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  • 1.
    af Geijerstam, Lukas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Magnusson, Andreas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Nordström, Ida
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Westerberg, Samuel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Zingmark Lien, Max
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    The Use of Surface Plasmon Resonance 2014-2024: A Review2024Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Surface plasmon resonance (SPR) is a label-free, versatile and highly sensitive method for studying molecular interactions in real time. It is widely used by industry and academia alike in fields ranging from Alzheimer’s disease research to detection of heavy metals. In this review, studies published during the last 10 years using Biacore or other SPR instruments were compiled and compared. Trends were also identified in the field. Amine coupling was found to be the most common ligand strategy for proteins, and most SPR research related to the field of medicine. Furthermore, three main purposes of an SPR experiment were identified: To determine the affinity between a pair of molecules, kinetics between a pair of molecules or to detect a certain molecule in a solution. The results presented are often related to these three purposes, and are most often presented and evaluated in terms of kinetic, affinity and sensitivity constants. SPR can be used for studying a broad range of molecular interactions, and an overview was obtained by dividing up the field into different parts based on molecular interactions and SPR methods. The study of molecular interactions using SPR was divided into protein-protein interactions (PPIs), antibody-antigen, protein-biomolecule interactions, interactions between proteins and small molecules, and non-conventional SPR methods. Non-conventional SPR methods include localized surface plasmon resonance (LSPR) and SPR imaging (SPRi), which are both based on the same optical sensing principles as SPR. 

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    The Use of Surface Plasmon Resonance 2014-2024: A Review
  • 2.
    Agdur, Angelica
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Characterization of enzyme decomposing biological macromolecules from a fish pathogen2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [sv]

    Saprolegnia parasitica är en av de mest skadliga oomycetpatogenerna som orsakar många problem inom vattenbruket. Den påverkar vuxen fisk, fiskägg och ungfisk. För närvarande finns det ingen effektiv och miljösäker behandling mot S. parasitica, vilket understryker vikten av att utveckla ett nytt sätt att kontrollera patogenet. Toxicitetsbelastningen kan minskas genom att ha en mycket specifik behandling. Att uppnå detta skulle kräva en förståelse för de fysiologiska och molekylära vägarna som är involverade i patogenutvecklingen, infektionsprocessen och värdspecificiteten, för att hitta målproteiner. Majoriteten av Saprolegniales forskning har koncentrerats på utsöndrade proteaser och intracellulära effektorer, men rollen av kolhydrataktiva enzymer (CAZymes) i infektionen har försummats. Kitinaser är CAZymer och mer specifikt hydrolyserar glykosidhydrolasenzymer beta-1,4-bindningar i kitin. Kitin är en strukturell polysackarid som finns i exoskelettet hos kräftdjur och epitelceller hos fiskfjäll. S. parasitica kan etablera infektionen genom användning av kitinaser. Målet med projektet är att hitta fler kitinaser som för närvarande är okarakteriserade proteiner av S. parasitica. Bioinformatiska tillvägagångssätt används för att förutsäga potentiella kitinaser, och ytterligare experimentell funktionell karakterisering av förutsagt protein. 

    Åtta okarakteriserade förutsagda proteiner av S. parasitica erhölls från sekvensanalys av kitinaser från familjen GH18 med Enzyme Commission-numret: 3.2.1.14 för att vara potentiella kitinaser. Av dessa åtta proteinsekvenser är två SPRG_10284 och SPRG_09577 de mest lovande. Dessa kan testas ytterligare experimentellt och båda förutspås vara lösliga proteiner. SPRG_10284 har framgångsrikt uttryckts i Escherichia coli stammar Rosetta 2 och BL21(DE3). Protokollen för proteinuttryck, extraktion och rening måste dock standardiseras för att erhålla en stor mängd lösligt protein.

  • 3.
    AI-Tamimi, Lejla
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Structural features underlying antigen presentation by the non-classical MHC class Ib molecule Qa-1b2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Blocking of the NKG2A receptor expressed on NK cells and CD8+ T cells with an anti-NKG2A antibody for elicitation of cytolytic activity, is a promising immune checkpoint in cancer immunotherapy. EXX1, a novel TCR-like antibody with specificity for the NKG2A ligand, Qa-1b - a murine non-classical MHC class Ib ortholog of HLA-E - has been assessed in tumor models in vitro. The antibody only engages with Qa-1b when it presents the dominant peptide Qdm, derived from the leader sequence of the classical MHC class Ia H-2D. This raises questions about the structural features of antigen presentation by Qa-1b, and the molecular parameters driving the specificity of the TCR-like antibody. The purpose of this study is to determine and compare the crystal structures of Qa-1b in complex with Qdm (AMAPRTLLL) and peptide 001 (AQAERTPEL). The Qa-1b heavy chain and mouse beta-2 microglobulin were recombinantly expressed in E.coli, refolded in the presence of respective peptide, purified using size exclusion chromatography and crystallized with the hanging drop vapor diffusion method. Thermal stability of the MHC/peptide complexes was assessed with nano differential scanning fluorimetry, implying a higher stability of Qa-1b/001. Crystals of the Qa-1b/Qdm and Qa-1b/001 were obtained with 8% PEG4000, 10 mM NiCl2, 0.1 M sodium acetate at pH 5.7, and 10% PEG4000, 10mM NiCl2 and 0.1 M sodium acetate at pH 6.0, respectively. The structure of Qa-1b/001 was resolved by molecular replacement at 2.43 Å, and the presence of negatively charged side chains that protrude from the binding groove, may imply that differences in electrostatic interactions between Qdm and 001 will determine antibody-binding. Further structural characterizations, of Qa-1b complexes with bound EXX1 are of great interest.

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  • 4.
    Akan, Pelin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Alexeyenko, Andrey
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Costea, Paul Igor
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hedberg, Lilia
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Werne Solnestam, Beata
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hallman, Jimmie
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Comprehensive analysis of the genome transcriptome and proteome landscapes of three tumor cell lines2012In: Genome Medicine, E-ISSN 1756-994X, Vol. 4, p. 86-Article in journal (Refereed)
    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.

  • 5. Altai, M.
    et al.
    Tsourma, M.
    Mitran, B.
    Honarvar, H.
    Perols, Anna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Robillard, M.
    Rossin, R.
    ten Hoeve, W.
    Sandstrom, M.
    Orlova, A.
    Karlström, Amelie Eriksson
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, V.
    Affibody-based bioorthogonal chemistry-mediated radionuclide pretargeting: proof-of-principle.2015In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, p. S246-S246Article in journal (Refereed)
  • 6. Altai, Mohamed
    et al.
    Vorobyeva, Anzhelika
    Tolmachev, Vladimir
    Eriksson Karlström, Amelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Westerlund, Kristina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Preparation of Conjugates for Affibody-Based PNA-Mediated Pretargeting2020In: Methods in Molecular Biology, Humana Press Inc. , 2020, p. 283-304Chapter in book (Refereed)
    Abstract [en]

    Affibody molecules are small engineered scaffold proteins suitable for in vivo tumor targeting. Radionuclide molecular imaging using directly radiolabelled affibody molecules provides excellent imaging. However, affibody molecules have a high renal reabsorption, which complicates their use for radionuclide therapy. The high renal reabsorption is a common problem for the use of engineered scaffold proteins for radionuclide therapy. Affibody-based PNA-mediated pretargeting reduces dramatically the absorbed dose to the kidneys and makes affibody-based radionuclide therapy possible. This methodology might, hopefully, solve the problem of high renal reabsorption for radionuclide therapy mediated by other engineered scaffold proteins. 

  • 7.
    Andersson, Klara
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Characterization of nsP-specific nanobodies targeting Chikungunya and Semliki Forest Virus2020Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Viral infections are constantly increasing and impose a large threat to the public health. Alphaviruses are responsible for several animal and human diseases and have a large medical importance with few treatments available today. Alphaviruses are small, spherical single stranded RNA viruses, and are most often transmitted by mosquito vectors. Alphaviruses contains a domain of nonstructural proteins that compose the replication machinery. The domain is crucial for viral replication to occur and is therefore an interesting target for antiviral therapy. With the focus on Chikungunya and Semliki Forest Virus this work investigates the events in the cells on molecular level during infections. To do this a panel of Camelid derived single domain antibodies are developed to target the nonstructural proteins of Chikungunya and Semliki Forest Virus. Binding of the produced nanobodies to the viral proteins was investigated by biochemical methods including immunoprecipitations, western blot, and ELISA. Cell lines that express nsP-specific nanobodies in the cytosol were employed for infection- and plaque assays with Semliki Forest Virus in order to determine the antiviral potential of the new nanobodies. Three of the nanobodies proved to bind two different nonstructural proteins of the viruses, providing opportunities for further investigations and a possible use of these nanobodies to identify viral vulnerabilities that could be exploited for antiviral intervention.

