Phototaxis signaling by the membrane complex of archaeal sensory rhodopsins and their transducers

Sensory rhodopsins I and II (SRI and SRII) are visual pigment-like phototaxis receptors in the archaeon Halobacterium salinarum. The receptor proteins each consist of a single polypeptide that folds into 7 $\alpha$-helical membrane-spanning segments forming an internal pocket where the chromophore retinal is bound. They transmit signals to their tightly bound transducer proteins, HtrI and HtrII, respectively, which in turn control a phosphotransfer pathway modulating the flagellar motors. SRI-HtrI mediates attractant responses to orange-light and repellent responses to UV light, while SRII-HtrII mediates repellent response to blue light. Experiments were designed to analyze the molecular processes in the SR-Htr complexes responsible for receptor activation, which previously had been shown by our laboratory to involve proton transfer reactions of the retinylidene Schiff base in the photoactive site, transfer of signals from receptor to transducer, and signaling specificity by the receptor-transducer complex.^ Site-directed mutagenesis and laser-flash kinetic spectroscopy revealed that His-166 in SRI (i) plays a role in the proton transfers both to and from the Schiffbase, either as a structurally critical residue or possibly as a direct participant, (ii) is involved in the modulation of SIU photoreaction kinetics by HtrI, and (iii) modulates the pKa of Asp-76, an important residue in the photoactive site, through a long-distance electrostatic interaction. Computerized cell tracking and motion analysis demonstrated that (iv) His-166 is crucial in phototaxis signaling: a spectrum of substitutions either eliminate signaling or greatly perturb the activation process that produces attractant and repellent signaling states of the receptor.^ The signaling states of SRI are communicated to HtrI, whose oligomeric structure and conformational changes were investigated by engineered sulfhydryl probes. It was found that signaling by the SRI-HtrI complex involves reversible conformational changes within a preexisting HtrI dimer, which is likely accomplished through a slight winding or unwinding of the two HtrT monomers via their loose coiled coil association. To elucidate which domains of the Htr dimers confer specificity for interaction with SRI or SRII, chimeras of HtrI and HtrII were constructed. The only determinant needed for functional and specific interaction with SRI or SRII was found to be the four transmembrane segments of the HtrI or HtrII dimers, respectively. The entire cytoplasmic parts of HtrI and HtrII, which include the functionally important signaling and adaptation domains, were interchangeable.^ These observations support a model in which SRI and SRII undergo conformational changes coupled to light-induced proton transfers in their photoactive sites, and that lateral helix-helix interactions with their cognate transducers' 4-helix bundle in the membrane relay these conformational changes into different states of the Htr proteins which regulate the down-stream phosphotransfer pathway. ^

Interim analysis of clinical trials: Simulation studies of fractional Brownian motion

Interim clinical trial monitoring procedures were motivated by ethical and economic considerations. Classical Brownian motion (Bm) techniques for statistical monitoring of clinical trials were widely used. Conditional power argument and α-spending function based boundary crossing probabilities are popular statistical hypothesis testing procedures under the assumption of Brownian motion. However, it is not rare that the assumptions of Brownian motion are only partially met for trial data. Therefore, I used a more generalized form of stochastic process, called fractional Brownian motion (fBm), to model the test statistics. Fractional Brownian motion does not hold Markov property and future observations depend not only on the present observations but also on the past ones. In this dissertation, we simulated a wide range of fBm data, e.g., H = 0.5 (that is, classical Bm) vs. 0.5< H <1, with treatment effects vs. without treatment effects. Then the performance of conditional power and boundary-crossing based interim analyses were compared by assuming that the data follow Bm or fBm. Our simulation study suggested that the conditional power or boundaries under fBm assumptions are generally higher than those under Bm assumptions when H > 0.5 and also matches better with the empirical results. ^

Cost analysis of opt-in consent procedures for the Texas statewide immunization information system, ImmTrac

Background. Childhood immunization programs have dramatically reduced the morbidity and mortality associated with vaccine-preventable diseases. Proper documentation of immunizations that have been administered is essential to prevent duplicate immunization of children. To help improve documentation, immunization information systems (IISs) have been developed. IISs are comprehensive repositories of immunization information for children residing within a geographic region. The two models for participation in an IIS are voluntary inclusion, or "opt-in," and voluntary exclusion, or "opt-out." In an opt-in system, consent must be obtained for each participant, conversely, in an opt-out IIS, all children are included unless procedures to exclude the child are completed. Consent requirements for participation vary by state; the Texas IIS, ImmTrac, is an opt-in system.^ Objectives. The specific objectives are to: (1) Evaluate the variance among the time and costs associated with collecting ImmTrac consent at public and private birthing hospitals in the Greater Houston area; (2) Estimate the total costs associated with collecting ImmTrac consent at selected public and private birthing hospitals in the Greater Houston area; (3) Describe the alternative opt-out process for collecting ImmTrac consent at birth and discuss the associated cost savings relative to an opt-in system.^ Methods. Existing time-motion studies (n=281) conducted between October, 2006 and August, 2007 at 8 birthing hospitals in the Greater Houston area were used to assess the time and costs associated with obtaining ImmTrac consent at birth. All data analyzed are deidentified and contain no personal information. Variations in time and costs at each location were assessed and total costs per child and costs per year were estimated. The cost of an alternative opt-out system was also calculated.^ Results. The median time required by birth registrars to complete consent procedures varied from 72-285 seconds per child. The annual costs associated with obtaining consent for 388,285 newborns in ImmTrac's opt-in consent process were estimated at $702,000. The corresponding costs of the proposed opt-out system were estimated to total $194,000 per year. ^ Conclusions. Substantial variation in the time and costs associated with completion of ImmTrac consent procedures were observed. Changing to an opt-out system for participation could represent significant cost savings. ^