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  • 101.
    Ettema, Thijs J. G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mitochondria in the second act2016In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 531, no 7592, p. 39-40Article in journal (Other academic)
    Abstract [en]

    A large phylogenomics study reveals that the symbiotic event that led to the emergence of organelles known as mitochondria may have occurred later in the evolution of complex cells than was thought.

  • 102. Ezzedine, Jade A.
    et al.
    Uwizeye, Clarisse
    Si Larbi, Grégory
    Villain, Gaelle
    Louwagie, Mathilde
    Schilling, Marion
    Hagenmuller, Pascal
    Gallet, Benoît
    Stewart, Adeline
    Petroutsos, Dimitris
    Université Grenoble Alpes.
    Devime, Fabienne
    Salze, Pascal
    Liger, Lucie
    Jouhet, Juliette
    Dumont, Marie
    Ravanel, Stéphane
    Amato, Alberto
    Valay, Jean-Gabriel
    Jouneau, Pierre-Henri
    Falconet, Denis
    Maréchal, Eric
    Adaptive traits of cysts of the snow alga Sanguina nivaloides unveiled by 3D subcellular imaging2023In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1, article id 7500Article in journal (Refereed)
    Abstract [en]

    Sanguina nivaloides is the main alga forming red snowfields in high mountains and Polar Regions. It is non-cultivable. Analysis of environmental samples by X-ray tomography, focused-ion-beam scanning-electron-microscopy, physicochemical and physiological characterization reveal adaptive traits accounting for algal capacity to reside in snow. Cysts populate liquid water at the periphery of ice, are photosynthetically active, can survive for months, and are sensitive to freezing. They harbor a wrinkled plasma membrane expanding the interface with environment. Ionomic analysis supports a cell efflux of K+, and assimilation of phosphorus. Glycerolipidomic analysis confirms a phosphate limitation. The chloroplast contains thylakoids oriented in all directions, fixes carbon in a central pyrenoid and produces starch in peripheral protuberances. Analysis of cells kept in the dark shows that starch is a short-term carbon storage. The biogenesis of cytosolic droplets shows that they are loaded with triacylglycerol and carotenoids for long-term carbon storage and protection against oxidative stress.

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  • 103.
    Farhat, Crystal Marian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. Karolinska Institute, Department of Oncology-pathology.
    Mechanistic study of one-carbon folate cycle enzymes in DNA repair: Determining the role of MTHFD1 and MTHFD2 in cancer cell survival2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Cancer cells are characterized by several hallmarks, two of which include metabolic reprogramming and genomic instability. Due to their high proliferation demand, cancer cells need to synthesize nucleotides at a higher pace compared to healthy cells. The one-carbon folate metabolic pathway helps transformed cells meet proliferation demands by mediating nucleotide/thymidylate biosynthesis and de novo purine synthesis, where key enzymes include methylenetetrahydrofolate dehydrogenase 1 and 2 (MTHFD1 and MTHFD2). MTHFD1 is the cytosolic enzyme of the 1C pathway and MTHFD2 is the mitochondrial enzyme contributing to the folate cycle through driving nucleotide biosynthesis. Here, we show that MTHFD2 also has a non-canonical function in DNA repair. This study aims to understand the contribution of MTHFD1/2 in the DNA damage response (DDR) in the presence of DNA double-strand breaks (DSBs) in colorectal cancer cells. It further aims to study the effects of MTHFD1/MTHFD2 inhibitors synthesized by the Helleday group. Knockdown (KD) of MTHFD1 /MTHFD2 using siRNA coupled with ionizing radiation (IR) and colony formation assays (CFA) were used to determine cell viability. Loss of cell viability and proliferation was observed upon mthfd2 gene KD. Changes in DDR protein induction were observed in MTHFD2 KD cells, using Western blot analysis. Confocal analysis was used to detect and quantify sites of DSBs in wildtype SW620 and MTHFD2 -/- cells post IR, where MTHFD2-/- cells were shown to sustain more damage, indicating reduced DNA repair capacity. Subcellular fractionation data revealed that MTHFD1/MTHFD2 inhibitors interacting with nuclear and cytosolic MTHFD proteins and inducing replication stress.

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  • 104.
    Fatsis-Kavalopoulos, Nikos
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Gradientech AB, Uppsala, Sweden.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Xie, Beichen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Hernández Vera, Rodrigo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Idevall Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution2019In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 19, no 6, p. 1071-1081Article in journal (Refereed)
    Abstract [en]

    The function and behaviour of any given cell in a healthy tissue, or in a tumor, is affected by interactions with its neighboring cells. It is therefore important to create methods that allow for reconstruction of tissue niches in vitro for studies of cell-cell signaling and associated cell behaviour. To this end we created the cell assembly generator (CAGE), a microfluidic device which enables the organization of different cell types into precise cell clusters in a flow chamber compatible with high-resolution microscopy. In proof-of-concept paracrine signalling experiments, 4-cell clusters consisting of one pancreatic -cell and three breast cancer cells were formed. It has previously been established that extracellular ATP induces calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol before it is cleared back into the ER via sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps. Here, ATP release from the -cell was stimulated by depolarization, and dynamic changes in Ca2+ levels in the adjacent cancer cells measured using imaging of the calcium indicator Fluo-4. We established that changes in the concentration of cytosolic Ca2+ in the cancer cells were proportional to the distance from the ATP-releasing -cell. Additionally, we established that the relationship between distance and cytosolic calcium changes were dependent on Ca2+-release from the ER using 5-cell clusters composed of one -cell, two untreated cancer cells and two cancer cells pretreated with Thapsigargin (to deplete the ER of Ca2+). These experiments show that the CAGE can be used to create exact cell clusters, which affords precise control for reductionist studies of cell-cell signalling and permits the formation of heterogenous cell models of specific tissue niches.

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  • 105. Finazzi, Giovanni
    et al.
    Petroutsos, Dimitris
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Physiology and Environmental Toxicology.
    Tomizioli, Martino
    Flori, Serena
    Sautron, Emeline
    Villanova, Valeria
    Rolland, Norbert
    Seigneurin-Berny, Daphné
    Ions channels/transporters and chloroplast regulation.2015In: Cell Calcium, ISSN 0143-4160, E-ISSN 1532-1991, Vol. 58, no 1, p. 86-97, article id S0143-4160(14)00157-2Article in journal (Refereed)
    Abstract [en]

    Ions play fundamental roles in all living cells and their gradients are often essential to fuel transports, to regulate enzyme activities and to transduce energy within and between cells. Their homeostasis is therefore an essential component of the cell metabolism. Ions must be imported from the extracellular matrix to their final subcellular compartments. Among them, the chloroplast is a particularly interesting example because there, ions not only modulate enzyme activities, but also mediate ATP synthesis and actively participate in the building of the photosynthetic structures by promoting membrane-membrane interaction. In this review, we first provide a comprehensive view of the different machineries involved in ion trafficking and homeostasis in the chloroplast, and then discuss peculiar functions exerted by ions in the frame of photochemical conversion of absorbed light energy.

  • 106.
    Florenza, Javier
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Jeevannavar, Aditya
    University of Turku, Department of Biology.
    Divne, Anna-Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Tamminen, Manu
    University of Turku, Department of Biology.
    Bertilsson, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab. Sveriges Lantbruksuniversitet, Institutionen för vatten och miljö.
    De novo assembled single-cell transcriptomes from aquatic phytoflagellates reveal a metabolically distinct cell populationManuscript (preprint) (Other academic)
    Abstract [en]

    Single-cell transcriptomics has rapidly become a standard tool for decoding cell identity, fate and interactions in mammalian model organisms. Adopting such techniques to uncover functional dynamics in aquatic single-celled organisms holds huge potential, but evidence of applicability to non-model, poorly understood microeukaryotes remains limited. In the present study, live Ochromonas triangulata cells from fast and slow growth phases were FACS-sorted based on food vacuole staining and chlorophyll fluorescence, and single-cell transcriptomic libraries were prepared following the Smart-seq2 protocol. In total, 744 transcriptomes were Illumina sequenced. Lacking a reference genome, transcriptomes were assembled de novo using Trinity and resulting transcripts were annotated by BLAST using the Swiss-Prot database. Following read mapping, differential gene expression was evaluated using DESeq2 and metabolic maps were generated based on pathways from the KEGG Orthology database. Clustering the read counts revealed the identity of the two expected transcriptional states corresponding to each growth phase as well as a third distinct cluster of cells present in both growth phases. This cryptic group showed extensive downregulation of genes in pathways associated with ribosome-functioning, CO2 fixation and core carbohydrate catabolism such as glycolysis, β oxidation of fatty acids and tricarboxylic acid cycle. Nevertheless, the biological underpinnings of this population, which would have remained unnoticed in an integrated approach, could not be clarified. Additionally, the possibility of using carry-over rRNA reads for taxonomic annotation was tested, verifying the identity of 88% of the O. triangulata cells. In conclusion, we demonstrate the power of single cell transcriptomics for metabolic mapping of microeukaryotes for which reference resources might be limited and thereby highlight its potential as a tool to gain access to microeukaryote dynamics in natural communities.