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  • 8.
    Andersson, Olivia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Engineering of the epidermal skin layer using recombinant spider silk2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The skin is the largest human organ, serving a fundamental role in protecting the human body from harmful external stimuli by acting as a physical and biological barrier. The human skin can be divided into three layers, the epidermal layer being of interest in this study. The human epidermis, mainly populated by keratinocytes, can in turn be divided into 5 cell layers, each distinguished by the keratinocyte differentiation state. In addition, laminins are of interest in this study. Laminins are large glycoproteins that are secreted, as heterotrimers, by e.g., keratinocytes. They are a major component of the basal lamina of in vivo skin and are involved in various signalling pathways and regulate the adhesion, migration, differentiation, and apoptosis of keratinocytes.

    Human skin equivalents (HSEs) aim to represent the human skin, allowing us to study its physiology and relation to disease. Although various biomaterials have been used to construct HSEs, many of them suffer from poor mechanical integrity, elasticity, fragile ECM structure and/or require functionalization. FN-4RepCT, a recombinantly produced spider silk protein, forms a biomaterial that overcomes these shortcomings. It contains four repetitive polyalanine blocks and four glycine-rich units, a C-terminal domain, and a fibronectin derived RGD motif. Protein solutions of FN-4RepCT can be used to produce 3D-scaffolds of various formats, that are protein-permeable, biodegradable, elastic, mechanically robust, and support cell proliferation. Within this project FN-silk membranes, with nanofibrillar structures, are formed under ambient conditions and are used to construct an epidermal equivalent integrated with laminins and keratinocytes. The method presented herein for forming such membranes, and coating them with key laminins, is easy and may be repeated without the use of complex laboratory equipment.

    The results presented herein provides further support for FN-4RepCT as a biomaterial within the field of tissue engineering. It is also demonstrated that laminins, combined with the silk, promote the proliferation and differentiation of keratinocytes, and that coating FN-silk membranes with laminins is beneficial for construction of a physiologically relevant epidermal equivalent. The epidermal constructs were evaluated with immunofluorescence staining of the keratinocyte differentiation markers keratin 5, keratin 10, and involucrin. The results reveal that the construct is built up by multiple cell layers with distinct morphology and expression of the aforementioned differentiation markers. Overall, it can be concluded that FN-4RepCT, in combination with laminins, is a promising option for tissue engineering of epidermal constructs.

  • 9.
    Aniander, Gustav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Investigation of localized B cell activation at stromal tumor microenvironment site by bispecific affinity proteins - AffimAbs2020Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Cancer is a number of diseases where the cells in the body start growing and dividing out ofcontrol. It is a widespread health issue, which requires novel treatments to combat effectively.Some novel strategies involve activating the body’s immune system and letting it attack thecancerous cells. This has been achieved through the use of engineered antibodies, normally a part of the immune system. Antibodies bind to different antigens and the areas that facilitate the binding are well known, allowing for manipulation of what the antibodies bind to. To activate the immune system, cell-surface receptors of T cells and B cells have been targeted. There has been some promise shown, but issues include systemic side effects, some of them severe. A proposed way to circumvent this is to try and only locally activate the immune system through the use of bispecific proteins. The idea behind this is to have specificity towards an activation target and another specificity towards some target of interest on cancer cells. One such class of bispecifics is AffimAbs, monoclonal antibodies (mAbs) with an affibody conjugated to them. An affibody is an affinity molecule derived from the staphylococcal protein A, with its main advantage being its small size of 7 kDa compared to an antibody at 150 kDa. This study investigates the ability of an AffimAb with specificity towards the cell-surface receptor CD40 expressed on B cells and specificity towards PDGFR-β, a cell-surface receptor found to be overexpressed in some types of cancer, to locally activate B cells. To investigate this a co-culture activation assay was designed and the separate parts validated. This included finding proper seeding density for PDGFR-β-expressing cells, establishing a protocol for isolating primary B cells, and validating binding ofconstructs towards targets. The results from the co-culture activation assay show that there is a potential higher activation for the AffimAb compared to a mAb, but the small scale of this study precludes it from being statistically significant. Further, larger assays need to be performed to show these results to be significant.

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    sammanfattning
  • 10.
    Ask, Per
    et al.
    Linköping University, Department of Biomedical Engineering, Physiological Measurements. Linköping University, The Institute of Technology.
    EDWALL, G
    Tibbling, Lita
    ESOPHAGEAL PH MEASUREMENTS USING AN ANTIMONY ELECTRODE1980In: Medical and Biological Engineering and Computing, ISSN 0140-0118, E-ISSN 1741-0444, Vol. 18, no 1Article in journal (Refereed)
  • 11.
    Ask, Per
    et al.
    Linköping University, Department of Biomedical Engineering, Physiological Measurements. Linköping University, The Institute of Technology.
    Öberg, Åke
    Linköping University, Department of Biomedical Engineering.
    Tibbling, Lita
    ESOPHAGEAL MANOMETRY - DETERMINATION OF BANDWIDTH REQUIREMENTS BY SIGNAL ANALYSIS1980In: Physics in Medicine and Biology, ISSN 0031-9155, E-ISSN 1361-6560, Vol. 25, no 5Article in journal (Refereed)
  • 12.
    Atif, Abdul Raouf
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering.
    Carter, Sarah-Sophia
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering.
    Deng, Hanlu
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Engqvist, Håkan
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering, Applied Material Science. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tenje, Maria
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering.
    Mestres, Gemma
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering.
    A Universal Microfluidic Platform for In Vitro Biomaterial Evaluation2022Conference paper (Other academic)
    Abstract [en]

    INTRODUCTION: Conventionally, the biological properties of biomaterials are evaluated using well plates. Although being a standardized method, it is static in terms of fluid flow and is far from the physiological conditions found in vivo. This work presents a versatile microfluidic system that allows for integration of different biomaterials (ceramic, metals and polymers) under dynamic conditions.

    METHODS: The Universal Biomaterial-on-Chip (UBOC) consisted of two separate 3D printed (Polylactic acid, Ultimaker 2+) structures: the upper layer which contains the channel through which medium can flow (Fig1A) and the bottom layer that holds and secures the biomaterial in place (Fig 1B). A glass coverslip was taped to the upper layer to tightly seal the channel. Subsequently, an oval Polydimethylsiloxane (PDMS) gasket (l=10mm,w=7mm, h=0.8mm) was inserted into the periphery of the channel in the upper layer. Furthermore, magnets (Ø=12mm, h=3mm) were glued on both sides of the bottom layer. To close the channel, two magnets were placed on the upper layer, causing attraction to the magnets in the bottom layer. The gasket would then directly interface with the biomaterial inside the bottom layer, creating a leak-free channel on its surface. MC3T3-E1 pre-osteoblasts were seeded in the UBOC platform (50,000 cells/cm2) on calcium-deficient hydroxyapatite (HA) (Ø=15mm) and clinical grade titanium (Ti) (Ø=12mm). The cells were cultured for a period of 5 days at a flow rate of 2 μl/min using supplemented MEM-α medium (Hyclone, 10% FBS, 1% Pen-Strep). On day 5, the cells were stained on-chip with Live/Dead stain (Calcein, Propidium Iodide and Hoechst) and subsequently imaged.

    RESULTS: HA and Ti samples were successfully integrated into the UBOC. Cells cultured on-chip displayed a high degree of viability and confluence on day 5 of culture on both HA and Ti substrates (Fig 2).

    DISCUSSION & CONCLUSIONS: UBOC presents the possibility for flexible in vitro biomaterial analysis as it allows for easy incorporation of flow to conventional cell culture regimes in a low-cost manner. Via this method,cells can be cultured on the biomaterial with exposure to fluid flow and controlled shear-stress. The platform is compatible with standard characterization methods, such as imaging and biochemical cell analysis. In addition, since the system is designed to be opened and closed, the biomaterial could be easily accessed, harvested and transferred to a regular tissue culture vessel,enabling standard off-chip biochemical assays and protocols to be performed for further analysis.

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    fulltext
  • 13.
    Augustinsson, Sebastian
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Mass Spectrometric Virus Detection with Multiplex Assay2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The purpose of project was to develop a multiplex assay capable of detecting antigens from SARSCoV-2, influenza and respiratory syncytial virus by utilizing a mass spectrometric approach involving antigen-specific binders. Binders were cloned, purified and biotinylated before being employed in an assay developed by though a targeted method involving the antigens. It was concluded to be possible for Avi-tagged binders to specifically bind antigen-derived peptide targets in the multiplex assay.

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    fulltext
  • 14.
    Bajhaiya, Amit K.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Univ Manchester, Fac Life Sci, Michael Smith Bldg,Oxford Rd, Manchester M13 9PT, Lancs, England.
    Moreira, Javiera Ziehe
    Pittman, Jon K.
    Transcriptional Engineering of Microalgae: Prospects for High-Value Chemicals2017In: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 35, no 2, p. 95-99Article in journal (Refereed)
    Abstract [en]

    Microalgae are diverse microorganisms that are of interest as novel sources of metabolites for various industrial, nutritional, and pharmaceutical applications. Recent studies have demonstrated transcriptional engineering of some metabolic pathways. We propose here that transcriptional engineering could be a viable means to manipulate the biosynthesis of specific high-value metabolic products.

