Patchy particles are patterned particles with at least one well-defined patch that can have highly directional and strongly anisotropic interactions with other particles or surfaces. Multiple theoretical studies point to interesting self-assembly of these particles into superstructures and, as a result, a multitude of possible applications. However, reliable synthetic methods for patchy particles, especially at the sub-micron level, are still a challenge and an active area of research. This work presents a novel synthesis route for making patchy particles at the sub-micron level that involves the use of capillary condensation. Colloidal silica particles of various sizes were synthesized and ordered into closely-packed arrays via evaporative self-assembly. Various chemical agents were capillary condensed into the voids of this assembly which, due to the face-centered cubic structure of the crystallized colloidal silica, produced distinct \patches" on the particle surface. The patches on these particles were successfully functionalized with gold nanoparticles. This method was shown to provide control over the patch size by modifying the silica particle radius, which thermodynamically changes the amount of capillary condensation. The patchy nature of the resultant particles was confirmed using infrared spectroscopy, scanning electron and optical microscopies, energy dispersive x-ray analysis and zeta potential measurements.

The concept of tiny machines capable of selectively transporting particles between two compartments by Brownian motion dates back to the 19th century when James Clerk Maxwell pondered the significance of a hypothetical ‘sorting demon’ being able to perform such a task adiabatically. This thesis report the design, synthesis and operation of a compartmentalized molecular machine in which the distribution of a Brownian particle, the macrocycle in a rotaxane, is controlled by using the lightinduced transmission of information to lower a kinetic barrier according to the location of the particle. For an ensemble of such machines the particle distribution is driven further and further away from equilibrium, providing a non-adiabatic realization of Maxwell’s pressure demon in molecular form. The nanomachine does not break the Second Law of Thermodynamics because the energy cost of the information transfer is met by externally supplied photons. As the molecular structure can be understood in chemical terms, it is possible in this experimental system to pin-point precisely how information is traded for energy. Intriguingly, the chemical mechanism can also be understood in terms of game theory. This is the first example of a synthetic molecular machine designed to operate via an information ratchet mechanism, where knowledge of the object’s position is used to control its transport away from equilibrium.

Filamentous bulking is a problem that has long plagued activated sludge (AS) wastewater treatment plants (WWTPs). Much research has looked at its prevention and control but there is still no solution. The sludge microbiological community is very complex and there are many factors that can affect bulking. Clay addition in scaled-down activated sludge systems was investigated at concentrations of 0.4, 2.0 and 5.0 g/L along with sequencing batch reactor (SBR) parameters when run with a synthetic wastewater (SWW). The 5.0g/L concentration exhibited positive results on settling in the form of modified SVI but appeared to cause no reduction in filament length. These preliminary investigations indicate that clay may help improve sludge settling but make no difference in the abundance of filamentous microorganisms. The SBRs exhibited trends in regards to running systems with a synthetic wastewater. A loss of volatile suspended solids (VSS), coupled with increase in sludge volume index (SVI), suggested a link between lack of non-VSS and settling ability. This has implications in the importance of non-VSS such as grit or clay in research performed using SWWs.

This creative project was a study of combining large-scale ceramics and polymer resin (Brandname: Bondo) vessels. This project came out of an earlier experiment combining metals and resin, and therefore the interest in combining clay and resin was a natural transition for me, since ceramics was my major focus. I had a definite interest in the challenge of marrying two very different materials; clay and resin, and by using this combination I would make vessels that were visually successful and engaged the viewer.The technical basis for all of the ceramic elements of the vessels is wheel throwing. The molds made for the resin parts of the piece were wheel thrown as well. This project was very unique and there was little documented technical information for me to rely on other than personal experimentation and ceramic knowledge.The visual elements of clay with its opaqueness, and resin with its translucent qualities offered great visual contrast. The use of color contrast between warm and cool colors became an important element of the pieces.From an expressive standpoint, I feel the form of the vessels is viewed as solid and confrontational, yet very compelling. The smooth surfaces and the lush vibrant colors compel the viewer to examine the forms. The pieces are commanding in size and strength, so the viewer is somewhat hesitant to view for too long. There is a visual push/pull, a contrast between these simple yet over-scaled layered forms that evokes an emotional push/pull as well. / Department of Art

