Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1

BACKGROUND: We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations. RESULTS: cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. CONCLUSION: Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

A three dimensional surface measurement and reconstruction system for respiratory function analysis

Respiration is a complex activity. If the relationship between all neurological and skeletomuscular interactions was perfectly understood, an accurate dynamic model of the respiratory system could be developed and the interaction between different inputs and outputs could be investigated in a straightforward fashion. Unfortunately, this is not the case and does not appear to be viable at this time. In addition, the provision of appropriate sensor signals for such a model would be a considerable invasive task. Useful quantitative information with respect to respiratory performance can be gained from non-invasive monitoring of chest and abdomen motion. Currently available devices are not well suited in application for spirometric measurement for ambulatory monitoring. A sensor matrix measurement technique is investigated to identify suitable sensing elements with which to base an upper body surface measurement device that monitors respiration. This thesis is divided into two main areas of investigation; model based and geometrical based surface plethysmography. In the first instance, chapter 2 deals with an array of tactile sensors that are used as progression of existing and previously investigated volumetric measurement schemes based on models of respiration. Chapter 3 details a non-model based geometrical approach to surface (and hence volumetric) profile measurement. Later sections of the thesis concentrate upon the development of a functioning prototype sensor array. To broaden the application area the study has been conducted as it would be fore a generically configured sensor array. In experimental form the system performance on group estimation compares favourably with existing system on volumetric performance. In addition provides continuous transient measurement of respiratory motion within an acceptable accuracy using approximately 20 sensing elements. Because of the potential size and complexity of the system it is possible to deploy it as a fully mobile ambulatory monitoring device, which may be used outside of the laboratory. It provides a means by which to isolate coupled physiological functions and thus allows individual contributions to be analysed separately. Thus facilitating greater understanding of respiratory physiology and diagnostic capabilities. The outcome of the study is the basis for a three-dimensional surface contour sensing system that is suitable for respiratory function monitoring and has the prospect with future development to be incorporated into a garment based clinical tool.

An evaluation of task analysis techniques for industrial process control

The concept of a task is fundamental to the discipline of ergonomics. Approaches to the analysis of tasks began in the early 1900's. These approaches have evolved and developed to the present day, when there is a vast array of methods available. Some of these methods are specific to particular contexts or applications, others more general. However, whilst many of these analyses allow tasks to be examined in detail, they do not act as tools to aid the design process or the designer. The present thesis examines the use of task analysis in a process control context, and in particular the use of task analysis to specify operator information and display requirements in such systems. The first part of the thesis examines the theoretical aspect of task analysis and presents a review of the methods, issues and concepts relating to task analysis. A review of over 80 methods of task analysis was carried out to form a basis for the development of a task analysis method to specify operator information requirements in industrial process control contexts. Of the methods reviewed Hierarchical Task Analysis was selected to provide such a basis and developed to meet the criteria outlined for such a method of task analysis. The second section outlines the practical application and evolution of the developed task analysis method. Four case studies were used to examine the method in an empirical context. The case studies represent a range of plant contexts and types, both complex and more simple, batch and continuous and high risk and low risk processes. The theoretical and empirical issues are drawn together and a method developed to provide a task analysis technique to specify operator information requirements and to provide the first stages of a tool to aid the design of VDU displays for process control.

Relieving the first bottleneck in the drug discovery pipeline:using array technologies to rationalize membrane protein production

The slow down in the drug discovery pipeline is, in part, owing to a lack of structural and functional information available for new drug targets. Membrane proteins, the targets of well over 50% of marketed pharmaceuticals, present a particular challenge. As they are not naturally abundant, they must be produced recombinantly for the structural biology that is a prerequisite to structure-based drug design. Unfortunately, however, obtaining high yields of functional, recombinant membrane proteins remains a major bottleneck in contemporary bioscience. While repeated rounds of trial-and-error optimization have not (and cannot) reveal mechanistic details of the biology of recombinant protein production, examination of the host response has provided new insights. To this end, we published an early transcriptome analysis that identified genes implicated in high-yielding yeast cell factories, which has enabled the engineering of improved production strains. These advances offer hope that the bottleneck of membrane protein production can be relieved rationally.

Time resolved bit error rate analysis of a fast switching tunable laser for use in optically switched networks

We investigate the use of different direct detection modulation formats in a wavelength switched optical network. We find the minimum time it takes a tunable sampled grating distributed Bragg reflector laser to recover after switching from one wavelength channel to another for different modulation formats. The recovery time is investigated utilizing a field programmable gate array which operates as a time resolved bit error rate detector. The detector offers 93 ps resolution operating at 10.7 Gb/s and allows for all the data received to contribute to the measurement, allowing low bit error rates to be measured at high speed. The recovery times for 10.7 Gb/s non-return-to-zero on–off keyed modulation, 10.7 Gb/s differentially phase shift keyed signal and 21.4 Gb/s differentially quadrature phase shift keyed formats can be as low as 4 ns, 7 ns and 40 ns, respectively. The time resolved phase noise associated with laser settling is simultaneously measured for 21.4 Gb/s differentially quadrature phase shift keyed data and it shows that the phase noise coupled with frequency error is the primary limitation on transmitting immediately after a laser switching event.

Optical sensor interrogation with a blazed fiber Bragg grating and a charge-coupled device linear array

We present what is to our knowledge the first comprehensive investigation of the use of blazed fiber Bragg gratings (BFBGs) to interrogate wavelength division multiplexed (WDM) in-fiber optical sensor arrays. We show that the light outcoupled from the core of these BFBGs is radiated with sufficient optical power that it may be detected with a low-cost charge-coupled device (CCD) array. We present thorough system performance analysis that shows sufficient spectral-spatial resolution to decode sensors with a WDM separation of 75 ρm, signal-to-noise ratio greater than 45-dB bandwidth of 70 nm, and drift of only 0.1 ρm. We show the system to be polarization-state insensitive, making the BFBG-CCD spectral analysis technique a practical, extremely low-cost, alternative to traditional tunable filter approaches.

Analysis and Optimization of Synthetic Aperture Ultrasound Imaging Using the Effective Aperture Approach

An effective aperture approach is used as a tool for analysis and parameter optimization of mostly known ultrasound imaging systems - phased array systems, compounding systems and synthetic aperture imaging systems. Both characteristics of an imaging system, the effective aperture function and the corresponding two-way radiation pattern, provide information about two of the most important parameters of images produced by an ultrasound system - lateral resolution and contrast. Therefore, in the design, optimization of the effective aperture function leads to optimal choice of such parameters of an imaging systems that influence on lateral resolution and contrast of images produced by this imaging system. It is shown that the effective aperture approach can be used for optimization of a sparse synthetic transmit aperture (STA) imaging system. A new two-stage algorithm is proposed for optimization of both the positions of the transmitted elements and the weights of the receive elements. The proposed system employs a 64-element array with only four active elements used during transmit. The numerical results show that Hamming apodization gives the best compromise between the contrast of images and the lateral resolution.

Time resolved bit error rate analysis of a fast switching tunable laser for use in optically switched networks

We investigate the use of different direct detection modulation formats in a wavelength switched optical network. We find the minimum time it takes a tunable sampled grating distributed Bragg reflector laser to recover after switching from one wavelength channel to another for different modulation formats. The recovery time is investigated utilizing a field programmable gate array which operates as a time resolved bit error rate detector. The detector offers 93 ps resolution operating at 10.7 Gb/s and allows for all the data received to contribute to the measurement, allowing low bit error rates to be measured at high speed. The recovery times for 10.7 Gb/s non-return-to-zero on–off keyed modulation, 10.7 Gb/s differentially phase shift keyed signal and 21.4 Gb/s differentially quadrature phase shift keyed formats can be as low as 4 ns, 7 ns and 40 ns, respectively. The time resolved phase noise associated with laser settling is simultaneously measured for 21.4 Gb/s differentially quadrature phase shift keyed data and it shows that the phase noise coupled with frequency error is the primary limitation on transmitting immediately after a laser switching event.

