1,058 research outputs found
Channel 40 news segment on E. Coli rapid detection method (April 1998)
No full textThis is a short Channel 40 news segment on Dr. Chun-Kwun Wun and Dr. Frank J. Torre development of a E. Coli Rapid Detection method. The segment is about two minutes and 20 seconds long. The segment was digitized from a VHS tape which over the years seem to have been damaged. Around a minute into the segment the sound disappears. The segment includes some interviews of Dr. Wun and Dr. Torre, some clips of the Mobility Channel Pathogen Detector being used, walking around a supermarket, and a segment in a meat packing company
The ABC: an overview
No full textThis paper presents an overview of Australia\u27s principal public broadcaster, the ABC, with reference to its origins, current composition and functions and its vision for a viable future within a changing media environment. The paper also makes reference to some of the controversies which have been integral to long-running discussion about the ABC - allegations of bias, political appointments to the broadcaster and the perennial question of funding adequacy.
Executive summary
This paper presents a brief overall picture of Australia’s principal public service broadcaster, the Australian Broadcasting Corporation (ABC).It describes the origins and development of the ABC and looks at its current structure, general operational policies and functions.
The paper also discusses a number of important issues which are often raised in discussions of public broadcasting in general, and in particular, in relation to the ABC. One of the most significant of these is the issue of how the ABC is funded. The paper provides a brief background to the current funding situation, noting the debate surrounding the adequacy of funding and what may be future funding outcomes. Discussion of this issue is supplemented by detailed funding tables presented in Appendix C. These tables show funding trends and provide information actual dollars and dollar adjusted to 2012–13 prices.
The ABC has regularly been the subject of criticism from a number of quarters; there have been frequent allegations of bias in reporting, inappropriate programming, political appointments to the ABC Board and mismanagement of funds. Recently the ABC has been accused also of neglecting its Charter obligations by withdrawing local services and outsourcing production.
The debates surrounding these and other similar issues are also considered in the paper. Arguments supporting and criticising the broadcaster are noted throughout. While no attempt is made to draw definitive conclusions about the future of the ABC, it is noted that the broadcaster continues to be admired and supported by a significant number of Australians who see it as a constant source of quality information and entertainment
Investigation of the Function and Regulation of ABC Transporters
No full textThe copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the authorATP-Binding-Cassette (ABC) transporters are primary active pumps that typically couple the binding and hydrolysis of ATP to the translocation of compounds across cellular membranes. Some, like ABCB1, ABCC1 and ABCC3, are polyspecific and can efflux clinically important drugs which may contribute to their therapeutic failure.
In this study I have investigated (1) the mechanism of ABC transporter function, (2) studied the potential for regulation by ubiquitin ligases (both using ABCB1 as a model), and (3) tested the involvement of ABCC1 and ABCC3 in autocrine signalling in cancer. (1) In 1966, Jardetzky et. al [1] proposed that membrane pumps function by exposing their ligand-binding pocket alternately on different sides of the membrane. For ABC transporters, this coupling of the aspect and affinity of the ligand-binding cavities of the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism but is poorly understood at the molecular level. Structure data suggest signals are transduced through intracellular loops of the TMDs which slot into grooves on the top surface of the NBDs. At the base of these grooves is the Q-loop. By analysing the function of Q-loop mutants in combination with ligand binding cavity mutants I have discovered that the Q-loops are crucial to the transport cycle and that they are required to couple ligand binding to conformational changes at the NBDs necessary to drive the transporter into an inward closed state.
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(2) ABCB1 is known to be a key component of chemical barrier separating the circulation from the cerebrospinal fluid. It has also been reported to transport β-amyloid across the lumenal membrane and into the circulation. Loss of ABCB1 from the barrier with age has therefore been suggested to play a role in Alzheimer’s Disease. The ubiquitin ligase Nedd4-1 has been implicated in the post-translational regulation of ABCB1 abundance in cells. Here, I report that ABCB1 can be ubiquitinated by Nedd4-1 in vitro and identify the residues modified (by mass spectrometry).
