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  • 1.
    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.

  • 2.
    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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  • 3.
    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, ISSN 1756-994X, 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.

  • 4. 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)
  • 5. 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. 

  • 6.
    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.

  • 7.
    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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  • 8.
    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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  • 9.
    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.

  • 10.
    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)
  • 11. 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)
  • 12.
    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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  • 13.
    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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  • 14.
    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)
  • 15. 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.

  • 16. 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)
  • 17.
    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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  • 18.
    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. 

  • 19.
    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.

  • 20.
    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.

  • 21.
    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.

  • 22.
    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.

  • 23.
    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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  • 24.
    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
  • 25.
    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.

  • 26.
    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.

  • 27.
    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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  • 28. 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)
  • 29.
    Hammar, Hugo
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Development of ultra-sensitive immunoassays for low abundance biomarkers in human plasma2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The SIngle Molecule Arrays technology (Simoa, Quanterix Corp) is an emerging approach enabling the detection of protein biomarkers in human body fluids. In contrast to other common or bead based assays, the system implements a digital read-out strategy capable of detecting singular captured protein molecules. The digital read-out system improves noteworthy sensitivity detecting below picomolar levels of proteins. For this reason the method is a prime candidate for detection and quantification of low abundance biomarkers. As a proof of concept study, here we apply the Simoa technology aiming to quantify twelve proteins associated with liver diseases. In particular we focused on the development of ultra-sensitive assays for targets for which other commercial assays were not available. The performance was tested at first using a pair of antibodies targeting the liver-derived protein IGFBP1. The assays showed an impressive lower limit of quantification of 5.6 fg/mL as compared to 2.6 pg/mL reported for ELISA assays. Further to this, we optimized the bead coupling protocol to reduce the amounts of antibody consumed. We achieved data from a 16-folds reduction in antibodies and observed relatively minor loss of assay performance. We obtained dilution-dependent curves with recombinant proteins or plasma samples for five candidate biomarkers for liver pathologies (HGF, HGFAC, FETUB, FGA, SPP1). A multiplexed Simoa assay targeting six cytokines was also applied for benchmarking the analysis of liver disease samples which elucidated some significant differences between liver disease groups. Further experiments will be required to fine tune and fully develop novel Simoa assays suitable for the detection of novel biomarkers circulating in blood.

  • 30.
    Hedberg, Elin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Selection and Characterisation of Affibody Molecules Intended for Drug Conjugates Targeting Cancer Cells2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Affibody molecules are small affinity proteins (6.5 kDa) suggested to substitute monoclonal antibodies in therapeutic applications, e.g., antibody-drug conjugates (ADCs) targeting biomarker proteins expressed on cancer cells. An affibody-drug conjugate (AffiDC) could be used to target these types of overexpressed proteins on cancer cells while offering attractive properties, such as rapid transportation and distribution in the body, as well as efficient tumour penetration. These AffiDCs could be used as a targeted cancer therapy for cancers that are yet to be treatable and curable, like urothelial cancers. 

    This study suggested the use of ABD-fused affibodies to target a novel cancer protein that has been shown to be overexpressed on cancer cells, including breast, pancreatic and urothelial cancer. Affibody candidates toward this novel target were selected from a recombinant library, of 1×1011 transformants, that is expressed using E. coli cell display system. The final candidates were subsequently biochemically characterized and assessed for affinity for the target. Three affibodies were finally identified and assessed in in vitro studies on mammalian cells, revealing two affibodies that appear to bind to the cell lines BT-474 and MCF-7 with KD ranging 10 to 100 nM.

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  • 31.
    Hedin, Blenda
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Characterization of novel bispecific ADAPTs selected for cancer-related targets2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Cancer is still one of the most common causes of death world-wide and in parallel there is a need to update the repertoire of therapies that withstand resistance of recurrent cancers. Since the introduction of antibody therapies as anti-cancer pharmaceuticals, recognized as immunotherapy in health care, it has been an increasing field in cancer therapy, as a more targeted treatment compared to chemotherapy. Despite the great success, immunotherapy rely on parenteral administration, partly due to poor tissue penetration. If the treatment is administered intravenously, specialized personnel is required, in addition to that it can be inconvenient for the patient. Also, pharmaceuticals based on antibodies often require costly production steps which yields a high-priced treatment.

