Doctoral Theses / Doktorsavhandlingar Institutionen för kemi och molekylärbiologi

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Biofilms in Nitrogen Removal - Bacterial Population Dynamics and Spatial Distribution
(2012-01-12) Almstrand, Robert
Efficient nitrogen removal at wastewater treatment plants (WWTPs) is necessary to avoid eutrophication of recipient waters. The most commonly used approach consists of aerobic nitrification and subsequent anaerobic denitrification resulting in the release of dinitrogen gas into the atmosphere. Nitrification is a two-step process performed by ammoniaoxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) often grown in biofilms at WWTPs. An alternative approach is anaerobic ammonium oxidation (anammox) where anammox bacteria convert ammonium and nitrite directly into dinitrogen gas. These groups of bacteria grow very slowly and are sensitive to perturbations, which may result in decreased efficiency or even breakdown of the process. Therefore, the ecology and activity of these bacteria and the structure of the biofilms in which they grow require detailed investigation to improve the understanding of nitrification and to facilitate the design of efficient nitrogen-removal strategies. To facilitate such studies of relevance for wastewater treatment, a nitrifying pilotplant was built where environmental conditions and especially ammonium concentrations could be controlled. In an experiment on model nitrifying trickling filters (NTFs), it was shown that biofilms subjected to intermittent feeding regimes of alternating high and low ammonium concentration in the water, could maintain a higher nitrification potential than biofilms constantly fed with low ammonium water. Such ammonium feed strategies can be used to optimize wastewater treatment performance. Different AOB populations within the N. oligotropha lineage were shown to respond differently to changes in environmental conditions, indicating microdiversity within this lineage which may be of importance for wastewater treatment. This diversity was further investigated through the development of new image analysis methods for analyzing bacterial spatial distribution in biofilms. The diversity within the N. oligotropha lineage was also reflected in the positioning of two such populations in the biofilm, where the vertical distribution patterns and relative positions compared to the NOB Nitrospira were different. In combination with a cryosectioning approach for retrieval of intact biofilm from small biofilm carrier compartments, the new image analysis methods showed a threedimensonal stratification of AOB-anammox biofilms. This may be of importance for mathematical modeling of such biofilms and the design of new biofilm carriers.
Symmetry of Halonium Complexes in Solution
(2012-01-13) Carlsson, Anna-Carin
In this thesis the symmetry of two interaction types involving electropositive halogens have been studied in solution; the N-X+-N halogen bond (X = Br or I), and the C-X+-C interaction of previously characterised, cyclic, 1,2-bridged halonium ions (X = Cl or Br), respectively. The three N-X+-N model structures included are bispyridine, 1,2-bis(pyridine-2-ylethynyl)benzene and 1,2-bis((4-methylpyridin-2-yl)ethynyl)benzene halonium triflate complexes. Model structures representing the C-X+-C interaction are the dimethylethylene- and ethylenehalonium ions. All structures included in this thesis are comprised of symmetrically arranged atoms, but have the possibility to exist as either a static, symmetric structure, or as two asymmetric, fast equilibrating tautomers. For a symmetric structure, the positive halogen is positioned with equal distances to the electron donor nitrogens/carbons. In asymmetric structures, the halogen is always closer to one of the nitrogens/carbons, and is consistently jumping between the two nitrogens/carbons. In this investigation the NMR spectroscopic method Isotopic Perturbation of Equilibrium (IPE) has been applied for distinguishing a single symmetric structure from rapidly, interconverting tautomers. The technique measures 13C NMR isotope shifts, nobs, resulting from unsymmetrical introduction of deuterium isotopes in the molecule for which the symmetry is in doubt. Based on the magnitudes, signs, and temperature-dependency of nobs obtained from 13C NMR spectra of a mixture of non-labelled and deuterium labelled molecules, the symmetry of the molecule being considered can be determined. The IPE NMR experiments revealed that all bis(pyridine)based halonium complexes were best represented as static, symmetric structures in dichloromethane. The symmetric N-X+-N arrangement was also shown to be independent of environmental factors, such as increased solvent polarity and tight binding of the counter ion. Thus, these observations indicated that the formation of a symmetric N-X+-N halogen bond is energetically favourable. The 15N and 13C chemical shifts of the pyridine rings revealed significantly stronger N-X+-N interaction for the iodonium complexes than for the corresponding bromonium complexes, suggesting a covalent character of the N-I+-N interaction and an ionic character of the N-Br+-N interaction. Strongest interaction was observed for the bispyridine halonium complexes, in which the N-N distances are freely adjustable to provide the most favourable interaction. Ionisation of 2,3-dihalobutane or 1,2-dihaloethane precursors in SbF5-SO2 at -80 C were attempted for generation of the desired ethylenehalonium ions. Both bromonium ions were characterised as asymmetric, equilibrating structures; the dimethylethylenebromonium ions from their nobs values, and the ethylenebromonium ion from the dynamic behaviour, typical for asymmetric structures in a slow equilibrium, of the signals shown in its 1H and 13C NMR spectra. The 1H NMR spectral pattern of the ethylenechloronium ion was also consistent with asymmetric structures in a slow equilibrium. The symmetry of the dimethylethylenechloronium ions could not be determined, as they, if formed at all, immediately rearranged. SO2 was revealed to be sufficiently nucleophilic to add to the cations formed. Hence, the source of the asymmetry observed is ascribed the labile addition of SO2 to either cyclic halonium ions or open beta-halocarbenium ions.
