Biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core

Lipopolysacharide (LPS) present on the outer leaflet of Gram-negative bacteria is important for the adaptation of the bacteria to the environment. Structurally, LPS can be divided into three parts: lipid A, core and O-polysaccharide (OPS). OPS is the outermost and also the most diverse moiety. When OPS is composed of identical sugar residues it is called homopolymeric and when it is composed of repeating units of oligosaccharides it is called heteropolymeric. Bacteria synthesize LPS at the inner membrane via two separate pathways, Lipid A-core via one and OPS via the other. These are ligated together in the periplasmic space and the completed LPS molecule is translocated to the surface of the bacteria. The genes directing the OPS biosynthesis are often clustered and the clusters directing the biosynthesis of heteropolymeric OPS often contain genes for i) the biosynthesis of required NDP-sugar precursors, ii) glycosyltransferases needed to build up the repeating unit, iii) translocation of the completed O-unit to the periplasmic side of the inner membrane (flippase) and iv) polymerization of the repeating units to complete OPS. The aim of this thesis was to characterize the biosynthesis of the outer core (OC) of Yersinia enterocolitica serotype O:3 (YeO3). Y. enterocolitica is a member of the Gram-negative Yersinia genus and it causes diarrhea followed sometimes by reactive arthritis. The chemical structure of the OC and the nucleotide sequence of the gene cluster directing its biosynthesis were already known; however, no experimental evidence had been provided for the predicted functions of the gene products. The hypothesis was that the OC biosynthesis would follow the pathway described for heteropolymeric OPS, i.e. a Wzy-dependent pathway. In this work the biochemical activities of two enzymes involved in the NDP-sugar biosynthesis was established. Gne was determined to be a UDP-N-acetylglucosamine-4-epimerase catalyzing the conversion of UDP-GlcNAc to UDP-GalNAc and WbcP was shown to be a UDP-GlcNAc- 4,6-dehydratase catalyzing the reaction that converts UDP-GlcNAc to a rare UDP-2-acetamido- 2,6-dideoxy-d-xylo-hex-4-ulopyranose (UDP-Sugp). In this work, the linkage specificities and the order in which the different glycosyltransferases build up the OC onto the lipid carrier were also investigated. In addition, by using a site-directed mutagenesis approach the catalytically important amino acids of Gne and two of the characterized glycosyltranferases were identified. Also evidence to show the enzymes involved in the ligations of OC and OPS to the lipid A inner core was provided. The importance of the OC to the physiology of Y. enterocolitica O:3 was defined by determining the minimum requirements for the OC to be recognized by a bacteriophage, bacteriocin and monoclonal antibody. The biological importance of the rare keto sugar (Sugp) was also shown. As a conclusion this work provides an extensive overview of the biosynthesis of YeO3 OC as it provides a substantial amount of information of the stepwise and coordinated synthesis of the Ye O:3 OC hexasaccharide and detailed information of its properties as a receptor.

Viral and cellular modulation of virus-induced apoptosis in experimental infection models

The aim of this study was to investigate herpes simplex virus type 1 (HSV-1)- and measles virus (MV)-induced cell death. HSV-1 with deletion in genes encoding infected cell protein (ICP)4 and protein kinase Us3 (d120) induced apoptosis and cathepsin activation in epithelial (HEp-2) and monocytic (U937) cells. Inhibition of cathepsin activity decreased the amount of d120-induced apoptosis indicating that d120-induced apoptosis could be cathepsin-mediated. Also, HSV-1 infection increased caspase activation suggesting that d120-induced apoptosis is probably caspase-mediated. Cystatin treatment decreased the activity of cathepsins and the replication of HSV-1 indicating that cathepsins contribute to HSV-1 infection. Interestingly, d120 induced also necroptosis in monocytic cells. This is the first report on necroptosis in HSV-1- infected cells. MV induced apoptosis in uninfected bystander T lymphocytes, probably via interaction of MV-infected monocytes with uninfected lymphocytes. The expression of death receptor Fas was clearly increased on the surface of lymphocytes. The number of apoptotic cells and the activation of cathepsins and caspases were increased in MVinfected U937 cells suggesting that MV-induced apoptosis could be cathepsin- and caspase-mediated. Cystatin treatment inhibited cathepsin activities but not MV-induced apoptosis. Besides HSV-1-induced apoptosis, innate immune responses were studied in HSV-1-infection. HSV-1 viruses with either ICP4 and Us3, or Us3 deletion only, increased the expression of Toll-like receptor (TLR)3 and stimulated its downstream pathways leading to increased expression of type I interferon gene and to functional interferons. These findings suggest that besides controlling apoptosis, HSV-1 ICP4 and Us3 genes are involved in the control of TLR3 response in infected cell.

