In this thesis the thermal- and photo-substitution behavior of polypyridyl ruthenium complexes is described at the surface of lipid bilayers and in homogeneous solutions. It is shown that the... Show moreIn this thesis the thermal- and photo-substitution behavior of polypyridyl ruthenium complexes is described at the surface of lipid bilayers and in homogeneous solutions. It is shown that the successive thermal binding and light-induced unbinding of the cationic ruthenium complex at the surface of the lipid bilayer requires negatively charged liposomes and ruthenium complexes containing moderately hindered N-N bidentate ligands. Our results in homogeneous solution show that changing the steric hindrance of the bidentate ligand influences both the photo- and thermal reactivities of these complexes, by altering the mechanism of the Ru-S bond formation. It is also shown that the Ru-S bond formation at the surface of negative lipid bilayers is faster than the same reaction in homogenous aqueous solutions, and a two-steps mechanism is proposed for the thermal coordination of ruthenium aqua complexes at membrane-embedded ligands. Furthermore, the application of ruthenium-functionalized liposomes in drug delivery is discussed. In vitro tests on cancer cell lines show that neutral liposomes functionalized with ruthenium compounds are more readily taken up by cancer cells than ruthenium-free liposomes. The liposome samples with ruthenium compounds are shown to be poorly cytotoxic in the dark. After light irradiation, the cytotoxicity increased at least up to five times for ruthenium complexes supported on non-PEGylated liposomes. Finally, the photoactivation of polypyridyl complexes with low-energy photons was studied using a photosensitization approach. A photosubstitution reaction was made faster upon yellow light irradiation than upon blue light irradiation by covalently linking a rhodamine B dye to the ruthenium complex. Show less
The aim of this thesis is to introduce the readers to the importance of bio-based products and their potential applications in chemical industries. Alternative routes to the widely used nylon... Show moreThe aim of this thesis is to introduce the readers to the importance of bio-based products and their potential applications in chemical industries. Alternative routes to the widely used nylon precursor caprolactam are discussed and compared with the current fossil-based synthesis. Furthermore, the aim of the research - the development of a novel synthesis route to caprolactam based on biomass - is reported; the envisaged reaction sequence includes the development of new catalytic chemistry, namely reductive amidation and hydroamidomethylation. Show less
In this thesis the behavior and functionality of peptide amphiphiles at the surface of bilayer vesicles is examined. By controlling the behavior of the surface bound peptides, I was able to... Show moreIn this thesis the behavior and functionality of peptide amphiphiles at the surface of bilayer vesicles is examined. By controlling the behavior of the surface bound peptides, I was able to construct assemblies which could: 1) release their content (triggered by pH), 2) fuse in a targeted and controlled manner or 3) dock to cells and zebrafish embryos Show less
CRANK is a suite that links different macromolecular X-ray crystallographic programs to solve macromolecular crystal structures automatically from experimental phasing data. In chapter 2, several... Show moreCRANK is a suite that links different macromolecular X-ray crystallographic programs to solve macromolecular crystal structures automatically from experimental phasing data. In chapter 2, several new algorithms implemented within CRANK increase the robustness and speed of the structure solution process. The new MULTICOMB program, discussed in chapter 3, provides a new phase combination algorithm for the density modification step of the structure solution process. MULTICOMB implements a novel advanced multivariate function that considers the single-wavelength anomalous diffraction (SAD) data directly, accounts for the correlation between the initial and density-modified maps and refines errors that can occur in a SAD experiment. Testing of these new algorithms with over 100 real data sets showed a dramatic improvement over state-of-the-art methods. These novel methods were also applied in solving the new structure of the DNA-binding protein Sso10a2 from Sulfolobus solfataricus reported in chapter 4. This structure provides insight to the observed differences in behaviour between Sso10a2 and its close homolog Sso10a. The last chapter of this work describes the crystallization conditions for a recombinant, fully glycosylated form of the human C1 inhibitor protein, which is involved in hereditary angioedema, a potentially life threatening condition. Show less
This thesis describes the application of a variety of different classical and paramagnetic NMR to different protein system. To this end cytochrome P450cam was assigned using classical techniques... Show moreThis thesis describes the application of a variety of different classical and paramagnetic NMR to different protein system. To this end cytochrome P450cam was assigned using classical techniques and paramagnetic NMR was used to investigate the solution state of this enzyme in complex with its binding partner, putidaredoxin. The solution structure of Avr2 was also solved using classical NMR spectroscopy techniques. Additionally, an algorithm for the assignment of protein nuclei on the basis pseudocontact shifts, PARAssign, is presented. An investigation of the dynamic behaviour of a paramagnetic tag attached to T4 lysozyme using relaxation dispersion is described. Show less
