Fluorescent protein based genetically encoded fluorescent reporters play an improtant role in understanding the cellular physiology by directly monitoring real-time cellular signaling pathways with fluorescent microscope. Quantitative analysis of Ca2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca2+-dependent signaling under physiological and pathological conditions. Here, we developed a novel class of genetically encoded indicators by designing a Ca2+ binding site in the enhanced green fluorescent protein (EGFP). One of them, CatchER (Calcium sensor for detecting high concentration in the ER), exhibits unprecedented Ca2+ release kinetics with an off-rate estimated at around 700 s-1 and appropriate Ca2+ binding affinity, likely due to local, Ca2+-induced conformational changes around the designed Ca2+ binding site and reduced chemical exchange between two chromophore states. CatchER reported considerable differences in ER Ca2+ dynamics and concentration among epithelial HeLa, kidney HEK 293, and muscle C2C12 cells, enabling us to monitor SR luminal Ca2+ in flexor digitorum brevis (FDB) muscle fibers to determine the mechanism of diminished SR Ca2+ release in aging mice. Moreover, the structure of CatchER has been investigated by nuclear magnetic resonance spectroscope (NMR) and high-resolution X-ray crystal structures to understand the novel mechanism of Ca2+ induced fluorescent enhancement of GFP. It is crucial to investigate the metal selectivity of Ca2+/Mg2+ of these metalloproteins to understand cellular physiology. The major Mg2+ binding sites of proteins have been reviewed and classified based on structural differences, and identified several key factors to determine Mg2+/Ca2+ selectivity with binding constants difference up to 104 in several types of metalloproteins. Thrombin is involved in numerous cellular signaling pathways and plays a crucial role in blood coagulation. I designed a novel class of single EGFP-based thrombin sensors by inserting a thirty-amino acid short peptide with a thrombin cleavage site into the fluorescent sensitive location of EGFP. These designed protease sensors exhibited optimized kcat/Km up to 104 magnitudes higher than that of small peptide based absorption indicator EGR-pNA. The measured Km value is in below 10 mM, in the same magnitude as that of natural thrombin substrate Fibrinogen A.

Cyanobacteria belong to the oldest organisms of our planet. They use photosynthesis to produce ATP and gain biomass from carbon dioxide. The cyanobacteria Nostoc punctiforme is a filamentous bacterium that consists of two different types of cells, vegetative cells and heterocysts. The type of cell it differentiates into depends on the media they grow in. In an ammonium-rich medium, the N.punctiforme consists of vegetative cells that differentiate into heterocysts when in the medium is changed to a low-concentration ammonium medium. The ammonium-binding nitrogenase in the heterocysts does not work in an oxidative environment. During oxidative stress, N.punctiforme produces Dps (DNA binding protein from starved cells) which protects DNA. In the heterocysts the nitrogenase produces hydrogen as a side product. The hypothesis is that Dps is cell specific. In order to study this protein, a fusion of the promotor of Dps and GFP (Green Flourescent Protein) was constructed. To detect GFP, optimization of a Western Blot (WB) for GFP was performed. Protein samples were analyzed in strains of N.punctiforme. In strain 12A, the production of GFP was visualized but the band was not specific. Several attempts of optimization of the WB procedure were performed, but none of them showed clear specific protein detection in the N.punctiforme strains. Further optimization of the WB protocol is needed.

Stem cells have great potential value for treating a number of diseases and conditions, including diabetes, Parkinson's, and spinal cord injuries. Applying stem cells for therapeutic purposes will require an in-depth understanding of their biology, not only of the genes they express, but also the functions of the proteins encoded by the genes. The goal of the project presented in this thesis was to develop a method for high-throughput analyses of protein localization in mouse stem cells. Localization information can provide insight into the functions and biological roles of proteins. <br /><br /> One means of studying protein localization involves creating proteins with a green fluorescent protein (GFP) reporter gene and analyzing their localization using fluorescence microscopy. The research outlined in this thesis focused on developing a system to create a large number of GFP-tagged proteins by constructing a cDNA?GFP fusion library. This involved exploring methods for optimizing cDNA synthesis, designing a retroviral vector (pBES23) for the expression of cDNA?GFP fusions in mouse stem cells, and constructing a cDNA?GFP fusion library in this vector using R1 mouse embryonic stem cell mRNA. The library constructed was not successfully delivered to target cells for GFP-tagged protein expression; it was therefore not possible to characterize protein localization in mouse stem cells. Suggestions are given as to how the methods used in this thesis might be optimized further.

Cold acclimation is a complex process where plants acquire increased freezing tolerance following exposure to low, non-freezing temperatures. Microtubules are dynamic components of the cytoskeleton that are essential for plant growth and development, and there are multiple lines of evidence indicating microtubules are involved in the acquisition of freezing tolerance. <p>The organization of microtubules (MTs) was tracked over the course of a cold acclimation period using GFP:TUB6 and fluorescent imaging tools. Experiments found that MTs undergo incomplete, transient disassembly following exposure to acclimating temperatures, which is accompanied by intranuclear tubulin accumulation and followed by MT reassembly. The importance of the observed changes to MT organization was examined with MT disrupting chemicals that caused reduced MT dynamics or induced transient MT disassembly similar to that of cold acclimation. Results of these experiments suggest that MT reorganization is important for cold acclimation, but the disassembly and reassembly do not directly control cold acclimation.<p>MT binding proteins are likely to play a key role in the low temperature response because they control MT activity and organization, participate in low temperature signal transduction pathways, and mediate interactions between various elements of this pathway. By employing a number of proteomics techniques we were able to identify 96 tubulin-binding proteins from untreated and short term cold acclimated Arabidopsis plants. Proteins both known to and predicted to bind to MTs and unexpected MT binding proteins were identified. The identified tubulin binding proteins have a range of cellular functions, including RNA transport and protein translation, stress responses, and functions related to various metabolic pathways, and cell growth and organization. <p>Exposure to low temperatures affected the binding of some of these proteins to MTs with the identified tubulin binding proteins potentially involved in the cold acclimation process and stress response through a number of possible pathways.<p>This study represents the first live cell imaging of MT reorganization in response to low temperatures and the first time microtubule binding proteins from whole plant protein extracts were identified using 1D gel LC-MS/MS analysis.

