The design of an array processor for pattern recognition studies

Thesis (B.S.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering, 1960.MIT copy bound with: The electrostatic synchronous motor for smear camera application / Saul Fox Stanten. 1960. -- Design and construction of a thermoelectric dew point measuring device / Tom Neil Thiele. 1960. -- A study of modification in the German noun phrase / Robert Symons Troth. 1960. -- A selective signalling device for paging or call systems / George M. Walsh. 1960. -- A dual channel transistor transmitter for sensory aid research / Stephen B. Weinstein. 1960. -- A low cost, solid state digital converter / John Edward Yates. 1960.Includes bibliographical references (leaf 45).by Herbert Martin Shanzer.B.S

From Biomimetic Ion Carriers to Helical Structures

Biomimetic chemistry aims at reproducing the functions of natural compounds with the simplest possible synthetic molecules. Our strategy in this endeavor involved: (i) first reproduction of elementary processes such as molecular recognition, mass- transport, electron-transport, and signaling, and (ii) subsequently integration of several of these properties into single molecules. We approached the problems of molecular recognition and mass- transport by concentrating on the design and synthesis of all-artificial iron(III)-carriers that mimic the properties of microbial siderophores (iron(III) carriers): (i) the capability to effectively bind iron(III), (ii) to interact with specific membrane receptors as their iron(III)-complexes, and (iii) to transport iron(III) into the cells’ interior. Conjugation of the synthetic carriers with fluorescent markers enabled us to couple molecular recognition with signaling and to thereby provide diagnostic tools for the identification of specific microorganisms. The knowledge gained in the course of this work was then applied to the synthesis of (i) triple-stranded binders that form helical, dinuclear complexes, and of (ii) helical structures where four elementary process are integrated into a single molecule to provide molecular »redox-switches«

Photoinduced Electron Transfer in Ruthenium Bipyridyl Complexes: Evidence for the Existence of a Cage with Molecular Oxygen

Ruthenium complexes with three bipyridyl ligands, one of which is modified by attaching one or two hydroxamic acids groups (Ru-1 and Ru-2, respectively), were synthesized. Using EPR spectroscopy, we have found that photoexcitation leads to formation of nitroxyl radicals. The nitroxyl radical concentration in Ru-2 increased dramatically in the presence of spin traps DMPO (5,5‘-dimethyl-1-pyrroline-N-oxide) and PBN (N-tert-butyl-α-phenylnitrone) characterized by strong affinity to superoxide radicals. We have attributed this behavior to the formation of a cage complex between Ru-2 and the superoxide radical. This paper concerns the study of cages formed between ruthenium complexes and molecular oxygen and the effect of functional groups attached to modified bipyridyl ligands on cage formation. The complex between Ru-2 and O_2 was formed in the ground state, probably with participation of the hydroxamic acid groups. The equilibrium constant of this complex was determined by EPR as K_(eq) ∼ 3 M^(-1). The formation of the Ru-2−O_2 complex is supported by the temperature-dependent rate of appearance of the EPR signal in the presence of PBN. Additional evidence comes from observation of paramagnetic shifts of the peaks in the 1H NMR spectrum of specific aromatic protons in the substituted bipyridyl ring upon exposure to O_2. Similar shifts were observed in the spectrum of Os-2, with osmium replacing ruthenium. Model compounds with functional groups that replace the hydroxamic acid or compounds without the metal center, but with the two hydroxamic acids, were synthesized. No shifts in the ^1H NMR spectra of these derivatives were observed in the presence of O_2. These results lead to the conclusion that both metal ions, Ru(II) or Os(II), and hydroxamic acid groups are essential components for the formation of the oxygen cage

Double nuclear coherence transfer (DONUT)-HYSCORE: A new tool for the assignment of nuclear frequencies in pulsed EPr experiments

