An anion transporter theory of the outer hair cell motor protein.

This thesis addresses the mechanism of electromotility in outer hair cells (OHCs) of the mammalian cochlea. Prestin, a protein densely packed in the lateral membrane is assumed to drive electromotility. A current hypothesis is that prestin is an incomplete transporter, shuttling chloride across the membrane without allowing it to dissociate at the extracellular surface. In this thesis kinetic models are formulated to show that this hypothesis cannot reproduce the previously published experimental data from electrical recordings. However an alternative model of prestin as an anion exchanger (modelled here as a chloride/sulphate exchanger) is formulated, which can reproduce many of the experimental observations. In this model the experimentally observed charge movements across the cell membrane are produced by the translocation of a chloride ion combined with some intrinsic charged residues. To further test the predictions of the model, patch clamp recordings were performed on dissociated OHCs, in the excised patch and whole-cell configurations. The OHC non-linear capacitance (NLC) depended on the concentration of intracellular chloride (Clj). When Clj was removed from internal and external solutions, a residual NLC (-15- 30%) was found, which was consistent with the predictions of the model for contaminant levels of Clj ( 10uM). Additionally the effect on the NLC of reducing Clj depended on the species of anion used to replace Clj. The largest effect was produced by replacement with sulphate, whilst the smallest effect was produced by replacement with glutamate. These findings support the model. Finally two potential causes for previous controversy in the literature were identified. 1) The NLC depended on the recording configuration when Clj was reduced below 1-1 OmM. 2) The dependence of the NLC on Clj was affected when Tris+ replaced Na+ as the major cation in solutions

Research supervision: faculty perspectives

Background: Research supervision is an increasingly important professional role of faculty members. There is now a growing body of research that explores factors which underpin good supervisory practice. Despite the progress in this area, there is scarcity of existing literature about the readiness of academic faculty who are involved in both undergraduate and postgraduate research supervision. One of the main obstacles in not being able to understand these issues is the lack of appropriate tools to measure research supervision skills. The aim of this thesis is to develop a valid and reliable scale to explore research supervision practices among health science faculty members, identify factors affecting the supervisory process and assess research supervisor’s readiness/preparedness towards guiding students’ research projects. Methods: A stepwise mixed methods study was carried out to develop and validate an instrument, the Research Supervision and Academic Readiness Scale (RSARS) that explored research supervision and academic readiness among health sciences faculty. The first stage (stage I) involved expert’s opinions, a focus group and a Delphi technique which generated appropriate items that were deemed necessary to be included in the instrument. The scale was piloted to identify the main dimensions/domains which included administrative and personal skills and interpersonal factors while looking for the internal consistency and the strengths of individual items. In stage II, a follow up in-depth qualitative study of the research supervisors’ experiences and practices was conducted with 18 participants from two universities to explore factors that influence academic supervision and to further refine the RSARS. Finally (stage III) was carried out to test the instrument in two different academic institutions representing two different contexts: King Saud Bin Abdul Aziz University for Health Sciences (KSAU-HS) and the University of Sydney (Sydney). To identify the underlying relationships between the items under each domain, Cronbach’s alpha were calculated and Exploratory Factor Analysis (EFA) performed to evaluate items step by step and 15 items were finally retained for the main study. Results: In stage I, the experts’ opinions highlighted the different questionnaire sections and domains. Findings from analysis of the focus group confirmed these domains and helped in refinement and additional items. The Delphi rounds helped in further items refinement and modification. Two rounds were considered adequate and all developed items were approved by 75% of the expert panel in agreement. The developed scale at this stage consisted of a total of thirty-eight items and Cronbach alpha of 0.98 showed item redundancy indicating the need for further review. In stage II (semi-structured interviews), the results were summarized into five main emergent themes, including institutional factors, supervisor/student interaction, professional development opportunities, motivational factors and challenges faced by supervisors. These issues are related to supervisors, students and their contexts. There was consensus among all participants regarding their motivation, challenges, and personal concerns when supervising research students. Rewards, recognition and time management were important factors expressed by the majority of the participants. Students’ personal characteristics such as enthusiasm, professional level and progress were of importance to the research supervision process. Contextual factors included clear institutional rules and regulations of supervision, valuing and recognition of supervisors, which were essential to majority of the supervisors. However, some structural differences were observed between the KSAU-HS and Sydney research supervisors. The majority of Sydney University participants had reservations about being co-supervisors rather than primary supervisors compared to KSAU-HS. In stage III, the RSARS was further developed and completed by a total of 235 participants as part of a survey including participant characteristics. There were 112/235 (47.7%) from KSAU-HS and 123/235 (52.3%) from the University of Sydney in the sample. The majority of KSAU-HS participants were males (p-value of 0.002); of younger age group (p-value of < 0.001), and had less teaching and supervision experience with a p-value of < 0.05 than their counterparts at Sydney. There was a highly significant difference between the two groups in the areas of initiating new studies or number of published papers with p-values of < 0.001. However, there were no significant differences between the two study areas with regard to the number of research students supervised (p-value < 0.36) or the number of times they were the primary supervisor (p-value of < 0.18). Also, there was variability in the supervisors’ personal skills and professionalism scores between the two study sites. Despite those differences, all study participants were in agreement with the need for more institutional support while also encouraging faculty enhancement activities for better supervision outcome. Conclusions: This study developed and validated a tool to assess the needs and readiness of research supervisors for individual assessments and faculty development interventions. The finding reports a range of validity evidence to support the use of the Research Supervision and Academic Readiness Scale (RSARA). This study highlighted that research supervision is influenced by multiple factors that need to be recognized and implemented for improving research supervisory skills. It is envisaged that this will have important implications for research supervisors' professional development. Future research is needed to further explore these factors from the perspective of supervisees as well as other relevant stakeholders

