Polymer Protected Gold Nanoparticles

Polymer protected gold nanoparticles have successfully been synthesized by both "grafting-from" and "grafting-to" techniques. The synthesis methods of the gold particles were systematically studied. Two chemically different homopolymers were used to protect gold particles: thermo-responsive poly(N-isopropylacrylamide), PNIPAM, and polystyrene, PS. Both polymers were synthesized by using a controlled/living radical polymerization process, reversible addition-fragmentation chain transfer (RAFT) polymerization, to obtain monodisperse polymers of various molar masses and carrying dithiobenzoate end groups. Hence, particles protected either with PNIPAM, PNIPAM-AuNPs, or with a mixture of two polymers, PNIPAM/PS-AuNPs (i.e., amphiphilic gold nanoparticles), were prepared. The particles contain monodisperse polymer shells, though the cores are somewhat polydisperse. Aqueous PNIPAM-AuNPs prepared using a "grafting-from" technique, show thermo-responsive properties derived from the tethered PNIPAM chains. For PNIPAM-AuNPs prepared using a "grafting-to" technique, two-phase transitions of PNIPAM were observed in the microcalorimetric studies of the aqueous solutions. The first transition with a sharp and narrow endothermic peak occurs at lower temperature, and the second one with a broader peak at higher temperature. In the first transition PNIPAM segments show much higher cooperativity than in the second one. The observations are tentatively rationalized by assuming that the PNIPAM brush can be subdivided into two zones, an inner and an outer one. In the inner zone, the PNIPAM segments are close to the gold surface, densely packed, less hydrated, and undergo the first transition. In the outer zone, on the other hand, the PNIPAM segments are looser and more hydrated, adopt a restricted random coil conformation, and show a phase transition, which is dependent on both particle concentration and the chemical nature of the end groups of the PNIPAM chains. Monolayers of the amphiphilic gold nanoparticles at the air-water interface show several characteristic regions upon compression in a Langmuir trough at room temperature. These can be attributed to the polymer conformational transitions from a pancake to a brush. Also, the compression isotherms show temperature dependence due to the thermo-responsive properties of the tethered PNIPAM chains. The films were successfully deposited on substrates by Langmuir-Blodgett technique. The sessile drop contact angle measurements conducted on both sides of the monolayer deposited at room temperature reveal two slightly different contact angles, that may indicate phase separation between the tethered PNIPAM and PS chains on the gold core. The optical properties of amphiphilic gold nanoparticles were studied both in situ at the air-water interface and on the deposited films. The in situ SPR band of the monolayer shows a blue shift with compression, while a red shift with the deposition cycle occurs in the deposited films. The blue shift is compression-induced and closely related to the conformational change of the tethered PNIPAM chains, which may cause a decrease in the polarity of the local environment of the gold cores. The red shift in the deposited films is due to a weak interparticle coupling between adjacent particles. Temperature effects on the SPR band in both cases were also investigated. In the in situ case, at a constant surface pressure, an increase in temperature leads to a red shift in the SPR, likely due to the shrinking of the tethered PNIPAM chains, as well as to a slight decrease of the distance between the adjacent particles resulting in an increase in the interparticle coupling. However, in the case of the deposited films, the SPR band red-shifts with the deposition cycles more at a high temperature than at a low temperature. This is because the compressibility of the polymer coated gold nanoparticles at a high temperature leads to a smaller interparticle distance, resulting in an increase of the interparticle coupling in the deposited multilayers.