Cyanobacteria are solar-powered cell factories that can be engineered to supply us with renewable fuels and chemicals. To do so robust and well-working biological parts and tools are necessary. Parts for controlling gene expression are of special importance in living systems, and specifically promoters are needed for enabling and simplifying rational design. Synthetic biology is an engineering science that incorporates principles such as decoupling, standardization and modularity to enable the design and construction of more advanced systems from simpler parts and the re-use of parts in new contexts. For these principles to work, cross-talk must be avoided and therefore orthogonal parts and systems are important as they are decoupled by definition. This work concerns the design and development of biological parts and tools that can enable synthetic biology in cyanobacteria. This encompasses parts necessary for the development of other systems, such as vectors and translational elements, but with a focus on transcriptional regulation. First, to enable the development and characterization of promoters in different cyanobacterial chassis, a broad-host-range BioBrick plasmid, pPMQAK1, was constructed and confirmed to function in several cyanobacterial strains. Then, ribosome binding sites, protease degradation tags and constitutive, orthogonal promoters were characterized in the model strain Synechocystis PCC 6803. These tools were then used to design LacI-regulated promoter libraries for studying DNA-looping and the behaviour of LacI-mediated loops in Synechocystis. Ultimately, this lead to the design of completely repressed LacI-regulated promoters that could be used for e.g. cyanobacterial genetic switches, and was used to design a destabilized version of the repressed promoter that could be induced to higher levels. Further, this promoter was used to implement an orthogonal transcriptional system based on T7 RNAP that was shown to drive different levels of T7 promoter transcription depending on regulation. Also, Gal4-repressed promoters for bacteria were engineered and examined in Escherichia coli as an initial step towards transferring them to cyanobacteria. Attempts were also made to implement a light-regulated one-component transcription factor based on Gal4. This work provides a background for engineering transcription and provides suggestions for how to develop the parts further.

A primary aim of Synthetic Biology is the design and implementation of biological systems that perform engineered functions. However, the assembly of double-stranded DNA molecules is a major barrier to this progress, as it remains time consuming and laborious. Here I present three improved methods for DNA assembly. The first is based on, and makes use of, BioBricks. The second method relies on overlap-extension PCR to assemble non-standard parts. The third method improves upon overlap extension PCR by reducing the number of steps and the time it takes to assemble DNA. Finally, I show how the PCR-based assembly methods presented here can be used, in concert, with in vivo homologous recombination in yeast to assemble as many as 19 individual DNA parts in one step. These methods will also be used to assemble an incoherent feedforward loop, gene regulatory network.

<p>Promising applications in the design of various biological systems hold critical implications as heralded in the rising field of synthetic biology. But, to achieve these goals, the ability to synthesize in situ DNA constructs of any size or sequence rapidly, accurately and economically is crucial. Today, the process of DNA oligonucleotide synthesis has been automated but the overall development of gene and genome synthesis technology has far lagged behind that of gene and genome sequencing. This has meant that numerous ideas go unfulfilled due to scale, cost and impediments in the quality of DNA due to synthesis errors. </p><p>This thesis presents the development of a multi-tool ensemble platform targeted at gene synthesis. An inkjet oligonucleotide synthesizer is constructed to synthesize DNA microarrays onto silica functionalized cylic olefin copolymer substrates. The arrays are married to microfluidic wells that provide a chamber to for enzymatic amplification and assembly of the DNA from the microarrays into a larger construct. Harvested product is then amplified off-chip and error corrected using a mismatch endonuclease-based reaction. This platform has the potential to be particularly low-cost since it employs standard phosphoramidite reagents and parts that are cheaper than optical and electrochemical systems. Genes sized 160 bp to 993 bp were successfully harvested and, after error correction, achieved up to 94% of intended functionality.</p> / Dissertation