Decoding Ca2+ signals: a question of timing

Receptor-stimulated Ca2+ signals come in several flavors. The Ca2+ signals can be decoded linearly or by integration of the response. How the duration of the signal conveyed by cytosolic Ca2+ concentration ([Ca2+]i) changes is regulated is not well understood. Liu et al. (Liu, Q., S.A. Walker, D. Gao, J.A. Taylor, Y.-F. Dai, R.S. Arkell, M.D. Bootman, H.L. Roderick, P.J. Cullen, and P.J. Lockyer. 2005. J. Cell Biol. 170:183–190) now report an example of decoding based on the differential regulation of Ras function by two Ca2+-sensitive Ras inhibitors: Ca2+-promoted Ras activator (CAPRI), which extends the duration of the effect of Ca2+ on Ras activity, and Ras GTPase activating-like protein (RASAL), which functions as a linear decoder of the Ca2+ signal

Scattering from supramacromolecular structures

We study theoretically the scattering imprint of a number of branched supramacromolecular architectures, namely, polydisperse stars and dendrimeric, hyperbranched structures. We show that polydispersity and nature of branching highly influence the intermediate wavevector region of the scattering structure factor, thus providing insight into the morphology of different aggregates formed in polymer solutions.Comment: 20 pages, 8 figures To appear in PR

Transient receptor potential canonical type 3 channels control the vascular contractility of mouse mesenteric arteries

Transient receptor potential canonical type 3 (TRPC3) channels are non-selective cation channels and regulate intracellular Ca2+ concentration. We examined the role of TRPC3 channels in agonist-, membrane depolarization (high K+)-, and mechanical (pressure)-induced vasoconstriction and vasorelaxation in mouse mesenteric arteries. Vasoconstriction and vasorelaxation of endothelial cells intact mesenteric arteries were measured in TRPC3 wild-type (WT) and knockout (KO) mice. Calcium concentration ([Ca2+]) was measured in isolated arteries from TRPC3 WT and KO mice as well as in the mouse endothelial cell line bEnd.3. Nitric oxide (NO) production and nitrate/nitrite concentrations were also measured in TRPC3 WT and KO mice. Phenylephrine-induced vasoconstriction was reduced in TRPC3 KO mice when compared to that of WT mice, but neither high K+- nor pressure-induced vasoconstriction was altered in TRPC3 KO mice. Acetylcholine-induced vasorelaxation was inhibited in TRPC3 KO mice and by the selective TRPC3 blocker pyrazole-3. Acetylcholine blocked the phenylephrine-induced increase in Ca2+ ratio and then relaxation in TRPC3 WT mice but had little effect on those outcomes in KO mice. Acetylcholine evoked a Ca2+ increase in endothelial cells, which was inhibited by pyrazole-3. Acetylcholine induced increased NO release in TRPC3 WT mice, but not in KO mice. Acetylcholine also increased the nitrate/nitrite concentration in TRPC3 WT mice, but not in KO mice. The present study directly demonstrated that the TRPC3 channel is involved in agonist-induced vasoconstriction and plays important role in NO-mediated vasorelaxation of intact mesenteric arteries.Fil: Yeon, Soo-In. Yonsei University College of Medicine; Corea del SurFil: Kim, Joo Young. Yonsei University College Of Medicine; . Yonsei University College of Medicine; Corea del SurFil: Yeon, Dong-Soo. Kwandong University College of Medicine; Corea del SurFil: Abramowitz, Joel. National Institute of Environmental Health Sciences; Estados UnidosFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. National Institute of Environmental Health Sciences; Estados UnidosFil: Muallem, Shmuel. National Institutes of Health; Estados UnidosFil: Lee, Young-Ho. Yonsei University College of Medicine; Corea del Su

Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle