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  • 101.
    Rykaczewska, Urszula
    et al.
    Karolinska Inst, Dept Mol Med & Surg, SE-17176 Stockholm, Sweden.;Karolinska Univ Hosp Solna, Stockholm, Sweden..
    Suur, Bianca E.
    Karolinska Inst, Dept Mol Med & Surg, SE-17176 Stockholm, Sweden.;Karolinska Univ Hosp Solna, Stockholm, Sweden..
    Rohl, Samuel
    Karolinska Inst, Dept Mol Med & Surg, SE-17176 Stockholm, Sweden.;Karolinska Univ Hosp Solna, Stockholm, Sweden..
    Razuvaev, Anton
    Karolinska Inst, Dept Mol Med & Surg, SE-17176 Stockholm, Sweden.;Karolinska Univ Hosp Solna, Stockholm, Sweden..
    Lengquist, Mariette
    Karolinska Inst, Dept Mol Med & Surg, SE-17176 Stockholm, Sweden.;Karolinska Univ Hosp Solna, Stockholm, Sweden..
    Sabater-Lleal, Maria
    Karolinska Univ Hosp Solna, Stockholm, Sweden.;Karolinska Inst, Dept Med Solna, Stockholm, Sweden.;Inst Recerca Hosp St Pau IIB St Pau, Unit Genom Complex Dis, Barcelona, Spain..
    van der Laan, Sander W.
    Univ Utrecht, Univ Med Ctr Utrecht, Cent Diagnost Lab, Pharm & Biomed Genet, Utrecht, Netherlands..
    Miller, Clint L.
    Univ Virginia, Dept Publ Hlth Sci, Ctr Publ Hlth Genom, Charlottesville, VA USA.;Stanford Univ, Sch Med, Div Cardiovasc Med, Stanford, CA 94305 USA..
    Wirka, Robert C.
    Stanford Univ, Sch Med, Div Cardiovasc Med, Stanford, CA 94305 USA..
    Kronqvist, Malin
    Karolinska Inst, Dept Mol Med & Surg, SE-17176 Stockholm, Sweden.;Karolinska Univ Hosp Solna, Stockholm, Sweden..
    Diez, Maria Gonzalez
    Karolinska Univ Hosp Solna, Stockholm, Sweden.;Karolinska Inst, Dept Med Solna, Stockholm, Sweden..
    Vesterlund, Mattias
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Stockholm, Sweden..
    Gillgren, Peter
    Karolinska Inst, Dept Clin Sci & Educ, Sodersjukhuset, Stockholm, Sweden.;Soder Sjukhuset, Dept Vasc Surg, Stockholm, Sweden..
    Odeberg, Jacob
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik. Karolinska Univ Hosp Solna, Stockholm, Sweden.;Karolinska Inst, Dept Med Solna, Stockholm, Sweden.;KTH, Sch Chem Biotechnol & Hlth CBH, Dept Prote, Sci Life Lab, Stockholm, Sweden..
    Lindeman, Jan H.
    Leiden Univ, Med Ctr, Dept Vasc Surg, Leiden, Netherlands..
    Veglia, Fabrizio
    IRCCS, Ctr Cardiol Monzino, Milan, Italy..
    Humphries, Steve E.
    UCL, Inst Cardiovasc Sci, Dept Med, Cardiovasc Genet, Rayne Bldg, London, England. Karolinska Inst, Inst Environm Med, Div Cardiovasc Epidemiol, Stockholm, Sweden..
    de Faire, Ulf
    Baldassarre, Damiano
    IRCCS, Ctr Cardiol Monzino, Milan, Italy.;Univ Milan, Dept Med Biotechnol & Translat Med, Milan, Italy..
    Tremoli, Elena
    IRCCS, Ctr Cardiol Monzino, Milan, Italy..
    Lehtio, Janne
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Stockholm, Sweden..
    Hansson, Goran K.
    Karolinska Univ Hosp Solna, Stockholm, Sweden.;Karolinska Inst, Dept Med Solna, Stockholm, Sweden..
    Paulsson-Berne, Gabrielle
    Karolinska Univ Hosp Solna, Stockholm, Sweden.;Karolinska Inst, Dept Med Solna, Stockholm, Sweden..
    Pasterkamp, Gerard
    Univ Med Ctr Utrecht, Div Heart & Lungs, Lab Expt Cardiol, Utrecht, Netherlands..
    Quertermous, Thomas
    Stanford Univ, Sch Med, Div Cardiovasc Med, Stanford, CA 94305 USA..
    Hamsten, Anders
    Karolinska Univ Hosp Solna, Stockholm, Sweden.;Karolinska Inst, Dept Med Solna, Stockholm, Sweden..
    Eriksson, Per
    Karolinska Univ Hosp Solna, Stockholm, Sweden.;Karolinska Inst, Dept Med Solna, Stockholm, Sweden..
    Hedin, Ulf
    Karolinska Inst, Dept Mol Med & Surg, SE-17176 Stockholm, Sweden.;Karolinska Univ Hosp Solna, Stockholm, Sweden..
    Matic, Ljubica
    Karolinska Inst, Dept Mol Med & Surg, SE-17176 Stockholm, Sweden.;Karolinska Univ Hosp Solna, Stockholm, Sweden..
    PCSK6 Is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Remodeling2020Inngår i: Circulation Research, ISSN 0009-7330, E-ISSN 1524-4571, Vol. 126, nr 5, s. 571-585Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Rationale: PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in human atherosclerotic plaques associated with smooth muscle cells (SMCs), inflammation, extracellular matrix remodeling, and mitogens. Objective: Here, we applied a systems biology approach to gain deeper insights into the PCSK6 role in normal and diseased vessel wall. Methods and Results: Genetic analyses revealed association of intronic PCSK6 variant rs1531817 with maximum internal carotid intima-media thickness progression in high-cardiovascular risk subjects. This variant was linked with PCSK6 mRNA expression in healthy aortas and plaques but also with overall plaque SMA+ cell content and pericyte fraction. Increased PCSK6 expression was found in several independent human cohorts comparing atherosclerotic lesions versus healthy arteries, using transcriptomic and proteomic datasets. By immunohistochemistry, PCSK6 was localized to fibrous cap SMA+ cells and neovessels in plaques. In human, rat, and mouse intimal hyperplasia, PCSK6 was expressed by proliferating SMA+ cells and upregulated after 5 days in rat carotid balloon injury model, with positive correlation to PDGFB (platelet-derived growth factor subunit B) and MMP (matrix metalloprotease) 2/MMP14. Here, PCSK6 was shown to colocalize and cointeract with MMP2/MMP14 by in situ proximity ligation assay. Microarrays of carotid arteries from Pcsk6(-/-) versus control mice revealed suppression of contractile SMC markers, extracellular matrix remodeling enzymes, and cytokines/receptors. Pcsk6(-/-) mice showed reduced intimal hyperplasia response upon carotid ligation in vivo, accompanied by decreased MMP14 activation and impaired SMC outgrowth from aortic rings ex vivo. PCSK6 silencing in human SMCs in vitro leads to downregulation of contractile markers and increase in MMP2 expression. Conversely, PCSK6 overexpression increased PDGFBB (platelet-derived growth factor BB)-induced cell proliferation and particularly migration. Conclusions: PCSK6 is a novel protease that induces SMC migration in response to PDGFB, mechanistically via modulation of contractile markers and MMP14 activation. This study establishes PCSK6 as a key regulator of SMC function in vascular remodeling.

  • 102. Samalova, Marketa
    et al.
    Melida, Hugo
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Vilaplana, Francisco
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bulone, Vincent
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Soanes, Darren M.
    Talbot, Nicholas J.
    Gurr, Sarah J.
    The beta-1,3-glucanosyltransferases (Gels) affect the structure of the rice blast fungal cell wall during appressorium-mediated plant infection2017Inngår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 19, nr 3, artikkel-id UNSP e12659Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative beta-1,3-glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72(+), which carry a putative carbohydrate-binding module, and the GH72(-)Gels, without this motif. We reveal that M. oryzae GH72(+) GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that Delta gel1 Delta gel3 Delta gel4 null mutant has a modified cell wall in which 1,3-glucans have a higher degree of polymerization and are less branched than the wild-type strain. The mutant showed significant differences in global patterns of gene expression, a hyper-branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium-mediated plant infection.