  • 15.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Lab-on-DVD: Optical Disk Drive-Based Platforms for Point-of-Care Diagnostics2018In: Frugal Innovation in Bioengineering for the Detection of Infectious Diseases / [ed] AK Chavali, R Ramji, Switzerland: Springer, 2018, 2, p. 23-38Chapter in book (Refereed)
    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.

  • 16.
    Bayati, Shaghayegh
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Anti-kinesin autoantibodies in ANCA-associated vasculitisManuscript (preprint) (Other academic)
  • 17.
    Beckman, S
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Giertz, Tobias
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Högqvist Bandefur, Hampus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Levin, Mattias
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Ridderström, Linnéa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Rosenblad, Elsa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Single-cell proteomics in blood samples2024Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Single-cell proteomics is a useful tool for measuring differences in cell populations for clinical trials. In this report we have conducted a literary review where we looked at 23 different single-cell proteomics methods and their advantages and disadvantages. We have looked at both mass spectrometry-based and affinity-based methods to find upcoming methods in the field of single-cell proteomics. Our findings show that there are multiple promising techniques that can be applied in different contexts. Moreover, we recommended combining different protocols, for instance Capillary zone electrophoresis (CZE) with a microfluidic platform or Optidrop with one of the barcoding methods for better results. When conducting this review it became clear that most methods could be improved by implementing software programs such as PEPerMINT and Infinity flow. Therefore, we encourage that such data acquisition and analysis methods are implemented to yield more accurate characterization and quantification of the single-cell proteome.

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    Rapport
  • 18. Beerenwinkel, N.
    et al.
    Greenman, C. D.
    Lagergren, Jens
    KTH, School of Computer Science and Communication (CSC), Computational Science and Technology (CST).
    Computational Cancer Biology: An Evolutionary Perspective2016In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 12, no 2, article id e1004717Article in journal (Refereed)
  • 19.
    Bergström, Ebba
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Optimization of immunotherapeutic relevant ABD-derived affinity proteins for prolonged serum half-life2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The market for protein-based drugs, or the so-called biopharmaceuticals, is a multibillion-dollar industry today. In the development of protein-based drugs it is common to use monoclonal antibodies (mAbs) due to their ability to bind to its target with high specificity. However, therapeutical development of mAbs is limited by its long and expensive production in mammalian expression system. An alternative to mAbs are the so-called alternative scaffolds which are small proteins that can be produced in bacteria at lower costs. Although a drawback with the latter proteins is their short serum half-life. A small scaffold protein, ABD-Derived Affinity ProTein (ADAPT) of approximate 7 kDa was earlier engineered to obtain bispecific affinity, to Human Serum Albumin (HSA), to extend its half-life, as well as to the pro-inflammatory cytokine, Interleukin 17c (IL17c). Unfortunately, it was shown that the simultaneous binding was not efficient enough for its desired purpose. The aim with this project was therefore to investigate if the previous mentioned binder could be optimized by multimerization and/or manipulation of the HSA binding site for an efficient half-life extension. By generating ten new designs of the ADAPT variants, it was observed that the new variants had stable alpha helical structures and an improved or similar melting temperature as the original variant. The evaluation of the target binding displayed an improved affinity to the target, IL17c, for two of the dimeric versions as well as for the trimer and a comparable affinity for two of the monomers with a manipulated HAS binding site. The interaction to HSA was comparable to the original ADAPT for all binders except from the monomers with impaired HSA binding and the dimer with two impaired HSA binding sites. The evaluation of the simultaneous binding showed that it was favored by dimerization when a distance between the two molecule and their binding surfaces was added. Moreover, it could also be seen that the order of binding events had an impact on the simultaneous binding.

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  • 20.
    Bergström, Sofia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Multiplexed antibody-based protein profiling in the pursuit of CSF biomarkers for neurodegenerative diseases2021Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    There is a desire for a transition from generic treatments designed for the average patient, towards more individual-based precision medicine. An increased knowledge about disease pathophysiology on a molecular level would be beneficial for this transition. The study of proteins can contribute with valuable insights into etiology and pathogenesis of different diseases and thereby aid the clinical assessment of patients and guide future treatments.

    Neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and frontotemporal dementia, are characterized by a progressive loss of function, and eventually death of neurons. Neurons allow the brain to communicate with the rest of the body, and a deteriorated function of neurons can result in problems with mobility or mental functions. Neurodegenerative diseases progress slowly over many years, with a long silent asymptomatic phase before symptom onset. It is hard to rebuild what is already lost, but disease-modifying treatments might be able to slow down or halt the deterioration of the brain. Therefore, there is a major research focus on investigating the early stages of disease pathogenesis in order to elucidate this critical phase in disease progression.

    The four papers included in this thesis focus on identifying altered protein profiles in cerebrospinal fluid from patients with neurodegenerative diseases. For this purpose, multiplexed antibody-based suspension bead arrays have been used. This method allows for hundreds of proteins to be analyzed in hundreds of samples in the same assay. Paper I focuses on Alzheimer’s disease and investigates the profiles of 200 proteins when comparing patients with controls. Six proteins were identified at altered levels and were further investigated in relation to the progression from mild cognitive impairment to Alzheimer’s disease. Paper II explores 100 protein profiles in relation to the core Alzheimer’s disease biomarkers in asymptomatic 70-year-olds to elucidate patterns preceding potential disease onset. Paper III investigates the transition to cognitive impairment in patients with Parkinson’s disease and explores potential associations between protein profiles and cognitive assessment tests. Finally, Paper IV explores panels of proteins in the context of frontotemporal dementia. Panels of proteins, instead of single biomarkers, have an increased potential to capture the range of biological processes within these types of complex and multifactorial diseases.

    Neurodegenerative diseases are often heterogeneous which puts high demands on the study design including an appropriate selection of study population. However, significant similarities are also present which makes it advantageous to have a broad perspective and work with several neurodegenerative disorders. This thesis presents the results from multiplexed antibody-based protein profiling as a contribution to a better understanding of neurodegenerative diseases.

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  • 21.
    Bergström, Sofia
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Remnestål, Julia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Olofsson, Jennie
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Markaki, Ioanna
    Karolinska Institutet.
    Carvalho, Stephanie
    Institut du Cerveau et de la Moelle épinière, Sorbonne Université.
    Corvol, Jean-Christophe
    Institut du Cerveau et de la Moelle épinière, Sorbonne Université.
    Kultima, Kim
    Uppsala Universitet.
    Kilander, Lena
    Uppsala Universitet.
    Löwenmark, Malin
    Uppsala Universitet.
    Ingelsson, Martin
    Uppsala Universitet.
    Blennow, Kaj
    Sahlgrenska University Hospital, University of Gothenburg.
    Zetterberg, Henrik
    Sahlgrenska University Hospital, University of Gothenburg; Department of Neurodegenerative Disease, UCL Institute of Neurology, London; UK Dementia Research Institute at UCL, London.
    Nellgård, Bengt
    Sahlgrenska University Hospital, University of Gothenburg.
    Brosseron, Frederic
    Universitätsklinikum, Bonn; German Center for Neurodegenerative Diseases (DZNE), Bonn.
    Heneka, Michael
    Universitätsklinikum, Bonn.
    Bosch, Beatriz
    University of Barcelona.
    Sanches-Valle, Raquel
    University of Barcelona.
    Månberg, Anna
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Svenningsson, Per
    Karolinska Institutet.
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Multi-cohort protein profiling reveals higher levels of six brain-enriched proteins in Alzheimer’s disease patientsManuscript (preprint) (Other academic)
  • 22. Björn, N.
    et al.
    Sigurgeirsson, Benjamin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. School of Engineering and Natural Sciences, University of Iceland, Reykjavík, Iceland.
    Svedberg, A.
    Pradhananga, Sailendra
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brandén, E.
    Koyi, H.
    Lewensohn, R.
    de Petris, L.
    Apellániz-Ruiz, M.
    Rodríguez-Antona, C.
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Gréen, Henrik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Clinical Pharmacology, Division of Drug Research, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden; Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden.
    Genes and variants in hematopoiesis-related pathways are associated with gemcitabine/carboplatin-induced thrombocytopenia2020In: The Pharmacogenomics Journal, ISSN 1470-269X, E-ISSN 1473-1150, Vol. 20, no 2, p. 179-191Article in journal (Refereed)
    Abstract [en]

