Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

Silica polymer bonding of stressed silica grains: An early growth of intergranular tensile strength

© 2015 Elsevier Ltd. All rights reserved.Laboratory tests on microscale are reported in which millimeter-sized amorphous silica cubes were kept highly compressed in a liquid environment of de-ionized water solutions with different silica ion concentrations for up to four weeks. Such an arrangement simulates an early evolution of bonds between two sand grains stressed in situ. In-house designed Grain Indenter-Puller apparatus allowed measuring strength of such contacts after 3-4 weeks. Observations reported for the first time confirm a long-existing hypothesis that a stressed contact with microcracks generates silica polymers, forming a bonding structure between the grains on a timescale in the order of a few weeks. Such structure exhibits intergranular tensile force at failure of 1-1.5 mN when aged in solutions containing silica ion concentrations of 200-to 500-ppm. The magnitude of such intergranular force is 2-3 times greater than that of water capillary force between the same grains.

ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation.

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.

ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat.

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPgammaS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.

Laminar Free Convection from a Vertically-Oriented Concentrated Photovoltaic Cell with Uniform Surface Heat Flux

This research project uses field measurements to investigate the cooling of a triple-junction, photovoltaic cell under natural convection when subjected to various amounts of insolation. The team built an experimental apparatus consisting of a mirror and Fresnel lens to concentrate light onto a triple-junction photovoltaic cell, mounted vertically on a copper heat sink. Measurements were taken year-round to provide a wide range of ambient conditions. A surface was then generated, in MATLAB, using Sparrow’s model for natural convection on a vertical plate under constant heat flux. This surface can be used to find the expected operating temperature of a cell at any location, given the ambient temperature and insolation. This research is an important contribution to the industry because it utilizes field data that represents how a cell would react under normal operation. It also extends the use of a well-known model from a one-sun environment to a multi-sun one.

Investigation and Development of a Fully 3D Tilt Capable Hybrid SPECT - CT System for Dedicated Breast Imaging

X-ray mammography has been the gold standard for breast imaging for decades, despite the significant limitations posed by the two dimensional (2D) image acquisitions. Difficulty in diagnosing lesions close to the chest wall and axilla, high amount of structural overlap and patient discomfort due to compression are only some of these limitations. To overcome these drawbacks, three dimensional (3D) breast imaging modalities have been developed including dual modality single photon emission computed tomography (SPECT) and computed tomography (CT) systems. This thesis focuses on the development and integration of the next generation of such a device for dedicated breast imaging. The goals of this dissertation work are to: [1] understand and characterize any effects of fully 3-D trajectories on reconstructed image scatter correction, absorbed dose and Hounsifeld Unit accuracy, and [2] design, develop and implement the fully flexible, third generation hybrid SPECT-CT system capable of traversing complex 3D orbits about a pendant breast volume, without interference from the other. Such a system would overcome artifacts resulting from incompletely sampled divergent cone beam imaging schemes and allow imaging closer to the chest wall, which other systems currently under research and development elsewhere cannot achieve.

The dependence of x-ray scatter radiation on object shape, size, material composition and the CT acquisition trajectory, was investigated with a well-established beam stop array (BSA) scatter correction method. While the 2D scatter to primary ratio (SPR) was the main metric used to characterize total system scatter, a new metric called ‘normalized scatter contribution’ was developed to compare the results of scatter correction on 3D reconstructed volumes. Scatter estimation studies were undertaken with a sinusoidal saddle (±15° polar tilt) orbit and a traditional circular (AZOR) orbit. Clinical studies to acquire data for scatter correction were used to evaluate the 2D SPR on a small set of patients scanned with the AZOR orbit. Clinical SPR results showed clear dependence of scatter on breast composition and glandular tissue distribution, otherwise consistent with the overall phantom-based size and density measurements. Additionally, SPR dependence was also observed on the acquisition trajectory where 2D scatter increased with an increase in the polar tilt angle of the system.