Design and Synthesis of Chalcone and Chromone Derivatives as Novel Anticancer Agents
(2012-01-23) Dyrager, Christine
This thesis comprises the design and synthesis of chalcone and chromone derivatives and their use in various biological applications, particularly as anticancer agents (targeting proteins associated with cancer pathogenesis) and as potential fluorophores for live-cell imaging. Conveniently, all structures presented were synthesized from commercially available 2 ́-hydroxyacetophenones. Different synthetic strategies were used to obtain an easily accessible chromone scaffold with appropriate handles that allows regioselective introduction of various substituents. Structural diversity was accomplished by using palladium-mediated reactions for the incorporation of suitable substituents for the generation of chromone derivatives that possess different biological activities. Challenging synthesis provided a series of fluorescent 2,6,8-trisubstituted 3- hydroxychromone derivatives with high quantum yields and molar extinction coefficients. Two of these derivatives were studied as fluorophores in live-cell imaging and showed rapid absorption, non-cytotoxic profiles and excellent fluorescent properties in a cellular environment. Synthetic chromone precursors, i.e. chalcones, and related dienones were evaluated as antiproliferative agents that interfere with the tubulin-microtubule equilibrium, crucial for cellular mitosis. It was shown that several of the synthesized compounds destabilize tubulin assembly. However, one of the compounds was instead found to stabilize tubulin to the same extent as the known anticancer drug docetaxel, thus representing the first chalcone with microtubule stabilizing activity. Molecular docking was used in order to theoretically investigate the interactions of the chalcones with ␣-tubulin mainly focusing on binding modes, potential interactions and specific binding sites. Structural-based design and extensive synthesis provided chromone-based derivatives that target two different MAP kinases (p38␣ and MEK1), involved in essential cellular signal transduction pathways. The study resulted in a series of highly selective ATP-competitive chromone-based p38␣ inhibitors with IC50 values in the nanomolar range. Among those, two derivatives also showed inhibition of p38 signaling in human breast cancer cells. Furthermore, molecular docking was used to study potential structural modifications on the chromone structure in order to obtain highly potent derivatives that selectively target the allosteric pocket on MEK1. Initial studies provided a first generation of non-ATP- competitive chromone derivatives that prevents the activation of MEK1 with micromolar activities.
Self-organization of nanoparticles - implications for interface biology
(2012-05-03) Lundgren, Anders
Cells bind to their surroundings via proteins displayed on the cell surface. These interactions support the cells and are important for many cellular processes, e.g. cell migration during morphogenesis, wound healing and cancer metastasis. There is a yet unmet need for simple and robust in vitro models mirroring the complex molecular organization found in natural tissue. In this thesis, protein-sized gold nanoparticles were used to introduce morphological and biochemical nanopatterns on material surfaces via nanoparticle self-assembly. These surfaces were used to explore the effect of protein organization and other nanoscopic parameters on cell response. In their simplest form, gold nanoparticles (in solution) are stabilized by negatively charged ions adsorbed onto their surfaces. It was shown that such nanoparticles, 10 nm in diameter, could self-organize on a dithiol modified gold surface under the influence of electrostatic double-layer forces. The distance between the adsorbed particles could be tuned by the ionic composition of the particle solution, which was described using classical DLVO-theory. A novel method to prepare surfaces with nanoparticle gradients, based on this mechanism, was introduced. Prepared surfaces were used as templates for the assembly of nanopatterns of chemical entities and proteins, with a periodicity in the sub 100 nm regime, by site-specific grafting of different molecules to the particle surfaces. Patterns with specific cell-binding proteins and peptides as well as synthetic polymers were realized and characterized with SEM, imaging SPR, QCM-D and TOF-SIMS. Gradient patterns were also assembled with multiple ligands, e.g. RGD-peptides and heparin, allowing the investigation of synergistic cell stimuli. Biochemical nanopatterns were evaluated in studies on human fibroblasts and endothelial cells, e.g. the cellular mobility was explored in response to different gradient stimuli. In a separate study, fimbria mediated adhesion of E. coli bacteria to nanoscopic adhesive domains was investigated. Surfaces decorated with gold nanoparticles were also shown to attenuate the complement protein cascade system via morphological alteration of adsorbed proteins. Altogether, concepts and methods presented in this thesis offer a route to systematically explore the interactions between biology and molecularly organized interfaces.
