Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

Merkel Cell Polyomavirus (MCPyV) genomes are clonally integrated in tumor tissues of approximately 85% of all Merkel cell carcinoma (MCC) cases, a highly aggressive tumor of the skin which predominantly afflicts elderly and immunosuppressed patients. All integrated viral genomes recovered from MCC tissue or MCC cell lines harbor signature mutations in the early gene transcript encoding for the large T-Antigen (LT-Ag). These mutations selectively abrogate the ability of LT-Ag to support viral replication while still maintaining its Rb-binding activity, suggesting a continuous requirement for LT-Ag mediated cell cycle deregulation during MCC pathogenesis. To gain a better understanding of MCPyV biology, in vitro MCPyV replication systems are required. We have generated a synthetic MCPyV genomic clone (MCVSyn) based on the consensus sequence of MCC-derived sequences deposited in the NCBI database. Here, we demonstrate that transfection of recircularized MCVSyn DNA into some human cell lines recapitulates efficient replication of the viral genome, early and late gene expression together with virus particle formation. However, serial transmission of infectious virus was not observed. This in vitro culturing system allows the study of viral replication and will facilitate the molecular dissection of important aspects of the MCPyV lifecycle

SADE : a structural active-object system drawing editor

Structural active-object systems (SAOSs) are transition-based object-oriented systems suitable as frameworks of various concurrent systems. AOSs are constructed from their component active objects by structural and hierarchical object composition (SHOC) like hardware systems. 1n this report, we demonstrate that the SAOS approach is effective in implementing a graphical editor. The SAOS Drawing Editor (SADE) is a SAOS program to aid creation of SAOS components, allowing its user to create, manipulate, and save graphical objects, which may be compound objects as well as such primitive objects as rectangles, lines, and ellipses. The implementation of SADE was simplified as structural active objects (SAOs) were used as its components including the canvas and the palette. Furthermore, since the graphical objects placed on the canvas are also SAOs, they can handle edit-operations for themselves.Keywords and Phrases: graphical editor, object-oriented programming, active-object system, structural composition, hierarchical composition, graphical user interface, com­pound object1993 best estimate for issue date based on available information

SARNS : the SAOS road network simulator

The SAOS Road Network Simulator (SARNS) is a graphical simulation program for transportation planning, implemented using the structural active-object system (SAOS) approach. A SAOS is an object-oriented concurrent system that consists of a collection of interacting structural active objects (SAOs), whose behaviors are determined by transition statements provided in their class definitions. Furthermore, SAOs can be structurally and hierarchically composed from their component SAOs, allowing various applications to be rapidly developed as SAOS programs. The active components used in transportation planning, such as vehicles and traffic signals, can be naturally modeled as SAOs. The typical composition of these components into a complete road network allows for the rapid prototyping of various road network configurations and vehicle generation scenarios. An interactive graphical user interface displaying the dynamic state of the simulation is an inherent part of a SARNS program. Such a graphical interface can be directly created from a design specification, either by hand or in the future by using a SAOS graphical editor.Key Words and Phrases: Transportation planning, computerized traffic simulation, object-oriented simulation, active-object systems, structural composition, hierarchical composition, rapid prototyping, graphical user interface**1993 best estimate for issue date and commencement year based on available information.*

The structural active-object system approach to AGV system editor implementation

The Structural Active-Object System (SAOS) approach allows rapid development of various concurrent systems by structural and hierarchical object composition (SHOC). We implemented, as a SAOS program, a graphical editing and simulation program for automatic guided vehicle (AGV) systems, which carry around parts in automated factories. It is difficult to predict the performance of an AGV system by analytical calculations, and hence a software tool that allows rapid construction and modification of a simulated AGV system is very useful. We can create an AGV system simply by placing track segments, stations, and AGVs on the graphical editor canvas. Simulation is automatically started when the first AGV is placed on the AGV track. Furthermore , the track layout can be dynamically modified during simulation, and the effects of the modifications can be immediately observed by the user.Key Words and Phrases: object-oriented programming, active object system, manufacturing control, AGV, FMS, graphical editor, structural composition, hierarchical composition