Online Ultrasound Measurements of Membrane Compaction

There are several filtration applications in the pulp and paper industry where the capacity and cost-effectiveness of processes are of importance. Ultrafiltration is used to clean process water. Ultrafiltration is a membrane process that separates a certain component or compound from a liquid stream. The pressure difference across the membrane sieves macromolecules smaller than 0.001-0.02 μm through the membrane. When optimizing the filtration process capacity, online information about the conditions of the membrane is needed. Fouling and compaction of the membrane both affect the capacity of the filtration process. In fouling a “cake” layer starts to build on the surface of the membrane. This layer blocks the molecules from sieving through the membrane thereby decreasing the yield of the process. In compaction of the membrane the structure is flattened out because of the high pressure applied. The higher pressure increases the capacity but may damage the structure of the membrane permanently. Information about the compaction is needed to effectively operate the filters. The objective of this study was to develop an accurate system for online monitoring of the condition of the membrane using ultrasound reflectometry. Measurements of ultrafiltration membrane compaction were made successfully utilizing ultrasound. The results were confirmed by permeate flux decline, measurements of compaction with a micrometer, mechanical compaction using a hydraulic piston and a scanning electron microscope (SEM). The scientific contribution of this thesis is to introduce a secondary ultrasound transducer to determine the speed of sound in the fluid used. The speed of sound is highly dependent on the temperature and pressure used in the filters. When the exact speed of sound is obtained by the reference transducer, the effect of temperature and pressure is eliminated. This speed is then used to calculate the distances with a higher accuracy. As the accuracy or the resolution of the ultrasound measurement is increased, the method can be applied to a higher amount of applications especially for processes where fouling layers are thinner because of smaller macromolecules. With the help of the transducer, membrane compaction of 13 μm was measured in the pressure of 5 bars. The results were verified with the permeate flux decline, which indicated that compaction had taken place. The measurements of compaction with a micrometer showed compaction of 23–26 μm. The results are in the same range and confirm the compaction. Mechanical compaction measurements were made using a hydraulic piston, and the result was the same 13 μm as obtained by applying the ultrasound time domain reflectometry (UTDR). A scanning electron microscope (SEM) was used to study the structure of the samples before and after the compaction.

Structural studies on enzymes of biotechnological and biomedical interest

Structural studies of proteins aim at elucidating the atomic details of molecular interactions in biological processes of living organisms. These studies are particularly important in understanding structure, function and evolution of proteins and in defining their roles in complex biological settings. Furthermore, structural studies can be used for the development of novel properties in biomolecules of environmental, industrial and medical importance. X-ray crystallography is an invaluable tool to obtain accurate and precise information about the structure of proteins at the atomic level. Glutathione transferases (GSTs) are amongst the most versatile enzymes in nature. They are able to catalyze a wide variety of conjugation reactions between glutathione (GSH) and non-polar components containing an electrophilic carbon, nitrogen or sulphur atom. Plant GSTs from the Tau class (a poorly characterized class) play an important role in the detoxification of xenobiotics and stress tolerance. Structural studies were performed on a Tau class fluorodifen-inducible glutathione transferase from Glycine max (GmGSTU4-4) complexed with GSH (2.7 Å) and a product analogue Nb-GSH (1.7 Å). The three-dimensional structure of the GmGSTU4-4-GSH complex revealed that GSH binds in different conformations in the two subunits of the dimer: in an ionized form in one subunit and a non-ionized form in the second subunit. Only the ionized form of the substrate may lead to the formation of a catalytically competent complex. Structural comparison between the GSH and Nb-GSH bound complexes revealed significant differences with respect to the hydrogen-bonding, electrostatic interaction pattern, the upper part of -helix H4 and the C-terminus of the enzyme. These differences indicate an intrasubunit modulation between the G-and Hsites suggesting an induced-fit mechanism of xenobiotic substrate binding. A novel binding site on the surface of the enzyme was also revealed. Bacterial type-II L-asparaginases are used in the treatment of haematopoietic diseases such as acute lymphoblastic leukaemia (ALL) and lymphomas due to their ability to catalyze the conversion of L-asparagine to L-aspartate and ammonia. Escherichia coli and Erwinia chrysanthemi asparaginases are employed for the treatment of ALL for over 30 years. However, serious side-effects affecting the liver and pancreas have been observed due to the intrinsic glutaminase activity of the administered enzymes. Structural studies on Helicobacter pylori L-asparaginase (HpA) were carried out in an effort to discover novel L-asparaginases with potential chemotherapeutic utility in ALL treatment. Detailed analysis of the active site geometry revealed structurally significant differences between HpA and other Lasparaginases that may be important for the biological activities of the enzyme and could be further exploited in protein engineering efforts.