Mechanistic Insights into Transition Metal Oxide Catalyzed Water Oxidation
(2012-05-14) Busch, Michael
A binuclear mechanism was proposed and evaluated by means of Density Functional Theory calculations. The central reaction steps were found to be the oxidation of the transition metal TM-OH moieties to TM=O and the subsequent intramolecular O-O bond formation between two TM=O groups. These step were employed as descriptors for the performance of a set of 3d transition metal oxides studied in a MgOx(OH)y test rig embedding. Two classes of oxygen evolution reaction (OER) catalysts were found to emerge at the TyrOH/TyrO reference potential. The first class, referred to as [+/-], which contains Mn(III-V), Co(II-IV) and Ni(II-IV), shows an endothermic oxidation step combined with exothermic O-O bond formation. The members of this class were argued to be active towards the (OER), i.e. the oxy moieties desorb spontaneously as O2 at the expense of high overpotentials. The second class, called [-/+], comprising V(III-V), Cr(III-V) and Fe(II-IV), was found to show opposite behavior. Thus, poor performance is expected due to a highly unfavorable O-O bond formation step. Improved performance was predicted by mixing [+/-] with [-/+] transition metal oxides. Intermediate behavior, meaning oxidation of the TM-OH moiety to TM=O at the TyrOH/TyrO potential combined with a thermoneutral O-O bond formation, is found for Ir(III-V) and Mn(II-IV). While the former displays high activity towards the water oxidation reaction the latter is argued to be inactive due to unfavorable kinetics. The idea of mixing transition metal oxides was generalized for mixed oxidation state systems and evaluated for a Mn(II-IV) Mn(III-V) system. Improved performance was found suggesting, that this reaction path is relevant for the (OER). The generality of the mechanism was shown by direct comparison with experimental findings on iridium oxide and RuO2.
Cellular Resilience and Fragility in Response to Environmental and Gene Expression Perturbations
(2012-08-30) Ottosson, Lars-Göran
Cells are constantly subjected to perturbations. Whether these are extracellular or intracellular, they can be detrimental to cellular fitness. The cell has evolved elaborate systems and mechanisms that allow it to remain functional in the face of disturbances. Cellular signal transduction can be summarised as the processes by which environmental stimuli is integrated with information on cellular status through the transmission of intracellular signals. This information is carried by specific proteins that operate jointly in signalling networks, or pathways. An important output of these pathways is to establish cellular responses to perturbations. To remain functional the signalling network must be robust to fluctuations in both environmental stimuli and levels of signalling components. In this thesis it is investigated to what extent cellular fitness is affected by gene overexpression of signalling components. A high degree of fragility to increases in gene dosage was observed. This stands in stark contrast to overall system resilience to deletions of the same components. Fragile nodes were also dispersed over different classes of signalling components as well as throughout the signalling networks. The observed fragility patterns were further demonstrated to be largely independent of environmental and genotypic fluctuations suggesting fragility to be a product of local network architecture. Cellular responses to the rare but toxic metalloid tellurite, in terms of gene-by-environment interactions, are also investigated. To genetically elucidate mechanisms of sensitivity and resistance to this compound a genome-wide collection of gene deletion mutants was screened in presence of tellurite. A metabolic pathway, the sulfate assimilation pathway, was found to be central to tellurite toxicity. Chemically related compounds were also shown to share a common toxicity mechanism. Quantitative biology is central to this thesis and high-throughput high-resolution measurement regimes for microbial growth have been applied to all studies included herein. Phenomics is introduced and the different types of phenotyping strategies applied to studies in this thesis are elaborated on.
Iron or Copper? - Method Development and Mechanistic Studies on Cross-Coupling Reactions
(2012-09-06) Larsson, Per-Fredrik
The area of catalysis has had an immeasurable impact on modern society. This has been acknowledged through several Nobel prizes during the 20th century ranging from Haber (1918) for the synthesis of ammonia to Richard F. Heck, Ei-ichi Negishi, and Akira Suzuki (2010) for palladium-catalyzed cross-coupling reactions. The development of efficient cross-coupling reactions has made this transformation a vital instrument in the method library of organic synthesis. Today, cross-coupling reactions are widely used in industrial applications in areas such as fine chemical production and pharmaceutical industry. With increasing demands on environmentally friendly and cheaper alternatives to the commonly used palladium and nickel catalysts several alternative metals have been evaluated. Among these, both iron and copper have experienced a revival during the past two decades. The iron-catalyzed cross-coupling reaction has proved successful for a range of transformations but the mechanistic picture behind these reactions is still not entirely comprehensive. Several important mechanistic features for this reaction are presented in this thesis based on experimental studies such as titration, kinetic, and competitive Hammett study. Several of these experimental results are supported by computational studies done by Dr. Kleimark. In the pursuit of finding alternative catalysts for known transformations one has to consider the presence of potential trace-metal contaminants. The case presented in this thesis concerns the “iron” catalyzed C-N cross-coupling reaction which turned out to be catalyzed by traces of copper present in the commercially available iron source. The discovery that many copper-catalyzed cross-coupling reactions could be run with much lower catalytic loadings than previously reported further strengthened the role of copper as a viable catalyst in cross-coupling reactions. Method development, kinetic studies and ligand synthesis for sub-mol% copper-catalyzed transformations are presented in this thesis. Keywords: iron, copper, transition metal, cross-coupling, reaction mechanism, kinetic investigation, Hammett study, sustainable catalysis, trace-metal, mass-transfer, ligand development, ligand scope.