(3) Lysophosphatidylinositol (LPI) is an autocrine metabolite produced by cancer cells that binds to the G-protein coupled transmembrane receptor GPR55 on the surface of cells. Stimulation of GPR55 activates a signalling cascade that induces proliferation and metastases of the cancer cells. How LPI is released from the cells was not known. In this study I show that ABCC1 and ABCC3, which are known to be expressed in ovarian and pancreatic cancers, can transport LPI into inside-out vesicles suggesting a new role for these “drug resistance” transporters in cancer biology
Diversity in management accounting practice through the ABC paradox : testing an institutional perspective
No full textIncludes bibliographical references (leaves 105-110).This study investigates the ability of Burns and Scapens' (2000) Institutional Framework, drawn from Old Institutional Economics, to explain diversity in management accounting practice. The framework contends that management accounting practices can shape, and be shaped by, the taken for granted ways of thinking (institutions) that exist within an organisation, and is offered in response to the perceived inability of neo-classical economics to explain diversity in management accounting practices (Burns & Scapens, 2000; Soin, Seal & Cullen, 2002; Scapens, 2006). This inability contributes to uncertainty regarding the value or relevance of the management accounting technique studied. One area in which this is particularly apparent is that of Activity Based Costing (ABC) where a paradox has emerged: while ABC is reported as being a superiour costing technique, the lack of widespread use thereof implies otherwise (Gosselin, 1997). This study tests the ability of the Institutional Framework to explain the change in management accounting practices that occurred in a medium sized South African university during the seven year period from 2000 to 2006. The case presents a situation where ABC might seem indicated, but instead a uniquely tailored response was devised. This case demonstrates the constraining influence of institutions on management accounting change and shows how institutions standing in opposition to change were altered through management processes, which is of practical relevance to management wishing to effect change successfully. Further, the study shows that that the relationship between economic considerations and institutional influences are not as suggested by the theory, and that the link between economic rationality and the institutional framework can be readily articulated
Mechanisms of the 40–70 Day Variability in the Yucatan Channel Volume Transport
No full textThe Yucatan Channel connects the Caribbean Sea with the Gulf of Mexico and is the main outflow region of the Caribbean Sea. Moorings in the Yucatan Channel show high-frequent variability in kinetic energy (50–100 days) and transport (20–40 days), but the physical mechanisms controlling this variability are poorly understood. In this study, we show that the short-term variability in the Yucatan Channel transport has an upstream origin and arises from processes in the North Brazil Current. To establish this connection, we use data from altimetry and model output from several high resolution global models. A significant 40–70 day variability is found in the sea surface height in the North Brazil Current retroflection region with a propagation toward the Lesser Antilles. The frequency of variability is generated by intrinsic processes associated with the shedding of eddies, rather than by atmospheric forcing. This sea surface height variability is able to pass the Lesser Antilles, it propagates westward with the background ocean flow in the Caribbean Sea and finally affects the variability in the Yucatan Channel volume transport.</p
The distribution and function of the ATP-sensitive potassium channel subunit Kir6.1 in cardiac and skeletal muscle cell lines.