Quantification of Proteins and Cells: Luminometric Nonspecific Particle-Based Methods

New luminometric particle-based methods were developed to quantify protein and to count cells. The developed methods rely on the interaction of the sample with nano- or microparticles and different principles of detection. In fluorescence quenching, timeresolved luminescence resonance energy transfer (TR-LRET), and two-photon excitation fluorescence (TPX) methods, the sample prevents the adsorption of labeled protein to the particles. Depending on the system, the addition of the analyte increases or decreases the luminescence. In the dissociation method, the adsorbed protein protects the Eu(III) chelate on the surface of the particles from dissociation at a low pH. The experimental setups are user-friendly and rapid and do not require hazardous test compounds and elevated temperatures. The sensitivity of the quantification of protein (from 40 to 500 pg bovine serum albumin in a sample) was 20-500-fold better than in most sensitive commercial methods. The quenching method exhibited low protein-to-protein variability and the dissociation method insensitivity to the assay contaminants commonly found in biological samples. Less than ten eukaryotic cells were detected and quantified with all the developed methods under optimized assay conditions. Furthermore, two applications, the method for detection of the aggregation of protein and the cell viability test, were developed by utilizing the TR-LRET method. The detection of the aggregation of protein was allowed at a more than 10,000 times lower concentration, 30 μg/L, compared to the known methods of UV240 absorbance and dynamic light scattering. The TR-LRET method was combined with a nucleic acid assay with cell-impermeable dye to measure the percentage of dead cells in a single tube test with cell counts below 1000 cells/tube.

EDTA- and DTPA-functionalized silica gel and chitosan adsorbents for the removal of heavy metals from aqueous solutions

Adsorbents functionalized with chelating agents are effective in removal of heavy metals from aqueous solutions. Important properties of such adsorbents are high binding affinity as well as regenerability. In this study, aminopolycarboxylic acid, EDTA and DTPA, were immobilized on the surface of silica gel, chitosan, and their hybrid materials to achieve chelating adsorbents for heavy metals such as Co(II), Ni(II), Cd(II), and Pb(II). New knowledge about the adsorption properties of EDTA- and DTPA-functionalizedadsorbents was obtained. Experimental work showed the effectiveness, regenerability, and stability of the studied adsorbents. Both advantages and disadvantages of the adsorbents were evaluated. For example, the EDTA-functionalized chitosan-silica hybrid materials combined the benefits of the silica gel and chitosan while at the same time diminishing their observed drawbacks. Modeling of adsorption kinetics and isotherms is an important step in design process. Therefore, several kinetic and isotherm models were introduced and applied in this work. Important aspects such as effect of error function, data range, initial guess values, and linearization were discussed and investigated. The selection of the most suitable model was conducted by comparing the experimental and simulated data as well as evaluating the correspondence between the theory behind the model and properties of the adsorbent. In addition, modeling of two-component data was conducted using various extended isotherms. Modeling results for both one- and twocomponent systems supported each other. Finally, application testing of EDTA- and DTPA-functionalized adsorbents was conducted. The most important result was the applicability of DTPA-functionalized silica gel and chitosan in the capturing of Co(II) from its aqueous EDTA-chelate. Moreover, these adsorbents were efficient in various solution matrices. In addition, separation of Ni(II) from Co(II) and Ni(II) and Pb(II) from Co(II) and Cd(II) was observed in two- and multimetal systems. Lastly, prior to their analysis, EDTA- and DTPA-functionalized silica gels were successfully used to preconcentrate metal ions from both pure and salty waters

Potkurilaitteen ohjausputken laserpinnoitus

Laserpinnoitus on additiivinen prosessi, jossa lasertehon avulla sulatetaan pinnoitusainetta pinnoitettavan kappaleen pintaan. Laserpinnoituksella on mahdollista aikaansaada tiivis sekä kova, kulutusta ja korroosiota kestävä pinnoite kappaleen pintaan. Tässä työssä kohteena oli potkurilaitteen ohjausputken laserpinnoitus. Kyseisessä kohteessa pinnoitteelta vaaditaan hyvää korroosion- ja kulumisen kestoa. Työn tavoitteena oli lisätä tietoisuutta laserpinnoitusprosessista ja siinä vaikuttavista tekijöistä. Myös materiaaliominaisuuksien vaikutuksia sekä pinnoitus- että pinnoitettavien aineiden osalta selvitettiin. Työhön sisältyi myös kokeellinen osuus, jossa tavoitteena oli löytää kustannustehokkain pinnoiteaine tutkittuun kohteeseen. Kokeissa ohjausputki- sekä levyaihioon tehtiin koepinnoituksia. Kokeiden ja edelleen materiaalitutkimuksien avulla tutkittiin neljää eri pinnoitusainetta, jotka olivat Stellitti 21, Inconel 625, AISI 316L ja AISI 431. Kovuus- ja taivutuskokeiden perusteella kyseisistä aineista vain Stelliitti 21 täytti ohjausputken pinnoitteelle asetetut vaatimukset. Näin ollen Stelliitti 21 valikoitui kustannustehokkaimmaksi pinnoitusaineeksi ohjausputkien laserpinnoitukseen.