Structural Insights at Sub-Ångstrom, Medium and Low Resolution: Crystallization of Trypsin, Bacterioferritin, Photosynthetic Reaction Center, and Photosynthetic Core Complex
(2012-09-28) Yuan Wahlgren, Weixiao
The catalytic action of serine proteases depends on the interplay of a nucleophile, a general base and a general acid. The catalytic triad is composed of serine, histidine and aspartate residues. The serine acts as a nucleophile while the histidine plays a dual role as the general base or acid at different steps of the reaction. However, the role of aspartate is unclear. I recovered an ultrahigh resolution (0.93 Å) X-ray structure of a complex formed between trypsin and a canonical inhibitor. At sub-ångstrom resolution, hydrogen atoms could be visualized, giving a clue to the protonation state of the catalytic residues. By comparing this with the theoretical electron density calculated by density theory functional, the protonation states of the catalytic histidine and aspartate are discussed. Hence, a refined mechanism for serine protease action is proposed in this thesis. Photosystem harvests energy from sunlight with near 100% quantum yield. To study light-induced structural changes of the photosynthetic reaction center from purple non-sulfur bacterium Blastochloris viridis using X-ray crystallography, robust protein crystals with tight crystal packing are prerequisite. In this thesis, lipidic-sponge phase crystallization method was used and yielded well diffracting crystals for structure determination. Crystals showed a type I packing and a 1.86 Å resolution structure was determined with four lipid molecules captured in the structure. Moreover, I demonstrated that an occupied QB binding site can be obtained by co-crystallizing with UQ2 using the sponge phase crystallization method. However, attempting to crystallize the reaction center-light harvesting 1 core complex, a 440 kDa membrane protein complex of total 54 putative subunits, it required different crystallization methods. Here, the resolution has been optimized to beyond 8 Å by using the lipidic bicelle crystallization method. Conflict between the free but potential toxic Fe(II) and the insolubility of Fe(III) led to the evolution of bacterioferritin in bacteria, which functions as an iron storage and detoxification protein. Bacterioferritin from Blastochloris viridis (Bv Bfr) was crystallized and the structure was solved to 1.58 Å resolution. With the combination of X-ray structure, redundancy PCR and tandem mass spectrometry, the previously unknown amino acid sequence of Bv Bfr was determined. Conformational states of the ferroxidase center which undergoes reorganization upon different soakings were trapped. One water-like small ligand coordinated to the Fe1 binding site was captured in the Fe(II)-soaked structure. By density functional theory calculations the character of this small legend was rationalized. In addition, the structure and mechanism of iron import of the protein was studied and discussed. Finally, the redox-state of the heme in the crystals with and without Fe(II)-soaking treatment was studied by single crystal UV-VIS microspectrophotometry, before and after the X-ray exposure.
Design, Synthesis, and Evaluation of Functionalized Chroman-4-one and Chromone Derivatives. Somatostatin receptor agonists and Sirt2 inhibitors
(2012-10-04) Fridén-Saxin, Maria
Peptides are involved in many physiological processes such as regulation of blood-pressure, food intake, pain transmission and blood-glucose levels. They consist of amino acids that are connected through amide bonds which make peptides hydrophilic and conformationally flexible. Peptides generally make poor oral drugs as amide bonds are easily cleaved by endogenous enzymes. One way to overcome the structural problems with peptides is to develop stabilized mimetics, so called peptidomimetics, via a scaffold approach. The amino acid side chains needed for activity are attached as substituents to the scaffold. In this thesis, chroman-4-ones and chromones have been used as scaffolds for the development of peptidomimetics. These frameworks are naturally occurring derivatives containing an oxa-pyran ring. Depending of the substitution pattern they show different biological effects. Synthetic modifications in the 2-, 3-, 6-, and 8-positions of chromones and chroman-4-ones have been conducted. This work has included the development of an efficient synthetic route to obtain 2-alkyl chroman-4-one derivatives. Via bromination in the 3-position of chroman-4-one, various substituents (NH2, Br, OAc, CN, CH2NHCbz) have been introduced either through substitution reactions or via a Sm-mediated Reformatsky reaction. By incorporation of the appropriate substituents on the chromone-4-one and the chromone scaffolds, the biological applications have included the development of β-turn mimetics of the peptide hormone somatostatin. This has resulted in two compounds with agonistic properties for two subtypes of somatostatin receptors. In addition, functionalized 2-alkyl substituted chroman-4-one and chromone derivatives were developed as selective inhibitors of the Silent information type 2 (Sirt2) enzyme. Sirt2 functions as a deacetylating enzyme using both histones and non-histone proteins (e.g. α-tubulin) as substrates. Sirt2 is located in the cytosol but enters the nucleus during mitosis. Evaluation of a number of chroman-4-one and chromone derivatives resulted in the identification of a series of novel Sirt2-selective inhibitors with IC50 values in the low µM range. Two chroman-4-one derivatives with 2-pyridylethyl substituents in the 2-position of the chroman-4-one showed significant reduction of the proliferation of breast and lung cancer cells using a fluorescent based assay. These results indicate that the synthesized chroman-4-one based Sirt2-selective inhibitors can be valuable in more detailed studies of the function of Sirt2 in cancer.