The dose delivered by any imaging system is of primary importance from the patient’s point of view, and therefore trajectory related differences in the dose distribution in a target volume were evaluated. Monte Carlo simulations as well as physical measurements using radiochromic film were undertaken using saddle and AZOR orbits. Results illustrated that both orbits deliver comparable dose to the target volume, and only slightly differ in distribution within the volume. Simulations and measurements showed similar results, and all measured dose values were within the standard screening mammography-specific, 6 mGy dose limit, which is used as a benchmark for dose comparisons.

Hounsfield Units (HU) are used clinically in differentiating tissue types in a reconstructed CT image, and therefore the HU accuracy of a system is very important, especially when using non-traditional trajectories. Uniform phantoms filled with various uniform density fluids were used to investigate differences in HU accuracy between saddle and AZOR orbits. Results illustrate the considerably better performance of the saddle orbit, especially close to the chest and nipple region of what would clinically be a pedant breast volume. The AZOR orbit causes shading artifacts near the nipple, due to insufficient sampling, rendering a major portion of the scanned phantom unusable, whereas the saddle orbit performs exceptionally well and provides a tighter distribution of HU values in reconstructed volumes.

Finally, the third generation, fully-suspended SPECT-CT system was designed in and developed in our lab. A novel mechanical method using a linear motor was developed for tilting the CT system. A new x-ray source and a custom made 40 x 30 cm2 detector were integrated on to this system. The SPECT system was nested, in the center of the gantry, orthogonal to the CT source-detector pair. The SPECT system tilts on a goniometer, and the newly developed CT tilting mechanism allows ±15° maximum polar tilting of the CT system. The entire gantry is mounted on a rotation stage, allowing complex arbitrary trajectories for each system, without interference from the other, while having a common field of view. This hybrid system shows potential to be used clinically as a diagnostic tool for dedicated breast imaging.

FANTASIO: a versatile experimental set-up to investigate jet-cooled molecules

The design of a new apparatus, named FANTASIO, for studying jet-cooled molecules is described. It includes, around the same supersonic expansion cell, a high resolution Fourier transform spectrometer with single or multipass optics, a tunable diode laser spectrometer with optional cavity ring-down facilities, and a quadrupole mass spectrometer. Performance and operational procedures are illustrated.

Time-dependent modelling and experimental validation of the metal/flux interface in a continuous casting mould

A finite volume computer model of the continuous casting process for steel flat products has been developed. In this first stage, the model concentrates on the hydrodynamic aspects of the process and in particular the dynamic behavior of the metal/slag interface. The model was validated against experimental measurements obtained in a water model apparatus.

Ghost/Machine: cinema in a digital age

Cinema, with its passive cinematic apparatus and linear narrative is often characterised as a contrast to new media narrative strategies, yet from Vertov’s Man with a Movie Camera to Mike Figgis’ TimeCode and Wong Kar Wei’s 2046 cinema provides narrative strategies and spatial conceptualisations which prefigure or are contiguous with new media environments. Both our perception of what cyberspace constitutes and the technology that actualises those perceptions arise out of and are driven by fantasy and desire. This paper will explore the metaphors used to represent and understand new media aesthetics through cinematic representations of new media environments. Two key themes relevant to new media aesthetics emerge. Irigaray, Haraway, and Grosz are used to explore the de-essentialising haptic and penetrative potential of new technologies and their ability to collapse the boundary between the body and the machine. The second fantasy, of new media as a liminal space that expresses the memorialising function of technology and its relation to mourning, is analysed using Benjamin, Burgin and Rutsky. These altered spaces and perceptions of the body and memory of the post-cinematic subject are illustrated through an analysis of Gondry’s Eternal Sunshine of the Spotless Mind and Jonze’s Being John Malkovich. [From the Author]

When deterrence works - and when it doesn't: a comparison of Sweden's prostitution law and UK's Dangerous Dogs Act