Cellular Membranes as a Playground for Semliki Forest Virus Replication Complex

All positive-strand RNA viruses utilize cellular membranes for the assembly of their replication complexes, which results in extensive membrane modification in infected host cells. These alterations act as structural and functional scaffolds for RNA replication, providing protection for the viral double-stranded RNA against host defences. It is known that different positive-strand RNA viruses alter different cellular membranes. However, the origin of the targeted membranes, the mechanisms that direct replication proteins to specific membranes and the steps in the formation of the membrane bound replication complex are not completely understood. Alphaviruses (including Semliki Forest virus, SFV), members of family Togaviridae, replicate their RNA in association with membranes derived from the endosomal and lysosomal compartment, inducing membrane invaginations called spherules. Spherule structures have been shown to be the specific sites for RNA synthesis. Four replication proteins, nsP1-nsP4, are translated as a polyprotein (P1234) which is processed autocatalytically and gives rise to a membrane-bound replication complex. Membrane binding is mediated via nsP1 which possesses an amphipathic α-helix (binding peptide) in the central region of the protein. The aim of this thesis was to characterize the association of the SFV replication complex with cellular membranes and the modification of the membranes during virus infection. Therefore, it was necessary to set up the system for determining which viral components are needed for inducing the spherules. In addition, the targeting of the replication complex, the formation site of the spherules and their intracellular trafficking were studied in detail. The results of current work demonstrate that mutations in the binding peptide region of nsP1 are lethal for virus replication and change the localization of the polyprotein precursor P123. The replication complex is first targeted to the plasma membrane where membrane invaginations, spherules, are induced. Using a specific regulated endocytosis event the spherules are internalized from the plasma membrane in neutral carrier vesicles and transported via an actin-and microtubule-dependent manner to the pericentriolar area. Homotypic fusions and fusions with pre-existing acidic organelles lead to the maturation of previously described cytopathic vacuoles with hundreds of spherules on their limiting membranes. This work provides new insights into the membrane binding mechanism of SFV replication complex and its role in the virus life cycle. Development of plasmid-driven system for studying the formation of the replication complex described in this thesis allows various applications to address different steps in SFV life cycle and virus-host interactions in the future. This trans-replication system could be applied for many different viruses. In addition, the current work brings up new aspects of membranes and cellular components involved in SFV replication leading to further understanding in the formation and dynamics of the membrane-associated replication complex.

Electron and proton transfer in NADH:ubiquinone oxidoreductase (Complex I) from Escherichia coli

Cells of every living organism on our planet − bacterium, plant or animal − are organized in such a way that despite differences in structure and function they utilize the same metabolic energy represented by electrochemical proton gradient across a membrane. This gradient of protons is generated by the series of membrane bound multisubunit proteins, Complex I, II, III and IV, organized in so-called respiratory or electron transport chain. In the eukaryotic cell it locates in the inner mitochondrial membrane while in the bacterial cell it locates in the cytoplasmic membrane. The function of the respiratory chain is to accept electrons from NADH and ubiquinol and transfer them to oxygen resulting in the formation of water. The free energy released upon these redox reactions is converted by respiratory enzymes into an electrochemical proton gradient, which is used for synthesis of ATP as well as for many other energy dependent processes. This thesis is focused on studies of the first member of the respiratory chain − NADH:ubiquinone oxidoreductase or Complex I. This enzyme has a boot-shape structure with hydrophilic and hydrophobic domains, the former of which has all redox groups of the protein, the flavin and eight to nine iron-sulfur clusters. Complex I serves as a proton pump coupling transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the membrane. So far the mechanism of energy transduction by Complex I is unknown. In the present study we applied a set of different methods to study the electron and proton transfer reactions in Complex I from Escherichia coli. The main achievement was the experiment that showed that the electron transfer through the hydrophilic domain of Complex I is unlikely to be coupled to proton transfer directly or to conformational changes in the protein. In this work for the first time properties of all redox centers of Complex I were characterized in the intact purified bacterial enzyme. We also probed the role of several conserved amino acid residues in the electron transfer of Complex I. Finally, we found that highly conserved amino acid residues in several membrane subunits form a common pattern with a very prominent feature – the presence of a few lysines within the membrane. Based on the experimental data, we suggested a tentative principle which may govern the redox-coupled proton pumping in Complex I.