Structural active object systems for mixed-mode simulation

A structural active-object system (SAOS) is a transition-based object-oriented system suitable for the design of various concurrent systems. A SAOS consists of a collection of interacting structural active objects (SAOs) whose behaviors are determined by the transi­tion statements provided in their class definitions. Furthermore, SAOs can be structurally and hierarchically composed from their component SAOs like hardware components. These features allow SAOs to model components for circuit simulation more naturally than pas­sive objects used in ordinary object-oriented programming, including digital, analog, and mixed-mode simulation. Each hardware object such as an AND gate or an integrator can be represented as a SAO. In addition, structural and hierarchical composition allows us to build complex components from elementary components. Prototype simulation programs with graphical user interfaces have been developed as SAOS programs for digital, analog, and mixed-mode circuit simulation.Key Words and Phrases: digital simulation, analog simulation, mixed-mode simulation, active-object system, concurrent object-oriented programming, structural composition, hi­erarchical composition, software IC, graphical user interface1993 best estimate for issue date based on available information

Conceptualising supply-side seasonality in tourism, a study of the temporal trading behaviours for small tourism businesses in Scotland

Seasonality in tourism is one of its most enduring features. During the past half century, the phenomenon has been studied extensively in order to gain insight into its dynamics. However, much of the empirical evidence has been developed from a demand-side perspective, focusing on the temporal travel behaviours and motivations of consumers. Conversely, relatively little attention has been paid to tourism's diverse supply-side elements, especially those at the destination. This study aims to redress that imbalance. It considers a key element of the destination mix in Scotland, the privately operated small tourism related business, specifically those who operate their business on a seasonal basis. Such businesses epitomise supply-side seasonality, yet their temporal operating behaviours and the underlying motivations and influences of these have evaded systematic examination. The thesis is therefore an attempt to aid understanding of the relationships between tourism seasonality and small business service provision. It represents an inductive, interpretivistic approach to the subject. In reviewing the tourism seasonality and small business literatures, it is argued that existing constructs of seasonality, entrepreneurialism and growth orientation, family business and 'lifestyle' business fail to shed light on the complexities of temporal trading among small businesses and indeed on the meanings of 'seasonality' from a supply-side perspective. Moreover, findings from an exploratory study and contextual literature reveal a variety of contextual factors that impinge on temporal trading behaviours. A nationwide survey of seasonally trading Scottish small businesses identifies distinct patterns of behaviour, influences and motivations according to type of business, demographic and contextual variables. Disposition and circumstance are identified as key formative elements in conceptualising supply-side temporal behaviours.Seasonality in tourism is one of its most enduring features. During the past half century, the phenomenon has been studied extensively in order to gain insight into its dynamics. However, much of the empirical evidence has been developed from a demand-side perspective, focusing on the temporal travel behaviours and motivations of consumers. Conversely, relatively little attention has been paid to tourism's diverse supply-side elements, especially those at the destination. This study aims to redress that imbalance. It considers a key element of the destination mix in Scotland, the privately operated small tourism related business, specifically those who operate their business on a seasonal basis. Such businesses epitomise supply-side seasonality, yet their temporal operating behaviours and the underlying motivations and influences of these have evaded systematic examination. The thesis is therefore an attempt to aid understanding of the relationships between tourism seasonality and small business service provision. It represents an inductive, interpretivistic approach to the subject. In reviewing the tourism seasonality and small business literatures, it is argued that existing constructs of seasonality, entrepreneurialism and growth orientation, family business and 'lifestyle' business fail to shed light on the complexities of temporal trading among small businesses and indeed on the meanings of 'seasonality' from a supply-side perspective. Moreover, findings from an exploratory study and contextual literature reveal a variety of contextual factors that impinge on temporal trading behaviours. A nationwide survey of seasonally trading Scottish small businesses identifies distinct patterns of behaviour, influences and motivations according to type of business, demographic and contextual variables. Disposition and circumstance are identified as key formative elements in conceptualising supply-side temporal behaviours

Structural active object system translator

A structural active-object systems (SAOS) is a, transition-based object-oriented system consisting of a collection of interacting structural active objects (SAOs), which can be structurally and hierarchically composed from their component SAOs. The behaviors of SAOs are determined by the transition statements provided in their class definitions. Var­ious concurrent systems such as simulation systems, graphical user interfaces and CAD systems can be implemented as SAOSs. SAOSs can be programmed in C++, but writing SAOS programs in C++ is tedious and time consuming. The SAOS description language (SAOSDL) is an extension of C++ with special constructs for transition statements and attribute initializations. SAOSDL simpli­fies SAOS programming by reducing program text sizes and enhancing understandability of programs. The SAOS translator (SAOST) translates SAOSDL programs into C++ pro­grams. In this report, the issues involved in translating SAOSDL special constructs into C++ and the implementation of SAOST ate discussed.1992 best estimate for issue date and commencement year based on available information

