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  • 1. Abramson, Alex
    et al.
    Frederiksen, Morten Revsgaard
    Vegge, Andreas
    Jensen, Brian
    Poulsen, Mette
    Mouridsen, Brian
    Jespersen, Mikkel Oliver
    Kirk, Rikke Kaae
    Windum, Jesper
    Hubálek, František
    Water, Jorrit J.
    Fels, Johannes
    Gunnarsson, Stefán B.
    Bohr, Adam
    Straarup, Ellen Marie
    Ley, Mikkel Wennemoes Hvitfeld
    Lu, Xiaoya
    Wainer, Jacob
    Collins, Joy
    Tamang, Siddartha
    Ishida, Keiko
    Hayward, Alison
    Herskind, Peter
    Buckley, Stephen T.
    Roxhed, Niclas
    KTH, School of Electrical Engineering and Computer Science (EECS), Intelligent systems, Micro and Nanosystems. MIT, Dept Chem Engn, 77 Massachusetts Ave, Cambridge, MA 02139 USA; MIT, David H Koch Inst Integrat Canc Res, 77 Massachusetts Ave, Cambridge, MA 02139 USA.
    Langer, Robert
    Rahbek, Ulrik
    Traverso, Giovanni
    Oral delivery of systemic monoclonal antibodies, peptides and small molecules using gastric auto-injectors2021In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 40, no 1, p. 103-109Article in journal (Refereed)
    Abstract [en]

    Oral administration provides a simple and non-invasive approach for drug delivery. However, due to poor absorption and swift enzymatic degradation in the gastrointestinal tract, a wide range of molecules must be parenterally injected to attain required doses and pharmacokinetics. Here we present an orally dosed liquid auto-injector capable of delivering up to 4-mg doses of a bioavailable drug with the rapid pharmacokinetics of an injection, reaching an absolute bioavailability of up to 80% and a maximum plasma drug concentration within 30 min after dosing. This approach improves dosing efficiencies and pharmacokinetics an order of magnitude over our previously designed injector capsules and up to two orders of magnitude over clinically available and preclinical chemical permeation enhancement technologies. We administered the capsules to swine for delivery of clinically relevant doses of four commonly injected medications, including adalimumab, a GLP-1 analog, recombinant human insulin and epinephrine. These multi-day dosing experiments and oral administration in awake animal models support the translational potential of the system. 

  • 2. Acero Sanchez, Josep Ll.
    et al.
    Joda, Hamdi
    Henry, Olivier Y. F.
    Solnestam, Beata W.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlén, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Laddach, Nadja
    Ramakrishnan, Dheeraj
    Riley, Ian
    Schwind, Carmen
    Latta, Daniel
    O'Sullivan, Ciara K.
    Electrochemical Genetic Profiling of Single Cancer Cells2017In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 89, no 6, p. 3378-3385Article in journal (Refereed)
    Abstract [en]

    Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present asimple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.

  • 3.
    Ahrenstedt, Lage
    et al.
    KTH, School of Biotechnology (BIO). KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Olksanen, Antti
    VTT Technical Research Centre of Finland.
    Salmien, Kristian
    VTT Technical Research Centre of Finland.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Paper dry strength improvement by xyloglucan addition: Wet-end application, spray coating and synergism with borate2008In: Holzforschung, ISSN 0018-3830, E-ISSN 1437-434X, Vol. 62, no 1, p. 8-14Article in journal (Refereed)
    Abstract [en]

    The polysaccharide xyloglucan as a wet-end additive improves paper properties. In the present study, paper strength improvement was analysed for dry handsheets made from chemical, mechanical and recycled pulps coated with xyloglucan in a spray application. Results are compared with sheets made from the same pulps treated with xyloglucan in the wet-end. Kraft pulp handsheets of bleached hardwood and softwood showed significant improvements of tensile, tear and Z-strength by xyloglucan spray treatment versus wet-end application, whereas handsheets of de-inked and thermomechanical pulp were improved only slightly. In both wet-end and spray applications, the effect of xyloglucan addition was intimately related to the presence of non-cellulosic components on the fibre surface. Further strength improvements were obtained for chemical pulps by addition of borax to the spray solution, which were likely to be due to the formation of borate-mediated xyloglucan cross-links. Spray coating of xyloglucan, with or without borax, thus represents a potential new application of this polysaccharide to increase paper dry strength.

  • 4.
    Akhtar, Ahmad Saleem
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Centrifugal microfluidics-based point of care diagnostics at resource limited settings2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Advancements in medical diagnostics have allowed us to understand the underlying mechanism and treat the root cause for many diseases which had been causing morbidity and mortality up until this point in human history. Furthermore, many of the standard diagnostic procedures have now been transformed to provide answers at or near the point-of-care. However, the effects of these positive developments have not trickled down to the parts of our society which are considered underdeveloped and lack the necessary infrastructure and facilities. Diagnostics in such resource limited settings still lag behind and fail to provide the requisite healthcare. 

    In order to translate the diagnostic solutions designed for central laboratories to resource limited settings, there are certain challenges that need to be addressed, such as portability, reduction in cost and ease-of-use, while keeping the sensitivity and specificity at the required level. The work presented in this thesis focuses on addressing some of these issues by using microfluidics to develop diagnostic platforms that are suitable to be used in resource limited settings. 

    In paper I, a very low-cost and simple centrifugal microfluidic platform was developed to be used in settings which do not have a reliable supply of electricity. The platform uses a smartphone as a source of power and the sensors of the phone for speed control.

    In paper II, a portable and low-cost diagnostic platform was developed for multiplexed detection of biomarkers based on centrifugal microfluidics. The platform uses colorimetric detection and a simple readout method which does not require a spectrophotometer for quantification.

    In paper III, a platform was developed for COVID-19 diagnostics which combines centrifugal microfluidics with a novel bead-based strategy for signal enhancement. The platform uses fluorescent detection with a smartphone readout and has the capability to process up to 20 samples at the same time.

    In paper IV, as a follow up of paper III, a more advanced platform was developed for COVID-19 diagnostics which allows the operator to carry out nucleic acid amplification in a completely automated manner, from adding the sample to getting the final result.

    In paper V, an alternative method for detection of SARS-CoV-2 was developed using electrochemical biosensing. This work combines the electrochemical technique with a flexible printed circuit board for a rapid, amplification-free and label-free detection of target SARS-CoV-2 sequences.

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    Ahmad_Thesis
  • 5.
    Akhtar, Ahmad Saleem
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lapins, Noa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Moura, João Martinho
    International Iberian Nanotechnology Laboratory.
    Paula, Luis
    International Iberian Nanotechnology Laboratory.
    Pedro, Adriano
    International Iberian Nanotechnology Laboratory.
    Martins, Fabio
    International Iberian Nanotechnology Laboratory.
    Mota, Duarte
    International Iberian Nanotechnology Laboratory.
    Pinto, Ines Fernandes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Martins, Marco
    International Iberian Nanotechnology Laboratory.
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Fully automated centrifugal microfluidic platform for COVID-19 detection using computer vision-based readoutManuscript (preprint) (Other academic)
    Abstract [en]

    COVID-19 pandemic made it evident that the world is unprepared for effectively tackling a pandemic resulting from an infectious disease. The conventional diagnostic methods for detection of infectious diseases were limited to centralized laboratories. As the burden of testing increased with the spread of the disease, the centralized testing facilities were strained for resources and personnel. These problems were further exacerbated in low- and middle-income countries where the health and transport infrastructure are not very well developed. To overcome this reliance on centralized testing and to facilitate decentralized testing, focus was shifted towards development of novel point-of-care diagnostic methods. We report the development of a fully automated centrifugal microfluidic platform that uses loop mediated isothermal amplification (LAMP) combined with computer vision-based readout for COVID-19 detection. The integrated platform allows sample to answer analysis at the push of a single button and can process 26 samples in 40 minutes. The platform performs a completely automated assay protocol involving heating, rotation and detection without the need for user intervention. A limit of detection of approximately 100 RNA copies in 10 µL reaction was achieved using RNA fragments spiked in water and similar results were obtained for artificial saliva samples. 

  • 6.
    Akhtar, Ahmad Saleem
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Soares, Ruben R. G.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Pinto, Ines Fernandes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Center for the Advancement of Integrated Medical and Engineering Sciences, AIMES.
    A portable and low-cost centrifugal microfluidic platform for multiplexed colorimetric detection of protein biomarkers2023In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 1245, article id 340823Article in journal (Refereed)
    Abstract [en]

    Cytokines play a very important role in our immune system by acting as mediators to put up a coordinated defense against foreign elements in our body. Elevated levels of cytokines in the body can signal to an ongoing response of the immune system to some abnormality. Thus, the quantification of a panel of cytokines can provide valuable information regarding the diagnosis of specific diseases and state of overall health of an individual. Conventional Enzyme Linked Immunosorbent Assay (ELISA) is the gold-standard for quantification of cytokines, however the need for trained personnel and expensive equipment limits its application to centralized laboratories only. In this context, there is a lack of simple, low-cost and portable devices which can allow for quantification of panels of cytokines at point-of-care and/or resource limited settings.

    Here, we report the development of a versatile, low-cost and portable bead-based centrifugal microfluidic platform allowing for multiplexed detection of cytokines with minimal hands-on time and an integrated colorimetric signal readout without the need for any external equipment. As a model, multiplexed colorimetric quantification of three target cytokines i.e., Tumor necrosis factor alpha (TNF-α), Interferon gamma (IFN-γ) and Interleukin-2 (IL-2) was achieved in less than 30 min with limits of detection in ng/mL range. The developed platform was further evaluated using spiked-in plasma samples to test for matrix interference. The ease of use, low-cost and portability of the developed platform highlight its potential to serve as a sample-to-answer solution for detection of cytokine panels in resource limited settings.