  • 103.
    Scheffel, Julia
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Kanje, Sara
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Borin, Jesper
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification2019Inngår i: mAbs, ISSN 1942-0862, E-ISSN 1942-0870Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    As reported here, we developed and optimized a purification matrix based on a Protein A-derived domain, Z(Ca), displaying calcium-dependent antibody binding. It provides an alternative to the acidic elution conditions of conventional Protein A affinity chromatography for purification of sensitive antibodies and other Fc-based molecules. We describe the multimerization of Z(Ca) to generate a chromatography resin with higher binding capacity. The highest order multimeric variant, Z(Ca)TetraCys, demonstrated a considerably high dynamic binding capacity (35 mg IgG/ml resin) while preserving the specificity for IgG. High recovery was obtained and host cell protein and DNA content in purified fractions proved to be comparable to commercial MabSelect SuRe and MabSelect PrismA. Various elution conditions for use of this domain in antibody purification were investigated. The purification data presented here revealed variations in the interaction of different subclasses of human IgG with Z(Ca)TetraCys. This resulted in diverse elution properties for the different IgGs, where complete elution of all captured antibody for IgG2 and IgG4 was possible at neutral pH. This optimized protein ligand and the proposed purification method offer a unique strategy for effective and mild purification of antibodies and Fc-fusion proteins that cannot be purified under conventional acidic elution conditions due to aggregation formation or loss of function.

  • 104.
    Schimpf, Ulrike
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Nachmann, Gilai
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Trombotto, Stehane
    Univ Lyon, Univ Claude Bernard Lyon 1, CNRS UMR 5223, IMP, F-69622 Villeurbanne, France..
    Houska, Petr
    Karolinska Univ Hosp, Transmed, ANOVA Androl, Sexual Med, Norra Stationsgatan 69, S-11364 Stockholm, Sweden.;Karolinska Inst, Norra Stationsgatan 69, S-11364 Stockholm, Sweden..
    Yan, Hongji
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Bjorndahl, Lars
    Karolinska Univ Hosp, Transmed, ANOVA Androl, Sexual Med, Norra Stationsgatan 69, S-11364 Stockholm, Sweden.;Karolinska Inst, Norra Stationsgatan 69, S-11364 Stockholm, Sweden..
    Crouzier, Thomas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Assessment of Oligo-Chitosan Biocompatibility toward Human Spermatozoa2019Inngår i: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 11, nr 50, s. 46572-46584Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The many interesting properties of chitosan polysaccharides have prompted their extensive use as biomaterial building blocks, for instance as antimicrobial coatings, tissue engineering scaffolds, and drug delivery vehicles. The translation of these chitosan-based systems to the clinic still requires a deeper understanding of their safety profiles. For instance, the widespread claim that chitosans are spermicidal is supported by little to no data. Herein, we thoroughly investigate whether chitosan oligomer (CO) molecules can impact the functional and structural features of human spermatozoa. By using a large number of primary sperm cell samples and by isolating the effect of chitosan from the effect of sperm dissolution buffer, we provide the first realistic and complete picture of the effect of chitosans on sperms. We found that CO binds to cell surfaces or/and is internalized by cells and affected the average path velocity of the spermatozoa, in a dose-dependent manner. However, CO did not affect the progressive motility, motility, or sperm morphology, nor did it cause loss of plasma membrane integrity, reactive oxygen species production, or DNA damage. A decrease in spermatozoa adenosine triphosphate levels, which was especially significant at higher CO concentrations, points to possible interference of CO with mitochondrial functions or the glycolysis processes. With this first complete and in-depth look at the spermicidal activities of chitosans, we complement the complex picture of the safety profile of chitosans and inform on further use of chitosans in biomedical applications.

  • 105.
    Shalaly, Nancy Dekki
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Aneiros, Eduardo
    Blank, Michael
    Mueller, Johan
    Nyman, Eva
    Blind, Michael
    Dabrowski, Michael A.
    Andersson, Christin V.
    Sandberg, Kristian
    Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers2015Inngår i: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 20, nr 9, s. 1112-1123Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl- channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyR1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyR1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyR1 and/or GlyR1. This aptamer potentiated whole-cell Cl- currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators.

  • 106.
    Sjöberg, Ronald
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    On Generation and Applications of High-Density Protein Microarrays2015Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Affinity proteomics has experienced rapid development over the last two decades and one of the most promising platforms to emerge are the protein microarrays. The combination of affinity reagents and miniaturisation enables assays for simultaneous high throughput and sensitive protein analysis. Due to the combination of these desrable properties, a multitude of protein array platforms for rapid and efficient study of proteomes and protein interactions are in use today. Although the protein microarray field has more than two decades of history to look back on the development of new protein microarray platforms continues to this day and beyond.

    In the paper I in this thesis, a microarray of eluates from dried blood spot samples collected from neonates were designed and utilised for detection of complement factor 3 (C3) deficiency. The data acquired from the microarrays platform were compared to C3 levels obtained through enzyme-linked immunosorbant assay (ELISA), and the microarray assay were found to separate the C3 deficient samples from the controls. The conclusion of this investigation was that the microarray platform would be suitable for high-throughput screening of C3 deficiency in neonates. Paper II outlines the work in developing a multiplex platform for validation of affinity reagents. A set of 398 affinity binders, originating from five research groups, were profiled against 432 antigens and representing both polyclonal rabbit antibodies, monoclonal mouse antibodies, and recombinant single-chain variable fragments. Approximately 50% of the binders were found to preferably recognise their intended target while 10% of the binders did not generate any, or low, signals with their respective targets. For paper III, a reverse phase array (RPPA) platform using fluorescence-based detection of IgA deficiency in over 2.000 samples where validated on a label-free detection system and ELISA. The data from the label-free platform and the RPPA were found agree well with each other while data from ELISA did with neither of them. It was found that the label-free platform proved to be well-suited for detection of IgA in serum. Paper IV describes one of the world’s largest protein microarrays containing 21.120 recombinant protein fragments. We describe some of the possible applications of these large-scale arrays, such as binding profiles for the validation of antibodies with 11.520 and 21.120 recombinant proteins, as well as screening for autoimmunity in human serum samples.

    Fulltekst (pdf)
    Thesis
  • 107.
    Sjöstedt, Evelina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Towards a deeper understanding of the human brain2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Identifying the proteome variation in different parts of the body provides fundamental molecular details, enabling further findings and mapping of tissue specific proteins. By combining quantitative transcriptomics with qualitative antibody based proteomics, the Human Protein Atlas (HPA) project aims to protein profile each human protein-coding gene. Genes with varying expression levels in the different tissue types are categorized as tissue elevated in one tissue compared to others, thus connecting genes to potential tissue specific functions. This thesis focuses on the most complex organ in the human body, the brain. With its billions of neurons specifically organized and interconnected, the ability of not only controlling the body but also responsible for higher cognitive functions, the brain is still not fully understood.

    In my search for brain important proteins, genes were classified at different stages based on expression levels. In Paper I and II the transcriptome of cerebral cortex was compared with peripheral organs to classify genes with elevated expression in the brain. Brain expression information was expanded by including external data (GTEx and FANTOM5) into the analysis, in Paper III. Thereafter, in Paper IV, the three datasets (HPA, GTEx and FANTOM5) were aligned and combined, enabling a consensus classification with an improved representation of the brain complexity. The most recent classification provided whole body gene expression profiles and out of the 19,670 protein-coding genes, 2,501 were expressed at elevated levels in the brain compared to the other tissue types. Twelve individual regions represented the brain as an organ, and were further analyzed and compared for regional classification of gene expression. One thousand genes showed regional variation in expression level, thus classified as regionally elevated within the brain. Interestingly, less than 500 of the genes classified as brain elevated on the whole body level, and were also regionally elevated in the brain. Many genes with regionally variable expression within the brain showed higher expression in a peripheral organ than in the brain when comparing whole body expression. Based on elevated expression in the brain or brain regions, more than 3,000 genes were suggested to be of high importance to the brain.

