<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).

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "August, 2008." Includes bibliographical references (leaves 140-147). Library also has microfilm. Ann Arbor, Mich. : ProQuest Information and Learning Company, [2008]. 1 microfilm reel ; 35 mm. Online version available on the World Wide Web. Library also has electronic version on CD-ROM

Thesis (M.S.)--California Polytechnic State University, 2009. / Mode of access: Internet. Title from PDF title page; viewed on February 18, 2009. Major professor: John Saghri, Ph.D. "Presented to the Electrical Engineering Department faculty of California Polytechnic State University, San Luis Obispo, California." "In partial fulfillment of the requirements for the degree [of] Master of Science in Electrical Engineering." "January 2009." Includes bibliographical references (p. 134-136). Also available on microfiche.

Thesis (M.E.)--University of Canterbury, 2007. / Typescript (photocopy). "February 2007." Includes bibliographical references (p. [85]-88). Also available via the World Wide Web.