  • 7. Albèr, C.
    et al.
    Brandner, B. D.
    Björklund, S.
    Billsten, P.
    Corkery, Robert
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Engblom, J.
    Effects of water gradients and use of urea on skin ultrastructure evaluated by confocal Raman microspectroscopy2013In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 11, p. 2470-2478Article in journal (Refereed)
    Abstract [en]

    The rather thin outermost layer of the mammalian skin, stratum corneum (SC), is a complex biomembrane which separates the water rich inside of the body from the dry outside. The skin surface can be exposed to rather extreme variations in ambient conditions (e.g. water activity, temperature and pH), with potential effects on the barrier function. Increased understanding of how the barrier is affected by such changes is highly relevant for regulation of transdermal uptake of exogenous chemicals. In the present study we investigate the effect of hydration and the use of a well-known humectant, urea, on skin barrier ultrastructure by means of confocal Raman microspectroscopy. We also perform dynamic vapor sorption (DVS) microbalance measurements to examine the water uptake capacity of SC pretreated with urea. Based on novel Raman images, constructed from 2D spectral maps, we can distinguish large water inclusions within the skin membrane exceeding the size of fully hydrated corneocytes. We show that these inclusions contain water with spectral properties similar to that of bulk water. The results furthermore show that the ambient water activity has an important impact on the formation of these water inclusions as well as on the hydration profile across the membrane. Urea significantly increases the water uptake when present in skin, as compared to skin without urea, and it promotes formation of larger water inclusions in the tissue. The results confirm that urea can be used as a humectant to increase skin hydration.

  • 8.
    Allerbring, E. B.
    et al.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Duru, A. D.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Uchtenhagen, H.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Madhurantakam, C.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Tomek, M. B.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Grimm, Sebastian
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Mazumdar, P. A.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Friemann, R.
    Univ Gothenburg, Dept Chem Biochem & Biophys, Gothenburg, Sweden..
    Sandalova, T.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    Uhlin, M.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Stockholm, Sweden..
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Achour, A.
    Karolinska Inst, Ctr Infect Med CIM, Dept Med Huddinge, Stockholm, Sweden..
    The unexpected T-cell recognition of an altered peptide ligand is driven by reversed thermodynamics and an alternative structural hotspot2011In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 135, p. 70-70Article in journal (Other academic)
  • 9. Alvarez, Francisco J.
    et al.
    Ryman, Kicki
    Hooijmaijers, Cornelis
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ljungdahl, Per O.
    Diverse Nitrogen Sources in Seminal Fluid Act in Synergy To Induce Filamentous Growth of Candida albicans2015In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 81, no 8, p. 2770-2780Article in journal (Refereed)
    Abstract [en]

    The pathogenic fungus Candida albicans is the leading cause of vulvovaginal candidiasis (VVC). VVC represents a major quality- of-life issue for women during their reproductive years, a stage of life where the vaginal epithelium is subject to periodic hormonally induced changes associated with menstruation and concomitant exposure to serum as well as potential intermittent contact with seminal fluid. Seminal fluid potently triggers Candida albicans to switch from yeastlike to filamentous modes of growth, a developmental response tightly linked to virulence. Conversely, vaginal fluid inhibits filamentation. Here, we used artificial formulations of seminal and vaginal fluids that faithfully mimic genuine fluids to assess the contribution of individual components within these fluids to filamentation. The high levels of albumin, amino acids, and N-acetylglucosamine in seminal fluid act synergistically as potent inducers of filamentous growth, even at atmospheric levels of CO2 and reduced temperatures (30 degrees C). Using a simplified in vitro model that mimics the natural introduction of seminal fluid into the vulvovaginal environment, a pulse of artificial seminal fluid (ASF) was found to exert an enduring potential to overcome the inhibitory efficacy of artificial vaginal fluid (AVF) on filamentation. These findings suggest that a transient but substantial change in the nutrient levels within the vulvovaginal environment during unprotected coitus can induce resident C. albicans cells to engage developmental programs associated with virulent growth.

  • 10.
    Alvez, Maria Bueno
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Edfors, Fredrik
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    von Feilitzen, Kalle
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zwahlen, Martin
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Mardinoglu, Adil
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. Centre for Host-Microbiome Interactions, Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College London, London, SE1 9RT, UK.
    Edqvist, Per Henrik
    Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Sjöblom, Tobias
    Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Lundin, Emma
    Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Rameika, Natallia
    Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Enblad, Gunilla
    Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Lindman, Henrik
    Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Höglund, Martin
    Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
    Hesselager, Göran
    Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
    Stålberg, Karin
    Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden.
    Enblad, Malin
    Department of Surgical Sciences, Uppsala University, Uppsala, Sweden.
    Simonson, Oscar E.
    Department of Surgical Sciences, Uppsala University, Uppsala, Sweden.
    Häggman, Michael
    Department of Surgical Sciences, Uppsala University, Uppsala, Sweden.
    Axelsson, Tomas
    Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
    Åberg, Mikael
    Department of Medical Sciences, Clinical Chemistry and SciLifeLab Affinity Proteomics, Uppsala University, Uppsala, Sweden.
    Nordlund, Jessica
    Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
    Zhong, Wen
    Science for Life Laboratory, Department of Biomedical and Clinical Sciences (BKV), Linköping University, Linköping, Sweden.
    Karlsson, Max
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gyllensten, Ulf
    Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Ponten, Fredrik
    Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Fagerberg, Linn
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
    Next generation pan-cancer blood proteome profiling using proximity extension assay2023In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1, article id 4308Article in journal (Refereed)
    Abstract [en]

    A comprehensive characterization of blood proteome profiles in cancer patients can contribute to a better understanding of the disease etiology, resulting in earlier diagnosis, risk stratification and better monitoring of the different cancer subtypes. Here, we describe the use of next generation protein profiling to explore the proteome signature in blood across patients representing many of the major cancer types. Plasma profiles of 1463 proteins from more than 1400 cancer patients are measured in minute amounts of blood collected at the time of diagnosis and before treatment. An open access Disease Blood Atlas resource allows the exploration of the individual protein profiles in blood collected from the individual cancer patients. We also present studies in which classification models based on machine learning have been used for the identification of a set of proteins associated with each of the analyzed cancers. The implication for cancer precision medicine of next generation plasma profiling is discussed.

  • 11.
    Andrup, Klara
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Development of a method to detect lysis and investigation if ozone has a lysing effect on Escherichia coli2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    This project was conducted at Sangair. The company is currently developing a medical device aimed at treating bacteremia with ozone. Bacteremia is a condition that occurs when bacteria enters the bloodstream, and can trigger sepsis and septic shock, potentially leading to death if untreated. Antibiotics have traditionally been the way to treat bacteremia, but the looming threat of antibiotic resistance worldwide threatens this way of treatment, and research into novel approaches to eradicate the bacteria needs to be done. As part of Sangairs development, an investigation was done to see how ozone interacts with bacteria. The project aimed to 1) develop a method to detect bacterial lysis and 2) use the method to detect if ozone had a lysing effect on the bacterial model organism Escherechia coli. To test the method, the BL21 strain of  E. coli was transformed with the pUC-19 plasmid to produce the reporter enzyme Beta-galactosidase. The Beta-galactosidase activity was then measured in a supernatant of sonicated bacteria, which confirmed the suitability of the method to detect bacteria cell lysis. To be able to see if ozone had a lysing effect, while still being able to measure Beta-galactosidase, it was found that the optimal setup for this was using an ozone concentration of 6 g/m3, a gas flow of 5 ml/min and a liquid flow of 25 ml/min. The results acquired with this setup indicated that ozone had a lysing effect on E. coli but more studies are needed to verify this. It was further investigated if Beta-galactosidase detection could be improved by addition of bovine serum albumin (BSA) to quench residual ozone in the samples, but the results showed that it did not have any effect on the Beta-galactosidase enzymatic activity. As a final experiment, endotoxins that were released upon treatment were also measured, and it was found that when bacteria are treated with ozone, lipopolysaccharides (LPS) and peptidoglycans are released, further confirming lysis of the bacterial cells. 

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    fulltext
  • 12.
    Asp, Julia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Profiling the Blood Proteome in Autoimmune Disease Using Proximity Extension Assay2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Autoimmune diseases are complex, chronic, inflammatory conditions characterized by dysregulation of the immune system, resulting in inflammation and damage to various tissues, cells and organs. These diseases significantly impact individuals’ quality of life and often contribute to increased mortality risk in the presence of comorbidities. However, due to the diverse array of symptoms associated with different autoimmune diseases, accurate diagnosis, prognosis, and treatment evaluation pose significant challenges. Thus, there is a pressing need for the discovery of novel biomarkers. 

    In this study, a comprehensive analysis of 944 plasma samples using the OlinkR Explore platform was conducted, generating data on 1463 unique proteins. Based on the expression data, associated proteins were identified for six selected autoimmune diseases, namely multiple sclerosis, myositis, rheumatoid arthritis, systemic sclerosis, Sjögren’s syndrome, and systemic lupus erythematosus, as well as some of their defined subgroups. These are prospective biomarkers and have the potential to aid in early diagnosis, therapeutic intervention, subgroup identification, disease differentiation, and disease prognosis. Notably, some of these proteins have not been previously associated with the specific diseases in the existing literature, especially not in plasma samples, thereby offering intriguing new perspectives for biomarker development. However, it is of great importance to conduct robust validation studies in independent cohorts to confirm the outcomes of this study. 

