| 1. |
- Ekblad, Torun, 1977-
(författare)
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Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are essential components in most processes in living organisms. The detection and quantification of specific proteins can be used e.g. as measures of certain physiological conditions, and are therefore of great importance. This thesis focuses on development of affinity-based bioassays for specific protein detection. The use of Affibody molecules for specific molecular recognition has been central in all studies in this thesis. Affibody molecules are affinity proteins developed by combinatorial protein engineering of the 58-residue protein A-derived Z domain scaffold. In the first paper, solid phase peptide synthesis is investigated as a method to generate functional Affibody molecules. Based on the results from this paper, chemical synthesis has been used throughout the following papers to produce Affibody molecules tailored with functional groups for protein detection applications in vitro and in vivo. In paper I, an orthogonal protection scheme was developed to enable site-specific chemical introduction of three different functional probes into synthetic Affibody molecules. Two of the probes were fluorophores that were used in a FRET-based binding assay to detect unlabeled target proteins. The third probe was biotin, which was used as an affinity handle for immobilization onto a solid support. In paper II, a panel of Affibody molecules carrying different affinity handles were synthesized and evaluated as capture ligands on microarrays. Paper III describes the synthesis of an Affibody molecule that binds to the human epidermal growth factor receptor type 2, (HER2), and the site-specific incorporation of a mercaptoacetyl-glycylglycylglycine (MAG3) chelating site in the peptide sequence to allow for radiolabeling with 99mTc. The derivatized Affibody molecule was found to retain its binding capacity, and the 99mTc-labeling was efficient and resulted in a stable chelate formation. 99mTc-labeled Affibody molecules were evaluated as in vivo HER2-targeting imaging agents in mice. In the following studies, reported in papers IV-VI, the 99mTc-chelating sequence was engineered in order to optimize the pharmacokinetic properties of the radiolabeled Affibody molecules and allow for high-contrast imaging of HER2-expressing tumors and metastatic lesions. The main conclusion from these investigations is that the biodistribution of Affibody molecules can be dramatically modified by amino acid substitutions directed to residues in the MAG3-chelator. Finally, paper VII is a report on the chemical synthesis and chemoselective ligation to generate a cross-linked HER2-binding Affibody molecule with improved thermal stability and tumor targeting capacity. Taken together, the studies presented in this thesis illustrate how peptide synthesis can be used for production and modification of small affinity proteins, such as Affibody molecules for protein detection applications.
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| 2. |
- Eriksson, Harald, 1977-, et al.
(författare)
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A novel phage cocktail inhibiting the growth of 99 β-lactamase carrying Klebsiella pneumoniae clinical isolates in vitro
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Klebsiella pneumoniae is a gram-negative bacterial pathogen, accountable for a variety of nosocomial infections in immunocompromised patients, open-wound infections and community-acquired pneumonia in elderly. K. pneumoniae strains harboring plasmid-mediated extended spectrum β-lactamase enzymes (ESBL) are resistant to all penicillin and cephalosoprins, whereas bacteria capable of producing carbapenemase enzymes (e.g. NDM, KPC and VIM) are resistant to virtually all β-lactam group antibiotics. The use of bacterial viruses lysing bacterial hosts (phage therapy) has been suggested as an alternative in fighting bacterial infections resistant to known antibiotics. In this study, we assembled a phage cocktail consisting of 6 novel lytic bacteriophages infecting K. pneumoniae. The phage cocktail was tested against 125 β-lactamase producing clinical isolates of K. pneumoniae and we found that at high titres, the cocktail was able to lyse 99 of these isolates in vitro.
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| 3. |
- Eriksson, Harald, 1977-, et al.
(författare)
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A suggested new bacteriophage genus, “Kp34likevirus”, within the Autographivirinae subfamily of Podoviridae
- 2015
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 7:4, s. 1804-1822
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Tidskriftsartikel (refereegranskat)abstract
- Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to theAutographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 (NC_013649), F19 (NC_023567) and NTUH-K2044-K1-1 (NC_025418). Bioinformatic analysis of these phage genomes show high conservation of gene arrangement and gene content, conserved catalytically active residues of their RNA polymerase, a common and specific lysis cassette, and form a joint cluster in phylogenetic analysis of their conserved genes. Also, we have performed biological characterization of the burst size, latent period, host specificity (together with KP34 and NTUH-K2044-K1-1), morphology, and structural genes as well as sensitivity testing to various conditions. Based on the analyses of these phages, the creation of a new phage genus is suggested within the Autographivirinae, called “Kp34likevirus” after their type phage, KP34. This genus should encompass the recently genome sequenced Klebsiella phages KP34, SU503, SU552A, F19 and NTUH-K2044-K1-1.
