Stem cells have the ability to reproduce themselves for a long period and differentiate into specific morphological and functional cells. The stem cells are an important material in the developmental biology, genomics, and transgenic methods, as well as in potential clinical applications, gene therapy and tissue engineering. The pluripotent stem cells will be a valuable source in numerous functional degenerated pathologies. Therefore, the objective of this research program was to establish transgenic porcine embryonic stem (pES) cell lines which can express green fluorescent protein (GFP) report gene stably for tracking after transplantation. We also developed a directed differentiation of pES into neural lineages and applied in rat Parkinson¡¦s disease model. Although the establishment of pluripotent ES cell lines from domestic species is much more difficult than that in murine species, our results had successfully isolated and established pES cell lines from pre-implantation blastocysts. Furthermore, we established the novel GFP-expressing pES cell lines (pES/GFP+), which were obtained by electroporation- mediated transfection with exogenous GFP gene. These pES/GFP+ cells exhibited pluripotent markers including Oct-4, AP, SSEA-4, TRA-1-60, and TRA-1-81 as that of human ES cells. The strategy of directed neural differentiation was to culture pES with neurogenic stimulators such as retinoic acid (RA), sonic hedgehog (SHH), and fibroblast growth factor (FGF). Upon directed differentiation toward neural differentiation, these pES-derived cells exhibited typical neuronal morphology and expressed neural lineage-specific markers such as nestin, NFL, MAP2, GFAP, A2B5, TH, ChAT, and GABA. These results showed the pES cells had the potential to differentiate into neural lineages. When pES/GFP+ cells were transplanted into the SD rat¡¦s brain, and their survival and development was determined by the non-invasive In Vivo Imaging System (IVIS 50), and the invasive fibered confocal Cellvizio® Imaging System (Cellvizio®). The results showed that fluorescent signals from pES/GFP+ cells on the injection site of SD rats¡¦ brain could be detected through the experimental period of 3 months. The level of fluorescent signals detected in treatment groups was two folds above that of the control group. Besides, the functional behavior recovery analysis by amphetamine-induced rotation test indicated the PD rat grafted with pES/GFP+ cells and their derived neural progenitors showed no significant recovery of rotation rate in these two treatments because a progressively increased relative rotation through 3 months duration. However, the relative rotation of PD rats grafted with the pES/GFP+-derived mature neurons, showed a stably decrease relative rotation and resulted in a functional recovery from Parkinsonian behavioral defects. Following 3 months completion of behavioral analyses, PD rats were sacrificed for immunohistochemical analysis. In the section of injected site without tumorgenesis and showed the survival and dopaminergic differentiation of grafted pES/GFP+ derived cells when stained with anti-TH and anti-DA. To our knowledge, there have been no reports of establishing GFP-expressing pES cell lines. These novel pES/GFP+ cell lines established in this study might serve as a non-rodent model and could benefit to the studies involving ES cell transplantation, cell replacement therapy, tissue regeneration, and actual approach for pre-clinical research due to their traceable capacity.

The green fluorescent protein chromophore exhibits remarkably different properties upon removal from the protective beta-barrel. This work focuses on the synthesis of these chromophores as wells studying the photophysics as to why they readily deactivate. Following these initial discoveries, these chromophores can be applied to many different environments providing a fluorescence "turn-on" and thus proving to be applicable in a number of different environments and fields.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 19, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. James E. Schoelz. Includes bibliographical references.

In 2008 the Nobel Prize in Chemistry was awarded to the scientists who revolutionized biomedical technology by isolating, characterizing, and pioneering the use of a green fluorescent protein (GFP) from a humble hydrozoan jellyfish. Now numbering in the hundreds of colors and applications, fluorescent protein (FP) tools have facilitated the explosion of biological knowledge elucidated by a technology that can label DNA or RNA, track protein expression, and identify protein interactions. The development of the large variety of FP biotechnology available today has been due to the need for expanded color palettes and applications, and more efficient functionality. Yet, as our understanding of the biochemical and spectral characteristics of these genetically-encoded, self-assembling proteins has expanded, our comprehension of the biological function of FPs in the host organisms has remained inadequate. While the need for novel FP laboratory applications still continues, the new focus in the field of fluorescent proteins is moving to also characterize their biological functions. In this research compilation, the identification of three groups of new fluorescent proteins from marine copepods and hydrozoans has provided a collection of eleven FPs exhibiting previously uncharacterized colors, and biochemical and structural features. The green FPs from copepods are the brightest wild-type FPs identified and support the hypothesized biological function of fluorescence as counter-shading in the marine environment where these animals live. The FPs from the siphonophore and anthoathecate jelly, both hydrozoan animals, are comprised of tandemly expressed fluorescent protein units, a solution to the oligomeric structure common to most FPs that suggests a novel structure-function relationship. The fluorescent proteins from Obelia reveal a novel hydrozoan cyan FP, previously uncharacterized higher-order structural complexes, and have initiated the work to describe the biological function of these proteins as potential regenerators of their internal bioluminescent light sources. All eleven fluorescent proteins may also be adapted for FP technology. / text

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 176-185). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis(M.S.)--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references.

Thesis (Ph. D.)--Oregon State University, 2004. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 136-140).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 89-98).

Metabolic flux analysis (MFA) is a powerful technique for quantifying the intracellular fluxes in central carbon metabolism. It relies on detection of stable isotope labelling from metabolites such as amino acids derived from protein. Current standard techniques are, however, unable to distinguish between different cell types in heterogeneous tissue. The aim of the thesis was to address this problem by developing and validating a strategy using green fluorescent protein (GFP) with cell type specific expression as a reporter protein for investigating the fluxes in specific cell types in the Arabidopsis thaliana root. The fundamental difficulty in applying a reporter protein strategy in a multicellular organism arises from the limited amount of recombinant protein expressed by the cells. The main novel contributions of the work in this thesis are threefold. First, a robust protocol for purification of GFP from the roots of Arabidopsis seedlings and for detection of reliable mass isotopomer distributions from the amino acids derived from GFP are described. Secondly, the reporter protein strategy is validated in this biological system with a focus on showing the data obtained by the use of the reporter protein is equal to that normally obtained from the total protein fraction. To expand on this, stable isotope labelling in isolated root hair cells is explored. These cells are easily isolated and show potential as a model system for cell type specific metabolism. Finally, the experimental data provide evidence for the feasibility of measuring data from specific cell types with appropriate mass spectrometric techniques. Analysis of cell type specific gene expression in this system suggests differences in the primary metabolism of different cell types cannot be ruled out without further investigation. Based on small scale in silico modelling described in this thesis, new solutions capable of providing data on sub-populations of cells are required, if central metabolism of the cell types differs significantly.