Glycoconjugates (a carbohydrate connected to a lipid, protein or other carbohydrate) play a key role in great variety of biological processes. The synthesis of these constructs is tightly regulated... Show moreGlycoconjugates (a carbohydrate connected to a lipid, protein or other carbohydrate) play a key role in great variety of biological processes. The synthesis of these constructs is tightly regulated by enzymes. Defects in these enzymes may result in an impaired degradation of the glycoconjugate. Consequently, the levels of glycoconjugates are increased and this may eventually lead to storage disorders such as Gaucher disease. The research described in this thesis focuses on the synthesis of chemical tools (activity-based probes, ABPs) to study the enzymes involved in the degradation of glycoconjugates. Using these probes, it was demonstrated that the activity of peptide N-glycanase (the enzyme that is responsible for the hydrolysis of N-linked glycoproteins)inhibitors is determined by its reactive group. Furthermore, the activity-based protein profiling strategy was used to study degradation of glycosy lated proteins. It appeared that deglycosylation of O-GlcNAclated proteins is not a prerequisite for proteasomal degradation. To study beta-glucosidases (enzymes that catalyze the hydrolysis of O-glycosidic linkages), ABPs based on cyclophellitol have been developed. Especially fluorescently labeled probes bind efficiently and selectively to beta-glucosidases. These probes have been used to investigate Gaucher disease. Both wild-type and mutant forms of the enzyme could be labeled in vitro and in living cells which allowed rapid identification activity of this glucosidase. Show less
This thesis described the interfacial properties of curved fluid interfaces mainly employing mean-field models. Investigations of Tolman's length in simple systems and systems in contact with a... Show moreThis thesis described the interfacial properties of curved fluid interfaces mainly employing mean-field models. Investigations of Tolman's length in simple systems and systems in contact with a hard wall are presented. Both the interfacial properties as well as the wetting behavior of phase-separated colloid-polymers are studied. Also the stability of the interface encountered in Pickering emulsions is investigated. Show less
The first half of this thesis describes the synthesis of several conformationally restricted alkylated and bicyclic sugar amino acids (SAAs). The second half of the thesis describes the application... Show moreThe first half of this thesis describes the synthesis of several conformationally restricted alkylated and bicyclic sugar amino acids (SAAs). The second half of the thesis describes the application of the SAAs and their intermediates presented in the first half, as components of tools applied for the probing of biological systems. Show less
With the help of modern techniques of imaging processing and computing, image data obtained by electron cryo-microscopy of biomolecules can be reconstructed to three-dimensional biological models... Show moreWith the help of modern techniques of imaging processing and computing, image data obtained by electron cryo-microscopy of biomolecules can be reconstructed to three-dimensional biological models at sub-nanometer resolution. These models allow answering urgent problems in life science, for instance, the pathways directing the self-recovery system of cell, which certainly has great significance for all our lives. To determine these models, there are two main electron microscopic methods available, corresponding to its two main modes of operation: 3DEM single particle reconstruction and electron diffraction. This thesis focuses on the research and methods of 3DEM and electron diffraction, and its practical application in solving the structure of a 50S ribosomal complex, which clarifies the mechanism of cell recovery in heat shock stress. Preliminary research on a novel structure determination method by using nano-crystals resulted in a novel software suite __ EDiff __ which is a program for unit cell parameter determination, indexing and so on. Show less
As nickel and platinum are in the same group of the periodic table, the Ni(111) and Pt(111) surfaces may be expected to show similar interaction with water and hydrogen. However in this thesis, we... Show moreAs nickel and platinum are in the same group of the periodic table, the Ni(111) and Pt(111) surfaces may be expected to show similar interaction with water and hydrogen. However in this thesis, we show these interactions for Ni(111) are quite different from those of Pt(111). Moreover, our results show that the Ni(111) surface is a unique surface with regards to its chemistry of water and hydrogen. Show less
The main goal of the research presented in this thesis is the synthesis of suitable structural and functional models for the enzyme [NiFe] hydrogenase, which can reduce protons into dihydrogen. A... Show moreThe main goal of the research presented in this thesis is the synthesis of suitable structural and functional models for the enzyme [NiFe] hydrogenase, which can reduce protons into dihydrogen. A brief survey of the roles of all the known nickel containing enzymes in biological systems with a focus on the [NiFe] hydrogenases. Structure, function, physicochemical and catalytic properties of the [NiFe] hydrogenase itself and of the reported model complexes are presented. Many new Nickel, [NiFe], [NiRu] and [NiCu] complexes have been synthesized and studied in view of better catalysts for proton electroreduction into dihydrogen. Show less
