The scope for this work was to develop rapid assays for enumerating microorganisms on gels, and to test whether fouling of gel surfaces is principally different from that of solid substrata. For this purpose, a standard set of different gels were selected, based on biocompatibility, polymer charge and gel strength. Bacterial adhesion to gels could be conveniently enumerated by first staining with SYBR Green I nucleic acid gel strain. Images were then collected using a confocal scanning laser microscope, followed by image analysis to determine the percent coverage of bacteria. Diatom adhesion could be qualified using a fluorescence scanner recording the fluorescent chlorophyll, showing a clear correlation between average fluorescence signals and cell density determined by counting. This method was successfully tested on glass, gels, a painted surface and an antifouling coated surface. Adhesion of the marine bacterium Pseudomonas sp. NCIMB 2021on gels decreased at higher shear rates. At low shear rates, adhesion varied significantly between different gels in the following descending order: alginate > agarose > chitosan > PVA-SbQ. Lowest cell coverage at all shear rates was recorded on the most hydrophobic gel, PVA-SbQ. Earlier work has shown that this organism adhere better to solid hydrophobic than solid hydrophilic surfaces. Thus, other properties than the surface free energy may be more important for bacterial adhesion to the gels. The marine diatom Amphora coffeaeformis was applied in the different adhesion arrays under different shear conditions. At high shear, cells adhered better to highly ionic polymer gels alginate and chitosan than to the low charge polymer gels agarose and PVA-SbQ. At very low shear, A. coeffeaeformis developed a film even on agarose equivalent to that on the charged polymer gels. Adhesion to PVA-SbQ remained low at all shear rates. As observed for solid substrate, low charge density led to reduced attachment. Settlement of Balanus amphitrite cypris larvae was tested at different polymer concentrations of the hydrogels. All gels inhibited cypris settlement compared to solid polystyrene controls. Gels consisting of 2.5% PVA-SbQ or 0.5% agarose showed the most promising antifouling properties. In all gel experiments, most of the non-settled larvae were able to settle when transferred and offered a suitable solid substratum. Results indicated that the gel strength was an important factor for cyprid settlement on gels, while the surface wettability seemed to be of minor importance. A few preliminary field experiments were carried out. These tests suggested that marine bacterial biofilm development is more readily on glass than on a PVA-SbQ gel surface, in accordance with monoculture lab experiments. However, similar amounts of photosynthetic organisms adhered to gels of agorose, alginate, chitosan and PVA-SbQ tested in an outdoor seawater basin during spring bloom. Finally, barnacle settlement was delayed on PVA-SbQ gels exposed in the open sea. After incubation for a full summer season, even those gels became as covered with marine fouling organisms as any other non-toxic surface. In conclusion, no universal antifouling effects of hydrogels were found. However, this work suggests that both adhesion of a bacterium and settlement of barnacle cypris larvae on gel surfaces may be principally different from solid substrata. Diatom adhesion, on the other hand, was lower on gels with a low charge density, as observed for solid substrata. In general, the most hydrophobic gel, PVA-SbQ, was the least attractive surface for all three organisms.

Proteins exist in a variety of isoforms with minor differences, mostly due to their glycosylation patterns, which can modulate their biological functions. It seems to be of clinical relevance to measure the isoform-distribution. Thesis describes a novel technology named Membrane-Assisted Isoform ImmunoAssay (MAIIA). This technique allows rapid (< 15 min.) isoform determination. It is based on a chromatographic separation combined with immunoassay detection. These steps are performed along a thin, disposable micro-porous chip in which capillary forces maintain the flow. By using anion-exchange as a chromatographic principle the technology has been utilized for the determination of transferrin isoforms in ten minutes. In one variant (the one-dimensional), selected isoforms (carbohydrate-deficient transferrin) are quantified. In a more elaborate variant (the two-dimensional) it was possible to determine the entire isoform profile of transferrin. Isoforms differing by only 0.1 pH unit in isoelectric point could be distinguished. The chromatography along the microporous bed of nitrocellulose showed very good separation performance with plate heights of 10-20 µm and only minor flow rate variations between individual devices. The quantitative determination of antibody-captured molecules was performed by using antibodies labelled with carbon black particles. Combined with a detection procedure by means of a flatbed scanner, a highly sensitive and specific immunoassay with a detection limit of 0.13 pM was obtained upon using IgE as a model analyte. This technology can thus be used to rapidly distinguish proteins with minor structure differences and specifically determine protein isoforms in complex environments, e.g., blood, down in the pM (10-12 M) concentration range.