    Chemotherapy-induced myelosuppression, including thrombocytopenia, is a recurrent problem during cancer treatments that may require dose alterations or cessations that could affect the antitumor effect of the treatment. To identify genetic markers associated with treatment-induced thrombocytopenia, we whole-exome sequenced 215 non-small cell lung cancer patients homogeneously treated with gemcitabine/carboplatin. The decrease in platelets (defined as nadir/baseline) was used to assess treatment-induced thrombocytopenia. Association between germline genetic variants and thrombocytopenia was analyzed at single-nucleotide variant (SNV) (based on the optimal false discovery rate, the severity of predicted consequence, and effect), gene, and pathway levels. These analyses identified 130 SNVs/INDELs and 25 genes associated with thrombocytopenia (P-value < 0.002). Twenty-three SNVs were validated in an independent genome-wide association study (GWAS). The top associations include rs34491125 in JMJD1C (P-value = 9.07 × 10−5), the validated variants rs10491684 in DOCK8 (P-value = 1.95 × 10−4), rs6118 in SERPINA5 (P-value = 5.83 × 10−4), and rs5877 in SERPINC1 (P-value = 1.07 × 10−3), and the genes CAPZA2 (P-value = 4.03 × 10−4) and SERPINC1 (P-value = 1.55 × 10−3). The SNVs in the top-scoring pathway “Factors involved in megakaryocyte development and platelet production” (P-value = 3.34 × 10−4) were used to construct weighted genetic risk score (wGRS) and logistic regression models that predict thrombocytopenia. The wGRS predict which patients are at high or low toxicity risk levels, for CTCAE (odds ratio (OR) = 22.35, P-value = 1.55 × 10−8), and decrease (OR = 66.82, P-value = 5.92 × 10−9). The logistic regression models predict CTCAE grades 3–4 (receiver operator characteristics (ROC) area under the curve (AUC) = 0.79), and large decrease (ROC AUC = 0.86). We identified and validated genetic variations within hematopoiesis-related pathways that provide a solid foundation for future studies using genetic markers for predicting chemotherapy-induced thrombocytopenia and personalizing treatments.

  • 23. Boldinova, Elizaveta O.
    et al.
    Stojkovic, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Khairullin, Rafil
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Kazan Volga Reg Fed Univ, Russia.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Makarova, Alena V.
    Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol2017In: PLOS ONE, E-ISSN 1932-6203, Vol. 12, no 9, article id e0184489Article in journal (Refereed)
    Abstract [en]

    Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37 degrees C. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

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  • 24.
    Bondza, Sina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Ridgeview Instruments AB, Vange, Sweden.
    Foy, Eleanor
    Univ Leeds, Leeds Inst Rheumat & Musculoskeletal Med, Leeds, W Yorkshire, England..
    Brooks, Jonathan
    Pfizer Inc, Cambridge, MA USA..
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Ridgeview Instruments AB, Vange, Sweden.
    Robinson, James
    Univ Leeds, Leeds Inst Rheumat & Musculoskeletal Med, Leeds, W Yorkshire, England..
    Richalet, Pascale
    BioRevera LLC, Arlington, MA USA..
    Buijs, Jos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Ridgeview Instruments AB, Vange, Sweden.
    Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells2017In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 8, article id 455Article in journal (Refereed)
    Abstract [en]

    Understanding molecular interactions on immune cells is crucial for drug development to treat cancer and autoimmune diseases. When characterizing molecular interactions, the use of a relevant living model system is important, as processes such as receptor oligomerization and clustering can influence binding patterns. We developed a protocol to enable time-resolved analysis of ligand binding to receptors on living suspension cells. Different suspension cell lines and weakly adhering cells were tethered to Petri dishes with the help of a biomolecular anchor molecule, and antibody binding was analyzed using LigandTracer. The protocol and assay described in this report were used to characterize interactions involving eight cell lines. Experiments were successfully conducted in three different laboratories, demonstrating the robustness of the protocol. For various antibodies, affinities and kinetic rate constants were obtained for binding to CD20 on both Daudi and Ramos B-cells, the T-cell co-receptor CD3 on Jurkat cells, and the Fc gamma receptor CD32 on transfected HEK293 cells, respectively. Analyzing the binding of Rituximab to B-cells resulted in an affinity of 0.7-0.9 nM, which is similar to values reported previously for living B-cells. However, we observed a heterogeneous behavior for Rituximab interacting with B-cells, which to our knowledge has not been described previously. The understanding of complex interactions will be facilitated with the possibility to characterize binding processes in real-time on living immune cells. This provides the chance to broaden the understanding of how binding kinetics relate to biological function.

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  • 25.
    Botella Bobadilla, Javier
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Silk Supported Hydrogels Towards Tissue Engineering Applications2024Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Hydrogels are considered as one of the most promising materials in the biomaterials field due to their wide range of applications, including drug development and tissue engineering. Despite their biocompatibility and ability to mimic native tissue, hydrogels lack an efficient barrier for cell co-culturing efforts, which restrains them from being an optimal choice in tissue barrier models. 

    In this thesis project, a recombinant spider silk protein is incorporated into hydrogels made of collagen and fibrinogen. The intent of this project is to evaluate the changes in material properties when compared to gels without silk. 

    The silk formation on the different gel types was verified using fluorescent and scanning electron microscopy. Permeation studies performed using reporter substances reveal a delay in the onset of permeation when the silk is present in the gels, along with lower permeation pass-through percentages, as the silk concentration used in the gels is increased. Confined compression studies also show an increase in the deformation force tolerated by PureCol collagen gels when the silk is present in the gel. 

    These findings serve as a promising starting point for the investigation of how silk can be combined with hydrogels. The enhancement of material properties displayed in hydrogels with silk can serve as the foundation for its use in co-culture tissue barrier models. The behavior of gels with silk with respect to permeation make it attractive for future research towards organ-on-chip and drug development applications.

  • 26. 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, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Centres, 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, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, 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)2010In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 28, no 7, p. 650-653Article in journal (Other academic)
  • 27.
    Brunsell, Richard
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Design, production and evaluation of cross linked target proteins to an affibody-based carrier framework aimed for affinity protein: antigen structure determination using single particle Cryo-EM2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Small proteins are difficult to study at high resolution with single-particle cryo-electron microscopy (cryo-EM). In general, sample properties such as large size (> 80 kDa), symmetry and rigidity are key to utilize this technology. To facilitate structural studies of small proteins as well, using cryo-EM, this project aims to incorporate a photo-inducible cross-link in a large and symmetric scaffold that is amenable for study, and covalently bind small proteins of interest to this scaffold. The scaffold in this project consists of rabbit muscle aldolase (157 kDa in tetrameric state) with an engineered affibody affinity protein (7 kDa) attached to the N-terminus of each aldolase monomer via a rigid helix fusion. The affibody-domain of the scaffold will be cross-linked to small proteins of structural interest, with a focus on a model target consisting of a second affibody with affinity for the affibody displayed on the aldolase scaffold.

    Photoconjugation of the affibody Zwt was performed to crosslink both the Fc of IgG and the anti-idiotypic affibody Z963, revealing that a methionine acceptor in the target is preferable but not necessary for UV crosslinking using BPA. Binding of affibodies rigidly displayed on of the scaffold to targets such as affibodies and antibody fragments was demonstrated , using surface plasmon resonance (SPR).

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  • 28.
    Börjesson, Anna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Automatization of Antithrombin III, Heparin Co-factor assay to STA R2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    In 1982, Atenativ was released to the market, a pharmaceutical today produced by Octapharma. The product is a concentrate of the protein Antithrombin III, an essential protein for dissolving blood clots in the coagulation pathway. The pharmaceutical is mainly used to treat patients with Antithrombin deficiency but are used for other purposes as well. The activity of the product is measured by an assay called “Antithrombin III, Heparin Co-factor assay” and is based on the assay principle described in the European pharmacopeia. The method is a kinetic method today performed by hand at Octapharma in a microtiter plate and is therefore strenuous and time-consuming for analysts. The method does also have a low capacity of only 4 samples and an automatization of the method would therefore be advantageous for the company. The automatized clotting instrument STA R would be an alternative for this automatization and is therefore tested in this report. In order to automate the method, a linear interval of the calibration curve would need to be tested. To find and evaluate the best fit, the pharmacopeia combistat statistical program were used. The program compared the sample with the calibration using the statistical evaluation model slope ratio and parallel line. Two calibrators were used, one plasma calibrator and one concentrate calibrator. The plasma standard gave a better R2 value compared to the concentrate standard but 20 % higher concentration of the samples compared to the real concentration and the concentrate standard. The precision of the instrument was tested by analyzing 6 samples and then looking at the relative standard deviation. The instrument did show a high precision of relative standard deviation of 1.13. The stability of the reagents was also evaluated by analyzing 5 samples, one hour after the other. The stability of the regents was approved. 4 Atenativ samples was analyzed by the instrument and compared with the results obtained from the manual method. The automated method did show a slightly lower concentration, this can be due to the samples used in the automated method were not directly dissolved, thus a loss of activity when being stored in the freezer for 1 – 2 months can occur. An automatization of the method is possible and would contribute to a less time-consuming, less strenuous and higher sample capacity method. This is needed to be future investigated. 

