| 1. |
- Felth, Jenny, 1979-
(författare)
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Studies of Cytotoxic Compounds of Natural Origin and their Mechanisms of Action
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer incidence is increasing and novel anticancer drugs with new mechanisms of action are essential for future chemotherapeutic treatment. Natural products have historically played an important role in the development of anti-cancer drugs and have potential to do so also in the future. In this thesis two classes of natural products are identified as possible drug lead candidates, and the mechanisms of their action are elucidated. Initially, in a screening of a compound library for cytotoxic effects in colon cancer cells, natural products with potent activity were identified. Based on their potency, and on previously reported activities in cancer cells, two main groups of compounds, cardiac glycosides (CGs) and gambogic acid (GA) analogues, were selected for further in-depth studies. The concentration-dependent cytotoxicity was confirmed in cell lines of different origin. Cardiac glycosides were mainly evaluated for their activity in colon cancer cells and in leukemic cells, whereas the GA analogues were studied using a resistance-based panel of ten human cancer cell lines. Using activity profiles and the ChemGPS-NP model, the compounds were compared, structurally and mechanistically, to standard chemotherapeutic drugs. The results from these analyses suggested that the CGs and the GA analogues act by mechanisms different from those of antimetabolites, alkylating agents, topoisomerase I and II inhibitors, or tubulin-active agents. By analysis of drug-induced gene expression, one GA analogue, dihydro GA, was identified as a possible inhibitor of the ubiquitin-proteasome system (UPS), and the CGs showed similarities to protein synthesis inhibitors. Starting from these hypotheses, we further investigated the mechanisms of actions on a molecular level. The results showed that GA and dihydro GA act as inhibitors of the 20S proteasome chymotrypsin activity, leading to accumulation of ubiquitinated proteins. The CGs were confirmed to inhibit protein synthesis in colon cancer cell lines. However, interestingly, in leukemia cell lines, it seemed that the CGs act through a different, yet unexplored, mechanism of action. The leukemic cells (pre-B and T-ALL) were particularly susceptible to the cytotoxic effects of CGs, including at concentrations that may be achievable in the clinic.
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| 2. |
- Aboye, Teshome Leta, 1975-
(författare)
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Engineering of the Ultra-stable Cystine Knot Framework of Microproteins : Design, Chemical Synthesis and Structural Studies
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ultra-stable cystine knotted microproteins, in which two disulfides and their connecting backbones form a circle that is penetrated by the third disulfide bonds, have attracted high interest due to their resistance to degradation in vitro and potential for the development of peptide drugs. This thesis gives new insights into engineering of that framework of microproteins, including approaches to their chemical synthesis, backbone engineering, structural and biological evaluations. Synthetic and oxidative folding approaches for bracelet cyclotides, a family of cyclic cystine knotted microproteins, was developed using a model peptide, cycloviolacin O2. Following assembly of the peptide chain, protected peptide was generated by mild cleavage that was subsequently thioesterified and cyclized in solution. The cyclic peptide was oxidatively folded under optimized conditions containing co-solvent and non-ionic detergent affording native cycloviolacin O2 as a major product. To gain further insights into the heterogeneity, efficiency and kinetics of cyclotides’ oxidative folding, the intermediates that accumulate in oxidative refolding pathways of all cyclotide subfamilies: Möbius, bracelet and the hybrid cyclotides were quantitatively determined under four different folding conditions. The results were used for defining major folding pathways, which indicated that Möbius cyclotides might accumulate heterogeneous folding intermediates with one-, two- and three-disulfides, whereas bracelet tend to accumulate a homogenous intermediate with three-disulfides, depending on the buffer systems used. Furthermore, to probe the internal factors contributing to inefficiency of oxidative folding, as well as undesired bioactivities of bracelet cyclotides (e.g., cytotoxic activity), polymer-hybridized cyclotides were designed by replacing non-conserved residues with small isosteric polymers. The designed hybrid analogs in which hybridization involved replacement of loop 3 with isosteric polymers showed improved synthetic and oxidative folding properties. The cytoxicity of a model hybrid designed with replacement of loop 3 and 5 exhibited no cytotoxic activity at concentration of 128-fold relative to that of native peptide. Furthermore, 1D and 2D 1H NMR analysis of this hybrid showed that it had well structured fold.