Two polysaccharide-degrading enzymes (endo-1,4-D-glucanase and β-mannanase) from blue mussel, Mytilus edulis, have been purified to homogeneity using a combination of several chromatographic steps. Each enzyme has been characterized with regard to its molecular weight, isoelectric point, pH and temperature stability, pH and temperature optimum and substrate specificity. The amino acid sequence of the endoglucanase has been determined at the protein level. The two enzymes are true blue mussel proteins as confirmed at the DNA level. The nucleotide sequences of synthesized cDNA from digestive gland and of genomic DNA from gill tissue were compared. Both genes contain introns, a property typical of eucaryotic organisms. Amino acid sequence based classification has revealed that the endoglucanase belongs to the glycoside hydrolase family 45, subfamily 2 while β-mannanase is a member of family 5. Both enzymes form insoluble inclusion bodies when expressed in Escherichia coli. Refolding attempts were unsuccessful. However, the β-mannanase was successfully expressed in the methylotropic yeast Pichia pastoris with an expression level above 100 mg/l in shaking culture. Crystals of the endoglucanase were made from the native protein and a dataset was collected to 1.85 Å resolution using an in-house rotating anode x-ray source. Crystals were also produced using recombinant β-mannanase and a dataset was collected to 1.4 Å resolution at the ESRF synchrotron beamline ID14-EH1. The three dimensional structure of the endoglucanase was solved by X-ray crystallography.

<p>A generalized quantum chemical approach for electron transport in molecular devices is developed. It allows to treat the devices where the metal electrodes and the molecule are either chemically or physically bonded on equal footing. Effects of molecular length and hydrogen bonding on the current-voltage (I-V) characteristics of molecular devices are discussed. An extension to include the vibration motions of the molecule has been derived and implemented. It provides the inelastic electron tunneling spectroscopy (IETS) of molecular devices with unprecedented accuracy, and reveals important information about the molecular structures that are not accessible in the experiment. The IETS is shown to be a powerful characterization tool for molecular devices.</p><p>An effective elongation method has been developed to study the electron transport in nanoand bio-electronic devices at hybrid density functional theory level. It enables to study electronic structures and transportation properties of a 40 nm long self-assembled conjugated polymer junction, a 21 nm long single-walled carbon nanotubes (SWCNT), and a 60 basepairs DNA molecule. It is the first time that systems consisting of more than 10,000 electrons have been described at such a sophisticated level. The calculations have shown that the electron transport in sub-22 nm long SWCNT and short DNA molecules is dominated by the coherent scattering through the delocalized unoccupied states. The derived length dependence of coherent electron transport in these nanostructured systems will be useful for the future experiments. Moreover, some unexpected behaviors of these devices have been discovered.</p>

Laccase is a blue multi-copper oxidase. It has a broad biotechnical potential which increases the interest to study the enzyme further. A laccase-encoding gene from the white-rot fungus Trametes versicolor (lcc2) was mutated using two different methods for random mutagenesis: error-prone PCR and a method based on an E.coli strain (ES1301 mutS) that introduces random mutations. For the error-prone PCR reaction, the vector pPICZB with the lcc2 gene inserted was used as template. The E. coli strain ES1301 mutS was transformed with the vector pBluescript SKII with the lcc2 gene as insert. The mutagenesis products were cloned into the Pichia pastoris expression vector pPICZB for transformation of P. pastoris SMD1168. The transformants were spread on agar plates containing zeocin. Laccase-secreting transformants were selected by their ability to oxidize the substrates ABTS [2,2´-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] and syringaldazine [N,N´-bis(3,5-dimethoxy-4-hydroxybenzylidene)hydrazine], the products of which give green and purple colour, respectively. Around 20 transformants from each of the mutagenesis methods were transformed to plates containing 1 mM ABTS or 1 mM syringaldazine. None of the transformants produced any colour. Control transformants (pPICZB with unmutated lcc2) were also spread on plates with either ABTS or syringaldazine. The transformants gave rise to green colour after 24 hours on the ABTS plates and to purple colour after 72 hours on the syringaldazine plates. Experimets with different chromogenic substrates indicated that ABTS and syringaldazine were best suited for screening of mutants. Remazol Brilliant Blue and Phenol Red are two substrates that after optimisation can serve as alternatives for the selection of laccase-secreting transformants.

The present thesis concerns theoretical studies of molecular interactions investigated by infrared and X-ray spectroscopic techniques, with emphasis on using these two techniques combined in pump-probe experiments. Four main types of studies are addressed: the use of near-edge X-ray absorption fine structure spectra (NEXAFS) to manifest through-bond and through-space interactions; the role of hydrogen bonding in the formation of X-ray photoelectron spectra as evidenced by simulations of the water dimer; the development of theory, with sample applications, for infrared X-ray pump-probe spectroscopy; and molecular dynamics simulations of light-induced fragmentation of water clusters. Ab initio calculations indicate that NEXAFS spectra give direct information about the through-bond and through-space interactions between vacant non-conjugated π* orbitals. It is found out that the X-ray photoelectron spectrum of the water dimer differs dramatically from the monomer spectrum in that two bands are observed, separated by the chemically shifted ionization potentials of the donor and the acceptor. The hydrogen bond is responsible for the anomalously strong broadening of these two bands. The studies show that X-ray core electron ionization of the water dimer driven by an infrared field is a proper technique to prove the proton transfered state contrary to conventional X-ray photoelectron spectroscopy. The physical aspects of the proposed new X-ray spectroscopic method - phase sensitive Infrared - X-Ray Pump-Probe Spectroscopy - are examined in detail using the wave packet technique in three applications; the NO molecule and the dynamics of proton transfer in core ionized water dimer and glyoxalmonoxime. It is found out that the phase of the infrared pump field strongly influences the trajectory of the nuclear wave packet on the ground state potential, which results in a phase dependence of the X-ray pump-probe spectra. A proper choice of the delay time of the X-ray pulse allows the direct observation of the X-ray transition in the proton transfered well of the core excited potential. It is found out that the glyoxalmonoxime molecule possesses an important feature; proton transfer accompanied by core hole hopping. Special attention is paid to the quantum control of the populations of vibrational level which is of crucial importance to shape the wave packet of desirable size. The wave packet technique becomes computationally very expensive when the number of nuclear degrees of freedom is large. Molecular dynamics is used instead in studies of light-induced nuclear kinetics in the water hexamer cluster. We predict a novel mechanism of the mechanical action of light on atoms and molecules. This mechanism is based on the rectification of the Lorentz force, which gives a unique opportunity of direct site selective mechanical action of light on atoms and molecules inside large systems like clusters or biomolecules. / <p>QC 20170222</p>

No description available.

Bioprocesses are industrial processes that use living cells to produce desired products. These processes are complex and require advanced monitoring and control in order to reach their highest potential. Software sensors are a potent tool for real time monitoring and control of bioprocesses. In this project, three novel software sensors were designed and tested in three fed-batch cultivations containing recombinant Escherichia coli producing green fluorescence protein (GFP). Special attention was given to the sensors for reacting to any stress triggered when production of the recombinant protein was initiated by an inducer molecule.   The sensors calculated the oxygen consumption per substrate, carbon emission per substrate and the sum of the specific growth rates of various acids. Their mathematical algorithms were derived from already existing software sensors and programmed on to computer software designed for monitoring and control.   The results showed that all three sensors gave stable signals as expected. Unfortunately they did not provide any useful information regarding the recombinant protein expression in this particular study. Complications such as measurement errors and incorrect system configurations also created difficulties. It is clear however, that the sensors work well and should be highly considered for further studies.

