MARRS in androgen-independent prostate carcinoma cell lines PC3 and DU145. Protein lysates from an array of cell lines were enriched for cytoplasmic and nuclear protein pools, in order to investigate the subcellular localisation of 1,25D3-MARRS and VDR. Further, we studied the effects of siRNA-mediated 1,25D3-MARRS depletion on cell proliferation and migration, as well as on gene expression of 1,25(OH)2-vitamin D3 metabolising enzymes and other genes of importance for 1,25(OH)2-vitamin D3-mediated signalling. The results of the present study indicate that depletion of 1,25D3-MARRS decreases proliferation in PC3 and DU145 prostate cancer cell lines, potentially via downregulation of c-Myc. 1,25D3-MARRS silencing also increased migration in PC3 cells, but not in DU145 cells, suggesting differential effects in different prostate cancer cell lines. In addition, significant effects of 1,25D3-MARRS silencing were found on CYP27B1 and CYP24A1, enzymes responsible for the regulation of cellular levels of 1,25(OH)2-vitamin D3. In summary, our data indicate that 1,25D3-MARRS can affect proliferation and/or migration in androgen-independent prostate cancer cells and may play a role for regulation and maintenance of adequate cellular 1,25(OH)2-vitamin D3 levels, either together with or independently of VDR.

  • 185.
    Khalil, Mahmoud I.
    et al.
    Beirut Arab Univ, Fac Sci, Dept Biol Sci, Beirut 11072809, Lebanon.;Alexandria Univ, Fac Sci, Dept Zool, Mol Biol Unit, Alexandria 21511, Egypt..
    Ali, Mohamad Moustafa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Holail, Jasmine
    Alfaisal Univ, Coll Med, Dept Biochem & Mol Med, Riyadh, Saudi Arabia.;Univ Bristol, Bristol Med Sch, Translat Hlth Sci, Bristol, England..
    Houssein, Marwa
    Beirut Arab Univ, Fac Sci, Dept Biol Sci, Beirut 11072809, Lebanon..
    Growth or death?: Control of cell destiny by mTOR and autophagy pathways2023In: Progress in Biophysics and Molecular Biology, ISSN 0079-6107, E-ISSN 1873-1732, Vol. 185, p. 39-55Article, review/survey (Refereed)
    Abstract [en]

    One of the central regulators of cell growth, proliferation, and metabolism is the mammalian target of rapamycin, mTOR, which exists in two structurally and functionally different complexes: mTORC1 and mTORC2; unlike m TORC2, mTORC1 is activated in response to the sufficiency of nutrients and is inhibited by rapamycin. mTOR complexes have critical roles not only in protein synthesis, gene transcription regulation, proliferation, tumor metabolism, but also in the regulation of the programmed cell death mechanisms such as autophagy and apoptosis. Autophagy is a conserved catabolic mechanism in which damaged molecules are recycled in response to nutrient starvation. Emerging evidence indicates that the mTOR signaling pathway is frequently activated in tumors. In addition, dysregulation of autophagy was associated with the development of a variety of human diseases, such as cancer and aging. Since mTOR can inhibit the induction of the autophagic process from the early stages of autophagosome formation to the late stage of lysosome degradation, the use of mTOR inhibitors to regulate autophagy could be considered a potential therapeutic option. The present review sheds light on the mTOR and autophagy signaling pathways and the mechanisms of regulation of mTOR-autophagy.

  • 186. Khorshidi, Mohammad Ali
    et al.
    Rajeswari, Prem Kumar Periyannan
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Jönsson, Håkan N.
    Andersson Svahn, Helene
    Automated analysis of dynamic behavior of single cells in picoliter droplets2014In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, p. 931-937Article in journal (Refereed)
  • 187.
    Kollberg Hedström, Tobias
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Molecular Mechanisms of Endophilin B1-Bax Macromolecular Complexes in Membrane Permeabilization and Cell Death2022Independent thesis Advanced level (degree of Master (Two Years)), 80 credits / 120 HE creditsStudent thesis
    Abstract [en]

    A crucial step during apoptosis is the accumulation of the pro-apoptotic protein Bax on the mitochondria where it triggers permeabilization of the outer membrane. This causes the release of cytochrome c into the cytosol and is considered a point of no return in programmed cell death. Endophilin B1, also known as Bax-interacting factor 1 (Bif-1) stimulates mitochondrial recruitment of Bax during apoptosis and loss of endophilin B1 is noted in many cancer types. Despite the importance of their interaction its role and function during cell death remains unclear. To examine the molecular mechanism behind their interaction this project aimed at solving the structure of endophilin B1-Bax complexes when bound to membrane mimicking platforms known as nanodiscs (NDs). NDs are composed of a lipid bilayer held together by a membrane scaffolding protein (MSP) that encircles the bilayer creating a disc-shaped structure. By designing NDs that resembles the mitochondrial outer membrane (MOM), this study intended to stimulate complex formation and stable binding to nanodiscs with the ambition of visualising their interaction using Cryo-EM. Due to difficulties of expressing and purifying Bax as well as time consuming optimization of ND assembly the final goal could not be reached. By establishing an optimized protocol for NDs using the MSP variant MSP2N2 and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) lipids as well as identifying challenges of expressing and purifying Bax this study lays ground for future structural studies that aims at elucidating the molecular mechanism behind the interaction.

