Dimeric ligands for G-protein coupled receptors that are involved in human reproduction, namely the gonadotropin releasing hormone receptor, the luteinizing hormone receptor and the follicle... Show moreDimeric ligands for G-protein coupled receptors that are involved in human reproduction, namely the gonadotropin releasing hormone receptor, the luteinizing hormone receptor and the follicle-stimulating hormone receptor, were synthesized and biologically evaluated. Show less
The study described in this thesis was conducted with the aim of developing lipophilic iminosugars as selective inhibitors for glucosylceramide synthase, glucocerbrosidase and _-glucosidase 2 that... Show moreThe study described in this thesis was conducted with the aim of developing lipophilic iminosugars as selective inhibitors for glucosylceramide synthase, glucocerbrosidase and _-glucosidase 2 that are enzymes involved in glucosylceramide metabolism. The study has resulted in many novel inhibitors of these three enzymes among which several that improve upon the inhibition profile of the lead compound in this study. The successful use of lipophilic iminosugars in type 2 diabetes models and the partial elucidation of their mechanism of action therein provide prospects for their development towards therapeutics for diabetes type 2. Show less
This thesis describes an investigation of the potential of electron diffraction for studying three dimensional sub-micro-crystals of proteins and pharmaceuticals. A prerequisite for using electron... Show moreThis thesis describes an investigation of the potential of electron diffraction for studying three dimensional sub-micro-crystals of proteins and pharmaceuticals. A prerequisite for using electron diffraction for structural studies is the predictable availability of tiny crystals. A method for growing such crystals using heterogeneous nucleation is demonstrated. The heterogeneous nucleant (in this case hair fibers) was serendipitously selected. Four different proteins (lysozyme, glucose isomerase, a Fab fragment and potato protease inhibitor) were shown to nucleate preferentially on the selected substrate and sub-micro crystals were grown. Further studies on the mechanism of heterogeneous nucleation using lysozyme as a test protein and different imaging techniques such as atomic force and fluorescent microscopy are also discussed. Sub-micron crystals of potato protease inhibitor and lysozyme were subject of electron diffraction studies. A detailed description of the diffraction experiments is presented. A special focus is given on the sample preparation procedure and in particular the vitrification and the cryo-preservation of the crystals. The preliminary results showed that the heterogeneously grown nano-crystals are well ordered and suitable for electron diffraction. The high beam sensitivity of the protein nano-crystals appeared to be the rate limiting step in the data collection, not allowing orientation of the crystals (a technique used in electron diffraction studies of inorganic crystals) or a 3D data collection of a single crystal (a technique used in X-ray protein crystallography). This suggested that new approaches for data collection and data analysis needed to be developed. Optimization of the diffraction data collection, as is described in this thesis allowed high diffraction resolution (up to 2.1_) to be obtained from vitrified lysozyme crystals. An algorithm for unit cell determination of randomly oriented diffraction patterns of different crystals is presented. The method was used for the analysis of the electron diffraction data acquired from lysozyme nano-crystals. The methods for collecting and analyzing electron diffraction data from lysozyme crystals were also confirmed in the case of penicilline type nano-crystals. The motivation behind these studies and the results obtained are discussed. In this case a crystalline powder sample was subjected to electron diffraction studies. Resolutions up to 0.8_ were obtained from oxacillin crystals and up to 1_ from penicillin G crystals. The unit cell parameters found from the analysis of electron diffraction data with the algorithm presented in the previous chapter were consistent with the unit cell parameters obtained by X-ray crystallography on the same two types of penicillin. Show less
The thesis describes the application of several different magnetic resonance (MR) techniques to study the effects of the progression of disease in a transgenic mouse model of Alzheimer's. Using MR... Show moreThe thesis describes the application of several different magnetic resonance (MR) techniques to study the effects of the progression of disease in a transgenic mouse model of Alzheimer's. Using MR imaging, the amyloid plaque deposition was visualized and the plaque load quantified in the same mice as they aged. Concurrently the transverse relaxation time (T2) was measured in affected brain regions and shown to decrease over time as plaque-load increased. To study the neurochemical profile in the mouse brain brain both one- (1D) and two-dimensional (2D) MR spectroscopic techniques were employed. 1D MRS is widely used in similar research, but has limited spectral resolution. To overcome this limitation, a 2D MRS technique was implemented and optimized for use in mouse brain. This technique, L-COSY, allowed the detection of several metabolites which were not visible using standard 1D MRS techniques. This technique was subsequently used to study the effects of Alzheimer's on the neurochemical profile. Observed changes were correlated with plaque deposition. Show less