Peroxidases in lignifying xylem of Norway spruce, Scots pine and silver birch

Lignin is a complex plant polymer synthesized through co-operation of multiple intracellular and extracellular enzymes. It is deposited to plant cell walls in cells where additional strength or stiffness are needed, such as in tracheary elements (TEs) in xylem, supporting sclerenchymal tissues and at the sites of wounding. Class III peroxidases (POXs) are secreted plant oxidoreductases with implications in many physiological processes such as the polymerization of lignin and suberin and auxin catabolism. POXs are able to oxidize various substrates in the presence of hydrogen peroxide, including lignin monomers, monolignols, thus enabling the monolignol polymerization to lignin by radical coupling. Trees produce large amounts of lignin in secondary xylem of stems, branches and roots. In this study, POXs of gymnosperm and angiosperm trees were studied in order to find POXs which are able to participate in lignin polymerization in developing secondary xylem i.e. are located at the site of lignin synthesis in tree stems and have the ability to oxidize monolignol substrates. Both in the gymnosperm species, Norway spruce and Scots pine, and in the angiosperm species silver birch the monolignol oxidizing POX activities originating from multiple POX isoforms were present in lignifying secondary xylem in stems during the period of annual growth. Most of the partially purified POXs from Norway spruce and silver birch xylem had highest oxidation rate with coniferyl alcohol, the main monomer in guaiacyl-lignin in conifers. The only exception was the most anionic POX fraction from silver birch, which clearly preferred sinapyl alcohol, the lignin monomer needed in the synthesis of syringyl-guaiacyl lignin in angiosperm trees. Three full-length pox cDNAs px1, px2 and px3 were cloned from the developing xylem of Norway spruce. It was shown that px1 and px2 are expressed in developing tracheids in spruce seedlings, whereas px3 transcripts were not detected suggesting low transcription level in young trees. The amino acid sequences of PX1, PX2 and PX3 were less than 60% identical to each other but showed up to 84% identity to other known POXs. They all begin with predicted N-terminal secretion signal (SS) peptides. PX2 and PX3 contained additional putative vacuolar localization determinants (VSDs) at C-terminus. Transient expression of EGFP-fusions of the SS- and VSD-peptides in tobacco protoplasts showed SS-peptides directed EGFP to secretion in tobacco cells, whereas only the PX2 C-terminal peptide seems to be a functional VSD. According to heterologous expression of px1 in Catharanthus roseus hairy roots, PX1 is a guaicol-oxidizing POX with isoelectric point (pI) approximately 10, similar to monolignol oxidizing POXs in protein extracts from Norway spruce lignifying xylem. Hence, PX1 has characteristics for participation to monolignol dehydrogenation in lignin synthesis, whereas the other two spruce POXs seem to have some other functions. Interesting topics in future include functional characterization of syringyl compound oxidizing POXs and components of POX activity regulation in trees.

Studies on diamond-like carbon and novel diamond-like carbon polymer hybrid coatings deposited with filtered pulsed arc discharge method

The main obstacle for the application of high quality diamond-like carbon (DLC) coatings has been the lack of adhesion to the substrate as the coating thickness is increased. The aim of this study was to improve the filtered pulsed arc discharge (FPAD) method. With this method it is possible to achieve high DLC coating thicknesses necessary for practical applications. The energy of the carbon ions was measured with an optoelectronic time-of-flight method. An in situ cathode polishing system used for stabilizing the process yield and the carbon ion energies is presented. Simultaneously the quality of the coatings can be controlled. To optimise the quality of the deposition process a simple, fast and inexpensive method using silicon wafers as test substrates was developed. This method was used for evaluating the suitability of a simplified arc-discharge set-up for the deposition of the adhesion layer of DLC coatings. A whole new group of materials discovered by our research group, the diamond-like carbon polymer hybrid (DLC-p-h) coatings, is also presented. The parent polymers used in these novel coatings were polydimethylsiloxane (PDMS) and polytetrafluoroethylene (PTFE). The energy of the plasma ions was found to increase when the anode-cathode distance and the arc voltage were increased. A constant deposition rate for continuous coating runs was obtained with an in situ cathode polishing system. The novel DLC-p-h coatings were found to be water and oil repellent and harder than any polymers. The lowest sliding angle ever measured from a solid surface, 0.15 ± 0.03°, was measured on a DLC-PDMS-h coating. In the FPAD system carbon ions can be accelerated to high energies (≈ 1 keV) necessary for the optimal adhesion (the substrate is broken in the adhesion and quality test) of ultra thick (up to 200 µm) DLC coatings by increasing the anode-cathode distance and using high voltages (up to 4 kV). An excellent adhesion can also be obtained with the simplified arc-discharge device. To maintain high process yield (5µm/h over a surface area of 150 cm2) and to stabilize the carbon ion energies and the high quality (sp3 fraction up to 85%) of the resulting coating, an in situ cathode polishing system must be used. DLC-PDMS-h coating is the superior candidate coating material for anti-soiling applications where also hardness is required.

