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  • 1.
    Agmo Hernández, Víctor
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Karlsson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Intrinsic Heterogeneity in Liposome Suspensions Caused by the Dynamic Spontaneous Formation of Hydrophobic Active Sites in Lipid Membranes2011In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 27, no 8, p. 4873-4883Article in journal (Refereed)
    Abstract [en]

    The spontaneous, dynamic formation of hydrophobic active sites in lipid bilayer membranes is studied and characterized. It is shown that the rates of formation and consumption of these active sites control at least two important properties of liposomes: their affinity for hydrophobic surfaces and the rate by which they spontaneously release encapsulated molecules. The adhesion and spreading of liposomes onto hydrophobic polystyrene nanoparticles and the spontaneous leakage of an encapsulated fluorescent dye were monitored for different liposome compositions employing Cryo-TEM, DLS, and fluorescence measurements. It was observed that an apparently homogeneous, monodisperse liposome suspension behaves as if composed by two different populations: a fast leaking population that presents affinity for the hydrophobic substrate employed, and a slow leaking population that does not attach immediately to it. The results reported here suggest that the proportion of liposomes in each population changes over time until a dynamic equilibrium is reached. It is shown that this phenomenom can lead to irreproducibility in, for example, spontaneous leakage experiments, as extruded liposomes leak much faster just after preparation than 24 h afterward. Our findings account for discrepancies in several experimental results reported in the literature. To our knowledge, this is the first systematic study addressing the issue of an existing intrinsic heterogeneity of liposome suspensions.

  • 2.
    Alexander, Michelle
    et al.
    Univ York, York YO10 5DD, N Yorkshire, England.;Univ Aberdeen, Sch Geosci, Dept Archaeol, Aberdeen AB24 3UF, Scotland..
    Ho, Simon Y. W.
    Univ Sydney, Sch Biol Sci, Sydney, NSW 2006, Australia..
    Molak, Martyna
    Polish Acad Sci, Museum & Inst Zool, PL-00679 Warsaw, Poland..
    Barnett, Ross
    Palaeogen & Bioarchaeol Res Network, Res Lab Archaeol, Oxford OX1 3QY, England..
    Carlborg, Örjan
    Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Dorshorst, Ben
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Virginia Tech, Dept Anim & Poultry Sci, Blacksburg, VA 24061 USA..
    Honaker, Christa
    Virginia Tech, Dept Anim & Poultry Sci, Blacksburg, VA 24061 USA..
    Besnier, Francois
    Inst Marine Res, Sect Populat Genet, N-5024 Bergen, Norway..
    Wahlberg, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Dobney, Keith
    Univ Aberdeen, Sch Geosci, Dept Archaeol, Aberdeen AB24 3UF, Scotland..
    Siegel, Paul
    Virginia Tech, Dept Anim & Poultry Sci, Blacksburg, VA 24061 USA..
    Andersson, Leif
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Swedish Univ Agr Sci, Dept Anim Breeding & Genet, S-75007 Uppsala, Sweden..
    Larson, Greger
    Palaeogen & Bioarchaeol Res Network, Res Lab Archaeol, Oxford OX1 3QY, England..
    Mitogenomic analysis of a 50-generation chicken pedigree reveals a rapid rate of mitochondrial evolution and evidence for paternal mtDNA inheritance2015In: Biology Letters, ISSN 1744-9561, E-ISSN 1744-957X, Vol. 11, no 10, article id 20150561Article in journal (Refereed)
    Abstract [en]

    Mitochondrial genomes represent a valuable source of data for evolutionary research, but studies of their short-term evolution have typically been limited to invertebrates, humans and laboratory organisms. Here we present a detailed study of 12 mitochondrial genomes that span a total of 385 transmissions in a well-documented 50-generation pedigree in which two lineages of chickens were selected for low and high juvenile body weight. These data allowed us to test the hypothesis of time-dependent evolutionary rates and the assumption of strict maternal mitochondrial transmission, and to investigate the role of mitochondrial mutations in determining phenotype. The identification of a non-synonymous mutation in ND4L and a synonymous mutation in CYTB, both novel mutations in Gallus, allowed us to estimate a molecular rate of 3.13 x 10(-7) mutations/site/year (95% confidence interval 3.75 x 10(-8)-1.12 x 10(-6)). This is substantially higher than avian rate estimates based upon fossil calibrations. Ascertaining which of the two novel mutations was present in an additional 49 individuals also revealed an instance of paternal inheritance of mtDNA. Lastly, an association analysis demonstrated that neither of the point mutations was strongly associated with the phenotypic differences between the two selection lines. Together, these observations reveal the highly dynamic nature of mitochondrial evolution over short time periods.

  • 3.
    Almgren, Mats
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Garamus, Vasil M
    Nordstierna, Lars
    Luc-Blin, Jean
    Stébé, Marie-José
    Nonideal mixed micelles of fluorinated and hydrogenous surfactants in aqueous solution: NMR and SANS studies of anionic and nonionic systems.2010In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 26, no 8, p. 5355-5363Article in journal (Refereed)
    Abstract [en]

    Contrast variation SANS and (19)F chemical shifts were measured for three mixed equimolar micelle systems: sodium perfluorooctanoate (SPFO) and sodiumdecylsulfate (SDeS) in 200 mM NaCl, lithium perfluorononanate (LiPFN) and lithium dodecylsulfate (LiDS) in 200 mM LiCl, and a nonionic system C(8)F(17)C(2)H(4)(OC(2)H(4))(9) and C(12)H(25)(OC(2)H(4))(8) in water, all at 25 degrees C. The chemical shift measurements allow the calculation of the average fraction of nearest neighbors of each kind around the reporter group (the trifluoromethyl group). A preference for like neighbors were found in all systems, smallest in the SDeS/SPFO system and largest in the nonionic system, but in all cases substantially smaller than expected at critical conditions. From the SANS measurements the width of the micelle composition distribution was obtained. For the ionic systems similar values were obtained, showing a broadening compared to ideal mixtures, but not broad enough for demixing or clearly bimodal distributions. In the nonionic system the width was estimated as sigma = 0.18 and 0.22 using two different evaluation methods. These values suggest that the system is close to critical conditions. The lower value refers to a direct modeling of the system, assuming an ellipsoidal shape and a Gaussian composition distribution. The modeling showed the nonionic mixed micelles to be prolate ellipsoids with axial ratio 2.2 and an aggregation number larger than 100, whereas the two ionic systems fitted best to oblate shapes (axial ratios 0.8 and 0.65 for SDeS/SPFO and LiDS/LiPFN, respectively) and aggregation numbers of 60 for both.

  • 4.
    Ammar, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Immunohistochemical studies on small arteries from women with polycystic ovary syndrome: Focus on makers for endothelial dysfunction2012Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Women with polycystic ovary syndrome (PCOS) have an increased risk of developing cardiovascular disease (CVD). Metabolic syndrome predominate which may lead to impaired insulin sensitivity and development of type II diabetes and hypertension. Endothelial dysfunction together with pro-inflammatory, pro-thrombotic and pro-oxidant environment serves as initial phase for CVD complications. The research group performs functional studies on isolated arteries with focus on endothelium dependent relaxation from PCOS women and age matched controls and the aim of the presented project was to compare markers for endothelial dysfunction using immunohistochemistry (IHC). Small arteries were isolated from subcutaneous fat biopsies from patients and controls and fixed in O.C.T. compound (-70C dry ice). The biopsies were sectioned in 8µm thin slices for further staining with primary antibodies, DAB as a chromogen and Mayer´s Hematoxylin. The primary antibody was selected for endothelial cell adhesion molecules PECAM-1, P-selectin, vWF and VCAM-1.    There was a tendency for stronger expression of P-selectin in arteries from PCOS women compared with controls, while in the controls a tendency was seen towards stronger expression of VCAM-1 and a weaker staining for vWF if compared with PCOS. There was no obvious difference for other stained markers between the groups. There were differences between tested markers in control groups and the PCOS women whit IHC staining as well as earlier functional tests. Tested markers reflected that endothelial dysfunction were expressed in the endothelium of isolated arteries from PCOS and control women, however further functional studies and IHC studies were warranted to quantify their contribution.

  • 5.
    Andaloussi, Mounir
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Henriksson, Lena M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wieckowska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Lindh, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Larsson, Anna M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Suresh, Surisetti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Iyer, Harini
    Srinivasa, Bachally R.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase2011In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 14, p. 4964-4976Article in journal (Refereed)
    Abstract [en]

    The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. alpha-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 mu M on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 mu g/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

  • 6.
    Andersson, Ken G.
    et al.
    KTH Royal Inst Technol, Div Prot Technol, SE-10691 Stockholm, Sweden.
    Oroujeni, Maryam
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Garousi, Javad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Ståhl, Stefan
    KTH Royal Inst Technol, Div Prot Technol, SE-10691 Stockholm, Sweden.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Löfblom, John
    KTH Royal Inst Technol, Div Prot Technol, SE-10691 Stockholm, Sweden.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR: 2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator2016In: International journal of oncology, ISSN 1791-2423, Vol. 49, no 6, p. 2285-2293Article in journal (Refereed)
    Abstract [en]

    The epidermal growth factor receptor (EGFR) is overexpressed in a number of malignant tumors and is a molecular target for several specific anticancer antibodies and tyrosine kinase inhibitors. The overexpression of EGFR is a predictive biomarker for response to several therapy regimens. Radionuclide molecular imaging might enable detection of EGFR overexpression by a non-invasive procedure and could be used repeatedly. Affibody molecules are engineered scaffold proteins, which could be selected to have a high affinity and selectivity to predetermined targets. The anti-EGFR ZEGFR:2377 affibody molecule is a potential imaging probe for EGFR detection. The use of the generator-produced radionuclide 99mTc should facilitate clinical translation of an imaging probe due to its low price, availability and favorable dosimetry of the radionuclide. In the present study, we evaluated feasibility of ZEGFR:2377 labeling with 99mTc using a peptide-based cysteine-containing chelator expressed at the C-terminus of ZEGFR:2377. The label was stable in vitro under cysteine challenge. In addition, 99mTc-ZEGFR:2377 was capable of specific binding to EGFR-expressing cells with high affinity (274 pM). Studies in BALB/C nu/nu mice bearing A431 xenografts demonstrated that 99mTc-ZEGFR:2377 accumulates in tumors in an EGFR-specific manner. The tumor uptake values were 3.6±1 and 2.5±0.4% ID/g at 3 and 24 h after injection, respectively. The corresponding tumor-to-blood ratios were 1.8±0.4 and 8±3. The xenografts were clearly visualized at both time-points. This study demonstrated the potential of 99mTc-labeled ZEGFR:2377 for imaging of EGFR in vivo.

  • 7. Aquilante, Francesco
    et al.
    Barone, V
    Roos, B O
    A theoretical investigation of valence and Rydberg electronic states of acrolein.2003In: Journal of Chemical Physics, ISSN 0021-9606, E-ISSN 1089-7690, Vol. 119, no 23, p. 12323-12334Article in journal (Refereed)
    Abstract [en]

    The main features of the ultraviolet spectrum of acrolein have been studied by a multireference perturbative treatment and by a time dependent density functional approach. The valence and Rydberg transition energies have been calculated and the assignment of the experimental bands has been clarified. The different relaxation trends of the three lowest singlet and triplet excited states have been analyzed by unconstrained geometry optimizations. This has allowed, in particular, the characterization of a twisted (3)(pipi*) state, which is crucial for the interesting photophysics and photochemistry of the acrolein molecule and, more generally, of the alpha,beta-enones. Solvatochromic shifts in aqueous solution have been investigated using a combined discrete/continuum approach based on the so called polarizable continuum model. The experimental trends are well reproduced by this approach and a closer degeneracy in the triplet manifold has been detected in solution with respect to gas phase.

  • 8. Aquilante, Francesco
    et al.
    Cossi, M
    Crescenzi, O
    Scalmani, G
    Barone, V
    Computation of the acetone ultraviolet spectrum in gas phase and in aqueous solution by a mixed discrete/continuum model.2003In: Molecular Physics, ISSN 0026-8976, E-ISSN 1362-3028, Vol. 101, no 13, p. 1945-1953Article in journal (Refereed)
    Abstract [en]

    The ultraviolet spectrum of acetone in vacuum and in aqueous solution has been computed by different quantum mechanical methods coupled to the polarizable continuum model (PCM) for describing bulk solvent effects. The results in vacuo show that the time-dependent density functional theory (TDDFT) approach using the PBE0 functional reproduces quite well the result obtained at the CASPT2 level. Supermolecule computations confirm that water molecules belonging to the first shell of polar groups ( here the carbonyl oxygen) must be explicitly included in the quantum mechanical treatment, whereas the effect of other solvent molecules ( which is far from being negligible) can be reliably described by the PCM. Moreover, statistical averaging effects have been taken into account by performing canonical molecular dynamics (MD) simulations followed by TDDFT quantum mechanical computations on representative clusters of increasing dimensions immersed in a polarizable continuum. The results show that the combined MD/DFT/PCM approach is reliable and effective, although the performances of the force field used in the MD simulations must be further investigated.

  • 9. Aquilante, Francesco
    et al.
    De Vito, Luca
    Ferré, Nicolas
    Chigo, Giovanni
    Malmqvist, Per-Åke
    Neogrády, Pavel
    Pedersen, Tjomas Bono
    Pitoňák, Michak
    Reiher, Markus
    Roos, Björn O
    Serrano-Andrés, Luis
    Miroslav, Urban
    Veryazov, Valera
    Lindh, Roland
    Department of Theoretical Chemistry, Lund University.
    Software news and update MOLCAS 7: The Next Generation2010In: Journal of Computational Chemistry, ISSN 0192-8651, E-ISSN 1096-987X, Vol. 31, no 1, p. 224-247Article, review/survey (Refereed)
    Abstract [en]

    Some of the new unique features of the MOLCAS quantum chemistry package version 7 are presented in this report. In particular, the Cholesky decomposition method applied to some quantum chemical methods is described. This approach is used both in the context of a straight forward approximation of the two-electron integrals and in the generation of so-called auxiliary basis sets. The article describes how the method is implemented for most known wave functions models: self-consistent field, density functional theory, 2nd order perturbation theory, complete-active space self-consistent field multiconfigurational reference 2nd order perturbation theory, and coupled-cluster methods. The report further elaborates on the implementation of a restricted-active space self-consistent field reference function in conjunction with 2nd order perturbation theory. The average atomic natural orbital basis for relativistic calculations, covering the whole periodic table, are described and associated unique properties are demonstrated. Furthermore, the use of the arbitrary order Douglas-Kroll-Hess transformation for one-component relativistic calculations and its implementation are discussed. This section especially focuses on the implementation of the so-called picture-change-free atomic orbital property integrals. Moreover, the ElectroStatic Potential Fitted scheme, a version of a quantum mechanics/molecular mechanics hybrid method implemented in MOLCAS, is described and discussed. Finally, the report discusses the use of the MOLCAS package for advanced studies of photo chemical phenomena and the usefulness of the algorithms for constrained geometry optimization in MOLCAS in association with such studies.

  • 10. Aquilante, Francesco
    et al.
    Jensen, K. P.
    Roos, Björn O.
    The allyl radical revisited: a theoretical study of the electronic spectrum.2003In: Chemical Physics Letters, ISSN 0009-2614, E-ISSN 1873-4448, Vol. 380, no 5-6, p. 689-698Article in journal (Refereed)
    Abstract [en]

    In this Letter, we report the electronic spectrum of the allyl radical, obtained with multiconfigurational perturbation theory (MS-CASPT2). The assignment of the spectrum is in accordance with experiment to within 0.2 eV. We have computed the complete first Rydberg series and the beginning of the second Rydberg series. A new valence-excited B-2(1) state has been found which has hitherto been hidden by Rydberg transitions. A rationalisation of the electronic spectrum is provided in terms of resonance forms in ground and excited states. This model shows that while a multiconfigurational wavefunction is necessary to qualitatively model the system, the large ionic character of the valence electronic states makes an accurate treatment of the dynamical correlation necessary for a quantitative description of the spectrum.

  • 11. Aquilante, Francesco
    et al.
    Malmqvist, Per-Åke
    Pedersen, Thomas Bondo
    Ghosh, Abhik
    Roos, Björn Olof
    Cholesky decomposition-based multiconfiguration second-order perturbation theory (CD-CASPT2): application to the spin-state energetics of Co-III(diiminato)(NPh).2008In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 4, no 5, p. 694-702Article in journal (Refereed)
    Abstract [en]

    The electronic structure and low-lying electronic states of a Co-III(diiminato)(NPh) complex have been studied using mulficonfigurational wave function theory (CASSCF/CASPT2) The results have been compared to those obtained with density functional theory. The best agreement with ab initio results is obtained with a modified B3LYP functional containing a reduced amount (15%) of Hartree-Fock exchange. A relativistic basis set with 869 functions has been employed in the most extensive ab initio calculations, where a Cholesky decomposition technique was used to overcome problems arising from the large size of the two-electron integral matrix. It is shown that this approximation reproduces results obtained with the full integral set to a high accuracy, thus opening the possibility to use this approach to perform multiconfigurational wave-function-based quantum chemistry on much larger systems relative to what has been possible until now.

