<p>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. </p><p>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.</p><p>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. </p><p>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. </p><p>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.</p><p>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. </p><p>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. </p>

<p>Brown seaweeds lack lignin and have a low cellulose content. Thus, seaweeds should be an easier material for biological degradation than land plants. However, seaweeds have a complex composition, and complete degradation of the material necessitates the presence of microorganisms with a broad substrate range. During anaerobic degradation of organic material, energy carriers such as methane and ethanol may be produced. This is a study of two particular species of brown seaweeds; <i>Laminaria hyperborea</i> and <i>Ascophyllum nodosum</i>, which are the most abundant Norwegian species and also the two species that are commercially harvested in Norway.</p><p>Most of the degradation studies were carried out in batch systems at pH 7 and at 35 °C. The digestion pattern of the seaweeds were studied by measuring gas production, alginate lyase activity, remaining alginate, the concentrations of uronic acids, VS, COD, mannitol, organic acids and polyphenols. NIR spectroscopy was applied as a new method for alginate quantification. Ethanol production was carried out at 30 °C at different pH, both in batch and continuous cultures. Gas production and concentrations of mannitol, laminaran, ethanol and organic acids were measured.</p><p>Methane is the end product of a mixed microbial community. However, it is the initial steps of hydrolysis and acidogenesis that are specific for the raw material. Alginate forms the major structural component of brown algae, and its degradation is catalysed by alginate lyases. Polyphenols proved to be the most important limiting factor in the biodegradation: the content of polyphenols was much higher in <i>A. nodosum</i> than <i>L. hyperborea</i>, and this led to a reduced biodegradability of <i>A. nodosum</i>. However, when the polyphenols were fixed with formaldehyde, this seaweed was also readily degraded. Manipulation of the content of polyphenols in <i>L. hyperborea</i> gave similar results. This toxic effect was probably caused by direct inhibition of the microbes, especially the methanogenic bacteria, and complexation reactions with algal material and enzymes. Generally, the guluronate content of the remaining alginate increased during biodegradation, probably due to the Ca-linked guluronate junction zones less accessible for alginate lyase. The main organic product of the acidogenesis was acetate, which was easily converted to methane. In this study, it was not attempted to optimise the methane yield.</p><p>Ethanol is an intermediate in the complete digestion of organic material and is produced by specific microbial strains. Thus, ethanol production should take place under controlled conditions to prevent contamination problems. The complex composition of seaweeds makes it a difficult substrate to ferment to ethanol by one or a few strains of microbes. In this work, laminaran and mannitol extracted from <i>L hyperborea</i> fronds were used as substrate for ethanol production. <i>A bacterium</i>, <i>Zymobacter palmae</i>, was able to produce ethanol from mannitol, but could not utilise laminaran. However, the yeast <i>Pichia angophorae</i> was able to produce ethanol from both substrates simultaneously. Some supply of oxygen was necessary for the fermentation of mannitol, while a too high aeration resulted in the production of organic acids.</p><p>Thus, it has been shown that both methane and ethanol can be produced from brown seaweeds. However, an optimisation of the processes will be necessary. Energy production from seaweeds will only be economic if the harvesting costs are low. It may be noted that wastes from the alginate industry may be considered a non-cost raw material for energy production.</p>

<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.</p>

<p>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.</p><p>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.</p><p>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. </p><p>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.</p><p>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.</p>

<p>Two polysaccharide-degrading enzymes (endo-1,4-D-glucanase and β-mannanase) from blue mussel, <i>Mytilus edulis</i>, 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. </p><p>Both enzymes form insoluble inclusion bodies when expressed in <i>Escherichia coli</i>. Refolding attempts were unsuccessful. However, the β-mannanase was successfully expressed in the methylotropic yeast <i>Pichia pastoris</i> 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>

