Cellular influence on protein folding

Advances in the study of protein folding and structure have greatly expanded our understanding and ability to predict and modify protein structure and, increasingly, even function. Attention has now turned to developing a better understanding of how protein structure, function, and folding interact to enable necessary biochemical processes in the cell. This work includes efforts to better understand the folding of model proteins in vitro, with an eye towards how this insight can inform questions about protein dynamics in more complex environments and protein-protein interactions. In addition, recent advances in NMR, mass spectrometry, fluorescence microscopy, and other techniques have enabled the study of protein folding in the cellular environment and have shown that the effect of the cell on protein folding is variable and difficult to predict. Researchers continue to develop new tools to investigate the effect of the cellular environment on more complicated biomolecular systems. This thesis is roughly divided into two sections: Chapters 1-2 discuss fast protein folding in vitro and Chapters 3-6 address the study of protein folding and folding mediated processes in the cell. Chapter 1 is a survey of the theory of protein folding and the major techniques and findings from the study of fast folding proteins in vitro, with a special emphasis on how this work informs our understanding of more complex protein dynamics. Chapter 2 characterizes dodine, a compound that combines the properties of chatropic denaturants and detergents. Chapter 3 is an introduction to how perturbative methods developed in vitro can be applied to study processes in the cell that are inaccessible by conventional steady-state measurements. Chapter 4 is a practical guide to improving the accuracy and reliability of Fluorescence Relaxation Imaging (FReI), our Fӧrster Resonance Energy Transfer (FRET) microscopy based technique for studying biomolecular kinetics in the cell. Chapter 5 describes the development and characterization of the fluorescent construct GPGK-tc and its use to study population-level variation of protein folding in E. coli. Finally, Chapter 6 examines the interaction of the chaperone Hsp70 with an unfolding substrate using a FRET-based binding assay both in vitro and in cells

Variable Selection in Covariate Dependent Random Partition Models: an Application to Urinary Tract Infection

Lower urinary tract symptoms can indicate the presence of urinary tract infection (UTI), a condition that if it becomes chronic requires expensive and time consuming care as well as leading to reduced quality of life. Detecting the presence and gravity of an infection from the earliest symptoms is then highly valuable. Typically, white blood cell (WBC) count measured in a sample of urine is used to assess UTI. We consider clinical data from 1341 patients in their first visit in which UTI (i.e. WBC ≥ 1) is diagnosed. In addition, for each patient, a clinical profile of 34 symptoms was recorded. In this paper, we propose a Bayesian nonparametric regression model based on the Dirichlet process prior aimed at providing the clinicians with a meaningful clustering of the patients based on both the WBC (response variable) and possible patterns within the symptoms profiles (covariates). This is achieved by assuming a probability model for the symptoms as well as for the response variable. To identify the symptoms most associated to UTI, we specify a spike and slab base measure for the regression coefficients: this induces dependence of symptoms selection on cluster assignment. Posterior inference is performed through Markov Chain Monte Carlo methods

Alloparental behaviour and long-term costs of mothers tolerating other members of the group in a plurally breeding mammal

Cooperative-breeding studies tend to focus on a few alloparental behaviours in highly cooperative species exhibiting high reproductive skew and the associated short-term, but less frequently long-term, fitness costs. We analysed a suite of alloparental behaviours (assessed via filming) in a kin-structured, high-density population of plurally breeding European badgers, Meles meles, which are not highly cooperative. Group members, other than mothers, performed alloparental behaviour; however, this was not correlated with their relatedness to within-group young. Furthermore, mothers babysat, allogroomed cubs without reciprocation, and allomarked cubs more than other members of the group (controlling for observation time). For welfare reasons, we could not individually mark cubs; however, the number observed pre-independence never exceeded that trapped. All 24 trapped cubs, in three filmed groups, were assigned both parents using 22 microsatellites. Mothers may breed cooperatively, as the time they babysat their assigned, or a larger, litter size did not differ. Furthermore, two mothers probably allonursed, as they suckled more cubs than their assigned litter size. An 18-year genetic pedigree, however, detected no short-term (litter size; maternal survival to the following year) or long-term (offspring breeding probability; offspring lifetime breeding success) fitness benefits with more within-group mothers or other members of the group. Rather, the number of other members of the group (excluding mothers) correlated negatively with long-term fitness. Mothers may tolerate other members of the group, as nonbreeders undertook more digging. Our study highlights that alloparental care varies on a continuum from that seen in this high-density badger population, where alloparenting behaviour is minimal, through to species where alloparental care is common and provides fitness benefits. (C) 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved

Preference versus performance: Investigating the dissociation between objective measures and subjective ratings of usability for schematic metro maps and intuitive theories of design

Three experiments are reported in which objective measures and subjective ratings of schematic metro map usability were investigated. Experiment 1 used a within-subjects design to compare octolinear and curvilinear Paris Metro maps. This replicated and extended Roberts et al. (2013); the curvilinear map was associated with faster journey planning times, and yet preference between the two was unrelated to this measure. In Experiment 2, nine matched versions of the London Underground map were rated for usability and attractiveness, and a clear octolinear bias was displayed. It was also possible to identify individuals who held a simplicity theory of effective design, versus an octolinearity theory. Experiment 3 investigated the relationship between usability ratings and journey planning times for three Berlin network maps, all optimized for simplicity of line trajectories. No differences in times were found, and yet usability ratings after experience at using the maps differed significantly, in line with the findings for the London designs in Experiment 2. Overall, the dissociation between objective measures of performance and subjective ratings of usability is robust, and appears to reflect expectations and prejudices concerning effective design. The octolinearity as a gold standard conjecture for achieving optimum usability continues to be refuted