Attenuation of acute inlammatory responses by surface nanotopography
(2012-10-04) Hulander, Mats
The interaction between biology and non-viable surfaces is crucial for many organisms and cells. For example, bacterial cells need to adhere to mineral surfaces in the soil, plants climb and adhere to walls and marine organisms produce adhesives to cling to underwater rocks etc. In the human body, tissue needs to firmly adhere to the mineral surface of bone, but also to foreign materials when for example a biomaterial is implanted. The knowledge of how biology interacts with surfaces is hence important and interesting in many aspects. Within seconds after implantation of a biomaterial, proteins from the immune complement and coagulation systems adsorbs to the surface with possible adverse consequences for the patient. To overcome this, chemical surface modifications are readily employed. However, recently the significance of surface nanotopography for the adsorption of proteins, and attachment of cells have been acknowledged. To facilitate research on the interactions between biology and nanostructured substrates novel experimental surfaces with defined nanotopography and surface chemistry were developed. The surfaces are fabricated by binding gold nanoparticles to a gold surface, using a non-lithographic method and standard laboratory equipment. The surface chemistry was evaluated using XPS and ToF-SIMS. On these surfaces, the effect of surface nanotopography on the activation of the immune complement and activation of blood platelets was studied using QCM-D, SEM and fluorescence microscopy. It was found that although nanostructured surfaces adsorbed greater amount of serum proteins, activation of the immune complement was attenuated by surface nanotopography. A suggested mechanism is that the curvature of the nanoparticles prevents interaction between complement proteins. It was also found that blood platelets were activated to a lower degree on nanostructured surfaces and were sensitive to changes in nanoparticle size and inter-particle distance. These nanostructures surfaces can hopefully facilitate research on protein/cell interactions on nanostructured surfaces.
Formation and Distribution of Marine Biogenic Halocarbons with Emphasis on Polar Regions
(2012-10-19) Karlsson, Anders
It is well-established that marine micro- and macroalgae form volatile halogenated compounds (halocarbons). Production occurs throughout the world ocean with strong regional sources in coastal areas. In the atmosphere, these compounds are converted to reactive inorganic halogen compounds, which degrade ozone. Biogenic halocarbons typically contain iodine or bromine, which have higher ozone depletion potentials than chlorine. The main pathway of halocarbon formation involves haloperoxidases, which reduce hydrogen peroxide, formed during photosynthesis, by oxidation of halide ions. Therefore, this is a universal process in the oceans, but there are large spatial and temporal variations. Estimations of the oceanic source of these compounds rely on accurate assessments of production and degradation rates, as well as on air-sea flux calculations. In this work, the distribution of biogenic halocarbons in the ice-covered central Arctic Ocean, and in the Pacific sector of the Southern Ocean, was investigated. Elevated concentrations were found under the sea ice, and even higher concentration in the ice. The production, attributed to sea ice algae, was highly variable. Degradation was also observed with half-lives as short as 1 day. Production was also found in snow, which could be attributed to biological activity. The studies were performed in a period when the sea ice was melting, and low nutrient levels indicated that a previous bloom had now been followed by heterotrophic activity. The fast degradation, presumably biotic since known abiotic reactions are much slower, showed the importance of this parameter when studying natural populations, as compared to culture studies. The highest concentrations in surface water and sea ice brine in the Arctic Ocean coincided with high concentrations of dissolved organic matter (DOM) carried by river water from the Russian rivers. It was hypothesized that this is a key parameter for the production of halocarbons in this region, as an organic substrate is needed for halocarbon production. In the Southern Ocean it was found that large regions may act as a sink for bromoform. This was highly dependent on the wind direction. When air was transported from coastal regions, under-saturation was measured in open ocean regimes. This has implications for the estimation of local sources to the atmosphere in the Southern Ocean, as the net air-sea flux may be lower due to these nearby sinks.
Selectivity of dopamine D1 and D2 receptor agonists – A combined computational approach
(2012-10-26) Malo, Marcus
Dopamine (DA) is an endogenous neurotransmitter acting in the central nervous system. DA plays a key role in many vital brain functions such as behavior, cognition, motor activity, learning, and reward. Dopamine receptors belong to the rhodopsin like family of G-protein coupled receptors (GPCRs). There are five subtypes of DA receptors (D1-D5), which are further divided into two main families based on sequence similarities and their coupling to intracellular signaling (D1- and D2-like receptors). Dopamine agonists mimic the effects of the natural neurotransmitter and it has been found that selective dopamine D2 or D1 and mixed D1/D2 agonists are useful in the treatment of Parkinson disease. As D2 (but not D1) agonists have shown undesirable dyskinetic effects it is of highest interest to understand the reasons behind D1/D2 agonist selectivity. This thesis is focused on the identification of structural features that determine the selectivity of D1 and D2 receptor agonists for their respective receptors. Selective pharmacophore models were developed for both receptors. The models were built by using projected pharmacophoric features that represent the main agonist interaction sites in the receptor, and excluded volumes where no heavy atoms are permitted. The sets of D1 and D2 ligands used for modeling were carefully selected from published sources and consist of structurally diverse, conformationally rigid full agonists as active ligands together with structurally related inactives. 3D receptor models in their agonist bound state were also generated for dopamine D1 and D2, in order to get improved insight into agonist binding. The constructed D1 and D2 agonist pharmacophore models were superimposed into their corresponding receptor model. The arrangement of pharmacophoric features were in agreement with the position of the agonist key interacting amino acids in the binding site, with exception of one hydrogen bond accepting/donating feature in the D2 model and the positioning of the excluded volumes in both models. Both pharmacophore models were refined to better reflect the shape of the binding pocket and had similar pharmacophore hit rate when screening the test sets of dopamine ligands. Several key factors for D1/D2 agonist selectivity were identified. In addition, a semi-empirical method to model transmembrane proteins with focus on the ligand binding site has been developed. The method was evaluated by generating a β1-adrenergic receptor model which had an RMSD of 1.6 Å for all heavy atoms in the binding site relative the crystal structure. A D2 receptor model with an agonist present was constructed, but this model was unable to discriminate actives from inactives in a docking study.
