Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation.

Identification of YAP as sperm-PAWP’s predominant binding partner in MII-arrested oocytes and the relevance of this WWI domain modular interaction to oocyte activation

PAWP, a candidate sperm-borne oocyte activating factor, induces oocyte activation and acts upstream of the calcium signalling pathway, however, PAWP’s downstream signalling pathway in oocyte cytoplasm remains to be uncovered. Data from our lab suggested that the interacting partner of PAWP, at least in the frog (Xenopus laevis) model may be YAP, a highly expressed protein in amphibian and mammalian oocytes. Therefore, the objectives of this study were to confirm that PAWP’s predominant binding partner in Xenopus laevis oocyte is YAP; to determine if mammalian oocyte activation is also dependent on PAWP-YAP interaction; and to verify that the PAWP-YAP interaction during oocyte activation is dependent on the WWI domain module. By immunohistochemistry, YAP was localized predominantly in the cytosol of metaphase II-arrested Xenopus laevis oocytes, where presumably the PAWP-YAP interaction occurs. Utilizing Far Western blotting, YAP was identified as the predominant binding partner of PAWP, in metaphase II-arrested frog (Xenopus laevis), swine (Sus scrofa) and mouse (mus musculus) oocytes. The specificity of this interaction was then tested on Far Western blotting of mouse ovarian and oocyte cytosolic extracts, by competition with both wild-type and point-mutated recombinant WWI domains derived from YAP. The removal of GST from the wild-type WWI-GST fusion protein was a requirement for effective blockage of WWI module interaction between PAWP and YAP. As expected, the mutated WWI domain was ineffective in inhibiting the PAWP-YAP interaction. To conclude, this study identified YAP as the predominant binding partner of PAWP in both amphibian and mammalian oocytes, and showed this interaction is dependent on the WWI modular interaction. The results allow us to test the functional relevance of this WWI modular interaction during oocyte activation in vivo, in the future.

Use of Extended Characteristics of Locomotion and Feeding Behavior for Automated Identification of Lame Dairy Cows.

This study was carried out to detect differences in locomotion and feeding behavior in lame (group L; n = 41; gait score ≥ 2.5) and non-lame (group C; n = 12; gait score ≤ 2) multiparous Holstein cows in a cross-sectional study design. A model for automatic lameness detection was created, using data from accelerometers attached to the hind limbs and noseband sensors attached to the head. Each cow's gait was videotaped and scored on a 5-point scale before and after a period of 3 consecutive days of behavioral data recording. The mean value of 3 independent experienced observers was taken as a definite gait score and considered to be the gold standard. For statistical analysis, data from the noseband sensor and one of two accelerometers per cow (randomly selected) of 2 out of 3 randomly selected days was used. For comparison between group L and group C, the T-test, the Aspin-Welch Test and the Wilcoxon Test were used. The sensitivity and specificity for lameness detection was determined with logistic regression and ROC-analysis. Group L compared to group C had significantly lower eating and ruminating time, fewer eating chews, ruminating chews and ruminating boluses, longer lying time and lying bout duration, lower standing time, fewer standing and walking bouts, fewer, slower and shorter strides and a lower walking speed. The model considering the number of standing bouts and walking speed was the best predictor of cows being lame with a sensitivity of 90.2% and specificity of 91.7%. Sensitivity and specificity of the lameness detection model were considered to be very high, even without the use of halter data. It was concluded that under the conditions of the study farm, accelerometer data were suitable for accurately distinguishing between lame and non-lame dairy cows, even in cases of slight lameness with a gait score of 2.5.

Test and evaluation of the AMF, Inc. and Fairchild Industries experimental safety vehicles. Volume II. ESV program summary. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Centromere identification and inactivation on a neo-Y chromosome fusion in threespine stickleback fish (Gasterosteus aculeatus)

Thesis (Ph.D.)--University of Washington, 2016-05

Identification of a major locus conferring resistance to powdery mildew (Erysiphe polygoni DC) in mungbean (Vigna radiata L. Wilczek) by QTL analysis

A major locus conferring resistance to the causal organism of powdery mildew, Erysiphe polygoni DC,, in mungbean (Vigna radiata L. Wilczek) was identified using QTL analysis with a population of 147 recombinant inbred individuals. The population was derived from a cross between 'Berken', a highly susceptible variety, and ATF 3640, a highly resistant line. To test for response to powdery mildew, F-7 and F-8 lines were inoculated by dispersing decaying mungbean leaves with residual conidia of E. polygoni amongst the young plants to create an artificial epidemic and assayed in a glasshouse facility. To generate a linkage map, 322 RFLP clones were tested against the two parents and 51 of these were selected to screen the mapping population. The 51 probes generated 52 mapped loci, which were used to construct a linkage map spanning 350 cM of the mungbean genome over 10 linkage groups. Using these markers, a single locus was identified that explained up to a maximum of 86% of the total variation in the resistance response to the pathogen.

