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  • 1.
    Akan, Pelin
    et al.
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Alexeyenko, Andrey
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Costea, Paul Igor
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hedberg, Lilia
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Werne Solnestam, Beata
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lundin, Sverker
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hallman, Jimmie
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Comprehensive analysis of the genome transcriptome and proteome landscapes of three tumor cell lines2012Inngår i: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 4, s. 86-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We here present a comparative genome, transcriptome and functional network analysis of three human cancer cell lines (A431, U251MG and U2OS), and investigate their relation to protein expression. Gene copy numbers significantly influenced corresponding transcript levels; their effect on protein levels was less pronounced. We focused on genes with altered mRNA and/or protein levels to identify those active in tumor maintenance. We provide comprehensive information for the three genomes and demonstrate the advantage of integrative analysis for identifying tumor-related genes amidst numerous background mutations by relating genomic variation to expression/protein abundance data and use gene networks to reveal implicated pathways.

  • 2. Altai, M.
    et al.
    Tsourma, M.
    Mitran, B.
    Honarvar, H.
    Perols, Anna
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Robillard, M.
    Rossin, R.
    ten Hoeve, W.
    Sandstrom, M.
    Orlova, A.
    Karlström, Amelie Eriksson
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, V.
    Affibody-based bioorthogonal chemistry-mediated radionuclide pretargeting: proof-of-principle.2015Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, s. S246-S246Artikkel i tidsskrift (Fagfellevurdert)
  • 3.
    Banerjee, Indradumna
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Russom, Aman
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Lab-on-DVD: Optical Disk Drive-Based Platforms for Point-of-Care Diagnostics2018Inngår i: Frugal Innovation in Bioengineering for the Detection of Infectious Diseases / [ed] AK Chavali, R Ramji, Switzerland: Springer, 2018, 2, s. 23-38Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    There is a growing demand for simple, affordable, reliable and quality-assured point-of-care (POC) diagnostics for use in resource-limited settings. Among the top ten leading causes of death worldwide, three are infectious diseases, namely, respiratory infections, HIV/AIDS and diarrheal diseases (World Health Organization 2012). Although high-quality diagnostic tests are available, these are often not available to patients in developing countries. While recent development in microfluidics and “lab-on-a-chip” devices has the potential to spur the development of protocols and affordable instruments for diagnosis of infectious disease at POC, integration of complex sample preparation and detection into automated molecular and cellular systems remain a bottleneck for implementation of these systems at resource-limited settings. Towards this, we describe here how low-cost optical drives can, with minor modifications, be turned into POC diagnostic platforms. A DVD drive is essentially a highly advanced and low-cost optical laser-scanning microscope, with the capability to deliver high-resolution images for biological applications. Furthermore, the inherent centrifugal force on rotational discs is elegantly used for sample preparation and integration. Hence, the merging of low-cost optical disc drives with centrifugal microfluidics is feasible concept for POC diagnostics, specifically designed to meet the needs at resource-limited settings.

  • 4. Beerenwinkel, N.
    et al.
    Greenman, C. D.
    Lagergren, Jens
    KTH, Skolan för datavetenskap och kommunikation (CSC), Beräkningsvetenskap och beräkningsteknik (CST).
    Computational Cancer Biology: An Evolutionary Perspective2016Inngår i: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 12, nr 2, artikkel-id e1004717Artikkel i tidsskrift (Fagfellevurdert)
  • 5. Bourbeillon, Julie
    et al.
    Orchard, Sandra
    Benhar, Itai
    Borrebaeck, Carl
    de Daruvar, Antoine
    Duebel, Stefan
    Frank, Ronald
    Gibson, Frank
    Gloriam, David
    Haslam, Niall
    Hiltker, Tara
    Humphrey-Smith, Ian
    Hust, Michael
    Juncker, David
    Koegl, Manfred
    Konthur, Zoltan
    Korn, Bernhard
    Krobitsch, Sylvia
    Muyldermans, Serge
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Palcy, Sandrine
    Polic, Bojan
    Rodriguez, Henry
    Sawyer, Alan
    Schlapshy, Martin
    Snyder, Michael
    Stoevesandt, Oda
    Taussig, Michael J.
    Templin, Markus
    Uhlén, Matthias
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    van der Maarel, Silvere
    Wingren, Christer
    Hermjakob, Henning
    Sherman, David
    Minimum information about a protein affinity reagent (MIAPAR)2010Inngår i: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 28, nr 7, s. 650-653Artikkel i tidsskrift (Annet vitenskapelig)
  • 6.
    Ding, Haozhong
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Altai, Mohamed
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden..
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, S-75123 Uppsala, Sweden..
    Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody-DM1 Drug Conjugates2019Inngår i: Cancers, ISSN 2072-6694, Vol. 11, nr 8, artikkel-id 1168Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderateto high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (Z(HER2:2891))(2) -ABD-MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-3-MC-DM1, or a hexaglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-6-MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (Z(HER2:2891))(2)-ABD-MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.

  • 7.
    Fasterius, Erik
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Exploring genetic heterogeneity in cancer using high-throughput DNA and RNA sequencing2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    High-throughput sequencing (HTS) technology has revolutionised the biomedical sciences, where it is used to analyse the genetic makeup and gene expression patterns of both primary patient tissue samples and models cultivated in vitro. This makes it especially useful for research on cancer, a disease that is characterised by its deadliness and genetic heterogeneity. This inherent genetic variation is an important aspect that warrants exploration, and the depth and breadth that HTS possesses makes it well-suited to investigate this facet of cancer.

