Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. 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New rôles and development rules for housing associations: rhe rise of design and build?

This paper summarises an initial report carried out by the Housing Business Research Group, of the University of Reading into Design and Build procurement and a number of research projects undertaken by the national federation of Housing Associations (NFHA), into their members' development programmes. The paper collates existing statistics from these sources and examines the way in which Design and Build procurement can be adapted for the provision of social housing. The paper comments on these changes and questions how risk averting the adopted strategies are in relation to long term housing business management issues arising from the quality of the product produced by the new system.

Design of a unified data with business rules storage model for OLTP and OLAP systems

This paper reviews the literature concerning the practice of using Online Analytical Processing (OLAP) systems to recall information stored by Online Transactional Processing (OLTP) systems. Such a review provides a basis for discussion on the need for the information that are recalled through OLAP systems to maintain the contexts of transactions with the data captured by the respective OLTP system. The paper observes an industry trend involving the use of OLTP systems to process information into data, which are then stored in databases without the business rules that were used to process information and data stored in OLTP databases without associated business rules. This includes the necessitation of a practice, whereby, sets of business rules are used to extract, cleanse, transform and load data from disparate OLTP systems into OLAP databases to support the requirements for complex reporting and analytics. These sets of business rules are usually not the same as business rules used to capture data in particular OLTP systems. The paper argues that, differences between the business rules used to interpret these same data sets, risk gaps in semantics between information captured by OLTP systems and information recalled through OLAP systems. Literature concerning the modeling of business transaction information as facts with context as part of the modelling of information systems were reviewed to identify design trends that are contributing to the design quality of OLTP and OLAP systems. The paper then argues that; the quality of OLTP and OLAP systems design has a critical dependency on the capture of facts with associated context, encoding facts with contexts into data with business rules, storage and sourcing of data with business rules, decoding data with business rules into the facts with the context and recall of facts with associated contexts. The paper proposes UBIRQ, a design model to aid the co-design of data with business rules storage for OLTP and OLAP purposes. The proposed design model provides the opportunity for the implementation and use of multi-purpose databases, and business rules stores for OLTP and OLAP systems. Such implementations would enable the use of OLTP systems to record and store data with executions of business rules, which will allow for the use of OLTP and OLAP systems to query data with business rules used to capture the data. Thereby ensuring information recalled via OLAP systems preserves the contexts of transactions as per the data captured by the respective OLTP system.

The duty of a steward to his lord : represented under several plain and distinct articles; wherein may be seen the indirect practices of several stewards, tending to lessen, and the several methods likely to improve their lords estates. To which is added an appendix shewing the way to plenty, proposed to the farmers; wherein are laid down general rules and directions for the management and improvement of a farm. Both design'd originally for the use of the several stewards and tenants of his grace the Duke of Buckingham, and now improv'd and publish'd for the general use and interest of all the nobility and gentry throughout England /

Mode of access: Internet.

Rules and Regulations Relating to the Safety of Design and Construction of Public School Buildings

Mode of access: Internet.

Problem solving in a middle school robotics design classroom

Little research has been conducted on how students work when they are required to plan, build and evaluate artefacts in technology rich learning environments such as those supported by tools including flow charts, Labview programming and Lego construction. In this study, activity theory was used as an analytic tool to examine the social construction of meaning. There was a focus on the effect of teachers’ goals and the rules they enacted upon student use of the flow chart planning tool, and the tools of the programming language Labview and Lego construction. It was found that the articulation of a teacher’s goals via rules and divisions of labour helped to form distinct communities of learning and influenced the development of different problem solving strategies. The use of the planning tool flow charting was associated with continuity of approach, integration of problem solutions including appreciation of the nexus between construction and programming, and greater educational transformation. Students who flow charted defined problems in a more holistic way and demonstrated more methodical, insightful and integrated approaches to their use of tools. The findings have implications for teaching in design dominated learning environments.