Nonlinear rheological characteristics of single species bacterial biofilms

Bacterial biofilms in natural and artificial environments perform a wide array of beneficial or detrimental functions and exhibit resistance to physical as well as chemical perturbations. In dynamic environments, where periodic or aperiodic flows over surfaces are involved, biofilms can be subjected to large shear forces. The ability to withstand these forces, which is often attributed to the resilience of the extracellular matrix. This attribute of the extracellular matrix is referred to as viscoelasticity and is a result of self-assembly and cross-linking of multiple polymeric components that are secreted by the microbes. We aim to understand the viscoelastic characteristic of biofilms subjected to large shear forces by performing Large Amplitude Oscillatory Shear (LAOS) experiments on four species of bacterial biofilms: Bacillus subtilis, Comamonas denitrificans, Pseudomonas fluorescens and Pseudomonas aeruginosa. We find that nonlinear viscoelastic measures such as intracycle strain stiffening and intracycle shear thickening for each of the tested species, exhibit subtle or distinct differences in the plot of strain amplitude versus frequency (Pipkin diagram). The biofilms also exhibit variability in the onset of nonlinear behaviour and energy dissipation characteristics, which could be a result of heterogeneity of the extracellular matrix constituents of the different biofilms. The results provide insight into the nonlinear rheological behaviour of biofilms as they are subjected to large strains or strain rates; a situation that is commonly encountered in nature, but rarely investigated

Studies of adeno-associated virus-induced DNA damage responses.

Adeno-associated virus (AAV) infection triggers a DNA damage response in the cell. This response is not induced by viral proteins but by the structure of AAV ssDNA being recognized by the cell as damaged DNA. The consequence of this is the killing of cells lacking p53 activity. We have observed that cells that lack p21 or pRb activity are also sensitive to AAV-induced cell death. We report that cells respond to AAV infection by activating two DNA damage-signaling cascades. The first activates the p84N5 protein, which in turn activates caspase-6, leading to cell death. The second cascade activates the p53-21-pRb pathway, which inhibits activation of the p84N5 protein and thus prevents cell death. The result of the antagonistic interaction between these two pathways is that cells that do not exhibit functional p53-p21-pRb signaling undergo apoptosis as a consequence of AAV infection. Cells with a functional p53- 21-pRb pathway are refractory to AAV-induced cell death. These results show that p53, although a pro-apoptotic protein, together with pRb and p21 proteins, is a member of an anti-apoptotic cellular mechanism. As such, these experiments reveal features that may be exploited to specifically kill cells that lack the p53-p21-pRb pathway, such as cancer cells. We also consider the role of DNA damage signaling in the restriction of AAV replication. We report that helper viruses may inhibit ATM, ATR and DNA-PK signaling and that this may be advantageous for AAV replication. Inhibition of ATM and DNA-PK in tissue culture cell lines led to an increase in AAV replication. We show that ATM, ATR and DNA-PK may be responsible for the hyperphosphorylation of replication protein A (RPA) in response to AAV infection. We speculate that hyperphosphorylated RPA may be a fundamental restriction factor for viral replication and delineate novel experimental tools to test such a hypothesis. The implications of these observations for oncolytic therapy are discussed

Processing and properties of thermoplastic composites containing cellulose nanocrystals or wood-based cellulose fibres

Cellulose nanocrystals (CNC) were surface modified with dialkylamines to increase the compatibility between the CNC and the polymeric matrix, and promising results were obtained, with a 300 % stiffness increase when the mixed dispersion was compression moulded on a laboratory scale. The manufacturing process was up-scaled using water-assisted mixing in a twin-screw extruder (TSE) followed by a second compounding step and injection moulding (IM). The composites were successfully produced using conventional melt-processing techniques but these did not show the same improvement in mechanical performance, probably due to the formation of CNC aggregates. There were indications of network formation when CNC was added, especially in the case of surface-modified CNC. Cellulose fibres and thermomechanical pulp were used as reinforcement in similar types of polymer matrices and the mixtures were similarly processed by TSE and IM. These materials were characterized with regard to appearance and durability. The discoloration of the composites due to excessive heat during processing did not significantly affect their mechanical properties, and the addition of the cellulose-based reinforcement to the polymer did not reduce its resistance to thermo-oxidative degradation compared to that of the pure matrix. In fact, the resistance to degradation was increased when lignin was present in the reinforcing element, showing a synergistic effect together with the added anti-oxidant.Superior properties were expected for the CNC composites compared to those of the larger cellulose fibre reinforcements, but in continuous production the stiffening effects were similar regardless of reinforcement type. These results confirm that the processing method and properties strongly affect the final properties of the composite