Thesis (Ph.D.)--York University, 2008. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references (leaves 183-207). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR39001

Thesis (M.S. in Bio. E.)--School of Biomedical Engineering, Georgia Institute of Technology, 2004. Directed by Cheng Zhu. / Includes bibliographical references (leaves 79-84).

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Selected nucleic acid binding species (aptamers) have been shown to undergo conformational changes in the presence of ligands, and have been adapted to function as biosensors. We were interested in whether the secondary structures of aptamers could be rationally engineered to undergo ligand dependent conformational changes. To this end, we used rational and computational design methods to generate a number of aptamer biosensors. First, we built upon previous work that showed that antisense oligonucleotides bearing reporter moieties could be used to denature aptamers. Upon addition of ligands, the conformational equilibrium is shifted towards release of the antisense oligonucleotide and a concomitant increase in fluorescence. We attempted to adapt this format to the potential detection of ricin, but were unsuccessful. In order to better evaluate rational designs, we attempted to use computational modeling methods. Again, aptamer biosensors have previously been engineered based on ligand-induced reorganization of secondary structure (as opposed to oligonucleotide displacement), a so-called 'slip-structure' model. We developed an algorithm to evaluate different lisp structures, predicted both aptamers and aptazymes that should have undergone ligand-dependent changes in conformation, and experimentally evaluated the computationally predicted sequences. A number of robust biosensors that could respond to the cytokine VegF and the small molecule flavin were discovered. The computational model was further adapted to an aptamer biosensor that underwent a larger conformational change upon ligand-binding, an antiswitch. In this model, binding of the ligand stabilizes one hairpin structure at the expense of a competing structure (as opposed to merely changing the register of the hairpin as in the previously described slip structure model). Again, we were able to computationally identify a number of antiswitches that upon synthesis were responsive to the ligand theophylline. Finally we again attempted to use rational design methods to optimize not just the degree of signal but also the kinetic performance of aptamer biosensors. To this end, we developed biosensors that signaled within seconds the presence of the coagulation protein thrombin. / text

No description available.

The optimisation of ligand-macromolecule interactions is fundamental to the design of therapeutic agents. The GRID method is a procedure for determining energetically favourable ligand binding sites on molecules of known structure using an empirical energy potential. In this thesis, it has been extended, tested, and then applied to the design of anti-influenza agents. In the GRID method, the energy of a hydrogen-bond is determined by a function which is dependent on the length of the hydrogen-bond, its orientation at the hydrogen-bond donor and acceptor atoms, and the chemical nature of these atoms. This function has been formulated in order to reproduce experimental observations of hydrogen-bond geometries. The reorientation of hydrogen atoms and lone-pair orbitals on the formation of hydrogen-bonds is calculated analytically. The experimentally observed water structures of crystals of four biological molecules have been used as model systems for testing the GRID method. It has been shown that the location of well-ordered waters can be predicted accurately. The ability of the GRID method to assist in the assignment of water sites during crystallographic refinement has been demonstrated. It has also been shown that waters in the active site of an enzyme may be both stabilized and displaced by a bound substrate. Ligands have been designed to block the highly conserved host cell receptor site of the influenza virus haemagglutinin in order to prevent the attachment of the virus to the host cells. The protein was mapped energetically by program GRID and specific ligand binding sites were identified. Ligands, which exploited these binding sites, were then designed using computer graphics and energy minimization techniques. Some of the designed ligands were peptides and these were synthesised and assayed. Preliminary results indicate that they may possess anti-influenza activity.

In the bone marrow, CD34 is the best currently available marker of human multipotential stem cells and is downregulated upon the commitment of cells to the myeloid pathway. CD33 is the earliest marker of stem cell commitment to the myeloid lineage but is down-regulated as myeloid cells mature to granulocytes while expression is retained on monocytes, dendritic cells and macrophages. CD34 is a mucosialin and CD33 a member of the immunoglobulin superfamily. To determine the functions of CD34 and CD33 in early haematopoiesis, soluble forms of CD34 and CD33 have been constructed by PCR based construction of extracellular domain-IgGlFc (ECDFc) fusion plasmids. These chimaeric proteins have been used to define the ligand binding of CD33 and CD34 in a range of assay systems including: screening cell lines by immunofluorescence and iodination analysis; immunohistochemical staining; screening of cDNA libraries transiently expressed in COS cells by "panning" for ligands; and phosphorylation studies to assess the potential of phosphoproteins to regulate these molecules and their ligands in vitro. Iodination demonstrated that CD33 binds increasing numbers of ligands heterophilically on erythroleukemic (K562) and promonocytic (U937) cell lines at sizes ranging from 54-69Kd and 97-1 lOKd in a differentiation-dependent manner. CD33 was established to associate with several src-like kinases while Fc-CD33 precipitates phosphoproteins in the same region in KG-la, K562 and U937. Novel divalent-cation-dependent CD34 ligands of 66Kd and HOKd were isolated on HUVEC and both CD34 and Fc-CD34 precipitated phosphoproteins from HUVEC. From panning studies, Fc-CD33 demonstrated low levels of binding to ICAM-1 and a cDNA product sharing homology to the 3' end of dystrophin, termed here as apo-dystrophin-4. Apo-4 appears to give rise to two major proteins, at least one of which may provide an in vitro ligand for CD33 and contain a 3' enhancer. Models for both CD33 phosphoprotein and ligand binding behaviour are presented.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2004. / Includes bibliographical references (leaves 137-145). / Also available by subscription via World Wide Web / xiv, 145 leaves, bound ill. (some col.) 29 cm

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on February 26, 2008) Vita. Includes bibliographical references.

Thesis (Ph.D.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/01/11 ). Includes bibliographical references.