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    Tobias Kollberg Hedström Master Thesis
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    Tobias Kollberg Hedström Popular Science Summary
  • 188.
    Kolliopoulos, Constantinos
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ali, Mohamad Moustafa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Castillejo-Lopez, Casimiro
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heldin, Paraskevi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence2022In: Cancers, ISSN 2072-6694, Vol. 14, no 15, article id 3747Article in journal (Refereed)
    Abstract [en]

    Simple Summary The hyaluronan receptor CD44 has an important role in glioblastoma multiforme (GBM) progression, but the precise mechanisms have not been elucidated. We have analyzed U251MG glioma cells, expressing CD44 or not, and grown in stem cell-like enriched spheres. Our results revealed that CD44 is important for cell growth and stemness, and for the prevention of senescence. Analysis by RNA sequencing revealed that CD44 is important for the interaction with the hyaluronan-enriched microenvironment. In addition, CD44 depletion impairs certain gene signatures, such as those for platelet-derived growth factor (PDGF) isoforms and PDGF receptors, as well as signatures related to hypoxia, glycolysis, and anti-tumor immune responses. Glioblastoma multiforme (GBM) is a lethal brain tumor, characterized by enhanced proliferation and invasion, as well as increased vascularization and chemoresistance. The expression of the hyaluronan receptor CD44 has been shown to correlate with GBM progression and poor prognosis. Here, we sought to elucidate the molecular mechanisms by which CD44 promotes GBM progression by knocking out (KO) CD44, employing CRISPR/Cas9 gene editing in U251MG cells. CD44-depleted cells exhibited an impaired proliferation rate, as shown by the decreased cell numbers, decreased Ki67-positive cell nuclei, diminished phosphorylation of CREB, and increased levels of the cell cycle inhibitor p16 compared to control cells. Furthermore, the CD44 KO cells showed decreased stemness and increased senescence, which was manifested upon serum deprivation. In stem cell-like enriched spheres, RNA-sequencing analysis of U251MG cells revealed a CD44 dependence for gene signatures related to hypoxia, the glycolytic pathway, and G2 to M phase transition. Partially similar results were obtained when cells were treated with the gamma-secretase inhibitor DAPT, which inhibits CD44 cleavage and therefore inhibits the release of the intracellular domain (ICD) of CD44, suggesting that certain transcriptional responses are dependent on CD44-ICD. Interestingly, the expression of molecules involved in hyaluronan synthesis, degradation, and interacting matrix proteins, as well as of platelet-derived growth factor (PDGF) isoforms and PDGF receptors, were also deregulated in CD44 KO cells. These results were confirmed by the knockdown of CD44 in another GBM cell line, U2990. Notably, downregulation of hyaluronan synthase 2 (HAS2) impaired the hypoxia-related genes and decreased the CD44 protein levels, suggesting a CD44/hyaluronan feedback circuit contributing to GBM progression.

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  • 189.
    Koltowska, Katarzyna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia.
    Okuda, Kazuhide S.
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia; Peter MacCallum Canc Ctr, Melbourne, Vic, Australia; Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic, Australia.
    Gloger, Marleen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Rondon-Galeano, Maria
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia.;Peter MacCallum Canc Ctr, Melbourne, Vic, Australia.;Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic, Australia..
    Mason, Elizabeth
    Peter MacCallum Canc Ctr, Melbourne, Vic, Australia.;Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic, Australia..
    Xuan, Jiachen
    Peter MacCallum Canc Ctr, Melbourne, Vic, Australia.;Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic, Australia..
    Dudczig, Stefanie
    Peter MacCallum Canc Ctr, Melbourne, Vic, Australia.;Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic, Australia..
    Chen, Huijun
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia..
    Arnold, Hannah
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Skoczylas, Renae
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Bower, Neil I.
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia..
    Paterson, Scott
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia.;Peter MacCallum Canc Ctr, Melbourne, Vic, Australia.;Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic, Australia..
    Lagendijk, Anne Karine
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia..
    Baillie, Gregory J.
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia..
    Leshchiner, Ignaty
    Harvard Med Sch, Massachusetts Gen Hosp, Harvard MIT Div Hlth Sci & Technol, Boston, MA 02115 USA.;Broad Inst MIT & Harvard, Cambridge, MA 02142 USA..
    Simons, Cas
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia.;Royal Childrens Hosp, Murdoch Childrens Res Inst, Parkville, Vic, Australia..
    Smith, Kelly A.
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia.;Univ Melbourne, Dept Anat & Physiol, Parkville, Vic, Australia..
    Goessling, Wolfram
    Harvard Med Sch, Massachusetts Gen Hosp, Harvard MIT Div Hlth Sci & Technol, Boston, MA 02115 USA..
    Heath, Joan K.
    Walter & Eliza Hall Inst Med Res, Epigenet & Dev Div, Parkville, Vic, Australia.;Univ Melbourne, Dept Med Biol, Parkville, Vic, Australia..
    Pearson, Richard B.
    Peter MacCallum Canc Ctr, Melbourne, Vic, Australia.;Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic, Australia.;Univ Melbourne, Dept Biochem & Mol Biol, Parkville, Vic, Australia.;Monash Univ, Dept Biochem & Mol Biol, Clayton, Vic, Australia..
    Sanij, Elaine
    Peter MacCallum Canc Ctr, Melbourne, Vic, Australia.;Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic, Australia.;Univ Melbourne, Dept Clin Pathol, Parkville, Vic, Australia.;St Vincents Inst Med Res, Fitzroy, Vic, Australia..
    Schulte-Merker, Stefan
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia.;WWU Munster, Inst Cardiovasc Organogenesis & Regenerat, Med Fac, Munster, Germany.;Hubrecht Inst KNAW, Utrecht, Netherlands.;Univ Med Ctr, Utrecht, Netherlands..
    Hogan, Benjamin M.
    Univ Queensland, Inst Mol Biosci, Div Genom Dev & Dis, Brisbane, Qld, Australia.;Peter MacCallum Canc Ctr, Melbourne, Vic, Australia.;Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic, Australia.;Univ Melbourne, Dept Anat & Physiol, Parkville, Vic, Australia.;Hubrecht Inst KNAW, Utrecht, Netherlands.;Univ Med Ctr, Utrecht, Netherlands..
    The RNA helicase Ddx21 controls Vegfc-driven developmental lymphangiogenesis by balancing endothelial cell ribosome biogenesis and p53 function2021In: Nature Cell Biology, ISSN 1465-7392, E-ISSN 1476-4679, Vol. 23, no 11, p. 1136-1147Article in journal (Refereed)
    Abstract [en]

    Hogan and colleagues report that the RNA helicase Ddx21 mediates Vegfc-stimulated lymphangiogenesis during zebrafish development through controlling rDNA transcription and ribosome biogenesis in endothelial cells. The development of a functional vasculature requires the coordinated control of cell fate, lineage differentiation and network growth. Cellular proliferation is spatiotemporally regulated in developing vessels, but how this is orchestrated in different lineages is unknown. Here, using a zebrafish genetic screen for lymphatic-deficient mutants, we uncover a mutant for the RNA helicase Ddx21. Ddx21 cell-autonomously regulates lymphatic vessel development. An established regulator of ribosomal RNA synthesis and ribosome biogenesis, Ddx21 is enriched in sprouting venous endothelial cells in response to Vegfc-Flt4 signalling. Ddx21 function is essential for Vegfc-Flt4-driven endothelial cell proliferation. In the absence of Ddx21, endothelial cells show reduced ribosome biogenesis, p53 and p21 upregulation and cell cycle arrest that blocks lymphangiogenesis. Thus, Ddx21 coordinates the lymphatic endothelial cell response to Vegfc-Flt4 signalling by balancing ribosome biogenesis and p53 function. This mechanism may be targetable in diseases of excessive lymphangiogenesis such as cancer metastasis or lymphatic malformation.

  • 190.
    Koning, Harmen Kornelis
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Boije: Zebrafish Neuronal Networks.
    Ahemaiti, Aikeremu
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Lagerström: Sensory circuits.
    Boije, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Boije: Zebrafish Neuronal Networks.
    A deep-dive into fictive locomotion - a strategy to probe cellular activity during speed transitions in fictively swimming zebrafish larvae2022In: BIOLOGY OPEN, ISSN 2046-6390, Vol. 11, no 3, article id bio059167Article in journal (Refereed)
    Abstract [en]

    Fictive locomotion is frequently used to study locomotor output in paralyzed animals. We have evaluated the character of swim episodes elicited by different strategies in zebrafish. Motor output was measured on both sides of a body segment using electrodes and a pipeline for synchronizing stimulation and recording, denoising data and peak-finding was developed. The optomotor response generated swims most equivalent to spontaneous activity, while electrical stimulation and NMDA application caused various artefacts. Our optimal settings, optomotor stimulation using 5-day-old larvae, were combined with calcium imaging and optogenetics to validate the setup's utility. Expression of GCaMP5G by the mnx1 promoter allowed correlation of calcium traces of dozens of motor neurons to the fictive locomotor output. Activation of motor neurons through channelrhodopsin produced aberrant locomotor episodes. This strategy can be used to investigate novel neuronal populations in a high-throughput manner to reveal their role in shaping motor output. This article has an associated First Person interview with the first author of the paper.

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  • 191.
    Koripella, Ravi Kiran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Chen, Yang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Peisker, Kristin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Koh, Cha San
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 36, p. 30257-30267Article in journal (Refereed)
    Abstract [en]

    Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome.