    To approach this problem, researchers have developed small affinity domains with the aim to increase tissue penetration while keeping a high specificity to its target. Albumin Binding Domain Derived Affinity Protein (ADAPT) is an example of a small affinity domain of only 7 kDa, which is based on albumin binding domain (ABD) from the streptococcal protein G. Recently, it was shown that the ADAPTs can be further engineered to bind albumin and another relevant target protein of interest simultaneously, which suggests a tolerable half-life in patient serum, alternative administration routes and lower production costs compared to antibody treatments. Furthermore, less side effects are expected due to higher specificity compared to chemotherapy.

    This work presents the characterization of novel ADAPT proteins that the target the cancer relatedproteins C-C motif ligand 7 (CCL7), vascular endothelial growth factor A (VEGF-A) and carcinoembryonic antigen related cell adhesion molecule 5 (CEACAM5). The new constructs were produced recombinantly in Escherichia coli (E. coli) and purified using affinity chromatography. Moreover, the results demonstrate bispecific binding with high affinity towards serum albumin and CCL7 and CEACAM5 respectively, while the ADAPT variants targeting VEGF-A remain to be further developed. Lastly, the importance of different amino acids for structural and binding properties of one CEACAM5 binder are stated. It reveals that the target binding relies on hydrophobic interactions which also can be connected to its poor structural attributes. Accordingly, this project adds new insights about the ADAPTs which can be useful in research towards future clinical applications aimed to improve cancer treatments.

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  • 32.
    Hober, Andreas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Development of novel affinity enrichment strategies for clinical applications using selected reaction monitoring2022Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins are key components of any living organism and an essential part of life itself. They can provide cells with structure and perform life-sustaining intracellular reactions. As organisms grow more complex, this task expands even further. The proteins’ areas of responsibility suddenly also include communication and coordination between cells and throughout entire organisms, such as the human body. Everything that can be touched and felt on a living organism is composed of millions and millions of proteins tightly packed together. They are even the molecules responsible for propagating the signals that make up the sense of feeling. Understanding the role of proteins in the complex system of life is essential for understanding what makes up a healthy human and what causes disease. This knowledge makes up the foundation of modern medicine, and to further this knowledge, allowing for new treatments and preventative interventions, the study of proteins is crucial. The large-scale study of proteins, proteomics, is an extensive field of research where a vast toolbox of technologies has been implemented. The foundation for this toolbox is made up of mass spectrometry- and affinity-based technologies.

    In this thesis, both mass spectrometry-based proteomics and affinity-based proteomics will be explored. The first part, Paper I and Paper II, describe the use of selected reaction monitoring for measuring proteins of clinical relevance in human blood plasma. The second part, Paper III and Paper IV, highlight the importance of validating reagents used for affinity-based proteomics and how this can be achieved in a high throughput manner. Lastly, Paper V showcases how a combined strategy, relying on both affinity-based proteomics and mass spectrometry-based proteomics, can capitalize on the best properties of each technology and how this combined strategy can even be utilized for diagnostic purposes.

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  • 33.
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

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

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

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    Doctoral Thesis Hofstrom 2013
  • 34.
    Hudson, Elton P.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Nikoshkov, Andrej
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics.
    Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force2012In: PLOS ONE, E-ISSN 1932-6203, Vol. 7, no 5, p. e37429-Article in journal (Refereed)
    Abstract [en]

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

  • 35.
    Idris, Tasnim Yasin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Affibody phage display selections for lipid nanoparticle and affibody-mediated transient CAR T-cell therapy2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy which has shown promising results in treating patients suffering from oncological malignancies. Despite the targeted immune response that can be achieved, elaborate manufacturing and procedure processes emphasise room for improvement. Engineered T-cells have illustrated a high persistence in patients, exposing them to the risk of systemic toxicity. In-vivo transient CAR T-cells using self-amplifying mRNA by delivery through affinity protein coated lipid nanoparticles (LNP) is proposed as a standardised and reversible alternative, allowing for dosing when needed.