    In summary, our findings highlight the potential utility of these proteomic plasma biomarkers in improving the early detection, subgroup characterization, and disease differentiation of autoimmune diseases. The identification of these proteins will hopefully stimulate further investigation in the field of biomarker research and potential advancements in personalized medicine. 

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    fulltext
  • 13.
    Barnkob, Rune
    et al.
    Tech Univ Denmark, Lyngby, Denmark .
    Iranmanesh, Ida
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Bruus, Henrik
    Tech Univ Denmark, Lyngby, Denmark .
    Measuring acoustic energy density in microchannel acoustophoresis using a simple and rapid light-intensity method2012In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 12, no 13, p. 2337-2344Article in journal (Refereed)
    Abstract [en]

    We present a simple and rapid method for measuring the acoustic energy density in microchannel acoustophoresis based on light-intensity measurements of a suspension of particles. The method relies on the assumption that each particle in the suspension undergoes single-particle acoustophoresis. It is validated by the single-particle tracking method, and we show by proper re-scaling that the re-scaled light intensity plotted versus re-scaled time falls on a universal curve. The method allows for analysis of moderate-resolution images in the concentration range encountered in typical experiments, and it is an attractive alternative to particle tracking and particle image velocimetry for quantifying acoustophoretic performance in microchannels.

  • 14. Basile, Walter
    et al.
    Sachenkova, Oxana
    Light, Sara
    Elofsson, Arne
    KTH, Centres, SeRC - Swedish e-Science Research Centre.
    High GC content causes orphan proteins to be intrinsically disordered2017In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 13, no 3, article id e1005375Article in journal (Refereed)
    Abstract [en]

    De novo creation of protein coding genes involves the formation of short ORFs from noncoding regions; some of these ORFs might then become fixed in the population These orphan proteins need to, at the bare minimum, not cause serious harm to the organism, meaning that they should for instance not aggregate. Therefore, although the creation of short ORFs could be truly random, the fixation should be subjected to some selective pressure. The selective forces acting on orphan proteins have been elusive, and contradictory results have been reported. In Drosophila young proteins are more disordered than ancient ones, while the opposite trend is present in yeast. To the best of our knowledge no valid explanation for this difference has been proposed. To solve this riddle we studied structural properties and age of proteins in 187 eukaryotic organisms. We find that, with the exception of length, there are only small differences in the properties between proteins of different ages. However, when we take the GC content into account we noted that it could explain the opposite trends observed for orphans in yeast (low GC) and Drosophila (high GC). GC content is correlated with codons coding for disorder promoting amino acids. This leads us to propose that intrinsic disorder is not a strong determining factor for fixation of orphan proteins. Instead these proteins largely resemble random proteins given a particular GC level. During evolution the properties of a protein change faster than the GC level causing the relationship between disorder and GC to gradually weaken.

  • 15.
    Battisti, Umberto Maria
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Univ Gothenburg, Dept Chem & Mol Biol, SE-41296 Gothenburg, Sweden..
    Monjas, Leticia
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. Univ Gothenburg, Dept Chem & Mol Biol, SE-41296 Gothenburg, Sweden..
    Akladios, Fady
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Univ Gothenburg, Dept Chem & Mol Biol, SE-41296 Gothenburg, Sweden..
    Matic, Josipa
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Univ Gothenburg, Dept Chem & Mol Biol, SE-41296 Gothenburg, Sweden..
    Andresen, Eric
    Univ Gothenburg, Dept Chem & Mol Biol, SE-41296 Gothenburg, Sweden..
    Nagel, Carolin H.
    Univ Gothenburg, Dept Chem & Mol Biol, SE-41296 Gothenburg, Sweden..
    Hagkvist, Malin
    Univ Gothenburg, Dept Chem & Mol Biol, SE-41296 Gothenburg, Sweden..
    Haversen, Liliana
    Univ Gothenburg, Dept Mol & Clin Med, SE-41345 Gothenburg, Sweden.;Sahlgrens Univ Hosp, SE-41345 Gothenburg, Sweden..
    Kim, Woonghee
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Boren, Jan
    Univ Gothenburg, Dept Mol & Clin Med, SE-41345 Gothenburg, Sweden.;Sahlgrens Univ Hosp, SE-41345 Gothenburg, Sweden..
    Mardinoglu, Adil
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. Kings Coll London, Fac Dent Oral & Craniofacial Sci, Ctr Host Microbiome Interact, London SE1 9RT, England..
    Grotli, Morten
    Univ Gothenburg, Dept Chem & Mol Biol, SE-41296 Gothenburg, Sweden..
    Exploration of Novel Urolithin C Derivatives as Non-Competitive Inhibitors of Liver Pyruvate Kinase2023In: Pharmaceuticals, E-ISSN 1424-8247, Vol. 16, no 5, article id 668Article in journal (Refereed)
    Abstract [en]

    The inhibition of liver pyruvate kinase could be beneficial to halt or reverse non-alcoholic fatty liver disease (NAFLD), a progressive accumulation of fat in the liver that can lead eventually to cirrhosis. Recently, urolithin C has been reported as a new scaffold for the development of allosteric inhibitors of liver pyruvate kinase (PKL). In this work, a comprehensive structure-activity analysis of urolithin C was carried out. More than 50 analogues were synthesized and tested regarding the chemical features responsible for the desired activity. These data could pave the way to the development of more potent and selective PKL allosteric inhibitors.

  • 16.
    Baumann, Martin J.
    KTH, School of Biotechnology (BIO).
    Xyloglucan-active enzymes: properties, structures and applications2007Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Cellulosic materials are the most abundant renewable resource in the world; plant cell walls are natural composite materials containing crystalline cellulose embedded in a matrix of hemicelluloses, structural proteins, and lignin. Xyloglucans are an important group of hemicelluloses, which coat and cross-link crystalline cellulose in the plant cell wall. In this thesis, structure-function relationships of a range of xyloglucan-active enzymes were examined.

    A paradigm for efficient enzymatic biomass utilization is the cellulosome of the anaerobic bacterium Clostridium thermocellum. The cellulosome is a high molecular weight complex of proteins with diverse enzyme activities, including the inverting xyloglucan endo-hydrolase CtXGH74A. The protein structure of CtXGH74A was solved in complex with xyloglucan oligosaccharides (XGOs) which stabilized disordered loops of the apo-structure. Further detailed kinetic and product analyses were used to conclusively demonstrate that CtXGH74A is an endo-xyloglucase that produces Glc4-based XGOs as limit digestion products.

    In comparison, the retaining glycoside hydrolase family 16 (GH16) contains hydrolytic endo-xyloglucanases as well as xyloglucan transglycosylases (XETs) from plants. To elucidate the determinants of the transglycosylase/hydrolysis ratio in GH16 xyloglucan-active enzymes, a strict transglycosylase, PttXET16-34 from hybrid aspen, was compared structurally and kinetically with the closely related hydrolytic enzyme NXG1 from nasturtium. A key loop extension was identified in NXG1, truncation of which yielded a mutant enzyme that exhibited an increased transglycosylase rate and reduced hydrolytic activity. Kinetic studies were facilitated by the development of new, sensitive assays using well-defined XGOs and a series of chromogenic XGO aryl-glycosides.

    A detailed understanding of GH16 xyloglucan enzymology has paved the way for the development of a novel chemo-enzymatic approach for biomimetic fiber surface modification, in which the transglycosylating activity of PttXET16-34 was employed. Aminoalditol derivates of XGOs were used as key intermediates to incorporate novel chemical functionality into xyloglucan, including chromophores, reactive groups, protein ligands, and initiators for polymerization reactions. The resulting modified xyloglucans were subsequently bound to a range of cellulose materials to radically alter surface properties. As such, the technology provides a novel, versatile toolkit for fiber surface modification.

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  • 17.
    Beckvid, Cecilia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Mapping genomic interactions in horse lung tissue using UMI-4C2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The horse is a highly athletic species, partly due to the thousands of years of selective breeding that has occurred since its domestication. Further, for the past 300 years, racehorses have been under an intense selection of traits associated with superior racing performance, which has further enhanced the athletic performance. Understanding the genetics behind this athletic performance is of importance for both the horse racing industry as well as for increasing the knowledge of certain conditions and diseases in horses as well as other species. A recent study identified a single nucleotide polymorphism (SNP) having significant associations with racing performance. In this degree project, the genomic region containing this SNP (22:46717860C>T) was further investigated through the mapping of its genomic interactions using targeted chromosome conformation capture with unique molecular identifiers (UMI-4C). Our hypothesis was that this genomic region interacts with and regulates a gene called EDN3. Previously prepared 3C DNA templates from horse lung tissue were sonicated, adapter-ligated, PCR amplified using primers targeting six different loci of interest, and sequenced. No direct interactions were found between the SNP and the EDN3 gene. However, multiple potential interactions were shared between them, suggesting that they interact with the same loci and might, therefore, indirectly interact with each other. Further, potential interactions were found between the SNP and loci within the genes GNAS and ZNF831, as well as between the SNP and the promoter of the gene LOC729296. However, the data obtained was scarce, and to be able to draw a clear conclusion regarding the interaction partners of the genomic region containing the SNP 22:46717860C>T, more data is required.