Repair of segmental bone defects with fiber-reinforced composite: a study of material development and an animal model on rabbits

The Repair of segmental defects in load-bearing long bones is a challenging task because of the diversity of the load affecting the area; axial, bending, shearing and torsional forces all come together to test the stability/integrity of the bone. The natural biomechanical requirements for bone restorative materials include strength to withstand heavy loads, and adaptivity to conform into a biological environment without disturbing or damaging it. Fiber-reinforced composite (FRC) materials have shown promise, as metals and ceramics have been too rigid, and polymers alone are lacking in strength which is needed for restoration. The versatility of the fiber-reinforced composites also allows tailoring of the composite to meet the multitude of bone properties in the skeleton. The attachment and incorporation of a bone substitute to bone has been advanced by different surface modification methods. Most often this is achieved by the creation of surface texture, which allows bone growth, onto the substitute, creating a mechanical interlocking. Another method is to alter the chemical properties of the surface to create bonding with the bone – for example with a hydroxyapatite (HA) or a bioactive glass (BG) coating. A novel fiber-reinforced composite implant material with a porous surface was developed for bone substitution purposes in load-bearing applications. The material’s biomechanical properties were tailored with unidirectional fiber reinforcement to match the strength of cortical bone. To advance bone growth onto the material, an optimal surface porosity was created by a dissolution process, and an addition of bioactive glass to the material was explored. The effects of dissolution and orientation of the fiber reinforcement were also evaluated for bone-bonding purposes. The Biological response to the implant material was evaluated in a cell culture study to assure the safety of the materials combined. To test the material’s properties in a clinical setting, an animal model was used. A critical-size bone defect in a rabbit’s tibia was used to test the material in a load-bearing application, with short- and long-term follow-up, and a histological evaluation of the incorporation to the host bone. The biomechanical results of the study showed that the material is durable and the tailoring of the properties can be reproduced reliably. The Biological response - ex vivo - to the created surface structure favours the attachment and growth of bone cells, with the additional benefit of bioactive glass appearing on the surface. No toxic reactions to possible agents leaching from the material could be detected in the cell culture study when compared to a nontoxic control material. The mechanical interlocking was enhanced - as expected - with the porosity, whereas the reinforcing fibers protruding from the surface of the implant gave additional strength when tested in a bone-bonding model. Animal experiments verified that the material is capable of withstanding load-bearing conditions in prolonged use without breaking of the material or creating stress shielding effects to the host bone. A Histological examination verified the enhanced incorporation to host bone with an abundance of bone growth onto and over the material. This was achieved with minimal tissue reactions to a foreign body. An FRC implant with surface porosity displays potential in the field of reconstructive surgery, especially regarding large bone defects with high demands on strength and shape retention in load-bearing areas or flat bones such as facial / cranial bones. The benefits of modifying the strength of the material and adjusting the surface properties with fiber reinforcement and bone-bonding additives to meet the requirements of different bone qualities are still to be fully discovered.