Developing a hybrid expert system program to aid in the design of plastic injection molding process

Experts in injection molding often refer to previous solutions to find a mold design similar to the current mold and use previous successful molding process parameters with intuitive adjustment and modification as a start for the new molding application. This approach saves a substantial amount of time and cost in experimental based corrective actions which are required in order to reach optimum molding conditions. A Case-Based Reasoning (CBR) System can perform the same task by retrieving a similar case which is applied to the new case from the case library and uses the modification rules to adapt a solution to the new case. Therefore, a CBR System can simulate human e~pertise in injection molding process design. This research is aimed at developing an interactive Hybrid Expert System to reduce expert dependency needed on the production floor. The Hybrid Expert System (HES) is comprised of CBR, flow analysis, post-processor and trouble shooting systems. The HES can provide the first set of operating parameters in order to achieve moldability condition and producing moldings free of stress cracks and warpage. In this work C++ programming language is used to implement the expert system. The Case-Based Reasoning sub-system is constructed to derive the optimum magnitude of process parameters in the cavity. Toward this end the Flow Analysis sub-system is employed to calculate the pressure drop and temperature difference in the feed system to determine the required magnitude of parameters at the nozzle. The Post-Processor is implemented to convert the molding parameters to machine setting parameters. The parameters designed by HES are implemented using the injection molding machine. In the presence of any molding defect, a trouble shooting subsystem can determine which combination of process parameters must be changed iii during the process to deal with possible variations. Constraints in relation to the application of this HES are as follows. - flow length (L) constraint: 40 mm < L < I 00 mm, - flow thickness (Th) constraint: -flow type: - material types: I mm < Th < 4 mm, unidirectional flow, High Impact Polystyrene (HIPS) and Acrylic. In order to test the HES, experiments were conducted and satisfactory results were obtained.

Ways of telling : tacit knowledge transfer for fashion design research

The knowledge and skills of fashion and textiles design have traditionally been transferred through the indenture of an apprentice to a master. This relationship relied heavily on the transfer of explicit methods of design and making but also on the transfer of tacit knowledge, explained by Michael Polanyi as knowledge that cannot be explicitly known. By watching the master and emulating his efforts in the presence of his example, the apprentice unconsciously picks up the rules of the art, including those which are not explicitly known to the master himself (Polanyi, 1962 p.53). However, it has been almost half a century since Michael Polanyi defined the tacit dimension as a state in which “we can know more than we can tell” (Polanyi, 1967 p.4) at a time when the accepted means of ‘telling’ was through academic writing and publishing in hardcopy format. The idea that tacit knowledge transfer involves a one to one relationship between apprentice and master would appear to have dire consequences for a discipline, such as fashion design, where there is no such tradition of academic writing. This paper counters this point of view by providing examples of strategies currently being employed in online environments (principally through ‘craft’) and explains how these methods might prove useful to support tacit knowledge transfer in respect to academic research within the field of fashion design, and in the wider academic community involved in creative practice research. A summary of the implications of these new ideas for contemporary fashion research will conclude the paper.

A Multifaceted Strategy for Implementation of the Ottawa Ankle Rules in Two Emergency Departments

Problem Despite widespread acceptance of the Ottawa ankle rules for assessment of acute ankle injuries, their application varies considerably. Design Before and after study. Background and setting Emergency departments of a tertiary teaching hospital and a community hospital in Australia. Key measures for improvement Documentation of the Ottawa ankle rules, proportion of patients referred for radiography, proportion of radiographs showing a fracture. Strategies for change Education, a problem specific radiography request form, reminders, audit and feedback, and using radiographers as “gatekeepers.” Effects of change Documentation of the Ottawa ankle rules improved from 57.5% to 94.7% at the tertiary hospital, and 51.6% to 80.8% at the community hospital (P<0.001 for both). The proportion of patients undergoing radiography fell from 95.8% to 87.2% at the tertiary hospital, and from 91.4% to 78.9% at the community hospital (P<0.001 for both). The proportion of radiographs showing a fracture increased from 20.4% to 27.1% at the tertiary hospital (P=0.069), and 15.2% to 27.2% (P=0.002) at the community hospital. The missed fracture rate increased from 0% to 2.9% at the tertiary hospital and from 0% to 1.6% at the community hospital compared with baseline (P=0.783 and P=0.747). Lessons learnt Assessment of case note documentation has limitations. Clinician groups seem to differ in their capacity and willingness to change their practice. A multifaceted change strategy including a problem specific radiography request form can improve the selection of patients for radiography.