The initial damage recognizing complex in bacterial nucleotide excision repair consists of two UvrA and two UvrB molecules. Of these proteins UvrB is the main damage recognition protein and is... Show moreThe initial damage recognizing complex in bacterial nucleotide excision repair consists of two UvrA and two UvrB molecules. Of these proteins UvrB is the main damage recognition protein and is capable of recognizing various structurally unrelated types of damage. To do so, the protein must recognize a common alteration of the DNA structure induced by these different damages. For UvrB sterical hindrance of the present DNA modification prevents it from passing behind a _-hairpin structure present in the protein, thereby conferring damage recognition. Upon stalling of the protein at the site of damage in this way we show that two nucleotides become extrahelical: the nucleotide directly 3__ to the lesion and its base-pairing partner in the non-damaged strand. In contrast to other repair enzymes however the damaged nucleotide itself does not become extrahelical. Flipping in one of the two DNA strands has been shown to be important in preventing stable binding of the protein to undamaged DNA. Flipping in the other DNA strand however is required for efficient 3__ incision by UvrC, the nuclease of the system. A second incision at the 5__ side of the damage by the same protein facilitates removal of the damage-containing DNA oligonucleotide. Show less
Toll-like receptors (TLRs) are receptors that continuously scour their direct surroundings for pathogen associated molecular patterns (PAMPs) of bacterial, viral or fungal origin. TLRs can be found... Show moreToll-like receptors (TLRs) are receptors that continuously scour their direct surroundings for pathogen associated molecular patterns (PAMPs) of bacterial, viral or fungal origin. TLRs can be found at cells that play a role in the immune system. Binding of the TLR with its corresponding ligand results in a signaling cascade, which initially activates the innate immune system and can eventually result in activation of the adaptive immune system. When used as well-defined adjuvants, their combination with antigen may increase the immunogenicity of the antigen itself. This so-called vaccine principle may lead to a desired long-lasting adaptive immune response or humoral memory. Currently a total of 11 TLRs are known in humans and their specific PAMPs are directly related to the location where the receptor resides. TLRs 1, 2, 4, 5, 6, 10 and 11 are situated on the outside of the cell membrane, where they come into direct contact with membrane components of pathogens. TLRs 3, 7, 8 and 9 can be found intracellularly and their ligands mainly consist of viral components released after cellular uptake and degradation of the invader. A large diversity of natural and synthetic TLR ligands is available. The newest generation of vaccines is based on synthetic TLR ligands having a well-defined chemical structure. The research described in this thesis is a first step towards the development of well-defined vaccines, able to trigger a controlled immune response by means of using TLR 2, 7 or 9. Discussed in detail are the synthesis and the immunological evaluation of ligands and conjugates aimed at activation of Toll-like receptors 2, 7 and 9. Some examples are the synthesis of a CpG DNA-peptide conjugate (TLR 9), a Pam3Cys-peptide conjugate (TLR 2) and a 7-hydro-8-oxo-adenine-peptide conjugate (TLR 7). After synthesis of the peptide (containing the antigen), several methods were applied to conjugate it to TLR2, 7 and 9 ligands. Resulting from the research performed, is that when well chosen, covalent coupling of a TLR ligand with an antigenic peptide results in an increase of cytokine production as well as antigen presentation, two of the most desired characteristics of a synthetic vaccine of this class. It is not unlikely, that future synthetic vaccines will be sharing these properties. Show less
This thesis focuses on bridging the gap between natural and artificial systems by the structural and structure-function characterization of two kinds of natural photosynthetic antenna systems, a... Show moreThis thesis focuses on bridging the gap between natural and artificial systems by the structural and structure-function characterization of two kinds of natural photosynthetic antenna systems, a pigment-protein complex i.e. the LH2 complex, and the protein-free chlorosome supramolecular light harvesters. Chlorosomes contain the largest numbers of chromophores for any antenna system known in nature and are very efficient ultra-fast light harvesters. They provide an optimal starting point for a novel class of artificial antenna arrays for ultra-rapid feeding of energy into photocatalytic devices. Show less