    The types of analyses that may be accomplished with HTS technologies are many, but they may be divided into two groups: those that analyse the DNA of the sample in question, and those that work on the RNA. While DNA-based methods give information regarding the genetic landscape of the sample, RNA-based analyses yield data regarding gene expression patterns; both of these methods have already been used to investigate the heterogeneity present in cancer. While RNA-based methods are traditionally used exclusively for expression analyses, the data they yield may also be utilised to investigate the genetic variation present in the samples. This type of RNA-based analysis is seldom performed, however, and valuable information is thus ignored.

    The aim of this thesis is the development and application of DNA- and RNA- based HTS methods for analysing genetic heterogeneity within the context of cancer. The present investigation demonstrates that not only may RNA-based sequencing be used to successfully differentiate different in vitro cancer models through their genetic makeup, but that this may also be done for primary patient data. A pipeline for these types of analyses is established and evaluated, showing it to be both robust to several technical parameters as well as possess a broad scope of analytical possibilities. Genetic variation within cancer models in public databases are evaluated and demonstrated to affect gene expression in several cases. Both inter- and intra-patient genetic heterogeneity is shown using the established pipeline, in addition to demonstrating that cancerous cells are more heterogeneous than their normal neighbours. Finally, two bioinformatic open source software packages are presented.

    The results presented herein demonstrate that genetic analyses using RNA-based methods represent excellent complements to already existing DNA-based techniques, and further increase the already large scope of how HTS technologies may be utilised.

  • 8.
    Fleetwood, Filippa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Klint, Susanne
    Hanze, Martin
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Gunneriusson, Elin
    Frejd, Fredrik
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity2014Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 4, s. 7518-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases.

  • 9. Garousi, J.
    et al.
    Anderson, Ken
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Dam, J. H.
    Olsen, B. B.
    Orlova, A.
    Buijs, J.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Thisgaard, H.
    Tolmachev, V.
    The use of radiocobalt as a label improves PET imaging of EGFR using DOTA-conjugated affibody molecules2015Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, s. S244-S244Artikkel i tidsskrift (Fagfellevurdert)
  • 10.
    Hofström, Camilla
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting2013Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets.

    In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR.

  • 11.
    Hudson, Elton P.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Nikoshkov, Andrej
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force2012Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 5, s. e37429-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

  • 12. Jeong, Yunjin
    et al.
    Svedberg, Gustav
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Réu, Pedro
    Lee, Yongju
    Song, Seo Woo
    Na, Hunjong
    Lee, Amos Chungwon
    Choi, Yeongjae
    Gantelius, Jesper
    Andersson Svahn, Helene
    Kwon, Sunghoon
    Solid-phase PCR on graphically encoded microparticles for multiplexed colorimetric detection of bacterial DNAManuskript (preprint) (Annet vitenskapelig)
  • 13.
    Johansson, Staffan
    et al.
    KTH, Skolan för elektro- och systemteknik (EES), Mikro- och nanosystemteknik.
    Eklund, Anders
    Umeå University.
    Malm, Jan
    Umeå University.
    Stemme, Göran
    KTH, Skolan för elektro- och systemteknik (EES), Mikro- och nanosystemteknik.
    Roxhed, Niclas
    KTH, Skolan för elektro- och systemteknik (EES), Mikro- och nanosystemteknik.
    A MEMS-based passive hydrocephalus shunt with adaptive flow characteristics2013Konferansepaper (Fagfellevurdert)
    Abstract [en]

    This paper reports a novel MEMS valve with adaptive flow characteristics for improved treatment of hydrocephalus, a disease that is characterized by elevated pressure in the cerebrospinal fluid (CSF) that surrounds the brain and spinal cord. In contrast to conventional valves with two ports, the valve presented here features a third port, called compensation port, which utilizes hydrostatic pressure to adapt CSF drainage based on body position. A prototype has been fabricated using standard MEMS manufacturing processes and the experimental evaluation successfully showed that the flow rate was adjustable with a varying hydrostatic pressure on the compensation port. Extracted data shows that flow rate was at near ideal values at both standing and laying body position showing an effective adaptation to body position. This is the first passive hydrocephalus valve intended for body position dependent CSF pressure regulation.

  • 14.
    Johansson, Staffan
    et al.
    KTH, Skolan för elektro- och systemteknik (EES), Mikro- och nanosystemteknik.
    Stemme, Göran
    KTH, Skolan för elektro- och systemteknik (EES), Mikro- och nanosystemteknik.
    Roxhed, Niclas
    KTH, Skolan för elektro- och systemteknik (EES), Mikro- och nanosystemteknik.
    A MEMS-based passive air flow regulator for handheld breath diagnostics2014Inngår i: Sensors and Actuators A-Physical, ISSN 0924-4247, E-ISSN 1873-3069, Vol. 215, s. 65-70Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    This paper reports on a passive MEMS-based flow regulator designed to maintain a steady flow during asthma diagnostics. A prototype consisting of six in-plane moving pistons that restrict the flow through six flow orifices has been fabricated from three wafers using standard silicon micromachining. The in-plane design enables relatively large flows and tuning of the flow and pressure range to specific application requirements by changing a wafer thickness. In particular, for FENO asthma monitoring, regulatory guidelines specifies that measurements should be made at steady flow of approximately 50 ml/s and within a pressure range of 1–2 kPa. Experimental evaluation of the prototype shows that the flow rate is controlled within a dynamic pressure range of 770 Pa compared to only 430 Pa for a dummy structure and that it can be achieved on a chip measuring only 2 mm × 2 mm × 4 mm. The evaluation also showed that condensation of exhaled air that expectedly occurs in the flow regulator at room temperature can be eliminated by local heating of the device to 40◦C.

  • 15.
    Kaczmarzyk, Danuta
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. Georg-August-University, Germany.
    Hudson, Elton P.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Fulda, Martin
    Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria2016Inngår i: AMB Express, ISSN 2191-0855, E-ISSN 2191-0855, Vol. 6, nr 1, s. 1-9, artikkel-id 7Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty acid (C10-C14) substrates in vitro. Furthermore, we used AAE15 to complement a Synechocystis aas deletion mutant and showed that the new strain preferentially incorporates supplied medium chain fatty acids into internal lipid molecules. Based on this data we propose that AAE15 can be utilized in metabolic engineering strategies for cyanobacteria that aim to produce compounds based on medium chain fatty acids.

  • 16. Kennedy, S
    et al.
    Fasterius, Erik
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Al-Khalili Szigyarto, Cristina
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Kolch, W
    et al.,
    Adaptive rewiring of protein-protein interactions and signal flow in the EGFR signaling network by mutant RASManuskript (preprint) (Annet vitenskapelig)
  • 17.
    Kroll, Jens
    et al.
    Westfälische Wilhelms-Universität Münster, Germany.
    Klinter, Stefan
    Westfälische Wilhelms-Universität Münster, Germany.
    Schneider, Cornelia
    Westfälische Wilhelms-Universität Münster, Germany.
    Voß, Isabella
    Westfälische Wilhelms-Universität Münster, Germany.
    Steinbüchel, Alexander
    Westfälische Wilhelms-Universität Münster, Germany.
    Plasmid addiction systems: Perspectives and applications in biotechnology2010Inngår i: Microbial Biotechnology, ISSN 1751-7907, E-ISSN 1751-7915, Vol. 3, nr 6, s. 634-657Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Biotechnical production processes often operate with plasmid-based expression systems in well-established prokaryotic and eukaryotic hosts such as Escherichia coli or Saccharomyces cerevisiae, respectively. Genetically engineered organisms produce important chemicals, biopolymers, biofuels and high-value proteins like insulin. In those bioprocesses plasmids in recombinant hosts have an essential impact on productivity. Plasmid-free cells lead to losses in the entire product recovery and decrease the profitability of the whole process. Use of antibiotics in industrial fermentations is not an applicable option to maintain plasmid stability. Especially in pharmaceutical or GMP-based fermentation processes, deployed antibiotics must be inactivated and removed. Several plasmid addiction systems (PAS) were described in the literature. However, not every system has reached a full applicable state. This review compares most known addiction systems and is focusing on biotechnical applications.

  • 18.
    Kuang, Qie
    et al.
    KTH, Skolan för teknik och hälsa (STH).
    Purhonen, Pasi
    Alander, Johan
    Svensson, Richard
    Hoogland, Veronika
    Winerdal, Jens
    Spahiu, Linda
    Ottosson-Wadlund, Astrid
    Jegerschold, Caroline
    KTH, Skolan för teknik och hälsa (STH).
    Morgenstern, Ralf
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Medicinsk teknik, Strukturell bioteknik.
    Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation2017Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikkel-id 7897Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.

  • 19.
    Lama, Lara
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Novel methods for improving rapid paper-based protein assays with gold nanoparticle detection2017Licentiatavhandling, monografi (Annet vitenskapelig)
    Abstract [en]

    This thesis describes methods for improving sensitivity in rapid singleplex and multiplex microarray assays. The assays utilize the optical characteristics of colloidal gold nanoparticles for the colorimetric detection of proteins.

    Multiplexed detection in sandwich immunoassays is limited by cross-reactivity between different detection antibodies. The cross-reactivity between antibodies can contribute to increased background noise - decreasing the Limit-of-Detection of the assay - or generate false positive signals. Paper I shows improved assay sensitivity in a multiplexed vertical flow assay by the application of ultrasonic energy to the gold nanoparticles functionalized with detection antibodies. The ultrasonication of the antibody conjugated gold nanoparticles resulted in a 10 000 fold increase in sensitivity in a 3-plex assay. COMSOL Multiphysics was used to simulate the acoustical energy of the probe used in Paper I for obtaining an indication of the size and direction of the forces acting upon the functionalized gold nanoparticles.

    In Paper II, it was studied if different gold nanoparticle conjugation methods and colorimetric signal enhancement of the gold nanoparticle conjugates could influence the sensitivity of a paper-based lateral flow microarray assay, targeting cardiac troponin T for the rapid diagnostics of acute myocardial infarction.

    Ultrasonication and signal enhancement of the detection gold nanoparticles has the potential of improving the sensitivity of paper based assays and expanding their potential future applications.

  • 20.
    Lama, Lara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Dias, Jorge T.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Gantelius, Jesper
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    A lateral flow cardiac troponin-T assay with colorimetric signal enhancementManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Cardiac troponin T (cTnT) is a biomarker for heart muscle damage such as in acute myocardial infarction (AMI). Its rapid assessment is needed to detect changes in the cTnT levels in blood for a quicker diagnosis of AMI. The sensitivity limit required to detect elevated levels of cTnT is 10 pg/mL, where the levels in the healthy population are 0.5-10 pg/mL. In this paper the detection of cardiac troponin T with a point-of-care lateral flow assay was investigated for the rapid diagnosis of AMI. It was studied by using different gold nanoparticle conjugation methods and colorimetric signal enhancement of detection gold nanoparticle conjugates could increase the sensitivity of a troponin T lateral flow microarray assay. The results indicate the same sensitivity range for the detection with gold nanoparticles functionalized with antibody by two different methods, and that the troponin T sandwich pair used might be essential for achieving a higher sensitivity. The signal enhancement increased the intensity signal of the detected cTnT on the array. The limit of detection of the assay changed from 10 μg/mL to 1 μg/mL for one conjugation method after signal enhancement but remained unchanged at 1 μg/mL for the other method.

