Development of techniques for mapping analytes and properties at interfaces

Scanning ion conductance microscopy (SICM) is a powerful and non-invasive tool which allows substrates to be probed at the nanoscale. Recently developed SICM functional mapping methodologies are capable of providing detailed information about interfacial properties and processes. Interaction of these techniques with complex active surfaces are not fully understood and much of the previous work in this area has been performed on ideal or model substrates. This thesis aims to extend the use of SICM functional mapping towards analysis of complex substrates and realworld samples. Through application of potential control programs and extensive finite element method (FEM) simulation, the interactions between the SICM probe and substrate can be elucidated to explain the ion current response. SICM delivery methodologies are applied to examine the effects of substrate charge and activity upon electroosmotic flow (EOF) in the nanopipette. This allows better informed selection of delivery parameters for the quantitative delivery of neutral species with SICM. Real-time tracking of active dissolution at an enamel surface at the nanoscale is achieved through the controlled delivery of protons and the effects of surface treatments upon the effective heterogeneous rate constant are calculated through FEM. Multiple functional mapping methodologies are combined to probe the bioelectrical properties of live bacteria and FEM is used to further understanding of the structural and dynamic properties of the bacterial cell envelope. Through the combination of experiments with theoretical modelling of the interactions occurring in SICM systems, this work also contributes to understanding of the fundamental processes underpinning SICM measurements

Multi-site rate control analysis identifies ribosomal scanning as the sole high-capacity/low-flux-control step in mRNA translation

Control of complex intracellular pathways such as protein synthesis is critical to organism survival, but is poorly understood. Translation of a reading frame in eukaryotic mRNA is preceded by a scanning process in which a subset of translation factors helps guide ribosomes to the start codon. Here, we perform comparative analysis of the control status of this scanning step that sits between recruitment of the small ribosomal subunit to the m7GpppG‐capped 5′end of mRNA and of the control exerted by downstream phases of polypeptide initiation, elongation and termination. We have utilized a detailed predictive model as guidance for designing quantitative experimental interrogation of control in the yeast translation initiation pathway. We have built a synthetic orthogonal copper‐responsive regulatory promoter (PCuR3) that is used here together with the tet07 regulatory system in a novel dual‐site in vivo rate control analysis strategy. Combining this two‐site strategy with calibrated mass spectrometry to determine translation factor abundance values, we have tested model‐based predictions of rate control properties of the in vivo system. We conclude from the results that the components of the translation machinery that promote scanning collectively function as a low‐flux‐control system with a capacity to transfer ribosomes into the core process of polypeptide production that exceeds the respective capacities of the steps of polypeptide initiation, elongation and termination. In contrast, the step immediately prior to scanning, that is, ribosome recruitment via the mRNA 5′ cap‐binding complex, is a high‐flux‐control step

Scanning ion conductance microscopy reveals differences in the ionic environments of gram-positive and negative bacteria

This paper reports on the use of scanning ion conductance microscopy (SICM) to locally map the ionic properties and charge environment of two live bacterial strains: the Gram-negative and the Gram-positive . SICM results find heterogeneities across the bacterial surface and significant differences among the Gram-positive and Gram-negative bacteria. The bioelectrical environment of the was found to be considerably more negatively charged compared to . SICM measurements, fitted to a simplified finite element method (FEM) model, revealed surface charge values of -80 to -140 mC m for the Gram-negative . The Gram-positive show a much higher conductivity around the cell wall, and surface charge values between -350 and -450 mC m were found using the same simplified model. SICM was also able to detect regions of high negative charge near , not detected in the topographical SICM response and attributed to the extracellular polymeric substance. To further explore how the cell wall structure can influence the SICM current response, a more comprehensive FEM model, accounting for the physical properties of the Gram-positive cell wall, was developed. The new model provides a more realistic description of the cell wall and allows investigation of the relation between its key properties and SICM currents, building foundations to further investigate and improve understanding of the Gram-positive cellular microenvironment

Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity

The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91$\textit{phox}$ and p22$\textit{phox}$ subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene $\textit{bc017643}$, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91$\textit{phox}$ and p22$\textit{phox}$. Consequently, $\textit{Eros}$-deficient mice quickly succumb to infection. $\textit{Eros}$ also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. $\textit{Eros}$ is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.D.C. Thomas was funded by a Wellcome Trust/CIMR Next Generation Fellowship, a National Institute for Health Research (NIHR) Clinical Lectureship, and a Starter Grant for Clinical Lecturers (Academy of Medical Sciences). K.G.C. Smith was funded by funded by the Medical Research Council (program grant MR/L019027) and is a Wellcome Investigator and a NIHR Senior Investigator. S. Clare and G. Dougan were funded by the Wellcome Trust (grant 098051). The Cambridge Institute for Medical Research is in receipt of a Wellcome Trust Strategic Award (079895). J.C.L is funded by a Wellcome Intermediate Clinical Fellowship 105920/2/14/2