Acidic dissolution of iron oxides and regeneration of a ceramic filter medium

The dewatering of iron ore concentrates requires large capacity in addition to producing a cake with low moisture content. Such large processes are commonly energy intensive and means to lower the specific energy consumption are needed. Ceramic capillary action disc filters incorporate a novel filter medium enabling the harnessing of capillary action, which results in decreased energy consumption in comparison to traditional filtration technologies. As another benefit, the filter medium is mechanically and chemically more durable than, for example, filter cloths and can, thus, withstand harsh operating conditions and possible regeneration better than other types of filter media. In iron ore dewatering, the regeneration of the filter medium is done through a combination of several techniques: (1) backwashing, (2) ultrasonic cleaning, and (3) acid regeneration. Although it is commonly acknowledged that the filter medium is affected by slurry particles and extraneous compounds, published research, especially in the field of dewatering of mineral concentrates, is scarce. Whereas the regenerative effect of backwashing and ultrasound are more or less mechanical, regeneration with acids is based on chemistry. The chemistry behind the acid regeneration is, naturally, dissolution. The dissolution of iron oxide particles has been extensively studied over several decades but those studies may not necessarily be directly applicable in the regeneration of the filter medium which has undergone interactions with the slurry components. The aim of this thesis was to investigate if free particle dissolution indeed correlates with the regeneration of the filter medium. For this purpose, both free particle dissolution and dissolution of surface adhered particles were studied. The focus was on acidic dissolution of iron oxide particles and on the study of the ceramic filter medium used in the dewatering of iron ore concentrates. The free particle dissolution experiments show that the solubility of synthetic fine grained iron oxide particles in oxalic acid could be explained through linear models accounting for the effects of temperature and acid concentration, whereas the dissolution of a natural magnetite is not so easily explained by such models. In addition, the kinetic experiments performed both support and contradict the work of previous authors: the suitable kinetic model here supports previous research suggesting solid state reduction to be the reaction mechanism of hematite dissolution but the formation of a stable iron oxalate is not supported by the results of this research. Several other dissolution mechanisms have also been suggested for iron oxide dissolution in oxalic acid, indicating that the details of oxalate promoted reductive dissolution are not yet agreed and, in this respect, this research offers added value to the community. The results of the regeneration experiments with the ceramic filter media show that oxalic acid is highly effective in removing iron oxide particles from the surface of the filter medium. The dissolution of those particles did not, however, exhibit the expected behaviour, i.e. complete dissolution. The results of this thesis show that although the regeneration of the ceramic filter medium with acids incorporates the dissolution of slurry particles from the surface of the filter medium, the regeneration cannot be assessed purely based upon free particle dissolution. A steady state, dependent on temperature and on the acid concentration, was observed in the dissolution of particles from the surface even though the limit of solubility of free iron oxide particles had not been reached. Both the regeneration capacity and efficiency, with regards to the removal of iron oxide particles, was found to be temperature dependent, but was not affected by the acid concentration. This observation further suggests that the removal of the surface adhered particles does not follow the dissolution of free particles, which do exhibit a dependency on the acid concentration. In addition, changes in the permeability and in the pore structure of the filter medium were still observed after the bulk concentration of dissolved iron had reached a steady state. Consequently, the regeneration of the filter medium continued after the dissolution of particles from the surface had ceased. This observation suggests that internal changes take place at the final stages of regeneration. The regeneration process could, in theory, be divided into two, possibly overlapping, stages: (1) dissolution of surface-adhered particles, and (2) dissolution of extraneous compounds from within the pore structure. In addition to the fundamental knowledge generated during this thesis, tools to assess the effects of parameters on the regeneration of the ceramic filter medium are needed. It has become clear that the same tools used to estimate the dissolution of free particles cannot be used to estimate the regeneration of a filter medium unless only a robust characterisation of the order of regeneration efficiency is needed.

Development of porous glass-fiber reinforced composite for bone implants. Evaluation of antimicrobial effect and implant fixation

Cranial bone reconstructions are necessary for correcting large skull bone defects due to trauma, tumors, infections and craniotomies. Traditional synthetic implant materials include solid or mesh titanium, various plastics and ceramics. Recently, biostable glass-fiber reinforced composites (FRC), which are based on bifunctional methacrylate resin, were introduced as novel implant solution. FRCs were originally developed and clinically used in dental applications. As a result of further in vitro and in vivo testing, these composites were also approved for clinical use in cranial surgery. To date, reconstructions of large bone defects were performed in 35 patients. This thesis is dedicated to the development of a novel FRC-based implant for cranial reconstructions. The proposed multi-component implant consists of three main parts: (i) porous FRC structure; (ii) bioactive glass granules embedded between FRC layers and (iii) a silver-polysaccharide nanocomposite coating. The porosity of the FRC structure should allow bone ingrowth. Bioactive glass as an osteopromotive material is expected to stimulate the formation of new bone. The polysaccharide coating is expected to prevent bacterial colonization of the implant. The FRC implants developed in this study are based on the porous network of randomly-oriented E-glass fibers bound together by non-resorbable photopolymerizable methacrylate resin. These structures had a total porosity of 10–70 volume %, of which > 70% were open pores. The pore sizes > 100 μm were in the biologically-relevant range (50-400 μm), which is essential for vascularization and bone ingrowth. Bone ingrowth into these structures was simulated by imbedding of porous FRC specimens in gypsum. Results of push-out tests indicated the increase in the shear strength and fracture toughness of the interface with the increase in the total porosity of FRC specimens. The osteopromotive effect of bioactive glass is based on its dissolution in the physiological environment. Here, calcium and phosphate ions, released from the glass, precipitated on the glass surface and its proximity (the FRC) and formed bone-like apatite. The biomineralization of the FRC structure, due to the bioactive glass reactions, was studied in Simulated Body Fluid (SBF) in static and dynamic conditions. An antimicrobial, non-cytotoxic polysaccharide coating, containing silver nanoparticles, was obtained through strong electrostatic interactions with the surface of FRC. In in vitro conditions the lactose-modified chitosan (chitlac) coating showed no signs of degradation within seven days of exposure to lysozyme or one day to hydrogen peroxide (H2O2). The antimicrobial efficacy of the coating was tested against Staphylococcus aureus and Pseudomonas aeruginosa. The contact-active coating had an excellent short time antimicrobial effect. The coating neither affected the initial adhesion of microorganisms to the implant surface nor the biofilm formation after 24 h and 72 h of incubation. Silver ions released to the aqueous environment led to a reduction of bacterial growth in the culture medium.