No full textATP-sensitive potassium channels (KAtp) are present in the plasma membrane of a number of tissues but are also present on endomembranes such as the endoplasmic reticulum (ER) and mitochondria. They are involved in a number of physiological and pathophysiological processes and form a link between cellular metabolism and membrane excitability. Ischaemic preconditioning describes the phenomenon in which a short period of ischaemia protects against a more prolonged one. The ability of potassium channel openers such as pinacidil and nicorandil can mimic this phenomenon, with inhibitors such as glibenclamide to abolish this response, led to the suggestion that the final effector in this process was the sarcolemmal Katp channel as it was able to shorten the cardiac action potential reducing the energy requirements of the cell. However, a number of pharmacological observations were not compatible with this hypothesis as diazoxide, which does not activate the sarcolemmal channel, was able to mimic preconditioning. The focus of research then turned to the potential involvement of a KAtp channel present in the mitochondrial inner membrane called the mitoKArp channel. The molecular identification of this channel would be important and there is controversial evidence to suggest that Kiro.l may be a major component of the mitoKATP channel. I examined the hypothesis that the localisation of Kir6.1 is functionally significant in cardiac and skeletal muscle because it generates important K+ flux in intracellular membranes such as the ER and perhaps mitochondria. Co-localisation studies showed that transfected Kiro.l was located in the ER with a small but significant proportion in mitochondria. However, Kir6.1 was ER retained and not trafficked to the plasma membrane when co-expressed with its regulatory subunit, the sulphonylurea receptor SUR1. Immunofluorescent staining also detected the presence of endogenous Kir6.1 in these cell lines using antibodies specific to Kir6.1. The distribution of Kir6.1 suggests that it may play a role in reactive oxygen species (ROS) production, calcium (Ca) handling in the ER and perhaps cellular respiration in mitochondria. ROS production is often associated with KAtp channel opening and protection against cell death at reperfusion. My results showed that diazoxide induced ROS production in C2C12, HepG2 and HEK293 cell lines with glibenclamide abolishing this effect. However, in the absence of Kir6.1, the same response was still observed. This suggests that Kir6.1 is not involved in the mechanism that is responsible for ROS production. The functional role of KAtp channels were also examined in mitochondria by measuring flavoprotein and NADH autofluorescence, an index of mitochondrial redox state and mitochondrial membrane potential (Au/m) in C2C12 cells and rat ventricular myocytes. In myocytes, flavoprotein oxidation increased when cells were treated with 3-nitroproprionic acid (3-NPA) and diazoxide. Glibenclamide did not reverse this effect. However, this phenomenon was absent in C2C12 cells. Given these observations, 3-NPA and diazoxide did not affect the Avj/m in C2C12 cells whereas glibenclamide caused mitochondrial depolarisation. The Aij/m could not be measured in myocytes. A large proportion of Kir6.1 resides in the ER and I examined whether Kir6.1 would alter ATP-induced Ca2+ transients. Upon ATP stimulation, C2C12 cells released Ca2+ from internal stores via the P2Y purinergic signalling pathway. The use of dominant negatives (DN) for Kir6.1 showed that ATP-induced Ca2+ transients were affected by the absence of Kir6.1. However, on closer inspection it was revealed that the presence of eGFP to identify transfected cells seriously perturbed the Fura-2 signal. In conclusion, the KAtp sensitive channel subunit Kir6.1 is predominantly distributed in the ER with a small but significant proportion in mitochondria. I also report that pharmacological compounds such as diazoxide and glibenclamide are not always truly 'selective' for the activation and inhibition of KAtp channels. I have not identified a specific role for Kir6.1 but my data suggests that Kir6.1 is not part of the mitoKATp channel and Kir6.1 is not involved in ROS production and mitochondrial function but it may still have a role in Ca2+ handling
Numerical investigation of heat transfer enhancement by using louvered-winglet vortex generators mounted in a solar air heater channel-type
No full textThe solar air heater (SAH) equipped with special modified surface geometries is one of the existing technologies capable of converting the solar energy into thermal energy with low associated pressure drop, and it has been used as heat source in a wide range of thermal applications which demand lower operating temperatures (about 30 to 80 °C). In the present research, the use of louvered-winglet longitudinal vortex generators as a passive heat transfer enhancement technique is proposed to numerically investigate the effect of the opened louvers have on the SAH thermo-hydraulic performance for: (i) Reynolds numbers of 5000 and 20,000, (ii) two different types of longitudinal vortex generators (delta winglets and rectangular winglets), (iii) aspect ratios of 2 and 6, (iv) angles of attack of 30° and 45°, (v) ratio between louver area and LVG area of 0.25 and 0.44, (vi) louver flap angles of 10°, 40°, and 60° and, (vii) louver opening direction–pointing to the adiabatic plate or to the absorber plate, both downstream of the main flow. The numerical simulations showed that louvered winglets mounted in common-flow-down configurations are thermo-hydraulically more efficient than the respective non-louvered winglets. Rectangular winglets and higher values of ratio between louver area and winglet area, louver flap angle, and aspect ratio promoted a better thermos-hydraulic performance of the channel. The effect of louver on performance of the SAH channel is more pronounced on rectangular winglet than delta winglet. Moreover, the winglets with louvers toward the adiabatic plate introduce less pressure loss to the SAH than the ones with louvers toward the absorber plate. Maximum thermal enhancement factor of 3.418 was achieved for Re = 20,000. With respect to flow patterns and heat transfer characteristics observed on louvered winglets, a new vortex structure generated by the louver openings is identified, in which is responsible to enhance the hot and cold flow mixing. Finally, the louver presents similar effect on thermo-hydraulic performance of the channel, independently of the Reynolds number.The Engineering Modeling and Applied Social Sciences Center (CECS) Federal University of ABC (UFABC), SPDepartment of Mechanical Engineering São Paulo State University (UNESP), SPDepartment of Mechanical Engineering São Paulo State University (UNESP), S
Structural Studies of a Urea Channel with Electron Microscopy
No full textThe Urea/Amide channel from Bacillus cereus (UACBc) was expressed in Escherichia
coli with a C-terminal hexa-histidine tag. The protein was purified in detergent as
confirmed by N-terminal sequencing. The purified protein in detergent was analysed
with single particle analysis processing and forms a particle consisting of a pair of
stacked discs with diameters of 120 Å with each disc representing an oligomer of
UACBc.