An investigation of the legal criteria governing differing site conditions disputes on construction contracts.

http://archive.org/details/investigationofl00teahN

Sexbots: a case for artificial ethical agents

Recent years have seen significant developments in Artificial Intelligence and we are now beginning to see some applications applied to sexual technologies. This has led to certain individuals and groups calling for a ban on the development of artificial sexual companions or “sexbots”. This is largely due to fears that commercialising sex in this way could could re-enforce existing gender inequalities, and sexual objectification. We argue that one possible risk mitigation strategy could be to develop ethical safeguards into sexual robots. In this paper we explore the problem of implementing artificial ethical agents through a review of the relevant literature. We begin by exploring the motivation to the problem, and the science fiction that has inspired it. Following this, the practical, and philosophical implementation challenges are discussed before providing an overview of the current state of the art. The paper concludes by introducing the grand challenges of the field

3’-5’ crosstalk contributes to transcriptional bursting

Transcription in mammalian cells is a complex stochastic process involving shuttling of polymerase between genes and phase-separated liquid condensates. It occurs in bursts, which results in vastly different numbers of an mRNA species in isogenic cell populations. Several factors contributing to “transcriptional bursting” have been identified, usually classified as intrinsic, i.e., local to single genes, or extrinsic, relating to the macroscopic state of the cell. However, each factor only accounts partially for the observed phenomenon, and some possible contributors have not been explored yet. We investigate processes at the 3’ and 5’ ends of a gene that enable reinitiation of transcription upon termination. Using an interdisciplinary approach, we measured the transcriptional bursting at a set of inducible transgenes with sufficient accuracy and precision to unveil significant patterns. We find that perturbation of polymerase shuttling typically reduces the average burst size and increases the burst frequency, thus limiting transcriptional noise. The observed noise patterns are reproduced by a generative model that captures major characteristics of the polymerase flux between a gene and a phase-separated compartment

How the perceived identity of a NPC companion influences player behavior

This paper explores how the perceived identity of a NonPlayerCharacter (NPC) effects a players behaviour in computer games. We explore whether the players will change their behaviour towards a synthetic in-game companion if it assumes different identities. Specifically, will the players change their behaviour if they interact with an identical artificial intelligence, assuming a guise of a human or robot companion. To investigate this question we developed a top-down, 2D on-line game where the player is given the objective of surviving successive waves of hostile opponents. As a secondary objective the player is asked to protect a unarmed male, female or robot companion. The intention is to explore whether the player is more protective over a known NPC assuming either a human or non-human identity. The results of our study indicate that superficially changing the identity of an AI companion can have a dramatic influence over the players behaviour. The players in this study are shown to be significantly more protective to human rather than robot companions, despite the underlying AI being identical. Moreover, our results highlight further differences between the male and female companions

Artificial synapse : spatiotemporal heterogeneities in dopamine electrochemistry at a carbon fiber ultramicroelectrode

An artificial synapse is developed that mimics ultramicroelectrode (UME) amperometric detection of single cell exocytosis. It comprises the nanopipette of a scanning ion conductance microscope (SICM), which delivers rapid pulses of neurotransmitter (dopamine) locally and on demand at >1000 defined locations of a carbon fiber (CF) UME in each experiment. Analysis of the resulting UME current-space-time data reveals spatiotemporal heterogeneous electrode activity on the nanoscale and submillisecond time scale for dopamine electrooxidation at typical UME detection potentials. Through complementary surface charge mapping and finite element method (FEM) simulations, these previously unseen variations in electrochemical activity are related to heterogeneities in the surface chemistry of the CF UME

Data for 3'-5' crosstalk contributes to transcriptional bursting

Transcription in mammalian cells is a complex stochastic process involving shuttling of polymerase between genes and phase-separated liquid condensates. It occurs in bursts, which results in vastly different numbers of an mRNA species in isogenic cell populations. Several factors contributing to “transcriptional bursting” have been identified, usually classified as intrinsic, i.e., local to single genes, or extrinsic, relating to the macroscopic state of the cell. However, each factor only accounts partially for the observed phenomenon, and some possible contributors have not been explored yet. We investigate processes at the 3’ and 5’ ends of a gene that enable reinitiation of transcription upon termination. Using an interdisciplinary approach, we measured the transcriptional bursting at a set of inducible transgenes with sufficient accuracy and precision to unveil significant patterns. We find that perturbation of polymerase shuttling typically reduces the average burst size and increases the burst frequency, thus limiting transcriptional noise. The observed noise patterns are reproduced by a generative model that captures major characteristics of the polymerase flux between a gene and a phase-separated compartment