  • 12. Aquilante, Francesco
    et al.
    Pedersen, Thomas Bondo
    Quartic scaling evaluation of canonical scaled opposite spin second-order Moller-Plesset correlation energy using Cholesky decompositions.2007In: Chemical Physics Letters, ISSN 0009-2614, E-ISSN 1873-4448, Vol. 449, no 4-6, p. 354-357Article in journal (Refereed)
    Abstract [en]

    The scaled opposite spin second-order Moller-Plesset (SOS-MP2) energy expression is reformulated using Cholesky decomposition of the amplitude matrix. The resulting algorithm requires an auxiliary basis or Cholesky representation of the two-electron integrals and shows fourth-order scaling with system size. Based on an analysis of operation counts, we estimate that the present approach is computationally advantageous compared to the analogous fourth-order algorithms that employ Laplace transforms.

  • 13. Aquilante, Francesco
    et al.
    Pedersen, Thomas Bondo
    Sanchez de Meras, Alfredo
    Koch, Henrik
    Fast noniterative orbital localization for large molecules.2006In: Journal of Chemical Physics, ISSN 0021-9606, E-ISSN 1089-7690, Vol. 125, no 17Article in journal (Refereed)
    Abstract [en]

    We use Cholesky decomposition of the density matrix in atomic orbital basis to define a new set of occupied molecular orbital coefficients. Analysis of the resulting orbitals (”Cholesky molecular orbitals”) demonstrates their localized character inherited from the sparsity of the density matrix. Comparison with the results of traditional iterative localization schemes shows minor differences with respect to a number of suitable measures of locality, particularly the scaling with system size of orbital pair domains used in local correlation methods. The Cholesky procedure for generating orthonormal localized orbitals is noniterative and may be made linear scaling. Although our present implementation scales cubically, the algorithm is significantly faster than any of the conventional localization schemes. In addition, since this approach does not require starting orbitals, it will be useful in local correlation treatments on top of diagonalization-free Hartree-Fock optimization algorithms.

  • 14.
    Baleani, Massimiliano
    et al.
    Laboratorio di Tecnologia Medica, Istituto Ortopedico Rizzoli, Bologna, Italy.
    Fognani, Roberta
    Laboratorio di Tecnologia Medica, Istituto Ortopedico Rizzoli, Bologna, Italy.
    Feresini, Chiara
    Centro Ceramico Bologna, Bologna, Italy.
    Öhman, Caroline
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Spagnolo, Claudia
    Laboratorio di Tecnologia Medica, Istituto Ortopedico Rizzoli, Bologna, Italy.
    Baruffaldi, Fabio
    Laboratorio di Tecnologia Medica, Istituto Ortopedico Rizzoli, Bologna, Italy.
    Real wet density of bone tissue: Does it depend on tissue type and subject?2014In: Proceedings of 7th World Congress of Biomechanics, 2014, 2014Conference paper (Refereed)
  • 15.
    Beckman Sundh, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Studies on Phosphohistidine Phosphatase 1: What? Where? Why?2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Phosphohistidine phosphatase 1 (PHPT1) is a small protein, consisting of 125 amino acids, that catalyzes the dephosphorylation of histidine but does not have any activity towards other phosphorylated amino acids. PHPT1 was identified in 2002, and is so far the only mammalian histidine phosphatase known, but still little is known about its physiological role. No mammalian histidine kinases have hitherto been identified.

    Phosphorylation is one of the most important ways in which the structure and activity of a protein may be changed after translation. Proteins are phosphorylated on the side chain of amino acid residues. When a hydroxyl is phosphorylated the result is a phosphoester and when a nitrogen is phosphorylated the result is a phosphoamidate. Histidine may be phosphorylated on either of the two nitrogens of the imidazole ring of the side chain. The resulting phosphoamidate bond is labile and rich in energy, which makes histidine phosphorylation highly reversible and flexible. However, histidine phosphorylation is less studied than that of the phosphoesters due to the acid lability of the phosphoamidate bond.

    The work described in this thesis was focused on further elucidating the physiological role of PHPT1. Amino acid residues of importance for the activity of PHPT1 were identified, and mutants with decreased phosphatase activity were produced. These mutants have been used in studies on the function of PHPT1. By using immunohistochemical methodology the localization of PHPT1 in both mouse and human tissues was determined, with mainly similar results. A general finding was that expression of PHPT1 was high in epithelial cells with short turnover time, indicating that PHPT1 may have an important role in proliferating cells. We have also developed a comparatively fast and simple screening method for determination of PHPT1 activity. Since research in this field has been hampered by the lack of efficient and practical methodology, hopefully this new method will be an asset in search of inhibitors for PHPT1, which in turn may be used for detection of the elusive mammalian histidine kinases, the finding of which may give major breakthroughs in the field.

    List of papers
    1. Mutational study of human phosphohistidine phosphatase: effect on enzymatic activity.
    Open this publication in new window or tab >>Mutational study of human phosphohistidine phosphatase: effect on enzymatic activity.
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    2005 (English)In: Biochem Biophys Res Commun, ISSN 0006-291X, Vol. 337, no 3, p. 887-91Article in journal (Refereed) Published
    Keywords
    Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Enzyme Activation, Evolution; Molecular, Humans, Molecular Sequence Data, Mutagenesis; Site-Directed, Mutation, Phosphoprotein Phosphatase/analysis/*chemistry/genetics/*metabolism, Protein Binding, Recombinant Proteins/analysis/chemistry/metabolism, Research Support; Non-U.S. Gov't, Sequence Homology; Amino Acid, Structure-Activity Relationship, Substrate Specificity
    Identifiers
    urn:nbn:se:uu:diva-80308 (URN)16219293 (PubMedID)
    Available from: 2006-05-05 Created: 2006-05-05 Last updated: 2012-05-30
    2. Immunohistochemical localization of phosphohistidine phosphatase PHPT1 in mouse and human tissues
    Open this publication in new window or tab >>Immunohistochemical localization of phosphohistidine phosphatase PHPT1 in mouse and human tissues
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    2009 (English)In: Upsala Journal of Medical Sciences, ISSN 0300-9734, E-ISSN 2000-1967, Vol. 114, no 2, p. 65-72Article in journal (Refereed) Published
    Abstract [en]

    Protein histidine phosphorylation accounts for about 6% of the total protein phosphorylation in eukaryotic cells; still details concerning histidine phosphorylation and dephosphorylation are limited. A mammalian 14-kDa phosphohistidine phosphatase, also denominated PHPT1, was found 6 years ago that provided a new tool in the study of phosphohistidine phosphorylation. The localization of PHPT1 mRNA by Northern blot analysis revealed high expression in heart and skeletal muscle. The main object of the present study was to determine the PHPT1 expression on protein level in mouse tissues in order to get further information on the physiological role of the enzyme. Tissue samples from adult mice and 14.5-day-old mouse embryos were processed for immunostaining using a PHPT1-specific polyclonal antibody. The same antibody was also provided to the Swedish human protein atlas project (HPR) (http://www.proteinatlas.org/index.php). The results from both studies were essentially consistent with the previously reported expression of mRNA of a few human tissues. In addition, several other tissues, including testis, displayed a high protein expression. A salient result of the present investigation was the ubiquitous expression of the PHPT1 protein and its high expression in continuously dividing epithelial cells.

    Keywords
    Phosphohistidine phosphatase, PHPT1, PHP, phosphohistidine, dephosphorylation, HPR-project
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-119851 (URN)10.1080/03009730802642337 (DOI)000265454800001 ()19396692 (PubMedID)
    Available from: 2010-03-02 Created: 2010-03-02 Last updated: 2017-12-12Bibliographically approved
    3.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
  • 16.
    Berggren, Gustav
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Mimicking Nature – Synthesis and Characterisation of Manganese Complexes of Relevance to Artificial Photosynthesis2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The development of efficient catalyst for water oxidation is of paramount importance to artificial photosynthesis, but before this can be achieved a deeper understanding of this reaction is essential. In nature this reaction occurs in a tetranuclear Mn-cluster which serves as the work-horse of oxygenic photosynthesis. This thesis summarises my efforts at developing molecular systems capable of mimicking this complex employing a biomimetic approach.

    Three different approaches towards this goal are described here-in. The first section describes a screening study, in which a number of manganese complexes were tested to see whether or not they were capable of catalysing the formation of dioxygen when treated with different oxidants (Papers I). For those reactions in which dioxygen formation was observed the reactions were repeated in labelled water and the incorporation of labelled O-atoms was studied by mass spectrometry. This allowed us to determine to what extent water was the source of the evolved dioxygen (Papers II-III).

    In Chapter three a reported catalyst and a derivative thereof is studied in depth. The influence of changes to the ligand on the oxygen–oxygen bond forming reaction could unfortunately not be reliably addressed, because of the instability of the complexes under “catalytic” conditions. Nevertheless, the study allowed us to revise the “carboxylate shift”-mechanism suggested in the literature (Papers IV-V).

    Chapter four describes the continuation of my work on ligands featuring the carboxylate ligand motif first introduced in Chapter three. In this study ligands containing multiple binding pockets were designed and synthesised (Paper VI).

    A better understanding of the mechanism in the natural water oxidising enzyme will facilitate the design of biomimetic complexes, this is discussed in Chapter five. In this work model complexes (Paper VII) are used to study the mechanism by which natures own water oxidising catalyst performs this reaction.

    List of papers
    1. Formation of stoichiometrically O-18-labelled oxygen from the oxidation of O-18-enriched water mediated by a dinuclear manganese complex: a mass spectrometry and EPR study
    Open this publication in new window or tab >>Formation of stoichiometrically O-18-labelled oxygen from the oxidation of O-18-enriched water mediated by a dinuclear manganese complex: a mass spectrometry and EPR study
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    2008 (English)In: Energy & Environmental Science, ISSN 1754-5692, E-ISSN 1754-5706, Vol. 1, no 6, p. 668-676Article in journal (Refereed) Published
    Abstract [en]

    Oxygen formation was detected for the oxidations of various multinuclear manganese complexes by oxone (HSO5-) in aqueous solution. To determine to what extent water was the source of the evolved O-2, (H2O)-O-18 isotope-labelling experiments coupled with membrane inlet mass spectrometry (MIMS) were carried out. We discovered that during the reaction of oxone with [Mn-2(OAc)(2)(bpmp)](+) (1), stoichiometrically labelled oxygen (O-18(2)) was formed. This is the first example of a homogeneous reaction mediated by a synthetic manganese complex where the addition of a strong chemical oxidant yields O-18(2) with labelling percentages matching the theoretically expected values for the case of both O-atoms originating from water. Experiments using lead acetate as an alternative oxidant supported this finding. A detailed investigation of the reaction by EPR spectroscopy, MIMS and Clark-type oxygen detection enabled us to propose potential reaction pathways.

    National Category
    Inorganic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-108518 (URN)10.1039/b811806j (DOI)000263888600006 ()1754-5692 (ISBN)
    Available from: 2009-09-21 Created: 2009-09-21 Last updated: 2017-12-13
    2. Sodium [1,2-bis(2-methyl-2-oxopropanamido)-benzene](tetrahydrofuran) manganese(III) methanol solvate
    Open this publication in new window or tab >>Sodium [1,2-bis(2-methyl-2-oxopropanamido)-benzene](tetrahydrofuran) manganese(III) methanol solvate
    Show others...
    2005 (English)In: Acta Crystallographica Section E-Structure Reports Online, Vol. 61, p. M1169-Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-75924 (URN)
    Available from: 2006-02-24 Created: 2006-02-24 Last updated: 2011-01-11
    3. Two tetranuclear Mn-complexes as biomimetic models of the oxygen evolving complex in Photosystem II. A synthesis, characterisation and reactivity study
    Open this publication in new window or tab >>Two tetranuclear Mn-complexes as biomimetic models of the oxygen evolving complex in Photosystem II. A synthesis, characterisation and reactivity study
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    2009 (English)In: Dalton Transactions, ISSN 1477-9226, E-ISSN 1477-9234, no 45, p. 10044-10054Article in journal (Refereed) Published
    National Category
    Inorganic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-108451 (URN)10.1039/b906175d (DOI)
    Available from: 2009-09-21 Created: 2009-09-18 Last updated: 2017-12-13
    4. Mechanistic Studies on the Water-Oxidizing Reaction of Homogeneous Manganese-Based Catalysts: Isolation and Characterization of a Suggested Catalytic Intermediate
    Open this publication in new window or tab >>Mechanistic Studies on the Water-Oxidizing Reaction of Homogeneous Manganese-Based Catalysts: Isolation and Characterization of a Suggested Catalytic Intermediate
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    2011 (English)In: Inorganic Chemistry, ISSN 0020-1669, E-ISSN 1520-510X, Vol. 50, no 8, p. 3425-3430Article in journal (Refereed) Published
    Abstract [en]

    The synthesis, isolation, and characterization of two high-valent manganese dimers with isomeric ligands are reported. The complexes are synthesized and crystallized from solutions of low-valent precursors exposed to tert-butyl hydroperoxide. The crystal structures display centrosymmetric complexesconsisting of Mn2 IV,IV(μ-O)2 cores, with one ligand coordinating to each manganese. The ligands coordinate with the diaminoethane backbone, the carboxylate, and one of the two pyridines, while the second pyridine is noncoordinating. The activity of these complexes, under water oxidation conditions, is discussed in light of a proposed mechanism for water oxidation, in which this type of complexes have been suggested as a key intermediate.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-108524 (URN)10.1021/ic102336a (DOI)000290457700034 ()21428420 (PubMedID)
    Available from: 2009-09-21 Created: 2009-09-21 Last updated: 2017-12-13
    5. Synthesis and characterisation of low valent Mn-complexes as models for Mn-catalases
    Open this publication in new window or tab >>Synthesis and characterisation of low valent Mn-complexes as models for Mn-catalases
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    2010 (English)In: Dalton Transactions, ISSN 1477-9226, E-ISSN 1477-9234, Vol. 39, no 45, p. 11035-11044Article in journal (Refereed) Published
    Abstract [en]

    In this work we report the synthesis of two novel manganese complexes, [L1(3)Mn(6)(II)](ClO4)(6) (1 center dot(ClO4)(6)) and [L2Mn(2)(II)(mu-OAc)(mu-Cl)](ClO4)(2) (2 center dot(ClO4)(2)), where L1(2-) is the 2,2'-(1,3-phenylenebis(methylene))bis-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)azanediyl)diacetic acid anion and L2 is N1,N1'-(1,3-phenylenebis(methylene))bis(N2,N2'-bis(pyridin-2-ylmethyl)ethane-1,2-diamine). The ligands Na(2)L1 and L2 are built on the same backbone, L2 only contains nitrogen donors, while two carboxylate arms have been introduced in Na(2)L1. The two complexes have been characterized by single-crystal X-ray diffraction, magnetic susceptibility, EPR spectroscopy, and electrochemistry. X-Ray crystallography revealed that 1 is a manganese(II) hexamer and 2 is a manganese(II) dimer featuring an unprecedented mono-mu-acetato, mono-mu-chlorido bridging motif. The ability of the complexes to catalyse H2O2 disproportionation, thereby acting as models for manganese catalases, has been investigated and compared to the activity of two other related manganese complexes. The introduction of carboxylate donors in the ligands, leading to increased denticity, resulted in a drop in H2O2 disproportionation activity.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-108525 (URN)10.1039/c0dt00165a (DOI)000284066100028 ()
    Available from: 2009-09-21 Created: 2009-09-21 Last updated: 2017-12-13
    6. Oxygen evolving reactions catalysed by synthetic manganese complexes: A systematic screening
    Open this publication in new window or tab >>Oxygen evolving reactions catalysed by synthetic manganese complexes: A systematic screening
    2007 (English)In: Dalton Transactions, ISSN 1477-9226, E-ISSN 1477-9234, no 38, p. 4258-4261Article in journal (Refereed) Published
    Abstract [en]

    A set of six multinuclear manganese complexes was screened for the ability to catalyse reactions yielding O(2) under coherent experimental conditions; we identify a much larger number of manganese compounds than previously known that catalyse oxygen formation.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-11551 (URN)10.1039/b710761g (DOI)000249705100004 ()17893814 (PubMedID)
    Available from: 2007-09-27 Created: 2007-09-27 Last updated: 2017-12-11
    7. Oxygen Evolving Reactions by Synthetic Manganese Complexes
    Open this publication in new window or tab >>Oxygen Evolving Reactions by Synthetic Manganese Complexes
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    2008 (English)In: Photosynthesis. Energy from the Sun: 14th International Congress on Photosynthesis, Springer, Netherlands , 2008Chapter in book (Other (popular science, discussion, etc.))
    Place, publisher, year, edition, pages
    Springer, Netherlands, 2008
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-108452 (URN)978-1-4020-6707-5 (ISBN)
    Available from: 2009-09-21 Created: 2009-09-18 Last updated: 2015-04-24
  • 17.
    Bergström, Gunnel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Ek, Pia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Dahlqvist, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Humble, Elisabet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Subtilisin-catalyzed removal of phosphorylated site of pig liver pyruvate kinase without inactivation of the enzyme1975In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 56, no 2, p. 288-291Article in journal (Refereed)
  • 18.
    Bergström, Gunnel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Ekman, Pia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Humble, Elisabet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Proteolytic modification of pig and rat liver pyruvate kinase including the phosphorylatable site1978In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 532, no 2, p. 259-267Article in journal (Refereed)
    Abstract [en]

    The phosphorylated or phosphate-accepting site of pyruvate kinase from pig and rat liver was removed without inactivation by incubation with subtilisin. At different time intervals the subtilisin was inactivated with phenylmethylsulfonyl fluoride and the amount of remaining phosphorylatable or phosphorylated sites of pyruvate kinase estimated by incubation with an excess of [32P]-ATP and protein kinase. It was found that to get the same rate of modification the subtilisin concentration required to modify unphosphorylated pyruvate kinase was approximately ten times higher than that used for removal of the phosphorylated site of phosphorylated site of phosphorylated enzyme. It was shown that the proteolytically-modified pyruvate kinase had an increased apparent Km for phosphoenolpyruvate without a change in V, when compared to unmodified unphosphorylated and phosphorylated pyruvate kinase. The removal of the phosphorylated site was not associated with loss of the allosteric sites for ATP and Fru-1,6-P2. The possibility that phosphorylation of the pyruvate kinase increases its degradation rate in vivo is briefly discussed.

