Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2

Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. However, the three-dimensional structure of this inactivated form has been unknown. We have used a lipid monolayer to obtain two-dimensional crystalline arrays of the unphosphorylated inactive form of smooth muscle heavy meromyosin suitable for structural studies by electron cryomicroscopy of unstained, frozen-hydrated specimens. The three-dimensional structure reveals an asymmetric interaction between the two myosin heads. The ATPase activity of one head is sterically “blocked” because part of its actin-binding interface is positioned onto the converter domain of the second head. ATPase activity of the second head, which can bind actin, appears to be inhibited through stabilization of converter domain movements needed to release phosphate and achieve strong actin binding. When the subfragment 2 domain of heavy meromyosin is oriented as it would be in an actomyosin filament lattice, the position of the heads is very different from that needed to bind actin, suggesting an additional contribution to ATPase inhibition in situ.

Interaction of the histone (H3-H4)2 tetramer of the nucleosome with positively supercoiled DNA minicircles: Potential flipping of the protein from a left- to a right-handed superhelical form.

We have studied the ability of the histone (H3-H4)2 tetramer, the central part of the nucleosome of eukaryotic chromatin, to form particles on DNA minicircles of negative and positive superhelicities, and the effect of relaxing these particles with topoisomerase I. The results show that even modest positive torsional stress from the DNA, and in particular that generated by DNA thermal fluctuations, can trigger a major, reversible change in the conformation of the particle. Neither a large excess of naked DNA, nor a crosslink between the two H3s prevented the transition from one form to the other. This suggested that during the transition, the histones neither dissociated from the DNA nor were even significantly reshuffled. Moreover, the particles reconstituted on negatively and positively supercoiled minicircles look similar under electron microscopy. These data agree best with a transition involving a switch of the wrapped DNA from a left- to a right-handed superhelix. It is further proposed, based on the left-handed overall superhelical conformation of the tetramer within the octamer [Arents, G., Burlingame, R. W., Wang, B. C., Love, W. E. & Moudrianakis, E. N. (1991) Proc. Natl.Acad. Sci. USA 88, 10148-10152] that this change in DNA topology is mediated by a similar change in the topology of the tetramer itself, which may occur through a rotation (or a localized deformation) of the two H3-H4 dimers about their H3-H3 interface. Potential implications of this model for nucleosome dynamics in vivo are discussed.

Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites.

DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme. It is specifically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of its derivatives are presently in clinical trial and exhibit remarkable anticancer activity. The present study is a further investigation of the molecular interactions between the drug and the enzyme-DNA complex. We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT), and compared its activity against calf thymus top1 in a DNA oligonucleotide containing a single top1 cleavage site with the activity of its nonalkylating analog, 7-ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT). In the presence of top1, 7-ClMe-MDO-CPT produced a DNA fragment that migrated more slowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT. Top1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C. This fragment was resistant to piperidine treatment and was also formed with an oligonucleotide containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base + 1). It was however cleaved by formic acid treatment followed by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-ClMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence that camptothecins inhibit top1 by binding at the enzyme-DNA interface.

Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.