A simplified PCR test for early detection of dwarf off-types in micropropagated Cavendish bananas (Musa spp. AAA)

The dwarf somaclonal variant is a major problem affecting micropropagation of the banana cultivar Williams (Musa spp. AAA; subgroup Cavendish). This problem arises from genetic changes that occur during the tissue culture process. Early identification of this problem is difficult and propagators must wait until plants are ex vitro in order to visualise the dwarfism phenotype. In this study, we have improved a SCAR-based molecular diagnostic technique, developed by Damasco et al. [Acta Hortic. 461 (1997) 157], for the early identification of dwarf off-types. We have included a positive internal control in a multiplex PCR and adapted the technique for use with small amounts of fresh in vitro leaf material as PCR template. The control product is a 500 bp fragment from 18S rRNA and is amplified in all tissues irrespective of phenotype. The use of small in vitro leaf material removing the need for genomic DNA extraction. (C) 2004 Elsevier B.V. All rights reserved.

The use of internal audit by Australian companies

Purpose – The purpose of this study is to explore the voluntary use of internal audit by Australian publicly listed companies and to identify factors that lead listed companies to have an internal audit function. Design/methodology/approach – Drawing on the Institute of Internal Auditors' definition of internal auditing, the paper predicts that internal audit use is associated with factors related to risk management, strong internal controls and strong corporate governance. To test the predictions, the study combines data from a survey of listed companies with information from corporate annual reports. The paper also provides descriptive information on the use of internal audit. Findings – The results indicate that only one-third of the sample companies use internal audit. While size appears to be the dominant driver, there is also a strong association between internal audit and the level of commitment to risk management. However, the study finds only weak support for an association between the use of internal audit and strong corporate governance. Research limitations/implications – A limitation of our study is that some of the variables in the model may not be good proxies for the factors being measured. Refinement of the model and the variables used provides an opportunity for future research. Practical implications – The limited use of internal audit by Australian companies has important implications for sound corporate governance. Originality/value – This is the first study that identifies factors associated with the use of internal audit by Australian listed companies.

Big 4 audit fee premiums for national and office-level industry leadership in the United Kingdom

The pricing of Big 4 industry leadership Is examined for a sample of U.K. publicly-listed companies, and adds to the evidence from the Australian and U.S. audit markets that city-specific industry leadership commands a fee premium. There is a significant fee premium for city-specific industry leaders relative to other Big 4 auditors, but no evidence that either the top-ranked or second-ranked firm nationally commands a fee premium relative to other Big 4 auditors, after controlling for city-level industry leadership. We also test for Big 4 fee premiums relative to non-Big 4 auditors and the U.K. data suggest a three-level hierarchy based on audit fee differentials: (1) Big 4 city-specific industry leaders have the largest fees; (2) other Big 4 auditors (noncity leaders) and second-tier national firms have comparable fees that are lower than Big 4 city leaders but larger than third-tier firms; and (3) third-tier accounting firms have the lowest fees.

The detection and identification of extraneous metals in material conveyance systems

The aim of this work was to design and build an equipment which can detect ferrous and non-ferrous objects in conveyed commodities, discriminate between them and locate the object along the belt and on the width of the belt. The magnetic induction mechanism was used as a means of achieving the objectives of this research. In order to choose the appropriate geometry and size of the induction field source, the field distributions of different source geometries and sizes were studied in detail. From these investigations it was found the square loop geometry is the most appropriate as a field generating source for the purpose of this project. The phenomena of field distribution in the conductors was also investigated. An equipment was designed and built at the preliminary stages of thework based on a flux-gate magnetometer with the ability to detect only ferrous objects.The instrument was designed such that it could be used to detect ferrous objects in the coal conveyors of power stations. The advantages of employing this detector in the power industry over the present ferrous metal electromagnetic separators were also considered. The objectives of this project culminated in the design and construction of a ferrous and non-ferrous detector with the ability to discriminate between ferrous and non-ferrous metals and to locate the objects on the conveying system. An experimental study was carried out to test the performance of the equipment in the detection of ferrous and non-ferrous objects of a given size carried on the conveyor belt. The ability of the equipment to discriminate between the types of metals and to locate the object on the belt was also evaluated experimentally. The benefits which can be gained from the industrial implementations of the equipment were considered. Further topics which may be investigated as an extension of this work are given.