Bone is the second most implanted tissue next to blood causing approximately 2.2 million people to receive bone grafts each year. Developing safe synthetic bone grafts allows quick and safe restoration of bone function while avoiding the surgical risks associated with bone autografting (self donation), and risks of disease transmission and immunogenic response associated with allografts (bone donated from other humans) and xenografts (grafts derived from animal tissue). This dissertation entails the study and development of a novel potential synthetic bone graft consisting of a composite of calcium-deficient hydroxyapatite (CdHAP) biomimetically deposited in a bacterial cellulose (BC) hydrogel. To determine which conditions provided optimum cellulose growth from Gluconacetobacter hansenii (ATCC 10821), a statistical analysis of the effects of different culture parameters was carried out. This utilized fractional factorial design which allowed a large number of factors to be tested using an abbreviated set of experiments. Statistical software was also used to process the data to determine which factors and factor interactions were significant to bacterial cellulose production. Purified bacterial cellulose was mineralized by sequential incubations in solutions of calcium chloride followed by sodium phosphate dibasic. This material was characterized using X-Ray Diffractometry, Fourier Transform Infrared Spectroscopy, Scanning Electron Microscopy, Electron Dispersive Spectroscopy, and mechanical testing. Characterization revealed that the CdHAP formed biomimetically in the BC in a manner similar to natural bone. It was also determined that the CdHAP had comparable structure, size, and composition to that found in natural bone. An ideal bone replacement material will degrade after stimulating new osseous tissue production. A chemical modification of the cellulose was carried out with periodate oxidation to render it degradable in-vivo. The BC structure was preserved as well as its ability to mineralize CdHAP after periodate oxidation. This composite was characterized and its capacity to degrade was analyzed by subjecting the samples to a simulated aqueous physiological environment. The last chapter discusses how the BC-CdHAP composite may have clinical use in the major bone grafting procedures currently being administered. Several surgeons were surveyed for this portion of the study to determine which grafts they used, the reasons for graft selection, and to obtain their opinion of the BC-CdHAP material. It was ultimately determined that the BC-CdHAP composite has ideal properties for dental bone grafting procedures including site extraction preservation and sinus lifts.

This dissertation consists of five parts, each describing a specific topic of unsaturated flow and transport. The first three parts describe the development of soil hydraulic function models with the effects of partial drainage by using fractal and probabilistic approaches. During drainage of a porous medium, both the pore size distribution and the connectivity of pores determine the drained pore volume as function of suction. New analytical expressions were presented for the water retention (part 1), intrinsic permeability (part 2), and relative permeability (part 3) functions. Predictions based on the analytical models are compared with estimates of the intrinsic permeability (k) derived from lattice Boltzmann method (LBM) simulations of saturated flow in virtual representations of classical (deterministic) and randomized Menger Sponges. Overall, the analytically predicted k values matched the k values from the LBM simulations with less than 14% error for deterministic sponges with minimum pore sizes ranging from 1/31 to 1/34. We presented a new approach that allows the prediction of relative permeability by direct use of measured water retention data without fitting. This new discrete model describes the drained pore space and permeability at different suctions incorporating the effect of both pore size distribution and connectivity among water-filled pores. We tested the performance of the new model by comparing its predictions of relative permeability to those of van Genuchten-Mualem (VG-M). Overall, the new method (RMSE=0.175, LRMSE=1.101) predicted the measured relative permeability data better than the VG-M model (RMSE=0.216, LRMSE=2.381). Part 4 presents analytical solutions of the advective solute transport in a macropore with simultaneous diffusion into an unbounded soil matrix. We obtained three sets of exact and approximate solutions for various boundary and initial conditions. Part 5 presents a moisture moment method to estimate unsaturated soil hydraulic properties. By analyzing the change in forces of two load cells that suspend either end of a soil column and the inlet water pressure, the unsaturated hydraulic conductivity and water retention functions are obtained. We applied the method to a sandy silt loam and the analyses show the method is promising.

