Transient-State Kinetic Studies of The Mechanisms of Dna Unwinding And Translocation of The E. Coli Recbc And Recbcd Helicases

This thesis presents mechanistic studies of the E. coli RecBC and RecBCD helicases using transient-state kinetic approaches to understand the relationship between DNA unwinding and ssDNA translocation. RecBC initiates unwinding from duplex DNA ends with pre-existing 5\u27-(dT)6 and 3\u27-(dT)6 ssDNA tails using a series of repeated rate-limiting steps with a rate of 345 ┬▒ 8 bp/sec and an average kinetic step-size of ~4 bp: 20 mM Mops-KOH, pH 7.0 at 25°C, 30 mM NaCl, 10 mM MgCl2, 5% glycerol, 1 mM 2-mercaptoethanol) while RecBCD unwinds these same DNA substrates with a rate of 745 ┬▒ 18 bp/sec using a more complicated mechanism which involves the loading of the 5\u27 ssDNA end onto the RecD subunit. After DNA unwinding, RecBC can continue to translocate along a ssDNA extension in the 3\u27 to 5\u27 direction with a rate of 909 ┬▒ 51 nt/sec, consistent with the directionality of the RecB motor subunit. Surprisingly, RecBC also possesses a secondary translocase activity that enables it to move along a ssDNA extension of the opposite DNA strand with a similar rate: 990 ┬▒ 49 nt/sec). Both translocase activities are coupled to ATP hydrolysis from the RecB motor, and the primary translocase is sensitive to the polarity of the ssDNA backbone while the secondary translocase is not. RecBC can also translocate along two non-complementary ssDNA extensions simultaneously using both translocase activities. During DNA unwinding, RecBC consumes an average of 0.95 ┬▒ 0.08 ATP/bp unwound. The primary translocase activity utilizes 0.81 ┬▒ 0.05 ATP/nt translocated while its secondary translocase activity is less tightly coupled and requires 1.12 ┬▒ 0.06 ATP/nt. Translocation along two non-complementary ssDNA extensions has an ATP coupling stoichiometry of 1.07 ┬▒ 0.09 ATP/nt. These data indicate that the large majority, possibly all, of the ATP hydrolyzed by RecBC during DNA unwinding is used to fuel RecBC translocation along the nucleic acid rather than to facilitate base pair melting. These results suggest that RecBC uses a two step active mechanism to unwind DNA by first destabilizing the duplex using its binding free energy in an ATP-independent process, followed by ATP-dependent translocation along the resulting ssDNA

Robust Tests in Genome-Wide Scans under Incomplete Linkage Disequilibrium

Under complete linkage disequilibrium (LD), robust tests often have greater power than Pearson's chi-square test and trend tests for the analysis of case-control genetic association studies. Robust statistics have been used in candidate-gene and genome-wide association studies (GWAS) when the genetic model is unknown. We consider here a more general incomplete LD model, and examine the impact of penetrances at the marker locus when the genetic models are defined at the disease locus. Robust statistics are then reviewed and their efficiency and robustness are compared through simulations in GWAS of 300,000 markers under the incomplete LD model. Applications of several robust tests to the Wellcome Trust Case-Control Consortium [Nature 447 (2007) 661--678] are presented.Comment: Published in at http://dx.doi.org/10.1214/09-STS314 the Statistical Science (http://www.imstat.org/sts/) by the Institute of Mathematical Statistics (http://www.imstat.org

A Cross-Validation Bandwidth Choice for Kernel Density Estimates with Selection Biased Data

AbstractThis paper studies the risks and bandwidth choices of a kernel estimate of the underlying density when the data are obtained fromsindependent biased samples. The main results of this paper give the asymptotic representation of the integrated squared errors and the mean integrated squared errors of the estimate and establish a cross-validation criterion for bandwidth selection. This kernel density estimate is shown to be asymptotically superior to many other intuitive kernel density estimates. The data-driven cross-validation bandwidth is shown to be asymptotically optimal in the sense of Stone (1984,Ann. Statist.12, 1285–1297). The finite sample properties of the cross-validation bandwidth are investigated through a Monte Carlo simulation

Design and Optimization of Low-power Level-crossing ADCs

This thesis investigates some of the practical issues related to the implementation of level-crossing ADCs in nanometer CMOS. A level-crossing ADC targeting minimum power is designed and measured. Three techniques to circumvent performance limitations due to the zero-crossing detector at the heart of the ADC are proposed and demonstrated: an adaptive resolution algorithm, an adaptive bias current algorithm, and automatic offset cancelation. The ADC, fabricated in 130 nm CMOS, is designed to operate over a 20 kHz bandwidth while consuming a maximum of 8.5 uW. A peak SNDR of 54 dB for this 8-bit ADC demonstrates a key advantage of level-crossing sampling, namely SNDR higher than the classic Nyquist limit

A Blockchain Model in Tamarin and Formal Analysis of Hash Time Lock Contract

Formal analysis and verification methods can aid the design and validation of security properties in blockchain based protocols. However, to generate a reasonable and correct verification, a proper model for the blockchain is needed. In this paper, we give a blockchain model in Tamarin. Based on our model we analyze and give a formal verification for the hash time lock contract, an atomic cross chain trading protocol. The result shows that our model is able to identify an underlying assumption for the hash time lock contract and that the model is useful for analyzing blockchain based protocols

The equilibrium model for the effect of temperature on enzymes: Insights and implications

A new, experimentally-validated “Equilibrium Model” describes the effect of temperature on enzymes, and provides a new mechanism for the reversible loss of enzyme activity with temperature. It incorporates two new, fundamental parameters that allow a complete description of the effect of temperature on enzyme activity: ΔHeq and Teq. ΔHeq emerges as an intrinsic and quantitative measure of enzyme eurythermal adaptation, while Teq, the equilibrium temperature, has fundamental and technological significance for our understanding of the effect of temperature on enzymatic reactions. For biotechnological purposes, these parameters need to be considered when enzymes are applied or engineered for activity at high temperatures

Enhanced anticancer activity of a combination of docetaxel and Aneustat (OMN54) in a patient-derived, advanced prostate cancer tissue xenograft model.

The current first-line treatment for advanced metastatic prostate cancer, i.e. docetaxel-based therapy, is only marginally effective. The aim of the present study was to determine whether such therapy can be improved by combining docetaxel with Aneustat (OMN54), a multivalent botanical drug candidate shown to have anti-prostate cancer activity in preliminary in vitro experiments, which is currently undergoing a Phase-I Clinical Trial. Human metastatic, androgen-independent C4-2 prostate cancer cells and NOD-SCID mice bearing PTEN-deficient, metastatic and PSA-secreting, patient-derived subrenal capsule LTL-313H prostate cancer tissue xenografts were treated with docetaxel and Aneustat, alone and in combination. In vitro, Aneustat markedly inhibited C4-2 cell replication in a dose-dependent manner. When Aneustat was combined with docetaxel, the growth inhibitions of the drugs were essentially additive. In vivo, however, the combination of docetaxel and Aneustat enhanced anti-tumor activity synergistically and very markedly, without inducing major host toxicity. Complete growth inhibition and shrinkage of the xenografts could be obtained with the combined drugs as distinct from the drugs on their own. Analysis of the gene expression of the xenografts using microarray indicated that docetaxel + Aneustat led to expanded anticancer activity, in particular to targeting of cancer hallmarks that were not affected by the single drugs. Our findings, obtained with a highly clinically relevant prostate cancer model, suggest, for the first time, that docetaxel-based therapy of advanced human prostate cancer may be improved by combining docetaxel with Aneustat