  • 18. Bell, E.
    et al.
    Lamminmäki, T.
    Alneberg, Johannes
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Andersson, Anders F.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Qian, C.
    Xiong, W.
    Hettich, R. L.
    Frutschi, M.
    Bernier-Latmani, R.
    Active sulfur cycling in the terrestrial deep subsurface2020In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 14, no 5, p. 1260-1272Article in journal (Refereed)
    Abstract [en]

    The deep terrestrial subsurface remains an environment where there is limited understanding of the extant microbial metabolisms. At Olkiluoto, Finland, a deep geological repository is under construction for the final storage of spent nuclear fuel. It is therefore critical to evaluate the potential impact microbial metabolism, including sulfide generation, could have upon the safety of the repository. We investigated a deep groundwater where sulfate is present, but groundwater geochemistry suggests limited microbial sulfate-reducing activity. Examination of the microbial community at the genome-level revealed microorganisms with the metabolic capacity for both oxidative and reductive sulfur transformations. Deltaproteobacteria are shown to have the genetic capacity for sulfate reduction and possibly sulfur disproportionation, while Rhizobiaceae, Rhodocyclaceae, Sideroxydans, and Sulfurimonas oxidize reduced sulfur compounds. Further examination of the proteome confirmed an active sulfur cycle, serving for microbial energy generation and growth. Our results reveal that this sulfide-poor groundwater harbors an active microbial community of sulfate-reducing and sulfide-oxidizing bacteria, together mediating a sulfur cycle that remained undetected by geochemical monitoring alone. The ability of sulfide-oxidizing bacteria to limit the accumulation of sulfide was further demonstrated in groundwater incubations and highlights a potential sink for sulfide that could be beneficial for geological repository safety.

  • 19.
    Bellinitzky, Matilda
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Studies on Fc-γ Receptor-IgG Interactions using a Biosensor Methodology2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    In the development of monoclonal antibody biosimilar products to be used as therapeutics, the Fc-receptor binding properties must be comparable to those of the innovator product. Hence, it is in the interest of biosimilar companies such as Xbrane Biopharma AB to de- velop analytical methods that support the assessment of this quality attribute throughout the cell line development and cell culture process development. In this study an antibody acting as an inhibitor to the, on tumor cells, overexpressed protein CD38, and with affinity for Fc gamma receptors located on immune effector cells will be of primary interest. An in- teraction involved in initiating antibody-mediated cell-dependent cytotoxicity, a mechanism of action utilized in therapeutic monoclonal antibodies. This study was therefore carried out, with the purpose to assess if the FcγRIIIa-IgG complex is suitable to be investigated with Bio-Layer Interferometry, more specifically, an Octet RED96E instrument. Thus, a methodology was created to provide useful kinetic parameters, such as the dissociation con- stant. The originator drug with differences in regards to shelf-life was utilized for a full affinity determination. In addition, a full affinity determination was also done with the an- tibody when deglycosylated.  Firstly, a decision was made regarding which bioassay format to use and then the optimal ligand concentration for the specific affinity determination was determined based on the obtained data. Moreover, the dissociation constant was obtained with both steady state analysis and full kinetics results. They were both reasonable when comparing to literature. The dissociation constant was very similar for the antibody despite variability in shelf-life. In conflict with literature data, deglycosylation of the antibody did not show a clear increase of the dissociation constant. Hence, did not indicate a negative impact on the affinity. However, the methodology requires further experiments. This in- cludes validation, comparison to other similar methods in regards to both economic and scientific aspects, analysis of the specific biosimilar not only the originator and evaluate the used data processing. Furthermore, it is necessary to further assess at what stage of the process development the attribute should be tested to adjust and evaluate the method based on those conditions.

  • 20.
    Bengtsson, Sofia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Identification of Monoclonal Antibodies:Peptide Mass Fingerprinting (PMF) with Matrix Assisted Laser Desorption/Ionization (MALDI), Time of Flight (ToF), Mass Spectrometry (MS) and Protein Peptide Mapping (PPM) with Capillary Electrophoresis (CE)2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The number of monoclonal antibodies used in pharmaceuticals is increasing sharply. These medicines are expensive, and the risk of counterfeiting is high. The need to develop a method for rapid and precise identification of monoclonal antibodies is therefore urgent. For identification, analyses were performed with Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) on nine monoclonal antibodies. The focus was to investigate whether significant physiochemical features and unique amino acid sequences were present and could be distinguished. Various analyses with MALDI-ToF-MS were used to both separate the monoclonal antibodies based on their physicochemical properties and annotate amino acid sequences containing key fragments. With the methods based on capillary electrophoresis, separation was also achieved. CZE is preferred over CGE as the amount of data obtained from CZE is greater and sample preparation is simpler. In summary, an identification process protocol was designed and is initiated with MALDI-ToF-MS analyses of reduced-form monoclonal antibodies against known references. A hypothesis is then formulated based on which antibodies look the most similar. Finally, these are analysed by CZE to determine the identity of the monoclonal antibody.

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  • 21.
    Bergstrand, Jan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Miao, Xinyan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Venugopal Srambickal, Chinmaya
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Auer, Gert
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, K7,Z1 00, S-17176 Stockholm, Sweden..
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Fast, streamlined fluorescence nanoscopy resolves rearrangements of SNARE and cargo proteins in platelets co-incubated with cancer cells2022In: Journal of Nanobiotechnology, E-ISSN 1477-3155, Vol. 20, no 1, article id 292Article in journal (Refereed)
    Abstract [en]

    Background Increasing evidence suggests that platelets play a central role in cancer progression, with altered storage and selective release from platelets of specific tumor-promoting proteins as a major mechanism. Fluorescence-based super-resolution microscopy (SRM) can resolve nanoscale spatial distribution patterns of such proteins, and how they are altered in platelets upon different activations. Analysing such alterations by SRM thus represents a promising, minimally invasive strategy for platelet-based diagnosis and monitoring of cancer progression. However, broader applicability beyond specialized research labs will require objective, more automated imaging procedures. Moreover, for statistically significant analyses many SRM platelet images are needed, of several different platelet proteins. Such proteins, showing alterations in their distributions upon cancer progression additionally need to be identified. Results A fast, streamlined and objective procedure for SRM platelet image acquisition, analysis and classification was developed to overcome these limitations. By stimulated emission depletion SRM we imaged nanoscale patterns of six different platelet proteins; four different SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) mediating protein secretion by membrane fusion of storage granules, and two angiogenesis regulating proteins, representing cargo proteins within these granules coupled to tumor progression. By a streamlined procedure, we recorded about 100 SRM images of platelets, for each of these six proteins, and for five different categories of platelets; incubated with cancer cells (MCF-7, MDA-MB-231, EFO-21), non-cancer cells (MCF-10A), or no cells at all. From these images, structural similarity and protein cluster parameters were determined, and probability functions of these parameters were generated for the different platelet categories. By comparing these probability functions between the categories, we could identify nanoscale alterations in the protein distributions, allowing us to classify the platelets into their correct categories, if they were co-incubated with cancer cells, non-cancer cells, or no cells at all. Conclusions The fast, streamlined and objective acquisition and analysis procedure established in this work confirms the role of SNAREs and angiogenesis-regulating proteins in platelet-mediated cancer progression, provides additional fundamental knowledge on the interplay between tumor cells and platelets, and represent an important step towards using tumor-platelet interactions and redistribution of nanoscale protein patterns in platelets as a basis for cancer diagnostics.

  • 22.
    Beven, Laure
    et al.
    Univ Bordeaux, Villenave Dornon, France ; INRA Villenave Dornon, France .
    Charenton, Claire
    Univ Bordeaux, Villenave Dornon, France ; INRA Villenave Dornon, France .
    Dautant, Alain
    Univ Bordeaux, Bordeaux, France ; IBMC, CNRS, Bordeaux, France.
    Bouyssou, Guillaume
    Univ Bordeaux, Villenave Dornon, France ; INRA Villenave Dornon, France .
    Labroussaa, Fabien
    Univ Bordeaux, Villenave Dornon, France ; INRA Villenave Dornon, France .
    Sköllermo, Anna
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Blanchard, Alain
    Univ Bordeaux, Villenave Dornon, France ; INRA Villenave Dornon, France .
    Sirand-Pugnet, Pascal
    Univ Bordeaux, Villenave Dornon, France ; INRA Villenave Dornon, France .
    Specific Evolution of F-1-Like ATPases in Mycoplasmas2012In: PLOS ONE, E-ISSN 1932-6203, Vol. 7, no 6, p. e38793-Article in journal (Refereed)
    Abstract [en]

    F1F0 ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F1F0 ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the alpha, beta, gamma and e subunits of F-1 ATPases and could form an F-1-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F-1-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F-1-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F-1-like structure is associated with a hypothetical X-0 sector located in the membrane of mycoplasma cells.

  • 23.
    Birgersson, Madeleine
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Role and mechanism of estrogen receptor beta in the ovary and colon2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Estrogen regulates a variety of important physiological functions in both males and females, where the regulation of female reproduction and the development of sexual organs are typical examples. The effects of estrogen are predominantly exerted via signaling through the two nuclear receptors estrogen receptor α (ERα) and β (ERβ), or the membrane G protein-coupled estrogen receptor 1 (GPER1). While estrogen signaling is important for human health, dysregulation of signaling can have adverse effects and impact the development and progression of a wide range of diseases including reproductive disorders and cancer.

    ERβ has been shown to be highly important for ovarian function by regulating folliculogenesis and ovulation but has also been implied to protect against the development of colorectal cancer (CRC) by mediating the effects of estrogen. Despite the known role of ERβ, there is a lack of mechanistic understanding regarding how ERβ acts under both normal conditions and during disease. The overall aim of this thesis was to characterize the function and molecular mechanism of endogenous ERβ and to decipher its role in the normal ovary as well as its impact on colitis and CRC development. To further understand the role of estrogen signaling in the colon, we also aimed to identify sex differences during CRC development. 

    In paper I we characterized the full cistrome of endogenous ovarian ERβ in the mouse and explored its transcriptional impact. We confirmed a direct role for ERβ in the regulation of essential ovarian functions and identified a novel crosstalk with the nuclear receptor LRH-1.  