Static and Dynamic Measurements of Neurotransmitters in Drosophila Brain
(2012-11-15) Berglund, Carina
Neurotransmitters, the substances neurons use for communication, and their precursors and metabolites are of obvious importance for the wellbeing of the individual and when the neurotransmitter balance is off it can lead to catastrophic suffering as in the addiction to drugs or in neurodegenerative diseases. By understanding how neurons communicate with the environment, treatment may be found to aid in the symptoms of unbalance. Drosophila melanogaster, the fruit fly, has been shown to be an excellent model for understanding neuronal processes and behaviors. Although the adult fly has a simpler nervous system than those of vertebrates, it is capable of higher-order brain functions, including aversive and appetitive learning, and recalling learned information from prior experiences. Invertebrate models, such as Drosophila melanogaster have been used previously to investigate neurochemical changes in the CNS associated with drug addiction as well as in the of study of neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease by Drosophila mutants. Many of the neurotransmitters associated with these diseases occur in minute amounts and can be difficult to detect in the small volume of the fly brain. As such, it is essential to develop analytical tools for these unique biological systems that can be quickley performed and accurately analyses the neuronal substances as well as requiring extremely small sample volume. Capillary electrophoresis and in vivo voltammetry are two methods that meet these requirements. In Paper I a new separation scheme for capillary electrophoresis was devised to allow resolution of 23 neurotransmitters, metabolites, and precursors. In fly homogenates a focus on six of the substances thought to be involved in the response to alcohol were identified. In Paper II the removal of the cuticles and eyes leaving only the brains further enhanced the separation profile of neurotransmitters from Paper I. In Paper III a method for sample preparation by freeze drying the Drosophila brains was presented. The use of freeze-dried samples offers a way to preserve the biological sample while making dissection of the tiny brain samples easier and faster. This provides more concentrated samples and with that higher signals and better detection limits. In Paper IV the effect of cocaine on the dopamine transporter was shown to be reduced by the ADHD drug methylphenidate using in vivo voltammetry.
Synthesis, Pharmacological Characterization and QSAR Modelling of 4-Phenylpiperidines and 4-Phenylpiperazines Effects on the dopaminergic neurotransmission in vivo
(2012-11-19) Pettersson, Fredrik
The endogenous neurotransmitter dopamine (DA) is involved in several functions that are controlled from the central nervous system (CNS), for example behaviour, memory, cognition and reward. A disturbed dopaminergic neurotransmission may lead to many severe conditions, such as schizophrenia, attention deficit hyperactivity disorder (ADHD) or Parkinson's disease (PD). The dopamine receptors belong to the G-protein coupled receptors (GPCRs) and are divided into five distinct subtypes (D1-D5). These subtypes can be either of the D1- or D2-types based on their effect on the production of cyclic adenosine monophosphate (cAMP). The most common dopaminergic receptor used as target for pharmaceuticals is by far the D2 receptor and drugs acting as full agonists, partial agonists and antagonists at this receptor have been developed. In the search for new dopaminergic ligands, a set of 4-phenylpiperidines and 4-phenylpiperazines have been synthesized and their effects have been tested in both in vivo and in vitro assays. Starting with the known partial agonist 3-(1-benzylpiperidin-4-yl)phenol, stepwise structural modifications of functional groups afforded mainly D2 antagonists but with a conserved preference for binding to the agonist binding site and fast dissociation rates from the receptor. However, further modifications, including changes of the position of the aromatic substituent, indicated that other targets than the D2 receptor was involved and binding affinity studies later concluded that some of these compounds had MAO A inhibiting properties. In order to fully elucidate what structural properties are related to the different pharmacological responses, QSAR models with physicochemical descriptors set against each respective response were acquired by means of partial least square (PLS) regression. Models with high predictivity (Q2>0.53) were obtained and the interpretation of these models has provided an improved understanding of how structural modifications in this chemical class affect the response both in vivo and in vitro. The structural motifs that were investigated included the position and physicochemical properties of the aromatic substituent as well as the heterocycle being a piperazine or a piperidine. All these properties turned out to be significant for the different responses in some aspect. In addition, a strong correlation between the affinities to the D2 receptor and to MAO A and the levels of the metabolite DOPAC in striatum has been established. This led us to the conclusion that it is primarily interactions with these two targets that lead to the in vivo response observed for this class of compounds.