Two-dimensional (2D) crystallisation produced highly aggregated crystals that
became suitable for high resolution imaging upon sonication to disperse them.
Using the 2D crystals for electron cryomicroscopy yielded images that upon
crystallographic processing and analysis suggested that the crystals had p6 symmetry
with an additional single p622 crystal indicating a possible double-layered crystal
form.
The images with p6 symmetry were merged to produce a 9 Å projection map showing
the protein forming a hexameric ring with 7 density features in each putative
monomer possibly representing the predicted 7 transmembrane helices of UACBc.
AFM and production of a negative stain three dimensional (3D) density map were
used to determine the thickness of the crystals and based on a mono-layered crystal
form, bioinformatic analysis and biochemical experiments to verify the oligomeric
state and topology, a model with the putative locations of the 7 predicted
transmembrane helices and their orientations with respect to each other has been
produced
Transport and catabolism of murein tripeptide in Escherichia coli K-12
No full textIn bacteria, extracellular peptides are not only a source of nutrients but also play important roles in cell-cell communication. The primary mode of uptake of these peptides in bacteria is via ATP Binding Cassette (ABC) transporters. The substrate-binding protein (SBP) of these transporters captures extracellular peptides and delivers them to membrane-bound components for transport. Bacterial peptide-binding SBPs that function in ABC transporters have evolved predominantly to function as generalists, recognising peptides of particular lengths but with low or no sequence specificity. However, within the peptide-specific SBPs are examples that appear to have evolved subsequently to recognise specific peptides.
Here we have provided the first biophysical evidence, using native electrospray mass spectrometry, for the binding of a specific peptide to an Escherichia coli SBP. This peptide is a cell wall component named murein tripeptide (Mtp, L-Ala--D-Glu-meso-Dap) and is transported by an ABC transporter containing the SBP MppA. We demonstrate that MppA recognises Mtp specifically and with high affinity (KD ~ 250 nM), as determined by protein fluorescence spectroscopy and isothermal titration calorimetry. The crystal structure of MppA in complex with Mtp has been solved in order to understand the structural basis of specific binding of Mtp to MppA. Comparison of the structure of MppA-Mtp with structures of general tripeptides bound to oligopeptide binding protein OppA, reveals close similarity in the protein chains which fold to form an enclosed interdomain pocket in which the respective peptides reside. The peptide ligands superimpose remarkably closely given the profound differences in their structure. Strikingly, the effect of the D-stereochemistry, which projects the side chain of residue 2 in Mtp in the direction of the main chain in a conventional tripeptide, is compensated by the side chain γ-linkage of the carboxylate of the D-Glu to the amino group of diaminopimelic acid. The resulting amide linkage mimicks the peptide bond between residues 2 and 3 of a conventional tripeptide. Specificity for Mtp is conferred by a series of ionic and polar residues of MppA which make charge-charge and dipole-charge interactions with the highly ionised peptide.