This thesis explores fundamental design strategies in the field of synthetic ion channel research from two different perspectives. In the first part the synthesis of complex, shape persistent and thermodynamically stable structures based on metal-ligand self-assembly is explored. The second part examines transport systems with dynamic transport behavior in response to chemical inputs which more closely mimic the dissipative assembly of Natural ion channels. In part one, two model systems, the ethylenediamine palladium(II) - 4,4’-bipyridine squares of Fujita and the trimeric bis(terpyridine) - iron(II) hexagonal macrocycles of Newkome, were targeted for structural modification towards becoming transport competent systems via improving the membrane partitioning characteristics of the final coordination compounds by increasing their lipophilicity. Modifications of the Fujita system involved the generation of two lipophilic 4,4’-bipyridines with addition of lipophilic groups of 13 and 17 carbon long alkyl chains respectively at the 3 and 3’ positions. After pursuing multiple unsuccessful synthetic routes the successful syntheses afforded the final lipophilic 4,4’-bipyridines in overall yields of 19 to 21% over two synthetic steps. Mixtures of the newly generated lipophilic 4,4’-bipyridines with a known lipophilic ethylenediamine palladium(II) “corner” exhibited evidence of self-assembly from NMR spectroscopy experiments however attempts at further characterization by ESI-MS and X-ray crystallography were unproductive. The putative self-assembled structures were inactive in HPTS vesicle assays but showed erratic conductance activity in bilayer clamp experiments. However, the magnitude of the conductance observed was not indicative of the passage of ions through the internal pore of the square complex. Modifications to the Newkome hexagons were aimed at generating overall neutral assemblies with external lipophilic groups. These modifications involved imparting a net -2 charge to the ligand via modifications to the terminal tridentate ligands so that upon coordination to octahedral metal centers in the +2 oxidation state the overall hexagonal complex would be neutrally charged. Two bis-polydentate ligands were generated; a dissymmetric molecule comprising one terpyridine and one dipicolinate tridentate ligand (TERPY-DPA) and a symmetrical molecule comprising two 2,2’-bipyridine-6-carboxylate tridentate ligands (BIPYA-BIPYA). The successful syntheses provided the desired trimethylsilylethyl ester protected compounds in yields of 9.2 and 7.5 % over 6 and 8 total synthetic steps for TERPY-DPA and BIPYA-BIPYA respectively. A new approach to metal-ligand complex formation by concomitant fluoride deprotection and assembly was demonstrated with a monomeric complex. Polymetallic complexes formed with a variety of transition metals based on colorimetric changes but the products were very intractable and resisted full structural or transport characterization. Part two develops a system potentially capable of exhibiting dissipative assembly of active transporters. A library of six thioester containing compounds structurally related to known active oligoester compounds was synthesized. The successful syntheses provided the desired compounds in overall yields of 1.0 to 17.7% over 11 to 13 total synthetic steps. The intramolecular cyclization - truncation and thioester exchange reactions central to the dissipative assembly strategy were explored using a model compound. The full length compounds showed transport activity via the HPTS vesicle assay that was significantly below that of the lead compound. Bilayer clamp experiments however, revealed significant transport activity for both the full length as well as the truncated thiol molecules. In the case of the latter the transport events had exceedingly high conductivity for such a small molecule. This unexpected activity for both the full length and truncated compounds, although different, prevented a full implementation of dissipative assembly of transport. / Graduate / 0490 / 0485 / 0494