  • 192.
    Kosek, David M
    et al.
    Department of Cell and Molecular Biology, Karolinska Institute , Biomedicum 9B, Solnavägen 9, 17177 Stockholm , Sweden.
    Banijamali, Elnaz
    Department of Medical Biochemistry and Biophysics, Karolinska Institute , Biomedicum 9B, Solnavägen 9, 17177 Stockholm , Sweden.
    Becker, Walter
    Department of Medical Biochemistry and Biophysics, Karolinska Institute , Biomedicum 9B, Solnavägen 9, 17177 Stockholm , Sweden.
    Petzold, Katja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Department of Medical Biochemistry and Biophysics, Karolinska Institute , Biomedicum 9B, Solnavägen 9, 17177 Stockholm , Sweden.
    Andersson, Emma R
    Department of Cell and Molecular Biology, Karolinska Institute , Biomedicum 9B, Solnavägen 9, 17177 Stockholm , Sweden.
    Efficient 3′-pairing renders microRNA targeting less sensitive to mRNA seed accessibility2023In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, no 20, p. 11162-11177Article in journal (Refereed)
    Abstract [en]

    MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression by binding to specific sites in mRNAs. Site recognition is primarily mediated by the seed region (nucleotides g2–g8 in the miRNA), but pairing beyond the seed (3′-pairing) is important for some miRNA:target interactions. Here, we use SHAPE, luciferase reporter assays and transcriptomics analyses to study the combined effect of 3′-pairing and secondary structures in mRNAs on repression efficiency. Using the interaction between miR-34a and its SIRT1 binding site as a model, we provide structural and functional evidence that 3′-pairing can compensate for low seed-binding site accessibility, enabling repression of sites that would otherwise be ineffective. We show that miRNA 3′-pairing regions can productively base-pair with nucleotides far upstream of the seed-binding site and that both hairpins and unstructured bulges within the target site are tolerated. We use SHAPE to show that sequences that overcome inaccessible seed-binding sites by strong 3′-pairing adopt the predicted structures and corroborate the model using luciferase assays and high-throughput modelling of 8177 3′-UTR targets for six miRNAs. Finally, we demonstrate that PHB2, a target of miR-141, is an inaccessible target rescued by efficient 3′-pairing. We propose that these results could refine predictions of effective target sites.

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  • 193.
    Kovachev, Petar Stefanov
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Banerjee, Debapriya
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Rangel, Luciana Pereira
    Univ Fed Rio de Janeiro, Fac Farm, BR-21941902 Rio De Janeiro, Brazil..
    Eriksson, Jonny
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Pedrote, Murilo M
    Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo de Meis, Inst Nacl Ciencia Tecnol Biol Estrutural & Bioima, BR-21941902 Rio De Janeiro, Brazil..
    Martins-Dinis, Mafalda Maria D C
    Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo de Meis, Inst Nacl Ciencia Tecnol Biol Estrutural & Bioima, BR-21941902 Rio De Janeiro, Brazil..
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Cordeiro, Yraima
    Univ Fed Rio de Janeiro, Fac Farm, BR-21941902 Rio De Janeiro, Brazil..
    Silva, Jerson L
    Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo de Meis, Inst Nacl Ciencia Tecnol Biol Estrutural & Bioima, BR-21941902 Rio De Janeiro, Brazil..
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Distinct modulatory role of RNA in the aggregation of the tumor suppressor protein p53 core domain.2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 22, p. 9345-9357Article in journal (Refereed)
    Abstract [en]

    Inactivation of the tumor suppressor protein p53 by mutagenesis, chemical modification, protein-protein interaction, or aggregation has been associated with different human cancers. Although DNA is the typical substrate of p53, numerous studies have reported p53 interactions with RNA. Here, we have examined the effects of RNA of varied sequence, length, and origin on the mechanism of aggregation of the core domain of p53 (p53C) using light scattering, intrinsic fluorescence, transmission electron microscopy, thioflavin-T binding, seeding, and immunoblot assays. Our results are the first to demonstrate that RNA can modulate the aggregation of p53C and full-length p53. We found bimodal behavior of RNA in p53C aggregation. A low RNA:protein ratio (∼1:50) facilitates the accumulation of large amorphous aggregates of p53C. By contrast, at a high RNA:protein ratio (≥1:8), the amorphous aggregation of p53C is clearly suppressed. Instead, amyloid p53C oligomers are formed that can act as seeds nucleating de novo aggregation of p53C. We propose that structured RNAs prevent p53C aggregation through surface interaction and play a significant role in the regulation of the tumor suppressor protein.

  • 194.
    Kozlova, Inna
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Cell Biology.
    Studies on Airway Surface Liquid in Connection with Cystic Fibrosis2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cystic fibrosis (CF) is one of the most common fatal inherited diseases, most prevalent among Caucasians. CF is caused by a mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), which helps to create sweat, digestive juices, and airway surface liquid (ASL).