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| 3. |
- Yeshak, Mariamawit Yonathan
(författare)
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Cyclotides : Tuning Parameters Toward Their Use in Drug Design
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cyclotides are plant proteins with a unique topology, defined as the cyclic cystine knot motif. The motif endows cyclotides with exceptional chemical and biological stability. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects. Hence, cyclotides have become potential candidates in the development of peptide-based drugs; either as scaffolds to stabilize susceptible peptide sequences or as drugs by their own right. In this thesis, important parameters that could be inputs toward this development have been tuned. An extraction protocol that can be extended to industrial scale production of the cyclotides from natural sources was developed; accordingly, a single maceration with hydroalcoholic solutions of medium polarity represented an optimum extraction method. Moreover, it was shown that investigating the cyclotide content of cyclotide-bearing plants from diverse environments is a promising approach for extending the knowledge of both structural and biological diversity of these proteins. Five novel cyclotides with new sequence diversity were isolated and characterized from a violet that grows on Ethiopian highlands at an altitude of 3400 m. One of the areas where the cyclotide framework has attracted interest is the development of stable antimicrobial peptides. A stability study was carried out to determine the stability of the cyclotide framework in a cocktail of bacterial proteases and serum where the native forms of tested cyclotides exhibited high stability profile. Understanding the modes of cyclotide-cell interaction is certainly an important factor for the potential development of cyclotide-based drugs. Cellular studies were carried out using the comet assay and microautoradiography. A bell-shaped dose response curve was obtained for the DNA damaging effect of the cyclotides in the comet assay, which was the first toxicological assay of its kind on this class of proteins. The microautoradiography study revealed that the cyclotides penetrate into the cells even at cytotoxic concentrations. From previous reports, it was known that the cyclotides interact with membranes; the cellular studies in this thesis added to this knowledge by clearly demonstrating that these proteins have multiple modes of action.
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| 4. |
- Lundberg, Eva, 1964-
(författare)
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Micro-Structure Modelling of Acoustics of Open Porous Material
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transportation is a large and growing part of the world’s energy consumption. This drives a need for reduced weight of rail vehicles, just as it does for road vehicles. In spite of weight reductions, the vehicle still has to provide the same level of acoustic comfort for the passengers. Porous materials, with more than 90% air, are often included in multi-layer vehicle panels, contributing to acoustic performance without adding much weight. Here the acoustic performance of open cell porous materials, with focus on flow resistivity, is evaluated based on simplified micro-structure models to investigate the effect of anisotropy on the performance In order to evaluate how the redistribution of material affects the flow resistivity, the porosity of the material is kept constant. Two micro-geometries are analysed and compared: the hexahedral model and the tetrakaidecahedron (Kelvin cell). For flow resistivity calculations the solid frame is assumed to be rigid. The models are elongated in one direction to study the influence of micro-structural anisotropy on the macro level flow resistivity. To keep porosity constant, two different approaches are investigated. The first approach is to let strut thickness be uniform and adjust the volume of the cell to a constant ratio compared to the isotropic case. The second approach is to let the strut volume, and cell volume, be constant. For an anisotropic hexahedral cell with uniform strut thickness, the flow resistivity increases substantially with increasing height to width ratio for the hexahedral model, while the flow resistivity for the tetrakaidecahedron model with uniform strut thickness decreases with increasing height to width ratio. For both geometries and constant strut volume, the average flow resistivity is close to the same constant value. For uniform strut thickness the relative volume of anisotropic to isotropic volume is very important.
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| 5. |
- Muhammad, Taj, 1982-
(författare)
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LL-37-derived cyclic antimicrobial drug leads : Design, synthesis, activity and different ways of creating them
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In an era where last-line antibiotics are failing, one of the powerful approaches to develop novel therapeutic agents is to turn back to nature in order to identify possible drug candidates. Among the potential candidates, antimicrobial peptides (AMPs) have garnered much attention as an antimicrobial. These are broad spectrum host defense molecules produced by all living organisms. LL-37 is such a multitask human defense peptide that mediates various host immune responses and also exerts antimicrobial activity. However, the direct use of this 37-amino acid long α-helical peptide is hampered by protease susceptibility, in particular for antimicrobial applications. A small 12-residues peptide, referred as KR-12, derived from LL-37, has been reported to have selective toxic effect on bacteria. Analogues of KR-12 were generated in the form of Alanine and Lysine scans to find out the positions important for improved activity and selectivity. Backbone-cyclised dimers based on KR-12 and KR-12 analogues, tethered by linkers of two to four amino acid residues, were synthesised to explore the concept of cyclisation, dimerisation and cross-linking as means to enhance peptide stability and activity. Antimicrobial activities of the linear peptides and cyclic dimers were assayed against human pathogens, in buffer and/or physiological conditions. Proteolytic stability, permeabilisation efficacy on microbial membranes and, their structures were also characterised. From Ala and Lys scans, it was possible to identify two key positions for the enhanced broad-spectrum antibacterial activity: replacement of Gln5 with Lys, and Asp9 with either Ala or Lys. In serum stability assay, KR-12 and analogues were found to be unstable. The backbone-cyclised KR-12 dimers showed improved antimicrobial activity and increased stability compared to monomeric KR-12. KR-12 monomers adopt a well-defined α-helical structure in membrane-mimicking environment, while cyclised dimers were unstructured in solution judged by NMR. The KR-12 (Q5K, D9A) cyclised dimers retained antimicrobial activity in physiological conditions. Circular dichroism showed that the cyclic dimer, cd4-PP, had 77% helical content when bound to lyso-phosphatidylglycerol micelles.Moreover, the limits of cyanobactin-macrocyclase PatGmac were explored to cyclise peptides larger than their natural substrates, namely the PawS derived peptide Sunflower Trypsin Inhibitor-1 (SFTI-1) and the cyclotide kalata B1. PatGmac was used very efficiently to cyclise SFTI-1. In addition, semi-pure butelase 1, isolated from Clitoria ternatea seeds, was immobilised on NHS column. The immobilised column was then used to produce substrates ranging from 16 to 34 varying length.