  • 107.
    Forsberg, Lars A.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Reply to Guo's commentary on: "Immune cells lacking Y chromosome show dysregulation of autosomal gene expression"2022In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 79, no 1, article id 30Article in journal (Refereed)
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  • 108.
    Fu, Cheng-Jie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology.
    Sheikh, Sanea
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology.
    Miao, Wei
    Andersson, Siv G. E.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Baldauf, Sandra L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology.
    Missing Genes, Multiple ORFs, and C-to-U Type RNA Editing in Acrasis kona (Heterolobosea, Excavata) Mitochondrial DNA2014In: Genome Biology and Evolution, ISSN 1759-6653, E-ISSN 1759-6653, Vol. 6, no 9, p. 2240-2257Article in journal (Refereed)
    Abstract [en]

    Discoba (Excavata) is an ancient group of eukaryotes with great morphological and ecological diversity. Unlike the other major divisions of Discoba (Jakobida and Euglenozoa), little is known about the mitochondrial DNAs(mtDNAs) of Heterolobosea. We have assembled a complete mtDNA genome from the aggregating heterolobosean amoeba, Acrasis kona, which consists of a single circular highly AT-rich (83.3%) molecule of 51.5 kb. Unexpectedly, A. kona mtDNA is missing roughly 40% of the protein-coding genes and nearly half of the transfer RNAs found in the only other sequenced heterolobosean mtDNAs, those of Naegleria spp. Instead, over a quarter of A. kona mtDNA consists of novel open reading frames. Eleven of the 16 protein-coding genes missing from A. kona mtDNA were identified in its nuclear DNA and polyA RNA, and phylogenetic analyses indicate that at least 10 of these 11 putative nuclear-encoded mitochondrial (NcMt) proteins arose by direct transfer from the mitochondrion. Acrasis kona mtDNA also employs C-to-U type RNA editing, and 12 homologs of DYW-type pentatricopeptide repeat (PPR) proteins implicated in plant organellar RNA editing are found in A. kona nuclear DNA. A mapping of mitochondrial gene content onto a consensus phylogeny reveals a sporadic pattern of relative stasis and rampant gene loss in Discoba. Rampant loss occurred independently in the unique common lineage leading to Heterolobosea + Tsukubamonadida and later in the unique lineage leading to Acrasis. Meanwhile, mtDNA gene content appears to be remarkably stable in the Acrasis sister lineage leading to Naegleria and in their distant relatives Jakobida.

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  • 109.
    Fujimoto, Toyoshi
    et al.
    Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Interleaflet Coupling, Pinning, and Leaflet Asymmetry: Major Players in Plasma Membrane Nanodomain Formation2017In: Frontiers in Cell and Developmental Biology, E-ISSN 2296-634X, Vol. 4, article id 155Article, review/survey (Refereed)
    Abstract [en]

    The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence.

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  • 110.
    Fukuda, Tomohiko
    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.
    Fukuda, Risa
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Koinuma, Daizo
    Univ Tokyo, Grad Sch Med, Dept Mol Pathol, Tokyo, Japan..
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Miyazono, Kohei
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Univ Tokyo, Grad Sch Med, Dept Mol Pathol, Tokyo, Japan..
    Heldin, Carl-Henrik
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    BMP2-induction of FN14 promotes protumorigenic signaling in gynecologic cancer cells2021In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 87, article id 110146Article in journal (Refereed)
    Abstract [en]

    We previously reported that bone morphogenetic protein (BMP) signaling promotes tumorigenesis in gynecologic cancer cells. BMP2 enhances proliferation of ovarian and endometrial cancer cells via c-KIT induction, and triggers epithelial-mesenchymal transition (EMT) by SNAIL and/or SLUG induction, leading to increased cell migration. However, the downstream effectors of BMP signaling in gynecological cancer cells have not been clearly elucidated. In this study, we performed RNA-sequencing of Ishikawa endometrial and SKOV3 ovarian cancer cells after BMP2 stimulation, and identified TNFRSF12A, encoding fibroblast growth factor-inducible 14 (FN14) as a common BMP2-induced gene. FN14 knockdown suppressed BMP2-induced cell proliferation and migration, confirmed by MTS and scratch assays, respectively. In addition, FN14 silencing augmented chemosensitivity of SKOV3 cells. As a downstream effector of BMP signaling, FN14 modulated both c-KIT and SNAIL expression, which are important for growth and migration of ovarian and endometrial cancer cells. These results support the notion that the tumor promoting effects of BMP signaling in gynecological cancers are partially attributed to FN14 induction.

  • 111.
    Garcia, Ignacio
    et al.
    Norwegian Radium Hosp, Inst Canc Res, Dept Mol Cell Biol, N-0379 Oslo, Norway.;Univ Oslo, Fac Med, Ctr Canc Cell Reprogramming, Inst Clin Med, N-0318 Oslo, Norway..
    Orellana-Munoz, Sara
    Norwegian Radium Hosp, Inst Canc Res, Dept Mol Cell Biol, N-0379 Oslo, Norway.;Univ Oslo, Fac Med, Ctr Canc Cell Reprogramming, Inst Clin Med, N-0318 Oslo, Norway..
    Ramos-Alonso, Lucia
    Univ Oslo, Fac Math & Nat Sci, Sect Biochem & Mol Biol, N-0316 Oslo, Norway.;Oslo Univ Hosp, Dept Microbiol, N-0372 Oslo, Norway..
    Andersen, Aram N.
    Norwegian Radium Hosp, Inst Canc Res, Dept Mol Cell Biol, N-0379 Oslo, Norway.;Univ Oslo, Fac Med, Ctr Canc Cell Reprogramming, Inst Clin Med, N-0318 Oslo, Norway.;Univ Oslo, Fac Math & Nat Sci, Sect Biochem & Mol Biol, N-0316 Oslo, Norway..
    Zimmermann, Christine
    Johannes Gutenberg Univ Mainz, Univ Med Ctr, Inst Virol, D-55131 Mainz, Germany..
    Eriksson, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bøe, Stig Ove
    Oslo Univ Hosp, Dept Microbiol, N-0372 Oslo, Norway..
    Kaferle, Petra
    Sorbonne Univ, PSL Res Univ, Inst Curie, CNRS,UMR3664, Paris, France..
    Papamichos-Chronakis, Manolis
    Univ Liverpool, Dept Mol Physiol, Liverpool L69 7BE, Merseyside, England.;Univ Liverpool, Cell Signalling Inst Syst Mol & Integrat Biol, Liverpool L69 7BE, Merseyside, England..
    Chymkowitch, Pierre
    Univ Oslo, Fac Math & Nat Sci, Sect Biochem & Mol Biol, N-0316 Oslo, Norway.;Oslo Univ Hosp, Dept Microbiol, N-0372 Oslo, Norway..
    Enserink, Jorrit M.
    Norwegian Radium Hosp, Inst Canc Res, Dept Mol Cell Biol, N-0379 Oslo, Norway.;Univ Oslo, Fac Med, Ctr Canc Cell Reprogramming, Inst Clin Med, N-0318 Oslo, Norway.;Univ Oslo, Fac Math & Nat Sci, Sect Biochem & Mol Biol, N-0316 Oslo, Norway..
    Kel1 is a phosphorylation-regulated noise suppressor of the pheromone signaling pathway2021In: Cell Reports, E-ISSN 2211-1247, Vol. 37, no 13, article id 110186Article in journal (Refereed)
    Abstract [en]

    Mechanisms have evolved that allow cells to detect signals and generate an appropriate response. The accuracy of these responses relies on the ability of cells to discriminate between signal and noise. How cells filter noise in signaling pathways is not well understood. Here, we analyze noise suppression in the yeast pheromone signaling pathway and show that the poorly characterized protein Kel1 serves as a major noise suppressor and prevents cell death. At the molecular level, Kel1 prevents spontaneous activation of the pheromone response by inhibiting membrane recruitment of Ste5 and Far1. Only a hypophosphorylated form of Kel1 suppresses signaling, reduces noise, and prevents pheromone-associated cell death, and our data indicate that the MAPK Fus3 contributes to Kel1 phosphorylation. Taken together, Kel1 serves as a phosphoregulated suppressor of the pheromone pathway to reduce noise, inhibit spontaneous activation of the pathway, regulate mating efficiency, and prevent pheromone-associated cell death.

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  • 112.
    Gaullier, Guillaume
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Univ Colorado, Dept Biochem, Boulder, CO 80309 USA; Univ Colorado, Howard Hughes Med Inst, Boulder, CO 80309 USA.
    Roberts, Genevieve
    Univ Colorado, Dept Biochem, Boulder, CO 80309 USA..
    Muthurajan, Uma M.
    Univ Colorado, Dept Biochem, Boulder, CO 80309 USA; Univ Colorado, Howard Hughes Med Inst, Boulder, CO 80309 USA.
    Bowerman, Samuel
    Univ Colorado, Dept Biochem, Boulder, CO 80309 USA; Univ Colorado, Howard Hughes Med Inst, Boulder, CO 80309 USA..
    Rudolph, Johannes
    Univ Colorado, Dept Biochem, Boulder, CO 80309 USA; Univ Colorado, Howard Hughes Med Inst, Boulder, CO 80309 USA..
    Mahadevan, Jyothi
    Univ Colorado, Dept Biochem, Boulder, CO 80309 USA..
    Jha, Asmita
    Univ Colorado, Dept Biochem, Boulder, CO 80309 USA..
    Rae, Purushka S.
    Univ Colorado, Dept Biochem, Boulder, CO 80309 USA..
    Luger, Karolin
    Univ Colorado, Dept Biochem, Boulder, CO 80309 USA.;Univ Colorado, Howard Hughes Med Inst, Boulder, CO 80309 USA..
    Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF12020In: PLOS ONE, E-ISSN 1932-6203, Vol. 15, no 11, article id e0240932Article in journal (Refereed)
    Abstract [en]

    Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are rel- evant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleo- somes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1•PARP2•nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.