  • 19.
    Blom, Elisabeth
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Karimi, Farhad
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Eriksson, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Hall, Håkan
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Synthesis and in vitro evaluation of 18F-β-carboline alkaloids as PET ligands2008In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 51, no 6, p. 277-282Article in journal (Refereed)
    Abstract [en]

    A one-step 18F-labelling strategy was used to prepare four 18F-labelled analogues of 7-methoxy-1-methyl-9H-β-carboline (harmine): 7-(2-[18F]fluoroethoxy)-1-methyl-9H-β-carboline (5), 7-(3-[18F]fluoro-propoxy)-1-methyl-9H-β-carboline (6), 7-[2-(2-[18F]fluoroethoxy)ethoxy]-1-methyl-9H-β-carboline (7), and 7-{2-[2-(2-[18F]fluoroethoxy)ethoxy]-ethoxy}-1-methyl-9H-β-carboline (8). These were synthesized as potential PET ligands for monoamine oxidase A. A solution of pure labelled compound in buffer was obtained in < 70 min from end of radionuclide production, with a decay-corrected yield of up to 23%. The average specific binding to MAO-A in rat brain, determined by autoradiography experiments, was highest for compounds 7 and 8 (89 ± 2 and 96 ± 1% respectively), which was obtained at < 1 nM radioligand concentration.

  • 20.
    Blom, Elisabeth
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Karimi, Farhad
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    [F-18]/F-19 exchange in fluorine containing compounds for potential use in F-18-labelling strategies2009In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 52, no 12, p. 504-511Article in journal (Refereed)
    Abstract [en]

    Exchange of [F-18]fluoride with F-19 in various organofluorine compounds in concentrations ranging from 0.06 to 56 mM was explored. We aimed to explore whether exchange reactions can be a potential useful labelling strategy, when there are no requirement of high specific radioactivity. Parameters such as solvents, temperature, conventional vs microwave heating, and the degree of fluorine load in some aromatic and alkyl compounds were investigated with regard to radiochemical yield and specific radioactivity. A series of fluorobenzophenones (1-6), 1-(4-fluorophenyl)ethanone (7), various activated and deactivated fluoro benzenes (8-16), N-(pentafluorophenyl)benzamide (17), (pentafluorophenyl)formamide (18), (tridecafluorohexyl) benzene (19) and tetradecafluorohexane (20) were subjected to [F-18]/F-19 exchange. To test this strategy to label biologically active molecules containing fluorine atoms in an aryl group, two analogues of WAY-100635 (21-22), Lapatinib (23), 2,5,6,7,8-pentafluoro-3-methyinaphthoquinone (24) and 1-(2,4-difluorophenyl)-3-(4-fluorophenyl)propan-l-one (25) were investigated. The multi-fluorinated molecules containing an electron-withdrawing group were successfully labelled at room temperature, whereas the monofluorinated, as well as those containing an electron-donating group, required heating for the exchange reaction to take place.

  • 21. Bradley, Jean-Claude
    et al.
    Guha, Rajarshi
    Lang, Andrew
    Lindenbaum, Pierre
    Neylon, Cameron
    Williams, Antony
    Willighagen, Egon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Beautifying Data in the Real World2009In: Beautiful Data: The Stories Behind Elegant Data Solutions / [ed] Toby Segaran & Jeff Hammerbacher, Sebastol, USA: O'Reilly , 2009, 1, p. 259-278Chapter in book (Other (popular science, discussion, etc.))
  • 22.
    Brännström, Nikolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Comparison of the natural variation of Tripeptidyl peptidase II activity in blood samples among healthy subjects2011Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Tripeptidyl peptidase II (TPPII) is a very large enzyme complex with a molecular weight of 5-6MDa and it has broad substrate specificity. It is located in the cytosol in most eukaryotic cells. TPPII which is an amino peptidase has exopeptidase activity, it removes tripeptides from the free N-terminus of oligopeptides and it is acting downstream of the proteasome. TPPII participates in a number of important processes in the cell: protein degradation, antigen presentation and apoptosis. In some tumor cells an increased expression of TPPII has been found which raise the question if TPPII can be used as a tumor marker in blood. The aim of this study was to compare the natural variation of the enzyme activity in blood samples among healthy subjects and also to see if the specific activity changed dependent on how the samples were stored after sampling. To do this an activity assay was used to measure the TPPII enzyme activity and the method of Bradford. Western blot was used to ensure that the right product, TPPII protein was detected. Finally qPCR was used to evaluate the feasibility of detecting TPPII mRNA in blood samples and to determine if mRNA levels correlated to the TPPII protein amount. The result showed a variation in enzyme activity among healthy subjects, a high activity in erythrocyte fractions compared to plasma and leukocyte fractions and also that storing the samples as lysate in -80C gave the least change in relative specific activity in comparison to the fresh blood cell fractions.

  • 23.
    Chi, Celestine N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bach, Anders
    University of Copenhagen.
    Gottschalk, Marie
    University of Copenhagen.
    Kristensen, S. Anders
    University of Copenhagen.
    Strømgaard, Kristian
    University of Copenhagen.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Deciphering the kinetic binding mechanism of dimeric ligands, using a potent plasma-stable dimeric inhibitor of postsynaptic density protein-95 as an example2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 36, p. 28252-28260Article in journal (Refereed)
    Abstract [en]

    Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity towards their targets due to their ability to bind simultaneously to two binding sites and are therefore very attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide of PDZ1-2 of PSD-95 using protein engineering in combination with fluorescence polarisation, isothermal titration calorimetry and stopped-flow fluorimetry. Our experiments demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and we also find evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand, but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.

  • 24. Chung, L. W.
    et al.
    Hayashi, S.
    Lundberg, Marcus
    Kyoto University.
    Nakatsu, T.
    Kato, H.
    Morokuma, K.
    Mechanism of efficient firefly bioluminescence via adiabatic transition state and seam of sloped conical intersection.2008In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 130, no 39, p. 12880-12881Article in journal (Refereed)
    Abstract [en]

    Firefly emission is a well-known efficient bioluminescence. However, the mystery of the efficient thermal generation of electronic excited states in firefly still remains unsolved, particularly at the atomic and molecular levels. We performed SA-CASSCF(12,12)/6-31G* and CASPT2(12,12)/6-31G*//SA-CASSCF(12,12)/6-31G* calculations to elucidate the reaction mechanism of bioluminescence from the firefly dioxetanone in the gas phase. Adiabatic transition state (TS) for the O-O bond cleavage and the minimum energy conical intersection (MECI) were located and characterized. The unique topology of MECI featuring a seam of a sloped conical intersection for the firefly dioxetanone, which was uncovered for the first time, emerges along the reaction pathway to provide a widely extended channel to diabatically access the excited-state from the ground state.

  • 25.
    Co, Michelle
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Pressurised Fluid Extraction of Bioactive Species in Tree Barks: Analysis using Hyphenated Electrochemical Mass Spectrometric Detection2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Analytical chemistry has developed throughout time to meet current needs. At present, the interest in biorefinery is growing, due to environmental awareness and the depletion of fossil resources. Biomass from agricultural and forestry industries has proven to be excellent raw material for different processes. Biorefinering valuable species such as bioactive species from biomass, without compromising the primary process of the biomass is highly desirable. Pressurised fluid extraction (PFE) using water and ethanol as a solvent was developed for extracting betulin from birch (Betula pendula) bark. Apart from betulin, stilbene glucosides such as astringin, isorhapontin and picied were also extracted from spruce (Picea abies) using PFE. PFE is an advanced technique that extracts at temperatures above the solvent’s atmospheric boiling point. The applied pressure in PFE is mainly to maintain the liquid state of the extraction solvent. Parameters such as type of solvent, temperature, and time affect the extraction selectivity and efficiency. Therefore it is necessary to comprehend these parameters in order to optimise extraction. The DPPH (1,1-diphenyl-2-picrylhydrazyl) assay was used to determine the antioxidant capacity and activity of the obtained bioactive species. The results showed high antioxidant capacity in bioactive species that were extracted at an elevated temperature, 180°C. Extraction and degradation occur simultaneously during the extraction. Hence, it is crucial to separate these two processes in order to obtain the actual value.

    An online hyphenated system of chromatographic separation electrochemical mass spectrometric detection was developed (LC-DAD-ECD-MS/MS). The electrochemical detector facilitates real-time monitoring of the antioxidant capacity and activity of each antioxidant and its oxidation products. This developed LC-DAD-ECD-MS/MS method enabled rapid screening of antioxidants and created a fingerprint map for their oxidation products. Characterisation and molecular elucidation of bioactive species were also performed. Degradation of bioactive species was investigated with the said online system and birch bark extract was compared with birch bark extracts that were hydrothermally treated. The obtained results showed some degradation of antioxidants at 180°C.

    In summary, the aim of this thesis was to develop analytical methods integrated with sustainable chemistry for extraction of bioactive species in biomass from the forestry industry. A novel online system using selective and sensitive detectors such as diode-array, electrochemical, and tandem mass spectrometry was developed to rapidly determine the antioxidant capacity and activity of antioxidants. Furthermore, tandem mass spectrometry enables identification of unknown bioactive species without the need of reference samples.

    List of papers
    1. Pressurized liquid extraction of betulin and antioxidants from birch bark
    Open this publication in new window or tab >>Pressurized liquid extraction of betulin and antioxidants from birch bark
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    2009 (English)In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 11, no 5, p. 668-674Article in journal (Refereed) Published
    Abstract [en]

    Pressurized hot (subcritical) water and ethanol were used to extract betulin and antioxidants from birch bark. Betulin was found to be the major compound (around 26% (w/w)), which was able to be extracted with ethanol (120 degrees C, 50 bar, 15 minutes) but not with water at any of the temperatures tested (40-180 degrees C, 50 bar). The obtained extraction result for betulin is supported by theoretical solvation parameter calculations. Furthermore, high antioxidant activity of the extract was obtained using both ethanol and water as solvent. The antioxidant activity, as determined by a DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, was found to be highest for the water extract of finely ground bark and it markedly increased with elevated extraction temperatures (90-180 degrees C). To elucidate if this was due to increased extraction efficiency or chemical reactions, a set of experiments was performed in which the samples were pre-treated with water at different temperatures before extraction. Results from liquid chromatography showed some differences in molecular composition between samples pre-treated at ambient and 180 degrees C, respectively. However, more detailed studies have to be performed to distinguish between hot-water extraction and reaction kinetics.

    Place, publisher, year, edition, pages
    The Royal Society of Chemistry, 2009
    National Category
    Analytical Chemistry
    Research subject
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-106795 (URN)10.1039/b819965e (DOI)000266004400012 ()
    Available from: 2009-07-03 Created: 2009-07-03 Last updated: 2017-12-13Bibliographically approved
    2. Extraction of Antioxidants from Spruce (Picea abies) Bark using Eco-Friendly Solvents
    Open this publication in new window or tab >>Extraction of Antioxidants from Spruce (Picea abies) Bark using Eco-Friendly Solvents
    Show others...
    2012 (English)In: Phytochemical Analysis, ISSN 0958-0344, E-ISSN 1099-1565, Vol. 23, no 1, p. 1-11Article in journal (Refereed) Published
    Abstract [en]

    Introduction-Antioxidants are known to avert oxidation processes and they are found in trees and other plant materials. Tree bark is a major waste product from paper pulp industries; hence it is worthwhile to develop an extraction technique to extract the antioxidants.

    Objective- To develop a fast and environmentally sustainable extraction technique for the extraction of antioxidants from bark of spruce (Picea abies) and also to identify the extracted antioxidants that are abundant in spruce bark.

    Methodology- A screening experiment that involved three different techniques, was conducted to determine the best technique to extract antioxidants.The antioxidant capacity of the extracts was determined with DPPH (2,2-diphenyl-2’-picrylhydrazyl) assay. Pressurised fluid extraction (PFE) turned out to be the best technique and a response surface design was therefore utilised to optimise PFE. Furthermore, NMR and HPLC-DAD-MS/MS were applied to identify the extracted antioxidants.

    Results- PFE using water and ethanol as solvent at 160 and 180°C, respectively, gave extracts of the highest antioxidant capacity. Stilbene glucosides such as isorhapontin, piceid and astringin were identified in the extracts.

    Conclusion-The study has shown that PFE is a fast and environmentally sustainable technique, using water and ethanol as solvent for the extraction of antioxidants from spruce bark.

    Place, publisher, year, edition, pages
    Wiley-Blackwell, 2012
    Keywords
    Accelerated solvent extraction, antioxidant, DPPH, ethanol, Picea abies, pressurised fluid extraction, supercritical fluid extraction, water
    National Category
    Other Basic Medicine
    Research subject
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-133267 (URN)10.1002/pca.1316 (DOI)000298260100001 ()
    Funder
    Swedish Research Council
    Available from: 2010-11-04 Created: 2010-11-04 Last updated: 2018-01-12Bibliographically approved
    3. Identification and Characterization of Polyphenolic Antioxidants Using On-Line Liquid Chromatography, Electrochemistry, and Electrospray Ionization Tandem Mass Spectrometry
    Open this publication in new window or tab >>Identification and Characterization of Polyphenolic Antioxidants Using On-Line Liquid Chromatography, Electrochemistry, and Electrospray Ionization Tandem Mass Spectrometry
    Show others...
    2009 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 81, no 21, p. 8968-8977Article in journal (Refereed) Published
    Abstract [en]

    It is demonstrated that electrochemistry (EC) coupled to liquid chromatography (LC) and electrospray ionization tandem mass spectrometry (LC/EC/ESI-MS/MS) can be used to rapidly obtain information about the antioxidant activity (i.e., oxidation potential) and capacity (i.e., amount) of polyphenolic compounds, including catechin, kaempferol, resveratrol, quercetin, and quercetin glucosides. The described on-line LC/EC/ESI-MS/MS method facilitates the detection and characterization of individual antioxidants based on a combination of the obtained m/z values for the antioxidants and their oxidation products, the potential dependences for the ion intensities, and correlations between the retention times in the LC, EC, and MS chromatograms. As these results provide patterns that can be used in rapid screening for antioxidants in complex samples, the method should be a valuable complement to chemical assays commonly used to determine the total antioxidant capacity of samples. It is shown that the antioxidant capacity for a mixture of polyphenolic compounds depends on the redox potential employed in the evaluation, and this should consequently be taken into account when comparing results from different total antioxidant capacity assays. It is also demonstrated that the inherent antioxidant capacities of phenolic compounds increase with an increasing number of hydroxyl groups and that the potential needed to oxidize the remaining hydroxyl groups increases successively upon oxidation of the compound. Unlike chemical assays, which generally do not provide any information about the identities of the compounds on the molecular level, the present screening method can be used to identify individual antioxidants, rank compounds with respect to their ease of oxidation, and to study the antioxidant capacity at any redox potential of interest.

    National Category
    Analytical Chemistry Inorganic Chemistry
    Research subject
    Analytical Chemistry; Inorganic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-99328 (URN)10.1021/ac901397c (DOI)000276191900046 ()
    Available from: 2009-03-12 Created: 2009-03-12 Last updated: 2018-06-26Bibliographically approved
    4. Degradation effects in the extraction of antioxidants from birch bark using water at elevated temperature and pressure
    Open this publication in new window or tab >>Degradation effects in the extraction of antioxidants from birch bark using water at elevated temperature and pressure
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    2012 (English)In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 716, p. 40-48Article in journal (Refereed) Published
    Abstract [en]

    Experiments with birch bark samples have been carried to enable a distinction between extraction and degradation effects during pressurised hot water extraction. Two samples, E80 and El 80, contained birch bark extracts obtained after extraction at 80 and 180 degrees C for up to 45 min, respectively. Two other samples, P80 and P180, were only extracted for 5 min at the two temperatures and were thereafter filtered and hydrothermally treated at 80 and 180 degrees C, respectively. During the latter treatment, samples were collected at different times to assess the stability of the extracted compounds. An offline DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, as well as a high performance liquid chromatographic separation coupled to an electrochemical detector, were used to determine the antioxidant capacity of the processed samples. The results obtained with the different techniques were compared to assess the yield of the extraction and degradation processes. In addition, an online hyphenated system comprising high performance liquid chromatography coupled to diode-array; electrochemical; and tandem mass spectrometric detection (HPLC-DAD-ECD-MS/MS) was used to study the compositions of the extracts in more detail. The results for the samples processed at 80 degrees C showed that the extraction reached a steady-state already after 5 min, and that the extracted compounds were stable throughout the entire extraction process. Processing at 180 degrees C, on the other hand, gave rise to partly degraded extracts with a multitude of peaks in both the diode array and electrochemical detectors, and a higher antioxidant capacity compared to for the extracts obtained at 80 degrees C. It is concluded that HPLC-DAD-ECD is a more appropriate technique for the determination of antioxidants than the DPPH assay. The mass spectrometric results indicate that one of the extracted antioxidants, catechin, was isomerised to its diastereoisomers; (+)-catechin, (-)-catechin, (+)-epicatechin, and (-)-epicatechin.