Study of interaction between Si(O,C) nanowires and the biological system

Nanomedicine is a new branch of medicine, based on the potentiality and intrinsic properties of nanomaterials. Indeed, the nanomaterials ( i.e. the materials with nano and under micron size) can be suitable to different applications in biomedicine. The nanostructures can be used by taking advantage of their properties (for example superparamagnetic nanoparticles) or functionalized to deliver the drug in a specific target, thanks the ability to cross biological barriers. The size and the shape of 1D-nanostructures (nanotubes and nanowires) have an important role on the cell fate: their morphology plays a key role on the interaction between nanostructure and the biological system. For this reason the 1D nanostructure are interesting for their ability to mime the biological system. An implantable material or device must therefore integrate with the surrounding extracellular matrix (ECM), a complex network of proteins with structural and signaling properties. Innovative techniques allow the generation of complex surface patterns that can resemble the structure of the ECM, such as 1D nanostructures. NWs based on cubic silicon carbide (3C-SiC), either bare (3C-SiC NWs) or surrounded by an amorphous shell (3C-SiC/SiO2 core/shell NWs), and silicon oxycarbide nanowires (SiOxCy NWs) can meet the chemical, mechanical and electrical requirements for tissue engineering and have a strong potential to pave the way for the development of a novel generation of implantable nano-devices. Silicon oxycarbide shows promising physical and chemical properties as elastic modulus, bending strength and hardness, chemical durability superior to conventional silicate glasses in aggressive environments and high temperature stability up to 1300 °C. Moreover, it can easily be engineered through functionalization and decoration with macro-molecules and nanoparticles. Silicon carbide has been extensively studied for applications in harsh conditions, as chemical environment, high electric field and high and low temperature, owing to its high hardness, high thermal conductivity, chemical inertness and high electron mobility. Also, its cubic polytype (3C) is highly biocompatible and hemocompatible, and some prototypes of biomedical applications and biomedical devices have been already realized starting from 3C-SiC thin films. Cubic SiC-based NWs can be used as a biomimetic biomaterial, providing a robust and novel biocompatible biological interface . We cultured in vitro A549 human lung adenocarcinoma epithelial cells and L929 murine fibroblast cells over core/shell SiC/SiO2, SiOxCy and bare 3C-SiC nanowire platforms, and analysed the cytotoxicity, by indirect and direct contact tests, the cell adhesion, and the cell proliferation. These studies showed that all the nanowires are biocompatible according to ISO 10993 standards. We evaluated the blood compatibility through the interaction of the nanowires with platelet rich plasma. The adhesion and activation of platelets on the nanowire bundles, assessed via SEM imaging and soluble P-selectin quantification, indicated that a higher platelet activation is induced by the core/shell structures compared to the bare ones. Further, platelet activation is higher with 3C-SiC/SiO2 NWs and SiOxCyNWs, which therefore appear suitable in view of possible tissue regeneration. On the contrary, bare 3C-SiC NWs show a lower platelet activation and are therefore promising in view of implantable bioelectronics devices, as cardiovascular implantable devices. The NWs properties are suitable to allow the design of a novel subretinal Micro Device (MD). This devices is based on Si NWs and PEDOT:PSS, though the well know principle of the hybrid ordered bulk heterojunction (OBHJ). The aim is to develop a device based on a well-established photovoltaic technology and to adapt this know-how to the prosthetic field. The hybrid OBHJ allows to form a radial p–n junction on a nanowire/organic structure. In addition, the nanowires increase the light absorption by means of light scattering effects: a nanowires based p-n junction increases the light absorption up to the 80%, as previously demonstrated, overcoming the Shockley-Queisser limit of 30 % of a bulk p-n junction. Another interesting employment of these NWs is to design of a SiC based epicardial-interacting patch based on teflon that include SiC nanowires. . Such contact patch can bridge the electric conduction across the cardiac infarct as nanowires can ‘sense’ the direction of the wavefront propagation on the survival cardiac tissue and transmit it to the downstream surivived regions without discontinuity. The SiC NWs are tested in terms of toxicology, biocompatibility and conductance among cardiomyocytes and myofibroblasts.

RGBD Human-Hand recognition for the Interaction with Robot-Hand

New low cost sensors and the new open free libraries for 3D image processing are permitting to achieve important advances for robot vision applications such as tridimensional object recognition, semantic mapping, navigation and localization of robots, human detection and/or gesture recognition for human-machine interaction. In this paper, a method to recognize the human hand and to track the fingers is proposed. This new method is based on point clouds from range images, RGBD. It does not require visual marks, camera calibration, environment knowledge and complex expensive acquisition systems. Furthermore, this method has been implemented to create a human interface in order to move a robot hand. The human hand is recognized and the movement of the fingers is analyzed. Afterwards, it is imitated from a Barret hand, using communication events programmed from ROS.