  • 21.
    Mahdessian, Diana
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik.
    Sullivan, D. P.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik.
    Danielsson, Frida
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Arif, Muhammad
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Zhang, Cheng
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Åkesson, Lovisa
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Gnann, Christian
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Shutten, Rutger
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Thul, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik.
    Carja, Oana
    Department of Genetics, Stanford University, Stanford, CA 94305, USA. ; Chan Zuckerberg Biohub, San Francisco, San Francisco, CA 94158, USA..
    Ayoglu, Burcu
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik.
    Mardinoglu, Adil
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. Centre for Host–Microbiome Interactions, Faculty of Dentistry, Oral & Craniofacial Sciences, King's College London, London, SE1 9RT, United Kingdom.
    Pontén, Fredrik
    Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Lindskog, Cecilia
    Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden..
    Lundberg, Emma
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik. Department of Genetics, Stanford University, Stanford, CA 94305, USA. ; Chan Zuckerberg Biohub, San Francisco, San Francisco, CA 94158, USA..
    Spatiotemporal dissection of the cell cycle regulated human proteomeManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Here we present a spatiotemporal dissection of proteome single cell heterogeneity in human cells, performed with subcellular resolution over the course of a cell cycle. We identify 17% of the human proteome to display cell-to-cell variability, of which we could attribute 25% as correlated to cell cycle progression, and present the first evidence of cell cycle association for 258 proteins. A key finding is that the variance, of many of the cell cycle associated proteins, is only partially explained by the cell cycle, which hints at cross-talk between the cell cycle and other signaling pathways. We also demonstrate that several of the identified cell cycle regulated proteins may be clinically significant in proliferative disorders. This spatially resolved proteome map of the cell cycle, integrated into the Human Protein Atlas, serves as a valuable resource to accelerate the molecular knowledge of the cell cycle and opens up novel avenues for the understanding of cell proliferation.

  • 22.
    McKee, Lauren S.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center. Univ British Columbia, Canada.
    Growth of Chitinophaga pinensis on Plant Cell Wall Glycans and Characterisation of a Glycoside Hydrolase Family 27 beta-L-Arabinopyranosidase Implicated in Arabinogalactan Utilisation2015Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 10, artikkel-id e0139932Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The genome of the soil bacterium Chitinophaga pinensis encodes a diverse array of carbohydrate active enzymes, including nearly 200 representatives from over 50 glycoside hydrolase (GH) families, the enzymology of which is essentially unexplored. In light of this genetic potential, we reveal that C. pinensis has a broader saprophytic capacity to thrive on plant cell wall polysaccharides than previously reported, and specifically that secretion of beta-L-arabinopyranosidase activity is induced during growth on arabinogalactan. We subsequently correlated this activity with the product of the Cpin_5740 gene, which encodes the sole member of glycoside hydrolase family 27 (GH27) in C. pinensis, CpArap27. Historically, GH27 is most commonly associated with alpha-D-galactopyranosidase and alpha-D-N-acetylgalactosaminidase activity. A new phylogenetic analysis of GH27 highlighted the likely importance of several conserved secondary structural features in determining substrate specificity and provides a predictive framework for identifying enzymes with the less common beta-L-arabinopyranosidase activity.

  • 23.
    Mikus, Maria
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johansson, Catharina
    Acevedo, Nathalie
    Nilsson, Peter
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Scheynius, Annika
    The antimicrobial protein S100A12 identified as a potential autoantigen in a subgroup of atopic dermatitis patientsManuskript (preprint) (Annet vitenskapelig)
  • 24.
    Nielsen, Jens
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Global regulation of yeast metabolism2015Inngår i: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 32, s. S33-S33Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    The yeast Saccharomyces cerevisiae is widely used for production of fuels, chemicals, pharmaceuticals andmaterials. Through metabolic engineering of this yeast a number of novel new industrial processes have beendeveloped over the last 10 years. Besides its wide industrial use, S. cerevisiae serves as an eukaryal modelorganism, and many systems biology tools have therefore been developed for this organism. Despite ourextensive knowledge of yeast metabolism and its regulation we are still facing challenges when we want tointegrate this information into mathematical models. In this presentation examples of studies on global regulationof yeast metabolism will be provided. This will include analysis of how yeast rewire its metabolism at the globallevel when exposed to various types of stress and how global regulators like Snf1 and Sir2 controls yeastmetabolism

  • 25.
    Nielsen, Jens
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Mathematical modeling of yeast: a driver for innovation in biotechnology and human medicine2015Inngår i: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 32, s. S50-S51Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    Saccharomyces cerevisiae is the most studied organism and is for this reason used widely as a model organismfor studying molecular mechanisms of relevance for human disease. Thus, several Nobel Prizes have been givento yeast researchers. Among many other discoveries studies of yeast resulted in identification of cyclins andcyclin dependent kinases that play a central role in the cell cycle of eukaryal cells and mapping of the proteinsecretory pathway in eukaryal cells. This yeast is also used for production of fuels, chemicals, pharmaceuticalsand materials, and the annual revenue derived from processes based on S. cerevisiae fermentations by farexceeds 200 billion EURO. Furthermore. through metabolic engineering of this yeast a number of novel newindustrial processes are under development resulting in an even more important role of this cell factory in thefuture. In order to advance our fundamental understanding of this important organism, but in human healthresearch and industrial biotechnology, it is important to advance our ability to integrate novel experimental datain quantitative framework. Mathematical models represent an excellent scaffold for this as they allowreconciliation of data and at the same time enable generation of novel hypothesis concerning specific molecularprocesses. Furthermore, in the field of industrial biotechnology mathematical models may be used for advancingmetabolic engineering, which will result in a reduction in development costs and hereby advance towards biosustainable production of fuels and chemicals