     

    Keywords
    Degradation, Pressurised fluid extraction, Antioxidants, DPPH assay, Electrochemical detection, Diode-array detection, Tandem mass spectrometry, Elevated temperature, Birch bark
    National Category
    Inorganic Chemistry
    Research subject
    Analytical Chemistry; Chemistry with specialization in Inorganic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-132344 (URN)10.1016/j.aca.2011.04.038 (DOI)000301092900008 ()
    Available from: 2010-11-04 Created: 2010-10-18 Last updated: 2017-12-12Bibliographically approved
  • 26.
    Co, Michelle
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Fagerlund, Amelie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Engman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Sunnerheim, Kerstin
    Department of Natural Sciences, Engineering and Mathematics, Mid Sweden University.
    Sjöberg, Per J. R
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Turner, Charlotta
    Dept of Organic Chemistry, Lund University.
    Extraction of Antioxidants from Spruce (Picea abies) Bark using Eco-Friendly Solvents2012In: Phytochemical Analysis, ISSN 0958-0344, E-ISSN 1099-1565, Vol. 23, no 1, p. 1-11Article in journal (Refereed)
    Abstract [en]

    Introduction-Antioxidants are known to avert oxidation processes and they are found in trees and other plant materials. Tree bark is a major waste product from paper pulp industries; hence it is worthwhile to develop an extraction technique to extract the antioxidants.

    Objective- To develop a fast and environmentally sustainable extraction technique for the extraction of antioxidants from bark of spruce (Picea abies) and also to identify the extracted antioxidants that are abundant in spruce bark.

    Methodology- A screening experiment that involved three different techniques, was conducted to determine the best technique to extract antioxidants.The antioxidant capacity of the extracts was determined with DPPH (2,2-diphenyl-2’-picrylhydrazyl) assay. Pressurised fluid extraction (PFE) turned out to be the best technique and a response surface design was therefore utilised to optimise PFE. Furthermore, NMR and HPLC-DAD-MS/MS were applied to identify the extracted antioxidants.

    Results- PFE using water and ethanol as solvent at 160 and 180°C, respectively, gave extracts of the highest antioxidant capacity. Stilbene glucosides such as isorhapontin, piceid and astringin were identified in the extracts.

    Conclusion-The study has shown that PFE is a fast and environmentally sustainable technique, using water and ethanol as solvent for the extraction of antioxidants from spruce bark.

  • 27. Cox, Nicholas
    et al.
    Ho, Felix M.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Pewnim, Naray
    Steffen, Ronald
    Smith, Paul J.
    Havelius, Kajsa G. V.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Hughes, Joseph L.
    Debono, Lesley
    Styring, Stenbjörn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Krausz, Elmars
    Pace, Ron J.
    The S-1 split signal of photosystem II: a tyrosine-manganese coupled interaction2009In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1787, no 7, p. 882-889Article in journal (Refereed)
    Abstract [en]

    Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q(A)(-) is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (<5%) oxidation of chlorophyll or carotenoid centers occurs for illumination temperatures 5-20 K. An optically silent electron donor to P680(+) is observed with the same decay kinetics as the S-1 split signal. Cryogenic donors to P680(+) seen are: (i) transient (t(1/2)similar to 150 s) tyrosine related species, including 'split signals' (similar to 15% total centers), (ii) reduced cytochrome b(559) (similar to 30-50% centers), and (iii) an organic donor, possibly an amino acid side chain, (similar to 30% centers).

  • 28.
    Dahlqvist, Ulla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Ek, Pia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Endogenous substrates of protein kinase in rat liver cell sap under different dietary conditions1978In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 540, no 1, p. 13-23Article in journal (Refereed)
    Abstract [en]

    Liver cell sap from normally fed rats, rats fed with a high-carbohydrate diet and fasted rats was chromatographed on DEAE-cellulose (pH 7.0). The chromatogram from each diet group was analyzed for pyruvate kinase activity and endogenous substrates of cyclic AMP-stimulated protein kinase. The materials were pooled into five phosphorylatable fractions, in each of which phosphate incorporation at 0.1 mM and 1.0 mM [32P]ATP in the presence of cyclic AMP and protein kinase was determined. For characterization of the phosphorylatable components, thin-layer gel chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis in detergent were used for determination of native and minimal molecular weights, respectively. Except for pyruvate kinase, eight components which incorporated at least 0.05 nmol of [32P]phosphate/g of liver were detected. The phosphorylation of four of them was stimulated by cyclic AMP. Their minimal molecular weights were 42000, 21000, 52000 and 49000. The component with a minimal molecular weight of 42000 seemed to have a native molecular weight of 160000. Both the 21000 and the 52000 component had a native molecular weight of about 110000-120000. The protein with a minimal molecular weight of 49000 could not be correlated with certainty to a native molecular weight. The proteins whose phosphorylation was not stimulated by cyclic AMP had minimal molecular weights of 54000, 39000, 34000 and 22000.

  • 29.
    Danfors, Torsten
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
    Sörensen, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Lubberink, Mark
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Medical Physics.
    Kumlien, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
    Relative Cerbral Blood Flow Measurement using dynamic Flumazenil-PET may Replace Fluorodeoxyglucose-PET in Epilepsy Surgical Investigations2012Article in journal (Other academic)
  • 30.
    Diesen, Jarle Sidney
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Organic Chemistry.
    Asymmetric Hydrogenations of Imines, Vinyl Fluorides, Enol Phosphinates and Other Alkenes Using N,P-Ligated Iridium Complexes2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The research described in this thesis is directed toward the efficient, enantioselective synthesis of chiral products that have useful functionality. This goal was pursued through catalytic asymmetric hydrogenation, a reaction class that selectively introduces one or two stereocenters into a molecule in an atom-efficient step. This reaction uses a small amount (often <1 mol%) of a chiral catalyst to impart stereoselectivity to the product formed. Though catalytic asymmetric hydrogenation is not a new reaction type, there remain many substrate classes for which it is ineffective. The present thesis describes efforts to extend the reaction to some of these substrates classes. Some of the products synthesized in these studies may eventually find use as building blocks for the production of chiral pharmaceuticals, agrochemicals, or flavouring or colouring agents. However, the primary and immediate aim of this thesis was to develop and demonstrate new catalysts that are rapid and effective in the asymmetric hydrogenation of a broad range of compounds.

    Paper I describes the design and construction of two new, related chiral iridium compounds that are catalysts for asymmetric hydrogenation. They each contain an N,P-donating phosphinooxazoline ligand that is held together by a rigid bicyclic unit. One of these iridium compounds catalyzed the asymmetric hydrogenation of acyclic aryl imines, often with very good enantioselectivities. This is particularly notable because acyclic imines are difficult to reduce with useful enantioselectivity. The second catalyst was useful for the asymmetric hydrogenation of two aryl olefins. In Paper II, the class of catalysts introduced into Paper I is expanded to include many more related compounds, and these are also applied to the asymmetric hydrogenation of prochiral imines and olefins. By studying a range of related catalysts that differ in a single attribute, we were able to probe how different parts of the catalyst affect the yield and selectivity of the hydrogenation reactions.

    Whereas iridium catalysts had been applied to the asymmetric hydrogenation of imines and largely unfunctionalized olefins prior to this work (with varied degrees of success), they had not been used to reduce fluoroolefins. Their hydrogenation, which is discussed in Paper III, was complicated by concomitant defluorination to yield non-halogenated alkanes. To combat this problem, several iridium-based hydrogenation catalysts were applied to the reaction. Two catalysts stood out for their ability to produce chiral fluoroalkanes in good enantioselectivity while minimizing the defluorination reaction, and one of these bore a phosphinooxazoline ligand of the type described in Papers I and II.

    Enol phosphinates are another class of olefins that had not previously been subjected to iridium-catalyzed asymmetric hydrogenation. They do, however, constitute an attractive substrate class, because the product chiral alkyl phosphinates can be transformed into chiral alcohols or chiral phosphines with no erosion of enantiopurity. Iridium complexes of the phosphinooxazoline ligands described in Papers I and II were extremely effective catalysts for the asymmetric hydrogenation of enol phosphinates. They produced alkyl phosphinates from di- and trisubstituted enol phosphinate, β-ketoester-derived enol phosphinates, and even purely alkyl-substituted enol phopshinates, in very high yields and enantioselectivities.

    List of papers
    1. Application of Phosphine-Oxazoline Ligands in Ir-Catalyzed Asymmetric Hydrogenation of Acyclic Aromatic N-Arylimines
    Open this publication in new window or tab >>Application of Phosphine-Oxazoline Ligands in Ir-Catalyzed Asymmetric Hydrogenation of Acyclic Aromatic N-Arylimines
    2004 In: Organic Letters, Vol. 6, no 21, p. 3825-3827Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-97350 (URN)
    Available from: 2008-05-13 Created: 2008-05-13Bibliographically approved
    2. Hydrogenation of Imines and Olefins Using Phosphine-Oxazoline Iridium Complexes as Catalysts
    Open this publication in new window or tab >>Hydrogenation of Imines and Olefins Using Phosphine-Oxazoline Iridium Complexes as Catalysts
    2006 In: Chemistry-A European Journal, Vol. 12, no 8, p. 2318-2328Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-97351 (URN)
    Available from: 2008-05-13 Created: 2008-05-13Bibliographically approved
    3. Iridium-Catalyzed Asymmetric Hydrogenation of Fluorinated Olefins Using N,P-Ligands: A struggle with hydrogenolysis and selectivity
    Open this publication in new window or tab >>Iridium-Catalyzed Asymmetric Hydrogenation of Fluorinated Olefins Using N,P-Ligands: A struggle with hydrogenolysis and selectivity
    2007 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 129, no 15, p. 4536-4537Article in journal (Refereed) Published
    Abstract [en]

    To broaden the substrate scope of asymmetric iridium-catalyzed hydrogenation, fluorine-functionalized olefins were synthesized and hydrogenated with iridium complexes. Preliminary results showed high levels of fluorine elimination together with low selectivity. The loss of vinylic fluorine at first seemed difficult to handle, but further studies revealed that a catalyst with an azanorbornyl scaffold in the ligand gave more promising results. With this in mind, a new ligand was developed. This gave among the best results published to date for fluorine asymmetric hydrogenation, yielding high conversion and very high ee's with very little fluorine elimination. Further increasing the selectivity, the trials also revealed that tetrasubstituted fluorine-containing olefins can be hydrogenated with high ee's, despite that this class of compounds has usually shown low reactivity in this reaction type.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-97352 (URN)10.1021/ja0686763 (DOI)000245739700016 ()17375924 (PubMedID)
    Available from: 2008-05-13 Created: 2008-05-13 Last updated: 2017-12-14Bibliographically approved
    4. Asymmetric Hydrogenation of Di and Trisubstituted Enol Phosphinates with N,P-Ligated Iridium Complexes
    Open this publication in new window or tab >>Asymmetric Hydrogenation of Di and Trisubstituted Enol Phosphinates with N,P-Ligated Iridium Complexes
    2008 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 130, no 16, p. 5595-5599Article in journal (Refereed) Published
    Abstract [en]

    The iridium-catalyzed asymmetric hydrogenation of various di- and trisubstituted enol phosphinates has been studied. Excellent enantioselectivities (up to >99% ee) and full conversion were observed for a range of substrates with both aromatic and aliphatic side chains. Enol phosphinates are structural analogues of enol acetates, and the hydrogenated alkyl phosphinate products can easily be transformed into the corresponding alcohols with conservation of stereochemistry. We have also hydrogenated, in excellent ee, several purely alkyl-substituted enol phosphinates, producing chiral alcohols that are difficult to obtain highly enantioselectively from ketone hydrogenations.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-97722 (URN)10.1021/ja711372c (DOI)000255041400050 ()
    Available from: 2008-11-11 Created: 2008-11-11 Last updated: 2017-12-14Bibliographically approved
  • 31.
    Dinér, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry.
    Catalytic asymmetric chiral lithium amide-promoted epoxide rearrangement: a NMR spectroscopic and kinetic investigation2010In: Tetrahedron: asymmetry, ISSN 0957-4166, E-ISSN 1362-511X, Vol. 21, no 21-22, p. 2733-2739Article in journal (Refereed)
    Abstract [en]

    The lithium amide derived from the chiral diamine (1R,3S,4S)-3-(1-pyrrolidinyl)methyl-2-azabicyclo[2.2.1]heptane, has been reported to catalytically deprotonate cyclohexene oxide and other epoxides, yielding chiral allylic alcohols in excellent enantiomeric excess. In this work, 6Li, 1H and 13 C NMR spectroscopy have been used to study the aggregation of the chiral lithium amide in THF and the influence on the aggregation by the addition of additives, such as 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU). The activated complex under catalytic deprotonation of cyclohexene oxide, that is, with excess Li-DBU and free DBU, is built from one monomer of the chiral lithium amide, one molecule of epoxide and one additional molecule of DBU. The reaction order (0.97) obtained for the bulk base Li-DBU shows an inverse dependence on the concentration, suggesting a deaggregation of the initial mixed dimer to a monomer-based transition state containing a monomer of the lithium amide.

  • 32.
    Dubois, Louise
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Björkelund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Evaluating real-time immunohistochemistry on multiple tissue samples, multiple targets and multiple antibody labeling methods2013In: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 6, p. 542-Article in journal (Refereed)
    Abstract [en]

    Background

    Immunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method.

    Results

    Three different antibodies labeled with fluorescent or radioactive labels were applied on nine different tissue samples from either human or mouse, and the results for all RT-IHC analyses distinctly show that the method is generally applicable. The collected binding curves showed that the majority of the antibody-antigen interactions did not reach equilibrium within 3 hours, suggesting that standardized protocols for immunohistochemistry are sometimes inadequately optimized. The impact of tissue size and thickness as well as the position of the section on the glass petri dish was assessed in order for practical details to be further elucidated for this emerging technique. Size and location was found to affect signal magnitude to a larger extent than thickness, but the signal from all measurements were still sufficient to trace the curvature. The curvature, representing the kinetics of the interaction, was independent of thickness, size and position and may be a promising parameter for the evaluation of e.g. biopsy sections of different sizes.

    Conclusions

    It was found that RT-IHC can be used for the evaluation of a number of different antibodies and tissue types, rendering it a general method. We believe that by following interactions over time during the development of conventional IHC assays, it becomes possible to better understand the different processes applied in conventional IHC, leading to optimized assay protocols with improved sensitivity.

  • 33.
    Edlund, Bror
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Andersson, Jill
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Titanji, Vincent
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Dahlqvist, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Ek, Pia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Zetterqvist, Örjan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Amino acid sequence at the phosphorylated site of rat liver pyruvate kinase1975In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 67, no 4, p. 1516-1521Article in journal (Refereed)
    Abstract [en]

    One dominating peptic phosphopeptide, Asx-Thr-Lys-Gly-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu-Arg-Arg-Ala-(32P)SerP-Val-Ala-Glx-Leu, was obtained from rat liver pyruvate kinase (type L) phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase from the same tissue. The sequence around the phosphorylated serine residue is similar to that of a corresponding but smaller peptic phosphopeptide previously isolated from pig liver (type L) pyruvate kinase, Leu-Arg-Arg-Ala-(32P)SerP-Leu.

  • 34.
    Edlund, Karolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Molecular Characterisation and Prognostic Biomarker Discovery in Human Non-Small Cell Lung Cancer2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Non-small cell lung cancer (NSCLC) constitutes a clinically, histologically, and genetically heterogeneous disease entity that represents a major cause of cancer-related death. Early-stage patients, who undergo surgery with curative intent, experience high recurrence rates and the effect of adjuvant treatment is modest. Prognostic biomarkers would be of particular relevance to guide intensified treatment depending on expected outcome and moreover often infer a biological role in tumourigenesis.

