NMR and in silico studies of fucosylated chondroitin sulfate (fCS) and its interactions with selectins

This thesis describes structural studies on the interactions between the fucosylated chondroitin sulfate (fCS) oligosaccharides and human proteins known as selectins. fCS is a carbohydrate obtained from sea cucumbers, that can be classified as a branched glycosaminoglycan (GAG). It has attracted much attention due to its anti-coagulant, anti-inflammatory, antimetastatic and anti-HIV properties and its structure was previously determined by NMR. Selectins constitute a family of proteins involved in cell adhesion processes, such as inflammation, attachment of viral particles and migration of tumour cells. fCS oligosaccharides have been shown to bind to selectins, which is likely a reason behind their biological activity. However, the mechanism of this interaction is currently unknown. The initial part of the thesis describes the experimental work on expression and purification of the recombinant L- and P-selectin constructs in Pichia pastoris, Escherichia coli and HEK 293 cells. The aim of these experiments was to produce two constructs for each selectin, a single domain construct, consisting of the C-type lectin domain only, and a double domain construct, consisting of both the C-type lectin and the EGF-like domains. The intention was that the recombinant proteins would be labelled with 13C and 15N to allow for the in-depth structural NMR studies on the fCS-selectin interaction. Various experimental approaches have been explored, including the use of different cell lines, modifications to construct design, as well as alterations to expression and purification conditions. Although it was not possible to produce soluble selectin constructs in either bacterial or yeast cells, protein expression tests in HEK293 cells, performed in collaboration with the Oxford Protein Production facility (OPPF), led to production of a soluble L-selectin construct, consisting of the L-selectin C-type lectin domain. The produced L-selectin construct, as well as two commercially available constructs of the Land P-selectin extracellular domains, were used in the Saturation Transfer Difference (STD) NMR experiments to provide new information about the nature of the fCS-selectin binding. The STD experiments allowed to identify the regions within the fCS oligosaccharides that are in direct contact with the protein and likely play an important role in this interaction. Experiments on different protein constructs allowed the comparison of fCS binding to P-selectin and to two different recombinant constructs of L-selectin. Results of these studies suggest that the binding occurs via a similar mechanism for both L- and P-selectins and that the fCS oligosaccharides bind to one-domain L-selectin construct with similar affinity as to a larger construct, consisting of the entire extracellular region of the protein. Alongside the experimental work, theoretical in silico studies on the fCS-selectin binding were undertaken as part of this project. The existing X-ray structures of selectin complexes were subjected to Molecular Dynamics (MD) simulations, which allowed to explore the dynamic behaviour of E-selectin upon binding to sialyl Lewis x (sLex). It was found that sLex forms a more favourable interaction with the extended conformation of E-selectin and that the protein in this conformation is characterised by a high degree of interdomain flexibility, with a new type of interdomain movement observed in the MD studies on this complex. In further in silico studies, the fCS oligosaccharides were docked to the existing P-selectin structures. The docking tests were performed on the computationally produced fCS trisaccharides with fucose branches either 2,4 or 3,4-sulfated. Results were evaluated with MD simulations and analysed in the light of current knowledge of selectin-ligand binding and the STD NMR experimental results. The in silico studies allowed to identify a subset of P-selectin residues that are likely involved in the interaction with fCS oligosaccharides in vivo. The conformational behaviour of P-selectin upon binding to fCS was also explored and it was found that the interdomain hinge is flexible during this interaction and allows transition from bent to extended conformational state. Finally, a new NMR method was developed to facilitate the studies of complex carbohydrates, incorporating the concepts of G-matrix Fourier Transform (GFT) NMR into 2D HSQC and 2D HSQC-TOCSY experiments. The method allows to separate peaks in the regions of high spectral overlap, providing information that can simplify the assignment process. The new experiments facilitated the structural evaluation of a sample containing a mixture of oligosaccharides resulting from the depolymerisation of fCS polysaccharide

