Quantification of the unsharp masking technique of image enhancement

The technique of unsharp masking is described and its use as an image enhancement technique discussed. A mathematical model for the masking process is developed; experimental testing and MTF measurements of the masked and sharpened images are made to test the validity of the mathematical model as a predictor of the mask and final image characteristics. The effect of contrast, mask unsharpness, and source spread function size on the resulting MTF are presented. Subjective evaluations are used to determine the visually optimum image. It is shown that the visually best image is not necessarily the one with the largest MTF value or area; suggestions are made for adjusting existing image quality specifications to incorporate the results of unsharp masking techniques

Investigating protein structure by means of mass spectrometry

The three-dimensional conformation of a protein is central to its biological function. Mass spectrometry (MS) has become an important tool for the study of various aspects of protein structure. This project investigates the use of MS for diagnosis of hemoglobinopathies, through primary structure identification, and for threedimensional protein structure analysis, through comparison to established methods and application to protein systems. Travelling-wave ion mobility mass spectrometry (TWIM-MS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIM-MS. Cross-sections were estimated for proteins studied, for charge states most indicative of native structure, and were found to be in good agreement with those calculated from published X-ray crystallography and nuclear magnetic resonance structures. These results illustrated that the TWIM-MS approach can provide biologically-relevant data on three-dimensional protein structure. TWIM-MS was then used to study the structural properties of the hemoglobin tetramer and its components. Results showed that globin monomers exist in similar conformations whether in apo- or holo- forms and that a heme-deficient dimer is unlikely to be a prerequisite for hemoglobin tetramer assembly. TWIM-MS was used to successfully differentiate between normal and sickle hemoglobin tetramers. The conformational changes occurring in VanS, a histidine kinase, upon autophosphorylation were investigated by TWIM-MS. Results provided insights into the mechanism of autophosphorylation. MS was used to follow the rate of the autophosphorylation and results obtained compared well with those from an established method. This demonstrated that MS offers a simple, reproducible alternative to conventional methods for the study of phosphorylation rates. MS was used to provide positive identification of a range of hemoglobinopathies caused by single point mutations. A high-throughput method was used to screen for hemoglobinopathies in South Asians with and without cardiovascular disease. Results showed a positive correlation between patients with hemoglobinopathies and those with cardiovascular disease

Investigating protein structure by means of mass spectrometry

The three-dimensional conformation of a protein is central to its biological function. Mass spectrometry (MS) has become an important tool for the study of various aspects of protein structure. This project investigates the use of MS for diagnosis of hemoglobinopathies, through primary structure identification, and for threedimensional protein structure analysis, through comparison to established methods and application to protein systems. Travelling-wave ion mobility mass spectrometry (TWIM-MS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIM-MS. Cross-sections were estimated for proteins studied, for charge states most indicative of native structure, and were found to be in good agreement with those calculated from published X-ray crystallography and nuclear magnetic resonance structures. These results illustrated that the TWIM-MS approach can provide biologically-relevant data on three-dimensional protein structure. TWIM-MS was then used to study the structural properties of the hemoglobin tetramer and its components. Results showed that globin monomers exist in similar conformations whether in apo- or holo- forms and that a heme-deficient dimer is unlikely to be a prerequisite for hemoglobin tetramer assembly. TWIM-MS was used to successfully differentiate between normal and sickle hemoglobin tetramers. The conformational changes occurring in VanS, a histidine kinase, upon autophosphorylation were investigated by TWIM-MS. Results provided insights into the mechanism of autophosphorylation. MS was used to follow the rate of the autophosphorylation and results obtained compared well with those from an established method. This demonstrated that MS offers a simple, reproducible alternative to conventional methods for the study of phosphorylation rates. MS was used to provide positive identification of a range of hemoglobinopathies caused by single point mutations. A high-throughput method was used to screen for hemoglobinopathies in South Asians with and without cardiovascular disease. Results showed a positive correlation between patients with hemoglobinopathies and those with cardiovascular disease.EThOS - Electronic Theses Online ServiceBiotechnology and Biological Sciences Research Council (Great Britain) (BBSRC)Waters CorporationGBUnited Kingdo

Gas Gains Over 104^4 and Optimisation using 55^{55}Fe X-rays in Low Pressure SF6_6 with a Novel Multi-Mesh ThGEM for Directional Dark Matter Searches

The Negative Ion Drift (NID) gas SF$_6$ has favourable properties for track reconstruction in directional Dark Matter (DM) searches utilising low pressure gaseous Time Projection Chambers (TPCs). However, the electronegative nature of the gas means that it is more difficult to achieve significant gas gains with regular Thick Gaseous Electron Multipliers (ThGEMs). Typically, the maximum attainable gas gain in SF$_6$ and other Negative Ion (NI) gas mixtures, previously achieved with an $^{55}$Fe X-ray source or electron beam, is on the order of $10^3$; whereas electron drift gases like CF$_4$ and similar mixtures are readily capable of reaching gas gains on the order of $10^4$ or greater. In this paper, a novel two stage Multi-Mesh ThGEM (MMThGEM) structure is presented. The MMThGEM was used to amplify charge liberated by an $^{55}$Fe X-ray source in 40 Torr of SF$_6$. By expanding on previously demonstrated results, the device was pushed to its sparking limit and stable gas gains up to $\sim$50000 were observed. The device was further optimised by varying the field strengths of both the collection and transfer regions in isolation. Following this optimisation procedure, the device was able to produce a maximum stable gas gain of $\sim$90000. These results demonstrate an order of magnitude improvement in gain with the NID gas over previously reported values and ultimately benefits the sensitivity of a NITPC to low energy recoils in the context of a directional DM search