  • 26.
    Nilvebrant, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    An albumin-binding domain as a scaffold for bispecific affinity proteins2012Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Protein engineering and in vitro selection systems are powerful methods to generate binding proteins. In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use both in basic and applied research. By means of combinatorial protein engineering and protein library technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the generation of affinity proteins. Using protein engineering, the albumin binding has been complemented with a new binding interface localized to the opposite surface of this three-helical bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains with ability to recognize several different target proteins were generated. In paper I, a bispecific albumin-binding domain was selected by phage display and utilized as a purification tag for highly efficient affinity purification of fusion proteins. The results in paper II show how protein engineering, in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives of proteins fused to it or potentially of the domain itself. When combined with a second targeting ability, a new molecular format with potential use in therapeutic applications is provided. The engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers III and IV are aimed in this direction. Over-expression of these receptors is associated with the development and progression of various cancers, and both are well-validated targets for therapy. Small bispecific binding proteins based on the albumin-binding domain could potentially contribute to this field. The new alternative protein scaffold described in this thesis is one of the smallest structured affinity proteins reported. The bispecific nature, with an inherent ability of the same domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding proteins may provide several advantages as compared to antibodies in several applications, particularly when a small size and an extended half-life are of key importance. 

  • 27.
    Nilvebrant, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Kuku, Gamze
    Björkelund, Hanna
    Nestor, Marika
    Selection and in vitro characterization of human CD44v6-binding antibody fragments2012Inngår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 59, nr 5, s. 367-380Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The cluster of differentiation (CD) 44v6 antigen has been suggested to be involved in tumor formation, invasion, and metastasis formation, and has been observed in a majority of primary and metastatic squamous cell carcinomas of the head and neck. Probes specifically binding to this region may be utilized as tools for the challenging tasks of early detection and targeted treatments of small residual disease. In this project, an epitope-guided phage display selection of human fragment antigen-binding (Fab) fragments with affinity to the v6 sequence was performed. A selected set of Fab fragments was shown to specifically recognize increasingly complex forms of the target sequence, both in the form of a short synthetic or recombinant peptide and in the context of a purified extracellular domain of CD44. The binding was independent of known v6-sequence variation and posttranslational modifications that are common in the CD44 protein family. Furthermore, real-time interaction measurements on antibody fragments labeled with 125I showed specific and high-affinity binding to the antigen present on cultured head and neck squamous cell carcinoma cells. There was no cross-reactivity toward cells that lack the target protein. As hypothesized, characterization of the interaction between Fab fragments and the targets using the mathematical tool Interaction Map revealed more heterogeneous interactions on cells than with pure proteins analyzed by surface plasmon resonance. One main candidate Fab fragment with optimal affinity for all forms of the target sequence was identified. The flexible recombinant source of the Fab fragments might aid the development of tailored molecules adapted for therapeutic or diagnostic applications in the future.

  • 28.
    Nybond, Susanna
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Réu, Pedro
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Rhedin, Samuel
    Svedberg, Gustav
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Alfvén, Tobias
    Gantelius, Jesper
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Svahn Andersson, Helene
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Adenoviral detection by recombinase polymerase amplification and vertical flow paper microarray.2019Inngår i: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, nr 4, s. 813-822Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Respiratory viral infections often mimic the symptoms of infections caused by bacteria; however, restricted and targeted administration of antibiotics is needed to combat growing antimicrobial resistance. This is particularly relevant in low-income settings. In this work, we describe the use of isothermal amplification of viral DNA at 37 °C coupled to a paper-based vertical flow microarray (VFM) setup that utilizes a colorimetric detection of amplicons using functionalized gold nanoparticles. Two oligonucleotide probes, one in-house designed and one known adenoviral probe were tested and validated for microarray detection down to 50 nM using a synthetic target template. Furthermore, primers were shown to function in a recombinase polymerase amplification reaction using both synthetic template and viral DNA. As a proof-of-concept, we demonstrate adenoviral detection with four different adenoviral species associated with respiratory infections using the paper-based VFM format. The presented assay was validated with selected adenoviral species using the in-house probe, enabling detection at 1 ng of starting material with intra- and inter-assay %CV of ≤ 9% and ≤ 13%. Finally, we validate our overall method using clinical samples. Based on the results, the combination of recombinase polymerase amplification, paper microarray analysis, and nanoparticle-based colorimetric detection could thus be a useful strategy towards rapid and affordable multiplexed viral diagnostics.