The design of environmental test rigs

Product reliability and its environmental performance have become critical elements within a product's specification and design. To obtain a high level of confidence in the reliability of the design it is customary to test the design under realistic conditions in a laboratory. The objective of the work is to examine the feasibility of designing mechanical test rigs which exhibit prescribed dynamical characteristics. The design is then attached to the rig and excitation is applied to the rig, which then transmits representative vibration levels into the product. The philosophical considerations made at the outset of the project are discussed as they form the basis for the resulting design methodologies. It is attempted to directly identify the parameters of a test rig from the spatial model derived during the system identification process. It is shown to be impossible to identify a feasible test rig design using this technique. A finite dimensional optimal design methodology is developed which identifies the parameters of a discrete spring/mass system which is dynamically similar to a point coordinate on a continuous structure. This design methodology is incorporated within another procedure which derives a structure comprising a continuous element and a discrete system. This methodology is used to obtain point coordinate similarity for two planes of motion, which is validated by experimental tests. A limitation of this approach is that it is impossible to achieve multi-coordinate similarity due to an interaction of the discrete system and the continuous element at points away from the coordinate of interest. During the work the importance of the continuous element is highlighted and a design methodology is developed for continuous structures. The design methodology is based upon distributed parameter optimal design techniques and allows an initial poor design estimate to be moved in a feasible direction towards an acceptable design solution. Cumulative damage theory is used to provide a quantitative method of assessing the quality of dynamic similarity. It is shown that the combination of modal analysis techniques and cumulative damage theory provides a feasible design synthesis methodology for representative test rigs.

System identification of linear vibrating structures

Methods of dynamic modelling and analysis of structures, for example the finite element method, are well developed. However, it is generally agreed that accurate modelling of complex structures is difficult and for critical applications it is necessary to validate or update the theoretical models using data measured from actual structures. The techniques of identifying the parameters of linear dynamic models using Vibration test data have attracted considerable interest recently. However, no method has received a general acceptance due to a number of difficulties. These difficulties are mainly due to (i) Incomplete number of Vibration modes that can be excited and measured, (ii) Incomplete number of coordinates that can be measured, (iii) Inaccuracy in the experimental data (iv) Inaccuracy in the model structure. This thesis reports on a new approach to update the parameters of a finite element model as well as a lumped parameter model with a diagonal mass matrix. The structure and its theoretical model are equally perturbed by adding mass or stiffness and the incomplete number of eigen-data is measured. The parameters are then identified by an iterative updating of the initial estimates, by sensitivity analysis, using eigenvalues or both eigenvalues and eigenvectors of the structure before and after perturbation. It is shown that with a suitable choice of the perturbing coordinates exact parameters can be identified if the data and the model structure are exact. The theoretical basis of the technique is presented. To cope with measurement errors and possible inaccuracies in the model structure, a well known Bayesian approach is used to minimize the least squares difference between the updated and the initial parameters. The eigen-data of the structure with added mass or stiffness is also determined using the frequency response data of the unmodified structure by a structural modification technique. Thus, mass or stiffness do not have to be added physically. The mass-stiffness addition technique is demonstrated by simulation examples and Laboratory experiments on beams and an H-frame.

Development of a co-culture model of the human lungs for toxicity testing and identification of biomarkers of inhalation toxicity