    Using phage display technology, selection of affibody molecules toward the three immune oncology proteins CD5, CD8 and CD19 was performed in four cycles. Monoclonal phage enzyme-linked immunosorbent assay (ELISA) and DNA sequencing identified seven putative candidates toward CD5, one putative candidate was isolated toward CD8, and three toward CD19. Surface plasmon resonance analysis (SPR) showed specific target binding of the CD5 candidate binders, while target binding could not be demonstrated for the CD8 and CD19 candidates. The identified CD5 binders could be conjugated to LNP for T-cell targeted delivery of self-amplifying mRNA encoding any CAR of interest.

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  • 36.
    Iseri, Emre
    KTH, School of Electrical Engineering and Computer Science (EECS), Intelligent systems, Micro and Nanosystems.
    Microfluidic Compartmentalization for Smart Materials, Medical Diagnostics and Cell Therapy2022Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The organisation of fluids in small compartments is ubiquitous in nature, such as in the cellular composition of all life. This work explores several engineering avenues where microscale fluid compartmentalization can bring novel material properties or novel functionality in life sciences or medicine. 

    Here, we introduce four unique compartmentalization methods: 1) 3D fluid self-organisation in microscaffolds (FLUID3EAMS), 2) 2D microcapillary arrays on a dipstick (Digital Dipstick), 3) a sliding microfluidic platform with cross-flow (Slip-X-Chip), and 4) compartmentalization by cutting of soft solid matter (Solidify & Cut). These methods were used in a wide range of applications. 

    Within the area of smart materials, we applied FLUID3EAMS to synthesize materials with temperature-tuneable permeability and surface energy and to establish, in a well-controlled fashion, tissue-like materials in the form of 3D droplet interface bilayer networks. Solidify & Cut was used to form soft composites with a new type of magnetic behaviour, rotation-induced ferromagnetism, that allows easy reprogramming of the magnetization of magnetopolymers. 

    Within the area of medical diagnostics, we applied Digital Dipstick to perform rapid digital bacterial culture in a dipstick format and obtained clinically relevant diagnostic results on samples from patients with a urinary tract infection. Furthermore, Slip-X-Chip enables particle concentration and washing as new functions in sliding microfluidic platforms, which significantly expands their potential application area. 

    Finally, within the area of cell therapy, we explored the microencapsulation of high concentrations of therapeutic cells and presented a novel technique to fabricate core-shell microcapsules by exploiting the superior material properties of spider silk membranes. 

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  • 37. Jeong, Yunjin
    et al.
    Svedberg, Gustav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Réu, Pedro
    Lee, Yongju
    Song, Seo Woo
    Na, Hunjong
    Lee, Amos Chungwon
    Choi, Yeongjae
    Gantelius, Jesper
    Andersson Svahn, Helene
    Kwon, Sunghoon
    Solid-phase PCR on graphically encoded microparticles for multiplexed colorimetric detection of bacterial DNAManuscript (preprint) (Other academic)
  • 38.
    Jernbom Falk, August
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    On the analysis of antibody repertoires2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The antibody repertoire is the ensemble of antibodies found in an individual at a given time. It displays high heterogeneity between individuals while being both largely temporally stable within an individual and rapidly responsive to immunological challenge. As distinct collections of antibodies within the repertoire contribute to the function and malfunction of the immune system, studying the many aspects of the antibody repertoire can give increased knowledge on antibody-mediated pathogen defense and autoimmune conditions.