  • 29.
    Oroujeni, Maryam
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Garousi, Javad
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Andersson, Ken G.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Mitran, Bogdan
    Uppsala Univ, Dept Med Chem, SE-75123 Uppsala, Sweden..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, SE-75123 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75003 Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Preclinical Evaluation of [Ga-68]Ga-DFO-ZEGFR:2377: A Promising Affibody-Based Probe for Noninvasive PET Imaging of EGFR Expression in Tumors2018Inngår i: CELLS, ISSN 2073-4409, Vol. 7, nr 9, artikkel-id 141Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Radionuclide imaging of epidermal growth factor receptor (EGFR) expression in tumors may stratify patients for EGFR-targeting therapies and predict response or resistance to certain treatments. Affibody molecules, which are nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging. In this study, maleimido derivative of desferrioxamine B (DFO) chelator was site-specifically coupled to the C-terminal cysteine of the anti-EGFR affibody molecule ZEGFR:2377, and the DFO-ZEGFR:2377 conjugate was labeled with the generator-produced positron-emitting radionuclide Ga-68. Stability, specificity of binding to EGFR-expressing cells, and processing of [Ga-68]Ga-DFO-ZEGFR:2377 by cancer cells after binding were evaluated in vitro. In vivo studies were performed in nude mice bearing human EGFR-expressing A431 epidermoid cancer xenografts. The biodistribution of [Ga-68]Ga-DFO-ZEGFR:2377 was directly compared with the biodistribution of [Zr-89]Zr-DFO-ZEGFR:2377. DFO-ZEGFR:2377 was efficiently (isolated yield of 73 +/- 3%) and stably labeled with Ga-68. Binding of [Ga-68]Ga-DFO-ZEGFR:2377 to EGFR-expressing cells in vitro was receptor-specific and proportional to the EGFR expression level. In vivo saturation experiment demonstrated EGFR-specific accumulation of [Ga-68]Ga-DFO-ZEGFR:2377 in A431 xenografts. Compared to [Zr-89]Zr-DFO-ZEGFR:2377, [Ga-68]Ga-DFO-ZEGFR:2377 demonstrated significantly (p < 0.05) higher uptake in tumors and lower uptake in spleen and bones. This resulted in significantly higher tumor-to-organ ratios for [Ga-68]Ga-DFO-ZEGFR:2377. In conclusion, [Ga-68]Ga-DFO-ZEGFR:2377 is a promising probe for imaging of EGFR expression.

  • 30.
    Ortiz, Cantin
    et al.
    Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
    Fernandez Navarro, Jose
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jurek, Aleksandra
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Märtin, Antje
    Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
    Lundeberg, Joakim
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Meletis, Konstantinos
    Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
    Molecular atlas of the adult mouse brainManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Brain maps are essential for integrating information and interpreting the structure-function relationship of circuits and behavior. We aimed to generate a systematic classification of the adult mouse brain organization based on unbiased extraction of spatially-defining features. Applying whole-brain spatial transcriptomics, we captured the gene expression signatures to define the spatial organization of molecularly discrete subregions. We found that the molecular code contained sufficiently detailed information to directly deduce the complex spatial organization of the brain. This unsupervised molecular classification revealed new area- and layer-specific subregions, for example in isocortex and hippocampus, and a new division of striatum. The whole-brain molecular atlas further supports the identification of the spatial origin of single neurons using their gene expression profile, and forms the foundation to define a minimal gene set - a brain palette – that is sufficient to spatially annotate the adult brain. In summary, we have established a new molecular atlas to formally define the identity of brain regions, and a molecular code for mapping and targeting of discrete neuroanatomical domains.

  • 31.
    Sialana, Fernando J.
    et al.
    Univ Vienna, Dept Pharmaceut Chem, Vienna, Austria.;Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria..
    Gulyassy, Peter
    Eotvos Lorand Univ, Inst Biol, Lab Prote, Budapest, Hungary..
    Majek, Peter
    Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria..
    Sjöstedt, Evelina
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Uppsala Univ, Dept Immunol Genet & Pathol, Sci Life Lab, Uppsala, Sweden..
    Kis, Viktor
    Eotvos Lorand Univ, Dept Anat Cell & Dev Biol, Budapest, Hungary..
    Muller, Andre C.
    Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria.;ThermoFisher Sci, Vienna, Austria..
    Rudashevskaya, Elena L.
    Med Univ Vienna, Inst Med Chem, Vienna, Austria.;ISAS, Leibniz Inst Analyt Wissensch, Dortmund, Germany..
    Mulder, Jan
    Uppsala Univ, Dept Neurosci, Sci Life Lab, Uppsala, Sweden..
    Bennett, Keiryn L.
    Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria..
    Lubec, Gert
    Univ Vienna, Dept Pharmaceut Chem, Vienna, Austria..
    Mass spectrometric analysis of synaptosomal membrane preparations for the determination of brain receptors, transporters and channels2016Inngår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, nr 22, s. 2911-2920Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The molecular composition of synaptic signal transduction machineries shapes synaptic neurotransmission. The repertoire of receptors, transporters and channels (RTCs) comprises major signaling events in the brain. RTCs are conventionally studied by candidate immunohistochemistry and biochemistry, which are low throughput with resolution greatly affected by available immunoreagents and membrane interference. Therefore, a comprehensive resource of synaptic brain RTCs is still lacking. In particular, studies on the detergent-soluble synaptosomal fraction, known to contain transporters and channels, are limited. We, therefore, performed sub-synaptosomal fractionation of rat cerebral cortex, followed by trypsin/chymotrypsin sequential digestion of a detergent-soluble synaptosomal fraction and a postsynaptic density preparation, stable-isotope tryptic peptide labeling and liquid chromatography mass spectrometry. Based on the current study, a total of 4784 synaptic proteins were submitted to the ProteomExchange database (PXD001948), including 274 receptors, 394 transporters/channels and 1377 transmembrane proteins. Function-based classification assigned 1781 proteins as probable drug targets with 834 directly linked to brain disorders. The analytical approach identified 499 RTCs that are not listed in the largest, curated database for synaptosomal proteins (SynProt). This is a threefold RTC increase over all other data collected to date. Taken together, we present a protein discovery resource that can serve as a benchmark for future molecular interrogation of synaptic connectivity.