Using a SMALP platform to determine a sub-nm single particle cryo-EM membrane protein structure

The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7 angstrom range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.Peer reviewe

The sensory features of a food cue influence its ability to act as an incentive stimulus and evoke dopamine release in the nucleus accumbens core

The sensory properties of a reward-paired cue (a Conditioned Stimulus; CS) may impact the motivational value attributed to the cue, and in turn influence the form of the conditioned response (CR) that develops. A cue with multiple sensory qualities, such as a moving lever-CS, may activate numerous neural pathways that process auditory and visual information, resulting in CRs that vary both within and between individuals. For example, CRs include approach to the lever-CS itself (rats that “sign-track;” ST), approach to the location of reward delivery (rats that “goal-track;” GT), or an “intermediate” combination of these behaviors. We found that the multimodal sensory features of the lever-CS were important to the development and expression of sign-tracking. When the lever-CS was covered, and thus could only be heard moving, STs continued to approach the lever location, but also started to approach the food cup during the CS period. While still predictive of reward, the auditory component of the lever-CS was a much weaker conditioned reinforcer than the visible lever-CS. This plasticity in behavioral responding observed in STs closely resembled behaviors normally seen in rats classified as “intermediates.” Furthermore, the ability of both the lever-CS and reward-delivery to evoke dopamine release in the nucleus accumbens was also altered by covering the lever – dopamine signaling in STs resembled neurotransmission observed in rats that normally only GT. These data suggest that while the visible lever-CS was attractive, wanted, and had incentive value for STs, when presented in isolation the auditory component of the cue was simply predictive of reward, lacking incentive salience. Therefore, the specific sensory features of cues may differentially contribute to responding and ensure behavioral flexibility

Demonstration of ThGEM-multiwire hybrid charge readout for directional dark matter searches

Sensitivities of current directional dark matter search detectors using gas time projection chambers are now constrained by target mass. A ton-scale gas TPC detector will require large charge readout areas. We present a first demonstration of a novel ThGEM-Multiwire hybrid charge readout technology which combines the robust nature and high gas gain of Thick Gaseous Electron Multipliers with lower capacitive noise of a one-plane multiwire charge readout in SF6 target gas. Measurements performed with this hybrid detector show an ion drift velocity of 139 ± 12 ms−1 in a reduced drift field E/N of 93 Td (10−17 V cm2) at a gas gain of 2470±160 in 20 Torr of pure SF6 target gas

Demonstration of radon removal from SF6 using molecular sieves

The gas SF6 has become of interest as a negative ion drift gas for use in directional dark matter searches. However, as for other targets in such searches, it is important that radon contamination can be removed as this provides a source of unwanted background events. In this work we demonstrate for the first time filtration of radon from SF6 gas by using a molecular sieve. Four types of sieves from Sigma-Aldrich were investigated, namely 3Å, 4Å, 5Å and 13X. A manufactured radon source was used for the tests. This was attached to a closed loop system in which gas was flowed through the filters and a specially adapted Durridge RAD7 radon detector. In these measurements, it was found that only the 5Å type was able to significantly reduce the radon concentration without absorbing the SF6 gas. The sieve was able to reduce the initial radon concentration of 3875 ± 13 Bqm−3 in SF6 gas by 87% when cooled with dry ice. The ability of the cooled 5Å molecular sieve filter to significantly reduce radon concentration from SF6 provides a promising foundation for the construction of a radon filtration setup for future ultra-sensitive SF6 gas rare-event physics experiments

Charge amplification in sub-atmospheric CF4:He mixtures for directional dark matter searches

Low pressure gaseous Time Projection Chambers (TPCs) are a viable technology for directional Dark Matter (DM) searches and have the potential for exploring the parameter space below the neutrino fog [1,2]. Gases like CF4 are advantageous because they contain flourine which is predicted to have heightened elastic scattering rates with a possible Weakly Interacting Massive Particle (WIMP) DM candidate [3,4,5]. The low pressure of CF4 must be maintained, ideally lower than 100 Torr, in order to elongate potential Nuclear Recoil (NR) tracks which allows for improved directional sensitivity and NR/Electron Recoil (ER) discrimination [6]. Recent evidence suggests that He can be added to heavier gases, like CF4, without significantly affecting the length of 12C and 19F recoils due to its lower mass. Such addition of He has the advantage of improving sensitivity to lower mass WIMPs [1]. Simulations can not reliably predict operational stability in these low pressure gas mixtures and thus must be demonstrated experimentally. In this paper we investigate how the addition of He to low pressure CF4 affects the gas gain and energy resolution achieved with a single Thick Gaseous Electron Multiplier (ThGEM)

PocketWATCH: design and operation of a multi-use test bed for water Cherenkov detector components in pure and gadolinium loaded water

The PocketWATCH facility is a unique multi-purpose test bed designed to replicate the conditions of large water Cherenkov detectors. Housed at the University of Sheffield, the facility consists of a light-tight 2000 L ultrapure water tank with purification and temperature control systems. Water temperature, resistivity, and UV attenuation in the tank are monitored and shown to be stable over time. The system is also shown to be compatible with a solution of 0.2% gadolinium sulfate, allowing further utility in testing equipment bound for the next generation neutrino and nucleon decay water Cherenkov particle detectors. The relevant water quality parameters are shown to be stable whilst running in Gd-mode, thereby providing a suitable test bed for hardware development in a realistic, ex situ environment