    The airways are covered with a thin layer of fluid, the airway surface liquid, in which the cilia bathe. Defective or absent CFTR leads to a defective water and ion transport in the epithelial cells, which results in viscous mucus, defective ciliary clearance, bacterial inflammation and tissue damage. The volume and composition of ASL are important in the pathogenesis of cystic fibrosis and it is therefore relevant to determine its composition. However, there are a number of difficulties in determining the ionic composition of the ASL due to its small volume. Literature data vary from very hypotonic to markedly hypertonic. These controversial data inspired the development of a simple method for determining the elemental composition of the ASL in different animal species and humans. Two techniques were developed to determine the composition of ASL, from which indirect information on chloride transport in airway epithelium can be obtained. In the first method, tissue is removed from the animals under anesthesia, frozen and analyzed in the frozen-hydrated state. In the second method, the ASL is collected with small dextran (Sephadex) beads; the dried beads are then analyzed by X-ray microanalysis. The Sephadex-bead method appears more accurate compared to the frozen-hydrated samples. Both methods were applied to collect tracheal and/or nasal fluid in pigs, normal and transgenic cystic fibrosis mice, the fluid covering the apical surface of normal bronchial cells (16HBE14o-) and a cystic fibrosis human bronchial cell line (CFBE41o-), and finally nasal fluid in healthy and diseased subjects. The ionic composition of the ASL was isotonic both in pigs and healthy human subjects. CF patients had much higher levels of Na and Cl ions than healthy subjects. The ASL under control conditions was hypotonic in mice and cell cultures, whereas the concentrations of Na and Cl ions in the species with the ΔF508 mutation or absent CFTR were significantly higher than in the corresponding controls. It was also demonstrated that the ionic composition of the ASL can be influenced by pharmacological treatment. The study confirms earlier findings that CFTR also is involved in bicarbonate transport. Mist tent therapy has been tested in the study of a treatment for CF patients, in order to hydrate the viscous mucus. But the effect of mist tent therapy on ion concentrations in the ASL appeared to be short-lived, although no patients became chronically colonized with pseudomonads while on nocturnal mist tent therapy.

    List of papers
    1. X-ray microanalysis of airway surface liquid in the mouse
    Open this publication in new window or tab >>X-ray microanalysis of airway surface liquid in the mouse
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    2005 In: American Journal of Physiology - Lung Cellular and Molecular Physiology, ISSN 1040-0605, Vol. 288, p. L874-L878Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-97732 (URN)
    Available from: 2008-11-06 Created: 2008-11-06Bibliographically approved
    2. Anaesthesia and the elemental content of mouse nasal fluid
    Open this publication in new window or tab >>Anaesthesia and the elemental content of mouse nasal fluid
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    Article in journal (Refereed) Submitted
    Identifiers
    urn:nbn:se:uu:diva-97733 (URN)
    Available from: 2008-11-06 Created: 2008-11-06Bibliographically approved
    3. X-ray microanalysis of apical fluid in cystic fibrosis airway epithelial cell lines
    Open this publication in new window or tab >>X-ray microanalysis of apical fluid in cystic fibrosis airway epithelial cell lines
    2006 In: Cellular Physiology and Biochemistry, ISSN 1015-8987, Vol. 17, no 1, p. 13-20Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-97734 (URN)
    Available from: 2008-11-06 Created: 2008-11-06Bibliographically approved
    4. Elemental composition of airway surface liquid in the pig determined by X-ray microanalysis
    Open this publication in new window or tab >>Elemental composition of airway surface liquid in the pig determined by X-ray microanalysis
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    2005 In: American Journal Respiratory Cell and Molecular Biology, ISSN 1044-1549, Vol. 32, no 1, p. 59-64Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-97735 (URN)
    Available from: 2008-11-06 Created: 2008-11-06Bibliographically approved
    5. Composition of nasal airway surface liquid in cystic fibrosis and other airway diseases determined by X-ray microanalysis
    Open this publication in new window or tab >>Composition of nasal airway surface liquid in cystic fibrosis and other airway diseases determined by X-ray microanalysis
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    2006 In: Microscopy Research and Technique, ISSN 1059-910X, Vol. 69, no 4, p. 271-276Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-97736 (URN)
    Available from: 2008-11-06 Created: 2008-11-06Bibliographically approved
    6. Effect of mist tent on the ion content of nasal fluid in patients with cystic fibrosis
    Open this publication in new window or tab >>Effect of mist tent on the ion content of nasal fluid in patients with cystic fibrosis
    Article in journal (Refereed) Submitted
    Identifiers
    urn:nbn:se:uu:diva-97737 (URN)
    Available from: 2008-11-06 Created: 2008-11-06Bibliographically approved
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  • 195.
    Krakovka, Sascha
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Deradicalising Giardiasis Treatment2022Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Giardia intestinalis is a unicellular parasite causing the disease giardiasis. This disease is mostly prevalent in low resource settings, mandating low-cost treatment options. Treatment is mainly based on antiparasitics from the classes of 5-nitroimidazoles and benzimidazoles, which both target parasite DNA and other cellular macromolecules based on radicals generated from the drugs. In recent years resistance to those classes and cross-resistance in between them has become a problem, hence alternative antigiardials are needed.

    In this thesis we enhanced our understanding of the crucial differentiation process of encystation, which produce the environmentally stable and infective form of the parasite, the cyst (Paper I). Resistance development has been slow in G. intestinalis until now and it has been shown that resistance to the main treatment option, metronidazole, can be lost after en- and excystation so an enhanced understanding of this process can help us to identify the cause of this loss of drug tolerance. In the second study (Paper II) we followed this up by analysing two metronidazole resistant lines and one revertant on their ability to produce infective cysts, while cross checking with growth rates, metronidazole resistance level, transcriptomics and proteomics. We found the resistant cells lines to have deeply disturbed cellular pathways with the main resistance mechanisms being a reduction of uptake, a reduction of activation rate and an upregulation of oxidative stress responses. Cyst production and growth rates were highly reduced giving those lines a clear disadvantage when no drug pressure is applied. Most changes, phenotypically and expression wise, were reset in the revertant. As next step we evaluated alternative antigiardials and their targets. In Paper III we characterised the giardial thymidine kinase, on which this parasite depends completely to supply thymidine for DNA synthesis. We identified a nucleoside analogue, azidothymidine, that is targeting this enzyme and efficiently inhibits growth and encystation of trophozoites both in vivo and in vitro. Azidothymidine is currently used in HIV treatment, has a good safety profile and is comparatively cheap, which makes it a good candidate for treatment of giardiasis. 