    This thesis presents a translational study approach to establish a well-characterised NSCLC frozen-tissue cohort and to obtain a profile of each specimen with regard to genome-wide copy number alterations, global gene expression levels and somatic mutations in selected cancer-related genes. Furthermore, the generation of a formalin-fixed, paraffin-embedded tissue microarray enabled validation of findings on the protein level using immunohistochemistry. The comprehensive molecular characterisation, combined with data on clinical parameters, enabled the analysis of biomarkers linked to disease outcome. In Paper I, single nucleotide polymorphism arrays were applied to assess copy number alterations in NSCLC and associations with overall survival in adenocarcinoma and squamous cell carcinoma were described. In Paper II, we evaluated expression levels of selected stromal proteins in NSCLC using immunohistochemistry and the adhesion molecule CD99 was identified as an outcome-related biomarker in two independent cohorts. Paper III presents a strategy for prognostic biomarker discovery based on gene expression profiling, meta-analysis, and validation of protein expression on tissue microarrays, and suggests the putative tumour suppressor CADM1 as a candidate biomarker. In Paper IV, we propose a prognostic role for tumour-infiltrating IGKC-expressing plasma cells in the local tumour microenvironment, indicating an involvement of the humoral immune response in anti-tumor activity. In Paper V, we combined next-generation deep sequencing with statistical analysis of the TP53 database to define novel parameters for database curation.

    In summary, this thesis exemplifies the benefits of a translational study approach, based on a comprehensive tumour characterisation, and describes molecular markers associated with clinical outcome in NSCLC.

    List of papers
    1. Gene Copy Number Aberrations Are Associated with Survival in Histologic Subgroups of Non-small Cell Lung Cancer
    Open this publication in new window or tab >>Gene Copy Number Aberrations Are Associated with Survival in Histologic Subgroups of Non-small Cell Lung Cancer
    Show others...
    2011 (English)In: Journal of Thoracic Oncology, ISSN 1556-0864, E-ISSN 1556-1380, Vol. 6, no 11, p. 1833-1840Article in journal (Refereed) Published
    Abstract [en]

    Introduction:

    Non-small cell lung cancer (NSCLC) is characterized by a multitude of genetic aberrations with unknown clinical impact. In this study, we aimed to identify gene copy number changes that correlate with clinical outcome in NSCLC. To maximize the chance to identify clinically relevant events, we applied a strategy involving two prognostically extreme patient groups.

    Methods:

    Short-term (<20 month; n = 53) and long-term survivors (>58 month; n = 47) were selected from a clinically well-characterized NSCLC patient cohort with available fresh frozen tumor specimens. The samples were analyzed using high-resolution single-nucleotide polymorphism array technology to assess gene copy number variations and array-based gene expression profiling. The molecular data were combined with information on clinical parameters.

    Results:

    Genetic aberrations were strongly associated with tumor histology. In adenocarcinoma (n = 50), gene copy number gains on chromosome 8q21-q24.3 (177 genes) were more frequent in long-term than in short-term survivors. In squamous cell carcinoma (n = 28), gains on chromosome 14q23.1-24.3 (133 genes) were associated with shorter survival, whereas losses in a neighboring region, 14q31.1-32.33 (110 genes), correlated with favorable outcome. In accordance with copy number gains and losses, messenger RNA expression levels of corresponding genes were increased or decreased, respectively.

    Conclusion:

    Comprehensive tumor profiling permits the integration of genomic, histologic, and clinical data. We identified gene copy number gains and losses, with corresponding changes in messenger RNA levels that were associated with prognosis in adenocarcinoma and squamous cell carcinoma of the lung.

    Keywords
    Lung cancer, Prognosis, SNP array, CGH, Gene expression profile
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-162455 (URN)10.1097/JTO.0b013e3182295917 (DOI)000296700400008 ()
    Available from: 2011-12-01 Created: 2011-11-30 Last updated: 2018-02-01Bibliographically approved
    2. CD99 is a novel prognostic stromal marker in non-small cell lung cancer
    Open this publication in new window or tab >>CD99 is a novel prognostic stromal marker in non-small cell lung cancer
    Show others...
    2012 (English)In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 131, no 10, p. 2264-2273Article in journal (Refereed) Published
    Abstract [en]

    The complex interaction between cancer cells and the microenvironment plays an essential role in all stages of tumourigenesis. Despite the significance of this interplay, alterations in protein composition underlying tumour-stroma interactions are largely unknown. The aim of this study was to identify stromal proteins with clinical relevance in non-small cell lung cancer (NSCLC). A list encompassing 203 stromal candidate genes was compiled based on gene expression array data and available literature. The protein expression of these genes in human NSCLC was screened using the Human Protein Atlas. Twelve proteins were selected that showed a differential stromal staining pattern (BGN, CD99, DCN, EMILIN1, FBN1, PDGFRB, PDLIM5, POSTN, SPARC, TAGLN, TNC, VCAN). The corresponding antibodies were applied on tissue microarrays, including 190 NSCLC samples, and stromal staining was correlated with clinical parameters. Higher stromal expression of CD99 was associated with better prognosis in the univariate (p=0.037) and multivariate (p=0.039) analysis. The association was independent from the proportion of tumour stroma, the fraction of inflammatory cells, and clinical and pathological parameters like stage, performance status and tumour histology. The prognostic impact of stromal CD99 protein expression was confirmed in an independent cohort of 240 NSCLC patients (p=0.008). Furthermore, double-staining confocal fluorescence microscopy showed that CD99 was expressed in stromal lymphocytes as well as in cancer associated fibroblasts. Based on a comprehensive screening strategy the membrane protein CD99 was identified as a novel stromal factor with clinical relevance. The results support the concept that stromal properties have an important impact on tumour progression.

    National Category
    Clinical Laboratory Medicine
    Research subject
    Pathology
    Identifiers
    urn:nbn:se:uu:diva-170991 (URN)10.1002/ijc.27518 (DOI)000309185300007 ()22392539 (PubMedID)
    Note

    Karolina Edlund and Cecilia Lindskog are shared first authors.

    Available from: 2012-03-14 Created: 2012-03-14 Last updated: 2018-02-01
    3. Biomarker discovery in non-small cell lung cancer: integrating gene expression profiling, meta-analysis and tissue microarray validation
    Open this publication in new window or tab >>Biomarker discovery in non-small cell lung cancer: integrating gene expression profiling, meta-analysis and tissue microarray validation
    Show others...
    2013 (English)In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 19, no 1, p. 194-204Article in journal (Refereed) Published
    Abstract [en]

    Background:

    Global gene expression profiling has been widely used in lung cancer research to identify clinically relevant molecular subtypes as well as to predict prognosis and therapy response. So far, the value of these multi-gene signatures in clinical practice is unclear and the biological importance of individual genes is difficult to assess as the published signatures virtually do not overlap

    Methods:

    Here we describe a novel single institute cohort, including 196 non-small lung cancers (NSCLC) with clinical information and long-term follow-up. Gene expression array data was used as a training set to screen for single genes with prognostic impact. The top 450 probe sets identified using a univariate Cox regression model (significance level p<0.01) were tested in a meta-analysis including five publicly available independent lung cancer cohorts (n=860).

    RESULTS:

    The meta-analysis revealed 14 genes that were significantly associated with survival (p<0.001) with a false discovery rate <1%. The prognostic impact of one of these genes, the cell adhesion molecule 1 (CADM1), was confirmed by use of immunohistochemistry on tissue microarrays from two independent NSCLC cohorts, altogether including 617 NSCLC samples. Low CADM1 protein expression was significantly associated with shorter survival, with particular influence in the adenocarcinoma patient subgroup.

    CONCLUSIONS:

    Using a novel NSCLC cohort together with a meta-analysis validation approach, we have identified a set of single genes with independent prognostic impact. One of these genes, CADM1, was further established as an immunohistochemical marker with a potential application in clinical diagnostics.

    National Category
    Clinical Laboratory Medicine
    Research subject
    Pathology
    Identifiers
    urn:nbn:se:uu:diva-183399 (URN)10.1158/1078-0432.CCR-12-1139 (DOI)000313051100021 ()23032747 (PubMedID)
    Available from: 2012-10-25 Created: 2012-10-25 Last updated: 2018-02-01Bibliographically approved
    4. The prognostic relevance of tumour-infiltrating plasma cells and immunoglobulin kappa C indicates an important role of the humoral immune response in non-small cell lung cancer
    Open this publication in new window or tab >>The prognostic relevance of tumour-infiltrating plasma cells and immunoglobulin kappa C indicates an important role of the humoral immune response in non-small cell lung cancer
    Show others...
    2013 (English)In: Cancer Letters, ISSN 0304-3835, E-ISSN 1872-7980, Vol. 333, no 2, p. 222-228Article in journal (Refereed) Published
    Abstract [en]

    A prognostic impact of immunoglobulin kappa C (IGKC) expression has been described in cancer. We analysed the influence of B-cell and plasma cell markers, as well as IGKC expression, in non-small lung cancer (NSCLC) using immunohistochemistry on a tissue microarray. IGKC protein expression was independently associated with longer survival, with particular impact in the adenocarcinoma subgroup. Moreover, a correlation was seen with CD138+ cells, but not with CD20. CD138 expression revealed a comparable association with survival. In conclusion, IGKC expression in stroma–infiltrating plasma cells is a prognostic marker in NSCLC, supporting emerging treatment concepts that exploit the humoral immune response.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-181908 (URN)10.1016/j.canlet.2013.01.036 (DOI)000318838700011 ()
    Available from: 2012-10-01 Created: 2012-10-01 Last updated: 2018-02-01Bibliographically approved
    5. Data-driven unbiased curation of the TP53 tumor suppressor gene mutation database and validation by ultradeep sequencing of human tumors
    Open this publication in new window or tab >>Data-driven unbiased curation of the TP53 tumor suppressor gene mutation database and validation by ultradeep sequencing of human tumors
    Show others...
    2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 24, p. 9551-9556Article in journal (Refereed) Published
    Abstract [en]

    Cancer mutation databases are expected to play central roles in personalized medicine by providing targets for drug development and biomarkers to tailor treatments to each patient. The accuracy of reported mutations is a critical issue that is commonly overlooked, which leads to mutation databases that include a sizable number of spurious mutations, either sequencing errors or passenger mutations. Here we report an analysis of the latest version of the TP53 mutation database, including 34,453 mutations. By using several data-driven methods on multiple independent quality criteria, we obtained a quality score for each report contributing to the database. This score can now be used to filter for high-confidence mutations and reports within the database. Sequencing the entire TP53 gene from various types of cancer using next-generation sequencing with ultradeep coverage validated our approach for curation. In summary, 9.7% of all collected studies, mostly comprising numerous tumors with multiple infrequent TP53 mutations, should be excluded when analyzing TP53 mutations. Thus, by combining statistical and experimental analyses, we provide a curated mutation database for TP53 mutations and a framework for mutation database analysis.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-175985 (URN)10.1073/pnas.1200019109 (DOI)000305511300070 ()22628563 (PubMedID)
    Available from: 2012-06-14 Created: 2012-06-14 Last updated: 2018-02-01Bibliographically approved
  • 35.
    Ek, Pia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dahlqvist, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Humble, Elisabet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3´, 5´-AMP-stimulated protein kinase1976In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 429, no 2, p. 374-382Article in journal (Refereed)
    Abstract [en]

    The kinetics of rat liver L-type pyruvate kinase (EC 2.7.1.40), phosphorylated with cyclic AMP-stimulated protein kinase from the same source, and the unphosphorylated enzyme have been compared. The effects of pH and various concentrations of substrates, Mg2+, K+ and modifiers were studied. In the absence of fructose 1, 6-diphosphate at pH 7.3, the phosphorylated pyruvate kinase appeared to have a lower affinity for phosphoenolpyruvate (K0.5=0.8 mM) than the unphosphorylated enzyme (K0.5=0.3 mM). The enzyme activity vs. phosphoenolpyruvate concentration curve was more sigmoidal for the phosphorylated enzyme with a Hill coefficient of 2.6 compared to 1.6 for the unphosphorylated enzyme. Fructose 1, 6-diphosphate increased the apparent affinity of both enzyme forms for phosphoenolpyruvate. At saturating concentrations of this activator, the kinetics of both enzyme forms were transformed to approximately the same hyperbolic curve, with a Hill coefficient of 1.0 and K0.5 of about 0.04 mM for phosphoenolpyruvate. The apparent affinity of the enzyme for fructose 1, 6-diphosphate was high at 0.2 mM phosphoenolpyruvate with a K0.5=0.06 muM for the unphosphorylated pyruvate kinase and 0.13 muM for the phosphorylated enzyme. However, in the presence of 0.5 mM alanine plus 1.5 mM ATP, a higher fructose 1, 6-diphosphate concentration was needed for activation, with K0.5 of 0.4 muM for the unphosphorylated enzyme and of 1.4 muM for the phosphorylated enzyme. The results obtained strongly indicate that phosphorylation of pyruvate kinase may also inhibit the enzyme in vivo. Such an inhibition should be important during gluconeogenesis.

  • 36.
    Ekman, Pia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Hermansson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Bergström, Gunnel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Rapid proteolytic removal of phosphopeptides and phosphorylatable sites from proteins in rat liver cell sap1978In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 86, no 2, p. 250-254Article in journal (Refereed)
  • 37.
    Elhamili, Anisa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Development of Capillary Electrophoresis Methods Coupled to Mass Spectrometry for Biomedical and Pharmaceutical Analysis2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The analysis of large intact proteins and complex biological samples containing drug molecules is a common complicated task for many scientists. However, due to the importance of these molecules, there is a growing interest in pharmaceutical and medicinal research to develop rapid, highly sensitive and efficient analytical techniques. The advantages of capillary electrophoresis (CE) in combination with mass spectrometry (MS) provide a powerful analytical tool. However, further improvement and development of these techniques are required to extend their utility and to meet the challenges of selected analytes. Thus, the scope of this thesis deals with the development of novel analytical methods to achieve efficient and high performance analysis of peptides, intact proteins, digests of complex samples and basic pharmaceutical drug compounds in biological matrices.

    Implementation of CE for routine analysis of proteins and complex samples is constrained by the partial adsorption to the capillary wall. Consequently, the use of surface modified capillaries is required to control the surface properties and prevent analyte adsorption. In this thesis, analyte adsorption was successfully prevented using tailored covalent cationic (M7C4I) and electrostatic cationic (PVPy-Me) coatings. Rapid and efficient separations of peptides, proteins and digests of complex samples such as cerebrospinal fluids were obtained with these coatings. The M7C4I coating showed a distinct ability to handle large intact proteins with a molecular size of over 0.5 MDa. The highest peak efficiencies and surprisingly high peak stacking effects were obtained by adding salts to the protein samples. The effect of salt additives on peak efficiencies of intact proteins was further demonstrated and compared using different surface modified capillaries. Additionally, rapid CE-ESI-MS quantification of pharmaceutical drug molecules in human plasma was performed after a SCX-SPE sample preparation method using the M7C4I coating. In conclusion, the results presented in this thesis show the strong potential of CE in combination with MS using electrospray ionization (ESI) for the analysis of peptides and large intact proteins and the applicability for clinical monitoring of the levels of pharmaceutical drug molecules in human plasma with high sensitivity and efficiency.

    List of papers
    1. Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4l) for capillary coatings
    Open this publication in new window or tab >>Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4l) for capillary coatings
    Show others...
    2008 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 29, no 8, p. 1619-1625Article in journal (Refereed) Published
    Abstract [en]

    A monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide (M7C4I), has been evaluated as a surface derivatization reagent for CE in combination with TOF MS for the analysis of proteins, peptides, and protein digests. The M7C4I piperazine, at alkaline pH, forms a covalent bond via alkylation of the ionized silanols producing a cationic surface with a highly stable and reversed EOF. The obtained surface yields rapid separations (less than 5 min) of peptides and proteins at acidic pH with high separation efficiencies (up to 1.1 X 10(6) plates/m for peptides and up to 1.8 x 10(6) plates/m for proteins) and no observed bleeding of the coating reagent into the mass spectrometer. The simplicity of the coating procedure also enables fast (2 min) regeneration of the surface, if necessary. This is useful in the analysis of complex samples in order to prevent possible memory effects. The potential of using M7C4I-coated capillaries for MS analysis of complex samples is demonstrated by the separation of peptides, proteins, and protein digests. Even more, the spectacular thing in which large intact proteins with molecular masses over 0.5 MDa could be separated. The coating showed good ability to handle these large proteins with high efficiency and retained peak shape as demonstrated by separation of IgG(1) (150 kDa) and thyroglobulin (669 kDa).

    Keywords
    Capillary electrophoresis, M7C4l, Peptides, Proteins, Protein digests, Time-of-flight
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-16217 (URN)10.1002/elps.200700737 (DOI)000255703100005 ()
    Available from: 2008-05-13 Created: 2008-05-13 Last updated: 2017-12-08Bibliographically approved
    2. Analysis of peptides using N-methylpolyvinylpyridium as silica surface modifier for CE-ESI-MS
    Open this publication in new window or tab >>Analysis of peptides using N-methylpolyvinylpyridium as silica surface modifier for CE-ESI-MS
    Show others...
    2010 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 31, no 7, p. 1151-1156Article in journal (Refereed) Published
    Abstract [en]

    In this study, the N-methylpolyvinylpyridinuim polymer has for the first time been used as a silica surface modifier for CE in combination with ESI MS (CE-ESI-MS). The compatibility for ESI-MS was demonstrated by the analysis of peptides and protein digests. The N-methylpolyvinylpyridium surface interacts electrostatically with the ionized silanol groups, giving a cationic surface with a reversed EOF. The surface modifier gave rapid and repeatable separations of peptides, proteins and protein digests at acidic pH for more than 4 h of continuous use. The CE separation yielded peak efficiencies of up to 4.3 x 10(5) plates/m. The surface coating is highly compatible with ESI and facilitates the separation and analysis of complex peptide mixtures as shown by the analysis of BSA digests.