Scripting versus emergence : issues for game developers and players in game environment design

This paper defines and discusses two contrasting approaches to designing game environments. The first, referred to as scripting, requires developers to anticipate, hand-craft and script specific game objects, events and player interactions. The second, known as emergence, involves defining general, global rules that interact to give rise to emergent gameplay. Each of these approaches is defined, discussed and analyzed with respect to the considerations and affects for game developers and game players. Subsequently, various techniques for implementing these design approaches are identified and discussed. It is concluded that scripting and emergence are two extremes of the same continuum, neither of which are ideal for game development. Rather, there needs to be a compromise in which the boundaries of action (such as story and game objectives) can be hardcoded and non-scripted behavior (such as interactions and strategies) are able to emerge within these boundaries.

How novices design business processes

Process modeling is an important design practice in organizational improvement projects. In this paper, we examine the design of business process diagrams in contexts where novice analysts only have basic design tools such as paper and pencils available, and little to no understanding of formalized modeling approaches. Based on a quasi-experimental study with 89 BPM students, we identify five distinct process design archetypes ranging from textual to hybrid and graphical representation forms. We examine the quality of the designs and identify which representation formats enable an analyst to articulate business rules, states, events, activities, temporal and geospatial information in a process model. We found that the quality of the process designs decreases with the increased use of graphics and that hybrid designs featuring appropriate text labels and abstract graphical forms appear well-suited to describe business processes. We further examine how process design preferences predict formalized process modeling ability. Our research has implications for practical process design work in industry as well as for academic curricula on process design.

Design for Manufacturing (DFM) – A case study of repetitive measures

Design for Manufacturing (DFM) is a highly integral methodology in product development, starting from the concept development phase, with the aim of improving manufacturing productivity and maintaining product quality. While Design for Assembly (DFA) is focusing on elimination or combination of parts with other components (Boothroyd, Dewhurst and Knight, 2002), which in most cases relates to performing a function and manufacture operation in a simpler way, DFM is following a more holistic approach. During DFM, the considerable background work required for the conceptual phase is compensated for by a shortening of later development phases. Current DFM projects normally apply an iterative step-by-step approach and eventually transfer to the developer team. Although DFM has been a well established methodology for about 30 years, a Fraunhofer IAO study from 2009 found that DFM was still one of the key challenges of the German Manufacturing Industry. A new, knowledge based approach to DFM, eliminating steps of DFM, was introduced in Paul and Al-Dirini (2009). The concept focuses on a concurrent engineering process between the manufacturing engineering and product development systems, while current product realization cycles depend on a rigorous back-and-forth examine-and-correct approach so as to ensure compatibility of any proposed design to the DFM rules and guidelines adopted by the company. The key to achieving reductions is to incorporate DFM considerations into the early stages of the design process. A case study for DFM application in an automotive powertrain engineering environment is presented. It is argued that a DFM database needs to be interfaced to the CAD/CAM software, which will restrict designers to the DFM criteria. Consequently, a notable reduction of development cycles can be achieved. The case study is following the hypothesis that current DFM methods do not improve product design in a manner claimed by the DFM method. The critical case was to identify DFA/DFM recommendations or program actions with repeated appearance in different sources. Repetitive DFM measures are identified, analyzed and it is shown how a modified DFM process can mitigate a non-fully integrated DFM approach.

Flexible airport terminal design : towards a framework

Flexibility is a key driver of any successful design, specifically in highly unpredictable environment such as airport terminal. Ever growing aviation industry requires airport terminals to be planned and constructed in such a way that will allow flexibility for future design, alteration and redevelopment. The concept of flexibility in terminal design is a relatively new initiative, where existing rules or guidelines are not adequate to assist designers. A shift towards flexible design concept would allow terminal buildings to be designed to accommodate future changes and to make passengers’ journey as simple, timely and hassle free as possible. Currently available research indicates that a theoretical framework on flexible design approach for airport terminals would facilitate the future design process. The generic principles of flexibility are investigated in the current research to incorporate flexible design approaches within the process of an airport terminal design. A conceptual framework is proposed herein, which is expected to ascertain flexibility to current passenger terminal facilities within their corresponding locations as well as in future design and expansion.