Topochemistry of physical and chemical pretreatments of biomass as investigated by FE-SEM, XPS and ToF-SIMS

Surface chemistry is of great importance in plant biomass engineering and applications. The surface chemical composition of biomass which includes lignin, carbohydrates and extractives influences its interactions with chemical agents, such as pulp processing/papermaking chemicals, or enzymes for different purposes. In this thesis, the changes in the surface chemical composition of lignocellulosic biomass after physical modification for the improvement of resulting paper properties and chemical treatment for the enhancement of enzymatic hydrolysis were investigated. Low consistency (LC) refining was used as physical treatment of bleached softwood and hardwood pulp samples, and the surface chemistry of refined samples was investigated. The refined pulp was analysed as whole pulp while the fines-free fibre samples were characterized separately. The fines produced in LCrefining contributed to an enlarged surface specific area as well as the change of surface coverage by lignin and extractives, as investigated by X-ray photoelectron spectroscopy (XPS). The surface coverage by lignin of the whole pulp decreased after refining while the surface coverage by extractives increased both for pine and eucalyptus. In the case of pine, the removal of fines resulted in reduction of the surface coverage by extractives, while the surface coverage by lignin increased on fibre sample (without fines). In the case of eucalyptus, the surface coverage by lignin of fibre samples decreased after the removal of fines. In addition, the surface distribution of carbohydrates, lignin and extractives of pine and eucalyptus samples was determined by Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). LC-refining increased the amounts of pentose, hexose and extractives on the surface of pine samples. ToF-SIMS also gave clear evidence about xylan deposition and reduction of surface lignin distribution on the fibre of eucalyptus. However, the changes in the surface chemical composition during the physical treatment has led to an increase in the adsorption of fluorescent whitening agents (FWAs) on fibres due to a combination of electro-static forces, specific surface area of fibres and hydrophobic interactions. Various physicochemical pretreatments were conducted on wood and non-wood biomass for enhancing enzymatic hydrolysis of polysaccharides, and the surface chemistry of the pretreated and enzymatically hydrolysed samples was investigated by field emission scanning electron microscopy (FE-SEM), XPS and ToF-SIMS. A hydrotrope was used as a relatively novel pretreatment technology both in the case of wood and non-wood biomass. For comparison, ionic liquid and hydrothermal pretreatments were applied on softwood and hardwood as well. Thus, XPS analysis showed that the surface lignin was more efficiently removed by hydrotropic pretreatment compared to ionic liquid or hydrothermal pretreatments. SEM analysis also found that already at room temperature the ionic liquid pretreatments were more effective in swelling the fibres compared with hydrotropic pretreatment at elevated temperatures. The enzymatic hydrolysis yield of hardwood was enhanced due to the decrease in surface coverage of lignin, which was induced by hydrotropic treatment. However, hydrotropic pretreatment was not appropriate for softwood because of the predominance of guaiacyl lignin structure in this material. In addition, the reduction of surface lignin and xylan during pretreatment and subsequent increase in cellulose hydrolysis by enzyme could be observed from ToF-SIMS results. The characterisation of the non-wood biomass (e.g. sugarcane bagasse and common reed) treated by hydrotropic method, alkaline and alkaline hydrogen peroxide pretreatments were carried out by XPS and ToF-SIMS. According to the results, the action for the removal of the surface lignin of non-wood biomass by hydrotropic pretreatment was more significant compared to alkaline and alkaline hydrogen peroxide pretreatments, although a higher total amount of lignin could be removed by alkaline and alkaline hydrogen peroxide pretreatment. Furthermore, xylan could be remarkably more efficiently removed by hydrotropic method. Therefore, the glucan yield achieved from hydrotropic treated sample was higher than that from samples treated with alkaline or alkaline hydrogen peroxide. Through the use of ToF-SIMS, the distribution and localization of lignin and carbohydrates on the surface of ignocelluloses during pretreatment and enzymatic hydrolysis could be detected, and xylan degradation during enzymatic hydrolysis could also be assessed. Thus, based on the results from XPS and ToF-SIMS, the mechanism of the hydrotropic pretreatment in improving the accessibility of enzymes to fibre and further ameliorating of the enzymatic saccharification could be better elucidated.

