2D Kinetics and Forced Dissociation of Selectin-ligand Bindings

Cell adhesion is crucial to many pathophysiological processes, such as inflammatory reaction and tumor metastasis. It is mediated by specific interactions between receptors and ligands, and provides the physical linkages among cells. For example, interactions between selectins and glycoconjugate ligands mediate leukocyte initially tethering to and subsequently rolling on vascular surfaces in sites of inflammation or injury, which is determined by their fast kinetic rates. To mediate cell adhesion, the interacting receptors and ligands must anchor to apposing surfaces of two cells or a cell and the substratum, i.e. , the so-called two-dimensional (2D) binding, which differs from interactions in the fluid phase, i.e. , the three-dimensional (3D) binding. How structural variations and surface environments of interacting molecules affect their 2D kinetics, and how external forces manipulate their dissociation has little been known quantitatively, and nowadays attracts more and more attentions.

Destruction of Chemical Agents in a Plasma Reactor with Working Gas of Hydrogen

Parametric Analysis for Monitoring 2D Kinetics of Receptor-Ligand binding

Thermal fluctuation approach is widely used to monitor association kinetics of surface-bound receptor-ligand interactions. Various protocols such as sliding standard deviation (SD) analysis (SSA) and Page's test analysis (PTA) have been used to estimate two-dimensional (2D) kinetic rates from the time course of displacement of molecular carrier. In the current work, we compared the estimations from both SSA and modified PTA using measured data from an optical trap assay and simulated data from a random number generator. Our results indicated that both SSA and PTA were reliable in estimating 2D kinetic rates. Parametric analysis also demonstrated that such the estimations were sensitive to parameters such as sampling rate, sliding window size, and threshold. These results furthered the understandings in quantifying the biophysics of receptor-ligand interactions.

IL-8-induced L-selectin shedding regulates its binding kinetics to PSGL-1

L-selectin plays a crucial role in inflammation cascade by initiating the tethering and rolling of leukocytes on endothelium wall. While many L-selectin molecules are rapidly shed from the cell surface upon activation, the remaining membrane-anchored L-selectin may still play an important role in regulating leukocyte rolling and adhesion with different binding kinetics. Here we developed an in vitro model to activate Jurkat cells via interlukin-8 (IL-8) and quantified the two-dimensional (2D) binding kinetics, using a micropipette aspiration assay, of membrane-anchored L-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) ligand coupled onto human red blood cells (RBCs). The data indicated that L-selectin shedding reduced the amount of membrane-anchored L-selectin and lowered both its reverse and forward rates. These results suggested that the rolling dynamics of activated leukocytes was determined by two opposite impacts: reducing the surface presentation would enhance the rolling but lowering the kinetic rates would decrease the rolling. This finding provides a new insight into understanding how L-selectin shedding regulates leukocyte rolling and adhesion.

Influence of HCl on CO and NO emissions in combustion

The influence of HCl on CO and NO emissions was experimentally investigated in an entrained flow reactor (EFR) and an internally circulating fluidized bed (ICFB). The results in EFR show the addition of HCl inhibits CO oxidation and NO formation at 1073 K and 1123 K. At the lower temperature (1073 K) the inhibition of HCl becomes more obvious. In ICFB, chlorine-containing plastic (PVC) was added to increase the concentration of HCl during the combustion of coal or coke. Results show that HCl is likely to enhance the reduction of NO and N2O. HCl greatly increases CO and CH4 emission in the flue gas. A detailed mechanism of CO/NO/HCl/SO2 system was used to model the effect of HCl in combustion. The results indicate that HCl not only promotes the recombination of radicals O, H, and OH, but also accelerates the chemical equilibration of radicals. The influence of HCl on the radicals mainly occurs at 800-1200 K. (C) 2009 Elsevier Ltd. All rights reserved.

THE FORMATION MECHANISM OF THE VITRIFIED SLAG IN A PLASMA ARC REACTOR FOR HAZARDOUS WASTE TREATMENT

Various hazardous wastes with additives have been vitrified to investigate the formation mechanism of the glassy slag by a 30 kW DC plasma-arc reactor developed by the Institute of Mechanics, Chinese Academy of Sciences. The average temperature in the reaction area is controlled at 1500°C. The chemical compositions of three sorts of fly ashes are analyzed by XRF (X-Ray Fluorescence). Fly ashes with vitrifying additives can be vitrified to form glassy slag, which show that the ratio of the whole oxygen ions to the whole network former ions in glass (R) is appropriate in the range of 2~3 to form durable vitrified slag. In this experiment, the arc power is controlled below 5 kW to inhibit waste evaporation. To enhance the effects of heat transfer to wastes, ferrous powder has been added into the graphite crucible, which aggregates as ingot below the molten silicate after vitrification. The slag fails to form glass if the quenching rate is less than 1 K/min. Therefore, the slag will break into small chips due to the sharp quenching rate, which is more than 100 K/sec.

Part I. Kinetics of coarsening of the gamma-prime precipitate in nickel-silicon alloys. Part II. Rate of crystallization of an amorphous Fe-P-C alloy

The coarsening kinetics of Ni₃ Si(γ') precipitate in a binary Ni-Si alloy containing 6.5 wt. % silicon was studied by magnetic techniques and transmission electronmicroscopy. A calibration curve was established to determine the concentration of silicon in the matrix. The variation of the Si content of the Ni-rich matrix as a function of time follows Lifshitz and Wagner theory for diffusion controlled coarsening phenomena. The estimated values of equilibrium solubility of silicon in the matrix represent the true coherent equilibrium solubilities.