Expressed sequence tags (ESTs) are derived from the developing tilapia brain was established in our lab. There are 9 transcripts were identified as thermosensitive/Neural development-related gene. The effects of different temperatures on the ontogenetic expression of these thermosensitive/Neural development-related gene during the critical period of brain sexual differentiation were investigated in the present study. The ontogenetic expression of inhibitor of DNA binding/differentiation protein 2 (Id2), thermosensitive/Neural development-related gene, were enhanced by both lower (20¢J) and higher (32¢J) temperatures before 10 days post-hatching. In this study, bioinformatics were searched for Id2, which is a gene with 738 bp of patial cDNA sequence, open reading frame (ORF) is 411bp, and deduced 137 amino acids of protein sequence. The protein of Id2 was expressed in a prokaryotic system, BL21 (Escherichia coli) and purified with Ni-NTA affinity chromatography. Also, the ORF of Id2 was cloned into pEGFP vector, and plasmid (pEGFP-Id2) was transfected into the eukaryotic system, mouse neuroblastoma cell (Neuro-2a cell). The distribution of Id2 expressed in the Neuro-2a cell was identified by fluorescence microscopy.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 141-152). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 141-152). Also available on the Internet.

Polyphenol oxidases (PPOs) are ubiquitous enzymes that oxidize phenols to quinones in the presence of molecular oxygen, often leading to tissue discolouration. They are sometimes considered as defense proteins but other functions, for example in phenolic compound biosynthesis, have also been found. In this thesis, bioinformatic searches were conducted to identify putative PPO genes from available genomes representing five Viridiplantae lineages: chlorophytes, bryophytes, lycophytes, monocotyledonous anthophytes and eudicotyledonous anthophytes. Duplicated PPO genes were found in most land plant genomes. A detailed investigation of the poplar (Populus trichocarpa) PPO gene family found nine genes that exhibit differential expression profiles during development and following stress, of which PtrPPO1 was the only significant wound-inducible PPO gene. A phylogenetic reconstruction of the poplar PPOs identified PtrPPO13 to be an unusual PPO homolog and it was studied in detail. Experimental evidence indicated that PtrPPO13 is expressed in most organs, and unlike most PPOs, is localized to the vacuole. Together, the phylogeny, gene expression and subcellular localization studies suggest that PPOs are likely to have variable physiological functions in plants and that PtrPPO13 is distinct from most typical PPOs. / Graduate / 0309

Current filter validation methods require 48 hours culture for results to become available, which creates time delays within the manufacturing process and quality control back-logs The thesis compares alternative methods for the production of filter challenge test data Within 24 hours to the desired test sensitivity, using bioluminescent and fluorescent genetically engineered strains of the test organism Brevundzmonas dzmznuta The recombinant strains were produced using a Tn5 transposon system, using a filter mating method. The genes cloned into the bacterial chromosome were the biolummescence lux_ genes, taken from the marme bacteria, Photorhabdus lummescens or Vzbno harveyz, and the gene encoding green fluorescent protein taken from the marine jelly fish Aequoria victoria The cloned strains were found to show no difference to the w1ld type strain With respect to their surface hydrophobicity, according to a bacterial adherence to hydrocarbons assay, and surface charge, according to an electro-static interaction chromatography method. Furthermore, the cell size according to Transmission Electron Microscopy was not significantly different to the wild type strain, which had cell dimensions of 1 05 x 0 52 Jlm The retention of cells by 0 45 mtcron rated filters, was shown to be not significantly different to the wild type All strains were retained by 0 2 Jlm filters These data confirmed that the cloned strains were suitable for challenge testing Four methods were used to detect microcolonies of the recombinant strains on filters. The advantage of the microcolony detection system was that it showed that the cells detected downstream of the filter were viable and culturable. The best detection method was with an epifluorescent microscope and the fluorescent strain after 24 hours, for which the sensitivity was 98.1 %. Two CCD camera systems were used to detect the bioluminescent strains on filters. The sensitivity of these systems were 80.1% and 83 9%, for the Nucleovision and Nightowl CCD camera systems, respectively, after 24 hours In addition, the Bwprobe photomultipherbased system was shown to achieve the detection sensitivity of one microcolony after 24 hours. Also, steps were made to study transcription Initiation signals for gene expression in fluorescent recombinant Brevundzmonas dzmmuta. Various putative promoter sequences were Identified in one fluorescent strain, using a DNA sequencmg method. These sequences showed homology to previously identified E colr and Brevundzmonas promoter sequences. Finally, an attempt was made to produce recombinant fluorescent and bioluminescent Acholeplasma lazdlawu, however this was unsuccessful and further work will be required to achieve this objective.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 199-213). Also available on the Internet.