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  • 113. Gerecht, A. C.
    et al.
    Šupraha, Luka
    Uppsala University, Disciplinary Domain of Science and Technology, Earth Sciences, Department of Earth Sciences, Palaeobiology.
    Edvardsen, B.
    Langer, G.
    Henderiks, Jorijntje
    Uppsala University, Disciplinary Domain of Science and Technology, Earth Sciences, Department of Earth Sciences, Palaeobiology.
    Phosphorus availability modifies carbon production in Coccolithus pelagicus (Haptophyta)2015In: Journal of Experimental Marine Biology and Ecology, ISSN 0022-0981, E-ISSN 1879-1697, Vol. 472, p. 24-31Article in journal (Refereed)
    Abstract [en]

    Abstract The coccolithophore Coccolithus pelagicus (Wallich) Schiller fixes CO2 into particulate organic carbon (POC) through photosynthesis and into particulate inorganic carbon (PIC) in the form of calcite. To examine the role of phosphorus (P) availability in the production of POC and PIC, C. pelagicus subsp. braarudii (Gaarder) Geisen et al. was grown in semi-continuous cultures at three initial phosphate concentrations (P-replete, 1, and 0.5 μM [P]). Reduced P-availability (1 and 0.5 μM [P]) decreased POC production, while PIC production only decreased when phosphate concentrations became growth limiting (0.5 μM [P]). This decrease has not been observed previously in batch cultures, highlighting the inadequacy of the batch culture approach with regard to determining carbon production. The reduction in growth rate by 50% at 0.5 μM [P] was accompanied by a doubling in cell volume (and POC). PIC production was halved, resulting in a lowered PIC to POC ratio. The average number of coccoliths per cell (and PIC content) remained the same among treatments, despite the significant change in cell size. Our data suggest that POC production in C. pelagicus is more sensitive towards a moderate reduction in phosphorus availability than PIC production. Once phosphorus availability limits cell division, however, phosphorus resources are invested into POC rather than PIC production. This reduces cell density and sinking rates, indicating that coccoliths do not act as ballast for reaching deeper nutrient-rich layers under nutrient limitation.

  • 114.
    Giacomello, Stefania
    et al.
    Stockholm Univ, Dept Biochemestry & Biophys, Stockholm, Sweden; SciLifeLab, Royal Inst Technol, Dept Gene Technol, Stockholm, Sweden.
    Asp, Michaela
    SciLifeLab, Royal Inst Technol, Dept Gene Technol, Stockholm, Sweden.
    Wärdell, Eva
    Karolinska Inst, Dept Med, Stockholm, Sweden.
    Reimegård, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Salmén, Fredrik
    SciLifeLab, Royal Inst Technol, Dept Gene Technol, Stockholm, Sweden.
    Grinnemo, Karl-Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Månsson-Broberg, Agneta
    Karolinska Inst, Dept Med, Stockholm, Sweden.
    Corbascio, Cecilia Österholm
    Karolinska Inst, Dept Med, Stockholm, Sweden.
    Sylvén, Christer
    Karolinska Inst, Dept Med, Stockholm, Sweden.
    Ståhl, Patrik
    SciLifeLab, Royal Inst Technol, Dept Gene Technol, Stockholm, Sweden.
    Corbascio, Matthias
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.
    Lundeberg, Joakim
    SciLifeLab, Royal Inst Technol, Dept Gene Technol, Stockholm, Sweden.
    New insights into the human heart development using a combined spatial and single-cell transcriptomics approach2018In: Human Genomics, ISSN 1473-9542 , E-ISSN 1479-7364 , Vol. 12, no Suppl 1, p. 50-51Article in journal (Other academic)
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  • 115.
    Giustacchini, Alice
    et al.
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Thongjuea, Supat
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Barkas, Nikolaos
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Woll, Petter S.
    Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Povinelli, Benjamin J.
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Booth, Christopher A. G.
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Sopp, Paul
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Norfo, Ruggiero
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Rodriguez-Meira, Alba
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Ashley, Neil
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Jamieson, Lauren
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England..
    Vyas, Paresh
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England..
    Anderson, Kristina
    Oslo Univ Hosp, Norwegian Radium Hosp, Dept Cellular Therapy, Oslo, Norway..
    Segerstolpe, Åsa
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.;Karolinska Inst, Integrated Cardio Metab Ctr, Huddinge, Sweden..
    Qian, Hong
    Karolinska Inst, Ctr Hematol & Regenerat Med, Dept Med, Stockholm, Sweden..
    Olsson-Strömberg, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Mustjoki, Satu
    Univ Helsinki, Dept Clin Chem & Hematol, Hematol Res Unit Helsinki, Helsinki, Finland.;Helsinki Univ Hosp, Comprehens Canc Ctr, Helsinki, Finland..
    Sandberg, Rickard
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.;Ludwig Inst Canc Res, Stockholm, Sweden..
    Jacobsen, Sten Eirik W.
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England.;Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.;Karolinska Inst, Ctr Hematol & Regenerat Med, Dept Med, Stockholm, Sweden.;Karolinska Univ Hosp, Stockholm, Sweden..
    Mead, Adam J.
    Univ Oxford, Weatherall Inst Mol Med, MRC Mol Hematol Unit, Oxford, England.;Univ Oxford, Weatherall Inst Mol Med, Haemopoiet Stem Cell Biol Lab, Oxford, England.;Churchill Hosp, NIHR Biomed Res Ctr, Oxford, England..
    Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia2017In: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 23, no 6, p. 692-+Article in journal (Refereed)
    Abstract [en]

    Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis.

  • 116.
    Griesche, Nadine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sanchez, Gonzalo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Hermans, Cedric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Idevall Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Cortical mitochondria regulate insulin secretion by local Ca2+ buffering in rodent beta cells2019In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 132, no 9, article id jcs228544Article in journal (Refereed)
    Abstract [en]

    Mitochondria play an essential role in regulating insulin secretion from beta cells by providing the ATP needed for the membrane depolarization that results in voltage-dependent Ca2+ influx and subsequent insulin granule exocytosis. Ca2+, in turn, is also rapidly taken up by the mitochondria and exerts important feedback regulation of metabolism. The aim of this study was to determine whether the distribution of mitochondria within beta cells is important for the secretory capacity of these cells. We find that cortically localized mitochondria are abundant in rodent beta cells, and that these mitochondria redistribute towards the cell interior following depolarization. The redistribution requires Ca2+-induced remodeling of the cortical F-actin network. Using light-regulated motor proteins, we increased the cortical density of mitochondria twofold and found that this blunted the voltage-dependent increase in cytosolic Ca2+ concentration and suppressed insulin secretion. The activity-dependent changes in mitochondria distribution are likely to be important for the generation of Ca2+ microdomains required for efficient insulin granule release.

  • 117. Groth, P
    et al.
    Orta, ML
    Elvers, I
    Majumder, MM
    Lagerqvist, A
    Helleday, T
    Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 14, p. 6585-6594Article in journal (Refereed)
  • 118.
    Grönholdt-Klein, Max
    et al.
    Karolinska Inst, Dept Neurosci, S-17177 Stockholm, Sweden.
    Altun, Mikael
    Karolinska Inst, Dept Lab Med, Huddinge, Sweden.
    Becklen, Meneca
    Karolinska Inst, Dept Neurosci, S-17177 Stockholm, Sweden.
    Kahm, Emelie Dickman
    Karolinska Inst, Dept Neurosci, S-17177 Stockholm, Sweden.
    Fahlström, Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Enblad: Neurosurgery. Karolinska Inst, Dept Neurosci, S-17177 Stockholm, Sweden.
    Rullman, Eric
    Karolinska Inst, Dept Lab Med, Huddinge, Sweden.
    Ulfhake, Brun
    Karolinska Inst, Dept Neurosci, S-17177 Stockholm, Sweden.
    Muscle atrophy and regeneration associated with behavioural loss and recovery of function after sciatic nerve crush2019In: Acta Physiologica, ISSN 1748-1708, E-ISSN 1748-1716, Vol. 227, no 3, article id e13335Article in journal (Refereed)
    Abstract [en]

    Aim To resolve timing and coordination of denervation atrophy and the re-innervation recovery process to discern correlations indicative of common programs governing these processes. Methods Female Sprague-Dawley (SD) rats had a unilateral sciatic nerve crush. Based on longitudinal behavioural observations, the triceps surae muscle was analysed at different time points post-lesion. Results Crush results in a loss of muscle function and mass (-30%) followed by a recovery to almost pre-lesion status at 30 days post-crush (dpc). There was no loss of fibres nor any significant change in the number of nuclei per fibre but a shift in fibres expressing myosins I and II that reverted back to control levels at 30 dpc. A residual was the persistence of hybrid fibres. Early on a CHNR -epsilon to -gamma switch and a re-expression of embryonic MyHC showed as signs of denervation. Foxo1, Smad3, Fbxo32 and Trim63 transcripts were upregulated but not Myostatin, InhibinA and ActivinR2B. Combined this suggests that the mechanism instigating atrophy provides a selectivity of pathway(s) activated. The myogenic differentiation factors (MDFs: Myog, Myod1 and Myf6) were upregulated early on suggesting a role also in the initial atrophy. The regulation of these transcripts returned towards baseline at 30 dpc. The examined genes showed a strong baseline covariance in transcript levels which dissolved in the response to crush driven mainly by the MDFs. At 30 dpc the naive expression pattern was re-established. Conclusion Peripheral nerve crush offers an excellent model to assess and interfere with muscle adaptions to denervation and re-innervation.