  • 29.
    Cardemil, Carina
    Department of Biomaterials, Institute of Clinical Sciences, The Sahlgrenska Academy at the University of Gothenburg, Göteborg, Sweden.
    Effects of antiresorptive agents on inflammation and bone regeneration in different osseous sites - experimental and clinical studies2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The biological mechanisms involved in bone regeneration in osteoporotic bone and the effect of antiresorptive drugs in relation to surgically inserted biomaterials are not fully understood. Improved osseointegration of titanium implants but also adverse effects of antiresorptive therapies, such as osteonecrotic jaw have been described in the literature. The aims of this research project were, firstly, to investigate and to understand the biological events determining bone regeneration and implant integration, after administration of antiresorptive agents; secondly, to determine the cellular and molecular patterns of bone regeneration at implants and synthetic bone substitutes under osteoporotic conditions and, thirdly, to determine how different skeletal sites are affected. The present research included a study of jawbone morphology and gene expression in patients treated with systemic bisphosphonates. When compared to controls, higher gene expression levels of IL-1β was observed in bisphosphonate treated patients with osteonecrosis while bisphosphonate treated patients without necrosis showed lower expression levels of caspase 8, an apoptosis marker involved in the immune response. In ovariectomised rats, zoledronic acid resulted in site-specific differences in the rate of osseointegration and also of gene expression involved in bone healing and regeneration. Strontium-doped calcium phosphate inserted in the rat femur induced lower expression of osteoclastic markers compared to hydroxyapatite and higher bone formation in the periphery of the defects. Whereas major structural changes were demonstrated in the long bones of the ovariectomised rat, less structural alterations were shown in the mandible. However, ovariectomy resulted in lower expression of genes coding for bone formation and angiogenesis in the mandible. In conclusion, the present study shows that the mandible is differently affected by experimentally induced estrogen deficiency than the long bones. Bisphosphonates, administered systemically to estrogen deficient animals, impair osseointegration in the mandible, at least partly related to a downregulation of genes important for the osteogenic process. These observations may have implications for understanding the mechanisms involved in the deranged bone healing observed in the jawbone of bisphosphonate treated patients.

  • 30.
    Cardemil, Carina
    et al.
    Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden, Department of Oral and Maxillofacial Surgery, Örebro University Hospital, Örebro, Sweden, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden .
    Elgali, Ibrahim
    Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden.
    Xia, Wei
    Applied Materials Science, Department of Engineering Sciences, Uppsala University, Uppsala, Sweden, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden.
    Emanuelsson, Lena
    Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden.
    Norlindh, Birgitta
    Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden.
    Omar, Omar
    Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden.
    Thomsen, Peter
    Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden .
    Strontium-doped calcium phosphate and hydroxyapatite granules promote different inflammatory and bone remodelling responses in normal and ovariectomised rats2013In: PLOS ONE, E-ISSN 1932-6203, Vol. 8, no 12, article id e84932Article in journal (Refereed)
    Abstract [en]

    The healing of bone defects may be hindered by systemic conditions such as osteoporosis. Calcium phosphates, with or without ion substitutions, may provide advantages for bone augmentation. However, the mechanism of bone formation with these materials is unclear. The aim of this study was to evaluate the healing process in bone defects implanted with hydroxyapatite (HA) or strontium-doped calcium phosphate (SCP) granules, in non-ovariectomised (non-OVX) and ovariectomised (OVX) rats. After 0 (baseline), six and 28d, bone samples were harvested for gene expression analysis, histology and histomorphometry. Tumour necrosis factor-α (TNF-α), at six days, was higher in the HA, in non-OVX and OVX, whereas interleukin-6 (IL-6), at six and 28d, was higher in SCP, but only in non-OVX. Both materials produced a similar expression of the receptor activator of nuclear factor kappa-B ligand (RANKL). Higher expression of osteoclastic markers, calcitonin receptor (CR) and cathepsin K (CatK), were detected in the HA group, irrespective of non-OVX or OVX. The overall bone formation was comparable between HA and SCP, but with topological differences. The bone area was higher in the defect centre of the HA group, mainly in the OVX, and in the defect periphery of the SCP group, in both non-OVX and OVX. It is concluded that HA and SCP granules result in comparable bone formation in trabecular bone defects. As judged by gene expression and histological analyses, the two materials induced different inflammatory and bone remodelling responses. The modulatory effects are associated with differences in the spatial distribution of the newly formed bone.

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    Strontium-doped calcium phosphate and hydroxyapatite granules promote different inflammatory and bone remodelling responses in normal and ovariectomised rats
  • 31.
    Carinelli, S.
    et al.
    Univ Autonoma Barcelona, Dept Quim, Grp Sensors & Biosensors, Bellaterra, Spain..
    Kühnemund, Malte
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Box 1031, SE-17I21 Solna, Sweden..
    Pividori, M. I.
    Univ Autonoma Barcelona, Dept Quim, Grp Sensors & Biosensors, Bellaterra, Spain..
    Yoctomole electrochemical genosensing of Ebola virus cDNA by rolling circle and circle to circle amplification2017In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 93, p. 65-71Article in journal (Refereed)
    Abstract [en]

    This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries.

  • 32.
    Carter, Sarah-Sophia D.
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering.
    Mestres, Gemma
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tenje, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Diez-Escudero, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    The effect of unidirectional and recirculating flow on the behavior of MC3T3-E1 preosteoblast-like cells in a microfluidic systemManuscript (preprint) (Other academic)
    Abstract [en]

    Microfluidic systems have been proposed as a promising tool to capture enhanced physiological relevance in an in vitro setting. Although offering new opportunities, maintaining cells in such systems differs significantly from culturing cells under conventional static in vitro conditions. In order to directly compare the results from these two set-ups and to make more conclusive statements regarding the differences between them, it is important to carefully consider which factors can affect cell behavior. In this work, we investigated the effect of the flow type, namely unidirectional and recirculating flow, on the behavior of MC3T3-E1 preosteoblast-like cells and compared this to cells cultured under standard static cell culture conditions. Cell proliferation and differentiation (i.e. alkaline phosphatase activity, extracellular collagen and mineral matrix deposition) were overall higher for the cells maintained under static conditions when compared to either of the microfluidic set-ups. It should however be noted that cell proliferation showed to be highly dependent on the frequency of medium renewal and the amount of medium used in the static conditions. In addition to that, we demonstrated that the use of differentiation medium resulted in higher proliferation than regular growth medium, regardless the culture system used. No clear differences in cell proliferation and differentiation were found between the cells exposed to unidirectional or recirculating flow. Interestingly, secreted IGF-I was higher in the microfluidic systems, where unidirectional flow seemed to enhance the secretion. Overall, our results demonstrated that in vitro cell culture conditions can drastically affect cell response and should therefore be carefully considered. 

  • 33.
    Celsing, Margareta
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Scale-down modeling of the upstream process for production of Affibody® molecules2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    One of the crucial properties of a biopharmaceutical in order to be chosen as a potential drug candidate is that it can be expressed properly and effectively at commercial scale. Though, as fermentation at production scale is not workable during the early phases of research and development, scale-down models of cultivation at large scale are commonly applied. By using cultivation systems with decreased working volumes, resource consumption can be minimized. During the research process, a large number of molecules are typically screened, increasing the need for a high-throughput cultivation method. Though, prior to implementation of a downscaled system, similar behavior in terms of key performance parameters such as cell growth and protein expression should be demonstrated across the scales.

    In this work performed at Affibody AB, 12 different Affibody®️ molecules were expressed using cultivation in stirred tank reactors (STRs), shake flasks and microtiter plate (MTP). STR fermentations were performed in an automated system with controlled and monitored substrate supply, pH and agitation. Shake flask cultivations were operated in batch mode with an autoinducing medium, and MTP cultivations were performed in wells with diffusion-based glucose addition. The expression levels of the Affibody®️ molecules in the three production methods were quantified using SDS-PAGE and/or affinity chromatography.

    Through linear regression analysis it was indicated that the correlation in expression levels (g/L) quantified through affinity chromatography between STRs and shake flasks was limited, with a coefficient of determination (R2) of 0.47. This was also the case when using SDS-PAGE data (R2=0.42). Comparison of SDS-PAGE-quantified product concentrations of STR and MTP cultivations resulted in a slightly larger linear correlation (R2=0.6), suggesting that MTP was a somewhat more representative scale-down model of STR cultivations than shake flask. Though, coefficient of variation (CV%) values of product titers between the replicate cultivations were considered outside of the acceptable range, which restrained the ability to perform proper correlation analysis. Altogether the results indicate that optimization of the cultivation methods would be favorable in order to increase correlation and robustness of the scale-down models.

