Structure to function : case studies of hotdog-fold superfamily thioesterases from Escherichia coli.

My doctoral research primarily focuses on the Escherichia coli hotdog-fold thioesterases ydiI and ybgC. The ydiI gene is colocalized in the operon ydiHIJ. A substrate screen provided evidence that YdiI prefers aryl-CoA substrates and discriminated against the analogous aryl-ACP, unlike the paralog YbdB which works on 2,4-DHB-EntB in the enterobactin synthesis pathway. A bioinformatic approach showed that in some bacteria, ydiI is colocalized with genes from the menaquinone pathway. YdiI was shown to catalyze the hydrolysis of DHNA-CoA with physiological relevance (kcat/KM ~ 105). Furthermore, the E. coli YdiI- strain was shown to have perturbed growth, in good agreement with other menaquinone enzyme knockout experiments. Taking into account the evidence provided, YdiI is likely the DHNA-CoA thioesterase in the menaquinone pathway. Within the substrate binding pocket, changes to the YdiI Val68 to the YbdB equivalent of Met resulted in perturbed catalytic efficiency towards lauroyl-CoA, however not towards its physiological substrate DHNA-CoA. The YbdB M68V mutant resulted in increased efficiency towards lauroyl-CoA and DHNA-CoA, seemingly making it a gatekeeper. Secondly, YdiI catalyzes the hydrolysis of acyl-CoA\u27s using a Glu63, His54, and Gln48 triad determined by mutagenesis experiments. The utilization of 18-O incorporation and rapid-quench techniques provided insight into YdiI\u27s catalytic mechanism. A single phase multiple turnover reaction and the incorporation of 18-O in single turnover reactions suggests that ydiI uses a general base catalytic mechanism where Glu63 is the activating residue. The second E. coli hotdog-fold thioesterase protein discussed within is YbgC. YbgC is encoded by the first ORF of the tol-pal gene cluster. Previous work on the H. influenzae homologue demonstrated affinity for short chain acyl-CoA\u27s and tandem affintity purificiation experiments showed E. coli YbgC co-purifies with ACP. No previous attempts to screen for acyl-ACP have been made. To screen YbgC for ACP activity, a method was developed to stoichiometrically construct acyl-ACP\u27s, using apoACP, BF1558 acyl-transferase and acyl-CoA. The E. coli YbgC demonstrated preference for long chain acyl-CoA\u27s and their analogous acyl-ACPs. To understand the nature of ACP binding to YbgC, SAXS analysis was carried out on the complex demonstrating that 2 ACP molecules bind to 1 dimer of YbgC

The effect of outlines, headings and summaries on the recall of informational text

The purpose of this study was to investigate various text-based ways of assisting Year Seven readers to enhance their recall of informational texts. The theory that was tested in this investigation is that text-based reading aids which secure the activation and rehearsal of relevant form and/or content schemata for written texts will enhance the recall of the content and structure of those texts. To investigate the comparative impact of the text conditions, 105 Year Seven students from a large primary school in a northern suburb of the Perth Metropolitan Area were ranked on the basis of their reading comprehension scores. They were systematically assigned to each of the five groups to create five approximately heterogeneous groups thus creating a stratified sample. Two separate experiments were conducted. The dependent variable for the first was the recall of content, while for the second, it was the ability to show a knowledge of the text’s structure. It was concluded that, for this sample of Year Seven students, the quantity of content recalled was enhanced to about the same significant degree by the use of in-text headings or the use of a pre-text outline when compared with the no text-based aids condition. Further, it was concluded that the use of in-text headings and a pre-text outline enhanced the recall of text content about as well as the use of in-text headings, a pre-text outline and a post-text summary. Both produced significantly enhanced recall when compared with the no text-based aids condition the in-text headings condition and the pre-text outline condition. As a result of experiment two, it was concluded that the recall of text structure was not enhanced by the use of in-text headings. It was further concluded that the use of a pre-text outline, the use of a pre-text outline with in-text headings, and the use of a pre-text outline with in-text headings and a post-text summary produced equal scores on the recall of text structure. All three of these conditions produced significantly enhanced recall of text structure when compared with the no text-based aids condition and the in-text headings condition. Since the pre-text outline was the variable common to all of these conditions, it was concluded that the use of a pre-text outline had a significant, beneficial effect upon the recall of text structure. It is clear from the findings of this study that the recall of the content and structure of informational texts by Year Seven students, like those in the sample used in this study could be significantly enhanced if texts were produced with carefully formulated pre-text outlines and in-text headings

Cotton movement and fluctuation

[8 unnumbered pages], 156 pages : illustrations ; 22 c

期外収縮後一過性収縮性増強の減衰過程を指数関数でカーブフィッティングすると心筋細胞内のカルシウム再循環率を過小評価する可能性がある：理論的・数学的解析

Marine cloud brightening (MCB) is one of several proposed solar radiation management (SRM) geoengineering schemes designed to ameliorate some of the undesirable effects of climate change, for example polar ice loss and associated increased sea levels. Satellite measurements over the last 40 years show a general reduction in polar sea ice area and thickness which is attributed to climate change. In our studies, HadGEM1, a fully coupled climate model, is used to predict changes in surface temperatures and ice cover as a result of implementing MCB in a double carbon dioxide concentration atmosphere. The meridional heat flux (MHF) is the mechanism within the earth system for the transport of energy from tropical to polar regions. This poleward transport of heat in a double carbon dioxide atmosphere amplifies the effects in polar regions, where it has a significant impact on both temperatures and ice cover. The results from this work show that MCB is capable of roughly restoring control temperatures and ice cover (where control is defined as 440 ppm carbon dioxide, a predicted 2020 level) in a double carbon dioxide atmosphere scenario. This work presents the first results on the impact of MCB on the MHF and the ability of the MCB scheme to restore the MHF to a control level

