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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Review Pages: Planning for Livable and Safe Cities: Energy, Pollution and the Degradation of the Urban Environment

Starting from the relationship between urban planning and mobility management, TeMA has gradually expanded the view of the covered topics, always remaining in the groove of rigorous scientific in-depth analysis. During the last two years a particular attention has been paid on the Smart Cities theme and on the different meanings that come with it. The last section of the journal is formed by the Review Pages. They have different aims: to inform on the problems, trends and evolutionary processes; to investigate on the paths by highlighting the advanced relationships among apparently distant disciplinary fields; to explore the interaction’s areas, experiences and potential applications; to underline interactions, disciplinary developments but also, if present, defeats and setbacks. Inside the journal the Review Pages have the task of stimulating as much as possible the circulation of ideas and the discovery of new points of view. For this reason the section is founded on a series of basic’s references, required for the identification of new and more advanced interactions. These references are the research, the planning acts, the actions and the applications, analysed and investigated both for their ability to give a systematic response to questions concerning the urban and territorial planning, and for their attention to aspects such as the environmental sustainability and the innovation in the practices. For this purpose the Review Pages are formed by five sections (Web Resources; Books; Laws; Urban Practices; News and Events), each of which examines a specific aspect of the broader information storage of interest for TeMA.

Influence of information about trip time variability, personal benefits, and environmental harm from cars versus public transportation on the choice of transportation mode

The influence of information about trip time variability, personal benefits, or environmental harm from cars or public transportation on commuting mode choice (car or subway) is examined in an experimental study. In addition to these experimentally manipulated variables, the influence of prior attitudes towards the subway was verified. The sample is made up of habitual users of the car to travel to work (N = 220, age M = 37.4, SD = 8.1, 63.2% women). The results show that providing information about the advantages of public transportation, as well as prior attitudes towards the subway, decrease the preference, choice, and perceived control of car use. Of the experimentally manipulated variables, information about the variability of trip time had the greatest influence. These results highlight the importance of taking into account these variables to implement institutional campaigns to reduce car use as transportation mode.

Interoperabilidade, processamento e análise visual de dados geográficos

Esta dissertação apresenta uma arquitectura interoperável que permite lidar com a obtenção, manipulação, processamento e análise de informação geográfica. A aplicação 30, implementada como parte da arquitectura, para além de permitir a visualização e manipulação de dados dentro de um ambiente 30, oferece métodos que permitem descobrir, aceder e usar geo-processos, disponíveis através de serviços Web. A interacção com o utilizador é também feita através uma abordagem que quebra a típica complexidade que a maioria dos Sistemas de Informação Geográfica apresenta. O recurso à programação visual reduz a complexidade do sistema, e permite aos operadores tirar proveito da localização e de uma abstracção de um processo complexo, onde as unidades de processamento são representadas no terreno através de componentes 30 que podem ser directamente manipuladas e ligadas de modo a criar encandeamentos complexos de processos. Estes processos podem também ser criados visualmente e disponibilizados online. ABSTRACT; This thesis presents an interoperable architecture mainly designed for manipulation, processing and geographical information analysis. The three-dimensional interface, implemented as part of the architecture, besides allowing the visualization and manipulation of spatial data within a 30 environment, offers methods for discovering, accessing and using geo-processes, available through Web Services. Furthermore, the user interaction is done through an approach that breaks the typical complexity of most Geographic information Systems. This simplicity is in general archived through a visual programming approach that allows operators to take advantage of location, and use processes through abstract representations. Thus, processing units are represented on the terrain through 30 components, which can be directly manipulated and linked to create complex process chains. New processes can also be visually created and deployed online.

Sources of Information about Dietary Fibre: A Cross-Country Survey

Problem statement: Dietary fibre (DF) has been recognized as a major determinant for improvement of health. Hence the means of information through which people become aware of its benefits are crucial. Research questions: Where do you usually find information about dietary fibre? What means of communication do you consider the most appropriate to encourage the consumption of dietary fibre? Purpose of the study: This work aimed at studying the sources of information about dietary fibre, as a means to educate people about aspects related to healthy eating. Factors such as gender, level of education, living environment or country were evaluated as to their effect on the selection of sources and preferences.   Research Methods: Descriptive cross-sectional study by means of a questionnaire, applied to a non-probabilistic sample of 6010 participants from 10 countries in different continents (Europe, Africa and America), answered after informed consent by all participants. For the analysis were used several descriptive statistics tools, crosstabs and chi square test to assess the relations between some of the variables under study. Findings: Mostly the information about DF comes from the internet, but television is recognized as suitable. Differences were found between genders, levels of education, living environments and countries. The internet (preferred source), got highest scores for Hungary, urban areas, university education and female gender. The radio (least scored source) was preferred in Egypt, for men and with lower education (primary school). Conclusions: People get information through the internet due to easy access. However, it is to some extent a risk given the impossibility to control de information made public on the internet. The role of health centres and hospitals as well as schools should definitely be increased, as a responsible way to ensure correct information.

Collecting valuable consumer data: Motivating consumers to disclose personal information

Collecting and analyzing consumer data is essential in today’s data-driven business environment. However, consumers are becoming more aware of the value of the information they can provide to companies, thereby being more reluctant to share it for free. Therefore, companies need to find ways to motivate consumers to disclose personal information. The main research question of the study was formed as “How can companies motivate consumers to disclose personal information?” and it was further divided into two subquestions: 1) What types of benefits motivate consumers to disclose personal information? 2) How does the disclosure context affect the consumers’ information disclosure behavior? The conceptual framework consisted of a classification of extrinsic and intrinsic benefits, and moderating factors, which were recognized on the basis of prior research in the field. The study was conducted by using qualitative research methods. The primary data was collected by interviewing ten representatives from eight companies. The data was analyzed and reported according to predetermined themes. The findings of the study confirm that consumers can be motivated to disclose personal information by offering different types of extrinsic (monetary saving, time saving, self-enhancement, and social adjustment) and intrinsic (novelty, pleasure, and altruism) benefits. However, not all the benefits are equally useful ways to convince the customer to disclose information. Moreover, different factors in the disclosure context can either alleviate or increase the effectiveness of the benefits and the consumers’ motivation to disclose personal information. Such factors include the consumer’s privacy concerns, perceived trust towards the company, the relevancy of the requested information, personalization, website elements (especially security, usability, and aesthetics of a website), and the consumer’s shopping motivation. This study has several contributions. It is essential that companies recognize the most attractive benefits regarding their business and their customers, and that they understand how the disclosure context affects the consumer’s information disclosure behavior. The likelihood of information disclosure can be increased, for example, by offering benefits that meet the consumers’ needs and preferences, improving the relevancy of the asked information, stating the reasons for data collection, creating and maintaining a trustworthy image of the company, and enhancing the quality of the company’s website.

Brief report: preliminary proposal of a conceptual model of a digital environment for developing mathematical reasoning in students with Autism Spectrum Disorders

There is clear evidence that in typically developing children reasoning and sense-making are essential in all mathematical learning and understanding processes. In children with autism spectrum disorders (ASD), however, these become much more significant, considering their importance to successful independent living. This paper presents a preliminary proposal of a digital environment, specifically targeted to promote the development of mathematical reasoning in students with ASD. Given the diversity of ASD, the prototyping of this environment requires the study of dynamic adaptation processes and the development of activities adjusted to each user’s profile. We present the results obtained during the first phase of this ongoing research, describing a conceptual model of the proposed digital environment. Guidelines for future research are also discussed.