  • 34.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; Hannover Medical School, Hannover, Germany.
    CRISPR-Cas9: how research on a bacterial RNA-guided mechanism opened new perspectives in biotechnology and biomedicine2015In: EMBO Molecular Medicine, ISSN 1757-4676, E-ISSN 1757-4684, Vol. 7, no 4, p. 363-365Article in journal (Refereed)
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  • 35.
    Chudinova, Ekaterina
    et al.
    Tomsk Polytechnic University, Tomsk, Russia.
    Surmeneva, Maria
    Tomsk Polytechnic University, Tomsk, Russia.
    Koptioug, Andrei
    Mid Sweden University, Faculty of Science, Technology and Media, Department of Quality Technology and Management, Mechanical Engineering and Mathematics.
    Skoglund, Per
    Mid Sweden University, Faculty of Science, Technology and Media, Department of Quality Technology and Management, Mechanical Engineering and Mathematics.
    Surmenev, Roman
    Tomsk Polytechnic University, Tomsk, Russia.
    Additive manufactured Ti6Al4V scaffolds with the RF-magnetron sputter deposited hydroxyapatite coating2016In: Journal of Physics: Conference Series, Institute of Physics Publishing (IOPP), 2016, Vol. 669, article id 012004Conference paper (Refereed)
    Abstract [en]

    Present paper reports on the results of surface modification of the additively manufactured porous Ti6Al4V scaffolds. Radio frequency (RF) magnetron sputtering was used to modify the surface of the alloy via deposition of the biocompatible hydroxyapatite (HA) coating. The surface morphology, chemical and phase composition of the HA-coated alloy were studied. It was revealed that RF magnetron sputtering allows preparing a homogeneous HA coating onto the entire surface of scaffolds.

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  • 36.
    Chudinova, Ekaterina
    et al.
    Tomsk Polytechnic University, Tomsk, Russia.
    Surmeneva, Maria
    Tomsk Polytechnic University, Tomsk, Russia.
    Koptyug, Andrey
    Mid Sweden University, Faculty of Science, Technology and Media, Department of Quality Technology and Management, Mechanical Engineering and Mathematics.
    Selezneva, Irina
    Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Puschino.
    Skoglund, Per
    Mid Sweden University, Faculty of Science, Technology and Media, Department of Quality Technology and Management, Mechanical Engineering and Mathematics.
    Syrtanov, M
    Tomsk Polytechnic University, Tomsk, Russia.
    Surmenev, Roman
    Tomsk Polytechnic University, Tomsk, Russia.
    In Vitro Assessment of Hydroxyapatite Coating on the Surface of Additive Manufactured Ti6Al4V Scaffolds2017In: Materials Science Forum, ISSN 0255-5476, E-ISSN 1662-9752, Vol. 879, p. 2444-2449Article in journal (Refereed)
    Abstract [en]

    Custom orthopedic and dental implants may be fabricated by additive manufacturing (AM), for example using electron beam melting technology. This study is focused on the modification of the surface of Ti6Al4V alloy coin-like scaffolds fabricated via AM technology (EBM®) by radio frequency (RF) magnetron sputter deposition of hydroxyapatite (HA) coating. The scaffolds with HA coating were characterized by Scanning Electron microscopy, X-ray diffraction. HA coating showed a nanocrystalline structure with the crystallites of an average size of 32±9 nm. The ability of the surface to support adhesion and the proliferation of human mesenchymal stem cells was studied using biological short-term tests in vitro. In according to in vitro assessment, thin HA coating stimulated the attachment and proliferation of cells. Human mesenchymal stem cells cultured on the HA-coated scaffold also formed mineralized nodules.

  • 37.
    Dietrich, Franciele
    et al.
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences.
    Hammerman, Malin
    Lund Univ, Sweden.
    Bernhardsson, Magnus
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences.
    Eliasson, Pernilla T.
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences.
    Effect of storage and preconditioning of healing rat Achilles tendon on structural and mechanical properties2021In: Scientific Reports, E-ISSN 2045-2322, Vol. 11, no 1, article id 958Article in journal (Refereed)
    Abstract [en]

    Tendon tissue storage and preconditioning are often used in biomechanical experiments and whether this generates alterations in tissue properties is essential to know. The effect of storage and preconditioning on dense connective tissues, like tendons, is fairly understood. However, healing tendons are unlike and contain a loose connective tissue. Therefore, we investigated if storage of healing tendons in the fridge or freezer changed the mechanical properties compared to fresh tendons, using a pull-to-failure or a creep test. Tissue morphology and cell viability were also evaluated. Additionally, two preconditioning levels were tested. Rats underwent Achilles tendon transection and were euthanized 12 days postoperatively. Statistical analyzes were done with one-way ANOVA or Students t-test. Tissue force and stress were unaltered by storage and preconditioning compared to fresh samples, while high preconditioning increased the stiffness and modulus (p &lt;= 0.007). Furthermore, both storage conditions did not modify the viscoelastic properties of the healing tendon, but altered transverse area, gap length, and water content. Cell viability was reduced after freezing. In conclusion, preconditioning on healing tissues can introduce mechanical data bias when having extensive tissue strength diversity. Storage can be used before biomechanical testing if structural properties are measured on the day of testing.

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  • 38.
    Ding, Haozhong
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, 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, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, 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 Conjugates2019In: Cancers, ISSN 2072-6694, Vol. 11, no 8, article id 1168Article in journal (Refereed)
    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.

  • 39.
    Drobin, Kimi
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Marczyk, Michal
    Yale Univ, Sch Med, Dept Internal Med, Yale Canc Ctr, 333 Cedar St, New Haven, CT 06511 USA.;Silesian Tech Univ, Dept Data Sci & Engn, Gliwice 44100, Poland..
    Halle, Martin
    Karolinska Inst, Dept Mol Med & Surg, Stockholm 17176, Sweden.;Karolinska Univ Hosp, Reconstruct Plast Surg, Stockholm 17176, Sweden..
    Danielsson, Daniel
    Karolinska Inst, Div ENT Dis, Dept Clin Sci Intervent & Technol, Stockholm 14186, Sweden.;Karolinska Univ Hosp, Dept Oral & Maxillofacial Surg, Stockholm 17176, Sweden..
    Papiez, Anna
    Silesian Tech Univ, Dept Data Sci & Engn, Gliwice 44100, Poland..
    Sangsuwan, Traimate
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Ctr Radiat Protect Res, Stockholm 10691, Sweden..
    Bendes, Annika
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hong, Mun-Gwan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Qundos, Ulrika
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Harms-Ringdahl, Mats
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Ctr Radiat Protect Res, Stockholm 10691, Sweden..
    Wersall, Peter
    Karolinska Univ Hosp, Dept Radiotherapy, Stockholm 17176, Sweden..
    Polanska, Joanna
    Silesian Tech Univ, Dept Data Sci & Engn, Gliwice 44100, Poland..
    Schwenk, Jochen M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Haghdoost, Siamak
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Ctr Radiat Protect Res, Stockholm 10691, Sweden.;Univ Caen Normandy, Adv Resource Ctr HADrontherapy Europe ARCHADE, Cimap Laria, Dept Med, Caen 14076, France..
    Molecular Profiling for Predictors of Radiosensitivity in Patients with Breast or Head-and-Neck Cancer2020In: Cancers, ISSN 2072-6694, Vol. 12, no 3, article id 753Article in journal (Refereed)
    Abstract [en]

    Nearly half of all cancers are treated with radiotherapy alone or in combination with other treatments, where damage to normal tissues is a limiting factor for the treatment. Radiotherapy-induced adverse health effects, mostly of importance for cancer patients with long-term survival, may appear during or long time after finishing radiotherapy and depending on the patient's radiosensitivity. Currently, there is no assay available that can reliably predict the individual's response to radiotherapy. We profiled two study sets from breast (n = 29) and head-and-neck cancer patients (n = 74) that included radiosensitive patients and matched radioresistant controls. We studied 55 single nucleotide polymorphisms (SNPs) in 33 genes by DNA genotyping and 130 circulating proteins by affinity-based plasma proteomics. In both study sets, we discovered several plasma proteins with the predictive power to find radiosensitive patients (adjusted p < 0.05) and validated the two most predictive proteins (THPO and STIM1) by sandwich immunoassays. By integrating genotypic and proteomic data into an analysis model, it was found that the proteins CHIT1, PDGFB, PNKD, RP2, SERPINC1, SLC4A, STIM1, and THPO, as well as the VEGFA gene variant rs69947, predicted radiosensitivity of our breast cancer (AUC = 0.76) and head-and-neck cancer (AUC = 0.89) patients. In conclusion, circulating proteins and a SNP variant of VEGFA suggest that processes such as vascular growth capacity, immune response, DNA repair and oxidative stress/hypoxia may be involved in an individual's risk of experiencing radiation-induced toxicity.

  • 40.
    Ekstedt, Elias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Fryckstedt, Inna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Hyllander, Hanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Jonsson, Josefin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Ring, Elin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Wærn, Felix
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    The future of viral vectors for gene therapy2021Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Gene therapy is a fast growing technology that offers treatments for genetic diseases. The method is based on introducing genetic material into a patient to replace the disease-causing gene, using a vector. This report examines the potential of some viral vectors for gene therapy, to give Bio-Works Technologies a recommendation on what the future market demands. Oncolytic viruses, vaccines and gene editing are not treated in the report as a delimitation. 