    In addition, this high-throughput approach to combine transcriptomics and protein profiles in tissues and cells further generated new knowledge in several different other aspects: better understanding of uncharacterized and “missing proteins” (Paper III), validation of an antibody improving classification of pituitary adenoma (Paper V) and in Paper VI the possibility to explore cancer specific expression in relation to clinical data and normal tissue expression.

    There are multiple diseases of the brain that are poorly understood on both a cellular and molecular level. While my work mainly focused on identifying and understanding the molecular organization of the normal brain, the ultimate goal of mapping and studying the normal expression baseline is to understand the molecular aspects of disease and identify ways to prevent, treat and cure diseases.

    Fulltekst (pdf)
    ES avhandling
  • 108.
    Sjöstedt, Evelina
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Sivertsson, Åsa
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Norradin, Feria Hikmet
    Department of Immunology, Genetics and Pathology, Uppsala University.
    Katona, Borbala
    Department of Immunology, Genetics and Pathology, Uppsala University.
    Näsström, Åsa
    Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Sweden.
    Vuu, Jimmy
    Department of Immunology, Genetics and Pathology, Uppsala university.
    Kesti, Dennis
    Department of Immunology, Genetics and Pathology, Uppsala University.
    Oksvold, Per
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Edqvist, Per-Henrik
    Department of Immunology, Genetics and Pathology, Uppsala University.
    Olsson, Ingmarie
    Department of Immunology, Genetics and Pathology, Uppsala University.
    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, Uppsala University.
    Integration of Transcriptomics and Antibody-Based Proteomics for Exploration of Proteins Expressed in Specialized Tissues2018Inngår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 17, nr 12, s. 4127-4137Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A large portion of human proteins are referred to as missing proteins, defined as protein-coding genes that lack experimental data on the protein level due to factors such as temporal expression, expression in tissues that are difficult to sample, or they actually do not encode functional proteins. In the present investigation, an integrated omics approach was used for identification and exploration of missing proteins. Transcriptomics data from three different sourcesthe Human Protein Atlas (HPA), the GTEx consortium, and the FANTOM5 consortiumwere used as a starting point to identify genes selectively expressed in specialized tissues. Complementing the analysis with profiling on more specific tissues based on immunohistochemistry allowed for further exploration of cell-type-specific expression patterns. More detailed tissue profiling was performed for >300 genes on complementing tissues. The analysis identified tissue-specific expression of nine proteins previously listed as missing proteins (POU4F1, FRMD1, ARHGEF33, GABRG1, KRTAP2-1, BHLHE22, SPRR4, AVPR1B, and DCLK3), as well as numerous proteins with evidence of existence on the protein level that previously lacked information on spatial resolution and cell-type- specific expression pattern. We here present a comprehensive strategy for identification of missing proteins by combining transcriptomics with antibody-based proteomics. The analyzed proteins provide interesting targets for organ-specific research in health and disease.

  • 109. Stenler, S.
    et al.
    Wiklander, O. P. B.
    Badal-Tejedor, M.
    Turunen, J.
    Nordin, J. Z.
    Hallengärd, D.
    Wahren, B.
    Andaloussi, S. E. L.
    Rutland, Mark W.
    KTH, Skolan för kemivetenskap (CHE), Kemi, Yt- och korrosionsvetenskap.
    Smith, C. I. E.
    Lundin, K. E.
    Blomberg, P.
    Micro-minicircle gene therapy: Implications of size on fermentation, complexation, shearing resistance, and expression2014Inngår i: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, Vol. 3, s. e140-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, supercoiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.

  • 110. Su, Yu-Ching
    et al.
    Hallström, Björn M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Bernhard, Sara
    Singh, Birendra
    Riesbeck, Kristian
    Impact of sequence diversity in the Moraxella catarrhalis UspA2/UspA2H head domain on vitronectin binding and antigenic variation2013Inngår i: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 15, nr 5, s. 375-387Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The nasopharyngeal pathogen Moraxella catarrhalis recruits vitronectin to subvert complement-mediated killing. Ubiquitous surface protein (UspA) 2 and its hybrid form UspA2H bind vitronectin at the highly diverse N-terminal head domain. Here we characterized the sequence diversity of the head domain in multiple M. catarrhalis clinical isolates (n = 51) with focus on binding of vitronectin. The head domain of the uspA2 genes from 40 isolates were clustered according to an N-terminal sequence motif of UspA2 (NTER2), i.e., NTER2A (55% of uspA2 variants), NTER2B (32.5%), NTER2C (5%), and finally a group without an NTER2 (7.5%). Isolates harbouring the uspA2H gene (n = 11) contained N-terminal GGG repeats. Vitronectin binding to isolates having UspA2 did not correlate to variation in the NTER2 motifs but occurred in UspA2H containing 6 or >= 11 of GGG repeats. Analyses of recombinant UspA2/UspA2H head domains of multiple variants showed UspA2-dependent binding to the C-terminal of vitronectin. Furthermore, polyclonal anti-UspA2 antibodies revealed that the head domain of the majority of Moraxella UspA2/2H was antigenically unrelated, whereas the full length molecules were recognized. In conclusion, the head domains of UspA2/2H have extensive sequence polymorphism without losing vitronectin-binding capacity promoting a general evasion of the host immune system.

  • 111. Summer, D.
    et al.
    Garousi, J.
    Oroujeni, M.
    Mitran, B.
    Andersson, Ken G.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Vorobyeva, A.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Orlova, A.
    Tolmachev, V.
    Decristoforo, C.
    Cyclic versus Noncyclic Chelating Scaffold for 89Zr-Labeled ZEGFR:2377 Affibody Bioconjugates Targeting Epidermal Growth Factor Receptor Overexpression2018Inngår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, nr 1, s. 175-185Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Zirconium-89 is an emerging radionuclide for positron emission tomography (PET) especially for biomolecules with slow pharmacokinetics as due to its longer half-life, in comparison to fluorine-18 and gallium-68, imaging at late time points is feasible. Desferrioxamine B (DFO), a linear bifunctional chelator (BFC) is mostly used for this radionuclide so far but shows limitations regarding stability. Our group recently reported on fusarinine C (FSC) with similar zirconium-89 complexing properties but potentially higher stability related to its cyclic structure. This study was designed to compare FSC and DFO head-to-head as bifunctional chelators for 89Zr-radiolabeled EGFR-targeting ZEGFR:2377 affibody bioconjugates. FSC-ZEGFR:2377 and DFO-ZEGFR:2377 were evaluated regarding radiolabeling, in vitro stability, specificity, cell uptake, receptor affinity, biodistribution, and microPET-CT imaging. Both conjugates were efficiently labeled with zirconium-89 at room temperature but radiochemical yields increased substantially at elevated temperature, 85 °C. Both 89Zr-FSC-ZEGFR:2377 and 89Zr-DFO-ZEGFR:2377 revealed remarkable specificity, affinity and slow cell-line dependent internalization. Radiolabeling at 85 °C showed comparable results in A431 tumor xenografted mice with minor differences regarding blood clearance, tumor and liver uptake. In comparison 89Zr-DFO-ZEGFR:2377, radiolabeled at room temperature, showed a significant difference regarding tumor-to-organ ratios. MicroPET-CT imaging studies of 89Zr-FSC-ZEGFR:2377 as well as 89Zr-DFO-ZEGFR:2377 confirmed these findings. In summary we were able to show that FSC is a suitable alternative to DFO for radiolabeling of biomolecules with zirconium-89. Furthermore, our findings indicate that 89Zr-radiolabeling of DFO conjugates at higher temperature reduces off-chelate binding leading to significantly improved tumor-to-organ ratios and therefore enhancing image contrast.