    There are several emergent techniques for assessing different properties of the antibody repertoire as well as determining distinct antibodies of interest in health and disease. The studies presented in this thesis use planar and bead-based arrays to investigate parts of the antibody repertoire consisting of antibodies against SARS-CoV-2 proteins in serological studies, as well as autoantibodies against the large collection of antigens in the Human Protein Atlas. Paper I explores the autoantibody repertoires of patients with psychosis using planar arrays of 42 000 antigens followed by targeted bead arrays and identifies associations to specific symptoms. Paper II defines the baseline serological characteristics of a longitudinal cohort using a then recently developed multiplex serological assay and gives an early description of COVID-19 symptomatology. Paper III investigates the four-month persistence and antigen diversity of antibodies against SARS-CoV-2 following infection. This work is continued in Paper IV which examines the persistence of the humoral and cellular response to infection and their protective effect against reinfection. Paper V connects these parts by exploring the autoantibody repertoire of this longitudinal cohort and identifying new-onset autoantibodies emerging at infection using arrays of human and viral antigens. It associates three new-onset autoantibodies to post-COVID-19 symptoms and demonstrates sequence similarity between human and viral epitopes, which may indicate molecular mimicry.

    Antibody repertoires are heterogeneous and multifaceted, requiring several methods for full comprehension. The present investigation encompasses the analysis of one facet using antigen arrays and contributes to knowledge on disease-associated autoantibody repertoires as well as the prevalence and persistence of the serological and autoantibody response emerging after viral infection. This work represents a small step towards the goal of understanding the full repertoire complexity. Emergent large-scale techniques combined with the herein described analysis are together poised to identify clinically relevant antigens and advance knowledge on the diversity and heterogeneity of the antibody repertoire.

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  • 39.
    Jiménez Requena, Albert
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Biomarker development for early diagnostics of Duchenne Muscular dystrophy2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Background: Duchenne muscular dystrophy is an X-linked chromosomal inheritedrecessive severe muscular dystrophy disorder, caused by a mutation in thedystrophin gene. Affected individuals undergo a progressive disease, where theyexperience a loss of muscle mass and consequently the loss of muscle function, overthe years, sorrowfully, with fatal ending.

    The screening of newborns using dried blood spots (DBS) could aid early diagnoseand initiation of treatment. DBS are a minimal invasive method of collecting bloodby disposing a small volume of blood, a droplet, on specially prepared filter paperfor subsequent analysis and storage[1].

    Objectives: The aim of this study is to optimize elution of proteins from DSS andexplore detection of biomarkers for DMD

    Methods: A total of 15 serum samples, collected at clinical sites were analyzedthrough DSS elution by BCA, SDS-PAGE electrophoresis and suspension bead arrayplatform to assess CA3 as a biomarker targeted for DMD detection.Results: The presented results show that there is a relation between theconcentration of Tween 20 detergent and the protein extract, as the higher theconcentration the higher the protein extracted was. As well as reported an evidentrift effect between 1 and 5% Tween 20. Although elution of proteins is overall highcertain proteins are prone to elute at high detergent concentration whereas othersnot. Results also showed that the bibliographic time of elution could be optimizedand reduced up to 4 hours of elution, in two 2-hour cycles.

    Conclusions: The developed elution method in combination with the bead-basedimmunoassay constitute a feasible method for rapid assessment of biomarkersabundance such as CA3. The optimization of the elution joint method is feasible andcustomizable for the selected biomarkers. 

  • 40.
    Johansson, Staffan
    et al.
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    Eklund, Anders
    Umeå University.
    Malm, Jan
    Umeå University.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    Roxhed, Niclas
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    A MEMS-based passive hydrocephalus shunt with adaptive flow characteristics2013Conference paper (Refereed)
    Abstract [en]

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

  • 41.
    Johansson, Staffan
    et al.
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    Roxhed, Niclas
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    A MEMS-based passive air flow regulator for handheld breath diagnostics2014In: Sensors and Actuators A-Physical, ISSN 0924-4247, E-ISSN 1873-3069, Vol. 215, p. 65-70Article in journal (Refereed)
    Abstract [en]