Photosynthesis in plants, algae and cyanobacteria releases oxygen and is referred to as oxygenic photosynthesis. Among all the photosynthetic reaction centers only PSII provides a redox potential... Show morePhotosynthesis in plants, algae and cyanobacteria releases oxygen and is referred to as oxygenic photosynthesis. Among all the photosynthetic reaction centers only PSII provides a redox potential sufficiently strong for water oxidation. While the electron flow in PSII is strictly asymmetric, in PSI different levels of bidirectionality indicate functional flexibility of this complex. Photo-CIDNP is non-Boltzmann nuclear magnetization caused by photochemical reactions and can be observed by NMR spectroscopy as strongly enhanced absorptive or emissive signals. In this thesis photo-CIDNP solid state NMR with selective isotope labeling is applied to get direct access to the heart of large PSII and PSI photosynthetic complexes in intact and isolated systems of Spirodela oligorrhiza and Synechocyctis. For the first time the direct observation of selective atoms within the heart of the PSII and PSI complexes by experiment s on entire plants and whole cells is reported. In this way the conservation of the electronic structure of the PSII electron donor at various levels of biological preparations have been addressed and the electron spin density (ρi) in the active cofactors of PSI has been constructed. In addition the functional heterogeneity of the PSI electron donor among different plant species was probed. Show less
While aging remains one of the most significant risk factors for development of Alzheimer disease (AD), increasing evidence strongly points to the potential roles of cerebrovascular and white... Show moreWhile aging remains one of the most significant risk factors for development of Alzheimer disease (AD), increasing evidence strongly points to the potential roles of cerebrovascular and white matter abnormalities in the disease development. A better understanding of the manner in which these abnormalities contribute to disease progression can be achieved by in vivo characterization of AD related pathologies. To this end, MR based techniques serve as effective non-invasive tools to longitudinally monitor changes in AD brain. In this thesis, a variety of MR based techniques were optimized and employed to longitudinally monitor the AD progression in transgenic mouse models of the disease at 9.4T and 17.6T. In Chapter 2, age-dependent blood flow alterations were examined in a Tg2576 mouse model of Alzheimer's disease using MR angiography at 17.6T. AD is linked to abnormalities in the vascular system. In Chapter 3, in vivo T2 changes were longitudinally monitored in the corpus callosum, of the Tg2576 mice. In Chapter 4, age-dependent regional brain T1 and T2 changes in healty mice were established at 17.6T. In vivo imaging of these mouse models at ultra-high magnetic field strengths can permit a better understanding of the underlying cellular mechanism of AD. Show less
The work described in this thesis focuses on the application of various NMR techniques to the study of interactions between proteind and small molecules and proteins and peptides, incluiding the... Show moreThe work described in this thesis focuses on the application of various NMR techniques to the study of interactions between proteind and small molecules and proteins and peptides, incluiding the well-established classical NMR approaches, and the precently developed paramagnetic NMR methods. computational tools have been used to complement and visualized the experimental data. Show less
The majority of the work presented in this thesis involves the design and synthesis of paramagnetic NMR probes, including lanthanoids caged probes and spin labels. An overview of the development of... Show moreThe majority of the work presented in this thesis involves the design and synthesis of paramagnetic NMR probes, including lanthanoids caged probes and spin labels. An overview of the development of different types of lanthanoids caged probes is given. Among all of the reported lanthanoid probes, the caged lanthanoid NMR probe version 5 (CLaNP-5) shows the largest paramagnetic effects due to the fact that it is tagged to proteins via two-point attachment and its free complex presents a single conformation. Although CLaNP-5 is successfully applied to study proteins and protein complexes, the net charge of Ln CLaNP-5 complex and the weak disulfide linker are the drawbacks. A new paramagnetic probe, CLaNP-7, was synthesized, for which the net charge of lanthanoids complexes was reduced to +1 by introducing p-nitrophenol. The __-tensor of CLaNP-7 is pH-dependent when a histidine residue is located close to the attachment site. It is proposed that the pH dependence is due to the fact that the histidine forms a hydrogen bond with a water that acts as the ninth ligand of the lanthanoid. In order to enhance the stability of the tag linkers, two approaches, thioether and bioorthogonal reactions, were investigated. The results showed that a new thio-reactive CLaNP-9 was successfully attached to protein and the reaction product was stable in the reductive conditions. Moreover, inhibitor-based paramagnetic probes were also synthesized and the co-crystal structures of protein with inhibitor-based probes were also presented. Show less