  • 32.
    Svedberg, Gustav
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Novel planar and particle-based microarrays for point-of-care diagnostics2019Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Point-of-care assays are easy-to-use, portable and inexpensive tests that can

    be used to aid diagnostics by measuring levels of disease-specific molecules

    in settings where access to advanced laboratory equipment and trained

    personnel are limited, such as at the patient's bedside or in low resource

    parts of developing countries. In order to achieve high multiplexing

    capacities, such assays can be based on planar microarrays consisting of

    spots immobilized on a flat surface or on particle-based microarrays based

    on populations of encoded particles. The aim of the work presented in this

    thesis is to develop new point-of-care amenable planar and particle-based

    microarrays that allow for highly multiplexed assays while maintaining low

    sample-to-result times, complexity and instrumentation requirements.

    Paper I demonstrates the use graphically encoded particles for colorimetric

    detection of autoantibodies using a consumer-grade flatbed scanner. Four

    graphical characters on the surface of each particle allows for millions of

    codes and the use of gold nanoparticles as a detection label allows both the

    code and the signal intensity to be read out in a single image.

    Paper II describes a signal enhancement method that increases the

    sensitivity of gold nanoparticle detection on planar microarrays. Using this

    method, detection of allergen-specific IgE can be carried out using a

    consumer-grade flatbed scanner instead of a more expensive fluorescence

    scanner without sacrificing assay performance.

    Paper III demonstrates the use of an isothermal DNA amplification method

    for detection of adenoviral DNA on a paper-based microarray. Using an

    isothermal amplification method eliminates the need for a thermocycler,

    reducing the instrumentation required for such detection.

    Paper IV shows the use of solid-phase PCR to amplify bacterial DNA directly

    on the surface of particles. This strategy reduces assay time by eliminating

    the need for separate amplification and hybridisation steps.

  • 33.
    Turanli, Beste
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Karagoz, Kubra
    Rutgers Canc Inst New Jersey, Dept Radiat Oncol, New Brunswick, NJ USA..
    Bidkhori, Gholamreza
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Sinha, Raghu
    Penn State Coll Med, Dept Biochem & Mol Biol, Hershey, PA 17033 USA..
    Gatza, Michael L.
    Rutgers Canc Inst New Jersey, Dept Radiat Oncol, New Brunswick, NJ USA..
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Mardinoglu, Adil
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Arga, Kazim Yalcin
    Marmara Univ, Dept Bioengn, Istanbul, Turkey..
    Multi-Omic Data Interpretation to Repurpose Subtype Specific Drug Candidates for Breast Cancer2019Inngår i: Frontiers in Genetics, ISSN 1664-8021, E-ISSN 1664-8021, Vol. 10, artikkel-id 420Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Triple-negative breast cancer (TNBC), which is largely synonymous with the basal-like molecular subtype, is the 5th leading cause of cancer deaths for women in the United States. The overall prognosis for TNBC patients remains poor given that few treatment options exist; including targeted therapies (not FDA approved), and multi-agent chemotherapy as standard-of-care treatment. TNBC like other complex diseases is governed by the perturbations of the complex interaction networks thereby elucidating the underlying molecular mechanisms of this disease in the context of network principles, which have the potential to identify targets for drug development. Here, we present an integrated "omics" approach based on the use of transcriptome and interactome data to identify dynamic/active protein-protein interaction networks (PPINs) in TNBC patients. We have identified three highly connected modules, EED, DHX9, and AURKA, which are extremely activated in TNBC tumors compared to both normal tissues and other breast cancer subtypes. Based on the functional analyses, we propose that these modules are potential drivers of proliferation and, as such, should be considered candidate molecular targets for drug development or drug repositioning in TNBC. Consistent with this argument, we repurposed steroids, anti-inflammatory agents, anti-infective agents, cardiovascular agents for patients with basal-like breast cancer. Finally, we have performed essential metabolite analysis on personalized genome-scale metabolic models and found that metabolites such as sphingosine-1-phosphate and cholesterol-sulfate have utmost importance in TNBC tumor growth.

  • 34.
    Vastesson, Alexander
    et al.
    KTH, Skolan för elektro- och systemteknik (EES), Mikro- och nanosystemteknik.
    Guo, Maoxiang
    KTH, Skolan för elektro- och systemteknik (EES), Mikro- och nanosystemteknik.
    Haraldsson, Tommy
    KTH, Skolan för elektro- och systemteknik (EES), Mikro- och nanosystemteknik.
    van der Wijngaart, Wouter
    KTH, Skolan för elektro- och systemteknik (EES), Mikro- och nanosystemteknik. KTH, Skolan för elektroteknik och datavetenskap (EECS), Mikro- och nanosystemteknik.
    Polymer Nanoliter Well Arrays for Liquid Storage and Rapid On-demand Electrochemical Release2018Inngår i: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 267, s. 111-118Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Polymer microfluidic systems are of increasing importance in several applications in biomedicine and biosensing. The integrated encapsulation, storage, and controlled release of small amounts of liquid in such systems remains an unresolved technical challenge. Here, we report two methods for the room-temperature and adhesive-free sealing of 1–330 nanoliter volumes of liquid in off-stoichiometry thiol-ene polymer well arrays by spontaneous bonding to 200 nm thin gold films. Sealed well arrays were stored for more than one month in a liquid environment with <10% liquid loss, and for more than one week in air with minimal loss. We demonstrated that controlling the electrical potential and polarity over encapsulated wells allowed for selecting one of two well opening mechanisms: slow anodic electrochemical etching, or rapid electrolytic gas pressure-induced bursting of the gold film. The results may find potential applications in diagnostic testing, in vivo drug delivery, or in spatio-temporal release of chemical compounds in biological assays.