Plants, algae and bacteria respond to light in various manners. The effect of light on the growth of plants is called photomorphogenesis and is regulated by the photoreceptor protein named... Show morePlants, algae and bacteria respond to light in various manners. The effect of light on the growth of plants is called photomorphogenesis and is regulated by the photoreceptor protein named phytochrome. Phytochrome is formed in the dark in its inactive red-absorbing (Pr) state and transformed upon absorption of red light to its physiologically active far-red-absorbing state called Pfr. This switching process is linked to a Z-to-E photoisomerization of its open-chain tetrapyrrole cofactor and is transduced to the protein surface modifying the interaction with other proteins in the signal chain. 1H, 15N and 13C cross polarization MAS NMR was used to investigate at atomic resolution both the Pr and Pfr states as well as the two intermediates of the Pfr-to-Pr reaction, Lumi-F and Meta-F. The Pfr state is characterized by a strong hydrogen-bonding at the carbonyl of ring D which leads to an increase in length and strength of the conjugation of the pi-system. The Pfr-to-Pr conversion occurs in two steps: the C15=C16 double bond is photoisomerized in Lumi-F, the rotation around the C14-C15 single bond takes place during the formation of Meta-F. The signal transduction is linked to a change of hydrogen-bonding interaction at the ring D nitrogen with the protein environment. Show less
Described in this thesis are the investigations into the chemistry of N-heterocyclic carbene (NHC) ligands and transition-metal complexes thereof. Specifically, a variety of N-heterocyclic carbene... Show moreDescribed in this thesis are the investigations into the chemistry of N-heterocyclic carbene (NHC) ligands and transition-metal complexes thereof. Specifically, a variety of N-heterocyclic carbene complexes of nickel were prepared, characterized and used as catalysts in three types of homogeneous catalytic processes. First, nickel(II) complexes of monodentate NHC ligands were successfully used as catalysts in the hydrosilylation of internal alkynes. Second, nickel(II) complexes bearing bidentate N-heterocyclic carbene ligands or functionalized NHC ligands were shown to be highly effective in the Kumada coupling of aryl halides with aryl Grignard reagents. In addition, the nickel-catalyzed Kumada coupling was investigated using quantum-chemical calculations. Finally, all three types of nickel complex were tested for their catalytic activity in the vinyl polymerization of norbornene. Furthermore, a silv er(I) NHC complex was isolated, characterized and used in the preparation of a nickel(II) NHC complex. Show less
This thesis contains the results of imaging of adult zebrafish by using different MR approaches. We present the first high resolution mMR images of adult zebrafish. To achieve high spatial... Show moreThis thesis contains the results of imaging of adult zebrafish by using different MR approaches. We present the first high resolution mMR images of adult zebrafish. To achieve high spatial resolution we used a magnetic field of 9.4T, in combination with strong magnetic field gradients (1000 mT/m) and specialized radio frequency coils. To support imaging of living fish, we designed a special flow-through setup for continuous flow of aerated water to support living zebrafish inside the magnet. Clear morphological proton images were obtained by T2-weighted RARE sequences revealing many anatomical details in the entire intact zebrafish in vivo. We successfully implemented MRS at 9.4T and obtained for the first time detailed composition of zebrafish brain in vivo. Our results in this thesis suggest that zebrafish brain has similar metabolite profile as the human brain, which proves that zebrafish is a go od model organism to study human brain disorders. This thesis demonstrates also the application of high resolution mMRI methods to track spontaneous tumors in stable transgenic zebrafish models expressing a RAS oncoprotein and lacking P53 (mitf:Ras::mitf:GFP X p53-/-). Tumors were successfully visualized at different locations in live zebrafish. Show less
Membrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug... Show moreMembrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. Using the recently developed Target Immobilized NMR Screening (TINS) approach, we screened 1,200 fragments for binding to the enzyme Disulphide bond forming protein B. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 uM, which could be categorized as cofactor binding inhibitors or mixed model inhibitors of both cofactor and substrate protein interaction. Our results establish the utility of fragment-based methods in the development of inhibitors of membrane proteins, making a wide variety3of important membrane bound pharmaceutical targets amenable to such an approach. We first tested the immobilization procedure on G protein coupled receptors and ion channels. Furthermore, we used Nanodiscs, an alternative solubilization strategy, to solubilize teh protein without detergents. This resulted in less broad NMR signals, less non-specific binding issues, and identification of the binders from the original screen, proving that the nanodisc solubilization technique is compatible with TINS. Show less