Many pieces of legislation have been implemented with the anticipation - or justification - that they will have a deterrent effect. Deterrence was clearly argued in the debate preceding the Swedish prostitution law prohibiting the purchase of sexual services, but less so regarding the Dangerous Dogs Act, which was a very rapid response to a particular moral panic. As it turned out, the Swedish law has had a deterrent effect on street prostitution in that 'respectable' buyers were deterred. It will be argued that it is this very 'respectability' that makes deterrence work in this case. Regarding the Dangerous Dogs Act, the owners of Pit Bulls and other banned breeds are not considered 'respectable' and the banning might have had the reversed effect - increasing the attraction of these dogs, rather than deterring the ownership. Apart from deterrence and its consequences, the rendering invisible of key actors - buyers and owners respectively - and the use of symbolic legislation to promote moral messages will also be considered. [From the Author]

Direct measurement of powder flavour adhesion on to a food surface using a novel adhesion tester

The firm adhesion of flavouring particles onto crisp surfaces during coating processes is a major concern in the snack production industry. Detachment of flavouring powders from products during handling and production stages can lead to substantial financial losses for the industry, in terms of variable flavour performance and extended cleaning down time of fugitive particle build-up on process equipment. Understanding the adhesion strength of applied bulk particulates used for flavouring formulations will help analysts to evaluate the efficiency of coating processes and potentially enable them to assess the adhesion strength of newly formulated flavouring powder prior to commitment to full scale plant trials. A rapid prototype of a novel adhesion tester has been designed and constructed. The apparatus operates according to the principle of impact force acting on particles attached to the surface of the food substrate. The main component is a circular plate to which four sample holders are attached and which is subjected to vertical travel down a guide shaft. Several flavouring powders have been tested extensively. By plotting the detachment versus impact force, the difference obtained between adhesion strength of different flavouring powders (which is a strong function of particle size) has been discussed.

Evaluating the process of borosilicate glass capillaries used for fabrication of in-vitro fertilization (IVF) micro-pipettes

In this paper we investigate a number of gas flames for fire polishing borosilicate glass capillaries used in the manufacturing of IVF micro-pipettes. Hydrofluoric acid (HF) was also used as an alternative to finish the pipette end. Glass micro tools in the IVF industry are drawn from hollow glass capillaries of diameter 1 mm. These capillaries are cut manually to a length of 100 mm from hollow glass rods resulting in sharp and chipped edges. These capillaries are held in a customised holder having padding of soft silicone or rubber. Sharp and uneven edges of these capillaries pick up particles of rubber or soft silicone shavings, rendering them ineffective for IVF treatments. The working range of borosilicate glass is 800-1,200 degrees C. The experiments involved analysis of fire polishing process for borosilicate glass capillaries using candle, butane, propane, 2350 butane propane, oxyacetylene gas flames, finding the optimum distance of the capillary relative to the flame, optimum time for which the capillary should be held in the flame and optimum region of the flame which gives the required temperature range. The results show that 2350 butane propane gas mix is optimum for fire polishing of borosilicate glass capillaries. The paper is concluded by comparing the results of fire polishing with the results of acid polishing, in which HF of 1.6% concentration is used to etch the ends of the borosilicate glass pipettes.

Assessing the environmental consequences of CO2 leakage from geological CCS: Generating evidence to support environmental risk assessment