Regulation of Set1-mediated methylation of Dam1

Eukaryotic genomes exist within a dynamic structure named chromatin in which DNA is wrapped around an octamer of histones forming the nucleosome. Histones are modified by a range of posttranslational modifications including methylation, phosphorylation, and ubiquitination, which are integral to a range of DNA-templated processes including transcriptional regulation. A hallmark for transcriptional activity is methylation of histone H3 on lysine (K) 4 within active gene promoters. In S. cerevisiae, H3K4 methylation is mediated by Set1 within the COMPASS complex. Methylation requires prior ubiquitination of histone H2BK123 by the E2-E3 ligases Rad6 and Bre1, as well as the Paf1 transcriptional elongation complex. This regulatory pathway exemplifies cross-talk in trans between posttranslational modifications on distinct histone molecules. Set1 has an additional substrate in the kinetochore protein Dam1, which is methylated on K233. This methylation antagonizes phosphorylation of adjacent serines by the Ipl1 Aurora kinase. The discovery of a second Set1 substrate raised the question of how Set1 function is regulated at the kinetochore. I hypothesized that transcriptional regulatory factors essential for H3K4 methylation at gene promoters might also regulate Set1-mediated methylation of Dam1K233. Here I show that the regulatory factors essential for COMPASS activity at gene promoters is also indispensable for the methylation of Dam1K233. Deletion of members of the COMPASS complex leads to loss of Dam1K233 methylation. In addition, deletion of Rad6, Bre1, or members of the Paf1 complex abolishes Dam1 methylation. The role of Rad6 and Bre1 in Dam1 methylation is dependent on H2BK123 ubiquitination, as mutation of K123 within H2B results in complete loss of Dam1 methylation. Importantly, methylation of Dam1K233 is independent of transcription and occurs at the kinetochore. My results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription at the kinetochore. My data provide the first example of cross-talk in trans between modifications on a histone and a non-histone protein. Additionally, my results indicate that several factors previously thought to be required for Set1 function at gene promoters are more generally required for the catalytic activity of the COMPASS complex regardless of substrate or cellular process

Exploration of the Psychological Factors Impacting Confidence in, and Comfort with, the Delivery of End-of-Life Care

Part 1: Systematic Review Abstract Objective To explore the emotional experience of physicians in acute settings when encountering end-of-life conversations and decision making. Method Thematic Synthesis of qualitative studies. Medline, PsycINFO, PubMed, BNI, and CIAHL were searched from 1985 to 2021 for studies published in English. Data extraction was informed by a framework created for assessing methodological quality by Polanin, Pigott, Espelage, & Grotpeter (2019) and adapted by Draper et al. (2019). Results Of 8429 papers identified, 17 were selected for review. Two themes containing 10 sub-themes described the emotional and psychological factors impacting the experience of end-of-life care, namely: a tension between desire and ability to communicate end-of-life news, and a conflict of hiding versus revealing self across several practical and emotional contexts. Conclusion Medical training is only a minor factor influencing how well a person copes with end-of-life care and may sometimes feed negative appraisals. Lack of support from senior colleagues, fear of criticism and a sense of perceived failure were linked to lower self-efficacy in end-of-life care. Beyond learning practical skills, physicians benefit from understanding the psychological factors impacting their experience and in building self-efficacy and observing senior colleagues processing strong and difficult emotions effectively. Clinical Implications Promoting personal reflection and sharing of the experiences encountered in end-of-life care, especially modelled from senior colleagues, may contribute to improvements in competence and reduce the impact of heroism, feelings of failure, and avoidance in practice. Part 2: Empirical Study Abstract Objective To investigate whether fear of failure (FOF) influences a clinician’s perception of their confidence and comfortableness with the delivery of end of life (EOL) care, controlling for gender, role, years of experience, and number of EOL conversations. Methods Cross-sectional questionnaire study, using the Performance Failure Appraisal Inventory, the Self-Efficacy in Palliative Care scale, the Thanatophobia Scale, and analysed using a two-step multiple regression. Recruitment was across two large NHS hospital trusts in the UK, and national UK professional networks. Results Participants included 104 doctors and 101 specialist nurses across 20 hospital specialties. The study validated the use of the PFAI and its subscales within a novel population. No. of EOL conversations, gender, and role impacted confidence and comfortableness with EOL care. Fearing loss of interest negatively impacted a clinician’s confidence in communicating with patients. Fear of devaluing one’s self-estimate negatively impacted confidence in decision-making, working with others, and self-efficacy. Conclusion Three aspects of FOF negatively impacted both doctors’ and nurses’ delivery of EOL care. Clinical Implications Further study should explore how FOF develops, sustaining factors, and other areas of clinical practice that FOF impacts, drawing also from FOF research outside the field of medicine. Techniques developed to manage FOF in other populations can now be investigated with a medical population

Line Broadening in Field Metal-poor Red Giant and Red Horizontal Branch Stars

We report 349 radial velocities for 45 metal-poor field red giant and red horizontal branch stars. We have have identified one new spectroscopic binary, HD 4306, and one possible such system, HD 184711. We also report 57 radial velocities for 11 of the 91 stars reported on previously by Carney et al. (2003). As was found in the previous study, radial velocity "jitter" is present in many of the most luminous stars. Excluding stars showing spectroscopic binary orbital motion, all 7 of the red giants with M(V) <= -2.0 display jitter, as well as 3 of the 14 stars with -2.0 <= M(V) <= -1.4. We have also measured line broadening in all of the new spectra, using synthetic spectra as templates. The most luminous red giants show significant line broadening, as do many of the red horizontal branch stars, and we discuss briefly possible causes.Comment: To appear in the Astronomical Journa