  • 119.
    Gucek, Alenka
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gandasi, Nikhil R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Omar-Hmeadi, Muhmmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Bakke, Marit
    Univ Bergen, Dept Biomed, Bergen, Norway.
    Doskeland, Stein O.
    Univ Bergen, Dept Biomed, Bergen, Norway.
    Tengholm, Anders
    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.
    Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis2019In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e41711Article in journal (Refereed)
    Abstract [en]

    Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2(-/-) (Rapgef4(-/-)) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

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  • 120.
    Guo, Jingtao
    et al.
    Univ Utah, Howard Hughes Med Inst, Dept Oncol Sci, Sch Med, Salt Lake City, UT 84112 USA;Univ Utah, Sch Med, Huntsman Canc Inst, Salt Lake City, UT 84112 USA;Univ Utah, Ctr Reconstruct Urol & Mens Hlth, Dept Surg Androl Urol, Androl Lab,Hlth Sci Ctr, Salt Lake City, UT 84112 USA.
    Nie, Xichen
    Univ Utah, Howard Hughes Med Inst, Dept Oncol Sci, Sch Med, Salt Lake City, UT 84112 USA;Univ Utah, Sch Med, Huntsman Canc Inst, Salt Lake City, UT 84112 USA.
    Giebler, Maria
    Univ Oxford, MRC Weatherall Inst Mol Med, Radcliffe Dept Med, Oxford OX3 9DS, England.
    Mlcochova, Hana
    Univ Oxford, MRC Weatherall Inst Mol Med, Radcliffe Dept Med, Oxford OX3 9DS, England.
    Wang, Yueqi
    Columbia Univ, Dept Comp Sci, New York, NY 10027 USA.
    Grow, Edward J.
    Univ Utah, Howard Hughes Med Inst, Dept Oncol Sci, Sch Med, Salt Lake City, UT 84112 USA;Univ Utah, Sch Med, Huntsman Canc Inst, Salt Lake City, UT 84112 USA.
    Kim, Robin
    Univ Utah, Dept Surg, Sect Transplantat, Sch Med, Salt Lake City, UT 84132 USA.
    Tharmalingam, Melissa
    Univ Edinburgh, MRC Ctr Reprod Hlth, Queens Med Res Inst, Edinburgh EH16 4TJ, Midlothian, Scotland;Royal Hosp Children & Young People, Edinburgh EH9 1LF, Midlothian, Scotland.
    Matilionyte, Gabriele
    Univ Edinburgh, MRC Ctr Reprod Hlth, Queens Med Res Inst, Edinburgh EH16 4TJ, Midlothian, Scotland;Royal Hosp Children & Young People, Edinburgh EH9 1LF, Midlothian, Scotland.
    Lindskog, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Carrell, Douglas T.
    Univ Utah, Ctr Reconstruct Urol & Mens Hlth, Dept Surg Androl Urol, Androl Lab,Hlth Sci Ctr, Salt Lake City, UT 84112 USA.
    Mitchell, Rod T.
    Univ Edinburgh, MRC Ctr Reprod Hlth, Queens Med Res Inst, Edinburgh EH16 4TJ, Midlothian, Scotland;Royal Hosp Children & Young People, Edinburgh EH9 1LF, Midlothian, Scotland.
    Goriely, Anne
    Univ Oxford, MRC Weatherall Inst Mol Med, Radcliffe Dept Med, Oxford OX3 9DS, England.
    Hotaling, James M.
    Univ Utah, Ctr Reconstruct Urol & Mens Hlth, Dept Surg Androl Urol, Androl Lab,Hlth Sci Ctr, Salt Lake City, UT 84112 USA.
    Cairns, Bradley R.
    Univ Utah, Howard Hughes Med Inst, Dept Oncol Sci, Sch Med, Salt Lake City, UT 84112 USA;Univ Utah, Sch Med, Huntsman Canc Inst, Salt Lake City, UT 84112 USA.
    The Dynamic Transcriptional Cell Atlas of Testis Development during Human Puberty2020In: Cell Stem Cell, ISSN 1934-5909, E-ISSN 1875-9777, Vol. 26, no 2, p. 262-+Article in journal (Refereed)
    Abstract [en]

    The human testis undergoes dramatic developmental and structural changes during puberty, including proliferation and maturation of somatic niche cells, and the onset of spermatogenesis. To characterize this understudied process, we profiled and analyzed single-cell transcriptomes of similar to 10,000 testicular cells from four boys spanning puberty and compared them to those of infants and adults. During puberty, undifferentiated spermatogonia sequentially expand and differentiate prior to the initiation of gametogenesis. Notably, we identify a common pre-pubertal progenitor for Leydig and myoid cells and delineate candidate factors controlling pubertal differentiation. Furthermore, pre-pubertal Sertoli cells exhibit two distinct transcriptional states differing in metabolic profiles before converging to an alternative single mature population during puberty. Roles for testosterone in Sertoli cell maturation, antimicrobial peptide secretion, and spermatogonial differentiation are further highlighted through single-cell analysis of testosterone-suppressed transfemale testes. Taken together, our transcriptional atlas of the developing human testis provides multiple insights into developmental changes and key factors accompanying male puberty.

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  • 121.
    Gupta, Deepesh Kumar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Du, Jian
    Kamranvar, Siamak A.
    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.
    Tension-induced cytokinetic abscission in human fibroblasts2018In: Oncotarget, E-ISSN 1949-2553Article in journal (Other academic)
  • 122.
    Gupta, Deepesh Kumar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kamranvar, Siamak A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Du, Jian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Jilin Univ, Hosp 1, Changchun, Jilin, Peoples R China.
    Liu, Liangwen
    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.
    Septin and Ras regulate cytokinetic abscission in detached cells2019In: Cell Division, E-ISSN 1747-1028, Vol. 14, no 1, article id 8Article in journal (Refereed)
    Abstract [en]

    Background Integrin-mediated adhesion is normally required for cytokinetic abscission, and failure in the process can generate potentially oncogenic tetraploid cells. Here, detachment-induced formation of oncogenic tetraploid cells was analyzed in non-transformed human BJ fibroblasts and BJ expressing SV40LT (BJ-LT) +/- overactive HRas. Results In contrast to BJ and BJ-LT cells, non-adherent BJ-LT-Ras cells recruited ALIX and CHMP4B to the midbody and divided. In detached BJ and BJ-LT cells regression of the cytokinetic furrow was suppressed by intercellular bridge-associated septin; after re-adhesion these cells divided by cytofission, however, some cells became bi-nucleated because of septin reorganization and furrow regression. Adherent bi-nucleated BJ cells became senescent in G1 with p21 accumulation in the nucleus, apparently due to p53 activation since adherent bi-nucleated BJ-LT cells passed through next cell cycle and divided into mono-nucleated tetraploids; the two centrosomes present in bi-nucleated BJ cells fused after furrow regression, pointing to the PIDDosome pathway as a possible mechanism for the p53 activation. Conclusions Several mechanisms prevent detached normal cells from generating tumor-causing tetraploid cells unless they have a suppressed p53 response by viruses, mutation or inflammation. Importantly, activating Ras mutations promote colony growth of detached transformed cells by inducing anchorage-independent cytokinetic abscission in single cells.

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  • 123.
    Gupta, Deepesh Kumar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University.
    Kamranvar, Siamak A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University.
    Du, Jian
    Lu, Liangwen
    Johansson, Staffan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fate of bi-nucleated cells: the role of septin and Ras2018Manuscript (preprint) (Other academic)
    Abstract [en]

    Integrin-mediated adhesion is required to complete cytokinesis, and failure in the process can generate tetraploid cells, which are potentially oncogenic. The effect of cell detachment on the cytokinesis process and on the following cell cycle was analyzed in the non-transformed human fibroblast cell line BJ and in BJ cells expressing SV40 LT (BJ-LT) +/- an oncogenic Ras mutant. In non-adherent BJ and BJ-LT cells, ALIX could not be recruited to the midbody (MB) and cytokinetic abscission did not occur. Based on the results from several approaches, this block was concluded to be overcome in the detached BJ-LT-Ras cells. Non-adherent BJ and BJ-LT cells maintained the septin-associated intercellular bridge (ICB) formed by cleavage furrow ingression, for more than 24 hours. After re-adhesion to fibronectin most such cells divided by cytofission due to tension exerted on the narrow bridge, while a minor fraction of the cell population instead became bi-nucleated because of regression of the intercellular bridge. Adherent bi-nucleated BJ-LT cells progressed through the cell cycle and at mitosis they divided into two mono-nucleated (4N) cells, while adherent bi-nucleated BJ cells were arrested in the G1 phase and became senescent. Thus, p53-dependent mechanism(s) prevented the formation of tetraploid cells from non-transformed bi-nucleated cells. The two centrosomes in the adherent bi-nucleated cells rapidly fused, indicating that p53 was activated via the PIDDosome mechanism. The results show that several mechanisms contribute to prevent detached normal cells from generating tumor-causing tetraploid cells, and that expression of an activating Ras mutation can promote cytokinesis in detached tumor cells. 

  • 124.
    Gupta, Shantanu
    et al.
    Univ Sao Paulo, Dept Ciencia Comp, Inst Matemat & Estat, Rua Matao 1010, BR-05508090 Sao Paulo, SP, Brazil..
    Panda, Pritam Kumar
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Hashimoto, Ronaldo F.
    Univ Sao Paulo, Dept Ciencia Comp, Inst Matemat & Estat, Rua Matao 1010, BR-05508090 Sao Paulo, SP, Brazil..
    Samal, Shailesh Kumar
    Karolinska Inst, Inst Environm Med, Unit Immunol & Chron Dis, S-17177 Stockholm, Sweden..
    Mishra, Suman
    KIIT Univ, Sch Biotechnol, Bhubaneswar 751024, India..
    Verma, Suresh K.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Mishra, Yogendra Kumar
    Univ Southern Denmark, Mads Clausen Inst, NanoSYD, Alsion 2, DK-6400 Sonderborg, Denmark..
    Ahuja, Rajeev
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Dynamical modeling of miR-34a, miR-449a, and miR-16 reveals numerous DDR signaling pathways regulating senescence, autophagy, and apoptosis in HeLa cells2022In: Scientific Reports, E-ISSN 2045-2322, Vol. 12, article id 4911Article in journal (Refereed)
    Abstract [en]

    Transfection of tumor suppressor miRNAs such as miR-34a, miR-449a, and miR-16 with DNA damage can regulate apoptosis and senescence in cancer cells. miR-16 has been shown to influence autophagy in cervical cancer. However, the function of miR-34a and miR-449a in autophagy remains unknown. The functional and persistent G1/S checkpoint signaling pathways in HeLa cells via these three miRNAs, either synergistically or separately, remain a mystery. As a result, we present a synthetic Boolean network of the functional G1/S checkpoint regulation, illustrating the regulatory effects of these three miRNAs. To our knowledge, this is the first synthetic Boolean network that demonstrates the advanced role of these miRNAs in cervical cancer signaling pathways reliant on or independent of p53, such as MAPK or AMPK. We compared our estimated probability to the experimental data and found reasonable agreement. Our findings indicate that miR-34a or miR-16 may control senescence, autophagy, apoptosis, and the functional G1/S checkpoint. Additionally, miR-449a can regulate just senescence and apoptosis on an individual basis. MiR-449a can coordinate autophagy in HeLa cells in a synergistic manner with miR-16 and/or miR-34a.

