Nuclear receptors are ligand activated transcription factors, where upon binding with small molecule ligands, these proteins are involved in the regulation of gene expression. To date there are approximately 48 human nuclear receptors known, involved in multiple biological and cellular processes, ranging from differentiation to maintenance of homeostasis. Due to their critical role in transcriptional regulation, these receptors are implicated in several diseases. Currently, 13% of prescribed drugs in the market are NR ligands for diseases such as cancer, diabetes and osteoporosis. In addition to drug discovery, the mechanism of function, mobility and trafficking of these receptors is poorly understood. Gaining insight into the relationship between the function and /or dysfunction of these receptors and their mobility will aid in a better understanding of the role of these receptors. The green fluorescent protein (GFP) has revolutionized molecular biology by providing the ability to monitor protein function and structure via fluorescence. The fluorescence contribution from this biological marker is the chromophore, formed from the polypeptide backbone of three amino acid residues, buried inside 11-stranded â-barrel protein. Synthesis of GFP derivatives of is based on the structure of the arylmethyleneimidazolidinone (AMI), creating a molecule that is only weakly fluorescent. Characterizing these AMI derivatives for other proteins can provide a powerful visualization tool for analysis of protein function and structure. This development could provide a very powerful method for protein analysis in vitro and in vivo. Development of such fluorescent ligands will prove beneficial for the nuclear receptors. In this work, libraries of AMIs derviatives were synthesized by manipulating various R groups around the core structure, and tested for their ability to serve as nuclear receptor ligands with the ability to fluoresce upon binding. The fluorogens are developed for steroidal and non-steroidal receptors, two general classes of nuclear receptors. Specific AMIs were designed and developed for steroid receptor estrogen receptor á (ERá). These ligands are showed to activate the receptor with an EC50 of value 3 ìM and the 10-fold activation with AMI 1 and AMI 2 in comparison to the 21-fold activation observed with natural ERá ligand, 17â-estradiol. These novel ligands were not able to display the fluorescence upon binding the receptor. However, fluorescence localized in nucleus was observed in case of another AMI derivative, AMI 10, which does not activate the receptor. Such ligands open new avenues for developing fluorescent probes for ERá that do not involve fluorescent conjugates attached to a known ERá ligand core. AMIs were also characterized for non-steroidal receptors,specifically the pregnane x receptor (PXR) and retinoic acid receptor á (RARá). To date, fluorogens which turn fluorescence upon binding and activate the receptor have not been developed for these receptors. With respect to PXR, several AMI derivatives were discovered to bind and activate this receptor with a fold-activation better than the known agonist, rifampicin. The best characterized AMI derivative, AMI 4, activates the receptor with an EC50 of value 6.3 ìM and the 154-fold activation in comparison to the 90-fold activation and an EC50 value of 1.3 ìM seen with rifamipicin. This ligand is not only able to activate PXR but also displays fluorescence upon binding to the receptor. The fluroscence pattern was observed around the nucleus. Besides AMI 4, 16 other AMI derivatives are identified that activate PXR with different activation profiles. Thus, a novel class of PXR ligands with fluorescence ability has been developed. The AMI derivatives able to bind and activate RAR, also displayed activation profiles that were comparable to the wild-type ligand, all trans retinoic acid. These ligands activated the receptor with an EC50 value of 220 nM with AMI 109 in comparison to an EC50 value of 0.8 nM with the natural ligand for RARá. When these ligands were tested for fluorescence in yeast, the yeast were able to fluoresce only in the presence of the receptor and the AMI derivative, indicating that these agonists also have the ability to fluoresce.

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.

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.

In the human genome, 43 BTB (Bric-à-brac, Tramtrack, and Broad Complex) containing BTB-Zinc Finger proteins have been identified, many of which are transcription factors involved in cancer and development. These BTB domains have been shown to form homodimers and heterodimers which raise DNA binding affinity and specificity for transcription factors. This project was to develop an efficient assay to systematically identify interactions between BTB domains. It combined a co-expression system, fluorescent protein tagging and Ni-NTA plate retention. It was concluded that fourteen analyzed BTB domains formed homodimers, but only certain BTB pairs formed heterodimers, such as BCL6 with Miz1 and Miz1 with RP58. To further understand the specificity of BTB domain interactions, more structural and sequence information is still needed. In conclusion, this assay provided a comprehensive detection method for BTB domain interaction mapping. The information generated provides candidates for further functional and structural studies.