    In conclusion, this study has focussed on several aspects of nitroimidazole resistance in G. intestinalis throughout the life cycle as well as repurposing of antibiotics from other drug classes that could be used to fill our arsenal of antigiardials with new alternatives.

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

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

    Place, publisher, year, edition, pages
    MDPIMDPI AG, 2021
    Keywords
    diarrhea, RNAseq, small intestine, protozoa, differentiation
    National Category
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-465062 (URN)10.3390/genes12121932 (DOI)000737881000001 ()34946882 (PubMedID)
    Available from: 2022-01-21 Created: 2022-01-21 Last updated: 2024-01-15Bibliographically approved
    2. Characterization of Metronidazole-Resistant Giardia intestinalis Lines by Comparative Transcriptomics and Proteomics
    Open this publication in new window or tab >>Characterization of Metronidazole-Resistant Giardia intestinalis Lines by Comparative Transcriptomics and Proteomics
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    2022 (English)In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 13, article id 834008Article in journal (Refereed) Published
    Abstract [en]

    Metronidazole (MTZ) is a clinically important antimicrobial agent that is active against both bacterial and protozoan organisms. MTZ has been used extensively for more than 60 years and until now resistance has been rare. However, a recent and dramatic increase in the number of MTZ resistant bacteria and protozoa is of great concern since there are few alternative drugs with a similarly broad activity spectrum. To identify key factors and mechanisms underlying MTZ resistance, we utilized the protozoan parasite Giardia intestinalis, which is commonly treated with MTZ. We characterized two in vitro selected, metronidazole resistant parasite lines, as well as one revertant, by analyzing fitness aspects associated with increased drug resistance and transcriptomes and proteomes. We also conducted a meta-analysis using already existing data from additional resistant G. intestinalis isolates. The combined data suggest that in vitro generated MTZ resistance has a substantial fitness cost to the parasite, which may partly explain why resistance is not widespread despite decades of heavy use. Mechanistically, MTZ resistance in Giardia is multifactorial and associated with complex changes, yet a core set of pathways involving oxidoreductases, oxidative stress responses and DNA repair proteins, is central to MTZ resistance in both bacteria and protozoa.

    Place, publisher, year, edition, pages
    Frontiers Media S.A.Frontiers Media SA, 2022
    Keywords
    diarrhea, antibiotic resistance, RNAseq, proteomics, small intestine, protozoa
    National Category
    Microbiology in the medical area Microbiology
    Identifiers
    urn:nbn:se:uu:diva-470211 (URN)10.3389/fmicb.2022.834008 (DOI)000760863500001 ()35222342 (PubMedID)
    Available from: 2022-03-22 Created: 2022-03-22 Last updated: 2024-01-17Bibliographically approved
    3. Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery
    Open this publication in new window or tab >>Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery
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    2022 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 298, no 6, article id 102028Article in journal (Refereed) Published
    Abstract [en]

    Giardiasis is a diarrheal disease caused by the unicellular parasite Giardia intestinalis, for which metronidazole is the main treatment option. The parasite is dependent on exogenous deoxyribonucleosides for DNA replication and thus is also potentially vulnerable to deoxyribonucleoside analogs. Here, we characterized the G. intestinalis thymidine kinase, a divergent member of the thymidine kinase 1 family that consists of two weakly homologous parts within one polypeptide. We found that the recombinantly expressed enzyme is monomeric, with 100-fold higher catalytic efficiency for thymidine compared to its second-best substrate, deoxyuridine, and is furthermore subject to feedback inhibition by dTTP. This efficient substrate discrimination is in line with the lack of thymidylate synthase and dUTPase in the parasite, which makes deoxy-UMP a dead-end product that is potentially harmful if converted to deoxy-UTP. We also found that the antiretroviral drug azidothymidine (AZT) was an equally good substrate as thymidine and was active against WT as well as metronidazole-resistant G. intestinalis trophozoites. This drug inhibited DNA synthesis in the parasite and efficiently decreased cyst production in vitro, which suggests that it could reduce infectivity. AZT also showed a good effect in G. intestinalis-infected gerbils, reducing both the number of trophozoites in the small intestine and the number of viable cysts in the stool. Taken together, these results suggest that the absolute dependency of the parasite on thymidine kinase for its DNA synthesis can be exploited by AZT, which has promise as a future medication effective against metronidazole-refractory giardiasis.