  • 112.
    Svedendahl, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Branneby, Cecilia
    Lindberg, Lina
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Reversed enantiopreference of an ω-transaminase by a single-point mutationManuskript (preprint) (Annet vitenskapelig)
  • 113. Sánchez, J. L. A.
    et al.
    Henry, O. Y. F.
    Joda, H.
    Solnestam, Beata Werne
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kvastad, Linda
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johansson, Erik
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lladach, N.
    Ramakrishnan, D.
    Riley, I.
    O'Sullivan, C. K.
    Multiplex PCB-based electrochemical detection of cancer biomarkers using MLPA-barcode approach2016Inngår i: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 82, s. 224-232Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Asymmetric multiplex ligation-dependent probe amplification (MLPA) was developed for the amplification of seven breast cancer related mRNA markers and the MLPA products were electrochemically detected via hybridization. Seven breast cancer genetic markers were amplified by means of the MLPA reaction, which allows for multiplex amplification of multiple targets with a single primer pair. Novel synthetic MLPA probes were designed to include a unique barcode sequence in each amplified gene. Capture probes complementary to each of the barcode sequences were immobilized on each electrode of a low-cost electrode microarray manufactured on standard printed circuit board (PCB) substrates. The functionalised electrodes were exposed to the single-stranded MLPA products and following hybridization, a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the amplified strand completed the genocomplex, which was electrochemically detected following substrate addition. The electrode arrays fabricated using PCB technology exhibited an excellent electrochemical performance, equivalent to planar photolithographically-fabricated gold electrodes, but at a vastly reduced cost (>50 times lower per array). The optimised system was demonstrated to be highly specific with negligible cross-reactivity allowing the simultaneous detection of the seven mRNA markers, with limits of detections as low as 25 pM. This approach provides a novel strategy for the genetic profiling of tumour cells via integrated "amplification-to-detection".

  • 114.
    Takwa, Mohamad
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Lipase Specificity and Selectivity: Engineering, Kinetics and Applied Catalysis2010Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The specificity and selectivity of the enzyme Candida antarctica lipase B (CALB) were studiedfor several substrates and applications.With help of molecular modeling, the active site of CALB was redesigned for the ring openingpolymerization of D,D‐lactide. Two mutants, with about 90‐fold increase in activity ascompared to the wild‐type enzyme, were created. Changing a glutamine into alanineaccounted for this increase in both mutants by creating a larger space in the acyl donorpocket. The new space made it possible to accommodate the bulky substrate and improvethe transition state‐active site complementarity during polymer chain propagation.The enantioselectivity of CALB towards secondary alcohols was engineered by rationalredesign of the stereoselectivity pocket in the enzyme active site. A larger space created by asingle point mutation resulted in an 8’300’000 times change in enantioselectivity towards 1‐phenylethanol and the enantiopreference was inverted into S‐preference. The activitytowards the S‐enantiomer increased 64’000 times in the mutant as related to the wild‐type.The solvent and temperature effects on the enantioselectivity were studied for severalsubstrates and revealed the importance of entropy in the change in enantioselectivity.Substrate selectivity is of great importance for the outcome of enzyme catalyzed polymersynthesis. Ring opening polymerization (ROP) of γ‐acyloxy‐ε‐caprolactones will result in apolyester chain with pendant functional groups. CALB was found to have activity not onlytowards the lactone but also towards the γ‐ester leading to rearrangement of the monomersyielding γ‐acetyloxyethyl‐γ‐butyrolactone. This selectivity between the lactone and the γ‐ester was dependent on the type of group in the γ position and determined the ratio ofpolymerization and rearrangement of the monomers. Molecular dynamics simulations wereused to gain molecular understanding of the selectivity between the lactone and γ‐ester.In order to obtain (meth)acrylate functional polyesters we investigated the use of 2‐hydroxyethyl (meth)acrylate (HEA and HEMA) as initiators for ring opening polymerization.We found that, in addition to the ring opening polymerization activity, CALB catalyzed thetransacylation of the acid moiety of the initiators. The selectivity of CALB towards thedifferent acyl donors in the reaction resulted in a mixture of polymers with different endgroups. A kinetic investigation of the reaction showed the product distribution with timewhen using HEA or HEMA with ε‐caprolactone or ω‐pentadecalactone.The high selectivity of CALB towards lactones over (meth)acrylate esters such as ethyleneglycol di(meth)acrylate was used to design a single‐step route for the synthesis ofdi(meth)acrylated polymers. By mixing ω‐pentadecalactone with the ethylene glycoldi(meth)acrylate and the enzyme in solvent free conditions, we obtained >95 % ofdi(meth)acrylated polypentadecalactone.Taking advantage of the high chemoselectivity of CALB, it was possible to synthesizepolyesters with thiol and/or acrylate functional ends. When using a thioalcohol as initiatorCALB showed high selectivity towards the alcohol group over the thiol group as acyl acceptorfor the ROP reaction. The enzymatic ability of catalyzing simultaneous reactions (ROP andtransacylation) it was possible to develop a single‐step route for the synthesis ofdifunctionalized polyesters with two thiol ends or one thiol and one acrylate end by mixingthe initiator, lactone and a terminator.

    Fulltekst (pdf)
    FULLTEXT01
  • 115.
    Tan, Tien-Chye
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Haltrich, Dietmar
    Divne, Christina
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Regioselective Control of beta-D-Glucose Oxidation by Pyranose 2-Oxidase Is Intimately Coupled to Conformational Degeneracy2011Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 409, nr 4, s. 588-600Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Trametes multicolor pyranose 2-oxidase (P2O) is a flavoprotein oxidase that oxidizes D-glucose at C2 to 2-keto-D-glucose by a highly regioselective mechanism. In this work, fluorinated sugar substrates were used as mechanistic probes to investigate the basis of regioselectivity in P2O. Although frequently used to study the mechanisms of glycoside hydrolases, our work provides the first example of applying these probes to sugar oxidoreductases. Our previous structure of the P2O mutant H167A in complex with the slow substrate 2-deoxy-2-fluoro-D-glucose showed a substrate-binding mode compatible with oxidation at C3. To accommodate the sugar, a gating segment, (FSY456)-F-454, in the substrate recognition loop partly unfolded to create a spacious and more polar active site that is distinct from the closed state of P2O. The crystal structure presented here shows that the preferred C2 oxidation where an ordered complex of P2O H167A with 3-deoxy-3-fluoro-D-glucose at 1.35 angstrom resolution was successfully trapped. In this semi-open C2-oxidation complex, the substrate recognition loop tightens to form an optimized substrate complex stabilized by interactions between Asp452 and glucose O4, as well as Tyr456 and the glucose O6 group, interactions that are not possible when glucose is positioned for oxidation at C3. The different conformations of the (FSY456)-F-454 gating segment in the semiopen and closed states induce backbone and side-chain movements of Thr169 and Asp452 that add further differential stabilization to the individual states. We expect the semi-open state (C2-oxidation state) and closed state to be good approximations of the active-site structure during the reductive half-reaction (sugar oxidation) and oxidative half-reaction (O-2 reduction).