    Keywords
    CE, ESI, MS, N-methylpolyvinylpyridinuim, peptides
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-125809 (URN)10.1002/elps.200900536 (DOI)000276811000005 ()20209570 (PubMedID)
    Available from: 2010-05-28 Created: 2010-05-28 Last updated: 2017-12-12Bibliographically approved
    3. The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries.
    Open this publication in new window or tab >>The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries.
    Show others...
    2009 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1216, no 17, p. 3613-3620Article in journal (Refereed) Published
    Abstract [en]

    The effect of adding alkali salts to protein samples for capillary electrophoretic (CE) analysis of intact proteins was studied. A high degree of peak stacking, even for large proteins, was found to occur when alkali salts were added to the sample. The addition of salt to the protein sample promotes a strong improvement in the peak efficiency of individual proteins giving up to 2.1 x 10(6) apparent plates/m. The concentration of salt required in the sample to reach optimal peak efficiency show dependency on both the molecular weight and molar concentration of the protein. However, adding salt will, at a sufficiently high concentration, cause a mixture of proteins to co-migrate to one very sharp peak. The observed sample stacking effect was obtained with a number of different surface modified silica capillaries indicating a general phenomenon and not surface coating specific.

    Keywords
    Capillary electrophoresis, alkali salt, intact protein analysis, coated capillary, stacking effect
    National Category
    Chemical Sciences
    Research subject
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-100628 (URN)10.1016/j.chroma.2008.12.037 (DOI)000265467200004 ()19150070 (PubMedID)
    Available from: 2009-04-03 Created: 2009-04-03 Last updated: 2017-12-13Bibliographically approved
    4. Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic coated capillaries
    Open this publication in new window or tab >>Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic coated capillaries
    2011 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, no 6-7, p. 647-658Article in journal (Refereed) Published
    Abstract [en]

    In this study, the extraction and CE-ESI-TOF-MS analysis of tricyclic antidepressant (TCA) drugs imipramine, desipramine, clomipramine and norclomipramine in human plasma has been optimized. The CE capillaries were modified with ω-iodo-alkyl ammonium salt (M7C4I coating) to reduce analyte adsorption to the silica wall. The use of a strong cation exchange (SCX) solid-phase extraction (SPE) column specifically designed for the extraction of basic drug species from biofluids gave very clean extracts with high and reproducible recoveries. The extraction recoveries were ranging between 87 and 91% with % RSD values of 0.5-1.7% (n=3). The obtained strong cation exchange-SPE extracts of the TCA in human plasma only contained the analytes of interest. The optimized CE separation conditions were obtained by adding ACN and acetic acid to the sample while using an aqueous BGE. The CE-ESI-TOF-MS analysis was performed within 6min for all TCA analytes under the optimized condition with peak efficiencies up to 1.4×105plates/m and an average % RSD of the migration times of the analytes of 0.3% (n=5). The presented method can readily be used for the extraction and quantification of basic drug species in human biological fluids and in pharmaceutical formulations.

    Keywords
    Capillary electrophoresis, Mass spectrometry, Tricyclic Antidepressant Drugs, Quantification, Human plasma
    National Category
    Other Basic Medicine
    Research subject
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-143784 (URN)10.1002/elps.201000566 (DOI)000288602000001 ()21341290 (PubMedID)
    Available from: 2011-01-25 Created: 2011-01-25 Last updated: 2018-01-12Bibliographically approved
    5. A method for quantitative analysis of an anticancer drug in human plasma with CE-ESI-TOF-MS
    Open this publication in new window or tab >>A method for quantitative analysis of an anticancer drug in human plasma with CE-ESI-TOF-MS
    2011 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, no 13, p. 1778-1785Article in journal (Refereed) Published
    Abstract [en]

    In this study, the extraction recoveries of an anticancer drug (Imatinib) from human plasma using a common liquid-liquid extraction (LLE) method and a new strong cation exchange (SCX) solid-phase extraction (SPE) column was investigated. The extracts were analyzed with CE coupled on-line to electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) using a monoquaternarized piperazine compound (M7C4I) for capillary coatings. Clean extracts with high and reproducible extraction recoveries ranging between 85 and 91% with % RSD values of 2.5% (n = 3) were obtained using the SCX-SPE columns. This can be compared with the recoveries obtained with the LLE method ranging between 30 and 35%. The CE-ESI-TOF-MS analysis was performed in = 0.997 and % RSD values of 0.5% (n = 3). The intra-day and inter-day assay variations were lower than 8%. The presented CE-ESI-TOF-MS method with the use of SCX-SPE columns yielded rapid, efficient and high extraction recoveries together with high sensitivity (LOD 5 ng/mL), selectivity and good linearity. Accordingly, the method can readily be used for accurate determination and therapeutic monitoring of the Imatinib blood levels for more effective patient treatment. In addition, it can be applied for the extraction, quantification and clinical assessments of metabolites of Imatinib and other basic pharmaceutical drug molecules in biological fluids or pharmaceutical dosage forms.

    Keywords
    Capillary electrophoresis, Human plasma, Imatinib, Mass spectrometry, Quantification, Theraputic drug monitoring.
    National Category
    Other Basic Medicine
    Research subject
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-143791 (URN)10.1002/elps.201100121 (DOI)000292971000027 ()
    Available from: 2011-01-25 Created: 2011-01-25 Last updated: 2018-01-12Bibliographically approved
  • 38.
    Elhamili, Anisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    A method for quantitative analysis of an anticancer drug in human plasma with CE-ESI-TOF-MS2011In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, no 13, p. 1778-1785Article in journal (Refereed)
    Abstract [en]

    In this study, the extraction recoveries of an anticancer drug (Imatinib) from human plasma using a common liquid-liquid extraction (LLE) method and a new strong cation exchange (SCX) solid-phase extraction (SPE) column was investigated. The extracts were analyzed with CE coupled on-line to electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) using a monoquaternarized piperazine compound (M7C4I) for capillary coatings. Clean extracts with high and reproducible extraction recoveries ranging between 85 and 91% with % RSD values of 2.5% (n = 3) were obtained using the SCX-SPE columns. This can be compared with the recoveries obtained with the LLE method ranging between 30 and 35%. The CE-ESI-TOF-MS analysis was performed in = 0.997 and % RSD values of 0.5% (n = 3). The intra-day and inter-day assay variations were lower than 8%. The presented CE-ESI-TOF-MS method with the use of SCX-SPE columns yielded rapid, efficient and high extraction recoveries together with high sensitivity (LOD 5 ng/mL), selectivity and good linearity. Accordingly, the method can readily be used for accurate determination and therapeutic monitoring of the Imatinib blood levels for more effective patient treatment. In addition, it can be applied for the extraction, quantification and clinical assessments of metabolites of Imatinib and other basic pharmaceutical drug molecules in biological fluids or pharmaceutical dosage forms.

  • 39.
    Elhamili, Anisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Samuelsson, Jörgen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic coated capillaries2011In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, no 6-7, p. 647-658Article in journal (Refereed)
    Abstract [en]

    In this study, the extraction and CE-ESI-TOF-MS analysis of tricyclic antidepressant (TCA) drugs imipramine, desipramine, clomipramine and norclomipramine in human plasma has been optimized. The CE capillaries were modified with ω-iodo-alkyl ammonium salt (M7C4I coating) to reduce analyte adsorption to the silica wall. The use of a strong cation exchange (SCX) solid-phase extraction (SPE) column specifically designed for the extraction of basic drug species from biofluids gave very clean extracts with high and reproducible recoveries. The extraction recoveries were ranging between 87 and 91% with % RSD values of 0.5-1.7% (n=3). The obtained strong cation exchange-SPE extracts of the TCA in human plasma only contained the analytes of interest. The optimized CE separation conditions were obtained by adding ACN and acetic acid to the sample while using an aqueous BGE. The CE-ESI-TOF-MS analysis was performed within 6min for all TCA analytes under the optimized condition with peak efficiencies up to 1.4×105plates/m and an average % RSD of the migration times of the analytes of 0.3% (n=5). The presented method can readily be used for the extraction and quantification of basic drug species in human biological fluids and in pharmaceutical formulations.

  • 40.
    Enweji, Nizar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dynamics of Resistant Plasmodium falciparum Parasites2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Persistence of drug resistant Plasmodium falciparum is a major problem to management and control malaria in endemic areas. The focus of this thesis was to study the dynamics of resistant P. falciparum parasites. The study was performed in two African countries: 1) Sudan: Asar village in eastern Sudan, here we examined the persistence of drug sensitive and resistant P. falciparum genotypes among individuals with single-clone and multiple clones infection during the dry season. We genotyped microsatellite loci in the vicinity of the dihydrofolate reductase gene (dhfr) and the dihydropteroate synthase gene (dhps). Microsatellite investigation showed that asymptomatic parasitemia persisted in some patients for several months throughout the dry season and into the next transmission season. In some samples mixed infections were detected, and we noted several cases where the microsatellite haplotype varied from month to month, suggesting turnover of different parasite populations in the blood. This demonstrates that even during asymptomatic infections there can be dynamics within the parasite population in an individual. In addition, we calculated the parasite density throughout the dry season to the next transmission season by using allele-specific quantitative PCR. Parasite density during the dry season fluctuated, but was generally lower than in the first transmission season. A significant difference (P<0.05) between dry and first transmission season was found in regard to the parasite density, whereas no significant difference was observed when dry and second transmission season were compared (P>0.05). 2) Ethiopia: West Arsi zone, one of the malaria endemic zones of the Oromia region. In the first study we determined the prevalence of asymptomatic malaria carriages from November-December 2012. According to PCR the prevalence of sub-microscopic P. falciparum carriage was 19.2%, microscopy-based prevalence was 3.7% while the prevalence was 6.9% using RDT. Based on this, PCR was considered a better tool for measuring Plasmodium prevalence than microscopy and RDT. A second study addressed the genetic diversity of chloroquine resistance (CQR) in P. falciparum by analysing four microsatellite markers in and around the pfcrt gene. Although CQ was withdrawn for more than a decade, 100% of the parasites still carried the Pfcrt K76T mutation. Only the CVIET haplotype was identified. Based on combinations of MS markers, seven different Ethiopian CQR variants (E1-E7) were identified. Both intronic and MS flanking the pfcrt gene showed low levels of diversity.

    List of papers
    1. Dynamics of asymptomatic malaria infections as revealed by microsatellite typing
    Open this publication in new window or tab >>Dynamics of asymptomatic malaria infections as revealed by microsatellite typing
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Other Medical Sciences not elsewhere specified
    Research subject
    Molecular Genetics
    Identifiers
    urn:nbn:se:uu:diva-230219 (URN)
    Available from: 2014-08-20 Created: 2014-08-20 Last updated: 2015-01-22Bibliographically approved
    2. Real-time quantitative PCR for determining Plasmodium falciparum parasite density in patients with asymptomatic infection in a seasonal transmission area.
    Open this publication in new window or tab >>Real-time quantitative PCR for determining Plasmodium falciparum parasite density in patients with asymptomatic infection in a seasonal transmission area.
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Other Medical Sciences not elsewhere specified
    Identifiers
    urn:nbn:se:uu:diva-230222 (URN)
    Available from: 2014-08-20 Created: 2014-08-20 Last updated: 2015-01-22
    3. Detection of a substantial number of sub-microscopic Plasmodium falciparum infections by polymerase chain reaction: a potential threat to malaria control and diagnosis in Ethiopia
    Open this publication in new window or tab >>Detection of a substantial number of sub-microscopic Plasmodium falciparum infections by polymerase chain reaction: a potential threat to malaria control and diagnosis in Ethiopia
    Show others...
    2013 (English)In: Malaria Journal, ISSN 1475-2875, E-ISSN 1475-2875, Vol. 12, p. 352-Article in journal (Refereed) Published
    Abstract [en]

    Background: Prompt and effective malaria diagnosis not only alleviates individual suffering, but also decreases malaria transmission at the community level. The commonly used diagnostic methods, microscopy and rapid diagnostic tests, are usually insensitive at very low-density parasitaemia. Molecular techniques, on the other hand, allow the detection of low-level, sub-microscopic parasitaemia. This study aimed to explore the presence of sub-microscopic Plasmodium falciparum infections using polymerase chain reaction (PCR). The PCR-based parasite prevalence was compared against microscopy and rapid diagnostic test (RDT). Methods: This study used 1,453 blood samples collected from clinical patients and sub-clinical subjects to determine the prevalence of sub-microscopic P. falciparum carriages. Subsets of RDT and microscopy negative blood samples were tested by PCR while all RDT and microscopically confirmed P. falciparum-infected samples were subjected to PCR. Finger-prick blood samples spotted on filter paper were used for parasite genomic DNA extraction. Results: The prevalence of sub-microscopic P. falciparum carriage was 19.2% (77/400) (95% CI = 15.4-23.1). Microscopy-based prevalence of P. falciparum infection was 3.7% (54/1,453) while the prevalence was 6.9% (100/1,453) using RDT alone. Using microscopy and PCR, the estimated parasite prevalence was 20.6% if PCR were performed in 1,453 blood samples. The prevalence was estimated to be 22.7% if RDT and PCR were used. Of 54 microscopically confirmed P. falciparum-infected subjects, PCR detected 90.7% (49/54). Out of 100 RDT-confirmed P. falciparum infections; PCR detected 80.0% (80/100). The sensitivity of PCR relative to microscopy and RDT was, therefore, 90.7% and 80%, respectively. The sensitivity of microscopy and RDT relative to PCR was 16.5 (49/299) and 24.2% (80/330), respectively. The overall PCR-based prevalence of P. falciparum infection was 5.6- and 3.3 fold higher than that determined by microscopy and RDT, respectively. None of the sub-microscopic subjects had severe anaemia, though 29.4% had mild anaemia (10-11.9 g/dl). Conclusions: Asymptomatic, low-density malaria infection was common in the study area and PCR may be a better tool for measuring Plasmodium prevalence than microscopy and RDT. The inadequate sensitivity of the diagnostic methods to detect substantial number of sub-microscopic parasitaemia would undoubtedly affect malaria control efforts, making reduction of transmission more difficult. RDT and microscopy-based prevalence studies and subsequent reports of reduction in malaria incidence underestimate the true pictures of P. falciparum infections in the community. PCR, on the other hand, seems to have reasonable sensitivity to detect a higher number of infected subjects with low and sub-microscopic parasite densities than RDTs or microscopy.

    Keywords
    Sub-microscopic carriage, Asymptomatic malaria, Microscopy, RDT, PCR, Ethiopia
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-216887 (URN)10.1186/1475-2875-12-352 (DOI)000329097000001 ()
    Available from: 2014-01-27 Created: 2014-01-27 Last updated: 2017-12-06Bibliographically approved
    4. High prevalence of pfcrt-CVIET haplotype in isolates from asymptomatic and symptomatic patients in south-central Oromia, Ethiopia
    Open this publication in new window or tab >>High prevalence of pfcrt-CVIET haplotype in isolates from asymptomatic and symptomatic patients in south-central Oromia, Ethiopia
    Show others...
    2014 (English)In: Malaria Journal, ISSN 1475-2875, E-ISSN 1475-2875, Vol. 13, p. 120-Article in journal (Refereed) Published
    Abstract [en]

    Background: As a result of extensive chloroquine resistance (CQR) in Plasmodium falciparum in late 1990s, Ethiopia replaced CQ with sulphadoxine-pyrimethamine (SP) as first-line drug, which in turn was replaced by artemisinin combination therapy in 2004. Plasmodium falciparum resistance to CQ is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene. Understanding diversity in the P. falciparum genome is crucial since it has the potential to influence important phenotypes of the parasite such as drug resistance. Limited data is available regarding the type of pfcrt mutant allelic type, the effect of CQ withdrawal and diversity of the parasite population in south-central Oromia, Ethiopia. Methods: Finger-pricked blood spotted on Whatman 3MM filter papers were collected from falciparum malaria patients. Parasite DNA was extracted from individual blood spots on the filter papers. The presence of K76T mutations was determined using nested PCR for all isolates. Complete sequencing of mutations in pfcrt 72-76 was done for a set of randomly selected resistant isolates. Four microsatellite (MS) markers were analysed to determine the heterozygosity. Results: Although CQ was withdrawn for more than a decade, 100% of the parasites still carried the pfcrt K76T mutation. All isolates were mutant at the K76T polymorphism. Based on combinations of MS markers, seven different Ethiopian CQR variants (E1-E7) were identified. Heterozygosity (He) for MS flanking the pfcrt chloroquine resistance allele ranged from 0.00 (mscrt -29, -29.268 kb) to 0.21 (mscrt -2, -2.814 kb). H-e ranged from 0.00 (msint 3, 0 kb) to 0.19 (msint 2, 0 kb) for MS within the pfcrt gene. Both intronic and MS flanking the pfcrt gene showed low levels of diversity. Conclusion: pfcrt CQR allele seems to be fixed in the study area. Of the different haplotypes associated with CQR, only the CVIET genotype was identified. No reversal to the wild-type has occurred in Ethiopia unlike in many Africa countries where CQR parasites declined after cessation of CQ use. Decreased diversity in CQR isolates surrounding pfcrt suggests CQ selection and homogenization among CQR parasite population. While mutation in msint 3 and mscrt -29 of the mutant pfcrt allele is being fixed, it seems that mutations in msint 2 and mscrt -2 are still evolving and may indicate the start of re-diversification of the population from a fixed 76 T population.