    In paper II we induced colitis-associated CRC (CAC) in intestinal epithelial-specific ERβ knockout mice and identified a protective effect by intestinal ERβ against tumor development in both male and female mice. We further characterized sex-dependent effects and proposed an underlying mechanism involving the regulation of TNFα/NFκB signaling. 

    In paper III we expanded the investigation of sex-dependent changes during chemically-induced colitis in wildtype mice and identified a sex-specific response related to inflammatory response. We further found that male mice have an enhanced response to induced colitis.

    In paper IV the transcriptome of colitis-induced tumors and their immune cell infiltration was explored in wildtype and intestinal epithelial-specific ERβ knockout mice of both sexes. This showed that sex differences in the transcriptome appear to be dependent on the expression of ERβ. Also, the identified ERβ-dependent changes in the tumor transcriptome of female mice were specifically related to immune response. We corroborated an impact of ERβ on the infiltration of immune cells, especially a reduction of regulatory T cell and NK cell recruitment. 

    In summary, this thesis provides new mechanistic understanding of the transcriptional role of ERβ in the normal ovary and in the colon microenvironment. This includes the discovery of crosstalk with LRH-1 in the ovary and NFκB in the colon. Our characterization provides a foundation to develop targeted therapies for improved fertility and chemoprevention in CRC. This thesis also highlights the importance of including both sexes in colitis and CRC research to advance our knowledge and improve treatment development.

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  • 24.
    Birgersson, Madeleine
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Holm, Matilda
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Stepanauskaite, Lina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Gallardo-Dodd, Carlos
    Hases, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Kutter, Claudia
    Archer, Amena
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Williams, Cecilia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Intestinal estrogen receptor beta modulates the tumor immune microenvironment in a mouse model of colitis-associated cancerManuscript (preprint) (Other academic)
    Abstract [en]

    Chronic inflammation promotes the development of colorectal cancer (CRC), as evidenced by patients with inflammatory bowel disease (IBD), and sex disparities are evident in CRC. The tumor microenvironment (TME) is composed of stromal cells and infiltrating immune cells that directly affect processes including antitumor immunity. We have previously shown that intestinal estrogen receptor beta (ERβ) protects against colitis and colitis-induced cancer (CAC) by modulating inflammatory signaling and that males are more sensitive to the induction of colitis and cancer. However, sex differences between tumors and the impact of ERβ the tumor immune microenvironment have not been investigated. In this study, we have analyzed colon samples from AOM/DSS-treated wild-type and ERβKOVil mice (that lack intestinal ERβ) and profiled the differences in the transcriptome and immune response to CAC on the basis of sex and ERβ expression. RNA-sequencing revealed differences in gene expression and enriched biological processes depending on sex and genotype, and the immune response to cancer appears altered between tumors from female WT and ERβKOVil mice. Immunostaining subsequently showed that tumors from ERβKOVil mice display significantly increased CD68+ macrophage infiltration, decreased CD3+ T cell infiltration, and, strikingly, impaired NK cell infiltration. Here, for the first time, we show that intestinal ERβ modulates the tumor immune microenvironment during CAC and that lack of intestinal ERβ appears to promote the formation of an immunosuppressive TME. Our findings indicate that activation of ERβ could be used to treat CRC, possibly together with immunotherapies, and provide a foundation for future studies investigating ERβ and immunity. 

  • 25.
    Birgersson, Madeleine
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. Department of Biosciences and Nutrition, Karolinska Institutet, 141 83 Huddinge, Sweden.
    Indukuri, Rajitha
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Antonson, Per
    Department of Biosciences and Nutrition, Karolinska Institutet, 141 83 Huddinge, Sweden.
    Nalvarte, Ivan
    Department of Biosciences and Nutrition, Karolinska Institutet, 141 83 Huddinge, Sweden.
    Archer, Amena
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics. Department of Biosciences and Nutrition, Karolinska Institutet, 141 83 Huddinge, Sweden.
    Williams, Cecilia
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics. Department of Biosciences and Nutrition, Karolinska Institutet, 141 83 Huddinge, Sweden.
    ERβ in Granulosa Cell Tumors and Its Clinical Potential2023In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Endocrinology, ISSN 1945-7170, Vol. 164, no 6Article, review/survey (Refereed)
    Abstract [en]

    Granulosa cell tumors (GCTs) are rare ovarian tumors comprising an adult and a juvenile subtype. They have a generally good prognosis, but the survival rate drastically declines in patients with late-stage or recurring tumors. Due to the rarity of GCTs, the tumor type is largely understudied and lacks a specific treatment strategy. Estrogen receptor beta (ERβ/ESR2) has been found to be highly expressed in GCTs, which could be of therapeutic importance since it can be targeted with small molecules. However, its role in GCTs is not known. In this review, we summarize the current knowledge about the action of ERβ in the ovary and discuss its prospective role in GCTs.

  • 26.
    Birgersson, Madeleine
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Indukuri, Rajitha
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Lindquist, Linnea
    Stepanauskaite, Lina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Luo, Qing
    Deng, Qiaolin
    Archer, Amena
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Williams, Cecilia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Ovarian ERβ cistrome and transcriptome reveal chromatin interaction with LRH-1Manuscript (preprint) (Other academic)
    Abstract [en]

    Background:  Estrogen receptor beta (ERβ, Esr2) plays a pivotal role in folliculogenesis and ovulation, yet its exact mechanism of action is mainly uncharacterized.

    Results: We here performed ChIP-sequencing of mouse ovaries followed by complementary RNA-sequencing of wild-type and ERβ knockout ovaries. By integrating the cistrome and transcriptome, we identify its direct target genes and enriched biological functions in the ovary. This demonstrates a strong impact on genes regulating organism development, cell migration, lipid metabolism, response to hypoxia, and response to estrogen. Cell-type deconvolution analysis of the bulk RNA-seq data revealed a decrease in luteal cells and an increased proportion of theca cells and a specific type of cumulus cells upon ERβ loss. Moreover, we identify a significant overlap with the gene regulatory network of liver receptor homolog 1 (LRH-1). ERβ and LRH-1 extensively bind to the same chromatin locations in granulosa cells and we corroborate simultaneous co-binding using ChIP re-ChIP, at the ERβ-repressed gene Greb1. At other shared sites (by ERβ-upregulated genes Cyp11a1 and Fkbp5), they do not bind simultaneously. Transactivation assay experimentation further show that ERβ and LRH-1 can inhibit their respective transcriptional activity at classical response elements.

    Conclusions: We characterize genome-wide ERβ chromatin binding and gene regulations which reveal extensive crosstalk between ERβ and LRH-1. We experimentally corroborate co-binding to target genes and impact on transactivation. Our data offer genome-wide mechanistic underpinnings of ovarian physiology and fertility.

  • 27.
    Björklund, Frida
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Subcellular mapping of cell types in healthy human pancreatic islets2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Pancreatic islets are composed of endocrine cells that secrete hormones essential for blood-glucose homeostasis. Prior research has revealed that the gene expression and functionality of human islet cells is heterogenous. However, it is not currently understood how the heterogeneity correlates to normal islet cell function and dysfunction in diabetes pathogenesis. Subsequently, an international collaborative project has been initiated to elucidate what constitutes islet cell heterogeneity from a transcriptional, proteomic, and functional perspective in both health and disease. In this study, a highly multiplex tissue image assay was developed to allow for the study of the localization and distribution of proteins previously identified to correlate with functional activity and heterogeneity in islet cells using the CO-Detection by indEXing (CODEX) platform. In total, 22 proteins were studied simultaneously of which 10 were specifically expressed in islet cells. These included generic pancreatic markers such as C-peptide (C-pep) marking insulin-secreting β-cells, glucagon (GCG) and somatostatin (SST), but also less well-characterized proteins such as Shisa like 2B (FAM159B) and Neural proliferation, differentiation and control 1 (NPDC1). The multiplex tissue imaging allowed for single-cell analysis of protein expression in human islet cells showing that most islet specific proteins were heterogeneously expressed. The observations made in this study serves as a validation to that the human islet microenvironment is highly complex due to islet cell heterogeneity. Additionally, the study demonstrated that multiplex tissue imaging has the potential to reveal novel cell types and interactions.

