Sequence specific complexation of b DNA at sites containing g,c base pairs

A series of eight related analogs of distamycin A has been synthesized. Footprinting and affinity cleaving reveal that only two of the analogs, pyridine-2- car box amide-netropsin (2-Py N) and 1-methylimidazole-2-carboxamide-netrops in (2-ImN), bind to DNA with a specificity different from that of the parent compound. A new class of sites, represented by a TGACT sequence, is a strong site for 2-PyN binding, and the major recognition site for 2-ImN on DNA. Both compounds recognize the G•C bp specifically, although A's and T's in the site may be interchanged without penalty. Additional A•T bp outside the binding site increase the binding affinity. The compounds bind in the minor groove of the DNA sequence, but protect both grooves from dimethylsulfate. The binding evidence suggests that 2-PyN or 2-ImN binding induces a DNA conformational change.

In order to understand this sequence specific complexation better, the Ackers quantitative footprinting method for measuring individual site affinity constants has been extended to small molecules. MPE•Fe(II) cleavage reactions over a 10^5 range of free ligand concentrations are analyzed by gel electrophoresis. The decrease in cleavage is calculated by densitometry of a gel autoradiogram. The apparent fraction of DNA bound is then calculated from the amount of cleavage protection. The data is fitted to a theoretical curve using non-linear least squares techniques. Affinity constants at four individual sites are determined simultaneously. The distamycin A analog binds solely at A•T rich sites. Affinities range from 10^(6)- 10^(7)M^(-1) The data for parent compound D fit closely to a monomeric binding curve. 2-PyN binds both A•T sites and the TGTCA site with an apparent affinity constant of 10^(5) M^(-1). 2-ImN binds A•T sites with affinities less than 5 x 10^(4) M^(-1). The affinity of 2-ImN for the TGTCA site does not change significantly from the 2-PyN value. At the TGTCA site, the experimental data fit a dimeric binding curve better than a monomeric curve. Both 2-PyN and 2-ImN have substantially lower DNA affinities than closely related compounds.

In order to probe the requirements of this new binding site, fourteen other derivatives have been synthesized and tested. All compounds that recognize the TGTCA site have a heterocyclic aromatic nitrogen ortho to the N or C-terminal amide of the netropsin subunit. Specificity is strongly affected by the overall length of the small molecule. Only compounds that consist of at least three aromatic rings linked by amides exhibit TGTCA site binding. Specificity is only weakly altered by substitution on the pyridine ring, which correlates best with steric factors. A model is proposed for TGTCA site binding that has as its key feature hydrogen bonding to both G's by the small molecule. The specificity is determined by the sequence dependence of the distance between G's.

One derivative of 2-PyN exhibits pH dependent sequence specificity. At low pH, 4-dimethylaminopyridine-2-carboxamide-netropsin binds tightly to A•T sites. At high pH, 4-Me_(2)NPyN binds most tightly to the TGTCA site. In aqueous solution, this compound protonates at the pyridine nitrogen at pH 6. Thus presence of the protonated form correlates with A•T specificity.

The binding site of a class of eukaryotic transcriptional activators typified by yeast protein GCN4 and the mammalian oncogene Jun contains a strong 2-ImN binding site. Specificity requirements for the protein and small molecule are similar. GCN4 and 2-lmN bind simultaneously to the same binding site. GCN4 alters the cleavage pattern of 2-ImN-EDTA derivative at only one of its binding sites. The details of the interaction suggest that GCN4 alters the conformation of an AAAAAAA sequence adjacent to its binding site. The presence of a yeast counterpart to Jun partially blocks 2-lmN binding. The differences do not appear to be caused by direct interactions between 2-lmN and the proteins, but by induced conformational changes in the DNA protein complex. It is likely that the observed differences in complexation are involved in the varying sequence specificity of these proteins.

Techniques for strength measurement at high pressures and strain-rates using transverse waves

The study of the strength of a material is relevant to a variety of applications including automobile collisions, armor penetration and inertial confinement fusion. Although dynamic behavior of materials at high pressures and strain-rates has been studied extensively using plate impact experiments, the results provide measurements in one direction only. Material behavior that is dependent on strength is unaccounted for. The research in this study proposes two novel configurations to mitigate this problem.

The first configuration introduced is the oblique wedge experiment, which is comprised of a driver material, an angled target of interest and a backing material used to measure in-situ velocities. Upon impact, a shock wave is generated in the driver material. As the shock encounters the angled target, it is reflected back into the driver and transmitted into the target. Due to the angle of obliquity of the incident wave, a transverse wave is generated that allows the target to be subjected to shear while being compressed by the initial longitudinal shock such that the material does not slip. Using numerical simulations, this study shows that a variety of oblique wedge configurations can be used to study the shear response of materials and this can be extended to strength measurement as well. Experiments were performed on an oblique wedge setup with a copper impactor, polymethylmethacrylate driver, aluminum 6061-t6 target, and a lithium fluoride window. Particle velocities were measured using laser interferometry and results agree well with the simulations.

The second novel configuration is the y-cut quartz sandwich design, which uses the anisotropic properties of y-cut quartz to generate a shear wave that is transmitted into a thin sample. By using an anvil material to back the thin sample, particle velocities measured at the rear surface of the backing plate can be implemented to calculate the shear stress in the material and subsequently the strength. Numerical simulations were conducted to show that this configuration has the ability to measure the strength for a variety of materials.

Recent progress in femtosecond holography

The recent advancements of femtosecond (fs) holography are introduced. The experimental requirements and the time resolution are presented. Applications of femtosecond holography to signal processing, and other femtosecond holographic techniques such as femtosecond holographic imaging and microprocessing are detailed. A potential alternative of femtosecond holography is proposed, based on the sectional interference of reference pulse with the time stretched signal pulse. (c) 2005 Elsevier B.V. All rights reserved.

A cocktail of thermally stable, chemically synthesized capture agents for the efficient detection of anti-gp41 antibodies from human sera and techniques

This thesis reports on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60°C.

Formal methods for control synthesis in partially observed environments : application to autonomous robotic manipulation

Modern robots are increasingly expected to function in uncertain and dynamically challenging environments, often in proximity with humans. In addition, wide scale adoption of robots requires on-the-fly adaptability of software for diverse application. These requirements strongly suggest the need to adopt formal representations of high level goals and safety specifications, especially as temporal logic formulas. This approach allows for the use of formal verification techniques for controller synthesis that can give guarantees for safety and performance. Robots operating in unstructured environments also face limited sensing capability. Correctly inferring a robot's progress toward high level goal can be challenging.

This thesis develops new algorithms for synthesizing discrete controllers in partially known environments under specifications represented as linear temporal logic (LTL) formulas. It is inspired by recent developments in finite abstraction techniques for hybrid systems and motion planning problems. The robot and its environment is assumed to have a finite abstraction as a Partially Observable Markov Decision Process (POMDP), which is a powerful model class capable of representing a wide variety of problems. However, synthesizing controllers that satisfy LTL goals over POMDPs is a challenging problem which has received only limited attention.

This thesis proposes tractable, approximate algorithms for the control synthesis problem using Finite State Controllers (FSCs). The use of FSCs to control finite POMDPs allows for the closed system to be analyzed as finite global Markov chain. The thesis explicitly shows how transient and steady state behavior of the global Markov chains can be related to two different criteria with respect to satisfaction of LTL formulas. First, the maximization of the probability of LTL satisfaction is related to an optimization problem over a parametrization of the FSC. Analytic computation of gradients are derived which allows the use of first order optimization techniques.

The second criterion encourages rapid and frequent visits to a restricted set of states over infinite executions. It is formulated as a constrained optimization problem with a discounted long term reward objective by the novel utilization of a fundamental equation for Markov chains - the Poisson equation. A new constrained policy iteration technique is proposed to solve the resulting dynamic program, which also provides a way to escape local maxima.

The algorithms proposed in the thesis are applied to the task planning and execution challenges faced during the DARPA Autonomous Robotic Manipulation - Software challenge.