  • 35.
    Wikman, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Friedman, Mikaela
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Pinitkiatisakul, S.
    Andersson, Christin
    KTH, Skolan för bioteknologi (BIO).
    Hemphill, A.
    Lovgren-Bengtsson, K.
    Lunden, A.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    General strategies for efficient adjuvant incorporation of recombinant subunit immunogens2005Inngår i: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 23, nr 17-18, s. 2331-2335Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptides or lipid tags to improve their capacity to be incorporated into an adjuvant formulation, e.g., immunostimulating complexes (iscoms). Recently, we also explored the strong interaction between biotin and streptavidin to achieve iscom association of recombinant immunogens. Plasmodium falciparum, Toxoplasma gondii and Neospora caninum antigens have served as model immunogens in the different studies. Generated fusion proteins have been found to be successfully incorporated into iscoms and high-titer antigen-specific antibody responses have been obtained upon immunization of mice. We believe that the different concepts presented, utilizing either hydrophobic peptide or lipid tags, or the recently explored biotin-streptavidin principle, offer convenient methods to achieve efficient adjuvant incorporation of recombinant immunogens.

  • 36.
    Yu, Shengze
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Alkharusi, Amira
    Sultan Qaboos Univ, Coll Med & Hlth Sci, Muscat, Oman..
    Norstedt, Gunnar
    Sultan Qaboos Univ, Coll Med & Hlth Sci, Muscat, Oman.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    An in vivo half-life extended prolactin receptor antagonist can prevent STAT5 phosphorylation2019Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, nr 5, artikkel-id e0215831Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Increasing evidence suggests that signaling through the prolactin/prolactin receptor axis is important for stimulation the growth of many cancers including glioblastoma multiforme, breast and ovarian carcinoma. Efficient inhibitors of signaling have previously been developed but their applicability as cancer drugs is limited by the short in vivo half-life. In this study, we show that a fusion protein, consisting of the prolactin receptor antagonist PrlRA and an albumin binding domain for half-life extension can be expressed as inclusion bodies in Escherichia coli and efficiently refolded and purified to homogeneity. The fusion protein was found to have strong affinity for the two intended targets: the prolactin receptor (K-D = 2.3 +/- 0.2 nM) and mouse serum albumin (K-D = 0.38 +/- 0.01 nM). Further investigation showed that it could efficiently prevent prolactin mediated phosphorylation of STAT5 at 100 nM concentration and above, similar to the PrlRA itself, suggesting a potential as drug for cancer therapy in the future. Complexion with HSA weakened the affinity for the receptor to 21 +/- 3 nM, however the ability to prevent phosphorylation of STAT5 was still prominent. Injection into rats showed a 100-fold higher concentration in blood after 24 h compared to PrlRA itself.

  • 37.
    Zandian, Arash
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Array-based Autoantibody Profiling and Epitope Mapping2017Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Antibodies are a class of proteins that are made by the immune system to recognize harmful organisms and molecules. Their exceptional capability of specifically recognizing molecules has been investigated for over a century and information thereof has been utilized for a variety of applications including vaccine and generation of therapeutic antibodies. Occasionally, instead of protecting the host against pathogens, antibodies can recognize constituents of the host and thereby cause an autoimmune reaction that eventually can lead to a disease. Therefore, it is of great interest to understand what the antibodies bind to and their specificities.

     

    The last decades of technical development and availability of protein and peptide microarrays have enabled large-scale profiling of antibodies and precise determination of their specificities through epitope mapping. In this thesis the aim was to use affinity proteomics tools to profile antibodies, determine their specificities, and discover potential associations of autoantigens to disease by analyzing blood-derived samples with microarray-based methods.

     

    In Paper I, 57 serum samples from patients with the suggested autoimmune disease narcolepsy, were analyzed on planar antigen microarrays with 10,846 human protein fragments. Verification on an independent sample collection consisting of serum samples from 176 individuals, revealed METTL22 and NT5C1A as two potential autoantigens. In Paper II, antibodies from 53 plasma samples from patients with first-episode psychosis, a condition suggested to have a partial autoimmune component, were analyzed on planar antigen microarrays with 2,304 human protein fragments. After a follow-up study of the patients, antibodies toward an antigen representing the three proteins, PAGE2, PAGE2B, PAGE5, was found associated to an increased risk of developing schizophrenia. In Paper III, serum and plasma samples from patients with the autoimmune diseases multiple sclerosis and narcolepsy, were epitope mapped on high-density peptide microarrays with approximately 2.2 million peptides. Technical and biological verification, by using other microarray technology and analyzing  samples from 448 patients, revealed one peptide for multiple sclerosis and narcolepsy, representing the proteins MAP3K7 and NRXN1, with higher antibody reactivity towards in each group, respectively. In Paper IV, purified polyclonal antibodies raised against a surface antigen found on malaria-infected erythrocytes, were profiled on the peptide microarrays representing all proteins found on malaria-infected erythrocytes derived from Plasmodium falciparum. Then, different Plasmodium falciparum strains were analyzed by immunofluorescence microscopy and western blots, using the epitope mapped antibodies. The performance of the immunoassays were compared to the identified epitopes, and validated by RNA sequencing.

     

    In conclusion, these investigations describe multiplex methods to identify and characterize antibodies, their disease association and epitopes. Follow-up studies are needed to determine their potential use and clinical value.

1 - 37 of 37
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