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| 6. |
- Melander, Erik, et al.
(författare)
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Differential Blood-Brain Barrier Transport and Cell Uptake of Cyclic Peptides In Vivo and In Vitro
- 2023
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 15:5
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Tidskriftsartikel (refereegranskat)abstract
- The blood-brain barrier (BBB) poses major challenges to drug delivery to the CNS. SFTI-1 and kalata B1 are cyclic cell-penetrating peptides (cCPPs) with high potential to be used as scaffolds for drug delivery. We here studied their transport across the BBB and distribution within the brain to gauge the potential of these two cCPPs as scaffolds for CNS drugs. In a rat model, SFTI-1 exhibited, for a peptide, high extent of BBB transport with a partitioning of unbound SFTI-1 across the BBB, K-p,K-uu,K-brain, of 13%, while only 0.5% of kalata B1 equilibrated across the BBB. By contrast, kalata B1, but not SFTI-1, readily entered neural cells. SFTI-1, but not kalata B1, could be a potential CNS delivery scaffold for drugs directed to extracellular targets. These findings indicate that differences between the BBB transport and cellular uptake abilities of CPPs are crucial in the development of peptide scaffolds.
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| 7. |
- Steffen, Karin, 1989-
(författare)
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Genomics and metabolomics in the North Atlantic deep-sea sponge Geodia barretti
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sponges are among the earliest diverging taxa in the animal tree of life. They are sessile, filter-feeding animals found in marine and freshwater habitats. Many species are characterized by a close, specific and consistent association with microbes, mainly Bacteria and Archaea. This feature has been known for a long time and is suggested to be a factor contributing to the rich and diverse chemical output of the sponges. This thesis explored the effect of the habitat, specifically water mass or depth on sponges, their associated microbes, and their combined chemical output. The focal species of this thesis was the North Atlantic deep-sea high microbial abundance (HMA) demosponge Geodia barretti.In Paper I, 16S rRNA gene amplicon sequencing and untargeted metabolomics were used to quantify variation in prokaryotic community composition and chemical output in three sponge species. Water masses structured the prokaryotic community composition in the HMA species G. barretti and Stryphnus fortis. The community composition of the low microbial abundance (LMA) sponge Weberella bursa was unaffected by depth. Untargeted metabolomic data was modelled by depth. This allowed for identification of individual compounds varying with depth. Among those compounds were many putative osmolytes as well as diketopiperazines. Bioactive peptides and brominated tryptophan derivatives were unaffected by depth.In Paper II the diversity of the barrettide peptide family was explored in DNA sequencing data and chemical profiles across a wide selection of sponge species and G. barretti in particular. Five new barrettides were predicted and one sequence, barrettide C, was confirmed by solid phase peptide synthesis and co-elution with a native extract, antifouling bioassays and NMR structure elucidation. The confidence gained from sequence analysis and validating predictions lead us to suggest barrettides are a family of antifouling peptides in G. barretti.In Paper III, a reduced representation sequencing approach was used to evaluate the Stacks de novo pipeline in HMA sponges with the help of a whole genome assembled for this purpose. With this data, gene flow and connectivity were investigated in G. barretti populations sampled across the North Atlantic. The de novo pipeline was found to assemble and retain many putatively microbial loci and should thus only be used with reservations in HMA sponges. However, regarding biological inferences, strong population structure was recovered despite the apparent contamination.
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