<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

<p>Synthetic biology facilitates both the design and fabrication of biological components and systems that do not already exist in the natural world. From an engineering point of view, synthetic biology is akin to building a complex machine by assembling simpler parts. Complex genetic machines can also be built by a modular and rational assembly of simpler biological parts. These biological machines can profoundly affect various cellular processes including the transcriptional machinery. In this thesis I demonstrate the utilization of biological parts according to synthetic biology principles to solve three distinct transcription-level problems: 1) How to efficiently select for transgene excision in induced pluripotent stem cells (iPSCs)? 2) How to eliminate transposase expression following piggyBac-mediated transgenesis? 3) How to reprogram cell lineage specification by the dCas9/gRNA transactivator-induced expression of endogenous transcription factors? </p><p>Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In the first study, we design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1&#945; promoter; b) enables easy determination of lentiviral titer; c) enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows for transgene excision in a colony format. With our design, a reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX) in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies.</p><p>Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed "cut-and-paste" mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In the second study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the transposase element is deactivated after transposition. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells.</p><p>Genome editing by the efficient CRISPR/Cas9 system shows tremendous promise with ease of customization and the capability to multiplex distinguishing it from other such technologies. Endogenous gene activation is another aspect of CRISPR/Cas9 technology particularly attractive for biotechnology and medicine. However, the CRISPR/Cas9 technology for gene activation leaves much room for improvement. In the final study of this thesis we show that the fusion of two transactivation (VP64) domains to Cas9 dramatically enhances gene activation to a level that is sufficient to achieve direct cell reprogramming. Targeted activation of the endogenous Myod1 gene locus with this system leads to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. </p><p>In conclusion, this dissertation demonstrates the power of utilizing biological parts in a rational and systematic way to rectify problems associated with cell fate reprogramming and transposon-based gene delivery. Through design of genetic constructs aided by synthetic biology principles, I aspire to make contributions to the related fields of cellular reprogramming, stem cell differentiation, genomics, epigenetics, cell-based disease models, gene therapy, and regenerative medicine.</p> / Dissertation

A microwave assisted methodology was developed to modify Merrifield resins (1-2% cross-linked containing 1.0-3.5 mmol Cl-/g) with different nominal molecular weights PEG (200-1000). The synthesis was also carried out by conventional heating to assess the differences between the two procedures. The most efficient synthesis was achieved by using microwave and by using PEG with molecular weight 200 and MR 2% crosslinked containing 1.25 mmol Cl -/g. The structural elucidation was carried out using Fourier transform infrared (FTIR) spectroscopy and elemental analyses. Upon pyrolsis-GC/MS analysis of the PEGylated MR, the PEG showed the tendency to undergo thermal degradation by the loss of a smaller PEG fragments. This observed degradation of PEG was less prominent during microwave assisted synthesis compared to conventional heating, in addition to faster reaction rates and higher yields. As expected, the PEGylated MR showed improved swelling properties in polar solvents. The chemical reactivity of the PEGylated Merrifield resin was confirmed by the esterification with pyruvic acid and by the substitution of hydroxyl group using thionyl chloride. In addition, the PEGylated MR was converted into (1) polymer-supported acid/base or redox indicator by the attachment of a blue organic dye - 2,6-dichloroindophenol (DCIP) through a nucleophilic substitution reaction and (2) beta-cyclodextrin trap, a water insoluble inclusion-complex, by immobilization of beta-cyclodextrin through cross-linking with 1,6-hexamethylene diisocyanate reagent.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23956号 / 農博第2505号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5391(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 准教授 黒田 浩一 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Levels of carbon dioxide in the atmosphere have been steadily increasing since the beginning of the Industrial Revolution in the 1800s. The earth's climate is sensitive to alterations in these levels of carbon dioxide and other greenhouse gases (GHG), with significant changes in climate predicted long term. The adoption of the Kyoto Protocol in 1997 heralded a new age in terms of greenhouse gas accounting and emissions responsibility, for all nations. In Australia, carbon emissions from the Land Use and Land Use Change and Forestry sector are responsible for a large proportion of the national total emissions. Radar remote sensing has demonstrated considerable potential in the estimation and mapping of vegetation biomass and subsequently carbon. The aim of this research is to investigate the potential of airborne polarimetric radar for quantifying and mapping the biomass and structural diversity of woodlands in semi-arid Australia. Initial investigation focussed on the physical structure of the woodland, which revealed that despite a diversity of woodland associations, the species diversity was relatively low. Both excurrent and decurrent growth forms were present, which subsequently resulted in varying allocation of biomass to the components (i.e., branches, trunks). In view of this, both empirical and modelling methodologies were explored. Empirical relationships were established between SAR backscatter and the total above ground biomass. Considerable scatter was present in these relationships, which was attributed to the large range of species and their associated structures. Comparison of actual and model simulations for C-, L- and P-band wavelengths, reveal that no significant difference existed for these wavelengths, except at CHH, and the cross-polarised data at L- and P-band. The study confirmed that microwaves at C-band interacted largely with the leaves and small branches, with scattering at VV polarization dominating. Compared to the lower frequencies, the return from the ground surface (as expected) was significant. The differences in scattering mechanisms (i.e., branch-ground versus trunk-ground) between excurrent and decurrent structures were due largely to the larger angular branches associated with Eucalyptus and Angophora species, which were absent from Callitris glaucophylla.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Adelaide, 2004. / Photocopy. Includes bibliographical references (leaves 283-293).