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| 4. |
- Eriksson, Harald, 1977-
(författare)
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Bacterial viruses targeting multi-resistant Klebsiella pneumoniae and Escherichia coli
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The global increase in antibiotic resistance levels in bacteria is a growing concern to our society and highlights the need for alternative strategies to combat bacterial infections. Bacterial viruses (phages) are the natural predators of bacteria and are as diverse as their hosts, but our understanding of them is limited. The current levels of knowledge regarding the role that phage play in the control of bacterial populations are poor, despite the use of phage therapy as a clinical therapy in Eastern Europe.The aim of this doctoral thesis is to increase knowledge of the diversity and characteristics of bacterial viruses and to assess their potential as therapeutic agents towards multi-resistant bacteria.Paper I is the product of de novo sequencing of newly isolated phages that infect and kill multi-resistant Klebsiella pneumoniae. Based on similarities in gene arrangement, lysis cassette type and conserved RNA polymerase, the creation of a new phage genus within Autographivirinae is proposed.Paper II describes the genomic and proteomic analysis of a phage of the rare C3 morphotype, a Podoviridae phage with an elongated head that uses multi-resistant Escherichia coli as its host.Paper III describes the study of a pre-made phage cocktail against 125 clinical K. pneumoniae isolates. The phage cocktail inhibited the growth of 99 (79 %) of the bacterial isolates tested. This study also demonstrates the need for common methodologies in the scientific community to determine how to assess phages that infect multiple serotypes to avoid false positive results.Paper IV studies the effects of phage predation on bacterial virulence: phages were first allowed to prey on a clinical K. pneumoniae isolate, followed by the isolation of phage-resistant bacteria. The phage resistant bacteria were then assessed for their growth rate, biofilm production in vitro. The virulence of the phage resistant bacteria was then assessed in Galleria mellonella. In the single phage treatments, two out of four phages showed an increased virulence in the in G. mellonella, which was also linked to an increased growth rate of the phage resistant bacteria. In multi-phage treatments however, three out of five phage cocktails decreased the bacterial virulence in G. mellonella compared to an untreated control.
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| 5. |
- Hammar, Karl, 1982-
(författare)
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Content Ontology Design Patterns: Qualities, Methods, and Tools
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ontologies are formal knowledge models that describe concepts and relationships and enable data integration, information search, and reasoning. Ontology Design Patterns (ODPs) are reusable solutions intended to simplify ontology development and support the use of semantic technologies by ontology engineers. ODPs document and package good modelling practices for reuse, ideally enabling inexperienced ontologists to construct high-quality ontologies. Although ODPs are already used for development, there are still remaining challenges that have not been addressed in the literature. These research gaps include a lack of knowledge about (1) which ODP features are important for ontology engineering, (2) less experienced developers' preferences and barriers for employing ODP tooling, and (3) the suitability of the eXtreme Design (XD) ODP usage methodology in non-academic contexts.This dissertation aims to close these gaps by combining quantitative and qualitative methods, primarily based on five ontology engineering projects involving inexperienced ontologists. A series of ontology engineering workshops and surveys provided data about developer preferences regarding ODP features, ODP usage methodology, and ODP tooling needs. Other data sources are ontologies and ODPs published on the web, which have been studied in detail. To evaluate tooling improvements, experimental approaches provide data from comparison of new tools and techniques against established alternatives.The analysis of the gathered data resulted in a set of measurable quality indicators that cover aspects of ODP documentation, formal representation or axiomatisation, and usage by ontologists. These indicators highlight quality trade-offs: for instance, between ODP Learnability and Reusability, or between Functional Suitability and Performance Efficiency. Furthermore, the results demonstrate a need for ODP tools that support three novel property specialisation strategies, and highlight the preference of inexperienced developers for template-based ODP instantiation---neither of which are supported in prior tooling. The studies also resulted in improvements to ODP search engines based on ODP-specific attributes. Finally, the analysis shows that XD should include guidance for the developer roles and responsibilities in ontology engineering projects, suggestions on how to reuse existing ontology resources, and approaches for adapting XD to project-specific contexts.
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| 6. |
- Landälv, Ludvig, 1982-, et al.