    Keywords
    pfcrt, Wild type, Drug resistance, Chloroquine, Plasmodium falciparum, Mutations, Heterozygosity, Microsatellite, Ethiopia
    National Category
    Infectious Medicine
    Identifiers
    urn:nbn:se:uu:diva-225094 (URN)10.1186/1475-2875-13-120 (DOI)000334807900004 ()
    Note

    Golassa and Enweji contributed equally to this work.

    Available from: 2014-05-27 Created: 2014-05-27 Last updated: 2017-12-05Bibliographically approved
  • 41.
    Eriksson, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Carlsson, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Blom, Elisabeth
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Sundin, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Velikyan, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Preclinical evaluation of a 68Ga-labeled biotin analogue for applications in islet transplantation2012In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 39, no 3, p. 415-421Article in journal (Refereed)
    Abstract [en]

    INTRODUCTION:

    Islet transplantation is a promising treatment for type 1 diabetes mellitus, but the fate of the cells after intraportal infusion is unclear. It is therefore imperative to develop novel techniques for noninvasive imaging and quantification of events following islet transplantation.

    METHODS:

    Small islet-like microbeads, avidin-covered agarose resins (AARs), were used as a model system for islet transplantation. Capability for specific [(68)Ga]Ga-DOTA-(PEG)(2)-biotin uptake and retention for either AARs or human islets conjugated with avidin by means of a heparin scaffold was studied in vitro. Biodistribution of the novel positron emission tomography (PET) tracer [(68)Ga]Ga-DOTA-(PEG)(2)-biotin was evaluated in mice treated by intraportal transplantation of AARs by μPET/computed tomography and ex vivo organ distribution and compared with control mice.

    RESULTS:

    AARs had high capability to bind [(68)Ga]Ga-DOTA-(PEG)(2)-biotin, close to 50% of administrated tracer/μl in vitro (>0.25 MBq/μl). Avidin-tagged human islets could bind on average 2.2% of administered tracer/μl. Specificity (>90%) and retention (>90% after 1 h) were high for both AARs and avidin-tagged islets. Hepatic tracer uptake and retention were increased in mice transplanted with AARs [standardized uptake value (SUV)=2.6] compared to the untreated group (SUV=1.4). In vivo uptake of tracer to AARs was blocked by preadministration of unlabeled biotin.

    CONCLUSIONS:

    Avidin-tagged islet-like objects can be tracked in hepatic volume after intraportal transplantation by using [(68)Ga]Ga-DOTA-(PEG)(2)-biotin and PET.

  • 42.
    Eriksson, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Espes, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Selvaraju, Ram K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Jansson, Emma
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Sörensen, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Lubberink, Mark
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Biglarnia, Alireza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
    Eriksson, Jan W
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical diabetology and metabolism.
    Sundin, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Ahlström, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Eriksson, Barbro
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Tumor Biology.
    Johansson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Carlsson, Per-Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Transplantation and regenerative medicine.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    The Positron Emission Tomography ligand [11C]5-Hydroxy-Tryptophan can be used as a surrogate marker for the human endocrine pancreas2014In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 63, no 10, p. 3428-3437Article in journal (Refereed)
    Abstract [en]

    In humans a well-developed serotonin system is localized to the pancreatic islets while being absent in exocrine pancreas. Assessment of pancreatic serotonin biosynthesis could therefore be used to estimate the human endocrine pancreas. Proof of concept was tested in a prospective clinical trial by comparisons of type 1 diabetic (T1D) patients, with extensive reduction of beta cells, with healthy volunteers (HV).C-peptide negative (i.e. insulin-deficient) T1D subjects (n=10) and HV (n=9) underwent dynamic Positron Emission Tomography with the radiolabeled serotonin precursor [(11)C]5-Hydroxy-Tryptophan ([(11)C]5-HTP).A significant accumulation of [(11)C]5-HTP was obtained in the pancreas of the HV, with large inter-individual variation. A substantial and highly significant reduction (66%) in the pancreatic uptake of [(11)C]5-HTP in T1D subjects was observed, and this was most evident in the corpus and caudal regions of the pancreas where beta-cells normally are the major constituent of the islets.[(11)C]5-HTP retention in the pancreas was reduced in T1D compared to non-diabetic subjects. Accumulation of [(11)C]5-HTP in the pancreas of both HV and subjects with T1D were in agreement with previously reported morphological observations on the beta cell volume implying that [(11)C]5-HTP retention is a useful non-invasive surrogate marker for the human endocrine pancreas.

  • 43. Eriksson, Olof
    et al.
    Korsgren, Olle
    Selvaraju, Ram Kumar
    Mollaret, Marjorie
    de Boysson, Yann
    Chimienti, Fabrice
    Altai, Mohamed
    Pancreatic imaging using an antibody fragment targeting the zinc transporter type 8: a direct comparison with radio-iodinated Exendin-4.2017In: Acta Diabetologica, ISSN 0940-5429, E-ISSN 1432-5233Article in journal (Refereed)
    Abstract [en]

    AIM: The zinc transporter 8 (ZnT8) has been suggested as a suitable target for non-invasive visualization of the functional pancreatic beta cell mass, due to both its pancreatic beta cell restricted expression and tight involvement in insulin secretion.

    METHODS: In order to examine the potential of ZnT8 as a surrogate target for beta cell mass, we performed mRNA transcription analysis in pancreatic compartments. A novel ZnT8 targeting antibody fragment Ab31 was radiolabeled with iodine-125, and evaluated by in vitro autoradiography in insulinoma and pancreas as well as by in vivo biodistribution. The evaluation was performed in a direct comparison with radio-iodinated Exendin-4.

    RESULTS: Transcription of the ZnT8 mRNA was higher in islets of Langerhans compared to exocrine tissue. Ab31 targeted ZnT8 in the cytosol and on the plasma membrane with 108 nM affinity. Ab31 was successfully radiolabeled with iodine-125 with high yield and > 95% purity. [(125)I]Ab31 binding to insulinoma and pancreas was higher than for [(125)I]Exendin-4, but could only by partially competed away by 200 nM Ab31 in excess. The in vivo uptake of [(125)I]Ab31 was higher than [(125)I]Exendin-4 in most tissues, mainly due to slower clearance from blood.

    CONCLUSIONS: We report a first-in-class ZnT8 imaging ligand for pancreatic imaging. Development with respect to ligand miniaturization and radionuclide selection is required for further progress. Transcription analysis indicates ZnT8 as a suitable target for visualization of the human endocrine pancreas.

  • 44.
    Eskhult, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry.
    Ulrich, Christian
    Björefors, Fredrik
    Nyholm, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry.
    Current oscillations during chronoamperometric and cyclic voltammetric measurements in alkaline Cu(II)-citrate solutions2008In: Electrochimica Acta, ISSN 0013-4686, E-ISSN 1873-3859, Vol. 53, no 5, p. 2188-2197Article in journal (Refereed)
    Abstract [en]

    It is demonstrated that current oscillations can be observed during chronoamperometric and cyclic voltammetric experiments in solutions containing 0.4 M CuSO4 and 1.2 M citrate at pH 11 and 50 degrees C. The oscillations, which are shown to originate from local variations in the pH, result in the deposition of nanostructured Cu and Cu2O materials. It is concluded that the current oscillations are analogous to the previously described potential oscillations obtained under controlled current conditions in alkaline Cu(II)-lactate, -tartrate and -citrate solutions. Rotating disk electrode results clearly show that the reduction of the Cu(II)-complexes is kinetically controlled and that the rate of the reduction increases with increasing pH and temperature. It is also shown that the presence of a cathodic peak on the anodic scan in the cyclic voltammograms can be used to identify the experimental conditions leading to the spontaneous current (or potential) oscillations. Electrochemical quartz crystal microbalance results indicate that the cathodic peak stems from an increased rate of the reduction of the Cu(II)-citrate complexes due to a rapid increase in the local pH. This causes Cu2O rather than Cu to be deposited which, however, results in a decrease in the local pH and a decreasing current. In situ ellipsometry data confirm that Cu2O deposition replaces that of Cu in the potential region of the cathodic peak. The present findings should facilitate syntheses of nanolayered materials based on spontaneous potential or current oscillations.

  • 45.
    Fagerlund, Amelie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Organic Chemistry.
    Sunnerheim, Kerstin
    Dimberg, Lena H.
    Radical-scavenging and antioxidant activity of avenanthramides2009In: Food Chemistry, ISSN 0308-8146, E-ISSN 1873-7072, Vol. 113, no 2, p. 550-556Article in journal (Refereed)
    Abstract [en]

    Avenanthramides are amides of cinnamoyl-anthranilic acids and, among cereals, are exclusively found in oats. This study investigated the structure-antioxidant activities of 15 avenanthramides with different substitution patterns in the two aromatic rings, seven of which were new avenanthramides synthesised and characterised in this study. Radical-scavenging activity was tested as reactivity towards 1,1-diphenyl-2-picrylhydrazyl (DPPH-). The activity increased with the number of radical-stabilising groups ortho to the phenolic hydroxy group. Both aromatic rings were independently important for activity, while conjugation across the amide bond was of minor importance. Antioxidant activity was determined as inhibition of linoleic acid oxidation. In contrast to the radical-scavenging activity, antioxidant activity was observed for most avenanthramides, and also for compounds with only one hydroxy group in either of the aromatic rings. Compared with alpha-tocopherol, the avenanthramides protected linoleic acid from oxidation to a smaller extent initially, but the effect lasted for a longer time.

  • 46.
    Ferraz, Natalia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
    Effect of Surface Nanotopography on Blood-Biomaterial Interactions2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Biologically inspired materials are being developed with the aim of improving the integration of medical implants and minimizing non-desirable host reactions. A promising strategy is the design of topographically patterned surfaces that resemble those found in the extracellular environment.

    Nanoporous alumina has been recognized as a potential biomaterial and as an important template for the fabrication of nanostructures.

    In this thesis in vitro studies were done to elucidate the role of alumina nanoporosity on the inflammatory response. Specifically, by comparing alumina membranes with two pore sizes (20 and 200 nm in diameter). Complement and platelet activation were evaluated as well as monocyte/macrophage behaviour.

    Whole blood was incubated with the alumina membranes and thereafter the biomaterial surfaces were evaluated in terms of protein and platelet adhesion as well as procoagulant properties. The fluid phase was analyzed for complement activation products and platelet activation markers. Besides, human mononuclear cells were cultured on the alumina membranes and cell adhesion, viability, morphology and release of pro-inflammatory cytokines were evaluated.

    The results indicated that nanoporous alumina with 200 nm pores promotes higher complement activation than alumina with 20 nm pores.

    In addition, platelet response to nanoporous alumina was found to be highly dependent on the material porosity, as reflected by differences in adhesion, PMP generation and procoagulant characteristics.

    A clear difference in monocyte/macrophage adhesion and activation was found between the two pore size alumina membranes. Few but highly activated cells adhered to the 200 nm membrane in contrast to many but less activated monocytes/macrophages on the 20 nm surface.

    The outcome of this work emphasizes that nanotopography plays an important role in the host response to biomaterials.

    Better understanding of molecular interactions on nano-level will undoubtedly play a significant role in biomaterial implant development and will contribute to design strategies for controlling specific biological events.

    List of papers
    1. Nanoporesize affects complement activation
    Open this publication in new window or tab >>Nanoporesize affects complement activation
    2008 (English)In: Journal of biomedical materials research. Part A, ISSN 1552-4965, Vol. 87, no 3, p. 575-81Article in journal (Refereed) Published
    Abstract [en]

    In the present study, we have shown the vast importance of biomaterial nanotexture when evaluating inflammatory response. For the first time in an in vitro whole blood system, we have proven that a small increase in nanoporesize, specifically 180 nm (from 20 to 200 nm), has a huge effect on the complement system. The study was done using nanoporous aluminiumoxide, a material that previously has been evaluated for potential implant use, showing good biocompatibility. This material can easily be manufactured with different pore sizes making it an excellent candidate to govern specific protein and cellular events at the tissue-material interface. We performed whole blood studies, looking at complement activation after blood contact with two pore size alumina membranes (pore diameters, 20 and 200 nm). The fluid phase was analyzed for complement soluble components, C3a and sC5b-9. In addition, surface adsorbed proteins were eluted and dot blots were performed to detect IgG, IgM, C1q, and C3. All results point to the fact that 200 nm pore size membranes are more complement activating. Significantly, higher values of complement soluble components were found after whole blood contact with 200 nm alumina and all studied proteins adsorbed more readily to this membrane than to the 20 nm pore size membrane. We hypothesize that the difference in complement activation between our two test materials is caused by the type and the amount of adsorbed proteins, as well as their conformation and orientation. The different protein patterns created on the two alumina membranes are most likely a consequence of the material topography.

    Keywords
    nanotopography, nanoporous alumina, complement, whole blood, protein adsorption
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-88357 (URN)10.1002/jbm.a.31818 (DOI)000260984800002 ()18186072 (PubMedID)
    Available from: 2009-01-30 Created: 2009-01-29 Last updated: 2009-07-13Bibliographically approved
    2. Influence of nanoporesize on platelet adhesion and activation
    Open this publication in new window or tab >>Influence of nanoporesize on platelet adhesion and activation
    2008 (English)In: Journal of materials science. Materials in medicine, ISSN 0957-4530, E-ISSN 1573-4838, Vol. 19, no 9, p. 3115-21Article in journal (Refereed) Published
    Abstract [en]

    In this study we have evaluated the influence of biomaterial nano-topography on platelet adhesion and activation. Nano-porous alumina membranes with pore diameters of 20 and 200 nm were incubated with whole blood and platelet rich plasma. Platelet number, adhesion and activation were determined by using a coulter hematology analyzer, scanning electron microscopy, immunocytochemical staining in combination with light microscopy and by enzyme immunoassay. Special attention was paid to cell morphology, microparticle generation, P-selectin expression and beta-TG production. Very few platelets were found on the 200 nm alumina as compared to the 20 nm membrane. The platelets found on the 20 nm membrane showed signs of activation such as spread morphology and protruding filipodia as well as P-selectin expression. However no microparticles were detected on this surface. Despite the fact that very few platelets were found on the 200 nm alumina in contrast to the 20 nm membrane many microparticles were detected on this surface. Interestingly, all microparticles were found inside circular shaped areas of approximately 3 mum in diameter. Since this is the approximate size of a platelet we speculate that this is evidence of transient, non-adherent platelet contact with the surface, which has triggered platelet microparticle generation. To the authors knowledge, this is the first study that demonstrates how nanotexture can influence platelet microparticle generation. The study highlights the importance of understanding molecular and cellular events on nano-level when designing new biomaterials.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-88354 (URN)10.1007/s10856-008-3449-7 (DOI)000256964300016 ()18414999 (PubMedID)
    Available from: 2009-01-30 Created: 2009-01-29 Last updated: 2017-12-14Bibliographically approved
    3. Procoagulant behavior and platelet microparticle generation on nanoporous alumina
    Open this publication in new window or tab >>Procoagulant behavior and platelet microparticle generation on nanoporous alumina
    2010 (English)In: Journal of biomaterials applications, ISSN 0885-3282, E-ISSN 1530-8022, Vol. 24, no 8, p. 675-692Article in journal (Refereed) Published
    Abstract [en]

    In the present work, we have investigated platelet microparticle(PMP) generation in whole blood after contact with nanoporous alumina.Alumina membranes with pore sizes of 20 and 200nm in diameter were incubated with whole blood and the number of PMP in the fluid phase was determined by flow cytometry. The role of the complement system in PMP generation was investigated using an analog of the potent complement inhibitor compstatin. Moreover, the procoagulant activity of the two pore size membranes were compared by measuring thrombin formation. Results indicated that PMP were not present in the fluid phase after whole blood contact with either of the alumina membranes. However, scanning electron microscope micrographs clearly showed the presence of PMP clusters on the 200nm pore size alumina, while PMP were practically absent on the 20nm membrane. We probed no influence of complement activation in PMP generation and adhesion and we hypothesize that other specific material-related protein–platelet interactions are taking place. A clear difference in procoagulant activity between the membranes could also be seen, 20nm alumina showed 100% higher procoagulant activity than 200nm membrane. By combining surface evaluation and flow cytometry analyses of the fluid phase, we are able to conclude that 200nm pore size alumina promotes PMP generation and adhesion while the 20nm membrane does not appreciably cause any release or adhesion of PMP, thus indicating a direct connection between PMP generation and nanoporosity.