Chemical derivatization of galactoglucomannan for functional materials

In the framework of the biorefinery concept researchers aspire to optimize the utilization of plant materials, such as agricultural wastes and wood. For most of the known processes, the first steps in the valorisation of biomass are the extraction and purification of the individual components. The obtained raw products by means of a controlled separation can consecutively be modified to result in biofuels or biogas for energy production, but also in value-added products such as additives and important building blocks for the chemical and material industries. Considerable efforts are undertaken in order to substitute the use of oil-based starting materials or at least minimize their processing for the production of everyday goods. Wood is one of the raw materials, which have gained large attention in the last decades and its composition has been studied in detail. Nowadays, the extraction of water-soluble hemicelluloses from wood is well known and so for example xylan can be obtained from hardwoods and O-acetyl galactoglucomannans (GGMs) from softwoods. The aim of this work was to develop water-soluble amphiphilic materials of GGM and to assess their potential use as additives. Furthermore, GGM was also applied as a crosslinker in the synthesis of functional hydrogels for the removal of toxic metals and metalloid ions from aqueous solutions. The distinguished products were obtained by several chemical approaches and analysed by nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy (FTIR), size exclusion chromatography (SEC), thermal gravimetric analysis (TGA), scanning electron microscope SEM, among others. Bio-based surfactants were produced by applying GGM and different fatty acids as starting materials. On one hand, GGM-grafted-fatty acids were prepared by esterification and on the other hand, well-defined GGM-block-fatty acid derivatives were obtained by linking amino-functional fatty acids to the reducing end of GGM. The reaction conditions for the syntheses were optimized and the resultant amphiphilic GGM derivatives were evaluated concerning their ability to reduce the surface tension of water as surfactants. Furthermore, the block-structured derivatives were tested in respect to their applicability as additives for the surface modification of cellulosic materials. Besides the GGM surfactants with a bio-based hydrophilic and a bio-based hydrophobic part, also GGM block-structured derivatives with a synthetic hydrophobic tail, consisting of a polydimethylsiloxane chain, were prepared and assessed for the hydrophobization of surface of nanofibrillated cellulose films. In order to generate GGM block-structured derivatives containing a synthetic tail with distinguished physical and chemical properties, as well as a tailored chain length, a controlled polymerization method was used. Therefore, firstly an initiator group was introduced at the reducing end of the GGM and consecutively single electron transfer-living radical polymerization (SET-LRP) was performed by applying three different monomers in individual reactions. For the accomplishment of the synthesis and the analysis of the products, challenges related to the solubility of the reactants had to be overcome. Overall, a synthesis route for the production of GGM block-copolymers bearing different synthetic polymer chains was developed and several derivatives were obtained. Moreover, GGM with different molar masses were, after modification, used as a crosslinker in the synthesis of functional hydrogels. Hereby, a cationic monomer was used during the free radical polymerization and the resultant hydrogels were successfully tested for the removal of chromium and arsenic ions from aqueous solutions. The hydrogel synthesis was tailored and materials with distinguished physical properties, such as the swelling rate, were obtained after purification. The results generated in this work underline the potential of bio-based products and the urge to continue carrying out research in order to be able to use more green chemicals for the manufacturing of biorenewable and biodegradable daily products.