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  • 125.
    Gupta, Shantanu
    et al.
    Univ Sao Paulo, Dept Ciencia Computacao, Inst Matemat & Estat, Rua Matao 1010, BR-05508090 Sao Paulo, SP, Brazil..
    Panda, Pritam Kumar
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Silveira, Daner A.
    Childrens Canc Inst, BR-90620110 Porto Alegre, Rio Grande do S, Brazil..
    Ahuja, Rajeev
    Uppsala University, Disciplinary Domain of Science and Technology, Earth Sciences, Department of Earth Sciences, Solid Earth Geology. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics, Condensed Matter Theory. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Materials Science. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Theoretical Physics. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics, Theoretical Magnetism. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Molecular and Condensed Matter Physics. Indian Inst Technol Ropar, Dept Phys, Rupnagar 140001, Punjab, India..
    Hashimoto, Ronaldo F.
    Univ Sao Paulo, Dept Ciencia Computacao, Inst Matemat & Estat, Rua Matao 1010, BR-05508090 Sao Paulo, SP, Brazil..
    Quadra-Stable Dynamics of p53 and PTEN in the DNA Damage Response2023In: Cells, E-ISSN 2073-4409, Vol. 12, no 7, article id 1085Article in journal (Refereed)
    Abstract [en]

    Cell fate determination is a complex process that is frequently described as cells traveling on rugged pathways, beginning with DNA damage response (DDR). Tumor protein p53 (p53) and phosphatase and tensin homolog (PTEN) are two critical players in this process. Although both of these proteins are known to be key cell fate regulators, the exact mechanism by which they collaborate in the DDR remains unknown. Thus, we propose a dynamic Boolean network. Our model incorporates experimental data obtained from NSCLC cells and is the first of its kind. Our network's wild-type system shows that DDR activates the G2/M checkpoint, and this triggers a cascade of events, involving p53 and PTEN, that ultimately lead to the four potential phenotypes: cell cycle arrest, senescence, autophagy, and apoptosis (quadra-stable dynamics). The network predictions correspond with the gain-and-loss of function investigations in the additional two cell lines (HeLa and MCF-7). Our findings imply that p53 and PTEN act as molecular switches that activate or deactivate specific pathways to govern cell fate decisions. Thus, our network facilitates the direct investigation of quadruplicate cell fate decisions in DDR. Therefore, we concluded that concurrently controlling PTEN and p53 dynamics may be a viable strategy for enhancing clinical outcomes.

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  • 126.
    Gustafsson, Ann-Sofie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Radiation response in human cells: DNA damage formation, repair and signaling2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Ionizing radiation induces a range of different DNA lesions. In terms of mutation frequency and mammalian cell survival, the most critical of these lesions is the DNA double-strand break (DSB). DSB left unrepaired or mis-repaired may result in chromosomal aberrations that can lead to permanent genetic changes or cell death. The complexity of the DNA damage and the capacity to repair the DSB will determine the fate of the cell. This thesis focuses on the DNA damage formation, repair and signaling after irradiation of human cells.

    Radiation with high linear energy transfer (LET) produces clustered damaged sites in the DNA that are difficult for the cell to repair. Within these clustered sites, non-DSB lesions are formed that can be converted into a DSB and add to the damage complexity and affect DSB repair and the measurement. Heat-labile sites in DNA are converted into DSB at elevated temperatures. We show that heat-released DSB are formed post-irradiation with high-LET ions and increase the initial yield of DSB by 30%-40%, which is similar to yields induced by low-LET radiation.

    DNA-PKcs, a central player in non-homologous end-joining (NHEJ), the major mammalian DSB repair pathway, has been found to be both up- and downregulated in different tumor types. In Paper II we show that low levels of DNA-PKcs lead to extreme radiosensitivity but, surprisingly, had no effect on the DSB repair. However, the fraction of cells in G2/M phase increased two-fold in cells with low levels of DNA-PKcs. The study continued in Paper IV, where cells were synchronized to unmask potential roles of DNA-PKcs in specific cell cycle phases. Irradiation of DNA-PKcs suppressed cells in the G1/S phase caused a delay in cell cycle progression and an increase in accumulation of G2 cells. Further, these cells showed defects in DNA repair, where a significant amount of 53BP1 foci remained after 72 h. This further strengthens the hypothesis that DNA-PKcs has a role in regulation of mitotic progression.

    Several cellular signaling pathways are initiated in response to radiation. One of these downstream signaling proteins is AKT. We identified an interaction between DNA-PKcs and AKT. Knockouts of both AKT1 and AKT2 impaired DSB rejoining after radiation and low levels of DNA-PKcs increased radiosensitivity and decreased DNA repair further.  

    List of papers
    1. Formation and repair of clustered damaged DNA sites in high LET irradiated cells
    Open this publication in new window or tab >>Formation and repair of clustered damaged DNA sites in high LET irradiated cells
    2015 (English)In: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 91, no 10, p. 820-826Article in journal (Refereed) Published
    Abstract [en]

    PURPOSE: Radiation with high linear energy transfer (LET) produces clustering of DNA double-strand breaks (DSB) as well as non-DSB lesions. Heat-labile sites (HLS) are non-DSB lesions in irradiated cells that may convert into DSB at elevated temperature during preparation of naked DNA for electrophoretic assays and here we studied the initial formation and repair of these clustered damaged sites after irradiation with high LET ions.

    MATERIALS AND METHODS: Induction and repair of DSB were studied in normal human skin fibroblast (GM5758) after irradiation with accelerated carbon and nitrogen ions at an LET of 125 eV/nm. DNA fragmentation was analyzed by pulsed-field gel electrophoresis (PFGE) and by varying the lysis condition we could differentiate between prompt DSB and heat-released DSB.

    RESULTS: Before repair (t = 0 h), the 125 eV/nm ions produced a significant fraction of heat-released DSB, which appeared clustered on DNA fragments with sizes of 1 Mbp or less. These heat-released DSB increased the total number of DSB by 30-40%. This increase is similar to what has been found in low-LET irradiated cells, suggesting that the relative biological effectiveness (RBE) for DSB induction will not be largely affected by the lysis temperature. After 1-2 hours repair, a large fraction of DSB was still unrejoined but there was essentially no heat-released DSB present.

    CONCLUSIONS: These results suggest that high LET radiation, as low LET gamma radiation, induces a significant fraction of heat-labile sites which can be converted into DSB, and these heat-released DSB may affect both induction yields and estimates of repair.

    Keywords
    Clustered damage; DNA damage; DNA DSB repair; DNA repair; double-strand breaks; High LET
    National Category
    Basic Medicine Radiology, Nuclear Medicine and Medical Imaging
    Identifiers
    urn:nbn:se:uu:diva-265135 (URN)10.3109/09553002.2015.1068463 (DOI)000365614800007 ()26136085 (PubMedID)
    Funder
    Swedish Cancer Society
    Available from: 2015-10-22 Created: 2015-10-22 Last updated: 2018-01-11Bibliographically approved
    2. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair
    Open this publication in new window or tab >>Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair
    2014 (English)In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 769, p. 1-10Article in journal (Refereed) Published
    Abstract [en]

    Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80-95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or gamma-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

    Keywords
    DNA repair, DNA-PKcs, Ionizing radiation, DNA-PK deficiency, NU7441
    National Category
    Medical Genetics
    Identifiers
    urn:nbn:se:uu:diva-237292 (URN)10.1016/j.mrfmmm.2014.06.004 (DOI)000343625700001 ()
    Available from: 2014-12-03 Created: 2014-12-01 Last updated: 2019-03-08Bibliographically approved
    3. 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
    Show others...
    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
    4. Role of DNA-PKcs in DNA damage response and cell cycle regulation
    Open this publication in new window or tab >>Role of DNA-PKcs in DNA damage response and cell cycle regulation
    (English)Manuscript (preprint) (Other academic)
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265136 (URN)
    Note

    The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. We have previously reported that suppression of DNA-PKcs sensitize cells to radiation without affecting repair. In the present study we used synchronized cells to unmask potential roles of DNA-PKcs in specific cell-cycle phases. siRNA was used to deplete DNA-PKcs to 10-15 % of normal levels and cell cycle progression and DSB repair was observed in synchronized cells irradiated at different cell cycle phases. Surprisingly, cells irradiated in G2 phase showed similar p-H3 frequency after 72 h as unirradiated control cells, irrespective of their DNA-PKcs status. When cells were irradiated in G1/S phase, a significant increase of mitotic cells in siDNA-PKcs treated cells was seen 72 h later. Further, irradiation in G1/ S phase caused initially (0-12h) the same number of DSB (53BP1 foci), however, over time (>24h) 53BP1 foci remained at relatively high levels in DNA-PKcs depleted cells, indicating presence of unrepaired DSB in the following G1. This suggest that DNA-PKcs has an important regulatory role in G1/S and a key function in mitosis.

    Available from: 2015-10-22 Created: 2015-10-22 Last updated: 2018-01-11
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  • 127.
    Gutiérrez-de-Terán, Hugo
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sallander, Jessica
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sotelo, Eddy
    Univ Santiago de Compostela, Fac Pharm, Ctr Res Biol Chem & Mol Mat CIQUS, Santiago De Compostela 15782, A Coruna, Spain.;Univ Santiago de Compostela, Fac Pharm, Dept Organ Chem, Santiago De Compostela 15782, A Coruna, Spain..
    Structure-Based Rational Design of Adenosine Receptor Ligands2017In: Current Topics in Medicinal Chemistry, ISSN 1568-0266, E-ISSN 1873-4294, Vol. 17, no 1, p. 40-58Article, review/survey (Refereed)
    Abstract [en]

    The family of adenosine receptors (ARs) is focus of several medicinal chemistry programs aimed to find new potent and selective drugs. Each receptor subtype has been proposed as a relevant drug target in the treatment of, e.g., cardiovascular or inflammatory diseases, asthma or Parkinson's disease. Until recently, most of these efforts have been dominated by ligand-based or empirical approaches. However, the latest advances in G protein-coupled receptor (GPCR) crystallography allowed for a thorough structural characterization of the A(2A)AR subtype, which has been crystalized with a number of agonists and antagonists. Consequently, the ligand discovery of AR ligands has been enriched with a number of structure-based approaches. These include the generation of higher-confident homology models for the remaining AR subtypes, virtual screening identification of novel chemotypes, structure-based lead-optimization programs, rationalization of selectivity profiles, or the structural characterization of novel binding sites that enable the design of novel allosteric modulators. Computational methodologies have importantly contributed to the success of these structure-based approaches, and the recent advances in the field are also analyzed in this review. We conclude that the design of adenosine receptor ligands has improved dramatically with the consideration of structure-based approaches, which is paving the way to a better understanding of the biology and pharmacological modulation of this relevant family of receptors.

  • 128.
    Gutter-Kapon, Lilach
    et al.
    Technion, Bruce Rappaport Fac Med, Canc & Vasc Biol Res Ctr, IL-31096 Haifa, Israel..
    Alishekevitz, Dror
    Technion, Bruce Rappaport Fac Med, Dept Cell Biol & Canc Sci, IL-31096 Haifa, Israel..
    Shaked, Yuval
    Technion, Bruce Rappaport Fac Med, Dept Cell Biol & Canc Sci, IL-31096 Haifa, Israel..
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Aronheim, Ami
    Technion, Bruce Rappaport Fac Med, Dept Mol Genet, IL-31096 Haifa, Israel..
    Ilan, Neta
    Technion, Bruce Rappaport Fac Med, Canc & Vasc Biol Res Ctr, IL-31096 Haifa, Israel..
    Vlodavsky, Israel
    Technion, Bruce Rappaport Fac Med, Canc & Vasc Biol Res Ctr, IL-31096 Haifa, Israel..
    Heparanase is required for activation and function of macrophages2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 48, p. E7808-E7817Article in journal (Refereed)
    Abstract [en]

    The emerging role of heparanase in tumor initiation, growth, metastasis, and chemoresistance is well recognized and is encouraging the development of heparanase inhibitors as anticancer drugs. Unlike the function of heparanase in cancer cells, very little attention has been given to heparanase contributed by cells composing the tumor microenvironment. Here we used a genetic approach and examined the behavior and function of macrophages isolated from wild-type (WT) and heparanase-knockout (Hpa-KO) mice. Hpa-KO macrophages express lower levels of cytokines (e.g., TNF alpha, IL1-beta) and exhibit lower motility and phagocytic capacities. Intriguingly, inoculation of control monocytes togetherwith Lewis lung carcinoma (LLC) cells into Hpa-KO mice resulted in nearly complete inhibition of tumor growth. In striking contrast, inoculating LLC cells together with monocytes isolated from Hpa-KO mice did not affect tumor growth, indicating that heparanase is critically required for activation and function of macrophages. Mechanistically, we describe a linear cascade by which heparanase activates Erk, p38, and JNK signaling in macrophages, leading to increased c-Fos levels and induction of cytokine expression in a manner that apparently does not require heparanase enzymatic activity. These results identify heparanase as a key mediator of macrophage activation and function in tumorigenesis and cross-talk with the tumor microenvironment.

  • 129.
    Guy, Lionel
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Saw, Jimmy Hser Wah
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Ettema, Thijs J. G.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    The Archaeal Legacy of Eukaryotes: A Phylogenomic Perspective2014In: Cold Spring Harbor Perspectives in Biology, E-ISSN 1943-0264, Vol. 6, no 10, p. a016022-Article in journal (Refereed)
    Abstract [en]

    The origin of the eukaryotic cell can be regarded as one of the hallmarks in the history of life on our planet. The apparent genomic chimerism in eukaryotic genomes is currently best explained by invoking a cellular fusion at the root of the eukaryotes that involves one archaeal and one or more bacterial components. Here, we use a phylogenomics approach to reevaluate the evolutionary affiliation between Archaea and eukaryotes, and provide further support for scenarios in which the nuclear lineage in eukaryotes emerged from within the archaeal radiation, displaying a strong phylogenetic affiliation with, or even within, the archaeal TACK superphylum. Further taxonomic sampling of archaeal genomes in this superphylum will certainly provide a better resolution in the events that have been instrumental for the emergence of the eukaryotic lineage.

  • 130.
    Göstring, Lovisa
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Lindegren, Sture
    Univ Gothenburg, Sahlgrenska Acad, Dept Radiat Phys, SE-41345 Gothenburg, Sweden.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Swedish Radiat Safety Author, SE-17116 Stockholm, Sweden.
    17AAG-induced internalisation of HER2- specific Affibody molecules2016In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 12, no 4, p. 2574-2580Article in journal (Refereed)
    Abstract [en]

    The geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) is known to induce internalisation and degradation of the otherwise internalisation-resistant human epidermal growth factor receptor 2 (HER2) receptor. In the present study, 17-AAG was used to increase internalisation of the HER2-specific Affibody molecule ABY-025. The cellular redistribution of halogen-labelled At-211-ABY-025 and radiometal-labelled In-111-ABY-025 following treatment with 17-AAG was studied. 17-AAG treatment of SKOV-3 human ovarian carcinoma and SKBR-3 human breast carcinoma cells to some extent shifted the localisation of In-111-ABY-025 from the cell surface to intracellular compartments in the two cell lines. ABY-025 labelled with the high-linear energy transfer emitter At-211 was also internalised to a higher degree; however, due to its physiological properties, this nuclide was excreted faster. The results indicate that 17-AAG may be used to facilitate cell-specific intracellular localisation of a suitable cytotoxic or radioactive agent coupled to ABY-025 in HER2-overexpressing cells.

  • 131.
    Hammar, Petter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Walldén, Mats
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ullman, Gustaf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Leroy, Prune
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation2014In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 46, no 4, p. 405-+Article in journal (Refereed)
    Abstract [en]

    Transcription factors mediate gene regulation by site-specific binding to chromosomal operators. It is commonly assumed that the level of repression is determined solely by the equilibrium binding of a repressor to its operator. However, this assumption has not been possible to test in living cells. Here we have developed a single-molecule chase assay to measure how long an individual transcription factor molecule remains bound at a specific chromosomal operator site. We find that the lac repressor dimer stays bound on average 5 min at the native lac operator in Escherichia coli and that a stronger operator results in a slower dissociation rate but a similar association rate. Our findings do not support the simple equilibrium model. The discrepancy with this model can, for example, be accounted for by considering that transcription initiation drives the system out of equilibrium. Such effects need to be considered when predicting gene activity from transcription factor binding strengths.

  • 132.
    Hammarström, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Studies on Heterologous Seeding in Drosophila Melanogaster2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    The full text will be freely available from 2026-06-30 05:09
  • 133. Han, Shunhua
    et al.
    Dias, Guilherme
    Basting, Preston J
    Nelson, Michael G
    Patel, Sanjai
    Marzo, Mar
    Bergman, Casey M
    Ongoing transposition in cell culture reveals the phylogeny of diverse Drosophila S2 sublines.2022In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 221, no 3, article id iyac077Article in journal (Refereed)
    Abstract [en]

    Cultured cells are widely used in molecular biology despite poor understanding of how cell line genomes change in vitro over time. Previous work has shown that Drosophila cultured cells have a higher transposable element content than whole flies, but whether this increase in transposable element content resulted from an initial burst of transposition during cell line establishment or ongoing transposition in cell culture remains unclear. Here, we sequenced the genomes of 25 sublines of Drosophila S2 cells and show that transposable element insertions provide abundant markers for the phylogenetic reconstruction of diverse sublines in a model animal cell culture system. DNA copy number evolution across S2 sublines revealed dramatically different patterns of genome organization that support the overall evolutionary history reconstructed using transposable element insertions. Analysis of transposable element insertion site occupancy and ancestral states support a model of ongoing transposition dominated by episodic activity of a small number of retrotransposon families. Our work demonstrates that substantial genome evolution occurs during long-term Drosophila cell culture, which may impact the reproducibility of experiments that do not control for subline identity.

  • 134. Han, Shunhua
    et al.
    Dias, Guilherme
    Basting, Preston J
    Viswanatha, Raghuvir
    Perrimon, Norbert
    Bergman, Casey M
    Local assembly of long reads enables phylogenomics of transposable elements in a polyploid cell line.2022In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, no 21, p. e124-, article id e124Article in journal (Refereed)
    Abstract [en]

    Animal cell lines often undergo extreme genome restructuring events, including polyploidy and segmental aneuploidy that can impede de novo whole-genome assembly (WGA). In some species like Drosophila, cell lines also exhibit massive proliferation of transposable elements (TEs). To better understand the role of transposition during animal cell culture, we sequenced the genome of the tetraploid Drosophila S2R+ cell line using long-read and linked-read technologies. WGAs for S2R+ were highly fragmented and generated variable estimates of TE content across sequencing and assembly technologies. We therefore developed a novel WGA-independent bioinformatics method called TELR that identifies, locally assembles, and estimates allele frequency of TEs from long-read sequence data (https://github.com/bergmanlab/telr). Application of TELR to a ∼130x PacBio dataset for S2R+ revealed many haplotype-specific TE insertions that arose by transposition after initial cell line establishment and subsequent tetraploidization. Local assemblies from TELR also allowed phylogenetic analysis of paralogous TEs, which revealed that proliferation of TE families in vitro can be driven by single or multiple source lineages. Our work provides a model for the analysis of TEs in complex heterozygous or polyploid genomes that are recalcitrant to WGA and yields new insights into the mechanisms of genome evolution in animal cell culture.