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  • 28.
    Bladh, Oscar
    et al.
    Department of Clinical Sciences, Karolinska Institutet, Danderyd Hospital, 1882 88 Stockholm, Sweden.
    Marking, Ulrika
    Department of Clinical Sciences, Karolinska Institutet, Danderyd Hospital, 1882 88 Stockholm, Sweden.
    Havervall, Sebastian
    Department of Clinical Sciences, Karolinska Institutet, Danderyd Hospital, 1882 88 Stockholm, Sweden.
    Norin, Nina Greilert
    Department of Clinical Sciences, Karolinska Institutet, Danderyd Hospital, 1882 88 Stockholm, Sweden.
    Aguilera, Katherina
    Department of Clinical Sciences, Karolinska Institutet, Danderyd Hospital, 1882 88 Stockholm, Sweden.
    Hober, Sophia
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Smed-Sörensen, Anna
    Division of Immunology and Allergy, Department of Medicine Solna, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
    Gordon, Max
    Department of Clinical Sciences, Karolinska Institutet, Danderyd Hospital, 1882 88 Stockholm, Sweden.
    Blom, Kim
    Department of Clinical Sciences, Karolinska Institutet, Danderyd Hospital, 1882 88 Stockholm, Sweden; Public Health Agency of Sweden, Solna, Sweden.
    Åberg, Mikael
    Department of Medical Sciences, Clinical Chemistry and SciLifeLab Affinity Proteomics, Uppsala University, Uppsala, Sweden.
    Klingström, Jonas
    Public Health Agency of Sweden, Solna, Sweden; Division of Molecular Medicine and Virology, Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden.
    Thålin, Charlotte
    Department of Clinical Sciences, Karolinska Institutet, Danderyd Hospital, 1882 88 Stockholm, Sweden.
    Mucosal immune responses following a fourth SARS-CoV-2 vaccine dose2023In: The Lancet Microbe, E-ISSN 2666-5247, Vol. 4, no 7, p. 488-Article in journal (Refereed)
  • 29.
    Blomfeldt, Thomas O. J.
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Kuktaite, Ramune
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Johansson, Eva
    Hedenqvist, Mikael S.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Mechanical Properties and Network Structure of Wheat Gluten Foams2011In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 12, no 5, p. 1707-1715Article in journal (Refereed)
    Abstract [en]

    This Article reports the influence of the protein network structure on the mechanical properties of foams produced from commercial wheat gluten using freeze-drying. Foams were produced from alkaline aqueous solutions at various gluten concentrations with or without glycerol, modified with bacterial cellulose nanosized fibers, or both. The results showed that 20 wt % glycerol was sufficient for plasticization, yielding foams with low modulus and high strain recovery. It was found that when fibers were mixed into the foams, a small but insignificant increase in elastic modulus was achieved, and the foam structure became more homogeneous. SEM indicated that the compatibility between the fibers and the matrix was good, with fibers acting as bridges in the cell walls. IR spectroscopy and SE-HPLC revealed a relatively low degree of aggregation, which was highest in the presence of glycerol. Confocal laser scanning microscopy revealed distinct differences in HMW-glutenin subunits and gliadin distributions for all of the different samples.

  • 30. Bollampalli, V. P.
    et al.
    Harumi Yamashiro, L.
    Feng, X.
    Bierschenk, D.
    Gao, Y.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Henriques-Normark, B.
    Nylén, S.
    Rothfuchs, A. G.
    BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming2015In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 11, no 10, article id e1005206Article in journal (Refereed)
    Abstract [en]

    The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4+ T cells at that site. We found EpCAMlow CD11bhigh migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4+ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.

  • 31. Bouzenzana, Jamel
    et al.
    Pelosi, Ludovic
    Briolay, Anne
    Briolay, Jerome
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Identification of the first Oomycete annexin as a (1 -> 3)-beta-D-glucan synthase activator2006In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 62, no 2, p. 552-565Article in journal (Refereed)
    Abstract [en]

    (1 -> 3)-beta-D-Glucans are major components of the cell walls of Oomycetes and as such they play an essential role in the morphogenesis and growth of these microorganisms. Despite the biological importance of (1 -> 3)-beta-D-glucans, their mechanisms of biosynthesis are poorly understood. Previous studies on (1 -> 3)-beta-D-glucan synthases from Saprolegnia monoica have shown that three protein bands of an apparent molecular weight of 34, 48 and 50 kDa co-purify with enzyme activity. However, none of the corresponding proteins have been identified. Here we have identified, purified, sequenced and characterized a protein from the 34 kDa band and clearly shown that it has all the biochemical properties of proteins from the annexin family. In addition, we have unequivocally demonstrated that the purified protein is an activator of (1 -> 3)-beta-D-glucan synthase. This represents a new type of function for proteins belonging to the annexin family. Two other proteins from the 48 and 50 kDa bands were identified as ATP synthase subunits, which most likely arise from contaminations by mitochondria during membrane preparation. The results, which are discussed in relation with the possible regulation mechanisms of (1 -> 3)-beta-D-glucan synthases, represent a first step towards a better understanding of cell wall polysaccharide biosynthesis in Oomycetes.

  • 32.
    Branneby, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry.
    Exploiting enzyme promiscuity for rational design2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area.

    Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity.

    In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant.

    In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type.

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  • 33.
    Brechmann, Nils A.
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Hägg, Alice
    BioPharmaceuticals R&D Cell Therapy Department, Research and Early Development, Cardiovascular, Renal, and Matabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, SE-431 83, Gothenburg, Sweden.
    Jansson, Märta
    BioPharmaceuticals R&D Cell Therapy Department, Research and Early Development, Cardiovascular, Renal, and Matabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, SE-431 83, Gothenburg, Sweden.
    Andersson, Christian X.
    BioPharmaceuticals R&D Cell Therapy Department, Research and Early Development, Cardiovascular, Renal, and Matabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, SE-431 83, Gothenburg, Sweden.
    Hicks, Ryan
    BioPharmaceuticals R&D Cell Therapy Department, Research and Early Development, Cardiovascular, Renal, and Matabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, SE-431 83, Gothenburg, Sweden.
    Hyllner, Johan
    BioPharmaceuticals R&D Cell Therapy Department, Research and Early Development, Cardiovascular, Renal, and Matabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, SE-431 83, Gothenburg, Sweden.
    Eriksson, Kristofer
    MAGic Bioprocessing (Lab-on-a-Bead AB) Uppsala, Sweden.
    Chotteau, Véronique
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Negative selection of human induced pluripotent stem cells (hiPSC)Manuscript (preprint) (Other academic)
  • 34.
    Burt, Ayleen
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Genetic engineering of an inducible helper vector for the production of Adeno-Associated Viruses (AAVs)2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The use of adeno-associated viruses (AAVs) is a prominent delivery method for gene therapy.  The potentials of AAV-gene therapy are nonetheless hindered by production challenges of these viral vectors. One of the main production methods of AAVs is the triple transfection of plasmids into mammalian cells. Triple transfection is expensive, may lead to unequal ratios of plasmids and is an all over tedious production method.  One of the plasmids transfected is the helper vector containing helper genes VA-RNA III, E4orf6 and E2A.  In order to minimize the number of plasmids needed to be transfected, these genes could be stably integrated into the mammalian cells. A setback is however the toxic properties of the helper genes. To circumvent  this, induction systems could be applied to the genes, silencing them during cell proliferation, and producing viruses only upon induction of a small molecule. Here, both toxic effects of individual helper genes and inducibility of a suitable induction system were evaluated. Furthermore, the study initiated the formation   of an inducible helper vector based on the set properties of the helper genes.

  • 35.
    Byström, Sanna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Affinity assays for profiling disease-associated proteins in human plasma2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Affinity-based proteomics offers opportunities for the discovery and validation of disease-associated proteins in human body fluids. This thesis describes the use of antibody-based immunoassays for multiplexed analysis of proteins in human plasma, serum and cerebrospinal fluid (CSF). This high-throughput method was applied with the objective to identify proteins associated to clinical variables. The main work in this thesis was conducted within the diseases of multiple sclerosis and malignant melanoma, as well as mammographic density, a risk factor for breast cancer.

    The suspension bead array (SBA) technology has been the main method for the work presented in this thesis (Paper I-IV). SBA assays and other affinity proteomic technologies were introduced for protein profiling of sample material obtained from clinical collaborators and biobanks. Perspectives on the validation of antibody selectivity by means of e.g. immuno-capture mass spectrometry are also provided.

    Paper I describes the development and application of a protocol for multiplexed pro- tein profiling of CSF. The analysis of 340 CSF samples from patients with multiple sclerosis and other neurological disease revealed proteins with potential association to disease progression (GAP43) and inflammation (SERPINA3). Paper II continued on this work with an extended investigation of more than 1,000 clinical samples and included both plasma and CSF collected from the same patients. Comparison of disease subtypes and controls revealed five plasma proteins of potential diagnostic relevance, such as IRF8 and GAP43. The previously reported associations for GAP43 and SERPINA3 in CSF was confirmed. Subsequent immunohistochemical analysis of post-mortem brain tissue revealed differential protein expression in disease affected areas. In Paper III, 150 serum samples from patients with cutaneous malignant melanoma were analyzed. Protein profiles from antibody bead arrays suggested three proteins (RGN, MTHFD1L, STX7) of differential abundance between patients with no disease recurrence and low tumor thickness (T-stage 1 and 2) compared to patients with high tumor thickness (T-stage 3 and 4) and disease recurrence. We observed MTHFD1L expression in tissue of a majority of patients, while expression of STX7 in melanoma tissue had been reported previously. Paper IV describes the analysis of protein in plasma in relation to mammographic breast density (MD), one of the strongest risk factors for the development of breast cancers. More than 1,300 women without prior history of breast cancer were screened. Linear associations to MD in two independent sample sets were found for 11 proteins, which are expressed in the breast and involved in tissue homeostasis, DNA repair, cancer development and/or progression in MD.

    In conclusion, this thesis describes the use of multiplexed antibody bead arrays for protein profiling of serum, plasma and CSF, and it shortlists disease associated proteins for further validation studies. 

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  • 36.
    Byström, Sanna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Eklund, Martin
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hong, Mun-Gwan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Fredolini, Claudia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Eriksson, Mikael
    Czene, Kamila
    Hall, Per
    Schwenk, Jochen. M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Gabrielson, Marike
    Affinity proteomic profiling of plasma for proteins associated to mammographic breast densityManuscript (preprint) (Other academic)
  • 37.
    Byström, Sanna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Fredolini, Claudia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Edqvist, Per-Henrik
    Nyaiesh, Etienne-Nicholas
    Drobin, Kimi
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, Matthias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Bergqvist, Michael
    Pontén, Fredrik
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Affinity proteomics exploration of melanoma identifies proteins in serum with associations to T-stage and recurrenceManuscript (preprint) (Other academic)
  • 38.
    Caraballo, Rémi
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Sakulsombat, Morakot
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Towards Dynamic Drug Design: Identification and Optimization of β-Galactosidase Inhibitors from a Dynamic Hemithioacetal System2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 11, p. 1600-1606Article in journal (Refereed)
    Abstract [en]

    A discovery strategy relying on the identification of fragments through resolution of a constitutional dynamic system, coupled to subsequent static ligand design and optimization, is demonstrated. The strategic design and synthesis of the best molecular fragments identified from a dynamic hemithioacetal system into static ligand structures yielded a range of -galactosidase inhibitors. Two series of structures mimicking the hemithioacetal motif were envisaged: thioglycosides and C-glycosides. Inhibition studies provided important structural information for the two groups, and 1-thiobenzyl--D-galactopyranoside demonstrated the best inhibitory effects.