Generating white light with LED technology requires a trick; one of them is to use color-converting (phosphor) layer that covers the LED chip. Suitable phosphors that can be excited with LEDs are... Show moreGenerating white light with LED technology requires a trick; one of them is to use color-converting (phosphor) layer that covers the LED chip. Suitable phosphors that can be excited with LEDs are scarce, necessitating research in this area. Complexes of the trivalent lanthanoids are interesting candidates, as their absorption properties can be tuned by modification of the organic part of the molecule, while the emission is line-like in nature and characteristic for the Ln(III) ion. In this thesis, several classes of Ln(III) coordination compounds are explored and their photophysical properties investigated. Several compounds with promising properties, such as a long-wavelength excitation maximum and a high photoluminescence quantum yield are reported. Show less
This PhD thesis focuses on fundamental aspects of protein-protein interactions. A multidisciplinary methodology for the detection and visualization of transient, lowly-populated encounter protein... Show moreThis PhD thesis focuses on fundamental aspects of protein-protein interactions. A multidisciplinary methodology for the detection and visualization of transient, lowly-populated encounter protein complexes is described. The new methodology combined paramagnetic NMR spectroscopy with computational methods (ensemble docking approach and Monte Carlo simulations) to provide a new model to describe the formation of a protein complex on the basis of the physical forces involved in the process, namely electrostatic and hydrophobic interactions. The formation of a productive protein complex is a stepwise process, in which the free components evolve to the final complex passing through a transient, lowly-populated encounter state. For a long time the first step of association was thought to be exclusively driven by long-range electrostatic interactions. Experimental evidences and theoretical studies questioned this assumption and suggested also a role of hydrophobic interactions in protein association. To study the contribution of the different forces we study the highly dynamic complex formed by plastocyanin and cytochrome f, two redox partners in oxygenic photosynthesis, for which both electrostatic and hydrophobic interactions were shown to contribute to the stabilization of the final complex. Through the combination of paramagnetic relaxation enhancement NMR techniques and computational methods we were able to visualize the presence of hydrophobic interactions in the encounter state and to elucidate the contribution of either electrostatic or hydrophobic forces to the formation of the encounter complex. Show less
The primary goal of this thesis is to study the potential role of electrochemistry in finding new routes for sustainable chemicals from biomass in aqueous-phase solutions. In order to assess the... Show moreThe primary goal of this thesis is to study the potential role of electrochemistry in finding new routes for sustainable chemicals from biomass in aqueous-phase solutions. In order to assess the potential of electrochemistry in biomass conversion, we developed an online HPLC system by using a fraction collector with a micrometer-sized sampling tip placed close to the working electrode, with the collected sample fractions subsequently analyzed in an offline HPLC system. To demonstrate this method, we applied it to the poly-ols, i.e. glycerol (C3H8O3), electro-oxidation on Au and Pt electrodes at different pH conditions, visualizing the concentration changes of glycerol and its reaction products in correspondence with the current measured in voltammetry, which brought us a concept to co-generate hydrogen and valuable chemicals (i.e. glyceraldehyde, glyceric acid, dihydroxyacetone, formic acid) with high selectivity depending on the applied potential. In addition, we aimed at single-cell synthesis of sorbitol from cellulosic material by generating glucose as an intermediate species. This thesis has demonstrated the separate reactions of cellobiose hydrolysis to glucose by acid and hydroxyl radical on an anode and glucose hydrogenation to sorbitol on a cathode in an electrolysis cell. Show less
This thesis describes the synthesis of oligonucleotide conjugates by means of the strain induced [2+3] dipolar cycloaddition is described. To this end, dibenzocyclooctyne containing phosphoramidite... Show moreThis thesis describes the synthesis of oligonucleotide conjugates by means of the strain induced [2+3] dipolar cycloaddition is described. To this end, dibenzocyclooctyne containing phosphoramidite building blocks were synthesized. These building blocks were subsequently used in the solid phase synthesis of RNA and DNA oligonucleotides yielding dibenzocyclooctyne containing oligonucleotides. These oligonucleotides were used in strain induced cycloadditions with a variety of azides e.g. fluorescent labels, oligosaccharides, oligopeptides and bovine serum albumin yielding the corresponding conjugates. Additionally, the synthesis of D-arabinose and D-ribose derived C-glycosides is described. These nucleoside derivatives were obtained from D-ribose and D-arabinose C-1 substituted hemi-ketals in BF3OEt2-triethyl silane mediated reductions. The results and mechanism regarding the stereoselectivity in these reductions are discussed. Show less
In this thesis the prospects of electron diffraction and imaging techniques are discussed to solve the structure of three dimensional nano-crystals. These crystals are beyond the scope of current X... Show moreIn this thesis the prospects of electron diffraction and imaging techniques are discussed to solve the structure of three dimensional nano-crystals. These crystals are beyond the scope of current X-ray techniques. Show less