The experimental particle-size distributions and average particle size were determined from dark field electron micrographs. The average particle size varies linearly with t^-1/3 as suggested by Lifshitz and Wagner. The experimental distributions of particle sizes differ slightly from the theoretical curve at the early stages of aging, but the agreement is satisfactory at the later stages. The values of diffusion coefficient of silicon, interfacial free energy and activation energy were calculated from the results of coarsening kinetics. The experimental value of effective diffusion coefficient is in satisfactory agreement with the value predicted by the application of irreversible the rmodynamics to the process of volume constrained growth of coherent precipitate during coarsening. The coherent γ' particles in Ni-Sialloy unlike those in Ni-Al and Ni-Ti seem to lose coherency at high temperature. A mechanism for the formation of semi-coherent precipitate is suggested.

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

Exploring the Kinetics of Domain Switching in Ferroelectrics for Structural Applications

The complex domain structure in ferroelectrics gives rise to electromechanical coupling, and its evolution (via domain switching) results in a time-dependent (i.e. viscoelastic) response. Although ferroelectrics are used in many technological applications, most do not attempt to exploit the viscoelastic response of ferroelectrics, mainly due to a lack of understanding and accurate models for their description and prediction. Thus, the aim of this thesis research is to gain better understanding of the influence of domain evolution in ferroelectrics on their dynamic mechanical response. There have been few studies on the viscoelastic properties of ferroelectrics, mainly due to a lack of experimental methods. Therefore, an apparatus and method called Broadband Electromechanical Spectroscopy (BES) was designed and built. BES allows for the simultaneous application of dynamic mechanical and electrical loading in a vacuum environment. Using BES, the dynamic stiffness and loss tangent in bending and torsion of a particular ferroelectric, viz. lead zirconate titanate (PZT), was characterized for different combinations of electrical and mechanical loading frequencies throughout the entire electric displacement hysteresis. Experimental results showed significant increases in loss tangent (by nearly an order of magnitude) and compliance during domain switching, which shows promise as a new approach to structural damping. A continuum model of the viscoelasticity of ferroelectrics was developed, which incorporates microstructural evolution via internal variables and associated kinetic relations. For the first time, through a new linearization process, the incremental dynamic stiffness and loss tangent of materials were computed throughout the entire electric displacement hysteresis for different combinations of mechanical and electrical loading frequencies. The model accurately captured experimental results. Using the understanding gained from the characterization and modeling of PZT, two applications of domain switching kinetics were explored by using Micro Fiber Composites (MFCs). Proofs of concept of set-and-hold actuation and structural damping using MFCs were demonstrated.

Programming chemical kinetics: engineering dynamic reaction networks with DNA strand displacement

Over the last century, the silicon revolution has enabled us to build faster, smaller and more sophisticated computers. Today, these computers control phones, cars, satellites, assembly lines, and other electromechanical devices. Just as electrical wiring controls electromechanical devices, living organisms employ "chemical wiring" to make decisions about their environment and control physical processes. Currently, the big difference between these two substrates is that while we have the abstractions, design principles, verification and fabrication techniques in place for programming with silicon, we have no comparable understanding or expertise for programming chemistry.

In this thesis we take a small step towards the goal of learning how to systematically engineer prescribed non-equilibrium dynamical behaviors in chemical systems. We use the formalism of chemical reaction networks (CRNs), combined with mass-action kinetics, as our programming language for specifying dynamical behaviors. Leveraging the tools of nucleic acid nanotechnology (introduced in Chapter 1), we employ synthetic DNA molecules as our molecular architecture and toehold-mediated DNA strand displacement as our reaction primitive.

Abstraction, modular design and systematic fabrication can work only with well-understood and quantitatively characterized tools. Therefore, we embark on a detailed study of the "device physics" of DNA strand displacement (Chapter 2). We present a unified view of strand displacement biophysics and kinetics by studying the process at multiple levels of detail, using an intuitive model of a random walk on a 1-dimensional energy landscape, a secondary structure kinetics model with single base-pair steps, and a coarse-grained molecular model that incorporates three-dimensional geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Our findings are consistent with previously measured or inferred rates for hybridization, fraying, and branch migration, and provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.

In Chapters 3 and 4, we identify and overcome the crucial experimental challenges involved in using our general DNA-based technology for engineering dynamical behaviors in the test tube. In this process, we identify important design rules that inform our choice of molecular motifs and our algorithms for designing and verifying DNA sequences for our molecular implementation. We also develop flexible molecular strategies for "tuning" our reaction rates and stoichiometries in order to compensate for unavoidable non-idealities in the molecular implementation, such as imperfectly synthesized molecules and spurious "leak" pathways that compete with desired pathways.

We successfully implement three distinct autocatalytic reactions, which we then combine into a de novo chemical oscillator. Unlike biological networks, which use sophisticated evolved molecules (like proteins) to realize such behavior, our test tube realization is the first to demonstrate that Watson-Crick base pairing interactions alone suffice for oscillatory dynamics. Since our design pipeline is general and applicable to any CRN, our experimental demonstration of a de novo chemical oscillator could enable the systematic construction of CRNs with other dynamic behaviors.