  • 39.
    Carlsson Norlin, Roxanne
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Epigenetic Profiling of Canine Brain2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The domestication of dog to wolf started around 35000 BP and is believed to be the oldest domestication event among both plants and animals. During this event, dogs have developed differences in morphological and behavioural traits to their ancestors, such as wider snouts, higher skulls, and lower tendencies to taking risks. It is now suggested that many of these differences can be explained by differences in active regulatory regions. The main objective of this thesis is to map chromatin interactions in the genome of wolf and dog brain tissues to annotate regulatory variants between the canine species. This will hopefully provide novel information regarding genomic changes mediating traits gained through domestication. We will perform Capture Hi-C (HiCap) on tissue samples of hypothalamus and prefrontal cortex of wolf and dog. HiCap is a method derived from the chromosome capture method 3C. In HiCap, the interacting regions of the DNA are crosslinked, ensuring that promoter-enhancer interactions will not be lost. These interacting regions are then ligated together, followed by sequencing library preparation. Subsequently, sequencing of these libraries will provide information of which promoters are actively regulated by enhancers in the nucleus. We successfully prepared Hi-C libraries for all tissues and animals. However, there were longer fragments in some libraries which can be removed. Due to lack of necessary probes for sequence capture, the laboratory work was cut short and no major results were obtained. By continuing the laboratory work, hopefully these libraries will result in novel insights in the domestication of the dog.

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  • 40. Carreras-Puigvert, J.
    et al.
    Zitnik, M.
    Jemth, A. -S
    Carter, M.
    Unterlass, J. E.
    Hallström, Björn M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Loseva, O.
    Karem, Z.
    Calderón-Montanõ, J. M.
    Lindskog, C.
    Edqvist, P. -H
    Matuszewski, D. J.
    Ait Blal, Hammou
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Berntsson, R. P. A.
    Häggblad, M.
    Martens, U.
    Studham, M.
    Lundgren, B.
    Wählby, C.
    Sonnhammer, E. L. L.
    Lundberg, Emma
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Stenmark, P.
    Zupan, B.
    Helleday, T.
    A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family2017In: Nature Communications, E-ISSN 2041-1723, Vol. 8, no 1, article id 1541Article in journal (Refereed)
    Abstract [en]

    The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.

  • 41.
    Cavallaro, Sara
    KTH, School of Engineering Sciences (SCI), Applied Physics, Photonics.
    Development of Techniques for Characterization, Detection and Protein Profiling of Extracellular Vesicles2021Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Nanosized extracellular vesicles (EVs, ∼30-2000 nm) have emerged as important mediators of intercellular communication, offering opportunities for both diagnostics and therapeutics. In particular, small EVs generated from the endolysosomal pathway (∼30-150 nm), referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics and treatment monitoring based on minimally invasive liquid biopsies. This is because exosomes carry valuable biological information (proteins, lipids, genetic material, etc.) reflecting their cells of origin. Using EVs as biomarkers or drug delivery agents in clinical applications requires a full understanding of their cellular origin, functions, and biological relevance. However, due to their small size and very high heterogeneity in molecular and physical features, the analysis of these vesicles is challenged by the limited detection ranges and/or accuracy of the currently available techniques. To overcome some of these challenges, this thesis focuses on developing different techniques for characterization, detection and protein profiling of EVs at both bulk and single particle levels. Specifically, the three methods investigated are scanning electron microscopy, electrokinetic sensing, and combined fluorescence - atomic force microscopy. 

    First, a protocol for scanning electron microscopy imaging of EVs was optimized to improve the throughput and image quality of the method while preserving the shape of the vesicles. Application of the developed protocol for analysis of EVs from human serum showed the possibility to use scanning electron microscopy for morphological analysis and high-resolution size-based profiling of EVs over their entire size range. Comparison with nanoparticle tracking analysis, a commonly used technique for EV size estimation, showed a superior sensitivity of scanning electron microscopy for particles smaller than 70-80 nm. Moreover, the study showed process steps that can generate artifacts resembling sEVs and ways to minimize them. 

    Secondly, a novel label-free electrokinetic sensor based on streaming current was developed, optimized and multiplexed for EV protein analysis at a bulk level. Using multiple microcapillary sensors functionalized with antibodies, the method showed the capacity for multiplexed detection of different surface markers on small EVs from non-small-cell lung cancer cells. The device performance in the multichannel configuration remained similar to the single-channel one in terms of noise, detection sensitivity, and reproducibility. The application of the technique for analysis of EVs isolated from lung cancer patients with different genomic alterations and after different applied treatments demonstrated the prospect of using EVs from liquid biopsies as a source of biomarker for cancer monitoring. Moreover, the results held promise for the application of the developed method in clinical settings. 

    Finally, to increase the understanding of EV subpopulations and heterogeneity, a platform combining fluorescence and atomic force microscopy was developed for multiparametric analysis of EVs at a single particle level. The use of a precise spot identification approach and an efficient vesicle capture protocol allowed to study and correlate for the first time the membrane protein composition, size and mechanical properties (Young modulus) on individual small EVs. The application of the technique to vesicles isolated from different cell lines identified both common and cell line-specific EV subpopulations bearing distinct distributions of the analyzed parameters. For example, a sEV population co-expressing all the three analyzed proteins in relatively high abundance, yet having average diameters of <100 nm and relatively low Young moduli was found in all cell lines. The obtained results highlighted the possibility of using the developed platform to help decipher unsolved questions regarding EV biology. 

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  • 42.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wang, Jingjiao
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Tolf, Erika
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wicklund, Linn
    Karolinska Institute, Department of Neurobiology, Care Sciences and Society.
    Hovatta, Outi
    Karolinska Institutet, Department of Clinical Science.
    Marutle, Amelia
    Karolinska Institute, Department of Neurobiology, Care Sciences and Society.
    Comparison of cultivation in Techne spinner, Bellco spinner, shake flask and T-flask of human embryonic stem cells2010In: Proceedings of the SBE's Second International Conference on Stem Cell Engineering, 2010Conference paper (Other academic)
    Abstract [en]

    The recent progress in regenerative medicine indicates that pluripotent human embryonic stem cells (hESCs) may hold great potential providing cellular models for drug development and screening, modelling diseases as well as aid in the development of future cell-based therapies for neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Crucial to the success of generating specialized cell populations, is an understanding of the mechanisms, which influence the control of cell growth and differentiation by extrinsic and intrinsic factors. Nowadays, a limitation for the use of hESCs is the lack of proliferation methods in large scale. The purpose of the present work was to study several cultivation systems, which could potentially provide large-scale cultivation processes suitable for human therapy applications. Pluripotent human embryonic stem cells (hESCs), isolated from the inner cell mass of the blastocyst, were cultivated undifferentiated as embryoids bodies, i.e. large spherical aggregates of cells, in absence of serum and feeder layer. The cell growth and culture behavior in T-falsk, orbitally agitated shake flask, Bellco stirred spinner and Techne stirred spinner were observed. In Bellco spinner, the cells were agitated by a rotating impeller providing a movement comparable to stirred bioreactors. In Techne spinner, a slow and gentle orbital movement provided by a rotating bulb-ended stirrer maintained the cells in suspension. The design of this latter spinner allowed lower shear stress in comparison to Bellco spinner and shake flask. It was observed that the cell growth was fastest in Techne spinner followed by cultivation in T-flask and then cultivation in shake flask. Cultivating in Bellco spinner resulted in embryoid dissociation and viability decrease after 14 days. A larger number of single cells, i.e. cells not growing in aggregates, was observed in the static T-flask culture compared to the agitated systems, i.e. shake flask, Bellco spinner or Techne spinner. Probably the agitation promoted the spontaneous aggregation of the cells in spheres. In particular the Techne spinner allowed the most perfect spherical form among the different compared systems. Finally it was observed that hypoxia with 4 % oxygen concentration improved significantly the growth in Techne spinner or T-flask in comparison with normoxia with 21 % oxygen concentration. It was concluded that cultivation in Techne spinner under hypoxia was the most favorable condition among the ones studied here. The agitation provided by Techne spinner improved the cell growth in comparison with static system (T-flask). However using the other agitated systems, shake flask and Bellco spinner, was not comparably beneficial to the cell growth and viability, probably due to the higher shear stress of these systems compared to Techne spinner.

  • 43.
    Costa Felicissimo, Viviane
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Infrared - X-ray pump probe spectroscopy2005Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The present thesis concerns theoretical studies of molecular interactions investigated by infrared and X-ray spectroscopic techniques, with emphasis on using the two technologies combined in pump probe experiments. Three main types of studies are addressed: the use of near-edge X-ray absorption fine structure spectra (NEXAFS) to manifest through-bond and through-space interactions; the role of hydrogen bonding on the formation of X-ray photoelectron spectra as evidenced by simulations of the water dimer; and the development of theory, with sample applications, for infrared X-ray pump probe spectroscopy - the main theme of the thesis.