The airway epithelium is the first point of contact in the lung for inhaled material, including infectious pathogens and particulate matter, and protects against toxicity from these substances by trapping and clearance via the mucociliary escalator, presence of a protective barrier with tight junctions and initiation of a local inflammatory response. The inflammatory response involves recruitment of phagocytic cells to neutralise and remove and invading materials and is oftern modelled using rodents. However, development of valid in vitro airway epithelial models is of great importance due to the restrictions on animal studies for cosmetic compound testing implicit in the 7th amendment to the European Union Cosmetics Directive. Further, rodent innate immune responses have fundamental differences to human. Pulmonary endothelial cells and leukocytes are also involved in the innate response initiated during pulmonary inflammation. Co-culture models of the airways, in particular where epithelial cells are cultured at air liquid interface with the presence of tight junctions and differentiated mucociliary cells, offer a solution to this problem. Ideally validated models will allow for detection of early biomarkers of response to exposure and investigation into inflammatory response during exposure. This thesis describes the approaches taken towards developing an in vitro epithelial/endothelial cell model of the human airways and identification biomarkers of response to exposure to xenobiotics. The model comprised normal human primary microvascular endothelial cells and the bronchial epithelial cell line BEAS-2B or normal human bronchial epithelial cells. BEAS-2B were chosen as their characterisation at air liquid interface is limited but they are robust in culture, thereby predicted to provide a more reliable test system. Proteomics analysis was undertaken on challenged cells to investigate biomarkers of exposure. BEAS-2B morphology was characterised at air liquid interface compared with normal human bronchial epithelial cells. The results indicate that BEAS-2B cells at an air liquid interface form tight junctions as shown by expression of the tight junction protein zonula occludens-1. To this author’s knowledge this is the first time this result has been reported. The inflammatory response of BEAS-2B (measured as secretion of the inflammatory mediators interleukin-8 and -6) air liquid interface mono-cultures to Escherichia coli lipopolysaccharide or particulate matter (fine and ultrafine titanium dioxide) was comparable to published data for epithelial cells. Cells were also exposed to polymers of “commercial interest” which were in the nanoparticle range (and referred to particles hereafter). BEAS-2B mono-cultures showed an increased secretion of inflammatory mediators after challenge. Inclusion of microvascular endothelial cells resulted in protection against LPS- and particle- induced epithelial toxicity, measured as cell viability and inflammatory response, indicating the importance of co-cultures for investigations into toxicity. Two-dimensional proteomic analysis of lysates from particle-challenged cells failed to identify biomarkers of toxicity due to assay interference and experimental variability. Separately, decreased plasma concentrations of serine protease inhibitors, and the negative acute phase proteins transthyretin, histidine-rich glycoprotein and alpha2-HS glycoprotein were identified as potential biomarkers of methyl methacrylate/ethyl methacrylate/butylacrylate treatment in rats.

Identification and relative quantification of tyrosine nitration in a model peptide using two-dimensional infrared spectroscopy

Nitration of tyrosine in proteins and peptides is a post-translational modification that occurs under conditions of oxidative stress. It is implicated in a variety of medical conditions, including neurodegenerative and cardiovascular diseases. However, monitoring tyrosine nitration and understanding its role in modifying biological function remains a major challenge. In this work, we investigate the use of electron-vibration-vibration (EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration in model peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiate between the neutral and deprotonated states of 3-nitrotyrosine, and we characterize their spectral signatures using information obtained from quantum chemistry calculations and simulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptide samples containing various levels of tyrosine nitration, and we use mass spectrometry to independently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is able to provide detailed spectroscopic information on peptide side-chain modifications and to detect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonant Raman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy. (Graph Presented).

Towards the in silico identification of class II restricted T-cell epitopes:a partial least squares iterative self-consistent algorithm for affinity prediction

Motivation: The immunogenicity of peptides depends on their ability to bind to MHC molecules. MHC binding affinity prediction methods can save significant amounts of experimental work. The class II MHC binding site is open at both ends, making epitope prediction difficult because of the multiple binding ability of long peptides. Results: An iterative self-consistent partial least squares (PLS)-based additive method was applied to a set of 66 pep- tides no longer than 16 amino acids, binding to DRB1*0401. A regression equation containing the quantitative contributions of the amino acids at each of the nine positions was generated. Its predictability was tested using two external test sets which gave r pred =0.593 and r pred=0.655, respectively. Furthermore, it was benchmarked using 25 known T-cell epitopes restricted by DRB1*0401 and we compared our results with four other online predictive methods. The additive method showed the best result finding 24 of the 25 T-cell epitopes. Availability: Peptides used in the study are available from http://www.jenner.ac.uk/JenPep. The PLS method is available commercially in the SYBYL molecular modelling software package. The final model for affinity prediction of peptides binding to DRB1*0401 molecule is available at http://www.jenner.ac.uk/MHCPred. Models developed for DRB1*0101 and DRB1*0701 also are available in MHC- Pred