This dissertation focuses on the development of a computationally efficient and fast method that incorporates the kinematics obtained from fluoroscopy and extends it to the prediction of the in-vivo contact mechanics at the femoro-tibial articulation in modern knee implants for the deep knee bend activity. In this endeavor, this dissertation deals with the use of an inverse dynamic rigid body model characterizing the slip and roll behavior observed in the femoro-polyethylene articulation and a coupled deformation model where the polyethylene in knee implants are modeled as hexahedral discrete element networks. The performance of this method is tested by comparing force predictions from a telemetric knee and finite element analysis. Finally, the method is applied to study the in vivo contact mechanics and mechanics of the quadriceps mechanism in six popular knee designs. During the deep knee bend activity, the contact force generally increased with flexion. However, the medial lateral forces were not equally distributed and the medial lateral force distribution generally varied from 60%- 40% at full extension to as high as 75%-25% at full flexion in some patients. Also, the magnitude of axial force in the superior-inferior direction was the highest and was found to contribute around 98%-99% of the total load acting at the femorotibial joint. The forces in the medio-lateral and antero-posterior directions were low and the maximum magnitude observed was around 0.5BW. The contact areas and contact pressures were much more sensitive to the geometries involved and the in vivo kinematics. Though no definite pattern was observed for the variation of the contact areas throughout flexion, the contact pressures increased in both condyles with increasing flexion. Also, the contact pressures on the medial condyle were higher than the contact pressures observed in the lateral condyle. The patellofemoral and the quadriceps force ratio increased with the increase in flexion while the patellar ligament and the quadriceps force ratio decreased with increasing flexion. In some patients at high flexion, the quadriceps force and as a result the patellofemoral, patellar ligament and the knee contact forces were found to decrease due to the wrapping of the quadriceps coupled with posterior movement of the femoral condyles leading to the increase in the quadriceps moment arm.

This dissertation embodies the diversity and creativity of my research, of which much has been peer-reviewed, published in archival quality journals, and presented nationally and internationally. Portions of the work described herein have been published in the fields of image processing, forensic anthropology, physical anthropology, biomedical engineering, clinical orthopedics, and microwave engineering. The problem studied is primarily that of developing the tools and technologies for a next-generation surgical navigation system. The discussion focuses on the underlying technologies of a novel microwave positioning subsystem and a bone analysis subsystem. The methodologies behind each of these technologies are presented in the context of the overall system with the salient results helping to elucidate the difficult facets of the problem. The microwave positioning system is currently the highest accuracy wireless ultra-wideband positioning system that can be found in the literature. The challenges in producing a system with these capabilities are many, and the research and development in solving these problems should further the art of high accuracy pulse-based positioning.

Mechanosensation in cells is a well known phenomenon that is associated with cellular responses to force. Our knowledge of the trigger mechanism of this phenomenon is, however, limited. Earlier studies in this field have used atomic simulations, which although being accurate, are limited in their feasibility in multi-length scenarios like a mechanosensitive channel that undergoes micro-level changes in the composition of the protein to cause a macro-level change in the state of a biological structure such as the muscle. Finite Element Analysis has been used in various engineering fields to study the mechanical response of complex structures. The current study is a step in utilizing the phenomenal capabilities of Finite Element Analysis in developing and studying a 3D model (Membrane-Channel) of a mechanosensitive channel of large conductance (MscL). A simplified CAD structure of Mycobacterium tuberculosis (TbMscL) was developed in the first stage of this study. The authenticity of this model was tested by applying two types of loading conditions, namely (i) In-plane stretch and (ii) Out-of-plane bending. The results obtained from the first step of analysis are in accordance with previous experimental data, which elucidates the fact that tension within the membrane guides the gating mechanism of the channel and not the curvature of the membrane. The second stage of the analysis involved the use of the same model to study the disease commotio cordis. This was achieved by calculating the loading conditions during the onset of the condition in the human heart and then transferring those conditions to the Membrane-Channel model developed in the first stage. The result showed that although the channel did not fully open but there was a significant change in the channel‟s radius that might cause the flow of ions, thereby changing the state of the channel. It is anticipated that this model will help future research in areas that conventionally have been difficult to model.