Alginate is an exopolysaccharide secreted by Pseudomonas aeruginosa and is a major component of biofilms that infect the lungs of cystic fibrosis patients. Ten proteins have been implicated in alginate polymerization, modification and export, and are believed to assemble into a multi-protein complex that spans the cell envelope and coordinates the synthesis and secretion of alginate. AlgK is a protein encoded in the alginate biosynthetic operon, which is required for the secretion of high-molecular weight alginate. This study describes structural and functional studies of AlgK to improve our understanding of AlgK’s role in alginate biosynthesis. To shed light on the function of AlgK, C14-palmitic acid labeling and sucrose gradient fractionation studies confirmed that AlgK is an outer membrane lipoprotein. Cellular fractionation experiments also found that AlgK is involved in the proper localization of AlgE, the alginate secretion pore in the outer membrane. The structure of AlgK was determined to 2.5 Å resolution by X-ray crystallography and revealed that the protein folds into 22 alpha-helices that pack into a flexible right-handed solenoid. Closer examination of the amino acid sequence revealed that AlgK carries 9.5 tetratricopeptide repeat (TPR)-like elements. Given the role that TPR motifs generally play in protein-protein interaction and the assembly of multi-protein complexes, the presence of these motifs in AlgK suggests that it can bind to one or more proteins. Based on the results presented in this study, we propose that AlgK acts as a scaffold for the assembly of the alginate secretion complex. By mapping highly conserved residues onto the surface of our model, three putative sites of protein-protein interaction were identified. We hypothesize that the N-terminus of AlgK binds to AlgE in the outer membrane, and the C-terminus of AlgK binds to periplasmic and/or inner membrane Alg proteins, thereby acting as a linker between the inner and outer membrane components of the alginate biosynthetic complex. We further hypothesize that together AlgE and AlgK constitute a novel exopolysaccharide secretin. The alginate biosynthetic complex appears to be distinct from the canonical capsular polysaccharide systems currently described.

<p><i>Lactococcus lactis,</i> prolidase (<i>Lla</i>prol) hydrolyzes Xaa-Pro dipeptides. Since Xaa-Pro is known as bitter peptides, <i>Lla</i>prol is potentially applicable to reduce bitterness of fermented foods. <i>Lla</i>prol shows allosteric behaviour and substrate inhibition, which are not reported in other prolidases. Computer models of <i>Lla</i>prol based on an X-ray structure of non-allosteric <i>Pyrococcus furiosus</i> prolidase showed that a loop structure (Loop^32-43) is located at the interface of the protomers of this homodimeric metallodipeptidase. This study investigated roles of four charged residues (Asp³⁶, His³⁸, Glu³⁹, and Arg⁴⁰) of Loop^32-43 in <i>Lla</i>prol using a combination of kinetic examinations of ten mutant enzymes and their molecular models. Deletion of the loop structure by Î36-40 mutant resulted in a loss of activity, indicating Loop^32-43 is crucial for the activity of <i>Lla</i>prol. D36S and H38S exhibited 96.2 % and 10.3 % activity of WT, whereas little activities (less than 1.0 % of WT activity) were observed for mutants E39S, D36S/E39S, R40S, R40E, R40K and H38S/R40S. These results implied that Glu³⁹ and/or Arg⁴⁰ play critical role(s) in maintaining the catalytic activity of <i>Lla</i>prol. These observations suggested that the loop structure is flexible and this attribute, relying on charge-charge interactions contributed by Arg⁴⁰, Glu³⁹ and Lys¹⁰⁸, is important in maintaining the activity of <i>Lla</i>prol. When the loop takes a conformation close to the active site (closed state), Asp³⁶ and His³⁸ at the tip of the loop can be involved in the catalytic reaction of <i>Lla</i>prol. The two active mutant prolidases (D36S and H38S) resulted in modifications of the unique characteristics; the allosteric behaviour was not observed for D36S, and H38S <i>Lla</i>prol showed no substrate inhibition. D36E/R293K, maintaining the negative charge of position 36 and positive charge of position 293, still possessed the allosteric behaviour, whereas the loss of the charges at these positions (D36S of this study and R293S of a previous study (Zhang et al., 2009 BBA-Proteins Proteom 1794, 968-975) eliminated the allosteric behaviour. These results indicated the charge-charge attraction between Asp³⁶ and Arg²⁹³ is important for the allostery of <i>Lla</i>prol. In the presence of either zinc or manganese divalent cations as the metal catalytic centre, D36S and H38S enzymes also showed different substrate preferences from WT <i>Lla</i>prol, implying the influence of Asp³⁶ and His³⁸ on the substrate binding. D36S and H38S also showed higher activities at pH 5.0 to 6.0, in which range WT <i>Lla</i>prol steeply decreased its activity, indicating Asp³⁶ and His³⁸ are involved in the active centre and influence the microenvironment of catalytic His²⁹⁶. The above observations are attributed to modifications of their local structure in the active centre since the temperature dependency and thermal denaturing temperature indicated little effects on the overall structure of the <i>Lla</i>prol mutants.</p> <p>From these results, we concluded that the unique behaviours of <i>Lla</i>prol are correlated to Loop^32-43 and Asp³⁶ and His³⁸ on it. When Loop^32-43 takes a closed conformation, Asp³⁶ interacts with Arg²⁹³ via charge-charge attraction to form an allosteric subsite. The saturation of the allosteric site with substrates further allowed the communications of His³⁸ with S₁ site residues to complete the active site. When the substrate concentration becomes higher than it is required to saturated productive S₁' site, His³⁸, Phe¹⁹⁰ and Arg²⁹³ would resemble the residue arrangement of S₁' site residues (His²⁹², Tyr³²⁹, and Arg³³⁷) and bind to the proline residue of substrates. This non-productive binding would prevent the conformational change of Loop^32-43, which further results in the substrate inhibition. For further confirmation of this mechanism, crystallographic studies will be conducted. In this thesis, we have indentified the conditions to produce crystals of <i>Lla</i>prol proteins.</p>