  • 116.
    Thomas, Cecilia Engel
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Häussler, Ragna S.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hong, Mun-Gwan
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics.
    Raverdy, V.
    Ctr Hosp Reg Univ Lille 2, Lille, France..
    Dale, Matilda
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Vinuela, A.
    Univ Geneva, Dept Genet Med & Dev, Sch Med, Geneva, Switzerland..
    Canouil, M.
    Univ Lille, CNRS, Inst Pasteur Lille, Lille, France..
    Dermitzakis, E. T.
    Univ Geneva, Dept Genet Med & Dev, Sch Med, Geneva, Switzerland..
    Froguel, P.
    Univ Lille, CNRS, Inst Pasteur Lille, Lille, France..
    Brunak, S.
    Tech Univ Denmark, Dept Bio & Hlth Informat, Lyngby, Denmark..
    Pattou, F.
    Ctr Hosp Reg Univ Lille 2, Lille, France..
    Schwenk, Jochen M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinity Proteomics.
    Individual effects of gastric bypass surgery on longitudinal blood protein profiles: an IMI DIRECT study2019Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 62, s. S271-S271Artikkel i tidsskrift (Annet vitenskapelig)
  • 117.
    Thrane, Kim
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Eriksson, Hanna
    Karolinska Inst, Dept Oncol Pathol, SE-17176 Stockholm, Sweden.;Karolinska Univ Hosp, Dept Oncol, SE-17176 Stockholm, Sweden..
    Maaskola, Jonas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hansson, Johan
    Karolinska Inst, Dept Oncol Pathol, SE-17176 Stockholm, Sweden.;Karolinska Univ Hosp, Dept Oncol, SE-17176 Stockholm, Sweden..
    Lundeberg, Joakim
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Spatially Resolved Transcriptomics Enables Dissection of Genetic Heterogeneity in Stage III Cutaneous Malignant Melanoma2018Inngår i: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 78, nr 20, s. 5970-5979Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cutaneous malignant melanoma (melanoma) is characterized by a high mutational load, extensive intertumoral and intratumoral genetic heterogeneity, and complex tumor microenvironment (TME) interactions. Further insights into the mechanisms underlying melanoma are crucial for understanding tumor progression and responses to treatment. Here we adapted the technology of spatial transcriptomics (ST) to melanoma lymph node biopsies and successfully sequenced the transcriptomes of over 2,200 tissue domains. Deconvolution combined with traditional approaches for dimensional reduction of transcriptome-wide data enabled us to both visualize the transcriptional landscape within the tissue and identify gene expression profiles linked to specific histologic entities. Our unsupervised analysis revealed a complex spatial intratumoral composition of melanoma metastases that was not evident through morphologic annotation. Each biopsy showed distinct gene expression profiles and included examples of the coexistence of multiple melanoma signatures within a single tumor region as well as shared profiles for lymphoid tissue characterized according to their spatial location and gene expression profiles. The lymphoid area in close proximity to the tumor region displayed a specific expression pattern, which may reflect the TME, a key component to fully understanding tumor progression. In conclusion, using the ST technology to generate gene expression profiles reveals a detailed landscape of melanoma metastases. This should inspire researchers to integrate spatial information into analyses aiming to identify the factors underlying tumor progression and therapy outcome. Significance: Applying ST technology to gene expression profiling in melanoma lymph node metastases reveals a complex transcriptional landscape in a spatial context, which is essential for understanding the multiple components of tumor progression and therapy outcome. (C) 2018 AACR.

  • 118. Varemo, Leif
    et al.
    Henriksen, Tora Ida
    Scheele, Camilla
    Broholm, Christa
    Pedersen, Maria
    Uhlen, Mathias
    Pedersen, Bente Klarlund
    Nielsen, Jens
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Type 2 diabetes and obesity induce similar transcriptional reprogramming in human myocytes2017Inngår i: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 9, artikkel-id 47Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Skeletal muscle is one of the primary tissues involved in the development of type 2 diabetes (T2D). The close association between obesity and T2D makes it difficult to isolate specific effects attributed to the disease alone. Therefore, here we set out to identify and characterize intrinsic properties of myocytes, associated independently with T2D or obesity. Methods: We generated and analyzed RNA-seq data from primary differentiated myotubes from 24 human subjects, using a factorial design (healthy/T2D and non-obese/obese), to determine the influence of each specific factor on genome-wide transcription. This setup enabled us to identify intrinsic properties, originating from muscle precursor cells and retained in the corresponding myocytes. Bioinformatic and statistical methods, including differential expression analysis, gene-set analysis, and metabolic network analysis, were used to characterize the different myocytes. Results: We found that the transcriptional program associated with obesity alone was strikingly similar to that induced specifically by T2D. We identified a candidate epigenetic mechanism, H3K27me3 histone methylation, mediating these transcriptional signatures. T2D and obesity were independently associated with dysregulated myogenesis, down-regulated muscle function, and up-regulation of inflammation and extracellular matrix components. Metabolic network analysis identified that in T2D but not obesity a specific metabolite subnetwork involved in sphingolipid metabolism was transcriptionally regulated. Conclusions: Our findings identify inherent characteristics in myocytes, as a memory of the in vivo phenotype, without the influence from a diabetic or obese extracellular environment, highlighting their importance in the development of T2D.

  • 119. Vicedomini, Riccardo
    et al.
    Vezzi, Francesco
    KTH, Skolan för datavetenskap och kommunikation (CSC). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Scalabrin, Simone
    Arvestad, Lars
    KTH, Skolan för datavetenskap och kommunikation (CSC), Beräkningsbiologi, CB. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Policriti, Alberto
    GAM-NGS: genomic assemblies merger for next generation sequencing2013Inngår i: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 14, s. S6-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: In recent years more than 20 assemblers have been proposed to tackle the hard task of assembling NGS data. A common heuristic when assembling a genome is to use several assemblers and then select the best assembly according to some criteria. However, recent results clearly show that some assemblers lead to better statistics than others on specific regions but are outperformed on other regions or on different evaluation measures. To limit these problems we developed GAM-NGS (Genomic Assemblies Merger for Next Generation Sequencing), whose primary goal is to merge two or more assemblies in order to enhance contiguity and correctness of both. GAM-NGS does not rely on global alignment: regions of the two assemblies representing the same genomic locus (called blocks) are identified through reads' alignments and stored in a weighted graph. The merging phase is carried out with the help of this weighted graph that allows an optimal resolution of local problematic regions. Results: GAM-NGS has been tested on six different datasets and compared to other assembly reconciliation tools. The availability of a reference sequence for three of them allowed us to show how GAM-NGS is a tool able to output an improved reliable set of sequences. GAM-NGS is also a very efficient tool able to merge assemblies using substantially less computational resources than comparable tools. In order to achieve such goals, GAM-NGS avoids global alignment between contigs, making its strategy unique among other assembly reconciliation tools. Conclusions: The difficulty to obtain correct and reliable assemblies using a single assembler is forcing the introduction of new algorithms able to enhance de novo assemblies. GAM-NGS is a tool able to merge two or more assemblies in order to improve contiguity and correctness. It can be used on all NGS-based assembly projects and it shows its full potential with multi-library Illumina-based projects. With more than 20 available assemblers it is hard to select the best tool. In this context we propose a tool that improves assemblies (and, as a by-product, perhaps even assemblers) by merging them and selecting the generating that is most likely to be correct.

  • 120.
    von Witting, Emma
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    The ADAPT scaffold as a tool for diagnostic imaging and targeted therapy2020Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Molecular recognition, or the specific interactions between a protein and its ligand, is central to biology and a key factor for many different clinical and technical applications. Despite antibodies being only one of many different affinity proteins, it has by far been the most successful. However, their large size and complex structure can be limiting in terms of cost and stability. Furthermore, their effector functions can sometimes be undesired or even detrimental. Over the past decades, many alternative affinity proteins have emerged to overcome some of these limitations.