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

  • 42.
    Kaczmarzyk, Danuta
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Georg-August-University, Germany.
    Hudson, Elton P.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Fulda, Martin
    Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria2016In: AMB Express, ISSN 2191-0855, E-ISSN 2191-0855, Vol. 6, no 1, p. 1-9, article id 7Article in journal (Refereed)
    Abstract [en]

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

  • 43.
    Kanagaraj, Loguprasanth
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Comparative proteomic analysis of the closely related honey samples by mass spectrometry2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Honey has been widely used as both medicine and food since olden times. It is produced by honey bees, who combine floral nectar with enzymes in their top aero-digestive system, and then concentrate it within the beehive through a process called dehydration. Depending on its floral source, honey can have a highly complex chemical composition. Carbohydrates play a predominant role in the composition of honey. Furthermore, major components of honey include organic acids, minerals, amino acids, vitamins, and proteins.

    Until recently, little was known about the honey proteome, as it had not been extensively explored due to the relatively small quantity of protein present in honey. This study describes the complete proteomic profile of three different closely related honey samples, highlighting their unique and shared proteins. An improved isolation method involving dialysis was employed for the effective extraction of proteins, and mass spectrometry-based proteomics was utilized, offering advantages such as selectivity, sensitivity, and the ability to analyze large datasets.

    The proteins identified have essential potential benefits for mankind, including antioxidant, wound healing, immunomodulatory, anti-aging, anti-cancer, anti-diabetic, hepato-renal protective, and neuroprotective activities associated with the major royal jelly proteins found in all honey samples. The distinctive functions of each honey sample are discussed in this research, providing insights for the development of therapeutic processes and products.

  • 44.
    Karrani, Suha
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Drug Testing in a FN-silk Network Model of Breast Cancer2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Breast cancer is a heterogeneous disease grouped into distinct subtypes based on histological and molecular classification. The diversity of breast cancer poses challenges in treatment, as patients exhibit varied responses to drugs. Individualized treatment based on the molecular classification of breast cancer has become a major focus in overcoming treatment failures. To identify subtype-specific drugs, it is essential to establish models that recapitulate the in vivo conditions. Traditional breast cancer models based on 2D cultures or 3D spheroids lack the crucial cell-extracellular matrix (ECM) interactions observed in tumors. A newly developed 3D model using the recombinant spider silk protein termed the FN-silk network, has demonstrated ECM-like interactions and maintenance of subtype-specific marker expression in breast cancer cells.

    This project hypothesized that a) it is possible to use the FN-silk network to culture cells into a tumor model to measure the cytotoxicity of antineoplastic agents and b) different culturing conditions will impact the drug response. To test these hypotheses, drug treatment experiments were conducted on the breast cancer cells MCF-7, SK-BR-3, and MDA-MB-231 cultured in 2D, spheroids, and FN-silk networks. To obtain a comprehensive assessment the drug response was evaluated using the parameters EC50, GR, NDR, and DSS.

    The study results suggest that the FN-silk network holds promise as a base for a model for evaluating the effects of antineoplastic agents. Moreover, comparing drug responses among breast cancer cells cultured in 2D, spheroid, and FN-silk networks revealed a significant impact of the different culturing conditions on drug response. The results also demonstrated that the GR, NDR, and DSS provide deeper insights into drug response beyond the EC50 calculations, which can have misleading results.

    This study concludes that the FN-silk network has the potential to support the establishment of a valuable tumor model for the development of personalized breast cancer treatments and thereby contribute to improved success rates in drug development.