Major histocompatibility complex and coronary artery disease: Special emphasis on Chlamydia pneumoniae, and periodontitis

Most of the genes in the MHC region are involveed in adaptive and innate immunity, with essential function in inflammatory reactions and in protection against infections. These genes might serve as a candidate region for infection and inflammation associated diseases. CAD is an inflammatory disease. The present set of studies was performed to assess whether the MHC region harbors genetic markers for CAD, and whether these genetic markers explain the CAD risk factors: e.g., C. pneumoniae, periodontitis, and periodontal pathogens. Study I was performed using two separate patient materials and age- and sex-matched healthy controls, categorizing them into two independent studies: the HTx and ACS studies. Both studies consistently showed the HLA-A3– B35– DR1 (35 ancestral haplotype) haplotype as a susceptible MHC genetic marker for CAD. HLA-DR1 alone was associated not only with CAD, but also with CAD risk factor diseases, e.g., diabetes mellitus, and hyperlipidemia. The ACS study further showed the HLA-B*07 and -DRB1*15 -related haplotype as a protective MHC haplotype for CAD. Study II showed that patients with CAD showed signs of chronic C. pneumoniae infection when compared to age- and sex-matched healthy controls. HLA-B*35 or -related haplotypes associated with the C. pneumoniae infection markers. Among these haplotype carriers, males and smokers associated with elevated C. pneumoniae infection markers. Study III showed that CAD patients with periodontitis had elevated serum markers of P. gingivalis and occurrence of the pathogen in saliva. LTA+496C strongly associated with periodontitis, while HLA-DRB1*01 with periodontitis and with the elevated serum antibodies of P. gingivalis. Study IV showed that the increased level of C3/C4 ratio was a new risk factor and was associated with recurrent cardiovascular end-points. The increased C3 and decreased C4 concentrations in serum explained the increased level of the C3/C4 ratio. Both the higher than cut-off value (4.53) and the highest quartile of the C3/C4 ratio were also associated with worst survival, increased end-points, and C4 null alleles. The presence of C4 null alleles associated with decreased serum C4 concentration, and increased C3/C4 ratio. In conclusion, the present studies show that the CAD susceptibility haplotype (HLA-A3− B35− DR1 -related haplotypes, Study I) partially explains the development of CAD in patients possessing several recognized and novel risk factors: diabetes mellitus, increased LDL, smoking, C4B*Q0, C. pneumnoiae, periodontitis, P. gingivalis, and complement C3/C4 ratio (Study II, III, and IV).

Complex trait of acute pancreatitis : Studies of candidate genes and adipokines

Acute pancreatitis (AP) is a common disease. Mild disease resolves spontaneously in a few days. Severe forms of the disease can lead to local complications, necrosis, and abscesses in and around the pancreas. Systemic inflammation in severe AP is associated with distant organ failures. The aim of this study is to identify genetically determined prognostic factors involved in the clinical features of AP. The study employs a candidate-gene approach, and the genes are involved in trysinogen activation in the initiation phase of the disease, as well as in the systemic inflammation as the disease proceeds. The last study examines adipokines, fat-derived hormones characterized with the capacity to modify inflammation. SPINK 1 is a gene coding trypsin activation inhibitor. Mutations N34S and P55N were determined by minisequencing methods in 371 AP patients and in 459 controls. The mutation N34S was more common in AP patients (7.8%) than in controls (2.6%). This suggests that SPINK 1 gene mutation N34S is a risk factor for AP. In the fourth study, in 12 matched pairs of patients with severe and mild AP, levels of adipokines, adiponectin, and leptin were evaluated. Plasma adipokine levels did not differ between patients with mild and severe AP. The results suggest that in AP, adipokine plasma levels are not factors predisposing to organ failures. This study identified the SPINK 1 mutation N34S to be a risk factor for AP in the general population. As AP is a multifactorial disease, and extensive genetic heterogeneity is likely, further identification of genetic factors in the disease requires larger future studies with more advanced genetic study models. Further identification of the patient characteristics associated with organ failures offers another direction of the study to achieve more detailed understanding of the severe form of AP.