The Synthesis and Use of Certain Pyridine Derivatives as Modulators of the G-protein Coupled Receptors mGlu5 and P2Y12
(2012-11-20) Bach, Peter
The glutamatergic mGlu5 receptor and the purinergic P2Y12 receptor are two important targets in the development of novel treatments of gastroesophageal reflux disease (GERD) and thrombosis, respectively. Synthesis was developed to investigate the structure-activity relationships (SAR) of a novel series of 2-alkynylpyridine derivatives as mGluR5 antagonists. This led to the discovery of antagonists with potency in the low-nanomolar range. High microsomal metabolism, possibly due to high lipophilicity, remained an issue. Further, SAR development for a series of ethyl 6-piperazinylnicotinates, featured by a urea linker, as antagonists of the P2Y12 receptor showed the 3-ethoxycarbonyl substituent as central to binding. The low aqueous solubility was addressed by variation of the linker which led to the discovery of sulfonylureas as P2Y12 antagonists. The chemical stability of the sulfonylurea compounds during prolonged storage in solution was found to be related to the sulfonyl urea linker and depended on the type of solvent and the substitution pattern of the sulfonyl urea functionality. Synthesis was developed to facilitate the replacement of the 2-methyl substituent on pyridine with more electron donating substituents and of the 3-ethoxycarbonyl substituent with 5-ethyl-oxazoles. Both strategies led to compounds with higher metabolic stability, but also with lower potency. Pair-wise comparison of compounds showed that a correctly positioned alkyl group, like in an ethyl ester or a 5-ethyl-oxazole, and a correctly positioned strong hydrogen bond acceptor both were required for binding. Chemical design was used to study how the regioselectivity Rsel for the 2-position depended on the character of the 3-substituent in the reaction of 3-substituted 2,6-dichloropyridines with 1-methylpiperazine. It was found that Rsel depended on neither of the parameters PI, MR, or p, but showed a statistically significant correlation with the Verloop steric parameter B1 (R2: 0.45, p = 0.006). This implied that 3-substituents that are bulky close to the pyridine ring directed the regioselectivity towards the 6-position. With R3 = -CO2CH3 a study of the solvent effect showed that Rsel could be predicted by the Kamlet-Taft equation: Rsel = 1.28990 + 0.03992 - 0.59417 - 0.46169* (R2 = 0.95; p = 1.9 x 10-10). The dependency on the solvatochromic  parameter meant that the 16:1 regioselectivity for the 2-isomer in DCM ( = 0.10) could be switched to a 2:1 selectivity for the 6-isomer in DMSO ( = 0.76).
Algorithms and Interaction Potentials: phase density, surface tension and carbon dioxide
(2012-12-14) Persson, Rasmus
This thesis is an amalgamation of articles (Papers I–V) by the author. We present a perturbation algorithm to calculate the phase density, and thus the partition function including its temperature dependence. It works for Hamiltonians that are not too dissimilar, for which an extra degree of freedom interpolating between the two is defined so that microcanonical sampling allows the calculation of the ratio between the phase densities at any energy. The method is illustrated on a number of problems of different dimensionalities. In Paper I, we consider an anharmonic Einstein crystal, the square-well tetradecamer, and liquid gold. In addition, we consider the one-dimensional rotor and the low-dimensional ideal gas in a homogeneous external field, two Hamiltonians that display a phase transition at well-defined critical energies. We consider the interaction of linear molecules and discuss two coarse-grained pair potentials for their description. In Paper III, one of these potentials has been parametrized for the vapor-liquid envelope of carbon dioxide using two adjustablesobtaining good agreement, but for a detailed description of the carbon dioxide dimer and trimer structures, such coarse-graining fails. As reported in Paper IV, conventional all-atom force field descriptions also fail in describing the experimental second and third virial coefficients but an all-atom description with coarse-grained, single-site anisotropic three-body dispersion and single-site electrostatic induction manages to reproduce them. In addition, we note that this simple anisotropic three-body dispersion correction is essential for predicting the correct relative stability of the experimental trimer conformations when combined with a literature parametrization of the dimer ab initio potential energy surface. We present a simple method for calculating the surface tension with respect to vacuum from cluster simulations, by relating the scalar pressure to the infinitesimal isothermal pressure-volume work and equating it with the expression from classical nucleation theory. We then discuss the effect of molecular polarization on the surface tension using this method, as well as study its effect on the second and third virial coefficients of the fluid of polarizable Stockmayer molecules. The surface tension increases with polarizability, but so does its rate of decrease with temperature. The Tolman length is found positive and largely insensitive to temperature but increases non-linearly with increasing molecular polarizability. We discuss the semi-empirical calculation of the crystal-water surface tension of the pharmaceutical bicalutamide as reported in Paper V and provide a slight modification of the procedure.
Regulation and transport mechanisms of eukaryotic aquaporins
(2013-01-11) Palmgren, Madelene
Aquaporins are found in all kingdoms of life where they are involved in water homeostasis. They are small transmembrane water conducting channels that belong to the ancient protein family Major Intrinsic Proteins (MIP). Early on in the evolution, a gene duplication event took place that divided the aquaporin family into two subgroups; orthodox aquaporins, which are strict water facilitators, and aquaglyceroporins that except for water also transport small uncharged solutes. The main questions that I have tried to address in this thesis are which regulatory mechanisms that are involved in aquaporin gating and to investigate transport differences in solute permeation. Specifically, we have investigated yeast and human aquaporins. To find answers to our questions, we have attempted to combine structural knowledge with functional analysis. A high resolution structure of P. pastoris orthodox Aqy1 to 1.15Å generated new knowledge of regulatory mechanisms and functions of the long N-terminus that is common among fungi. We suggest that Aqy1 is gated by phosphorylation and by mechanosensation. An important functional role of Aqy1 in rapid freeze thaw cycles could be demonstrated. During this work, a single deletion strain was generated that now serves as the primary aquaporin expression platform in our laboratory. Fps1 is a regulated glycerol facilitator that is important for yeast osmo-regulation. The regulatory mechanism is still not known but here we show that a suppressor mutation within the transmembrane region restrict glycerol by its transmembrane core. Thereby, we suggest that post translational modifications in the regulatory domains of N- and C-termini fine tunes glycerol flux through Fps1. The aquaglyceroporins are classified as having a dual transport function, namely being capable of facilitating the movement of both water and glycerol over the plasma membrane. In this study, we can clearly show that there are major differences in the substrate specificity and efficiency between the different aquaglyceroporins and that small changes affect the transport efficiency and specificity of the channels.