  • 135. Han, Shunhua
    et al.
    Dias, Guilherme
    Dias, Guilherme B
    Luhur, Arthur
    Zelhof, Andrew C
    Bergman, Casey M
    Transposable element profiles reveal cell line identity and loss of heterozygosity in Drosophila cell culture.2021In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 219, no 2, article id iyab113Article in journal (Refereed)
    Abstract [en]

    Cell culture systems allow key insights into biological mechanisms yet suffer from irreproducible outcomes in part because of cross-contamination or mislabeling of cell lines. Cell line misidentification can be mitigated by the use of genotyping protocols, which have been developed for human cell lines but are lacking for many important model species. Here, we leverage the classical observation that transposable elements (TEs) proliferate in cultured Drosophila cells to demonstrate that genome-wide TE insertion profiles can reveal the identity and provenance of Drosophila cell lines. We identify multiple cases where TE profiles clarify the origin of Drosophila cell lines (Sg4, mbn2, and OSS_E) relative to published reports, and also provide evidence that insertions from only a subset of long-terminal repeat retrotransposon families are necessary to mark Drosophila cell line identity. We also develop a new bioinformatics approach to detect TE insertions and estimate intra-sample allele frequencies in legacy whole-genome sequencing data (called ngs_te_mapper2), which revealed loss of heterozygosity as a mechanism shaping the unique TE profiles that identify Drosophila cell lines. Our work contributes to the general understanding of the forces impacting metazoan genomes as they evolve in cell culture and paves the way for high-throughput protocols that use TE insertions to authenticate cell lines in Drosophila and other organisms.

  • 136.
    Haq, Syed Razaul
    et al.
    Stockholm Univ, Dept Mol BioSci, Wenner Gren Inst, Svante Arrhenius V 20C, S-10691 Stockholm, Sweden..
    Survery, Sabeen
    Stockholm Univ, Dept Mol BioSci, Wenner Gren Inst, Svante Arrhenius V 20C, S-10691 Stockholm, Sweden..
    Hurtig, Fredrik
    Stockholm Univ, Dept Mol BioSci, Wenner Gren Inst, Svante Arrhenius V 20C, S-10691 Stockholm, Sweden..
    Lindas, Ann-Christin
    Stockholm Univ, Dept Mol BioSci, Wenner Gren Inst, Svante Arrhenius V 20C, S-10691 Stockholm, Sweden..
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    NMR resonance assignment and dynamics of profilin from Heimdallarchaeota2020In: Scientific Reports, E-ISSN 2045-2322, Vol. 10, no 1, article id 15867Article in journal (Refereed)
    Abstract [en]

    The origin of the eukaryotic cell is an unsettled scientific question. The Asgard superphylum has emerged as a compelling target for studying eukaryogenesis due to the previously unseen diversity of eukaryotic signature proteins. However, our knowledge about these proteins is still relegated to metagenomic data and very little is known about their structural properties. Additionally, it is still unclear if these proteins are functionally homologous to their eukaryotic counterparts. Here, we expressed, purified and structurally characterized profilin from Heimdallarchaeota in the Asgard superphylum. The structural analysis shows that while this profilin possesses similar secondary structural elements as eukaryotic profilin, it contains additional secondary structural elements that could be critical for its function and an indication of divergent evolution.

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  • 137.
    Hatorangan, Marcelinus R.
    et al.
    Swedish Univ Agr Sci, Uppsala BioCtr, Dept Plant Biol, S-75007 Uppsala, Sweden.;Linnean Ctr Plant Biol, S-75007 Uppsala, Sweden..
    Laenen, Benjamin
    Stockholm Univ, Dept Ecol Environm & Plant Sci, Sci Life Lab, S-11418 Stockholm, Sweden..
    Steige, Kim A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology. Stockholm Univ, Dept Ecol Environm & Plant Sci, Sci Life Lab, S-11418 Stockholm, Sweden..
    Slotte, Tanja
    Stockholm Univ, Dept Ecol Environm & Plant Sci, Sci Life Lab, S-11418 Stockholm, Sweden..
    Kohler, Claudia
    Swedish Univ Agr Sci, Uppsala BioCtr, Dept Plant Biol, S-75007 Uppsala, Sweden.;Linnean Ctr Plant Biol, S-75007 Uppsala, Sweden..
    Rapid Evolution of Genomic Imprinting in Two Species of the Brassicaceae2016In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 28, no 8, p. 1815-1827Article in journal (Refereed)
    Abstract [en]

    Genomic imprinting is an epigenetic phenomenon occurring in mammals and flowering plants that causes genes to adopt a parent-of-origin-specific mode of expression. While the imprinting status of genes is well conserved in mammals, clear estimates for the degree of conservation were lacking in plants. We therefore analyzed the genome-wide imprinting status of Capsella rubella, which shared a common recent ancestor with Arabidopsis thaliana similar to 10 to 14 million years ago. However, only similar to 14% of maternally expressed genes (MEGs) and similar to 29% of paternally expressed genes (PEGs) in C. rubella were commonly imprinted in both species, revealing that genomic imprinting is a rapidly evolving phenomenon in plants. Nevertheless, conserved PEGs exhibited signs of selection, suggesting that a subset of imprinted genes play an important functional role and are therefore maintained in plants. Like in Arabidopsis, PEGs in C. rubella are frequently associated with the presence of transposable elements that preferentially belong to helitron and MuDR families. Our data further reveal that MEGs and PEGs differ in their targeting by 24-nucleotide small RNAs and asymmetric DNA methylation, suggesting different mechanisms establishing DNA methylation at MEGs and PEGs.

  • 138.
    Hedén Gynnå, Arvid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Induction kinetics of the lac operon: Studied by single molecule methods2014Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The repression of the E. coli lac operon seems to be more efficient than the current theoretical model allows for. Specifically, it is more quiet than expected during the replication of the chromosome. I have induced cells during short periods and counted the number of protein products from the operon to determine if there is a delay in activation of transcription that could account for the discrepancy. The results are compatible with a delay of 10-20 s, but the delay could not be conclusively proven. Furthermore, it has been investigated if the mechanism behind the delay might be differential localization of the lac operon with and without induction. It is shown that the lac operon is more often located in the periphery of the cell and in the internucleoid region when induced. These might be regions where genes are higher expressed, giving a delay in expression after de-repression before the gene is transported there.

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  • 139.
    Heldin, Carl-Henrik
    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.
    Tony Pawson (1952-2013)2014In: Growth Factors, ISSN 0897-7194, E-ISSN 1029-2292, Vol. 32, no 6, p. 174-175Article in journal (Other (popular science, discussion, etc.))
  • 140.
    Heldin, Carl-Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lennartsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Westermark, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Involvement of platelet-derived growth factor ligands and receptors in tumorigenesis2018In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 283, no 1, p. 16-44Article, review/survey (Refereed)
    Abstract [en]

    Platelet-derived growth factor (PDGF) isoforms and their receptors have important roles during embryogenesis, particularly in the development of various mesenchymal cell types in different organs. In the adult, PDGF stimulates wound healing and regulates tissue homeostasis. However, overactivity of PDGF signalling is associated with malignancies and other diseases characterized by excessive cell proliferation, such as fibrotic conditions and atherosclerosis. In certain tumours, genetic or epigenetic alterations of the genes for PDGF ligands and receptors drive tumour cell proliferation and survival. Examples include the rare skin tumour dermatofibrosarcoma protuberance, which is driven by autocrine PDGF stimulation due to translocation of a PDGF gene, and certain gastrointestinal stromal tumours and leukaemias, which are driven by constitute activation of PDGF receptors due to point mutations and formation of fusion proteins ofthe receptors, respectively. Moreover, PDGF stimulates cells in tumour stroma and promotes angiogenesis as well as the development of cancer-associated fibroblasts, both of which promote tumour progression. Inhibitors of PDGF signalling may thus be of clinical usefulness in the treatment of certain tumours.

  • 141.
    Heldin, Carl-Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lu, Benson
    Salk Inst Biol Studies, Gene Express Lab, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA..
    Evans, Ron
    Salk Inst Biol Studies, Gene Express Lab, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA..
    Gutkind, J. Silvio
    Natl Inst Dent & Craniofacial Res, NIH, Bethesda, MD 20892 USA..
    Signals and Receptors2016In: Cold Spring Harbor Perspectives in Biology, E-ISSN 1943-0264, Vol. 8, no 4, article id a005900Article in journal (Refereed)
    Abstract [en]

    Communication between cells in a multicellular organism occurs by the production of ligands (proteins, peptides, fatty acids, steroids, gases, and other low-molecular-weight compounds) that are either secreted by cells or presented on their surface, and act on receptors on, or in, other target cells. Such signals control cell growth, migration, survival, and differentiation. Signaling receptors can be single-span plasma membrane receptors associated with tyrosine or serine/threonine kinase activities, proteins with seven transmembrane domains, or intracellular receptors. Ligand-activated receptors convey signals into the cell by activating signaling pathways that ultimately affect cytosolic machineries or nuclear transcriptional programs or by directly translocating to the nucleus to regulate transcription.