In this thesis new fluorescent platinum(II) compounds are presented. All these new compounds were designed to overcome the cisplatin-resistance, using bulky ligands surrounding the platinum(II) ion... Show moreIn this thesis new fluorescent platinum(II) compounds are presented. All these new compounds were designed to overcome the cisplatin-resistance, using bulky ligands surrounding the platinum(II) ion. To study their biological activity two cell lines were chosen, the human ovarian carcinoma sensitive to cisplatin (A2780), and its resistant counterpart (A2780R). This thesis is divided in two parts: Part I: New fluorescent platinum(II) compounds containing 9-anthryl pyridyl enone as carrier ligand (chapters 2,3 and 4), and Part II: A new fluorescent platinum(II) compound containing N,N__-bis-(anthracen-9-ylmethyl)propane-1,3-diamine as a carrier ligand (chapter 5, 6 and 7). Show less
In this PhD thesis, I have described the novel time-resolved sum-frequency generation (TR-SFG) spectroscopic technique that I developed during the course of my PhD research and used it study the... Show moreIn this PhD thesis, I have described the novel time-resolved sum-frequency generation (TR-SFG) spectroscopic technique that I developed during the course of my PhD research and used it study the ultrafast vibrational, structural and orientational dynamics of water molecules at model biological membranes - key towards understanding the dynamic hydrogen-bonded structure of water interfacial with model biological membranes. The TR-SFG technique developed, follows a pump-probe experimental scheme whereby an intense IR laser pulse excites molecular vibrations and the Sum Frequency Generation (SFG) pulse is used to probe the dynamics of surface molecules as they relax back to the ground state, as a function of the time delay between the excitation and probe pulses. The rate and mechanism of vibrational relaxation (lifetime dynamics) helps in understanding the effects of local molecular structure and hydrogen bonding around these surface molecules. Show less
Verschillende biologische systemen, zoals het 50S ribosomale Hsp15 complex, een schistosomiasis diagnostisch antigen bindend antilichaam fragment (Fab 54-5C10-A), het zuurstof producerende enzym... Show moreVerschillende biologische systemen, zoals het 50S ribosomale Hsp15 complex, een schistosomiasis diagnostisch antigen bindend antilichaam fragment (Fab 54-5C10-A), het zuurstof producerende enzym chloriet dismutase (Cld) en het belangrijke regulatoreiwit van microtubule dynamiek (EB1) zijn onderzocht met geavanceerde biochemische technieken. De structurele informatie verkregen met electronenmicroscopie (EM), kleine hoek verstrooiing van R_ntgenstraling (SAXS) en R_ntgendiffractie aan individuele eiwitkristallen werd aangevuld met informatie uit andere biofysische en biochemische methoden, zoals kolom chromatografie, gel elektroforese, oppervlakte plasmon resonantie (SPR), massa spectrometrie (MS) en electron paramagnetische resonantie (EPR). Hoewel de in dit proefschrift beschreven biologische systemen uiteenlopen wat betreft chemische samenstelling, oorsprong en biologische functie, is er juist ook gekeken naar een gemeenschappelijk kenmerk: ge_nduceerde conformatieveranderingen binnen het eiwit, eiwit-RNA of eiwitcarbohydraat complex. Show less
In this thesis new strategies towards biologically active oligosaccharides are described. In addition a detailed mechanistic study is performed to investigate the stereodirecting capacity of... Show moreIn this thesis new strategies towards biologically active oligosaccharides are described. In addition a detailed mechanistic study is performed to investigate the stereodirecting capacity of glycosyl C-5 substituents in systems that were devoid of any other stereodirecting factors. The postulated mechanism described here can aid in the design of glycosylation strategies. Show less
The interactions between proteins are of central importance for virtually every process in a living cell. It has long been a mystery how two proteins associate to form a complex in a complicated... Show moreThe interactions between proteins are of central importance for virtually every process in a living cell. It has long been a mystery how two proteins associate to form a complex in a complicated cellular context. Recently, it was found that an intermediate state called encounter state, of a protein complex, exists briefly before a final protein complex is formed. In the encounter state, one protein is rolling over on the surface of its partner, searching for the optimal orientation. In my PhD thesis, a transient electron transfer complex formed between a heme protein and an iron-sulfur protein was found to be trapped in this intermediate state, existing as a pure encounter complex. Thus, characterization of this dynamic complex by nuclear magnetic resonance spectroscopy advances our understanding of the general mechanism of protein-protein interaction Show less