The Brazilian Pantanal, a continuous tropical wetland located in the center of South America, has been recognized as one of the largest and most important wetland ecosystems globally. The Pantanal exhibits a high biodiversity of flora and fauna species, and many threatened habitats. The spatial distribution of these habitats influence the distribution, abundance and interactions of animal species, and the change or destruction of habitat may cause alteration of key biological processes. The Pantanal may be divided into several distinct subregions based on geology and hydrology: flooding in these subregions is distinctly seasonal, but the timing, amplitude and duration of inundation vary considerably as a result of both the delayed release of floodwaters and regional rainfall patterns. Given the ecological importance of the Pantanal wetland ecosystem, the primary goal of this research was to utilize a dual season set of L-band (ALOS/PALSAR) and C-band (RADARSAT-2 and ENVISAT/ASAR) imagery, a comprehensive set of ground reference data, and a hierarchical object-oriented approach. This primary goal was achieved through two main research tasks. The first task was to define the diverse habitats of the Lower Nhecolândia subregion of the Pantanal at both a fine spatial resolution (12.5 m), and a relatively medium spatial resolution (50 m), thus evaluating the accuracy of the differing spatial resolutions for land cover classification of the highly spatially heterogeneous subregion. The second task was to define on a regional scale, using the 50 m spatial resolution imagery, the wetland habitats of each of the hydrological subregions of the Pantanal, thereby producing a final product covering the entire Pantanal ecosystem. The final classification maps of the Lower Nhecolândia subregion resulted in overall accuracies of 83% and 72% for the 12.5 m and 50 m spatial resolutions, respectively, and defined seven land cover classes. In general, the highest degree of confusion for both fine and medium resolution classifications related to issues of 1) scale of habitats, for instance, capões, cordilheiras, and lakes, in relation to spatial resolution of the imagery, and 2) issues relating to variable flooding patterns in the subregion, and 3) arbitrary class membership rules. The 50 m spatial resolution classification of the entire Pantanal wetland resulted in an overall accuracy of 80%, and defined ten land cover classes. Given the analysis of the comparison of fine and relatively medium spatial resolution classifications of the Lower Nhecolândia subregion, I conclude that significant improvements in accuracy can be achieved with the finer spatial resolution dataset, particularly in subregions with high spatial heterogeneity in land cover. The produced habitat spatial distribution maps will provide vital information for determining refuge zones for terrestrial species, connectivity of aquatic habitats during the dry season, and crucial baseline data to aid in monitoring changes in the region, as well as to help define conservation strategies for habitat in this critically important wetland. / Graduate / 0366 / tevans@uvic.ca

This thesis describes the development of an ion channel system exhibiting dissipative assembly characteristics. In this system an active transporter based on an oligoester fragment terminated in a thioester of 6-aminohexanoic acid (HO2C-Hex-Adip-OctS-Hex-NH2) undergoes thioester cleavage to form a thiol terminated oligoester (HO2C-Hex-ADip-Oct-SH). This fragment was expected to be inactive for ion transport but previous work showed high activity in planar bilayer experiments. In this work, the high activity was shown to be due to the oxidized form of the thiol, the disulfide HO2C-ADip-Oct-SS-Oct-ADip-Hex-CO2H. Air oxidation was found to be quite rapid for the thiol based on ESI-MS (negative ion) and HPLC analysis. Under anaerobic conditions, the thiol fragment was an inactive species for ion transport. In situ air oxidation initiated transport activity associated with the disulfide. The transporter HO2C-Hex-Adip-Oct-Hex-NH2 was active in planar bilayer experiments and was compared to the disulfide via activity grids. The activity of these two compounds was shown to be distinct from each other by conductance and channel duration differences. The activity of HO2C-Hex-Adip-Oct-Hex-NH2 was shown to die off in a period of 30 minutes at pH 8.2. Techniques were developed to stimulate and monitor activity and bilayer quality so that an inactive condition could be confirmed. The addition of Pr-S-Hex-NH3+-Cl as a fuel was shown to extend the activity of HO2C-Hex-Adip-Oct-Hex-NH2 by eight-fold in 1M CsCl electrolyte. Previous work had established the capability of thioester exchange reactions by a reaction between Pr-S-Hex-NH3+-Cl and benzyl thiol in a homogenous solution. The extended activity indicated that the same process may occur in a heterogeneous bilayer system. An inactive system created by the die-off in activity of HO2C-Hex-Adip-Oct-S-Hex-NH2 was treated with Pr-S-Hex-NH3+-Cl to regenerate activity. This cycle could be repeated once the activity died off again. All these findings are consistent with the dissipative assembly of a membrane transport system. / Graduate / 0490