    Place, publisher, year, edition, pages
    Elsevier, 2022
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-479890 (URN)10.1016/j.jbc.2022.102028 (DOI)000811813400005 ()35568200 (PubMedID)
    Funder
    Swedish Research Council, 2019-01242Swedish Research Council, 201805814
    Note

    Title in thesis list of papers: Giardia intestinalis thymidine kinase: a high-affinity enzyme crucial for DNA synthesis and an Achilles heel exploitable for drug discovery

    Available from: 2022-07-05 Created: 2022-07-05 Last updated: 2024-05-27Bibliographically approved
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  • 196.
    Kurylo, Chad M.
    et al.
    Weill Cornell Med, Dept Physiol & Biophys, New York, NY 10065 USA.
    Parks, Matthew M.
    Weill Cornell Med, Dept Physiol & Biophys, New York, NY 10065 USA.
    Juette, Manuel F.
    Weill Cornell Med, Dept Physiol & Biophys, New York, NY 10065 USA.
    Zinshteyn, Boris
    Johns Hopkins Univ, Sch Med, Dept Mol Biol & Genet, Baltimore, MD 21205 USA;Johns Hopkins Univ, Sch Med, Howard Hughes Med Inst, Baltimore, MD 21205 USA.
    Altman, Roger B.
    Weill Cornell Med, Dept Physiol & Biophys, New York, NY 10065 USA.
    Thibado, Jordana K.
    Weill Cornell Med, Dept Physiol & Biophys, New York, NY 10065 USA.
    Vincent, C. Theresa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Weill Cornell Med, Dept Physiol & Biophys, New York, NY 10065 USA;Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden.
    Blanchard, Scott C.
    Weill Cornell Med, Dept Physiol & Biophys, New York, NY 10065 USA;Weill Cornell Med, Triinst Training Program Chem Biol, New York, NY 10065 USA.
    Endogenous rRNA Sequence Variation Can Regulate Stress Response Gene Expression and Phenotype2018In: Cell Reports, E-ISSN 2211-1247, Vol. 25, no 1, p. 236-248.e6Article in journal (Refereed)
    Abstract [en]

    Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, ReIA and ReIE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology.

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  • 197.
    Kutashev, Konstantin O.
    et al.
    Masaryk Univ, CEITEC, Mendel Ctr Plant Genom & Prote, Kamenice 5, Brno 62500, Czech Republic.;Masaryk Univ, Fac Sci, Natl Ctr Biomol Res, Lab Funct Genom & Proteom, Kotlarska 2, Brno 61137, Czech Republic..
    Franek, Michal
    Masaryk Univ, CEITEC, Mendel Ctr Plant Genom & Prote, Kamenice 5, Brno 62500, Czech Republic..
    Diamanti, Klev
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Polish Acad Sci, Inst Comp Sci, PL-01248 Warsaw, Poland..
    Olsinova, Marie
    BioCEV Imaging Methods Core Facil, Prumyslova 595, Vestec 25250, Czech Republic..
    Dvorackova, Martina
    Masaryk Univ, CEITEC, Mendel Ctr Plant Genom & Prote, Kamenice 5, Brno 62500, Czech Republic..
    Nucleolar rDNA folds into condensed foci with a specific combination of epigenetic marks2021In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 105, no 6, p. 1534-1548Article in journal (Refereed)
    Abstract [en]

    Arabidopsis thaliana 45S ribosomal genes (rDNA) are located in tandem arrays called nucleolus organizing regions on the termini of chromosomes 2 and 4 (NOR2 and NOR4) and encode rRNA, a crucial structural element of the ribosome. The current model of rDNA organization suggests that inactive rRNA genes accumulate in the condensed chromocenters in the nucleus and at the nucleolar periphery, while the nucleolus delineates active genes. We challenge the perspective that all intranucleolar rDNA is active by showing that a subset of nucleolar rDNA assembles into condensed foci marked by H3.1 and H3.3 histones that also contain the repressive H3K9me2 histone mark. By using plant lines containing a low number of rDNA copies, we further found that the condensed foci relate to the folding of rDNA, which appears to be a common mechanism of rDNA regulation inside the nucleolus. The H3K9me2 histone mark found in condensed foci represents a typical modification of bulk inactive rDNA, as we show by genome-wide approaches, similar to the H2A.W histone variant. The euchromatin histone marks H3K27me3 and H3K4me3, in contrast, do not colocalize with nucleolar foci and their overall levels in the nucleolus are very low. We further demonstrate that the rDNA promoter is an important regulatory region of the rDNA, where the distribution of histone variants and histone modifications are modulated in response to rDNA activity.