Biosynthesis and analysis of plant oxylipins

The term oxylipin is applied to the generation of oxygenated products of polyunsaturated fatty acids that can arise either through non-enzymatic or enzymatic processes generating a complex array of products, including alcohols, aldehydes, ketones, acids and hydrocarbon gases. The biosynthetic origin of these products has revealed an array of enzymes involved in their formation and more recently a radical pathway. These include lipoxygenases and α-dioxygenase that insert both oxygen atoms in to the acyl chain to initiate the pathways, to specialised P450 monooxygenases that are responsible for their downstream processing. This latter group include enzymes at the branch points such as allene oxide synthase, leading to jasmonate signalling, hydroperoxide lyase, responsible for generating pathogen/pest defensive volatiles and divinyl ether synthases and peroxygenases involved in the formation of antimicrobial compounds. The complexity of the products generated raises significant challenges for their rapid identification and quantification using metabolic screening methods. Here the current developments in oxylipin metabolism are reviewed together with the emerging technologies required to expand this important field of research that underpins advances in plant-pest/pathogen interactions.

Field programmable gate array based target detection and gesture recognition

The move from Standard Definition (SD) to High Definition (HD) represents a six times increases in data, which needs to be processed. With expanding resolutions and evolving compression, there is a need for high performance with flexible architectures to allow for quick upgrade ability. The technology advances in image display resolutions, advanced compression techniques, and video intelligence. Software implementation of these systems can attain accuracy with tradeoffs among processing performance (to achieve specified frame rates, working on large image data sets), power and cost constraints. There is a need for new architectures to be in pace with the fast innovations in video and imaging. It contains dedicated hardware implementation of the pixel and frame rate processes on Field Programmable Gate Array (FPGA) to achieve the real-time performance. ^ The following outlines the contributions of the dissertation. (1) We develop a target detection system by applying a novel running average mean threshold (RAMT) approach to globalize the threshold required for background subtraction. This approach adapts the threshold automatically to different environments (indoor and outdoor) and different targets (humans and vehicles). For low power consumption and better performance, we design the complete system on FPGA. (2) We introduce a safe distance factor and develop an algorithm for occlusion occurrence detection during target tracking. A novel mean-threshold is calculated by motion-position analysis. (3) A new strategy for gesture recognition is developed using Combinational Neural Networks (CNN) based on a tree structure. Analysis of the method is done on American Sign Language (ASL) gestures. We introduce novel point of interests approach to reduce the feature vector size and gradient threshold approach for accurate classification. (4) We design a gesture recognition system using a hardware/ software co-simulation neural network for high speed and low memory storage requirements provided by the FPGA. We develop an innovative maximum distant algorithm which uses only 0.39% of the image as the feature vector to train and test the system design. Database set gestures involved in different applications may vary. Therefore, it is highly essential to keep the feature vector as low as possible while maintaining the same accuracy and performance^

Development of calix[4]arene-functionalized microcantilever array sensing system for the rapid, sensitive and simultaneous detection of metal ions in aqueous solutions

The work described in this thesis was conducted with the aim of: 1) investigating the binding capabilities of calix[4]arene-functionalized microcantilevers towards specific metal ions and 2) developing a new16-microcantilever array sensing system for the rapid, and simultaneous detection of metal ions in fresh water. Part I of this thesis reports on the use of three new bimodal calix[4]arenes (methoxy, ethoxy and crown) as potential host/guest sensing layers for detecting selected ions in dilute aqueous solutions using single microcantilever experimental system. In this work it was shown that modifying the upper rim of the calix[4]arenes with a thioacetate end group allow calix[4]arenes to self-assemble on Au(111) forming complete highly ordered monolayers. It was also found that incubating the microcantilevers coated with 5 nm of Inconel and 40 nm of Au for 1 h in a 1.0 M solution of calix[4]arene produced the highest sensitivity. Methoxy-functionalized microcantilevers showed a definite preference for Ca²⁺ ions over other cationic guests and were able to detect trace concentration as low as 10⁻¹² M in aqueous solutions. Microcantilevers modified with ethoxy calix[4]arene displayed their highest sensitivity towards Sr²⁺ and to a lesser extent Ca²⁺ ions. Crown calix[4]arene-modified microcantilevers were however found to bind selectively towards Cs⁺ ions. In addition, the counter anion was also found to contribute to the deflection. For example methoxy calix[4]arene-modified microcantilever was found to be more sensitive to CaCl₂ over other water-soluble calcium salts such as Ca(NO₃)₂ , CaBr₂ and CaI₂. These findings suggest that the response of calix[4]arene-modified microcantilevers should be attributed to the target ionic species as a whole instead of only considering the specific cation and/or anion. Part II presents the development of a 16-microcantilever sensor setup. The implementation of this system involved the creation of data analysis software that incorporates data from the motorized actuator and a two-axis photosensitive detector to obtain the deflection signal originating from each individual microcantilever in the array. The system was shown to be capable of simultaneous measurements of multiple microcantilevers with different coatings. A functionalization unit was also developed that allows four microcantilevers in the array to be coated with an individual sensing layer one at the time. Because of the variability of the spring constants of different cantilevers within the array, results presented were quoted in units of surface stress unit in order to compare values between the microcantilevers in the array.