Analysis of multiple bacterial genomes revealed that mppA is often situated near a gene called mpaA which encodes a putative amidase enzyme which is thought to hydrolyze murein tripeptide. This genetic organization is conserved among -proteobacteria and suggests that mppA and mpaA function together and are a part of peptide catabolic pathway. The MpaA enzyme from E. coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. MpaA was found to hydrolyze Mtp efficiently but it did not cleave murein tetrapeptide. Replacement of meso-Dap by L-Lys in the tripeptide resulted in much lower efficiency. Mass spectrometric analysis of purified MpaA suggested that it co-purifies with a zinc ion and exists as a dimer in solution. The first crystal structure of an MpaA protein from Vibrio harveyi was solved to 2.17Å. The structural fold of V. harveyi MpaA was similar to eukaryotic carboxypeptidases. Analysis of the structure showed an obvious dimer interface between two monomers of MpaA and presence of a zinc ion in the active centre of each monomer consistent with the mass spectrometric data. The structure provides the basis for future modelling and mutational studies for extensive functional characterization of MpaA
Characterisation of the vibrio cholerae antibiotic resistance var operon
No full textThe discovery and use of antibiotics in the chemotherapy of bacterial infections has revolutionised medicine as it is today. Unfortunately, the progressive use of antibiotics has promoted the evolution of bacterial defences against these mediators and thus the emergence of antibiotic resistance. Multidrug resistance (MDR) in bacterial pathogens has grown with such rapid progression that it now threatens to compromise the effective chemotherapy of a plethora of diseases. This thesis aspires to elucidate the molecular resistance mechanisms adopted by these bacteria, in order to expand our knowledge and to assist in the development of new therapeutic approaches to circumvent these mechanisms. On this basis, this thesis presents insights into a novel Vibrio cholerae antibiotic resistance, var, operon that encodes a metallo- β -lactamase (Mßl), VarG, and a tripartite ATP-binding cassette-type (ABC-type) transport system, VarACDEF that has substrate specificities for antimicrobial peptides and macrolide antibiotics. Mßls are fast emerging as a primary resistance mechanism, possibly as a consequence of the introduction of newer ß-lactam antibiotics such as the carbapenems in response to increasing Gram-negative bacterial resistance. Fascinatingly, the ABC transporter, through secondary structure predictions, has been envisaged to adopt a tripartite structure similar to the MDR transporter, AcrAB-TolC, from the resistance nodulation and cell division (RND) family. The structural characterisation of this system would be the first such tripartite system to be elucidated and may bring new insights into how Gram-negative bacteria may have evolved to tackle the issue that threatens its existence. The resistance mechanisms in the var Operon are believed to be under the control of a LysR-type transcriptional regulatory protein (LTTR), VarR. LTTR proteins form one of the largest transcriptional regulatory families with extremely diverse functions ranging from amino acid biosynthesis to CO(_2) fixation. VarR binds to three distinct promoter regions, varRG, varGA and varBC located upstream and adjacent to VarG, VarA an AcrA-like membrane fusion protein and VarC a TolC-like outer membrane protein, respectively. VarR has also been shown to act as a repressor at the varRG promoter region in the absence of its substrate. Interestingly, the mechanism of regulation by VarR is strikingly similar to the well documented LTTR, AmpR and serine ß-lactamase AmpC system that are found in many pathogenic bacteria. It could be that V. cholerae has evolved from this regular system and developed a ß-lactamase that would prove more beneficial in light of current selective pressures. Contrary to LTTRs being notoriously recalcitrant to purification due to their low solubility, this thesis reports the successful purification and crystallisation of full-length VarR in the presence and absence of its cognate promoter DNA. Elucidating the structural characteristics of VarR would be the first such regulator associated with MDR in the LTTR family. This would advance the knowledge on the only currently existing full-length crystal structure of a LTTR, CbnR, and will provide further insights into how structural conformations may lead to dissociation from the promoter and induction of gene expression. Understanding the mechanism by which VarR induces expression of these resistance mechanisms is paramount for future strategies to prevent the emergence of MDR microorganisms. Although these mechanisms of MDR maybe elucidated in V. cholerae, the evolutionary relatedness and conservation of structure and function in all families will enable this information to be related to similar systems in alternative bacterial species
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