Systematizing Confidence in Open Research and Evidence (SCORE)

Assessing the credibility of research claims is a central, continuous, and laborious part of the scientific process. Credibility assessment strategies range from expert judgment to aggregating existing evidence to systematic replication efforts. Such assessments can require substantial time and effort. Research progress could be accelerated if there were rapid, scalable, accurate credibility indicators to guide attention and resource allocation for further assessment. The SCORE program is creating and validating algorithms to provide confidence scores for research claims at scale. To investigate the viability of scalable tools, teams are creating: a database of claims from papers in the social and behavioral sciences; expert and machine generated estimates of credibility; and, evidence of reproducibility, robustness, and replicability to validate the estimates. Beyond the primary research objective, the data and artifacts generated from this program will be openly shared and provide an unprecedented opportunity to examine research credibility and evidence

Seasonal complementary in pollinators of soft-fruit crops

Understanding the relative contributions of wild and managed pollinators, and the functional contributions made by a diverse pollinator community, is essential to the maintenance of yields in the 75% of our crops that benefit from insect pollination. We describe a field study and pollinator exclusion experiments conducted on two soft-fruit crops in a system with both wild and managed pollinators. We test whether fruit quality and quantity is limited by pollination, and whether different pollinating insects respond differently to varying weather conditions. Both strawberries and raspberries produced fewer marketable fruits when insects were excluded, demonstrating dependence on insect pollinators. Raspberries had a short flowering season which coincided with peak abundance of bees, and attracted many bees per flower. In contrast, strawberries had a much longer flowering season and appeared to be much less attractive to pollinators, so that ensuring adequate pollination is likely to be more challenging. The proportion of high-quality strawberries was positively related to pollinator abundance, suggesting that yield was limited by inadequate pollination on some farms. The relative abundance of different pollinator taxa visiting strawberries changed markedly through the season, demonstrating seasonal complementarity. Insect visitors responded differently to changing weather conditions suggesting that diversity can reduce the risk of pollination service shortfalls. For example, flies visited the crop flowers in poor weather and at the end of the flowering season when other pollinators were scarce, and so may provide a unique functional contribution. Understanding how differences between pollinator groups can enhance pollination services to crops strengthens the case for multiple species management. We provide evidence for the link between increased diversity and function in real crop systems, highlighting the risks of replacing all pollinators with managed alternatives

Reprogramming alveolar macrophage responses to TGF-β reveals CCR2+ monocyte activity that promotes bronchiolitis obliterans syndrome

Bronchiolitis obliterans syndrome (BOS) is a major impediment to lung transplant survival and is generally resistant to medical therapy. Extracorporeal photophoresis (ECP) is an immunomodulatory therapy that shows promise in stabilizing BOS patients, but its mechanisms of action are unclear. In a mouse lung transplant model, we show that ECP blunts alloimmune responses and inhibits BOS through lowering airway TGF-β bioavailability without altering its expression. Surprisingly, ECP-treated leukocytes were primarily engulfed by alveolar macrophages (AMs), which were reprogrammed to become less responsive to TGF-β and reduce TGF-β bioavailability through secretion of the TGF-β antagonist decorin. In untreated recipients, high airway TGF-β activity stimulated AMs to express CCL2, leading to CCR2+ monocyte-driven BOS development. Moreover, we found TGF-β receptor 2-dependent differentiation of CCR2+ monocytes was required for the generation of monocyte-derived AMs, which in turn promoted BOS by expanding tissue-resident memory CD8+ T cells that inflicted airway injury through Blimp-1-mediated granzyme B expression. Thus, through studying the effects of ECP, we have identified an AM functional plasticity that controls a TGF-β-dependent network that couples CCR2+ monocyte recruitment and differentiation to alloimmunity and BOS

No Association between Personality and Candidate Gene Polymorphisms in a Wild Bird Population

Consistency of between-individual differences in behaviour or personality is a phenomenon in populations that can have ecological consequences and evolutionary potential. One way that behaviour can evolve is to have a genetic basis. Identifying the molecular genetic basis of personality could therefore provide insight into how and why such variation is maintained, particularly in natural populations. Previously identified candidate genes for personality in birds include the dopamine receptor D4 (DRD4), and serotonin transporter (SERT). Studies of wild bird populations have shown that exploratory and bold behaviours are associated with polymorphisms in both DRD4 and SERT. Here we tested for polymorphisms in DRD4 and SERT in the Seychelles warbler (Acrocephalus sechellensis) population on Cousin Island, Seychelles, and then investigated correlations between personality and polymorphisms in these genes. We found no genetic variation in DRD4, but identified four polymorphisms in SERT that clustered into five haplotypes. There was no correlation between bold or exploratory behaviours and SERT polymorphisms/haplotypes. The null result was not due to lack of power, and indicates that there was no association between these behaviours and variation in the candidate genes tested in this population. These null findings provide important data to facilitate representative future meta-analyses on candidate personality genes