This thesis encloses quantum chemical calculations and applications of a response function formalism recently implemented within the framework of density functional theory. The optical and magnetic properties of copper(II) molecular systems are the main goal of this work. In this work, the visible and near-infrared electronic transitions, which have shown a key role in studies on electronic structure and structure-function relationships of copper compounds, were investigated in order to explore the correlation of the positions and intensities of these transitions with the geometrical structures and their molecular distortions. The evaluation of solvent effects on the absorption spectra were successfully achieved, providing accurate and inedit computational insight of these effects for copper(II) complexes. Electron Paramagnetic Resonance (EPR) parameters, that is, the electronic g tensor and the hyperfine coupling constants, are powerful spectroscopic properties for investigating paramagnetic systems and were thoroughly analysed in this work in different molecular systems. Relativistic corrections generated by spin-orbit interactions or by scalar relativistic effects were taken into account in all calculations. In addition, we have designed a methodology for accurate evaluation of the electronic g tensors and hyperfine coupling tensors as well as for evaluation of solvent effects on these properties. It is found that this methodology is able to provide reliable and accurate results for EPR parameters of copper(II) molecular systems. The spin polarization effects on EPR parameters of square planar copper(II) complexes were also considered, showing that these effects give rise to significant contributions to the hyperfine coupling tensor, whereas the electronic g tensor of these complexes are only marginally affected by these effects. The evaluation of the leading-order relativistic corrections to the electronic g tensors of molecules with a doublet ground state has been also taken into account in this work. As a first application of the theory, the electronic g tensors of dihalogen anion radicals X$_2^-$ (X=F,~Cl,~Br,~I) have been investigated and the obtained results indicate that the spin--orbit interaction is responsible for the parallel component of the g tensor shift, while both the leading-order scalar relativistic and spin--orbit corrections are of minor importance for the perpendicular component of the g tensor in these molecules since they effectively cancel each other. Overall, both optical and magnetic results show quantitative agreements with experiments, indicating that the methodologies employed form a practical way in study of copper(II) molecular systems including those of biological importance. / QC 20100714

In this thesis, single-particle cryo-electron microscopy (cryo-EM) was used to study the structure of three macromolecular assemblies: the two hemocyanin isoforms from Rapana thomasiana, the Pyrococcus furiosus chaperonin, and the ribosome from Escherichia coli. Hemocyanins are large respiratory proteins in arthropods and molluscs. Most molluscan hemocyanins exist as two distinct isoforms composed of related polypeptides. In most species the two isoforms differ in terms of their oligomeric stability, and thus we set out to investigate the two Rapana thomasiana hemocyanins (RtH) in order to explain this behaviour. Our findings showed that the two RtHio forms are identical at the experimental resolution. Furthermore, three previously unreported connections that most likely contribute to the oligomeric stability were identified. Chaperonins are double-ring protein complexes that assist the folding process of nascent, non-native polypeptide chains. The chaperonin from the hyperthermophilic archaea Pyrococcus furiosus belongs to Group II chaperonins, and unlike most othergroup II chaperonins it appears to be homo-oligomeric. The 3D reconstruction of the Pyrococcus furiousus chaperonin revealed a di-octameric structure in a partially closed/open state, something in between the closed folding-active state and the open substrate-accepting state. The ribosome is the molecular machine where protein synthesis takes place. In bacteria there is a unique RNA molecule called transfer-messenger RNA (tmRNA) that together with its helper protein SmpB rescues ribosomes trapped on defective messenger RNAs (mRNAs) through a process called trans-translation. tmRNA is about 4 times the size of a normal tRNA, and it is composed of a tRNA-like domain (TLD) that is connected to the mRNA-like domain (MLD) by several pseudoknots (PKs) and RNA helices. During trans-translation, tmRNA utilize its TLD to receive the incomplete polypeptide from the peptidyl-tRNA in the ribosomal P site of the stalledribosome. Subsequently, its MLD is used to tag the incomplete polypeptide with adegradation signal. When tmRNA enters a stalled ribosome the MLD and pseudoknots form a highly structured arc that encircles the beak of the small ribosomal subunit. Byutilizing maximum-likelihood based methods for heterogeneity analysis we could observe the Escherichia coli ribosome in a number of different tmRNA·SmpB-boundstates. The cryo-EM map of the post-accommodated state revealed that the TLD·SmpBpart of the tmRNA·SmpB complex mimics native tRNAs in the A site of stalled ribosomes. The density map also showed that the tmRNA arc remains well structuredand that it is still attached to the beak of the small ribosomal subunit. Thereconstructions of the double-translocation tmRNA-bound ribosome complex showed that the pseudoknots of tmRNA still form an arc, and that they are located at positions similar to the ones assigned for the pseudoknots in the post-accommodated state. In addition, the tmRNA arc exists in two states; one stable and highly structured and another more flexible and disorganized. / QC 20100708