Quantifying epistemic actions in human-computer interaction

As digital systems move away from traditional desktop setups, new interaction paradigms are emerging that better integrate with users’ realworld surroundings, and better support users’ individual needs. While promising, these modern interaction paradigms also present new challenges, such as a lack of paradigm-specific tools to systematically evaluate and fully understand their use. This dissertation tackles this issue by framing empirical studies of three novel digital systems in embodied cognition – an exciting new perspective in cognitive science where the body and its interactions with the physical world take a central role in human cognition. This is achieved by first, focusing the design of all these systems on a contemporary interaction paradigm that emphasizes physical interaction on tangible interaction, a contemporary interaction paradigm; and second, by comprehensively studying user performance in these systems through a set of novel performance metrics grounded on epistemic actions, a relatively well established and studied construct in the literature on embodied cognition. The first system presented in this dissertation is an augmented Four-in-a-row board game. Three different versions of the game were developed, based on three different interaction paradigms (tangible, touch and mouse), and a repeated measures study involving 36 participants measured the occurrence of three simple epistemic actions across these three interfaces. The results highlight the relevance of epistemic actions in such a task and suggest that the different interaction paradigms afford instantiation of these actions in different ways. Additionally, the tangible version of the system supports the most rapid execution of these actions, providing novel quantitative insights into the real benefits of tangible systems. The second system presented in this dissertation is a tangible tabletop scheduling application. Two studies with single and paired users provide several insights into the impact of epistemic actions on the user experience when these are performed outside of a system’s sensing boundaries. These insights are clustered by the form, size and location of ideal interface areas for such offline epistemic actions to occur, as well as how can physical tokens be designed to better support them. Finally, and based on the results obtained to this point, the last study presented in this dissertation directly addresses the lack of empirical tools to formally evaluate tangible interaction. It presents a video-coding framework grounded on a systematic literature review of 78 papers, and evaluates its value as metric through a 60 participant study performed across three different research laboratories. The results highlight the usefulness and power of epistemic actions as a performance metric for tangible systems. In sum, through the use of such novel metrics in each of the three studies presented, this dissertation provides a better understanding of the real impact and benefits of designing and developing systems that feature tangible interaction.

A multimodal tablet-based interface for designing and reviewing 3D engineering models

The ability to view and interact with 3D models has been happening for a long time. However, vision-based 3D modeling has only seen limited success in applications, as it faces many technical challenges. Hand-held mobile devices have changed the way we interact with virtual reality environments. Their high mobility and technical features, such as inertial sensors, cameras and fast processors, are especially attractive for advancing the state of the art in virtual reality systems. Also, their ubiquity and fast Internet connection open a path to distributed and collaborative development. However, such path has not been fully explored in many domains. VR systems for real world engineering contexts are still difficult to use, especially when geographically dispersed engineering teams need to collaboratively visualize and review 3D CAD models. Another challenge is the ability to rendering these environments at the required interactive rates and with high fidelity. In this document it is presented a virtual reality system mobile for visualization, navigation and reviewing large scale 3D CAD models, held under the CEDAR (Collaborative Engineering Design and Review) project. It’s focused on interaction using different navigation modes. The system uses the mobile device's inertial sensors and camera to allow users to navigate through large scale models. IT professionals, architects, civil engineers and oil industry experts were involved in a qualitative assessment of the CEDAR system, in the form of direct user interaction with the prototypes and audio-recorded interviews about the prototypes. The lessons learned are valuable and are presented on this document. Subsequently it was prepared a quantitative study on the different navigation modes to analyze the best mode to use it in a given situation.

Minimal instrumentation may compromise failure diagnosis with an ecological interface

Interfaces designed according to ecological interface design (EID) display higher-order relations and properties of a work domain so that adaptive operator problem solving can be better supported under unanticipated system conditions. Previous empirical studies of EID have assumed that the raw data required to derive and communicate higher-order information would be available and reliable. The present research examines the relative advantages of an EID interface over a conventional piping-and-instrumentation diagram (PID) when instrumentation is maximally or only minimally adequate. Results show an interaction between interface and the adequacy of the instrumentation. Failure diagnosis performance with the EID interface with maximally adequate instrumentation is best overall. Performance with the EID interface drops more drastically from maximally to minimally adequate instrumentation than does performance with the PID interface, to the point where the EID interface with minimally adequate instrumentation supports nonsignificantly worse performance than does the equivalent PID interface. Actual or potential applications of this research include design of instrumentation and displays for complex industrial processes.

Reflective web interface agent

Web interface agent is used with web browsers to assist users in searching and interactions with the WWW. It is used for a variety of purposes, such as web-enabled remote control, web interactive visualization, and e-commerce activities. User may be aware or unaware of its existence. The intelligence of interface agent consists in its capability of learning and decision-making in performing interactive functions on behalf of a user. However, since web is an open system environment, the reasoning mechanism in an agent should be able to adapt changes and make decisions on exceptional situations, and therefore use meta knowledge. This paper proposes a framework of Reflective Web Interface Agent (RWIA) that is to provide causal connections between the application interfaces and the knowledge model of the interface agent. A prototype is also implemented for the purpose of demonstration.