    Place, publisher, year, edition, pages
    SAGE, 2010
    Keywords
    nanoporous alumina, nanotopography, platelets, platelet microparticles, procoagulant activity, compstatin
    National Category
    Chemical Sciences
    Research subject
    Immunology; Materials Science
    Identifiers
    urn:nbn:se:uu:diva-130496 (URN)10.1177/0885328209338639 (DOI)000277806100001 ()19581322 (PubMedID)
    Available from: 2010-09-08 Created: 2010-09-08 Last updated: 2017-12-12Bibliographically approved
    4. Time sequence of blood activation by nanoporous alumina: Studies on platelets and complement system
    Open this publication in new window or tab >>Time sequence of blood activation by nanoporous alumina: Studies on platelets and complement system
    2010 (English)In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 73, no 12, p. 1101-1109Article in journal (Refereed) Published
    Abstract [en]

    In the present work the time sequence of blood activation by alumina membranes with different porosities (20 and 200 nm in diameter) was studied. The membranes were incubated with whole blood from 2 min to 4 h. Platelet adhesion and activation in addition to complement activation were monitored at different time points. Evaluation of platelet adhesion and activation was done by determining the change in platelet number and the levels of thrombospondin-1 in the fluid phase. Scanning electron microscopy studies were done to further evaluate platelet adhesion and morphology. Immunocytochemical staining was used to evaluate the presence of CD41 and CD62P antigens on the material surface. Complement activation was monitored by measuring C3a and sC5b-9 in plasma samples by means of enzyme immunoassays. Both alumina membranes displayed similar complement activation time profiles, with levels of C3a and sC5b-9 increasing with incubation time. A statistically significant difference between the membranes was found after 60 min of incubation. Platelet activation characteristics and time profile were different between the two membranes. Platelet adhesion increased over time for the 20 nm surface, while the clusters of microparticles on the 200 nm surface did not appreciably change during the course of the experiment. The release of thrombospondin-1 increased with time for both membranes, however much later for the 200 nm alumina (240 min) as compared to the 20 nm membrane (60 min). The surface topography of the alumina most probably influence protein transition rate, which in turn affects material-platelet activation kinetics.

    Place, publisher, year, edition, pages
    Wiley-Liss Inc., 2010
    Keywords
    nanotopography, biomaterial, whole blood, thrombospondin-1, platelet microparticles
    National Category
    Other Basic Medicine
    Research subject
    Immunology; Materials Science
    Identifiers
    urn:nbn:se:uu:diva-110620 (URN)10.1002/jemt.20854 (DOI)000284063800005 ()
    Available from: 2009-11-18 Created: 2009-11-18 Last updated: 2018-01-12Bibliographically approved
    5. Nanoporosity of alumina surfaces induces different patterns of activation in adhering monocytes/macrophages
    Open this publication in new window or tab >>Nanoporosity of alumina surfaces induces different patterns of activation in adhering monocytes/macrophages
    2010 (English)In: International Journal of Biomaterials, ISSN 1687-8787, E-ISSN 1687-8795, Vol. 2010, p. 402715-Article in journal (Refereed) Published
    Abstract [en]

    The present study shows that alumina nanotopography affects monocyte/macrophage behaviour. Human mononuclear cells cultured on alumina membranes with pore diameters of 20 and 200 nm were evaluated in terms of cell adhesion, viability, morphology and release of pro-inflammatory cytokines. After 24 hours, cell adhesion was assessed by means of light microscopy and cell viability by measuring LDH release. The inflammatory response was evaluated by quantifying interleukin-1ß and tumour necrosis factor-α. Finally, scanning electron microscopy was used to study cell morphology. Results showed pronounced differences in cell number, morphology and cytokine release depending on the nanoporosity. Few but highly activated cells were found on the 200 nm porous alumina, while relatively larger number of cells was found on the 20 nm porous surface. However, despite their larger number, the cells adhering on the 20 nm surface exhibited reduced pro-inflammatory activity. It can be speculated that the difference in surface topography may lead to distinct protein adsorption patterns and therefore to different degree of cell activation. The data of this paper emphasize the role played by the material nanotexture in dictating cell responses and implies that nanotopography could be exploited for controlling the inflammatory response to implants.

    Keywords
    macrophages, nanoporous alumina, biomaterials, nanotopography, inflammatory response
    National Category
    Other Basic Medicine
    Research subject
    Immunology
    Identifiers
    urn:nbn:se:uu:diva-110623 (URN)10.1155/2010/402715 (DOI)21234322 (PubMedID)
    Available from: 2009-11-18 Created: 2009-11-18 Last updated: 2018-01-12Bibliographically approved
  • 47.
    Ferraz, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
    Hong, Jaan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Santin, Matteo
    School of Pharmacy & Biomolecualr Sciences, University of Brighton.
    Karlsson Ott, Marjam
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
    Nanoporosity of alumina surfaces induces different patterns of activation in adhering monocytes/macrophages2010In: International Journal of Biomaterials, ISSN 1687-8787, E-ISSN 1687-8795, Vol. 2010, p. 402715-Article in journal (Refereed)
    Abstract [en]

    The present study shows that alumina nanotopography affects monocyte/macrophage behaviour. Human mononuclear cells cultured on alumina membranes with pore diameters of 20 and 200 nm were evaluated in terms of cell adhesion, viability, morphology and release of pro-inflammatory cytokines. After 24 hours, cell adhesion was assessed by means of light microscopy and cell viability by measuring LDH release. The inflammatory response was evaluated by quantifying interleukin-1ß and tumour necrosis factor-α. Finally, scanning electron microscopy was used to study cell morphology. Results showed pronounced differences in cell number, morphology and cytokine release depending on the nanoporosity. Few but highly activated cells were found on the 200 nm porous alumina, while relatively larger number of cells was found on the 20 nm porous surface. However, despite their larger number, the cells adhering on the 20 nm surface exhibited reduced pro-inflammatory activity. It can be speculated that the difference in surface topography may lead to distinct protein adsorption patterns and therefore to different degree of cell activation. The data of this paper emphasize the role played by the material nanotexture in dictating cell responses and implies that nanotopography could be exploited for controlling the inflammatory response to implants.

  • 48.
    Ferraz, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
    Karlsson Ott, Marjam
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
    Hong, Jaan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Time sequence of blood activation by nanoporous alumina: Studies on platelets and complement system2010In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 73, no 12, p. 1101-1109Article in journal (Refereed)
    Abstract [en]

    In the present work the time sequence of blood activation by alumina membranes with different porosities (20 and 200 nm in diameter) was studied. The membranes were incubated with whole blood from 2 min to 4 h. Platelet adhesion and activation in addition to complement activation were monitored at different time points. Evaluation of platelet adhesion and activation was done by determining the change in platelet number and the levels of thrombospondin-1 in the fluid phase. Scanning electron microscopy studies were done to further evaluate platelet adhesion and morphology. Immunocytochemical staining was used to evaluate the presence of CD41 and CD62P antigens on the material surface. Complement activation was monitored by measuring C3a and sC5b-9 in plasma samples by means of enzyme immunoassays. Both alumina membranes displayed similar complement activation time profiles, with levels of C3a and sC5b-9 increasing with incubation time. A statistically significant difference between the membranes was found after 60 min of incubation. Platelet activation characteristics and time profile were different between the two membranes. Platelet adhesion increased over time for the 20 nm surface, while the clusters of microparticles on the 200 nm surface did not appreciably change during the course of the experiment. The release of thrombospondin-1 increased with time for both membranes, however much later for the 200 nm alumina (240 min) as compared to the 20 nm membrane (60 min). The surface topography of the alumina most probably influence protein transition rate, which in turn affects material-platelet activation kinetics.

  • 49.
    Fondell, Amelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Two-Step Targeting for Effective Radionuclide Therapy: Preclinical Evaluation of 125I-labelled Anthracycline Delivered by Tumour Targeting Liposomes2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    For the treatment of cancer, Auger-electron emitting radionuclides are strongly dependent on their close proximity to DNA to utilize the local therapeutic potential of the Auger electrons. This thesis investigates a two-step targeting approach that uses targeting liposomes for the delivery of an Auger-electron emitter, 125I, coupled to a DNA-binding compound, Comp1, to the tumour-cell DNA. In the first step the liposome targets overexpressed cell-surface receptors. Receptors belonging to epidermal growth factor receptor (EGFR) family are overexpressed in a number of different cancers and are therefore suitable targets. The second step is transportation of the radionuclide to the cell nucleus utilizing a DNA-binding compound. The DNA-binder used in this thesis is a daunorubicin derivative called Comp1. Papers I and II are in vitro characterizations of the targeting liposomes. Both EGFR- and HER2-targeting liposomes delivered 125I-Comp1 receptor specifically to tumour cells, and were efficient in decreasing growth of cultured tumour cells. Paper II also included a biodistribution of 125I-Comp1 delivered by HER2-targeting liposomes in tumour-bearing mice. The results gave a time-dependent uptake in tumours differed from when non-targeting liposomes encapsulating 125I-Comp1 were given. Paper III investigates the therapeutic effect of 125I-Comp1 delivered by HER2-targeting liposomes, in an animal model that mimics a situation of disseminated tumour cells in the abdomen. 125I-Comp1 delivered by HER2-targeting liposomes effectively prolonged survival of the mice in a dose-dependent relation. Several mice in the groups receiving the highest doses were tumour-free at the end of the study. Paper IV compares different lipid compositions of the liposomes with respect to leakage, cellular uptake and therapeutic efficacy of delivered 125I-Comp1on cultured cells. Liposomes containing sphingomyelin or dihydrosphingomyelin retained drug more efficiently and exhibited more receptor specific delivery properties than distearoylglycerophosphatidylcholine (DSPC) containing liposomes. However, it was the DSPC-containing liposomes that displayed best growth inhibition on cultured tumour cells. The thesis concludes that 125I-Comp1 delivered by targeting liposomes is a promising candidate for effective radionuclide therapy.

    List of papers
    1. Nuclisome: a novel concept for radionuclide therapy using targeting liposomes
    Open this publication in new window or tab >>Nuclisome: a novel concept for radionuclide therapy using targeting liposomes
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    2010 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 37, no 1, p. 114-123Article in journal (Refereed) Published
    Abstract [en]

    PURPOSE: For the treatment of cancer, the therapeutic potential of short-range, low-energy Auger-electron emitters, such as (125)I, is getting progressively wider recognition. The potency of Auger-electron emitters is strongly dependent on their location in close vicinity to DNA. We have developed a new two-step targeting strategy to transport (125)I into cancer-cell nuclei using PEG-stabilized tumour-cell targeting liposomes named "Nuclisome-particles". METHODS: In the present study, epidermal growth factor (EGF) was used as a tumour-cell-specific agent to target the EGF-receptor (EGFR) and the liposomes were loaded with (125)I-Comp1, a recently synthesized daunorubicin derivative. RESULTS: As analysed with cryo-TEM, the derivative precipitates inside liposomes at a drug-to-lipid molar ratio of 0.05:1. Receptor-specific uptake in cultured U-343MGaCl2:6 tumour cells of EGFR-targeting liposomes increased with time while non-specific and receptor-blocked uptake remained low. Nuclisome-particles were able to target single U-343MGaCl2:6 cells circulating in human blood during 4 h, with low uptake in white blood cells, as demonstrated in an ex vivo system using a Chandler loop. Autoradiography of targeted cells indicates that the grains from the radiolabelled drug are mainly co-localized with the cell nuclei. The successful targeting of the nucleus is shown to provide high-potency cell killing of cultured U-343MGaCl2:6 cells. At the concentration used, Nuclisome-particles were up to five orders of magnitude more effective in cell killing than EGFR-targeting liposomes loaded with doxorubicin. CONCLUSION: The results thus provide encouraging evidence that our two-step targeting strategy for tumour cell DNA has the potential to become an effective therapy against metastasizing cancer cells in the bloodstream.

    Keywords
    Targeting liposomes, EGF, Radionuclide therapy, Anthracyclines, Auger-electron emitter
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-125921 (URN)10.1007/s00259-009-1225-7 (DOI)000272615700014 ()19662408 (PubMedID)
    Available from: 2010-05-31 Created: 2010-05-31 Last updated: 2017-12-12Bibliographically approved
    2. In vitro evaluation and biodistribution of HER2-targeted liposomes loaded with an 125I-labelled DNA-intercalator
    Open this publication in new window or tab >>In vitro evaluation and biodistribution of HER2-targeted liposomes loaded with an 125I-labelled DNA-intercalator
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    2011 (English)In: Journal of drug targeting (Print), ISSN 1061-186X, E-ISSN 1029-2330, Vol. 19, no 9, p. 846-855Article in journal (Refereed) Published
    Abstract [en]

    Background: Increasing attention is currently focussed on the issue of finding strategies for the delivery of Auger-electron emitting radionuclides into tumour cell nuclei. Nuclear localisation is a prerequisite for these radionuclides, since their radiotoxic properties are functional only in close vicinity to DNA.

    Purpose: In this study we investigated tumour-cell uptake and cell killing ability in vitro, as well as in vivo biodistribution of an 125I-labelled anthracycline derivative administered by means of HER2-targeted liposomes.

    Methods: Anthracycline derivative Comp1 was radiolabelled with Auger-emitting 125I and encapsulated in liposomes (DSPC:Chol:DSPE-PEG) using pH-gradient loading. Single-chain fragment F5 was anchored to the liposomes as targeting device for HER2. Uptake and specificity of 125I-Comp1 delivered via targeting and non-targeting liposomes were analysed in cultured HER2-overexpressing SKOV3 and SKBR3 cells. Cell-killing efficacy was evaluated in SKOV3 cells and biodistribution for up to 48 hours was studied after intra-peritoneal injection in tumour-bearing female Balb/c nu/nu mice.

    Results: 125I-Comp1 was specifically taken up by the cultured cells when administered by means of HER2-targeted liposomes and a clear dose-effect correlation in survival of cells was seen with increasing specific activity. The biodistribution studies revealed that 125I-Comp1 accumulated in tumours when distributed using HER2-targeted liposomes and that this effect was absent when using non-targeting liposomes.

    Conclusion: The HER2-targeted liposomes possess the properties needed to bring about tumour-specific delivery and therapeutic effect of 125I-Comp1.

     

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-147028 (URN)10.3109/1061186X.2011.589436 (DOI)000295889800013 ()
    Available from: 2011-02-23 Created: 2011-02-23 Last updated: 2017-12-11Bibliographically approved
    3. Experimental radionuclide therapy of HER2-expressing xenografts using two-step targeting Nuclisome-particles
    Open this publication in new window or tab >>Experimental radionuclide therapy of HER2-expressing xenografts using two-step targeting Nuclisome-particles
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    2012 (English)In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 53, no 3, p. 480-487Article in journal (Refereed) Published
    Abstract [en]

    The therapeutic potential of Auger-electron emitting radionuclides is strongly dependent on their close vicinity to DNA, since the energy deposition is mainly localized within a few cubic nanometers around the site of decay. Thus, apart from specificity, successful tumor therapy relies on a nuclear delivery strategy. We recently presented a two-step targeting strategy to transport Auger-electron-emitting radionuclides into the cell nucleus by means of nuclide-filled liposomes (Nuclisome particles), that is, polyethylene glycol-stabilized, tumor-cell-targeting liposomes loaded with (125)I-labeled anthracyclines. In the present study, the survival of mice intraperitoneally inoculated with human HER2-expressing SKOV-3 tumor cells and treated with HER2-targeting Nuclisome particles was studied.

    METHODS:

    BALB/c nu/nu mice were inoculated with 10(7) SKOV-3 cells intraperitoneally and thereafter directly injected with Nuclisome particles with increasing specific radioactivity. Groups of 10-12 mice were treated with 0.01 MBq/mouse up to 2 MBq/mouse, and survival was monitored and compared with that in control groups (n = 33). Organs were analyzed for HER2 expression and radiotoxic effects histologically. Absorbed doses were estimated using dose factors from the online Radiation Dose Assessment Resource model.

    RESULTS:

    The results showed a clear correlation between administered radioactive dose and survival. No such dose-dependent survival was observed for mice treated with Nuclisome particles lacking HER2-targeting ability. With HER2-targeting Nuclisome particles, a significant increase in survival, compared with that of untreated control mice, could already be seen at an administered activity of 0.1 MBq/mouse (P = 0.0301). At the highest activity administered, 2 MBq/mouse (P < 0.0001), 70% of the mice survived the study and most were tumor-free. Neither macroscopic nor microscopic radiotoxic side effects were observed. Dosimetric calculations, assuming nonreceptor targeting, revealed that the radioactive doses to normal tissues were low.

    CONCLUSION:

    Taken together the results show that with successful targeting to the tumor-cell nucleus it is possible to obtain a therapeutic effect from Auger-electron-emitting radionuclides administered at radioactive doses low enough to spare normal tissue from radiotoxic side effects.

    National Category
    Other Basic Medicine
    Identifiers
    urn:nbn:se:uu:diva-158992 (URN)10.2967/jnumed.111.096891 (DOI)000301194300046 ()22323773 (PubMedID)
    Available from: 2011-09-20 Created: 2011-09-20 Last updated: 2018-01-12Bibliographically approved
    4. Influence of liposome composition on cellular drug delivery and therapeutic effect mediated by Nuclisome-particles
    Open this publication in new window or tab >>Influence of liposome composition on cellular drug delivery and therapeutic effect mediated by Nuclisome-particles