    Ab initio calculations indicate that NEXAFS spectra give direct information about the through-bond and through-space interactions between vacant non-conjugated π* orbitals. It is found that the X-ray photoelectron spectrum of the water dimer differs strongly from the monomer spectrum in that two bands are observed, separated by the chemically shifted ionization potentials of the donor and the acceptor. The hydrogen bond is responsible for the anomalously strong broadening of these two bands. The studies show that X-ray core electron ionization of the water dimer driven by an infrared field is a proper technique to prove the proton transfered state contrary to conventional X-ray photoelectron spectroscopy. Our simulations of infrared X-ray pump-probe spectra were carried out using wave packet propagation techniques.

    The physical aspects of the proposed new X-ray spectroscopic method - phase sensitive Infrared - X-ray pump probe spectroscopy - are examined in detail in two sample applications - on the NO molecule and on the dynamics of proton transfer in core ionized water dimer. It is found that the phase of the infrared pump field strongly influences the trajectory of the nuclear wave packet on the ground state potential. This results in a phase dependence of the X-ray pump probe spectra. A proper choice of the delay time of the X-ray pulse allows to directly observe the X-ray transition in the proton transfered well of the core excited potential.

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  • 44.
    Dahl, Leo
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kotliar, Ilana B.
    Rockefeller Univ, Lab Chem Biol & Signal Transduct, 1230 York Ave, New York, NY 10065 USA.;Triinst PhD Program Chem Biol, New York, NY 10065 USA..
    Bendes, Annika
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Dodig-Crnkovic, Tea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fromm, Samuel
    Stockholm Univ, Sci Life Lab, Dept Biochem & Biophys, S-17165 Solna, Sweden..
    Elofsson, Arne
    Stockholm Univ, Sci Life Lab, Dept Biochem & Biophys, S-17165 Solna, Sweden..
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Sakmar, Thomas P.
    Rockefeller Univ, Lab Chem Biol & Signal Transduct, 1230 York Ave, New York, NY 10065 USA.;Karolinska Inst, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, S-17164 Solna, Sweden..
    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.
    Multiplexed selectivity screening of anti-GPCR antibodies2023In: Science Advances, E-ISSN 2375-2548, Vol. 9, no 18, article id eadf9297Article in journal (Refereed)
    Abstract [en]

    G protein-coupled receptors (GPCRs) control critical cellular signaling pathways. Therapeutic agents including anti-GPCR antibodies (Abs) are being developed to modulate GPCR function. However, validating the selectivity of anti-GPCR Abs is challenging because of sequence similarities among individual receptors within GPCR sub-families. To address this challenge, we developed a multiplexed immunoassay to test >400 anti-GPCR Abs from the Human Protein Atlas targeting a customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. We found that-61% of Abs tested were selective for their intended target,-11% bound off -target, and-28% did not bind to any GPCR. Antigens of on-target Abs were, on average, significantly longer, more disordered, and less likely to be buried in the interior of the GPCR protein than the other Abs. These results provide important insights into the immunogenicity of GPCR epitopes and form a basis for designing therapeu-tic Abs and for detecting pathological auto-Abs against GPCRs.

  • 45.
    Dahlsson Leitao, Charles
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Rinne, S. S.
    Mitran, B.
    Vorobyeva, A.
    Andersson, Ken G.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, School of Engineering Sciences (SCI).
    Tolmachev, V.
    Ståhl, Stefan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Orlova, A.
    Molecular Design of HER3-Targeting Affibody Molecules: Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68 Ga-Labeled Tracers2019In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 20, no 5Article in journal (Refereed)
    Abstract [en]

    Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)₃-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)₃-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)₃-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)₃-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)₃-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.

  • 46. Delsing, Louise
    et al.
    Donnes, Pierre
    Sanchez, Jost
    Clausen, Maryam
    Voulgaris, Dimitrios
    KTH, School of Electrical Engineering and Computer Science (EECS), Intelligent systems, Micro and Nanosystems.
    Falk, Anna
    Herland, Anna
    KTH, School of Electrical Engineering and Computer Science (EECS), Intelligent systems, Micro and Nanosystems.
    Brolen, Gabriella
    Zetterberg, Henrik
    Hicks, Ryan
    Synnergren, Jane
    Barrier properties and transcriptome expression in human iPSC‐derived models of the blood–brain barrier2018In: Stem Cells, ISSN 1066-5099, E-ISSN 1549-4918, Vol. 36, no 12, p. 1816-1827Article in journal (Refereed)
    Abstract [en]

    Cell-based models of the blood-brain barrier (BBB) are important for increasing the knowledge of BBB formation, degradation and brain exposure of drug substances. Human models are preferred over animal models because of interspecies differences in BBB structure and function. However, access to human primary BBB tissue is limited and has shown degeneration of BBB functions in vitro. Human induced pluripotent stem cells (iPSCs) can be used to generate relevant cell types to model the BBB with human tissue. We generated a human iPSC-derived model of the BBB that includes endothelial cells in coculture with pericytes, astrocytes and neurons. Evaluation of barrier properties showed that the endothelial cells in our coculture model have high transendothelial electrical resistance, functional efflux and ability to discriminate between CNS permeable and non-permeable substances. Whole genome expression profiling revealed transcriptional changes that occur in coculture, including upregulation of tight junction proteins, such as claudins and neurotransmitter transporters. Pathway analysis implicated changes in the WNT, TNF, and PI3K-Akt pathways upon coculture. Our data suggest that coculture of iPSC-derived endothelial cells promotes barrier formation on a functional and transcriptional level. The information about gene expression changes in coculture can be used to further improve iPSC-derived BBB models through selective pathway manipulation.

  • 47. Ding, Mei
    et al.
    Tegel, Hanna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Sivertsson, Åsa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Snijder, Arjan
    Ormö, Mats
    Strömstedt, Per-Erik
    Davies, Rick
    Holmberg Schiavone, Lovisa
    Secretome-Based Screening in Target Discovery2020In: SLAS Discovery, ISSN 2472-5552, Vol. 25, no 6, p. 535-551Article in journal (Refereed)
  • 48.
    Ding, Weiqi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Droplet microfluidics for detection and sorting of photosynthetically produced sucrose2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Droplet microfluidics is a rapidly growing interdisciplinary field that combines microfluidics and droplet technology to create and manipulate tiny droplets in microscale channels, enabling the performance of various assays and screenings due to its low cost and high throughput. Cyanobacteria as model organisms for photosynthesis are attractive for biotechnology applications owing to fast growth, minimal nutritional requirements, genetic accessibility and photoautotrophy. However, when facing the task of screening a large number of strains, the conventional microtiter plate approach proves to be ever resource-intensive and time-consuming. Flow cytometry, on the other hand, is limited to the assessment of individual cells based on intracellular or surface-bound fluorescence markers. Droplet microfluidics technology, in this context, effectively bridges this gap through its high throughput and versatility. In this project, a droplet microfluidic workflow which combines several microfluidics devices to encapsulate, incubate, assay, detect and sort sucrose excreting stains of the cyanobacterium Synechocystis sp. PCC 6803, has been developed, optimized and validated based on a fluorescence assay. This droplet microfluidic-based method for detection of sucrose will facilitate the screening of mutant library as well as metabolic engineering studies.

  • 49.
    Drobin, Kimi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Antibody-based bead arrays for high-throughput protein profiling in human plasma and serum2018Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Affinity-based proteomics utilizes affinity binders to detect target proteins in a large-scale manner. This thesis describes a high-throughput method, which enables the search for biomarker candidates in human plasma and serum. A highly multiplexed antibody-based suspension bead array is created by coupling antibodies generated in the Human Protein Atlas project to color-coded beads. The beads are combined for parallel analysis of up to 384 analytes in patient and control samples. This provides data to compare protein levels from the different groups.

    In paper I osteoporosis patients are compared to healthy individuals to find disease-linked proteins. An untargeted discovery screening was conducted using 4608 antibodies in 16 cases and 6 controls. This revealed 72 unique proteins, which appeared differentially abundant. A validation screening of 91 cases and 89 controls confirmed that the protein autocrine motility factor receptor (AMFR) is decreased in the osteoporosis patients.

    Paper II investigates the risk proteome of inflammatory bowel disease (IBD). Antibodies targeting 209 proteins corresponding to 163 IBD genetic risk loci were selected. To find proteins related to IBD or its subgroups, sera from 49 patients with Crohn’s disease, 51 with ulcerative colitis and 50 matched controls were analyzed. From these targeted assays, the known inflammation-related marker serum amyloid protein A (SAA) was shown to be elevated in the IBD cases. In addition, the protein laccase (multi-copper oxidoreductase) domain containing 1 (LACC1) was found to be decreased in the IBD subjects.

    In conclusion, assays using affinity-based bead arrays were developed and applied to screen human plasma and serum samples in two disease contexts. Untargeted and targeted screening strategies were applied to discover disease-associated proteins. Upon further validation, these potential biomarker candidates could be valuable in future disease studies.

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  • 50.
    Drobin, Kimi
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Assadi, Ghazaleh
    Hong, Mun-Gwan
    Andersson, Eni
    Fredolini, Claudia
    Forsström, Björn
    Reznichenko, Anna
    Akhter, Tahmina
    Ek, Weronica
    Bonfiglio, Ferdinando
    Berner Hansen, Mark
    Sandberg, Kristian
    Greco, Dario
    Repsilber, Dirk
    Schwenk, Jochen
    D'Amato, Mauro
    Halfvarson, Jonas
    Targeted analysis of serum proteins encoded at known inflammatory bowel disease risk lociManuscript (preprint) (Other academic)
123456 1 - 50 of 271
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