The thesis project is devoted to the __search of antineoplastic ruthenium/platinum/copper complexes containing also intercalator ligands; such intercalators may show efficient DNA-cleaving and DNA... Show moreThe thesis project is devoted to the __search of antineoplastic ruthenium/platinum/copper complexes containing also intercalator ligands; such intercalators may show efficient DNA-cleaving and DNA-binding properties__. The studies have been carried out using a variety of techniques. Study of the in vitro cytotoxicity against human tumour cell lines along with cellular uptake has been the pivotal point of this research. Synthetic methodologies of the new complexes have been designed to establish structure-activity relation in several series of related complexes. Platinum complexes of derivatized-phenanthroline, pyridine and pyrimidine, have been synthesized and studied in detail for their biological activity. These complexes are different in structure and in overall charges, and were studied to elucidate the effects on the activity profile. A self-activating copper-complex formed from a unique amino-phenol ligand (Hpyramol) inspired the synthesis of platinum and ruthenium analogues. Biological studies including cellular uptake, conformational changes (CD and UV) and DNA cleavage have been performed to interpret the changes in activity profile upon various metal additions. Ruthenium complexes are known as suitable candidates for anticancer (specially as antimetastatic drugs) agents. A group of ruthenium(III) complexes has been studied for their anticancer activity and their DNA-binding properties. The extensive area of homo- or heterometallic dinuclear compounds opens up an interesting challenge towards the synthesis of complexes. The studies of different combinations viz., Ru-Ru, Cu-Cu and Pt-Pt, have been performed to design new series of anticancer complexes. Show less
The design of novel anticancer agents is one of the most active fields in Medicinal Chemistry, as the number of effective drugs for treatment of cancer is still very limited. This demand for new... Show moreThe design of novel anticancer agents is one of the most active fields in Medicinal Chemistry, as the number of effective drugs for treatment of cancer is still very limited. This demand for new drugs is even higher, considering the high cancer-prevalence rate in our society. The therapeutic application of metal complexes is an under-developed area of research and basic principles in the development of metallopharmaceuticals are lacking, or at least just recently being discovered. Metal-containing agents may offer unique therapeutic opportunities. However, significant obstacles, including potential metal accumulations and toxicities, require further research before a promising metal compound may be introduced in the clinic. In particular several ruthenium and gold coordination compounds have shown promising application as anticancer agents. In these terms, this thesis project, performed in the Coordination and Bioinorganic Chemistry group in the Leiden Institute of Chemistry, comprises the design, synthesis, detailed characterization (i.e. elemental analysis, UV-Vis spectroscopy, IR, far-IR, NMR, mass spectroscopy and X-ray single-crystal structure determination) and also the biological evaluation of novel gold compounds and ruthenium compounds. The promising cytotoxic activity developed for several of these compounds and the findings from this research are providing a better understanding of chemistry of Ru(III) and Ru(II) and Au(III) coordination compounds and their structure-activity relationships and may lead to the development of improved ruthenium and gold-chemotherapeutic drugs. Show less
Photochemically induced dynamic nuclear polarization (photo-CIDNP) is non-Boltzmann nuclear magnetization which can be observed by magic angle spinning NMR spectroscopy as enhanced absorptive or... Show morePhotochemically induced dynamic nuclear polarization (photo-CIDNP) is non-Boltzmann nuclear magnetization which can be observed by magic angle spinning NMR spectroscopy as enhanced absorptive or emissive signals. In solids, photo-CIDNP has been observed since its discovery in 1994 in various photosynthetic reaction centers. In natural photosynthetic charge separation, electron-electron interactions are fine-tuned to lead to highly efficient electron transfer. Nanosecond laser-flash photo-CIDNP magic-angle spinning NMR allows for determination of the nuclear polarizations and hyperfine interactions with atomic selectivity and with a resolution of a few microseconds. The build-up of nuclear polarization in reaction centers of Rhodobacter sphaeroides is found to depend on the presence and lifetimes of the molecular triplet states of the donor and carotenoid. Time-resolved 13C photo-CIDNP MAS NMR spectroscopy is used to map the electronic structure of the donor. In the dark state, maximum electron density is localized in the center of the special pair. In contrast, in the light state, the maximum of the electron spin density is localized at the periphery of the two cofactors. The balance of electron spin density between the two bacteriochlorophyll cofactors is shifted in favor of the L branch of the protein by the ratio of 7:3. We show that the asymmetry is induced by both geometric differences between the two cofactors and non-covalent interactions with the protein. Show less
This thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact... Show moreThis thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact that the absorption spectrum of the protein__s active site varies upon changing its redox state. This change can be translated into a change in the fluorescence intensity of a label that is covalently linked to the protein on the basis of F_rster Resonance Energy Transfer (FRET). With our method we could show that different redox proteins and enzymes can be studied down to the single molecule level. This exciting finding opens the door to the study of various redox enzymes and to monitor specific substances such as for example nitrite. Depending on the function of the enzyme under investigation a wide range of substrates can be monitored. Another example is the development of an oxygen sensor by employing proteins that are capable of binding oxygen. The findings presented in this thesis might be significant for applications in oxygen sensing and, more generally, in the fast growing field of biosensing Show less
The biological processes that are the basis of all life forms are mediated largely by protein-protein interactions. The protein complexes involved in these interactions can be categorised by their... Show moreThe biological processes that are the basis of all life forms are mediated largely by protein-protein interactions. The protein complexes involved in these interactions can be categorised by their affinity, which results in a range from static to transient complexes. Electron transfer complexes, which have to combine high turn-over with specificity are typically transient complexes. The transient complexes under investigation in this work are all part of the photosynthetic redox chain, in which electrons are transferred from membrane bound cytochrome f (cytf) to either plastocyanin (Pc) or cytochrome c6 (cytc6). This work describes the investigation of several aspects of transient protein-protein interactions that are applicable to many other transient complexes. The use of NMR as a tool of choice for these studies has proven it an invaluable technique that provides insight in rules that govern the world of transient complex formation. New approaches such as PRE and the creation of ensembles of structures are investigated. They will hopefully lead to a full understanding of dynamics and the variety of ways that transient complexes use dynamics to perform their function in the cell. Show less
Sodium alanate (NaAlH4) is a prototype system for storage of hydrogen in chemical form. However, a key experimental finding, that early transition metals (TMs) like Ti, Zr, and Sc are good... Show moreSodium alanate (NaAlH4) is a prototype system for storage of hydrogen in chemical form. However, a key experimental finding, that early transition metals (TMs) like Ti, Zr, and Sc are good catalysts for hydrogen release and re-uptake, while traditional hydrogenation catalysts like Pd and Pt are poor catalysts for NaAlH4, has so far gone unexplained. We have performed density functional theory calculations at the PW91 generalised gradient approximation level on Ti, Zr, Sc, Pd, and Pt interacting with the (001) surface of nanocrystalline NaAlH4, employing a cluster model of the complex metal hydride. A key difference between Ti, Zr, and Sc on the one hand, and Pd and Pt on the other hand is that exchange of the early TM atoms with a surface Na ion, whereby Na is pushed on to the surface, is energetically preferred over surface absorption in an interstitial site, as found for Pd and Pt. The theoretical findings are consistent with a crucial feature of the TM catalyst being that it can be transported with the reaction boundary as it moves into the bulk, enabling the starting material to react away while the catalyst eats its way into the bulk, and effecting a phase separation between a Na-rich and a Al-rich phase. In addition, the role of different active species such as Ti2 and TiH2 has been studied using the same model. The results imply that Ti2 and TiH2 are more stable in the subsurface region of the cluster than on the surface. Calculations were performed on the decomposition of two calcium alanates, to determine zero-point energy corrected enthalpies of dehydrogenation for these compounds, and to determine whether destabilization of LiBH4 by CaH2 might improve the performance of this material. Show less