At the start of the industrial revolution (circa 1750) the atmospheric concentration of carbon dioxide (CO2) was around 280 ppm. Since that time the burning of fossil fuel, together with other industrial processes such as cement manufacture and changing land use, has increased this value to 400 ppm, for the first time in over 3 million years. With CO2 being a potent greenhouse gas, the consequence of this rise for global temperatures has been dramatic, and not only for air temperatures. Global Sea Surface Temperature (SST) has warmed by 0.4–0.8 °C during the last century, although regional differences are evident (IPCC, 2007). This rise in atmospheric CO2 levels and the resulting global warming to some extent has been ameliorated by the oceanic uptake of around one quarter of the anthropogenic CO2 emissions (Sabine et al., 2004). Initially this was thought to be having little or no impact on ocean chemistry due to the capacity of the ocean’s carbonate buffering system to neutralise the acidity caused when CO2 dissolves in seawater. However, this assumption was challenged by Caldeira and Wickett (2005) who used model predictions to show that the rate at which carbonate buffering can act was far too slow to moderate significant changes to oceanic chemistry over the next few centuries. Their model predicted that since pre-industrial times, ocean surface water pH had fallen by 0.1 pH unit, indicating a 30% increase in the concentration of H+ ions. Their model also showed that the pH of surface waters could fall by up to 0.4 units before 2100, driven by continued and unabated utilisation of fossil fuels. Alongside increasing levels of dissolved CO2 and H+ (reduced pH) an increase in bicarbonate ions together with a decrease in carbonate ions occurs. These chemical changes are now collectively recognised as “ocean acidification”. Concern now stems from the knowledge that concentrations of H+, CO2, bicarbonate and carbonate ions impact upon many important physiological processes vital to maintaining health and function in marine organisms. Additionally, species have evolved under conditions where the carbonate system has remained relatively stable for millions of years, rendering them with potentially reduced capacity to adapt to this rapid change. Evidence suggests that, whilst the impact of ocean acidification is complex, when considered alongside ocean warming the net effect on the health and productivity of the oceans will be detrimental.

Flexible C : N ratio enhances metabolism of large phytoplankton when resource supply is intermittent

Phytoplankton cell size influences particle sinking rate, food web interactions and biogeographical distributions. We present a model in which the uptake, storage and assimilation of nitrogen and carbon are explicitly resolved in different-sized phytoplankton cells. In the model, metabolism and cellular C :N ratio are influenced by the accumulation of carbon polymers such as carbohydrate and lipid, which is greatest when cells are nutrient starved, or exposed to high light. Allometric relations and empirical data sets are used to constrain the range of possible C : N, and indicate that larger cells can accumulate significantly more carbon storage compounds than smaller cells. When forced with extended periods of darkness combined with brief exposure to saturating irradiance, the model predicts organisms large enough to accumulate significant carbon reserves may on average synthesize protein and other functional apparatus up to five times faster than smaller organisms. The advantage of storage in terms of average daily protein synthesis rate is greatest when modeled organisms were previously nutrient starved, and carbon storage reservoirs saturated. Small organisms may therefore be at a disadvantage in terms of average daily growth rate in environments that involve prolonged periods of darkness and intermittent nutrient limitation. We suggest this mechanism is a significant constraint on phytoplankton C :N variability and cell size distribution in different oceanic regimes.

The origin and evolution of synaptic proteins - choanoflagellates lead the way.

The origin of neurons was a key event in evolution, allowing metazoans to evolve rapid behavioral responses to environmental cues. Reconstructing the origin of synaptic proteins promises to reveal their ancestral functions and might shed light on the evolution of the first neuron-like cells in metazoans. By analyzing the genomes of diverse metazoans and their closest relatives, the evolutionary history of diverse presynaptic and postsynaptic proteins has been reconstructed. These analyses revealed that choanoflagellates, the closest relatives of metazoans, possess diverse synaptic protein homologs. Recent studies have now begun to investigate their ancestral functions. A primordial neurosecretory apparatus in choanoflagellates was identified and it was found that the mechanism, by which presynaptic proteins required for secretion of neurotransmitters interact, is conserved in choanoflagellates and metazoans. Moreover, studies on the postsynaptic protein homolog Homer revealed unexpected localization patterns in choanoflagellates and new binding partners, both which are conserved in metazoans. These findings demonstrate that the study of choanoflagellates can uncover ancient and previously undescribed functions of synaptic proteins.