  • 198.
    Kwak, Hee-Jin
    et al.
    Chungbuk Natl Univ, Coll Nat Sci, Dept Biol Sci & Biotechnol, Cheongju 28644, Chungbuk, South Korea.;Hebrew Univ Jerusalem, Alexander Silberman Inst Life Sci, Fac Sci, Dept Ecol Evolut & Behav, IL-9190401 Jerusalem, Israel..
    Jimenez, Brenda Irene Medina
    Uppsala University, Disciplinary Domain of Science and Technology, Earth Sciences, Department of Earth Sciences, Palaeobiology. Chungbuk Natl Univ, Coll Nat Sci, Dept Biol Sci & Biotechnol, Cheongju 28644, Chungbuk, South Korea..
    Park, Soon Cheol
    Chung Ang Univ, Coll Nat Sci, Dept Life Sci, Seoul 06974, South Korea..
    Kim, Jung-Hyeuk
    Chungbuk Natl Univ, Coll Nat Sci, Dept Biol Sci & Biotechnol, Cheongju 28644, Chungbuk, South Korea.;Natl Inst Wildlife Dis Control & Prevent, Wildlife Dis Response Team, Incheon 22689, South Korea..
    Jeong, Geon-Hwi
    Chungbuk Natl Univ, Coll Nat Sci, Dept Biol Sci & Biotechnol, Cheongju 28644, Chungbuk, South Korea..
    Jeon, Mi-Jeong
    Natl Inst Biol Resources, Environm Res Complex, Incheon 22689, South Korea..
    Kim, Sangil
    Harvard Univ, Museum Comparat Zool, Cambridge, MA 02138 USA.;Harvard Univ, Dept Organism & Evolutionary Biol, Cambridge, MA 02138 USA..
    Kim, Jung-Woong
    Chung Ang Univ, Coll Nat Sci, Dept Life Sci, Seoul 06974, South Korea..
    Weisblat, David A.
    Univ Calif Berkeley, Dept Mol & Cell Biol, 385 Weill Hall, Berkeley, CA 94720 USA..
    Cho, Sung-Jin
    Chungbuk Natl Univ, Coll Nat Sci, Dept Biol Sci & Biotechnol, Cheongju 28644, Chungbuk, South Korea..
    Slit-Robo expression in the leech nervous system: insights into eyespot evolution2023In: Cell & Bioscience, ISSN 2045-3701, Vol. 13, article id 70Article in journal (Refereed)
    Abstract [en]

    Background: Slit and Robo are evolutionarily conserved ligand and receptor proteins, respectively, but the number of slit and robo gene paralogs varies across recent bilaterian genomes. Previous studies indicate that this ligand-receptor complex is involved in axon guidance. Given the lack of data regarding Slit/Robo in the Lophotrochozoa compared to Ecdysozoa and Deuterostomia, the present study aims to identify and characterize the expression of Slit/Robo orthologs in leech development.

    Results: We identified one slit (Hau-slit), and two robo genes (Hau-robo1 and Hau-robo2), and characterized their expression spatiotemporally during the development of the glossiphoniid leech Helobdella austinensis. Throughout segmentation and organogenesis, Hau-slit and Hau-robo1 are broadly expressed in complex and roughly complementary patterns in the ventral and dorsal midline, nerve ganglia, foregut, visceral mesoderm and/or endoderm of the crop, rectum and reproductive organs. Before yolk exhaustion, Hau-robo1 is also expressed where the pigmented eye spots will later develop, and Hau-slit is expressed in the area between these future eye spots. In contrast, Hau-robo2 expression is extremely limited, appearing first in the developing pigmented eye spots, and later in the three additional pairs of cryptic eye spots in head region that never develop pigment. Comparing the expression of robo orthologs between H. austinensis and another glossiphoniid leech, Alboglossiphonia lata allows to that robo1 and robo2 operate combinatorially to differentially specify pigmented and cryptic eyespots within the glossiphoniid leeches.

    Conclusions: Our results support a conserved role in neurogenesis, midline formation and eye spot development for Slit/Robo in the Lophotrochozoa, and provide relevant data for evo-devo studies related to nervous system evolution.

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  • 199.
    Könberg, Erika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Developing and validating assay-ready HEK-Blue CD40L cells: For a more flexible and faster screening of Affibody® molecules2022Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Cell assay that evaluates a biologics’ potency and efficacy is an important part in the discovery and development of drug candidates. However, it requires regular maintenance of cell cultures, and the cell assays can only be performed when the cells have reached 70-80% confluency. By instead using assay-ready cells, the drugs can be screened at any time by simply thawing the cells. This creates a more flexible assay, while saving time, labor and materials in addition to removing day-to-day variability. In this report, the freezing conditions for HEK-Blue™ CD40L cells are evaluated using the assay-ready cell method compared to a continuous culture. Via colorimetric detection, the CD40 receptors’ activation can be determined and a dose-response curve of a CD40 agonist can be produced. The optimal freezing condition for the assay-ready cells were determined to be 10% DMSO and a cell concentration between 1-30 million cells/mL. After reproducibility, robustness and screening tests, it could be concluded that the method generally produced results that had no significant difference to a continuous culture. Some of the assay-ready cells display a higher background which can affect the value of the efficacy. The source of the background will have to be evaluated in future studies. The potency, on the other hand, is stable regardless of cell method or high background.

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    fulltext
  • 200. Lalle, Marco
    et al.
    Camerini, Serena
    Cecchetti, Serena
    Sayadi, Ahmed
    Department of Biochemical Sciences, University of Rome “Sapienza”, Rome, Italy.
    Crescenzi, Marco
    Pozio, Edoardo
    Interaction network of the 14-3-3 protein in the ancient protozoan parasite Giardia duodenalis.2012In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 5, p. 2666-2683Article in journal (Refereed)
    Abstract [en]

    14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.

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