Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Two novel and highly related C₂H₂ zinc finger proteins (CTIP1/BCL11A/EVI9 and CTIP2/BCL11B/Rit1) have been implicated in COUP-TF signaling, etiology of myeloid and lymphoid malignancies, and hematopoietic cell development. However, the precise cellular function(s) and the contribution of these proteins to neoplastic processes and hematopoietic cell development remain unknown. The goal of the studies described herein was to elucidate the molecular mechanisms underlying the transcriptional repression mediated by these proteins to understand their biological properties, and ultimately, their cellular function(s). CTIP proteins repressed transcription of a reporter gene in a TSA-insensitive manner, suggesting that this repression mechanism(s) may not involve TSA-sensitive histone deacetylation catalyzed by member(s) of class I and II HDACs. One possible mechanism is that CTIP proteins may exert ISA-insensitive histone deacetylation catalyzed by TSA-insensitive HDAC(s), such as SIRT1, to repress transcription. In deed, SIRT1 was found to interact with CTIP proteins both in vitro and in mammalian cells, and was recruited to the promoter template in a CTIP-dependent manner. The proline-rich regions of CTIP proteins and the sirtuin homology domain of SIRT1 were found to be essential for mediating CTIPs•SIRT1 interactions. Moreover, column chromatography revealed that SIRT1 and CTIP2 were components of a large complex in Jurkat cell nuclear extracts. Based on the findings that SIRT1 associates with CTIP proteins in mammalian cells, SIRT1 may underlie the transcriptional repression activity of CTIP proteins. The following results support the hypothesis that SIRT1 may underlie the mechanism(s) of CTIP-mediated transcriptional repression. First, CTIP-mediated transcriptional repression was inhibited, at least partially, by nicotinamide, an inhibitor of the NAD⁺-dependent, TSA-insensitive HDACs. Second, the decrease in levels of acetylated histones H3 and/or H4 at the promoter region of a reporter gene was observed upon overexpression of CTIP proteins, and this effect was inhibited, at least partially, by nicotinamide. Third, endogenous SIRT1 was recruited to the promoter template of a reporter gene in mammalian cells upon overexpression of CTIP proteins. Fourth, SIRT1 enhanced the transcriptional repression mediated by CTIP proteins and this enhancement required the catalytic activity of SIRT1. Finally, SIRT1 enhanced the deacetylation of template-associated histones H3 and/or H4 in CTIP-transfected cells. In summary, results described herein strongly suggest that CTIP-mediated transcriptional repression involves the recruitment of SIRT1 to the template, at which the TSA-insensitive, but nicotinamide-sensitive histone deacetylase catalyzes deacetylation of promoter-associated histones H3 and/or H4. These results contribute additional understanding to the molecular mechanisms underlying transcriptional activity of CTIP proteins, which might be helpful for identification and characterization of the target genes under the control of CTIP proteins in cells of hematopoietic system and/or the central nervous system. / Graduation date: 2004

The Hepatitis B virus (HBV) chronically infects 350 million individuals worldwide, leading to mortality by end-stage liver disease, liver cirrhosis, and hepatocellular carcinoma. The vaccine to prevent HBV infection is highly effective but is not extensively available in endemic areas, resulting in high infection rates. Nucleoside analogue treatment of HBV has allowed for higher rates of viral clearance in infected individuals, but most patients must remain on therapy long term and viral resistance to the drugs is growing. The HBV viral genome is an episome in the nucleus of infected hepatocytes. It is called covalently closed circular (ccc) DNA and is highly stable, has a long half-life, and is the template for all viral transcription and progeny production. Nucleoside analogues do not directly target cccDNA, therefore many patients experience rebound when antiviral therapy is stopped. I have designed novel DNA binding proteins called zinc finger proteins (ZFPs) to specifically bind to the cccDNA in infected cells and inhibit viral transcription. Seven ZFPs targeting the model duck HBV (DHBV) and ten ZFPs targeting HBV were developed. Kinetic analyses of the purified ZFPs were performed, characterizing their specificity and binding properties. Using the DHBV tissue culture model system, I have demonstrated that the DHBV-specific ZFPs can specifically inhibit transcription from the viral template, resulting in reduced viral RNA, protein products and progeny virions. The DHBV-specific ZFPs were tested in primary duck hepatocytes (PDH) and in vivo in the Pekin duck model. ZFPs failed to express in PDH transduced by baculovirus vectors when DHBV was present in the cells. In vivo gene delivery of the ZFPs was carried out by portal vein injection of chitosan-based nanospheres. Unfortunately, non-specific reductions in viral levels masked any direct effect by the ZFPs. Testing of the HBV-specific ZFPs in tissue culture was hindered by a lack of transfectable cell culture model. A number of different transfection methods were tested to express the HBV-specific ZFPs, all without success. Further work is being carried out using baculovirus vectors to deliver the HBV-specific ZFPs to HBV-harbouring cell lines and HBV-infected scid-Alb/uPA chimeric mice with human liver cells. / Virology