    Viral vectors have different biological properties and require different purification methods, making them suitable for different applications in gene therapy. In the purification of the viruses it can be challenging to obtain a high purity and large-scale manufacturing. One major drawback with most purification methods is that they are not specific to just one virus, which leads to contaminants in the solution and lower purity. The viral vectors handled in the report are the adenovirus, adeno-associated virus, gammaretrovirus, lentivirus, alpharetrovirus, foamy virus, herpes simplex virus and baculovirus. These were chosen as they are relevant vectors for gene therapy and stay within the scope of the report.

    Lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) will dominate the gene therapy field in the coming years. This is based on the information that the use of AAVs and LVs in clinical trials have increased in recent years, while the other vectors mentioned above have slightly decreased or show no apparent change. However, challenges still remain in the purification processes. Ligands used in affinity chromatography for purification of AAVs are effective at removing most contaminants, but cannot distinguish between empty and loaded capsids, which can induce immune response when used clinically. This is the main challenge when purifying AAVs. The empty capsids can be removed with ion exchange chromatography, which results in higher purity but also lower recovery. There is no specific purifying method for LVs, therefore a lentivirus-specific affinity ligand, such as an antibody ligand, would be beneficial for the purification and manufacturing procedure. 

    In addition to AAVs and LVs, baculoviral vectors and foamy viral vectors show great potential in a long-term perspective but they only have been researched in preclinical studies. Moreover, herpes simplex viral vectors and adenoviral vectors show potential in cancer treatments or as vaccines rather than in augmentation gene therapy.

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  • 41.
    Ericson, Ossian
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry.
    Development of proteincarbohydrate hydrogels for cosmetic and pharmaceutical applications2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    A new kind of carbohydrate-binding protein, found in a soil-derived Bacteroidetes species, Chitinophaga pinensis, displays a high binding specificity for branched polysaccharides.

    The biological function of the protein, provisionally named “F-protein”, is still unknown; however, two examples from this new protein family have already been partly characterised. The protein family seems to have the ability to specifically bind branched polysaccharide chains, and each protein can bind multiple polysaccharide chains as it has multiple binding sites. This enables cross-linking of polysaccharides to form hydrogel materials. In this project we have investigated the carbohydrate binding of two F-domains from the Cpin 2580 gene that also includes a glycoside hydrolase family 18 (GH18) chitinase domain and found them to primarily bind to 1-6 linked β-glucans. We have found the GH18 domain’s chitinase activity to be negatively impacted in its naturally occurring F-domain “sandwich” configuration, hinting at a biological non-catalytic role for this domain. We have discovered unexpected chitin binding for the full complex of two Fdomains together with GH18. We have successfully formed hydrogels from scleroglucan at low concentration, using four recombinant proteins containing either of the F-domains, and shown that gels form both with and without an appended GH18 domain. We have investigated the evolutionary context of the F-domain through PSI-BLAST of the full Cpin 2580 protein and the Fdomain sequence alone. We have found the F-domain to occur predominantly appended to a glycoside hydrolase catalytic subunit with and without a secretion domain targeting the protein to the type IX secretion system (T9SS). We have also found it to occur with an alginate lyase subunit, in this case without a T9SS tail. We have theorised a biological role for the Cpin 2580 gene as an adhesion lectin targeting potentially a fungal cell wall, and a biological micro environment dominated by decomposing microbial biomass as carbohydrate source. The sequence analyses presented here will hopefully guiding future efforts to explore for additional F-domain containing genes in nature.

  • 42.
    Eriksson, Ella
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Investigation of possible improvements of the stability of bispecific ADAPT proteins2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The aim of the project was to identify positions and amino acids that contribute to improved structure and stability of bispecific ADAPT proteins. During the 20 weeks project period, different amino acid substitutions were analysed to evaluate the effect on the three-helical structure and stability of bispecific ADAPTs targeting human serum albumin (HSA) and tumor necrosis factor α (TNFα). Furthermore, the study also included identification of which amino acid substitutions that affect the simultaneous binding ability of the anti-TNFα ADAPT. The amino acids substitutions that demonstrated improved stability was further evaluated in two other bispecific ADAPT proteins targeting epithelial cell adhesion molecule (EpCAM), in terms of structure and stability.

    The TNFα-targeting ADAPT variants was produced in Escherichia coli (E. coli), purified through affinity chromatography using a HSA-coupled matrix and was further analysed and evaluated using SDS-PAGE, circular dichrosim, size-exclusion chromatography and surface plasmon resonance to detect expression levels, yields, thermal stability, secondary structure, and simultaneous binding to TNFα and HSA. Furthermore, the production, purification and evaluation were redone with other bispecific ADAPTs targeting EpCAM, to be able to draw more general conclusions. The outcome showed which amino acids substitutions in the scaffold that improve the structure and stability of the TNFα- and EpCAM-binding ADAPT protein variants, respectively.

    Some of the ADAPT variants targeting TNFα showed improved stability and increased melting temperature. One of the variants with most potential from these mutants was ADAPT_TNFα5_F21K, both able to refold after heat treatment and demonstrated a higher melting temperature in the same order as the original binder. The variant bound HSA but not TNFα, thus consequently was not able to bind TNFα and HSA simultaneously. The variants ADAPT_TNFα5_V17I and ADAPT_TNFα5_M22Q both demonstrated a clear alpha-helix structure, were able to refold after heat treatment and demonstrated simultaneous binding to TNFα and HSA. The melting temperature for ADAPT_TNFα5_V17I was the same as for the original binder (59°C) and ADAPT_TNFα5_M22Q showed a decreased melting temperature (45°C) compared to the original binder. The amino acid substitutions that improve the stability of the original binder was combined and two variants withthese mutations were designed. Unfortunately, these variants could not express in E. coli cells and were not able to be produced. For the EpCAM targeting mutants one variant, ADAPT_EpCAM_02_X11N, showed huge improvements of the stability and structure compared to the original binder ADAPT_EpCAM_02. This variant improved the melting temperature with 24°C compared to the original binder and was able to refold after heat treatment, which the original binder did not have the ability to do. However, ADAPT_EpCAM_02_X11N was not able to simultaneously bind EpCAM and HSA, demonstrating that the mutation also had an effect on the binding ability. In the variant ADAPT_EpCAM_08 the mutation Y5I improved the melting temperature with 14°C compared to the original binder and was able to refold after thermal denaturation. However, the simultaneous binding to EpCAM and HSA was negatively affected.

    The project results have contributed to better understanding of the bispecific ADAPT proteins, which enables further development of the scaffold. The amino acid positions in the scaffold that showed to be important for ADAPT structure and stability will be used in the design of a new ADAPT-library, from which new binders with improved structure and stability hopefully can be selected, which might have the potentially to be used as future therapeutics.

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  • 43.
    Fasterius, Erik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Exploring genetic heterogeneity in cancer using high-throughput DNA and RNA sequencing2018Doctoral thesis, comprehensive summary (Other academic)
    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.

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    PhD Thesis Erik Fasterius
  • 44.
    Fernandez Navarro, Jose
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Croteau, Deborah L.
    Laboratory of Molecular Gerontology, National Institute on Aging, Baltimore, MD 21224, USA.
    Jurek, Aleksandra
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andrusivova, Zaneta
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Yang, Beiming
    Laboratory of Molecular Gerontology, National Institute on Aging, Baltimore,MD 21224, USA.
    Wang, Yue
    Laboratory of MolecularGerontology, NationalInstitute on Aging, Baltimore,MD 21224, USA.
    Ogedegbe, Benjamin
    Laboratory of MolecularGerontology, NationalInstitute on Aging, Baltimore,MD 21224, USA.
    Riaz, Tahira
    Unit for Genome Dynamics,Department of Microbiology,University of Oslo and OsloUniversity Hospital, 0372Oslo, Norway.
    Støen, Mari
    Unit for Genome Dynamics,Department of Microbiology,University of Oslo and OsloUniversity Hospital, 0372Oslo, Norway.
    Desler, Claus
    Center for Healthy Aging,Department of Cellular andMolecular Medicine,University of Copenhagen,2200 Copenhagen, Denmark.
    Rasmussen, Lene Juel
    Center for Healthy Aging,Department of Cellular andMolecular Medicine,University of Copenhagen,2200 Copenhagen, Denmark.
    Tønjum, Tone
    Unit for Genome Dynamics,Department of Microbiology,University of Oslo and OsloUniversity Hospital, 0372Oslo, Norway.
    Galas, Marie-Christine
    University of Lille, Inserm,CHU Lille, UMR-S 1172 -Centre de RechercheJean-Pierre AUBERTNeurosciences et Cancer,59000 Lille, France.
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Bohr, Vilhelm A.
    Unit for Genome Dynamics,Department of Microbiology,University of Oslo and OsloUniversity Hospital, 0372Oslo, Norway.
    Spatial Transcriptomics Reveals Genes Associated with Dysregulated Mitochondrial Functions and Stress Signaling in Alzheimer Disease2020In: iScience, ISSN 2589-0042, Vol. 23, no 10, article id 101556Article in journal (Refereed)
    Abstract [en]

    Cellular Neuroscience; Omics; Transcriptomics Alzheimer disease (AD) is a devastating neurological disease associated with progressive loss of mental skills and cognitive and physical functions whose etiology is not completely understood. Here, our goal was to simultaneously uncover novel and known molecular targets in the structured layers of the hippocampus and olfactory bulbs that may contribute to early hippocampal synaptic deficits and olfactory dysfunction in AD mice. Spatially resolved transcriptomics was used to identify high-confidence genes that were differentially regulated in AD mice relative to controls. A diverse set of genes that modulate stress responses and transcription were predominant in both hippocampi and olfactory bulbs. Notably, we identify Bok, implicated in mitochondrial physiology and cell death, as a spatially downregulated gene in the hippocampus of mouse and human AD brains. In summary, we provide a rich resource of spatially differentially expressed genes, which may contribute to understanding AD pathology.