    The Albumin Binding Domain (ABD), originally present on the surface of certain bacterial cells, has previously been subjected to combinatorial protein engineering for the generation of ADAPTs (ABD Derived Affinity ProTeins) that bind to different targets. One of these, the ADAPT6, targets HER2 and has shown great promise as a tracer for radionuclide molecular imaging for diagnosis and stratification of HER2 positive patients. The work in this thesis has aimed to optimise the ADAPT6 tracer further and also describes the first-inhuman clinical trial for imaging of HER2-overexpressing breast cancer. The results establish that ADAPT6 is safe and well-tolerated by patients and able to detect primary tumours as well as metastases with very high contrast already 2 hours after injection. However, the high kidney uptake associated with its fast blood clearance prevents further use of ADAPT6 also in a therapeutic setting. By engineering the ADAPT6 to prolong its circulatory half-life and reduce the kidney uptake, this thesis has also aimed to explore the therapeutic potential of this molecule. As a first step towards this goal, the ADAPT6 was genetically fused to an ABD to allow for binding to a patient’s own serum albumin and hence avoid the same extent of renal filtration. Indeed, when evaluated in mice, fusion to ABD increased the retention in circulation by more than 200-fold and exhibited a dramatically decreased renal activity. Treatment of tumour-bearing mice with the ABD-fused ADAPT6 conjugated to a cytotoxic radionuclide significantly prolonged survival by more than two-fold and was not associated with any observable toxicity. Finally, this thesis also describes a novel combinatorial library from which several bispecific ADAPTs have been identified, binding to both albumin and other clinically relevant targets simultaneously. This miniature bispecific scaffold offers an opportunity to combine the benefits associated with small size such as good tissue extravasation and alternative administration routes while still maintaining a sufficient in vivo half-life.

    Fulltekst (pdf)
    Thesis_EmmavonWitting
  • 121. von Witting, Emma
    et al.
    Lindbo, Sarah
    Lundqvist, Magnus
    Möller, Marit
    Wisniewski, Andreas
    Kanje, Sara
    Rockberg, Johan
    Tegel, Hanna
    Åstrand, Mikael
    Uhlén, Mathias
    Hober, Sophia
    ADAPT as a single-domain bispecific scaffold capable of albumin-associated haf-life extension for therapeutic applicationsManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Albumin-binding fusion partners are frequently used as a means for the in vivo half-life extension of small therapeutic molecules that would normally be cleared very rapidly from circulation. However, in applications where small size is key, fusion to an additional molecule can be disadvantageous. ABD-Derived Affinity ProTeins (ADAPTs) are a type of scaffold proteins based on one of the albumin binding domains of streptococcal Protein G, with newly introduced binding specificities against numerous targets. Here we engineered this scaffold further and showed that this domain, as small as 6 kDa, can harbor two distinct binding surfaces and utilize them to interact with two targets simultaneously. These novel ADAPTs were developed to bind to both serum albumin as well as another clinically relevant target, thus circumventing the need for an albumin binding fusion partner. To accomplish this, we designed a novel phage display library and used it to successfully select for single-domain bispecific binders towards a panel of targets: TNFa, PSA (Prostate Specific Antigen), CRP (C-reactive protein), renin, angiogenin and insulin. Apart from successfully identifying bispecific binders for all targets, we also demonstrated the formation of the ternary complex of the ADAPT together with albumin and each of the four targets TNFa, PSA, angiogenin and insulin. This simultaneous binding of albumin and other targets presents an opportunity to combine the advantages of small molecules with those of larger ones allowing for lower cost of goods and non-invasive administration routes while still maintaining a sufficient in vivo half-life.

  • 122.
    Vunk, Helian
    KTH, Skolan för bioteknologi (BIO).
    Nästa generations plasmadiagnostik med immunanriktning och riktad proteomik2016Independent thesis Advanced level (degree of Master (Two Years)), 20 poäng / 30 hpOppgave
    Fulltekst (pdf)
    fulltext
  • 123.
    Wiklund, Martin
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Green, Roy
    Univ Southampton, Hants, England .
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Acoustofluidics 14: Applications of acoustic streaming in microfluidic devices2012Inngår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 12, nr 14, s. 2438-2451Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    In part 14 of the tutorial series "Acoustofluidics - exploiting ultrasonic standing wave forces and acoustic streaming in microfluidic systems for cell and particle manipulation", we provide a qualitative description of acoustic streaming and review its applications in lab-on-a-chip devices. The paper covers boundary layer driven streaming, including Schlichting and Rayleigh streaming, Eckart streaming in the bulk fluid, cavitation microstreaming and surface-acoustic-wave-driven streaming.

  • 124.
    Williams, Cecilia
    et al.
    University of Houston, United States; Karolinska Institutet, Sweden .
    Helguero, Luisa
    Karolinska Institutet, Sweden .
    Edvardsson, Karin
    University of Houston, United States; Karolinska Institutet, Sweden .
    Haldosén, Lars-Arne
    Karolinska Institutet, Sweden .
    Gustafsson, Jan-Ake
    University of Houston, United States; Karolinska Institutet, Sweden .
    Gene expression in murine mammary epithelial stem cell-like cells shows similarities to human breast cancer gene expression.2009Inngår i: Breast cancer research : BCR, ISSN 1465-542X, Vol. 11, nr 3, s. R26-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    INTRODUCTION: Mammary stem cells are bipotential and suggested to be the origin of breast cancer development, but are elusive and vaguely characterized. Breast tumors can be divided into subgroups, each one requiring specific treatment. To determine a possible association between mammary stem cells and breast cancer, a detailed characterization of the transcriptome in mammary stem cells is essential.

    METHODS: We have used a murine mammary epithelial stem-like cell line (HC11) and made a thorough investigation of global gene-expression changes during stepwise differentiation using dual-color comparative microarray technique. Subsequently, we have performed a cross-species comparison to reveal conserved gene expression between stem cells and subtype-specific and prognosis gene signatures, and correlated gene expression to in vivo mammary gland development.

    RESULTS: Our analysis of mammary stem-like and stepwise cell differentiation, and an in-depth description of our findings in a breast cancer perspective provide a unique map of the transcriptomic changes and a number of novel mammary stem cell markers. We correlate the alterations to in vivo mammary gland differentiation, and describe novel changes in nuclear receptor gene expression. Interestingly, our comparisons show that specific subtypes of breast cancers with poor prognosis and metastasizing capabilities show resemblance to stem-like gene expression.

    CONCLUSIONS: The transcriptional characterization of these mammary stem-like cells and their differentiation-induced gene expression patterns is here made widely accessible and provides a basis for research on mammary stem-like cells. Our comparisons suggest that some tumors are more stem-like than others, with a corresponding worse prognosis. This information would, if established, be important for treatment decisions. We also suggest several marker candidates valuable to investigate further.

  • 125.
    Winkler, Thomas
    et al.
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Feil, Michael
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Stronkman, Eva Francisca Gerhardine Johanna
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Matthiesen, Isabelle
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Herland, Anna
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Low-cost microphysiological systems: feasibility study of a tape-based barrier-on-chip for small intestine modeling.2020Inngår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 20, nr 7, s. 1212-1226Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We see affordability as a key challenge in making organs-on-chips accessible to a wider range of users, particularly outside the highest-resource environments. Here, we present an approach to barrier-on-a-chip fabrication based on double-sided pressure-sensitive adhesive tape and off-the-shelf polycarbonate. Besides a low materials cost, common also to PDMS or thermoplastics, it requires minimal (€100) investment in laboratory equipment, yet at the same time is suitable for upscaling to industrial roll-to-roll manufacture. We evaluate our microphysiological system with an epithelial (Caco-2/BBe1) barrier model of the small intestine, studying the biological effects of permeable support pore size, as well as stimulation with a common food compound (chili pepper-derived capsaicinoids). The cells form tight and continuous barrier layers inside our systems, with comparable permeability but superior epithelial polarization compared to Transwell culture, in line with other perfused microphysiological models. Permeable support pore size is shown to weakly impact barrier layer integrity as well as the metabolic cell profile. Capsaicinoid response proves distinct between culture systems, but we show that impacted metabolic pathways are partly conserved, and that cytoskeletal changes align with previous studies. Overall, our tape-based microphysiological system proves to be a robust and reproducible approach to studying physiological barriers, in spite of its low cost.