(författare)
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Phase evolution of radio frequency magnetron sputtered Cr-rich (Cr,Zr)(2)O-3 coatings studied by in situ synchrotron X-ray diffraction during annealing in air or vacuum
- 2019
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Ingår i: Journal of Materials Research. - : CAMBRIDGE UNIV PRESS. - 0884-2914 .- 2044-5326. ; 34:22, s. 3735-3746
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Tidskriftsartikel (refereegranskat)abstract
- The phase evolution of reactive radio frequency (RF) magnetron sputtered Cr0.28Zr0.10O0.61 coatings has been studied by in situ synchrotron X-ray diffraction during annealing under air atmosphere and vacuum. The annealing in vacuum shows t-ZrO2 formation starting at similar to 750-800 degrees C, followed by decomposition of the alpha-Cr2O3 structure in conjunction with bcc-Cr formation, starting at similar to 950 degrees C. The resulting coating after annealing to 1140 degrees C is a mixture of t-ZrO2, m-ZrO2, and bcc-Cr. The air-annealed sample shows t-ZrO2 formation starting at similar to 750 degrees C. The resulting coating after annealing to 975 degrees C is a mixture of t-ZrO2 and alpha-Cr2O3 (with dissolved Zr). The microstructure coarsened slightly during annealing, but the mechanical properties are maintained, with no detectable bcc-Cr formation. A larger t-ZrO2 fraction compared with alpha-Cr2O3 is observed in the vacuum-annealed coating compared with the air-annealed coating at 975 degrees C. The results indicate that the studied pseudo-binary oxide is more stable in air atmosphere than in vacuum.
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| 7. |
- van der Valk, Ralf J P, et al.
(författare)
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A novel common variant in DCST2 is associated with length in early life and height in adulthood.
- 2015
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Ingår i: Human molecular genetics. - : Oxford University Press (OUP). - 1460-2083 .- 0964-6906. ; 24:4, s. 1155-68
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Tidskriftsartikel (refereegranskat)abstract
- Common genetic variants have been identified for adult height, but not much is known about the genetics of skeletal growth in early life. To identify common genetic variants that influence fetal skeletal growth, we meta-analyzed 22 genome-wide association studies (Stage 1; N = 28 459). We identified seven independent top single nucleotide polymorphisms (SNPs) (P < 1 × 10(-6)) for birth length, of which three were novel and four were in or near loci known to be associated with adult height (LCORL, PTCH1, GPR126 and HMGA2). The three novel SNPs were followed-up in nine replication studies (Stage 2; N = 11 995), with rs905938 in DC-STAMP domain containing 2 (DCST2) genome-wide significantly associated with birth length in a joint analysis (Stages 1 + 2; β = 0.046, SE = 0.008, P = 2.46 × 10(-8), explained variance = 0.05%). Rs905938 was also associated with infant length (N = 28 228; P = 5.54 × 10(-4)) and adult height (N = 127 513; P = 1.45 × 10(-5)). DCST2 is a DC-STAMP-like protein family member and DC-STAMP is an osteoclast cell-fusion regulator. Polygenic scores based on 180 SNPs previously associated with human adult stature explained 0.13% of variance in birth length. The same SNPs explained 2.95% of the variance of infant length. Of the 180 known adult height loci, 11 were genome-wide significantly associated with infant length (SF3B4, LCORL, SPAG17, C6orf173, PTCH1, GDF5, ZNFX1, HHIP, ACAN, HLA locus and HMGA2). This study highlights that common variation in DCST2 influences variation in early growth and adult height.
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| 8. |
- Örmälä-Odegrip, Anni-Maria, et al.
(författare)
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Evolution of virulence in Klebsiella pneumoniae treated with phage cocktails
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The worldwide emergence and spread of multidrug-resistant bacteria is a major concern in modern medicine, and threatens the once established control over bacterial infections. Phage therapy has been suggested as one potential solution to the problem of finding new antibacterial agents. Bacteria are known to evolve resistance against bacteriophages but in many cases phage-resistance comes with a cost on bacterial virulence in multicellular hosts. We investigated how the virulence of a clinical isolate of K. pneumoniae Kpn524 evolves in response to exposure to phage cocktails in the phage-resistant bacteria that would potentially survive the phage treatment. We found that the exposure to multiple phages was linked to lowered virulence in the phage-resistant bacteria, when measured in vivo with Galleria mellonella. However, two phages were found to increase the bacterial virulence when they were administered on the bacteria individually, and this was associated with an increased growth rate. Across all treatments, biofilm production was negatively correlated with virulence, whereas growth rate had a positive correlation with bacterial virulence. Our findings suggest that bacterial virulence is attenuated in the presence of multiple phages, possibly due to a trade-off between phage resistance andrate of replication. However, this is dependent on the composition of the phage cocktail.This is the first study to report increased bacterial virulence associated with exposure tolytic bacteriophages and our results call for meticulous consideration when choosingphages for phage cocktails, as phages with certain identity could have detrimentally adverse effects on the success of the treatment.
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