Advanced functionality for radio analysis in the Offline software framework of the Pierre Auger Observatory.

The advent of the Auger Engineering Radio Array (AERA) necessitates the development of a powerful framework for the analysis of radio measurements of cosmic ray air showers. As AERA performs "radio-hybrid" measurements of air shower radio emission in coincidence with the surface particle detectors and fluorescence telescopes of the Pierre Auger Observatory, the radio analysis functionality had to be incorporated in the existing hybrid analysis solutions for fluorescence and surface detector data. This goal has been achieved in a natural way by extending the existing Auger Offline software framework with radio functionality. In this article, we lay out the design, highlights and features of the radio extension implemented in the Auger Offline framework. Its functionality has achieved a high degree of sophistication and offers advanced features such as vectorial reconstruction of the electric field, advanced signal processing algorithms, a transparent and efficient handling of FFTs, a very detailed simulation of detector effects, and the read-in of multiple data formats including data from various radio simulation codes. The source code of this radio functionality can be made available to interested parties on request. (C) 2011 Elsevier B.V. All rights reserved.

Acoustic and Magnetic Techniques for the Isolation and Analysis of Cells in Microfluidic Platforms

Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management.

The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis.

The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments.

Global Value Chain Analysis: A Primer, 2nd Edition

The first edition of Global Value Chain Analysis: A Primer was released five years ago (May 2011) in order to provide an overview of the key concepts and methodological tools used by Duke University’s Center on Globalization, Governance & Competitiveness (Duke CGGC) a university-based research center that focuses on innovative applications of the GVC framework, which was developed by Duke CGGC’s founding director, Gary Gereffi. The Second Edition of Global Value Chain Analysis: A Primer (July 2016) retains a simple, expository style and use of recent research examples in order to offer an entry point for those wishing to better understand and use the GVC framework as a tool to analyze how local actors (firms, communities, workers) are linked to and affected by major transformations in the global economy. The GVC framework focuses on structural shifts in global industries, anchored by the core concepts of governance and upgrading. This Second Edition highlights some of the refinements in these concepts, and introduces a number of new illustrations drawing from recent Duke CGGC research. The bibliography offers a sampling of the broad array of studies available on the Duke CGGC website and in related academic publications. We hope this work stimulates continued interest in and use of the GVC framework as a tool to promote more dynamic, inclusive and sustainable development outcomes for all economies and the local actors within them.

Transcriptomic Analysis of the Host Response and Innate Resilience to Enterotoxigenic Escherichia coli Infection in Humans.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause of diarrhea. Though usually self-limited, it can be severe and debilitating. Little is known about the host transcriptional response to infection. We report the first gene expression analysis of the human host response to experimental challenge with ETEC. METHODS: We challenged 30 healthy adults with an unattenuated ETEC strain, and collected serial blood samples shortly after inoculation and daily for 8 days. We performed gene expression analysis on whole peripheral blood RNA samples from subjects in whom severe symptoms developed (n = 6) and a subset of those who remained asymptomatic (n = 6) despite shedding. RESULTS: Compared with baseline, symptomatic subjects demonstrated significantly different expression of 406 genes highlighting increased immune response and decreased protein synthesis. Compared with asymptomatic subjects, symptomatic subjects differentially expressed 254 genes primarily associated with immune response. This comparison also revealed 29 genes differentially expressed between groups at baseline, suggesting innate resilience to infection. Drug repositioning analysis identified several drug classes with potential utility in augmenting immune response or mitigating symptoms. CONCLUSIONS: There are statistically significant and biologically plausible differences in host gene expression induced by ETEC infection. Differential baseline expression of some genes may indicate resilience to infection.