  • 45. Kennedy, S
    et al.
    Fasterius, Erik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Al-Khalili Szigyarto, Cristina
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Kolch, W
    et al.,
    Adaptive rewiring of protein-protein interactions and signal flow in the EGFR signaling network by mutant RASManuscript (preprint) (Other academic)
  • 46.
    Klaesson, Helena
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Developing a 3D Co-Culture Model of Breast Cancer2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Breast cancer is the leading cause of cancer deaths among women worldwide, affecting millions each year. The incidence of breast cancer is increasing and there is a need for efficient preclinical models for drug discovery and the study of cancer biology. Breast cancer is a heterogeneous disease and can be stratified based on the expression of histological markers and gene signature. Current methods for studying cancer and drug efficacy mainly rely on two-dimensional (2D) cell cultures. The drawback with these models is that they do not correctly mimic the complex in vivo conditions of the tissue, lacking for instance the contact and support from the extracellular matrix (ECM). Three-dimensional (3D) cell cultures, which can mimic the support of the ECM, could provide a better preclinical model to study cancer biology and drug efficacy. Furthermore, the tumor microenvironment is complex and involves tumor cells as well as cells from the surrounding stroma. Stromal components, such as fibroblasts and endothelial cells, highly impact tumor progression, migration, and invasiveness, thus of importance for potential targets for cancer therapies. 

    The aim of this project was to develop a novel 3D breast cancer co-culture model containing breast cancer cells, fibroblasts, and endothelial cells to represent the complexity of the tumor microenvironment. The model is based on Biosilk, a recombinant spider silk harboring the RGD motif, which can form a scaffold for 3D cell growth. Three different breast cancer cell lines were used to represent the three main breast cancer subtypes (i.e., MCF-7 (luminal-like), SK-BR-3 (HER2-overexpression), and MDA-MB-231 (basal-like). The cell lines HDFn and HDMEC were used to represent fibroblasts and endothelial cells, respectively. The protocol to form the scaffold was optimized for the content of tumoral cells and media compatibility, the latter being done by monitoring cell proliferation using Alamar blue. Afterward, the specificity of biomarkers unique for each cell line was confirmed at the transcriptional level using reverse transcription-quantitative PCR (RT-qPCR). Such markers were subsequently used to analyze the 

    Biosilk co-culture constructs using immunofluorescence. Such final steps revealed that, for MDA-MB-231 and MCF-7, it was possible to grow cancer and stromal cells (i.e., fibroblasts and endothelial) within the Biosilk 3D scaffold.

  • 47.
    Kroll, Jens
    et al.
    Westfälische Wilhelms-Universität Münster, Germany.
    Klinter, Stefan
    Westfälische Wilhelms-Universität Münster, Germany.
    Schneider, Cornelia
    Westfälische Wilhelms-Universität Münster, Germany.
    Voß, Isabella
    Westfälische Wilhelms-Universität Münster, Germany.
    Steinbüchel, Alexander
    Westfälische Wilhelms-Universität Münster, Germany.
    Plasmid addiction systems: Perspectives and applications in biotechnology2010In: Microbial Biotechnology, ISSN 1751-7907, E-ISSN 1751-7915, Vol. 3, no 6, p. 634-657Article, review/survey (Refereed)
    Abstract [en]

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

  • 48.
    Kuang, Qie
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Purhonen, Pasi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Alander, Johan
    Karolinska Institutet.
    Svensson, Richard
    Karolinska Institutet.
    Hoogland, Veronika
    Karolinska Institutet.
    Winerdal, Jens
    Karolinska Institutet.
    Spahiu, Linda
    Karolinska institutet.
    Ottosson-Wadlund, Astrid
    Karolinska Insitutet.
    Jegerschöld, Caroline
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Department of Biosciences and Nutrition, Karolinska Institutet.
    Morgenstern, Ralf
    Karolinska Institutet.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Medical Engineering, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation2017In: Scientific Reports, E-ISSN 2045-2322, Vol. 7, article id 7897Article in journal (Refereed)
    Abstract [en]

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

  • 49.
    Lama, Lara
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Novel methods for improving rapid paper-based protein assays with gold nanoparticle detection2017Licentiate thesis, monograph (Other academic)
    Abstract [en]

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

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

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

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

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  • 50.
    Lama, Lara
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Dias, Jorge T.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    A lateral flow cardiac troponin-T assay with colorimetric signal enhancementManuscript (preprint) (Other academic)
    Abstract [en]

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

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