Ultrastructure of Biofilms Formed by Bacteria from Industrial Processes

Microorganisms exist predominantly as sessile multispecies communities in natural habitats. Most bacterial species can form these matrix-enclosed microbial communities called biofilms. Biofilms occur in a wide range of environments, on every surface with sufficient moisture and nutrients, also on surfaces in industrial settings and engineered water systems. This unwanted biofilm formation on equipment surfaces is called biofouling. Biofouling can significantly decrease equipment performance and lifetime and cause contamination and impaired quality of the industrial product. In this thesis we studied bacterial adherence to abiotic surfaces by using coupons of stainless steel coated or not coated with fluoropolymer or diamond like carbon (DLC). As model organisms we used bacterial isolates from paper machines (Meiothermus silvanus, Pseudoxanthomonas taiwanensis and Deinococcus geothermalis) and also well characterised species isolated from medical implants (Staphylococcus epidermidis). We found that coating of steel surface with these materials reduced its tendency towards biofouling: Fluoropolymer and DLC coatings repelled all four biofilm formers on steel. We found great differences between bacterial species in their preference of surfaces to adhere as well as their ultrastructural details, like number and thickness of adhesion organelles they expressed. These details responded differently towards the different surfaces they adhered to. We further found that biofilms of D. geothermalis formed on titanium dioxide coated coupons of glass, steel and titanium, were effectively removed by photocatalytic action in response to irradiation at 360 nm. However, on non-coated glass or steel surfaces irradiation had no detectable effect on the amount of bacterial biomass. We showed that the adhesion organelles of bacteria on illuminated TiO2 coated coupons were complety destroyed whereas on non-coated coupons they looked intact when observed by microscope. Stainless steel is the most widely used material for industrial process equipments and surfaces. The results in this thesis showed that stainless steel is prone to biofouling by phylogenetically distant bacterial species and that coating of the steel may offer a tool for reduced biofouling of industrial equipment. Photocatalysis, on the other hand, is a potential technique for biofilm removal from surfaces in locations where high level of hygiene is required. Our study of natural biofilms on barley kernel surfaces showed that also there the microbes possessed adhesion organelles visible with electronmicroscope both before and after steeping. The microbial community of dry barley kernels turned into a dense biofilm covered with slimy extracellular polymeric substance (EPS) in the kernels after steeping in water. Steeping is the first step in malting. We also presented evidence showing that certain strains of Lactobacillus plantarum and Wickerhamomyces anomalus, when used as starter cultures in the steeping water, could enter the barley kernel and colonise the tissues of the barley kernel. By use of a starter culture it was possible to reduce the extensive production of EPS, which resulted in a faster filtration of the mash.

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.

External signal-activated liposomal drug delivery systems

Modern drug discovery gives rise to a great number of potential new therapeutic agents, but in some cases the efficient treatment of patient may not be achieved because the delivery of active compounds to the target site is insufficient. Thus, drug delivery is one of the major challenges in current pharmaceutical research. Numerous nanoparticle-based drug carriers, e.g. liposomes, have been developed for enhanced drug delivery and targeting. Drug targeting may enhance the efficiency of the treatment and, importantly, reduce unwanted side effects by decreasing drug distribution to non-target tissues. Liposomes are biocompatible lipid-based carriers that have been studied for drug delivery during the last 40 years. They can be functionalized with targeting ligands and sensing materials for triggered activation. In this study, various external signal-assisted liposomal delivery systems were developed. Signals can be used to modulate drug permeation or release from the liposome formulation, and they provide accurate control of time, place and rate of activation. The study involved three types of signals that were used to trigger drug permeation and release: electricity, heat and light. Electrical stimulus was utilized to enhance the permeation of liposomal DNA across the skin. Liposome/DNA complex-mediated transfections were performed in tight rat epidermal cell model. Various transfection media and current intensities were tested, and transfection efficiency was evaluated non-invasively by monitoring the concentration of secreted reporter protein in cell culture medium. Liposome/DNA complexes produced gene expression, but electrical stimulus did not enhance the transfection efficiency significantly. Heat-sensitive liposomal drug delivery system was developed by coating liposomes with biodegradable and thermosensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate polymer. Temperature-triggered liposome aggregation and contents release from liposomes were evaluated. The cloud point temperature (CP) of the polymer was set to 42 °C. Polymer-coated liposome aggregation and contents release were observed above CP of the polymer, while non-coated liposomes remained intact. Polymer precipitates above its CP and interacts with liposomal bilayers. It is likely that this induces permeabilization of the liposomal membrane and contents release. Light-sensitivity was introduced to liposomes by incorporation of small (< 5 nm) gold nanoparticles. Hydrophobic and hydrophilic gold nanoparticles were embedded in thermosensitive liposomes, and contents release was investigated upon UV light exposure. UV light-induced lipid phase transitions were examined with small angle X-ray scattering, and light-triggered contents release was shown also in human retinal pigment epithelial cell line. Gold nanoparticles absorb light energy and transfer it into heat, which induces phase transitions in liposomes and triggers the contents release. In conclusion, external signal-activated liposomes offer an advanced platform for numerous applications in drug delivery, particularly in the localized drug delivery. Drug release may be localized to the target site with triggering stimulus that results in better therapeutic response and less adverse effects. Triggering signal and mechanism of activation can be selected according to a specific application.

Characterisation and processing of amorphous binary mixtures with low glass transition temperature

The number of drug substances in formulation development in the pharmaceutical industry is increasing. Some of these are amorphous drugs and have glass transition below ambient temperature, and thus they are usually difficult to formulate and handle. One reason for this is the reduced viscosity, related to the stickiness of the drug, that makes them complicated to handle in unit operations. Thus, the aim in this thesis was to develop a new processing method for a sticky amorphous model material. Furthermore, model materials were characterised before and after formulation, using several characterisation methods, to understand more precisely the prerequisites for physical stability of amorphous state against crystallisation. The model materials used were monoclinic paracetamol and citric acid anhydrate. Amorphous materials were prepared by melt quenching or by ethanol evaporation methods. The melt blends were found to have slightly higher viscosity than the ethanol evaporated materials. However, melt produced materials crystallised more easily upon consecutive shearing than ethanol evaporated materials. The only material that did not crystallise during shearing was a 50/50 (w/w, %) blend regardless of the preparation method and it was physically stable at least two years in dry conditions. Shearing at varying temperatures was established to measure the physical stability of amorphous materials in processing and storage conditions. The actual physical stability of the blends was better than the pure amorphous materials at ambient temperature. Molecular mobility was not related to the physical stability of the amorphous blends, observed as crystallisation. Molecular mobility of the 50/50 blend derived from a spectral linewidth as a function of temperature using solid state NMR correlated better with the molecular mobility derived from a rheometer than that of differential scanning calorimetry data. Based on the results obtained, the effect of molecular interactions, thermodynamic driving force and miscibility of the blends are discussed as the key factors to stabilise the blends. The stickiness was found to be affected glass transition and viscosity. Ultrasound extrusion and cutting were successfully tested to increase the processability of sticky material. Furthermore, it was found to be possible to process the physically stable 50/50 blend in a supercooled liquid state instead of a glassy state. The method was not found to accelerate the crystallisation. This may open up new possibilities to process amorphous materials that are otherwise impossible to manufacture into solid dosage forms.

Looking-Glass Reflections : Norms in translating Lewis Carroll

This thesis in the field of translation studies focusses on the role of norms in the work of a literary translator. Norms are seen as guidelines for the translator; they also reflect the way the target audience either accepts or rejects the translation. Thus they are of an intersubjective nature. The theoretical background of the study is based on the views on norms of Andrew Chesterman and Gideon Toury. The writer makes use of her own editing and publishing experience, as well as her experience in translating Lewis Carroll, considering these in respect to theoretical views of norms and vice versa. The aim is also to bring to light some of the tacit knowledge related to translating, editing and publishing in Finland. The study has three angles. First, the writer introduces the norms of Finnish literary translation as gathered from her own working experience. The sources from which these norms arise and which affect them are briefly described. Six central translation norms emerge; they are described and exemplified through cases of Carroll translation. Secondly, a small-scale questionnaire study is presented. This was carried out in order to sound the normative views of other translators and to limit the role of subjectivity. The views of the informants largely support the set of norms presented by the writer, although the norms of quotability and harmony between translation and illustration do not arise. Instead, the answers give rise to a seventh, ethical norm, which is labelled the norm of integrity. Thirdly, there is a brief presentation of Lewis Carroll, his Alice books and their translation history in Finland. The retranslation hypothesis and the motives of retranslation are considered in the light of the work of Outi Paloposki and Kaisa Koskinen. The final part of the thesis plunges into actual translation work. It includes one and a half chapters of Through the Looking-Glass (Alicen seikkailut peilintakamaassa) as translated into Finnish by the writer. The translation commentary first discusses a number of recurring and general translation points; the running commentary then analyses 77 individual translation solutions and their justifications. The writer uses introspection as a way of reflecting on her own translation process, its decisive points and the role of norms therein. Keywords: Translation studies, Carroll, norms