Formation, ageing and thermal properties of secondary organic aerosol
(2013-02-07) Emanuelsson, Eva
In order to properly represent and predict the effects of aerosol in climate systems, an accurate description of their formation and properties is needed. This thesis describes work done to increase the knowledge of processes and properties of atmospherically relevant secondary organic aerosol (SOA) from both biogenic and anthropogenic origin. The common theme for these projects is the use of a Volatility Tandem Differential Mobility Analyser (VTDMA) setup, which in combination with other observations has generated insight into both detailed chemical mechanisms and physical processes that eventually could be suitable for testing in air quality or climate models. During the course of this work, the experimental facility the Gothenburg Flow Reactor for Oxidation Studies at low Temperatures (G-FROST) and the VTDMA setup, as well as a corresponding data evaluation methodology, have been improved and refined. Thermal properties could be linked to both formation and ageing processes of SOA. Using a VTDMA setup, where the thermal characteristics of SOA were measured at a range of evaporation temperatures, a sigmoidal fit to the data enabled parameterisation of their volatility properties. The parameters extracted were e.g. the temperature corresponding to a volume fraction remaining of 0.5 (TVFR0.5) and the slope factor (SVFR), which are measures of the general volatility and the volatility distribution of the condensed phase products, respectively. A higher TVFR0.5 indicates lower volatility, while an increase of SVFR states a broader distribution of vapour pressures. The response of these parameters from changes in experimental conditions could be linked to processes occurring both in the gaseous and the condensed phase. In photo-chemical experiments, the change in TVFR0.5 and SVFR could be described using the OH dose. The gas phase processes were found to be very important for SOA ageing, driven mainly by OH radical exposure in the outdoor chamber SAPHIR. However, processes in the condensed phase, such as plausible non oxidative ageing processes and non-liquid behaviour of SOA particles, were also observed. Detailed studies of ozonolysis of the boreal forest monoterpenes β-pinene and limonene were enabled by precise control of reaction conditions using the G-FROST. The experimental findings in response to e.g. water and radical conditions emphasized the difference in ozonolysis reaction paths between endo- and exocyclic compounds. The results support the recently suggested decomposition of the stabilized Criegee Intermediate via the hydroperoxide channel in ozonolysis of β-pinene.
On the formation of small molecules by radiative association
(2013-03-05) Antipov, Sergey
This thesis is devoted to the theoretical investigation of radiative association, which is one of the processes contributing to formation of molecules in the interstellar medium. The formation of the CN, SiN, SiP and CO molecules through radiative association of the corresponding atoms in their ground electronic states is studied by the means of classical and quantum dynamical calculations. In all cases the Born–Oppenheimer approximation is employed. The corresponding rate coefficients are calculated and the Breit–Wigner theory is used to properly account for the resonance contributions. Some common features of the radiative association process for the considered systems are discovered. For example, a drop in magnitude of the cross sections at high energy and, consequently, the high-temperature rate coefficients is observed. Also, is is shown that in the absence of a potential barrier the semi-classical formalism provides a good estimate for the rate coefficients. A pronounced isotope effect is discovered for the formation of CO by radiative association of C(3P) and O(3P) atoms. It is shown that the presence of a potential barrier on the A1Π electronic state of carbon monoxide leads to different formation channels for the 12CO and 13CO isotopologues at low temperatures. The role of spin-orbit and rotational couplings in radiative association of C(3P) and N(4S) atoms is investigated. Couplings among doublet electronic states of the CN radical are considered, giving rise to a 6-state model of the process. Comparison of the energy-dependent rate coefficients calculated with and without spin-orbit and rotational couplings shows that these have a strong effect on the resonance structure and low-energy baseline of the rate coefficient.
Structural studies of aquporins in human kidney and plant
(2013-03-22) Frick, Anna
Membrane proteins are key players in our biology and are links between the inside and the outside of the cell, allowing for signal transduction and transport of molecules. Aquaporins are membrane protein channels that allow water to pass in and out of the cell. Since all life depend on water, their function is vital for any type of organism. Although aquaporins are very similar, they have small but important differences in their structure and function. Understanding these subtle dissimilarities helps us understand the fundamentals of our biology and is also essential if aquaporins are to be used as drug targets. This thesis has investigated the structure and function of two aquaporins from different species; human and spinach. The spinach aquaporin SoPIP2;1 has become the structural model for gated plant aquaporins. In this thesis, structural and functional data is presented that gives further insights into the gating mechanism controlled by the physiological signals phosphorylation, pH and divalent cations. In addition, the mechanism behind the activation of SoPIP2;1 by mercury, commonly regarded as an aquaporin blocker, has been studied. Human Aquaporin 2 is crucial for the kidneys ability to concentrate primary urine, and its malfunction leads to nephrogenic diabetes insipidus. An X-ray crystallographic structure to 2.95Å is presented, which show that AQP2 is markedly different also from its most closely related homologues. These differences are mainly focused on loop D and the C-terminus and can be related to binding of Cd2+ in the structure. We present data that Cd2+ could correspond to Ca2+ in vivo, and discuss the role of the C-terminal helix as a protein interaction partner. In addition, mutations leading to nephrogenic diabetes insipidus are studied in the structural context.

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