  • 45.
    Fleetwood, Filippa
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Klint, Susanne
    Hanze, Martin
    KTH, School of Biotechnology (BIO), Protein Technology.
    Gunneriusson, Elin
    Frejd, Fredrik
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity2014In: Scientific Reports, E-ISSN 2045-2322, Vol. 4, p. 7518-Article in journal (Refereed)
    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.

  • 46.
    Forsgren, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Brohede, Ulrika
    Sandvik AB, Stockholm.
    Strømme, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Engqvist, Håkan
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Co-loading of bisphosphonates and antibiotics to a biomimetic hydroxyapatite coating2011In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 33, no 6, p. 1265-1268Article in journal (Refereed)
    Abstract [en]

    We have incorporated bisphosphonates and antibiotics simultaneously into a biomimetic hydroxyapatite implant coating aiming to use the interaction between drug-molecules and hydroxyapatite to enable local release of the two different substances to obtain a dual biological effect. A sustained release over for 43 h of antibiotics (cephalothin) was achieved without negative interference from the presence of the bisphosphonate (clodronate) which, in turn, successfully bonded to the coating surface. To our knowledge, this is the first study that indicates the possibility to simultaneously incorporate both antibiotics and bisphosphonates to an implant coating, a strategy that is believed to improve implant stability and reduce implant-related infections.

  • 47.
    Friberg, Oscar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Engineered imaging scaffolds for cryo-EM of small proteins of interest2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Determining structures of proteins is important to understand protein functions, and a rapidly evolving technique in this field is cryogen electron microscopy. However, size limitations are preventing wider applications of the technique because small proteins have poor signal to noise ratios and are not possible to distinguish in single-particle images. The hypothesis of this project is that it is possible to image very small proteins, bypassing the conventional size limitations of single-particle cryo-EM, by utilizing a carrier protein-scaffold (Putrescine Aminotransferase; YgjG) connected through helical fusion to an affibody (Zwt) that can bind to a small protein of interest. The complex provides a sufficient size, symmetry, and rigidity for successful electron microscopy also of the non-covalently bound small protein of interest. To characterise the proposed scaffold, thermal stability through CD, binding of target protein in SPR, purity through SEC and experiments towards proof-of-concept in cryo-EM will be performed. The small protein of interest to be imaged in the proof-of-concept setup is another affibody, called Z963, that would be the smallest protein ever solved with cryo-EM. The results show that the investigated tetrameric protein scaffold is a highly stable protein (Tm~85oC) that can tolerate affibody fusion with retained binding function of multiple sites. The protein can be recombinantly expressed and purified in high yield and forms tetramers also when fused to affibody. The cryo-EM results are still pending, but promising grids have been created and in an initial particle selection clear 2-D classes that also reveal the small bound protein of interest have been generated. To conclude, biophysical characterization indicates that YgjG is a promising base structure for an imaging scaffold and preliminary single-particle cryo-EM analyses show that the proposed strategy to investigate structures of small proteins of interest is feasible.

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  • 48. Garousi, J.
    et al.
    Anderson, Ken
    KTH, School of Biotechnology (BIO), Protein Technology.
    Dam, J. H.
    Olsen, B. B.
    Orlova, A.
    Buijs, J.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Thisgaard, H.
    Tolmachev, V.
    The use of radiocobalt as a label improves PET imaging of EGFR using DOTA-conjugated affibody molecules2015In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, p. S244-S244Article in journal (Refereed)
  • 49.
    Garousi, Javad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    von Witting, Emma
    Borin, Jesper
    Vorobyeva, Anzhelika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Research Centrum for Oncotheranostics, Research School of Chemistry and Applied Biomedical Sciences, Research Tomsk Polytechnic University, Tomsk, Russia.
    Altai, Mohamed
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Vorontsova, Olga
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Konijnenberg, Mark W.
    Oroujeni, Maryam
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics. Uppsala University, Science for Life Laboratory, SciLifeLab. Research Centrum for Oncotheranostics, Research School of Chemistry and Applied Biomedical Sciences, Research Tomsk Polytechnic University, Tomsk, Russia.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Research Centrum for Oncotheranostics, Research School of Chemistry and Applied Biomedical Sciences, Research Tomsk Polytechnic University, Tomsk, Russia.
    Hober, Sophia
    Radionuclide therapy using ABD-fused ADAPT scaffold protein: Proof of Principle2021In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 266, article id 120381Article in journal (Refereed)
    Abstract [en]

    Molecular recognition in targeted therapeutics is typically based on immunoglobulins. Development of engineered scaffold proteins (ESPs) has provided additional opportunities for the development of targeted therapies. ESPs offer inexpensive production in prokaryotic hosts, high stability and convenient approaches to modify their biodistribution. In this study, we demonstrated successful modification of the biodistribution of an ESP known as ADAPT (Albumin-binding domain Derived Affinity ProTein). ADAPTs are selected from a library based on the scaffold of ABD (Albumin Binding Domain) of protein G. A particular ADAPT, the ADAPT6, binds to human epidermal growth factor receptor type 2 (HER2) with high affinity. Preclinical and early clinical studies have demonstrated that radiolabeled ADAPT6 can image HER2-expression in tumors with high contrast. However, its rapid glomerular filtration and high renal reabsorption have prevented its use in radionuclide therapy. To modify the biodistribution, ADAPT6 was genetically fused to an ABD. The non-covalent binding to the host's albumin resulted in a 14-fold reduction of renal uptake and appreciable increase of tumor uptake for the best variant, 177Lu-DOTA-ADAPT6-ABD035. Experimental therapy in mice bearing HER2-expressing xenografts demonstrated more than two-fold increase of median survival even after a single injection of 18 MBq 177Lu-DOTA-ADAPT6-ABD035. Thus, a fusion with ABD and optimization of the molecular design provides ADAPT derivatives with attractive targeting properties for radionuclide therapy.

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  • 50.
    Geraldine Guex, Anne
    et al.
    Empa, Switzerland; Empa, Switzerland.
    Poxson, David
    Linköping University, Department of Science and Technology, Laboratory of Organic Electronics. Linköping University, Faculty of Science & Engineering.
    Simon, Daniel
    Linköping University, Department of Science and Technology, Laboratory of Organic Electronics. Linköping University, Faculty of Science & Engineering.
    Berggren, Magnus
    Linköping University, Department of Science and Technology, Laboratory of Organic Electronics. Linköping University, Faculty of Science & Engineering.
    Fortunato, Giuseppino
    Empa, Switzerland.
    Rossi, Rene M.
    Empa, Switzerland.
    Maniura-Weber, Katharina
    Empa, Switzerland.
    Rottmar, Markus
    Empa, Switzerland.
    Controlling pH by electronic ion pumps to fight fibrosis2021In: Applied Materials Today, ISSN 2352-9407, Vol. 22, article id 100936Article in journal (Refereed)
    Abstract [en]

    Fibrosis and scar formation is a medical condition observed under various circumstances, ranging from skin wound healing to cardiac deterioration after myocardial infarction. Among other complex interdependent phases during wound healing, fibrosis is associated with an increased fibroblast to myofibroblast transition. A common hypothesis is that decreasing the pH of non-healing, alkaline wounds to a pH range of 6.0 to 6.5 increases healing rates. A new material-based strategy to change the pH by use of electronic ion pumps is here proposed. In contrast to passive acidic wound dressings limited by non-controlled delivery kinetics, the unique electronic ion pump design and operation enables a continuous regulation of pH by H+ delivery over prolonged durations. In an in vitro model, fibroblast to myofibroblast differentiation is attenuated by lowering the physiological pH to an acidic regime of 6.62 +/- 0.06. Compared to differentiated myofibroblasts in media at pH 7.4, gene and protein expression of fibrosis relevant markers alpha-smooth muscle actin and collagen 1 is significantly reduced. In conclusion, myofibroblast differentiation can be steered by controlling the pH of the cellular microenvironment by use of the electronic ion pump technology as new bioelectronic drug delivery devices. This technology opens up new therapeutic avenues to induce scar-free wound healing. (C) 2021 The Authors. Published by Elsevier Ltd.

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