Computational modeling of stented coronary arteries

The application of computational fluid dynamics (CFD) and finite element analysis (FEA) has been growing rapidly in the various fields of science and technology. One of the areas of interest is in biomedical engineering. The altered hemodynamics inside the blood vessels plays a key role in the development of the arterial disease called atherosclerosis, which is the major cause of human death worldwide. Atherosclerosis is often treated with the stenting procedure to restore the normal blood flow. A stent is a tubular, flexible structure, usually made of metals, which is driven and expanded in the blocked arteries. Despite the success rate of the stenting procedure, it is often associated with the restenosis (re-narrowing of the artery) process. The presence of non-biological device in the artery causes inflammation or re-growth of atherosclerotic lesions in the treated vessels. Several factors including the design of stents, type of stent expansion, expansion pressure, morphology and composition of vessel wall influence the restenosis process. Therefore, the role of computational studies is crucial in the investigation and optimisation of the factors that influence post-stenting complications. This thesis focuses on the stent-vessel wall interactions followed by the blood flow in the post-stenting stage of stenosed human coronary artery. Hemodynamic and mechanical stresses were analysed in three separate stent-plaque-artery models. Plaque was modeled as a multi-layer (fibrous cap (FC), necrotic core (NC), and fibrosis (F)) and the arterial wall as a single layer domain. CFD/FEA simulations were performed using commercial software packages in several models mimicking the various stages and morphologies of atherosclerosis. The tissue prolapse (TP) of stented vessel wall, the distribution of von Mises stress (VMS) inside various layers of vessel wall, and the wall shear stress (WSS) along the luminal surface of the deformed vessel wall were measured and evaluated. The results revealed the role of the stenosis size, thickness of each layer of atherosclerotic wall, thickness of stent strut, pressure applied for stenosis expansion, and the flow condition in the distribution of stresses. The thicknesses of FC, and NC and the total thickness of plaque are critical in controlling the stresses inside the tissue. A small change in morphology of artery wall can significantly affect the distribution of stresses. In particular, FC is the most sensitive layer to TP and stresses, which could determine plaque’s vulnerability to rupture. The WSS is highly influenced by the deflection of artery, which in turn is dependent on the structural composition of arterial wall layers. Together with the stenosis size, their roles could play a decisive role in controlling the low values of WSS (<0.5 Pa) prone to restenosis. Moreover, the time dependent flow altered the percentage of luminal area with WSS values less than 0.5 Pa at different time instants. The non- Newtonian viscosity model of the blood properties significantly affects the prediction of WSS magnitude. The outcomes of this investigation will help to better understand the roles of the individual layers of atherosclerotic vessels and their risk to provoke restenosis at the post-stenting stage. As a consequence, the implementation of such an approach to assess the post-stented stresses will assist the engineers and clinicians in optimizing the stenting techniques to minimize the occurrence of restenosis.

Ultraviolet light protection and weathering properties of wood-polypropylene composites

The main aim of this thesis is to study the effect of pigments on the weathering properties of wood-polypropylene composites (WPC). The studied properties are color change, water absorption, thickness swelling and Charpy impact strength. The impact of weathering and UV exposure on WPCs was studied by using pigments and minerals as protective agents. The study shows that the pigments and/or mineral fillers can be used to improve the weathering properties of WPCs. The effect of pigments was found to vary with the type of pigment and the method of weathering. The black pigment, an inorganic carbon black master-batch, was found to be the most effective one in reduction of the discoloration of WPCs. By preventing discoloration, and further reducing the degradation of the surface of the WPC, the pigments were found to reduce the decrease in the impact strength after weathering. As well as UV protection, the moisture resistance is a significant factor affecting the durability of WPCs. The addition of mineral fillers was found to improve the moisture-related properties, such as water absorption and thickness swelling, of WPC significantly. According to the findings, addition of pigments and mineral fillers to wood-polypropylene composites appears to be beneficial: color stability and moisture resistance can be enhanced especially in outdoor weathering. The combined effect of black pigment (carbon black master-batch) and wollastonite as a mineral filler was found to bring about the most effective properties against weathering.

An EDTA-β-cyclodextrin adsorbent for the adsorption of rare earth elements and its application in preconcentration of ultratrace rare earth elements from seawater

The objectives of this work were synthesizing an EDTA-β-CD adsorbent and investigating its adsorption potential and applications in preconcentration of REEs from aqueous phase. The adsorption capacity of EDTA-β-CD was investigated. The adsorption studies were performed by batch techniques both in one- and multi-component systems. The effects of pH, contact time and initial concentration were evaluated. The analytical detection methods and characterization methods were presented. EDTA-β-CD adsorbent was synthesized successfully with high EDTA coverage. The maximum REEs uptake was 0.310 mmol g-1 for La(III), 0.337 mmol g-1 for Ce(III) and 0.353 mmol g-1 for Eu(III), respectively. The kinetics of REEs onto EDTA-β-CD fitted well to pseudo-second-order model and the adsorption rate was affected by intra-particle diffusion. The experimental data of one component studies fitted to Langmuir isotherm model indicating the homogeneous surface of the adsorbent. The extended Sips model was applicable for the isotherm studies in three-component system. The electrostatic interaction, chelation and complexation were all involved in the adsorption mechanism. The preconcentration of RE ions and regeneration of EDTA-β-CD were successful. Overall, EDTA-β-CD is an effective adsorbent in adsorption and preconcentration of REEs.