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- Albers, Michael Franz, 1987-
(författare)
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Synthesis and investigation of bacterial effector molecules
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During infections, bacterial microorganisms initiate profound interactions with mammalian host cells. Usually defense mechanisms of the host destroy intruding bacteria in rapid manner. However, many bacterial pathogens have evolved in a way to avoid these mechanisms. By use of effector molecules, which can be small organic molecules or proteins with enzymatic activity, the host is manipulated on a molecular level. Effectors mediating post-translational modifications (PTMs) are employed by many pathogens to influence the biological activity of host proteins. In the presented thesis, two related PTMs are investigated in detail: Adenylylation, the covalent transfer of an adenosine monophosphate group from adenosine triphosphate onto proteins, and phosphocholination, the covalent transfer of a phosphocholine moiety onto proteins. Over the past years, enzymes mediating these modifications have been discovered in several pathogens, especially as a mechanism to influence the signaling of eukaryotic cells by adenylylating or phosphocholinating small GTPases. However, the development of reliable methods for the isolation and identification of adenylylated and phosphocholinated proteins remains a vehement challenge in this field of research. This thesis presents general procedures for the synthesis of peptides carrying adenylylated or phosphocholinated tyrosine, threonine and serine residues. From the resulting peptides, mono-selective polyclonal antibodies against adenylylated tyrosine and threonine have been raised. The antibodies were used as tools for proteomic research to isolate unknown substrates of adenylyl transferases from eukaryotic cells. Mass spectrometric fragmentation techniques have been investigated to ease the identification of adenylylated proteins. Furthermore, this work presents a new strategy to identify adenylylated proteins. Additionally, small effector molecules are involved in the regulation of infection mechanisms. In this work, the small molecule LAI-1 (Legionella autoinducer 1) from the pathogen Legionella pneumophila, the causative agent of the Legionnaire’s disease, was synthesised together with its amino-derivatives. LAI-1 showed are a clear pharmacological effect on the regulation of the life cycle of L. pneumophila, initiating transmissive traits like motility and virulence. Furthermore, LAI-1 was shown to have an effect on eukaryotic cells as well. Directed motility of the eukaryotic cells was significantly reduced and the cytoskeletal architecture was reorganised, probably by interfering with the small GTPase Cdc42.
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| 2. |
- Beckman Sundh, Ulla, 1953-
(författare)
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Studies on Phosphohistidine Phosphatase 1 : What? Where? Why?
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Hedberg, Christian, 1975-
(författare)
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Design, Synthesis, Mechanistic Rationalization and Application of Asymmetric Transition-Metal Catalysts
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes mechanistic studies, rational ligand design, and synthesis of asymmetric transition metal catalysts. The topics addressed concerned [Papers I-VII]:[I] The asymmetric addition of diethyl zinc to N-(diphenylphosphinoyl)benzalimine catalyzed by bicyclic 2-azanorbornyl-3-methanols was studied. An efficient route to both diastereomers of new bicyclic 2-azanorbornyl-3-methanols with an additional chiral center was developed, in the best case 97% ee was obtained with these ligands. The experimental results were rationalized by a computational DFT-study.[II] An aza-Diels-Alder reaction of cyclopentadiene with chiral heterocyclic imines derived from (S)-1-phenylethylamine and different heteroaromatic aldehydes was developed. The cycloaddition proved to be highly diastereoselective and offers a very rapid access to possible biologically active compounds and interesting precursors for chiral (P,N)-ligands. [III] A convenient and high-yielding method for the preparation of (R)-tolterodine, utilizing a catalytic asymmetric Me-CBS reduction was developed. Highly enantio-enriched (R)-6-methyl-4-phenyl-3,4-dihydrochromen-2-one (94% ee) was recrystallized to yield practically enantiopure material (ee >99%) and converted to (R)-tolterodine in a four-step procedure. [IV] The reaction mechanism of the iridium-phosphanooxazoline-catalyzed hydrogenation of unfunctionalized olefins has been studied by means of DFT-calculations (B3LYP) and kinetic experiments. The calculations suggest that the reaction involves an unexpected IrIII-IrV catalytic cycle facilitated by coordination of a second equivalent of dihydrogen. On the basis of the proposed catalytic cycle, calculations were performed on a full system with 88 atoms. These calculations were also used to explain the enantioselectivity displayed by the catalyst.[V and VI] A new class of chiral (P,N)-ligands for the Ir-catalyzed asymmetric hydrogenation of aryl alkenes was developed. These new ligands proved to be highly efficient and tolerate a broad range of substrates. The enantiomeric excesses are, so far, the best reported and can be rationalized using the proposed selectivity model.[VII] The complex formed between the quincorine-amine, containing both a primary and a quinuclidine amino function, and [Cp*RuCl]4 catalyzes the hydrogenation of aromatic and aliphatic ketones in up to 90% ee approx. 24-times faster than previously reported Ru-diamine complexes. The reason for the lower but opposite stereoselectivity seen with the quincoridine-amine, as compared to the quincorine-amine, was rationalized by a kinetic and computational study of the mechanism. The theoretical calculations also revealed a significantly lower activation barrier for the alcohol mediated split of dihydrogen, as compared to the non-alchol mediated process. A finding of importance also for the diphosphine/diamine mediated enantioselective hydrogenation of ketones.
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