XXV. mezinárodní kolokvium o regionálních vědách

Title in English: 25th International Colloquium on Regional Sciences: Conference proceedings The conference proceedings consists of papers presented at the 25th International Colloquium on Regional Sciences that was organized by Department of Regional Economics and Administration FEA MU. It contains 57 articles arranged by topic. The individual articles deal with e.g. socioeconomic disparities among regions, regional policy, territory attractiveness, tourism, or regional public administration

Synthesis of nanostructured ZnFe2O4 as a sorbent for H2S removal from syngas

The use of sorption properties of materials as sorbents for removing unwanted substances from gasified solid or liquid fuels (coal, oil, natural gas and biomass) in the heating plant industry seems to be more suitable than existing technology. In this diploma work, ZnFe2O4 is studied as a possible sorbent for the removal of H2S from gasified fuel. ZnFe2O4 has been prepared by various methods in the form of nanoparticles and nanocomposites. The prepared samples were characterized by powder X- ray diffraction, electron microscopy, BET measurement and thermal analysis. Further, the sorption activity of the samples was studied with regard to H2S. The results were evaluated with respect to the possible application of the prepared materials in heating plant industry. Keywords: ZnFe2O4, H2S absorption, sol-gel method, sol-gel auto-combustion method, SBA-15

Causes of Failure of Nation - Building in South Vietnam

The USA started nation-building in South Vietnam in 1954 after the Geneva Agreements. The USA wanted to build a state which would prevent expansion of communism to other states in Asia. This program failed and finally, in 1976 South Vietnam and North Vietnam were reunited into a single communist Vietnam. The goal of this bachelor thesis is to identify factors that caused nation-building in South Vietnam to fail. The causes of failure of nation-building are classified in three main levels - American policy in South Vietnam, South Vietnam and extern factors like the Democratic Republic of Vietnam, Soviet Union and People's Republic of China

Surface modification of silicon oxide

Katedra anorganické chemieDepartment of Inorganic ChemistryFaculty of SciencePřírodovědecká fakult

Microwave-assisted extraction of pectin from unutilized pumpkin biomass

Microwave-assisted extraction was employed to extract polysaccharides from pumpkin biomass and compared to the usual extraction by conventional heating. The effect of microwave heating time, liquid/solid (L/S) ratio and extraction temperature on the yield and molecular mass of extracted pectin from the seeded oil pumpkin biomass was investigated. Heating times ranged from 2 to 10 min, L/S ratio from 30/1 to 50/1 and temperature from 80 to 120 °C. The response surface methodology was used to optimize the effects of processing parameters to isolate pectin with medium molecular weight and with the highest yield. The retained conditions were as follows: liquid/solid ratio 50/1, microwave heating time of 10 min and a temperature of 102.2 °C. Among the studied factors, the liquid/solid ratio had the greatest influence on yield and molecular mass, respectively. Application of microwave heating in the extraction of pumpkin biomass gave a pectin with medium Mw and dramatically reduced extraction time in comparison to traditional hot acid extraction. Microwave is an excellent tool for extraction and modification of polysaccharides in one step

Structural properties of the extracellular biopolymer (β-D-xylo-α-D-mannan) produced by the green microalga Gloeocystis vesiculosa Nägeli.

Many species of microalgae produce a relatively diverse range of metabolites that are interesting for biotechnological applications, and among them exopolysaccharides attract attention due to their structural complexity, biological activities, biodegradability or biocompatibility. An exopolysaccharide of high molecular weight (Mp) of 6.8 × 105 g/mol was obtained by cultivation of the freshwater green coccal microalga Gloeocystis vesiculosa Nägeli 1849 (Chlorophyta). Chemical analyses revealed a dominance of Manp (63.4 wt%), Xylp and its 3-O-Me-derivative (22.4 wt%), and Glcp (11.5 wt%) residues. The results of the chemical and NMR analyses showed an alternating branched 1,2- and 1,3-linked α-D-Manp backbone terminated by a single β-D-Xylp and its 3-O-methyl derivative at O2 of the 1,3-linked α-D-Manp residues. The α-D-Glcp residues were found mainly as 1,4-linked and to a lesser extent as the terminal sugar, indicating partial contamination of β-D-xylo-α-D-mannan with amylose (∼10 wt%) in G. vesiculosa exopolysaccharide