αα'-trehalose 6,6'-dibehenate in non-phospholipid-based liposomes enables direct interaction with trehalose, offering stability during freeze-drying

Trehalose is a well known protector of biostructures like liposomes and proteins during freeze-drying, but still today there is a big debate regarding its mechanism of action. In previous experiments we have shown that trehalose is able to protect a non-phospholipid-based liposomal adjuvant (designated CAF01) composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6-dibehenate (TDB) during freeze-drying [D. Christensen, C. Foged, I. Rosenkrands, H.M. Nielsen, P. Andersen, E.M. Agger, Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying, Biochim. Biophys. Acta, Biomembr. 1768 (2007) 2120-2129]. Furthermore it was seen that TDB is required for the stabilizing effect of trehalose. Herein, we show using the Langmuir-Blodgett technique that a high concentration of TDB present at the water-lipid interface results in a surface pressure around 67 mN/m as compared to that of pure DDA which is approximately 47 mN/m in the compressed state. This indicates that the attractive forces between the trehalose head group of TDB and water are greater than those between the quaternary ammonium head group of DDA and water. Furthermore, addition of trehalose to a DDA monolayer containing small amounts of TDB also increases the surface pressure, which is not observed in the absence of TDB. This suggests that even small amounts of trehalose groups on TDB present at the water-lipid interface associate free trehalose to the liposome surface, presumably by hydrogen bonding between the trehalose head groups of TDB and the free trehalose molecules. Hence, for CAF01 the TDB component not only stabilizes the cationic liposomes and enhances the immune response but also facilitates the cryo-/lyoprotection by trehalose through direct interaction with the head group of TDB. Furthermore the results indicate that direct interaction with liposome surfaces is necessary for trehalose to enable protection during freeze-drying.

Accessing networked services : a user interface design problem

This research investigates the general user interface problems in using networked services. Some of the problems are: users have to recall machine names and procedures to. invoke networked services; interactions with some of the services are by means of menu-based interfaces which are quite cumbersome to use; inconsistencies exist between the interfaces for different services because they were developed independently. These problems have to be removed so that users can use the services effectively. A prototype system has been developed to help users interact with networked services. This consists of software which gives the user an easy and consistent interface with the various services. The prototype is based on a graphical user interface and it includes the following appJications: Bath Information & Data Services; electronic mail; file editor. The prototype incorporates an online help facility to assist users using the system. The prototype can be divided into two parts: the user interface part that manages interactlon with the user; the communicatIon part that enables the communication with networked services to take place. The implementation is carried out using an object-oriented approach where both the user interface part and communication part are objects. The essential characteristics of object-orientation, - abstraction, encapsulation, inheritance and polymorphism - can all contribute to the better design and implementation of the prototype. The Smalltalk Model-View-Controller (MVC) methodology has been the framework for the construction of the prototype user interface. The purpose of the development was to study the effectiveness of users interaction to networked services. Having completed the prototype, tests users were requested to use the system to evaluate its effectiveness. The evaluation of the prototype is based on observation, i.e. observing the way users use the system and the opinion rating given by the users. Recommendations to improve further the prototype are given based on the results of the evaluation. based on the results of the evah:1ation. . .'. " "', ':::' ,n,<~;'.'

Individual differences in human-computer interaction

This thesis initially presents an 'assay' of the literature pertaining to individual differences in human-computer interaction. A series of experiments is then reported, designed to investigate the association between a variety of individual characteristics and various computer task and interface factors. Predictor variables included age, computer expertise, and psychometric tests of spatial visualisation, spatial memory, logical reasoning, associative memory, and verbal ability. These were studied in relation to a variety of computer-based tacks, including: (1) word processing and its component elements; (ii) the location of target words within passages of text; (iii) the navigation of networks and menus; (iv) command generation using menus and command line interfaces; (v) the search and selection of icons and text labels; (vi) information retrieval. A measure of self-report workload was also included in several of these experiments. The main experimental findings included: (i) an interaction between spatial ability and the manipulation of semantic but not spatial interface content; (ii) verbal ability being only predictive of certain task components of word processing; (iii) age differences in word processing and information retrieval speed but not accuracy; (iv) evidence of compensatory strategies being employed by older subjects; (v) evidence of performance strategy differences which disadvantaged high spatial subjects in conditions of low spatial information content; (vi) interactive effects of associative memory, expertise and command strategy; (vii) an association between logical reasoning and word processing but not information retrieval; (viii) an interaction between expertise and cognitive demand; and (ix) a stronger association between cognitive ability and novice performance than expert performance.