Applications of stochastic simulation in two-stage multiple comparisons with the best problem and time average variance constant estimation

In this dissertation, we study two problems. In the first part, we consider the two-stage methods for comparing alternatives using simulation. Suppose there are a finite number of alternatives to compare, with each alternative having an unknown parameter that is the basis for comparison. The parameters are to be estimated using simulation, where the alternatives are simulated independently. We develop two-stage selection and multiple-comparison procedures for simulations under a general framework. The assumptions are that each alternative has a parameter estimation process that satisfies a random- time-change central limit theorem (CLT), and there is a weakly consistent variance estimator (WCVE) for the variance constant appearing in the CLT. The framework encompasses comparing means of independent populations, functions of means, and steady-state means. One problem we consider of considerable practical interest and not handled in previous work on two-stage multiple-comparison procedures is comparing quantiles of alternative populations. We establish the asymptotic validity of our procedures as the prescribed width of the confidence intervals or indifference-zone parameter shrinks to zero. Also, for the steady-state simulation context, we compare our procedures based on WCVEs with techniques that instead use standardized time series methods. In the second part, we propose a new technique of estimating the variance parameter of a wide variety of stochastic processes. This new technique is better than the existing techniques for some standard stochastic processes in terms of bias and variance properties, since it reduces bias at the cost of no significant increase in variance

Combinatorial structures for design of wireless sensor networks

Combinatorial designs are very effective tools for managing keys in an infrastructure where power and memory are two major constraints. None of the present day wireless technologies takes the advantage of combinatorial designs. In this paper, we have proposed a general framework using combinatorial designs which will enable the participating devices to communicate securely among themselves with little memory and power overhead. The scheme caters for different kinds of user requirements and allows the designer to choose different combinatorial designs for different parts or levels of the network. This general framework will find application in all wireless radio technologies, typically WPANs and WLANs. This is a hitherto unexplored technique in wireless technologies

Binding and conformation of denatured horseradish peroxidase during E. coli ribosome mediated folding

Denatured horseradish peroxidase (HRP) refolded in the presence of intact 70S E. coli ribosome. Fluorescence spectroscopic evidence of direct physical association between the ribosome particles and the denatured HRP during refolding has been detected. The efficiency of energy transfer from the single tryptophan (Trp) to the heme moiety and the quenching patterns of the Trp fluorescence by iodide and acrylamide differed with time while folding in the presence and absence of ribosome. An estimate of the binding of denatured fluorescein-conjugated HRP with ribosome was obtained from polarization measurements (Kd = 41 nM)

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

<div><p>Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA <i>in vivo</i> and in in vitro translation with <i>E</i>.<i>coli</i> S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here.</p></div

<i>E</i>. <i>coli</i> ribosomal RNA (rRNA) structure derived from PDB structure 3J7Z.

<p><b>(A)</b> Domain V of rRNA is shown in cyan; two r-proteins L4 and L22 are marked that forms narrowest constriction at the peptide exit tunnel; CCA-end of the P-site tRNA is colored yellow; rRNA nucleotides that release unfolded protein slowly are marked red (based on our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170333#pone.0170333.ref012" target="_blank">12</a>]); rRNA helices that constitute important bridges B2a/B2b, B3 –that join 50S to 30S are marked green (RRF interacts with this region to dissociate 70S). <b>(B)</b> Close-up view showing rRNA nucleotides numbering that release unfolded protein slowly (red) are in close proximity to the rRNA helices constituting B2a/B2b, B3 bridges (green); CCA-end of tRNA is marked yellow.</p

Dissociation of ribosomal subunits analyzed using fluorescence light scattering (at 20°C) and 5–20% sucrose density gradient centrifugation.

<p><b>(A)</b>Percent (%) dissociations of 70S are plotted as a function of time by: unfolded bovine carbonic anhydrase (UNP) (■); RRF+EFG-GTP+IF3 (●); decapeptide VGDANPALQK (▲); Native bovine carbonic anhydrase (▼). <b>(B)</b>Percent (%) dissociations of 70S by: RRF+EFG-GTP+IF3 (■); UNP+RRF+EFG-GTP+IF3 (●); UNP+EFG-GTP (▲); UNP+RRF (▼) are plotted against time. <b>(C)</b>Percent (%) dissociations of 70S by: UNP+EFG-GMPPNP (■); EFG-GMPPNP (●); EFG-GTP (▲) are plotted against time. <b>(D)</b>Dissociation rate constants (<i>k;</i> s^-1) are derived from the single exponential fits of the respective graphs and plotted as bar graphs against the corresponding combination of factors indicated in the figure. Error bars (s.d.) are propagated from three independent experiments for each combination of factors. <b>(E)</b> P-values for the dissociation rate constants (<i>k</i>) are calculated from three independent experiments for the respective combination of factors as indicated in Fig D and plotted here. Results showing statistical significance at <i>p</i>< 0.05. <b>(F)</b> Sucrose density gradient centrifugation showing dissociation of 70S by: unfolded protein (■); deca-peptide VGDANPALQK (●). The profile of 70S, 50S, 30S (▲) ran in a parallel sucrose gradient; and only untreated 70S (▼) ran in another gradient in parallel, are shown. <b>(G)</b> Sucrose density gradient centrifugation showing dissociation of 70S by: the combinations of RRF, EFG-GTP and IF3 (■);UNP, RRF, EFGGTP and IF3 (●); UNP and EFG-GTP (▲);UNP and RRF(▼).</p