  • 126. Wulff, Ragna Peterson
    et al.
    Lundqvist, Joakim
    Rutsdottir, Gudrun
    Hansson, Andreas
    Stenbaek, Anne
    Elmlund, Dominika
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Elmlund, Hans
    Jensen, Poul Erik
    Hansson, Mats
    The Activity of Barley NADPH-Dependent Thioredoxin Reductase C Is Independent of the Oligomeric State of the Protein: Tetrameric Structure Determined by Cryo-Electron Microscopy2011Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, nr 18, s. 3713-3723Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Thioredoxin and thioredoxin reductase can regulate cell metabolism through redox regulation of disulfide bridges or through removal of H2O2. These two enzymatic functions are combined in NADPH-dependent thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase domain fused with a C-terminal thioredoxin domain. Rice NTRC exists in different oligomeric states, depending on the absence or presence of its NADPH cofactor. It has been suggested that the different oligomeric states may have diverse activity. Thus, the redox status of the chloroplast could influence the oligomeric state of NTRC and thereby its activity. We have characterized the oligomeric states of NTRC from barley (Hard rum vulgare L.). This also includes a structural model of the tetrameric NTRC derived from cryo-electron microscopy and single-particle reconstruction. We conclude that the tetrameric NTRC is a dimeric arrangement of two NTRC homodimers. Unlike that of rice NTRC, the quaternary structure of barley NTRC complexes is unaffected by addition of NADPH. The activity of NTRC was tested with two different enzyme assays. The N-terminal part of NTRC was tested in a thioredoxin reductase assay. A peroxide sensitive Mg-protoporphyrin IX monomethyl ester (MPE) cyclase enzyme system of the chlorophyll biosynthetic pathway was used to test the catalytic ability of both the N- and C-terminal parts of NTRC. The different oligomeric assembly states do not exhibit significantly different activities. Thus, it appears that the activities are independent of the oligomeric state of barley NTRC.

  • 127. Zabotina, Olga
    et al.
    Malm, Erik
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Drakakaki, Georgia
    Bulone, Vincent
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Raikhel, Natasha
    Identification and Preliminary Characterization of a New Chemical Affecting Glucosyltransferase Activities Involved in Plant Cell Wall Biosynthesis2008Inngår i: MOLECULAR PLANT, ISSN 1674-2052, Vol. 1, nr 6, s. 977-989Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Chemical genetics as a part of chemical genomics is a powerful and fast developing approach to dissect biological processes that may be difficult to characterize using conventional genetics because of gene redundancy or lethality and, in the case of polysaccharide biosynthesis, plant flexibility. Polysaccharide synthetic enzymes are located in two main compartments-the Golgi apparatus and plasma membrane-and can be studied in vitro using membrane fractions. Here, we first developed a high-throughput assay that allowed the screening of a library of chemicals with a potential effect on glycosyltransferase activities. Out of the 4800 chemicals screened for their effect on Golgi glucosyltransferases, 66 compounds from the primary screen had an effect on carbohydrate biosynthesis. Ten of these compounds were confirmed to inhibit glucose incorporation after a second screen. One compound exhibiting a strong inhibition effect (ID 6240780 named chemical A) was selected and further studied. it reversibly inhibits the transfer of glucose from UDPglucose by Golgi membranes, but activates the plasma membrane-bound callose synthase. The inhibition effect is dependent on the chemical structure of the compound, which does not affect endomembrane morphology of the plant cells, but causes changes in cell wall composition. Chemical A represents a novel drug with a great potential for the study of the mechanisms of Golgi and plasma membrane-bound glucosyltransferases.

  • 128. Zamani, Leila
    et al.
    Lundqvist, Magnus
    Zhang, Ye
    Åberg, Magnus
    Edfors, Fredrik
    Bidkhori, Gholamreza
    Lindahl, Anna
    Mie, Axel
    Mardinoglu, Adil
    Rockberg, Johan
    Chotteau, Veronique
    High cell density perfusion culture has a maintained exoproteome and metabolomeManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Chinese hamster ovary (CHO) cells are the workhorse to produce recombinant proteins in the biopharmaceutical industry using mammalian cells and are commonly cultured in either fed-batch or perfusion mode. The optimization of the complex biological systems used in such processes is extremely challenging. Multi-omics approaches can reveal otherwise unknown characteristics of these systems and identify culture parameters that can be manipulated to optimize the cultivation process. Here we have ap- plied both metabolomic and proteomic profiling to a monoclonal antibody (mAb) production operated in perfusion mode to explore how cell biology and reactor environment change as the cell density reaches ≥ 200 x 106 cells/mL. The extracellular metabolic composition obtained in perfusion mode was also com- pared to fed-batch, which showed a more stable profile for perfusion despite a far larger range of viable cell densities. The proteomics data showed an increase of structural proteins as the cell density increased, and both the proteomic and metabolic results showed signs of oxidative stress and changes in glutathione metabolism at very high cell densities. The methodology presented herein could be a powerful tool for optimizing cultivation processes and recombinant protein production.

  • 129.
    Zandian, Arash
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Häggmark-Månberg, Anna
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ayoglu, Burcu
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy2017Inngår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 16, nr 3, s. 1300-1314Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The underlying molecular mechanisms of autoimmune diseases are poorly understood. To unravel the autoimmune processes across diseases, comprehensive and unbiased analyses of proteins targets recognized by the adaptive immune system are needed. Here we present an approach starting from high-density peptide arrays to characterize autoantibody repertoires and to identify new autoantigens. A set of ten plasma and serum samples from subjects with multiple sclerosis, narcolepsy, and without any disease diagnosis were profiled on a peptide array representing the whole proteome, hosting 2.2 million 12-mer peptides with a six amino acid lateral shift. On the basis of the IgG reactivities found on these whole-proteome peptide micro arrays, a set of 23 samples was then studied on a targeted array with 174 000 12-mer peptides of single amino acid lateral shift. Finally, verification of IgG reactivities was conducted with a larger sample set (n = 448) using the bead-based peptide microarrays. The presented workflow employed three different peptide microarray formats to discover and resolve the epitopes of human autoantibodies and revealed two potentially new autoantigens: MAP3K7 in multiple sclerosis and NRXN1 in narcolepsy. The presented strategy provides insights into antibody repertoire reactivity at a peptide level and may accelerate the discovery and validation of autoantigens in human diseases.

  • 130.
    Zelenin, Sergey
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hansson, Jonas
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ardabili, Sahar
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ramachandraiah, Harisha
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Russom, Aman
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Microfluidic-based isolation of bacteria from whole blood for sepsis diagnostics2015Inngår i: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 37, nr 4, s. 825-830Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Blood-stream infections (BSI) remain a major health challenge, with an increasing incidence worldwide and a high mortality rate. Early treatment with appropriate antibiotics can reduce BSI-related morbidity and mortality, but success requires rapid identification of the infecting organisms. The rapid, culture-independent diagnosis of BSI could be significantly facilitated by straightforward isolation of highly purified bacteria from whole blood. We present a microfluidic-based, sample-preparation system that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.

  • 131.
    Zhang, Shao-jie
    et al.
    Chinese Acad Sci, Kunming Inst Zool, State Key Lab Genet Resources & Evolut, Kunming 650223, Yunnan, Peoples R China.;Yunnan Univ, State Key Lab Conservat & Utilizat Bio Resource Y, Kunming 650091, Yunnan, Peoples R China..
    Wang, Guo-Dong
    Chinese Acad Sci, Kunming Inst Zool, State Key Lab Genet Resources & Evolut, Kunming 650223, Yunnan, Peoples R China.;Chinese Acad Sci, Ctr Excellence Anim Evolut & Genet, Kunming 650223, Yunnan, Peoples R China..
    Ma, Pengcheng
    Chinese Acad Sci, Kunming Inst Zool, State Key Lab Genet Resources & Evolut, Kunming 650223, Yunnan, Peoples R China..
    Zhang, Liang
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Yin, Ting-Ting
    Chinese Acad Sci, Kunming Inst Zool, State Key Lab Genet Resources & Evolut, Kunming 650223, Yunnan, Peoples R China..
    Liu, Yan-hu
    Chinese Acad Sci, Kunming Inst Zool, State Key Lab Genet Resources & Evolut, Kunming 650223, Yunnan, Peoples R China..
    Otecko, Newton O.
    Chinese Acad Sci, Kunming Inst Zool, State Key Lab Genet Resources & Evolut, Kunming 650223, Yunnan, Peoples R China..
    Wang, Meng
    Yunnan Univ, State Key Lab Conservat & Utilizat Bio Resource Y, Kunming 650091, Yunnan, Peoples R China..
    Ma, Ya-ping
    Yunnan Univ, State Key Lab Conservat & Utilizat Bio Resource Y, Kunming 650091, Yunnan, Peoples R China..
    Wang, Lu
    Yunnan Univ, State Key Lab Conservat & Utilizat Bio Resource Y, Kunming 650091, Yunnan, Peoples R China..
    Mao, Bingyu
    Chinese Acad Sci, Kunming Inst Zool, State Key Lab Genet Resources & Evolut, Kunming 650223, Yunnan, Peoples R China.;Chinese Acad Sci, Ctr Excellence Anim Evolut & Genet, Kunming 650223, Yunnan, Peoples R China..
    Savolainen, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Zhang, Ya-Ping
    Chinese Acad Sci, Kunming Inst Zool, State Key Lab Genet Resources & Evolut, Kunming 650223, Yunnan, Peoples R China.;Yunnan Univ, State Key Lab Conservat & Utilizat Bio Resource Y, Kunming 650091, Yunnan, Peoples R China..
    Genomic regions under selection in the feralization of the dingoes2020Inngår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 11, nr 1, artikkel-id 671Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Dingoes are wild canids living in Australia, originating from domestic dogs. They have lived isolated from both the wild and the domestic ancestor, making them a unique model for studying feralization. Here, we sequence the genomes of 10 dingoes and 2 New Guinea Singing Dogs. Phylogenetic and demographic analyses show that dingoes originate from dogs in southern East Asia, which migrated via Island Southeast Asia to reach Australia around 8300 years ago, and subsequently diverged into a genetically distinct population. Selection analysis identifies 50 positively selected genes enriched in digestion and metabolism, indicating a diet change during feralization of dingoes. Thirteen of these genes have shifted allele frequencies compared to dogs but not compared to wolves. Functional assays show that an A-to-G mutation in ARHGEF7 decreases the endogenous expression, suggesting behavioral adaptations related to the transitions in environment. Our results indicate that the feralization of the dingo induced positive selection on genomic regions correlated to neurodevelopment, metabolism and reproduction, in adaptation to a wild environment.

  • 132.
    Zhang, Wang
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH). Karolinska institutet.
    Development of novel molecular and microfluidics tools for identification and characterization of latent HIV-1 reservoir2019Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The existence of latent HIV-1 reservoir (LR) in all HIV-1 infected patients serves as a major obstacle to completely cure HIV-1 infection. However, up to now there is still no available assay that provides an accurate measurement of the reservoir size. This thesis aims to address this challenge from different aspects with several novel technologies, using both molecular and microfluidics-based tools. To find a proper tool to identify the latent HIV-1 reservoir, in Paper I and II, LIPS assay, RNAflow, and RNAscope assay were optimized and evaluated for indirect and direct detection of latent HIV-1 reservoir. The results indicated the LIPS method might not be sufficient for latent HIV-1 reservoir detection, although it has been proposed to quantify the latent HIV-1 reservoir indirectly. Furthermore, the optimized RNAscope technique performed better than RNAflow for transcription and translation competent latent HIV-1 reservoir identification. The RNAscope was also found to be independent of the HIV-1 subtype and can be applied to patient samples at single cell level. As there are currently no available surface biomarkers for latent HIV-1 reservoir, in Paper III, transcriptomics and proteomics-based analysis method for high-throughput selection of potential biomarker were established and applied to different patient groups. Twelve membrane protein-coding genes were identified as downregulated in the patient group who were hypothesized to have lower latent reservoir. These proteins might have the potential to be used as surface biomarkers for latent HIV-1 reservoir. CD4+ T cells, monocyte/macrophages, and natural killer cells are believed to be the primary source for HIV-1 reservoirs in peripheral blood. In paper IV, a microfluidic chip was developed to simultaneously isolate these three mononuclear leukocyte cell types directly from whole blood. The microfluidic method reduces the sample volume requirement and is a promising tool for latent HIV-1 reservoir study. Together, though further improvement and clinical verification are necessary, the work in this thesis has contributed to the advancement of latent HIV-1 reservoir characterization and may facilitate future development of the latent HIV-1 reservoir targeting and clearance methods with the ultimate goal – to cure HIV-1 infection.

    Fulltekst (pdf)
    fulltext
  • 133.
    Zhang, Wang
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi. karolinska institutet.
    Aljadi, Zenib
    Neogi, Ujjwal
    Russom, Aman
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Microfluidic based immunoaffinity mononuclear leukocytes isolation from whole bloodManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    CD4+ T cells, monocyte/macrophages and natural killer cells are believed to be the main source for HIV-1 reservoirs in peripheral blood. However, despite the potential these subsets of providing a wealth of new information about immune function and host pathology, current HIV latency studies are often based on PBMCs or only CD4+ T cells, mainly due to the lack of appropriate cell subset isolation methods. We present here a microfluidic chip-based method to capture and enrich the three mononuclear cells sub-population peripheral leukocyte sub-populations: CD4+ lymphocytes, natural killer cells and monocytes; using a single source of whole blood (volume < 200 μL) on a single integrated platform, within a time frame of 20 min. The single step isolation method can be used for downstream proteomics and genomics analysis to study the aberrations in these cell types’ functions in critical diseases such as HIV.

  • 134.
    Zhang, Ye
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Stobbe, Per
    Silvander, Christian Orrego
    Chotteau, Veronique
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor2015Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 213, s. 28-41Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 x 106 viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 C to 29 C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.

  • 135.
    Åstrand, Mikael
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Engineering strategies for ABD-derived affinity proteins for therapeutic and diagnostic applications2016Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Small stable protein domains are attractive scaffolds for engineering affinity proteins due to their high tolerance to mutagenesis without loosing structural integrity. The albuminbinding domain is a 5 kDa three-helix bundle derived from the bacterial receptor Protein G with low-nanomolar affinity to albumin. In this thesis, the albumin-binding domain is explored as a scaffold for engineering novel affinity proteins with the possible benefit of combining a prolonged serum half-life with specific targeting in a single small scaffold protein. Previously, a library was created by randomizing surface-exposed residues in order to engineer affinity to a new target antigen in addition to the inherent albumin affinity. Here, phage display selections were separately performed against the tumor antigens ERBB2 and ERBB3. The ERBB3 selection resulted in a panel of candidates that were found to have varying affinities to ERBB3 in the nanomolar range, while still retaining a high affinity to albumin. Further characterization concluded that the clones also competed for binding to ERBB3 with the natural activating ligand Heregulin. The selections against ERBB2 resulted in sub-nanomolar affinities to ERBB2 where the binding site was found to overlap with the antibody Trastuzumab. The binding sites on ABD to albumin and either target were found in both selections to be mutually exclusive, as increased concentrations of albumin reduced the level of binding to ERBB2 or ERBB3. An affinity-matured ERBB2 binder, denoted ADAPT6, which lacked affinity to albumin was evaluated as a radionuclide-labeled imaging tracer for diagnosing ERBB2-positive tumors. Biodistribution studies in mice showed a high renal uptake consistent with affinity proteins in the same size range and the injected ADAPT quickly localized to the implanted tumor. High contrast images could be generated and ERBB2-expressing tissue could be distinguished from normal tissue with high contrast, demonstrating the feasibility of the scaffold for use as diagnostic tool. In a fourth study, affinity maturation strategies using staphylococcal cell-surface display were evaluated by comparing two replicate selections and varying the stringency. A sub-nanomolar target concentration was concluded to be inappropriate for equilibrium selection as the resulting output was highly variable between replicates. In contrast, equilibrium sorting at higher concentrations followed by kinetic-focused off-rate selection resulted in high output overlap between attempts and a clear correlation between affinity and enrichment.

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    fulltext
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