  • 142.
    Heldin, Carl-Henrik
    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.
    Moustakas, Aristidis
    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. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Signaling Receptors for TGF-beta Family Members2016In: Cold Spring Harbor Perspectives in Biology, E-ISSN 1943-0264, Vol. 8, no 8, article id a022053Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor beta (TGF-beta) family members signal via heterotetrameric complexes of type I and type II dual specificity kinase receptors. The activation and stability of the receptors are controlled by posttranslational modifications, such as phosphorylation, ubiquitylation, sumoylation, and neddylation, as well as by interaction with other proteins at the cell surface and in the cytoplasm. Activation of TGF-beta receptors induces signaling via formation of Smad complexes that are translocated to the nucleus where they act as transcription factors, as well as via non-Smad pathways, including the Erk1/2, JNK and p38 MAP kinase pathways, and the Src tyrosine kinase, phosphatidylinositol 30-kinase, and Rho GTPases.

  • 143.
    Heldin, Carl-Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Ludwig Institute for Cancer Research.
    Scott, John
    Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington 98195, USA.
    Tony Pawson 1952-20132013In: Nature Cell Biology, ISSN 1465-7392, E-ISSN 1476-4679, Vol. 15, no 10, article id 1134Article in journal (Other (popular science, discussion, etc.))
  • 144.
    Hellman, Stina
    et al.
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, SLU, POB 7028, S-75007 Uppsala, Sweden..
    Martin, Frida
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, SLU, POB 7028, S-75007 Uppsala, Sweden..
    Tyden, Eva
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, SLU, POB 7028, S-75007 Uppsala, Sweden..
    Sellin, Mikael E.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Norman, Albin
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, SLU, POB 7028, S-75007 Uppsala, Sweden..
    Hjertner, Bernt
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, SLU, POB 7028, S-75007 Uppsala, Sweden..
    Svedberg, Pia
    Vidilab AB, POB 33, S-74521 Enkoping, Sweden..
    Fossum, Caroline
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, SLU, POB 7028, S-75007 Uppsala, Sweden..
    Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection2024In: Veterinary research (Print), ISSN 0928-4249, E-ISSN 1297-9716, Vol. 55, no 1, article id 25Article in journal (Refereed)
    Abstract [en]

    Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.

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  • 145.
    Hellsten, Sofie V.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Lekholm, Emilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ahmad, Tauseef
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Fredriksson, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    The gene expression of numerous SLC transporters is altered in the immortalized hypothalamic cell line N25/2 following amino acid starvation2017In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 7, no 2, p. 249-264Article in journal (Refereed)
    Abstract [en]

    Amino acids are known to play a key role in gene expression regulation,and in mammalian cells, amino acid signaling is mainly mediated via twopathways, the mammalian target of rapamycin complex 1 (mTORC1) pathwayand the amino acid responsive (AAR) pathway. It is vital for cells tohave a system to sense amino acid levels, in order to control protein andamino acid synthesis and catabolism. Amino acid transporters are crucialin these pathways, due to both their sensing and transport functions. Inthis large-scale study, an immortalized mouse hypothalamic cell line (N25/2)was used to study the gene expression changes following 1, 2, 3, 5 or 16 hof amino acid starvation. We focused on genes encoding solute carriers(SLCs) and putative SLCs, more specifically on amino acid transporters.The microarray contained 28 270 genes and 86.2% of the genes wereexpressed in the cell line. At 5 h of starvation, 1001 genes were upregulatedand 848 genes were downregulated, and among these, 47 genes from theSLC superfamily or atypical SLCs were found. Of these, 15 were genesencoding amino acid transporters and 32 were genes encoding other SLCsor atypical SLCs. Increased expression was detected for genes encodingamino acid transporters from system A, ASC, L, N, T, xc-, and y+. UsingGO annotations, genes involved in amino acid transport and amino acidtransmembrane transporter activity were found to be most upregulated at3 h and 5 h of starvation.

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  • 146.
    Hellsten, Sofie V
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tripathi, Rekha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ceder, Mikaela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fredriksson, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nutritional Stress Induced by Amino Acid Starvation Results in Changes for Slc38 Transporters in Immortalized Hypothalamic Neuronal Cells and Primary Cortex Cells2018In: Frontiers in Molecular Biosciences, E-ISSN 2296-889X, Vol. 5, article id 45Article in journal (Refereed)
    Abstract [en]

    Amino acid sensing and signaling is vital for cells, and both gene expression and protein levels of amino acid transporters are regulated in response to amino acid availability. Here, the aim was to study the regulation of all members of the SLC38 amino acid transporter family, Slc38a1-11 , in mouse brain cells following amino acid starvation. We reanalyzed microarray data for the immortalized hypothalamic cell line N25/2 subjected to complete amino acid starvation for 1, 2, 3, 5, or 16 h, focusing specifically on the SLC38 family. All 11 Slc38 genes were expressed in the cell line, and Slc38a1, Slc38a2, and Slc38a 7 were significantly upregulated at 5 h and most strongly at 16 h. Here, protein level changes were measured for SLC38A7 and the orphan family member SLC38A11 which has not been studied under different amino acid starvation condition at protein level. At 5 h, no significant alteration on protein level for either SLC38A7 or SLC38A11 could be detected. In addition, primary embryonic cortex cells were deprived of nine amino acids, the most common amino acids transported by the SLC38 family members, for 3 h, 7 h or 12 h, and the gene expression was measured using qPCR. Slc38a1, Slc38a2, Slc38a5, Slc38a6, Slc38a9, and Slc38a10 were upregulated, while Slc38a3 and Slc38a7 were downregulated. Slc38a8 was upregulated at 5 h and downregulated at 12 h. In conclusion, several members from the SLC38 family are regulated depending on amino acid levels and are likely to be involved in amino acid sensing and signaling in brain.

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  • 147.
    Herbert, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Synchronization of the circadian rhythm in a cell line used for testing neurodevelopmental toxicity.2024Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
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  • 148.
    Hernández Vera, Rodrigo
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Fatsis-Kavalopoulos, Nikos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Gradientech AB, Uppsala Science Park, Uppsala, Sweden.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Modular microfluidic systems cast from 3D-printed molds for imaging leukocyte adherence to differentially treated endothelial cultures2019In: Scientific Reports, E-ISSN 2045-2322, Vol. 9, article id 11321Article in journal (Refereed)
    Abstract [en]

    Microfluidic systems are very useful for in vitro studies of interactions between blood cells and vascular endothelial cells under flow, and several commercial solutions exist. However, the availability of customizable, user-designed devices is largely restricted to researchers with expertise in photolithography and access to clean room facilities. Here we describe a strategy for producing tailor-made modular microfluidic systems, cast in PDMS from 3D-printed molds, to facilitate studies of leukocyte adherence to endothelial cells. A dual-chamber barrier module was optimized for culturing two endothelial cell populations, separated by a 250 μm wide dividing wall, on a glass slide. In proof-of-principle experiments one endothelial population was activated by TNFα, while the other served as an internal control. The barrier module was thereafter replaced with a microfluidic flow module, enclosing both endothelial populations in a common channel. A suspension of fluorescently-labeled leukocytes was then perfused through the flow module and leukocyte interactions with control and tnfα-treated endothelial populations were monitored in the same field of view. Time-lapse microscopy analysis confirmed the preferential attachment of leukocytes to the TNFα-activated endothelial cells. We conclude that the functionality of these modular microfluidic systems makes it possible to seed and differentially activate adherent cell types, and conduct controlled side-by-side analysis of their capacity to interact with cells in suspension under flow. Furthermore, we outline a number of practical considerations and solutions associated with connecting and switching between the microfluidic modules, and the advantages of simultaneously and symmetrically analyzing control and experimental conditions in such a microfluidic system.

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  • 149.
    Herre, Melanie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cedervall, Jessica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mackman, Nigel
    UNC Blood Res Ctr, Dept Med, Div Hematol, Chapel Hill, NC USA..
    Olsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Neutrophil extracellular traps in the pathology of cancer and other inflammatory diseases2023In: Physiological Reviews, ISSN 0031-9333, E-ISSN 1522-1210, Vol. 103, no 1, p. 277-312Article, review/survey (Refereed)
    Abstract [en]

    Neutrophil extracellular trap (NET) formation, first described in 2004 as a previously unknown strategy of neutrophils to fight microbes, has attracted an increasing interest in the research community. NETs are formed when neutrophils externalize their decondensed chromatin together with content from their azurophilic granules. In addition to their role in defense against microbes, NETs have been implicated as mediators of pathology in sterile inflammation, such as cancer and autoimmunity, and their potential as therapeutic targets is actively explored. However, targeting of NETs is challenging since the beneficial effects of their removal need to be balanced against the potential harmful loss of their function in microbial defense. Moreover, depending on the stimuli or species, NETs can be formed via distinct mechanisms and are not always made up of the same components, making direct comparisons between various studies challenging. This review focuses on the role of NETs in cancer-associated pathology, such as thrombosis, organ dysfunction, and metastasis. Different strategies to target NETs, by either preventing their formation or degrading existing ones, are also discussed.

  • 150. Hewitt, Graeme
    et al.
    Borel, Valerie
    Segura-Bayona, Sandra
    Takaki, Tohru
    Ruis, Phil
    Bellelli, Roberto
    Lehmann, Laura C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sommerova, Lucia
    Vancevska, Aleksandra
    Tomas-Loba, Antonia
    Zhu, Kang
    Cooper, Christopher
    Fugger, Kasper
    Patel, Harshil
    Goldstone, Robert
    Schneider-Luftman, Deborah
    Herbert, Ellie
    Stamp, Gordon
    Brough, Rachel
    Pettitt, Stephen
    Lord, Christopher J.
    West, Stephen C.
    Ahel, Ivan
    Ahel, Dragana
    Chapman, J. Ross
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Boulton, Simon J.
    Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD2021In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 81, no 4, p. 767-783.e11Article in journal (Refereed)
    Abstract [en]

    Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.

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