A two-dimensional experiment, termed DONUT-HYSCORE (double nuclear coherence transfer hyperfine sublevel correlation) designed to obtain correlations between nuclear frequencies belonging to the same electron spin manifold is presented. The sequence employed is π/2-τ1-π/2-t1-π-τ2- π-t2-π/2τ1-echo, and the echo is measured as a function of t1 and t2 whereas τ1 and τ2 are held constant. It is complementary to the standard HYSCORE experiment which generates correlations between nuclear frequencies belonging to different M(s) manifolds and is particularly useful for 14N nuclei. The experiment is first demonstrated on a single crystal of copper- doped l-histidine hydrochloride monohydrate where the modulations are induced by a single 14N nucleus, the remote nitrogen in the imidazole group. HYSCORE and DONUTHY-SCORE experiments were carried out on two crystal orientations. In the first, one Cu2+ site contributes to the echo and all six nuclear frequencies together with the expected correlation were observed. In the second, 12 frequencies corresponding to two Cu2+ ions at different crystallographic sites appeared and all expected correlations were detected as well. This rather trivial example demonstrates that the DONUT-HYSCORE pulse sequence indeed generates correlations within the M(s) manifolds. The value of the DONUT-HYSCORE experiment is demonstrated on a frozen solution of a vanadyl complex with a bis-hydroxamate ion binder (VO-RL515). The modulations in this complex arise from the two InN nuclei in the hydroxamate groups, and orientation-selective three-pulse ESEEM (electron spin-echo envelope modulation) spectra showed a number of well-resolved peaks. An unambiguous assignment of all peaks and their orientation dependences could not be achieved through HYSCORE alone because at certain orientations frequencies of one of the M(s) manifolds were absent or overlapped with those of the other manifold. The application of the DONUT-HYSCORE experiment provided new correlations that led to the complete assignment of the ESEEM frequencies, thus paving the way for future systematic spectral simulations for the determination of the best-fit Hamiltonian parameters. This example shows that, in the case that the HYSCORE experiment cannot distinguish between two sets of frequencies belonging to the same M(s) manifold in different centers (or orientations) because signals from the other manifold are missing or overlapping, the DONUT-HYSCORE becomes most valuable

Targeted Chromosomal Insertion of Large DNA into the Human Genome by a Fiber-Modified High-Capacity Adenovirus-Based Vector System

A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes)

Exploitation of Herpesvirus Immune Evasion Strategies to Modify the Immunogenicity of Human Mesenchymal Stem Cell Transplants

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected. METHODOLOGY/PRINCIPAL FINDINGS: We aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients' natural killer (NK) cells were depleted prior to cell transplantation. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable

Writing in Britain and Ireland, c. 400 to c. 800

No abstract available

Induction of antigen-specific tolerance through hematopoietic stem cell-mediated gene therapy: the future for therapy of autoimmune disease?

Based on the principle that immune ablation followed by HSC-mediated recovery purges disease-causing leukocytes to interrupt autoimmune disease progression, hematopoietic stem cell transplantation (HSCT) has been increasingly used as a treatment for severe autoimmune diseases. Despite clinically-relevant outcomes, HSCT is associated with serious iatrogenic risks and is suitable only for the most serious and intractable diseases. A further limitation of autologous HSCT is that relapse rates can be high, suggesting disease-causing leukocytes are incompletely purged or the environmental and genetic determinants that drive disease remain active. Incorporation of antigen-specific tolerance approaches that synergise with autologous HSCT could reduce or prevent relapse. Further, by reducing the requirement for highly toxic immune-ablation and instead relying on antigen-specific tolerance, the clinical utility of HSCT could be significantly diversified. Substantial progress has been made exploring HSCT-mediated induction of antigen-specific tolerance in animal models but studies have focussed on primarily on prevention of autoimmune diseases. However, as diagnosis of autoimmune disease is often not made until autoimmune disease is well developed and populations of autoantigen-specific pathogenic effector and memory T cells have become well established, immunotherapies must be developed to address effector and memory T-cell responses which have traditionally been considered the key impediment to immunotherapy. Here, focusing on T-cell mediated autoimmune diseases we review progress made in antigen-specific immunotherapy using HSCT-mediated approaches, induction of tolerance in effector and memory T cells and the challenges for progression and clinical application of antigen-specific ‘tolerogenic’ HSCT therapy