Amorphous selenium (a-Se) and its alloys are important photoconductor materials used in direct conversion flat panel digital x-ray detectors. The performance of these detectors is determined, in part, by the electronic transport properties of the a-Se photoconductor layer namely, the charge carrier mobility m and the deep trapping lifetime t. The product of the mobility and the lifetime mt, referred to as the charge carrier range, determines the average distance that photo-generated charge will travel before being removed from the transport band by deep localized states in the mobility gap of the semiconductor. The loss of carriers to these deep states reduces the amount of charge collected per unit of x-ray exposure, and, hence, limits the x-ray sensitivity of the detector. Two experimental techniques that may be used to measure the transport properties of holes and electrons in high resistivity semiconductors are described in this thesis. The Time-of-Flight (TOF) transient photoconductivity technique is used to evaluate the charge carrier mobility by measuring the time required for the charge carriers to transit a fixed distance under the influence of an applied electric field. The Interrupted-Field Time-of-Flight (IFTOF) technique is used to determine the charge carrier deep trapping time; the drift of the injected carriers is temporarily interrupted at a position in the sample by removing the applied field. When the field is reapplied the number of charge carriers has decreased due to trapping events. The carrier lifetime is determined from the dependence of the fraction of recovered charge carriers before and after the interruption with the interruption time. <p> TOF and IFTOF measurements were carried out on a number of samples of vacuum deposited selenium alloy x-ray photoconductors. Device quality photoconductor films are fabricated by evaporating a-Se source material that has been alloyed with a small quantitiy of As (~0.3 at. %) and doped with a halogen (typically Cl) in the p.p.m. range. The dependence of the carrier range on the composition of the photoreceptor film was accurately measured using both TOF and IFTOF measurements. It was found that the transport properties of the film could be controlled by suitably adjusting the composition of the alloy. Combined IFTOF and TOF measurements were also performed on several samples to examine the effects of trapped electrons on the hole transport properties in a-Se films. It was found that drifting holes recombine with the trapped electrons, and that this process could be described by a Langevin recombination process. This finding is important for the correct modeling of amorphous selenium digital x-ray detector designs. Finally, the effects of x-ray exposure on a-Se films were examined. A temporary reduction in the effective hole lifetime was observed due to an increase in the number of hole capture centers following an x-ray exposure. The capture coefficient between free holes and the x-ray induced hole capture centers was measured using combined TOF and IFTOF measurements. It was shown that this capture process was governed by the Langevin recombination mechanism. From these observations it was concluded that trapped electrons from a previous x-ray exposure act as recombination centers for subsequently generated holes, thereby reducing the effective hole lifetime in the sample.

Living organisms constantly respond to changing environmental conditions, and some changes can be far from optimal for many organisms. Insects represent the majority of species in many ecosystems and play an important role in bioaccumulation and biotransformation of environmental contaminants such as selenium and arsenic. Some insectivorous predators feeding on these insects are highly sensitive to such elements resulting in reduced growth, reproductive failures and low population numbers. The mechanisms of selenium and arsenic uptake through the food chain are poorly understood. The determination of chemical speciation is a prerequisite for a mechanistic understanding of a contaminants bioavailability and toxicity to an organism. Synchrotron-based X-ray absorption spectroscopy was used to identify the chemical form of selenium and arsenic in insects in both the field and laboratory conditions. Insects living in streams near Hinton, Alberta affected by coal mine activities were examined for selenium speciation. Results showed higher percentages of inorganic selenium in primary consumers, detritivores and filter feeders than in predatory insects. Selenides and diselenides constitute a major fraction of selenium in these insects. In another field setting, speciation of selenium was studied in insects attacking selenium hyperaccumulating plant <i>Astragalus bisulcatus</i>. The effect of selenate and arsenate alone and the combined effects of selenate and arsenate on insects and parasitoids were monitored using a laboratory-reared moth (<i>Mamestra configurata</i>). Hosts receiving selenium biotransformed selenate to organic selenides and diselenides, which were transferred to the parasitoids in the third trophic level. Arsenic fed larvae biotransformed dietary arsenate to yield predominantly trivalent arsenic coordinated with three aliphatic sulfurs. Larvae receiving arsenate used a novel six-coordinated arsenic form as an excretory molecule in fecal matter and cast skin. X-ray absorption spectroscopy imaging with micro X-ray fluorescence imaging on selenate and arsenate fed larvae revealed highly localized selenium and arsenic species, zinc and copper within the gut. The results provide insights into how the insects cope with their toxic cargo, including how selenium and arsenic are biotransformed into other chemical forms and how they can be eliminated from the insects. The implication of selenium and arsenic species in the diet of predators and detritivores is discussed.

For high-resolution X-ray imaging scintillator applications, we have prepared and optically characterized divalent samarium doped fluorochlorozirconate (FCZ:Sm2+) glasses and glass-ceramics. Sm2+ doped FCZ glasses were obtained by adding a reducing agent, NaBH4 into the initial melt to convert some of the Sm3+ to Sm2+. However, the Sm2+ concentration at most was estimated to be only approximately 0.003 %. The as-prepared glass samples were further heat treated to obtain glass-ceramics; the nucleation and growth of BaCl2 nanocrystals were confirmed by powdered X-ray diffraction (XRD) experiments. Depending on the heat treatment conditions (temperature and time), the average nanocrystal size varies from 8 to 170 nm, and the sample contains BaCl2 nanocrystals with the orthorhombic and/or hexagonal structure. The optical absorption spectra for our glass-ceramic samples suggested the substitution of Sm2+ ions into the BaCl2 lattice site. The FCZ:Sm2+ glass-ceramics samples showed strong fluorescence in the red region of spectrum (approximately 8 times that of an as-prepared glass), and the transparency can be very high (transmittance > 80 % for samples with thickness about 0.5 mm) and can be equivalent to that of an as-prepared glass . These two results promise potential as a high-resolution X-ray scintillator due to the emission wavelength range and high transparency. Extensive studies of photoluminescence (PL) spectra at low temperatures (12 -- 200 K) for FCZ:Sm2+ glass-ceramics suggested useful indicators of the crystal structure and average size of embedded BaCl2 nanocrystals. A detailed analysis of the optical spectra has lead to the identification of the origin of the emission peaks and the location of Sm ions at specific crystallographic sites. X-ray induced luminescence (XL) studies have suggested a strong dependence of the fluorescence intensity on the concentration of Sm2+ ions. In addition, for more efficient fluorescence, a sample should be heat treated in a hydrogen containing atmosphere (e.g. H2 + Ar gas), and the heat treatment conditions should be such that the nanocrystals grow in the hexagonal structure.

Alginate is a popular biomaterial in tissue engineering. When crosslinked with calcium ions (Ca2+), alginate forms a hydrogel which provides necessary mechanical support as a scaffold. The material properties as well as the biological properties of alginate scaffold are of great importance. In this thesis, the aim is to use traditional methods, such as scanning electron microscopy (SEM) and light microscopy, and emerging X-ray imaging techniques, such as micro-computed tomography (micro-CT) and synchrotron radiation (SR) X-ray imaging, to characterize the alginate scaffolds. Firstly, the material properties of freeze-dried alginate scaffolds were evaluated using micro-CT, as it is a non-destructive and non-invasive imaging method, and can provide three-dimensional information. Alginate scaffolds made with different sodium alginate concentrations and frozen to different temperatures were scanned and analyzed in micro-CT. Results indicated that lower freezing temperature and higher sodium alginate concentration lead to smaller pore size and porosity. Secondly, cell culture experiments were carried out to study the biological properties and the interactions of alginate hydrogel with cells. A Schwann cell line was either blended with alginate solution before crosslinking with calcium chloride (CaCl2) or put around alginate gel in the culture dish. Light microscopy of sectioned slices showed that cells surrounding the alginate gel could not grow into the gel, while cells blended with alginate solution before crosslinking could proliferate inside the hydrogel. Cells grown inside a thin slice of alginate gels appeared to be in better condition and were larger in size and also grew in clusters. Thirdly, in order to image soft tissue buried inside alginate gels, such as brain slices, novel imaging methods based on synchrotron radiation (SR) were applied, such as absorption and phase contrast imaging, diffraction-enhanced imaging (DEI) and also combined with computed tomography (CT). Synchrotron-based monochromatic X-ray imaging proved to be good at distinguish objects of similar density, especially biological soft tissue samples, even without any staining material, such as osmium tetroxide (OsO4). These three pieces of research work show the potential in applying the emerging X-ray imaging in soft tissue engineering.

There are a significant number of microwave applications, where improvement of such qualities as manufacturing costs, size, weight, power consumption, etc. have attracted much research interest. In order to meet these requirements, new technologies can be actively involved in fabrication of microwave components with improved characteristics. One such fabrication technology is called LIGA (a German acronym with an English translation of lithography, electroforming, and moulding) that allows fabrication of high aspect ratio (tall) structures, and only recently is receiving growing attention in microwave component fabrication.<p> The characteristics of high aspect ratio microstrip and coplanar waveguide (CPW) transmission lines are investigated in this thesis. Very low impedance high aspect ratio CPW transmission lines can be realized. A high aspect ratio microstrip folded half wavelength open loop resonator is introduced. Effective configurations for external and bypass gap coupling with open loop resonators are given. Filters with transmission zeros in the stopband, consisting of high aspect ratio single mode open loop resonators are presented to demonstrate the advantages of high aspect ratio structures in realizing lower external quality factors or tight coupling. The transmission zeros are created by novel coupling routings. Some of the filters are fabricated and the filter responses are measured to validate high aspect ratio coupling structures. High aspect ratio diplexers with increased channel isolation are also designed by appropriately combining filters with transmission zeros.<p> A wideband bandpass filter design method, based on the electromagnetic bandgap (EBG) concept is introduced in this thesis. The wideband filters are miniaturized as a result of using the EBG concept in design. An EBG based wideband filter consisting of unit cells that are realized by using high aspect ratio CPW stepped impedance resonators is also presented. The main advantage of this approach is that the high aspect ratio CPW structures make short unit cells practically realizable, resulting in compact filter structure.

The Drosophila mutant fumble has a defect in mitochondrially targeted pantothenate kinase (PANK) and exhibits a movement disorder in the females. The human disease pantothenate kinase associated neurodegeneration (PKAN) has the same genetic defect and a neurodegenerative phenotype as well as iron accumulation in the brain. We have found that fumble females accumulate almost 2 fold more iron in the heads than wildtype. Dietary iron supplementation increases the iron accumulation in the heads further. The small isoform of malvolio (MVL), a homologue of mammalian NRAMP iron transporters, is expressed in the heads of flies. Its expression is upregulated in the fumble females, as well as in dietary iron supplemented wildtype flies. Unlike in the wildtype, dietary iron supplementation leads to a downregulation of MVL in the fumble flies. Although iron levels were elevated in fumble, ferritin expression was relatively unchanged and remained unchanged in the heads of fumble and wildtype with dietary iron supplementation. <p>The Drosophila mutant malvolio was used to determine how iron metabolism is affected when the MVL gene is defective. Iron levels were unchanged in malvolio relative to its parental strain (w1118) with or without dietary iron supplementation. Despite similar iron levels, a small decrease in ferritin expression was found in malvolio relative to w1118, and dietary iron increased ferritin expression in malvolio. However ferritin expression decreased in the parental strain of malvolio after iron supplementation. <p>Most of the iron in the Drosophila heads was in the form of goethite and ferrihydrite. The presence of iron oxides implies that this iron is in a mineralized storage form, likely ferritin. Dietary iron supplementation induced the appearance of ferric phosphates in fumble, malvolio, and wildtype. The subcellular location of this iron is unknown. It may be non-transferrin bound iron in the hemolymph, or a cytosolic intermediate in the labile iron pool. Also of note was the presence of transferrin-bound iron in wildtype heads on normal diet that was not seen after iron supplementation or in the heads of the fumble mutant. The presence in fumble of the kind of ferrihydrite characteristic of the mitochondrial protein frataxin may indicate that iron is accumulating in mitochondria.<p>The upregulation of MVL in the fumble mutant is of significant interest because it is the first protein involved in iron metabolism found to be altered with mitochondrial PANK deficiency. A disruption in MVL could be relevant to the brain iron accumulation in fumble and could be a treatment target for human PKAN.

Alginate is an exopolysaccharide secreted by Pseudomonas aeruginosa and is a major component of biofilms that infect the lungs of cystic fibrosis patients. Ten proteins have been implicated in alginate polymerization, modification and export, and are believed to assemble into a multi-protein complex that spans the cell envelope and coordinates the synthesis and secretion of alginate. AlgK is a protein encoded in the alginate biosynthetic operon, which is required for the secretion of high-molecular weight alginate. This study describes structural and functional studies of AlgK to improve our understanding of AlgK’s role in alginate biosynthesis. To shed light on the function of AlgK, C14-palmitic acid labeling and sucrose gradient fractionation studies confirmed that AlgK is an outer membrane lipoprotein. Cellular fractionation experiments also found that AlgK is involved in the proper localization of AlgE, the alginate secretion pore in the outer membrane. The structure of AlgK was determined to 2.5 Å resolution by X-ray crystallography and revealed that the protein folds into 22 alpha-helices that pack into a flexible right-handed solenoid. Closer examination of the amino acid sequence revealed that AlgK carries 9.5 tetratricopeptide repeat (TPR)-like elements. Given the role that TPR motifs generally play in protein-protein interaction and the assembly of multi-protein complexes, the presence of these motifs in AlgK suggests that it can bind to one or more proteins. Based on the results presented in this study, we propose that AlgK acts as a scaffold for the assembly of the alginate secretion complex. By mapping highly conserved residues onto the surface of our model, three putative sites of protein-protein interaction were identified. We hypothesize that the N-terminus of AlgK binds to AlgE in the outer membrane, and the C-terminus of AlgK binds to periplasmic and/or inner membrane Alg proteins, thereby acting as a linker between the inner and outer membrane components of the alginate biosynthetic complex. We further hypothesize that together AlgE and AlgK constitute a novel exopolysaccharide secretin. The alginate biosynthetic complex appears to be distinct from the canonical capsular polysaccharide systems currently described.

The Canadian Penning Trap (CPT) mass spectrometer at the Argonne National Laboratory makes precise mass measurements of both stable and unstable nuclides. To date, more than 60 radioactive isotopes having half-lives as short as one second have been measured with the CPT with a mass precision approaching 10 ppb. This thesis will present measurements made of nuclides along the rp-process path, which describes a process resulting from a series of rapid proton-capture reactions in an astrophysical environment. One possible site for the rp-process mechanism is an x-ray burst which results from the rapid accretion of hydrogen and helium from one star onto the surface of its neutron star binary companion. Mass measurements are required as key inputs to network calculations used to describe the rp-process in terms of the abundances of the nuclides produced, the light-curve profile of the x-ray bursts, and the energy produced. This thesis will describe the CPT apparatus, explain the method used to make precise mass measurements, and present the masses of the "waiting-point" nuclides ⁶⁸Se and ⁶⁴Ge. The mass measurement results, when used in x-ray burst models, confirm both ⁶⁸Se and ⁶⁴Ge as waiting-point nuclides which delay the rp-process by approximately 30 s and 7 s respectively. / October 2005