Potassium ion channels play a key role in the generation and propagation of action potentials. The S4-S5 linker peptide (L45) is believed to be responsible for the anesthetic/alcohol response of voltage-gated K+ channels. We investigated this region to define the structural basis of 1-alkanol binding site in dShaw2 K+ channel. L45 peptides derived from dShaw2 and hKv3.4 K+ channel, which, if part of the complete channel, demonstrate different sensitivity to 1-alcohols. Specifically, dShaw2 is alcohol sensitive and hKv3.4 is alcohol resistant. Structural analysis of L45 with NMR and CD suggested a direct correlation between alpha-helicity and the inhibition of dShaw2 channel by 1-butanol. We used CD and NMR to determine the structure of L45 peptides in micelles and vesicles. We measured spin-lattice relaxation time (T1) and determined the location and surface accessibility of L45 in micelles. These experiments confirm that L45 of dShaw2 adopts an α-helical conformation, partially buried in the membrane and parallel to the surface. The binding and accumulation of rev proteins to an internal loop of RRE (rev responsive element) of unspliced mRNA precursors is a key step of propagation of human immunodeficiency (HIV) virus. Molecules that interfere with this process can be expected to show anti-HIV activity. Our work is based on an assumption that zinc fingers could compete with rev proteins, therefore impeding the life cycle of HIV and stopping its infection. We studied the influence of different cations, anions, and the concentration of salts and osmolytes on the binding affinity with Polyacrylamide Gel Electrophoresis (PAGE) and Isothermal Titration Calorimetry (ITC). We conclude that the types of anions and/or cations and their concentrations affect the enthalpy and entropy of the binding interacitons. Using a gel assay, we confirm that there are three products in RNA-Protein reaction, and both EDTA and salts (and their concentrations) in the gel or samples interfere with RNA-protein complex mobility.

RNA - protein interactions constitute a vital part of numerous biochemical processes. In the HIV life cycle, the interaction of the viral protein Rev and the Rev Responsive Element (RRE), a part of unspliced HIV RNA, is crucial for the propagation of infectious virions. Intervention of this interaction disrupts the viral life cycle. Rev - RRE interaction initially occurs at a high affinity binding site localized to a relatively small stem loop structure called RREIIB. This binding event has been well characterized by a variety of biochemical, enzymatic and structural studies. Our collaborators have previously demonstrated the efficacy of zinc finger proteins, generated by phage display, in the specific targeting of RREIIB. We have shown that the binding of these zinc finger proteins is restricted to the bulge in stem loop IIB that Rev also targets. Currently these proteins bind RREIIB with dissociation constants in the nanomolar range. We have employed a wide assortment of biophysical techniques such as gel shift assays, circular dichroism, isothermal titration calorimetry and NMR structural studies to further investigate this interaction. Several mutants of the zinc finger protein and the RNA were also studied to delineate the parts of the protein secondary structure as well as the role of specific side chains in this interaction. We have generated a solution structure of the RREIIBTR RNA bound zinc finger protein, ZNF29G29R, which displayed the highest affinity to this RNA. This has allowed us to shed further light on the molecular basis of this RNA - protein interaction and provides input for further refinement in our structure guided phage display.

In this thesis, we proposed an innovation ideal that is employment of laser to extract finger feature, and constructed laser speckle recognition systems for this kind of feature. When projecting laser on the object surface, the speckle could be obtained to represent the characteristic of object surface by collecting scattered light. Two measurement of scattered light was adopted. First is laser signal recording the strength of scattered light when laser scan across the finger. The second is laser speckle image which is demonstrated when projecting the laser on the fingerprint and simultaneously collecting the scatter light by CCD. We proposed two recognition systems for laser signal and laser speckle. Besides, the proposed laser speckle fingerprint recognition system combines biometric detection, it can accurately distinguish biometric and non-biometric speckle. Experimental results demonstrate that proposed laser speckle recognition systems are feasible and with excellent ability of identity verification.

Smart materials have been widely used for control actuation. A robotic hand can be equipped with artificial tendons and sensors for the operation of its various joints mimicking human-hand motions. The motors in the robotic hand could be replaced with novel electroactive-polymer (EAP) actuators. In the three-finger gripper proposed in this paper, each finger can be actuated individually so that dexterous handling is possible, allowing precise manipulation. In this dissertation, a microscale position-control system using a novel EAP is presented. A third-order model was developed based on the system identification of the EAP actuator with an AutoRegresive Moving Average with eXogenous input (ARMAX) method using a chirp signal input from 0.01 Hz to 1 Hz limited to 7 ± V. With the developed plant model, a digital PID (proportional-integral-derivative) controller was designed with an integrator anti-windup scheme. Test results on macro (0.8-mm) and micro (50-μm) step responses of the EAP actuator are provided in this dissertation and its position tracking capability is demonstrated. The overshoot decreased from 79.7% to 37.1%, and the control effort decreased by 16.3%. The settling time decreased from 1.79 s to 1.61 s. The controller with the anti-windup scheme effectively reduced the degradation in the system performance due to actuator saturation. EAP microgrippers based on the control scheme presented in this paper will have significant applications including picking-and-placing micro-sized objects or as medical instruments. To develop model-based control laws, we introduced an approximated linear model that represents the electromechanical behavior of the gripper fingers. Several chirp voltage signal inputs were applied to excite the IPMC (ionic polymer metal composite) fingers in the interesting frequency range of [0.01 Hz, 5 Hz] for 40 s at a sampling frequency of 250 Hz. The approximated linear Box-Jenkins (BJ) model was well matched with the model obtained using a stochastic power-spectral method. With feedback control, the large overshoot, rise time, and settling time associated with the inherent material properties were reduced. The motions of the IPMC fingers in the microgripper were coordinated to pick, move, and release a macro- or micro-part. The precise manipulation of this three-finger gripper was successfully demonstrated with experimental closed-loop responses.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy