Thapsigargin modulates osteoclastogenesis through the regulation of RANKL-induced signaling pathways and reactive oxygen species production

Introduction: Apoptosis and differentiation are among the consequences of changes in intracellular Ca2+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation. Materials and Methods: Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-kappa B and activator protein-1 (AP-1) activity, Western blotting for phosphoextracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure. Results: At concentrations that caused a rapid rise in intracellular Ca2+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca2+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-kappa B activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation. Conclusion: Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca2+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca2+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.

Deceleration affects anticipatory and reactive components of triggered postural responses

Understanding the physiological and psychological factors that contribute to healthy and pathological balance control in man has been made difficult by the confounding effects of the perturbations used to test balance reactions. The present study examined how postural responses were influenced by the acceleration-deceleration interval of an unexpected horizontal translation. Twelve adult males maintained balance during unexpected forward and backward surface translations with two different acceleration-deceleration intervals and presentation orders (serial or random). SHORT perturbations consisted of an initial acceleration (peak acceleration 1.3 m s(-2); duration 300 ms) followed 100 ms later by a deceleration. LONG perturbations had the same acceleration as SHORT perturbations, followed by a 2-s interval of constant velocity before deceleration. Surface and intra-muscular electromyography (EMG) from the leg, trunk, and shoulder muscles were recorded along with motion and force plate data. LONG perturbations induced larger trunk displacements compared to SHORT perturbations when presented randomly and larger EMG responses in proximal and distal muscles during later (500-800 ms) response intervals. During SHORT perturbations, activity in some antagonist muscles was found to be associated with deceleration and not the initial acceleration of the support surface. When predictable, SHORT perturbations facilitated the use of anticipatory mechanisms to attenuate early (100-400 ms) EMG response amplitudes, ankle torque change and trunk displacement. In contrast, LONG perturbations, without an early deceleration effect, did not facilitate anticipatory changes when presented in a predictable order. Therefore, perturbations with a short acceleration-deceleration interval can influence triggered postural responses through reactive effects and, when predictable with repeated exposure, through anticipatory mechanisms.

EphA4 regulates central nervous system vascular formation

Molecules involved in axon guidance have recently also been shown to play a role in blood vessel guidance. To examine whether axon guidance molecules, such as the EphA4 receptor tyrosine kinase, might also play a role in development of the central nervous system (CNS) vasculature and repair following CNS injury, we examined wild-type and EphA4 null mutant (-/-) mice. EphA4-/- mice exhibited an abnormal CNS vascular structure in both the cerebral cortex and the spinal cord, with disorganized branching and a 30% smaller diameter. During development, EphA4 was expressed on endothelial cells. This pattern of expression was not maintained in the adult. After spinal cord injury in wild-type mice, expression of EphA4 was markedly up-regulated on activated astrocytes, many of which were tightly associated with blood vessels. In EphA4-/- spinal cord following injury, astrocytes were not as tightly associated with blood vessels as the wild-type astrocytes. In uninjured EphA4-/- mice, the blood-brain barrier (BBB) appeared normal, but it showed prolonged leakage following spinal cord injury. These results support a role for EphA4 in CNS vascular formation and guidance during development and an additional role in BBB repair.

Clinically active Crohn's disease in the presence of a low C-reactive protein

Objective. Clinical interest in C-reactive protein (CRP) - a component of the innate immune system - has focused mainly on its worth as an indicator of disease activity. There has been a resurgence of interest in CRP in the Crohn's disease ( CD) literature because several trials of new treatments for active CD have been characterized by both a large proportion of patients with low CRP ( < 10 mg/l) at entry to the trials and by a negative therapeutic outcome. It is therefore of interest to study the clinical characteristics of patients who are thought to have at the same time both active CD and a low CRP. Material and methods. Patients were prospectively recruited as part of the Brisbane IBD clinical and research programme. Subjects were included in the low CRP group only if there were complete datasets for CRP on all occasions of active CD, and CRP was < 10 mg/l. Active disease was defined as CD activity index (CDAI) > 200. The low CRP group was compared with patients in the raised CRP group for a range of clinical variables as well as the major NOD2 variants. Results. There were data sets for 223 CD patients, with a mean disease duration of 12 years. Of these, 22 patients fulfilled the criteria for low CRP. The low CRP group ( group 1) showed significant differences for disease site (p < 0.01) and for BMI (p = 0.006) compared to the raised CRP group ( group 2). Specifically, group 1 had a predominance of pure ileal disease (95% versus 53%) and lack of pure colonic disease (0% versus 24%) compared to group 2, and their BMI was significantly lower (20.3 kg/m(2) versus 25.0 kg/m(2)). Groups 1 and 2 did not differ with respect to Vienna behaviour at diagnosis, smoking, appendicectomy, extra-intestinal manifestations of CD, or NOD2 SNP variants. There was a trend for low CRP patients with previous ileal resection to evolve to a stricturing phenotype. Fat wrapping was noted in 11/13 (85%) of low CRP patients undergoing ileal resections. Conclusions. Patients with CD and a persistently low CRP in the face of active disease were characterized by an almost exclusive ileal disease distribution and a low BMI, compared to those with a raised CRP. These patients had a similar frequency and distribution of NOD2/CARD15 variants. Stricturing ( v inflammatory or penetrating) behaviour may explain some low CRP. Despite the abnormally low BMI, fat wrapping was noted in the majority of low CRP patients undergoing ileal resection.

Defenses against oxidative stress in Neisseria gonorrhoeae: A system tailored for a challenging environment

Neisseria gonorrhoeae is a host-adapted pathogen that colonizes primarily the human genitourinary tract. This bacterium encounters reactive oxygen and reactive nitrogen species as a consequence of localized inflammatory responses in the urethra of males and endocervix of females and also of the activity of commensal lactobacilli in the vaginal flora. This review describes recent advances in the understanding of defense systems against oxidative stress in N. gonorrhoeae and shows that while some of its defenses have similarities to the paradigm established with Escherichia coli, there are also some key differences. These differences include the presence of a defense system against superoxide based on manganese ions and a glutathione-dependent system for defense against nitric oxide which is under the control of a novel MerR-like transcriptional regulator. An understanding of the defenses against oxidative stress in N. gonorrhoeae and their regulation may provide new insights into the ways in which this bacterium survives challenges from polymorphonuclear leukocytes and urogenital epithelial cells.

Optimization of integrated chemical-biological degradation of a reactive azo dye using response surface methodology

The integrated chemical-biological degradation combining advanced oxidation by UV/H2O2 followed by aerobic biodegradation was used to degrade C.I. Reactive Azo Red 195A, commonly used in the textile industry in Australia. An experimental design based on the response surface method was applied to evaluate the interactive effects of influencing factors (UV irradiation time, initial hydrogen peroxide dosage and recirculation ratio of the system) on decolourisation efficiency and optimizing the operating conditions of the treatment process. The effects were determined by the measurement of dye concentration and soluble chemical oxygen demand (S-COD). The results showed that the dye and S-COD removal were affected by all factors individually and interactively. Maximal colour degradation performance was predicted, and experimentally validated, with no recirculation, 30 min UV irradiation and 500 mg H2O2/L. The model predictions for colour removal, based on a three-factor/five-level Box-Wilson central composite design and the response surface method analysis, were found to be very close to additional experimental results obtained under near optimal conditions. This demonstrates the benefits of this approach in achieving good predictions while minimising the number of experiments required. (c) 2006 Elsevier B.V. All rights reserved.

Combining influence maps and cellular automata for reactive game agents

Agents make up an important part of game worlds, ranging from the characters and monsters that live in the world to the armies that the player controls. Despite their importance, agents in current games rarely display an awareness of their environment or react appropriately, which severely detracts from the believability of the game. Some games have included agents with a basic awareness of other agents, but they are still unaware of important game events or environmental conditions. This paper presents an agent design we have developed, which combines cellular automata for environmental modeling with influence maps for agent decision-making. The agents were implemented into a 3D game environment we have developed, the EmerGEnT system, and tuned through three experiments. The result is simple, flexible game agents that are able to respond to natural phenomena (e.g. rain or fire), while pursuing a goal.

Continuous action system refinement

Action systems are a framework for reasoning about discrete reactive systems. Back, Petre and Porres have extended these action systems to continuous action systems, which can be. used to model hybrid systems. In this paper we define a refinement relation, and develop practical data refinement rules for continuous action systems. The meaning of continuous action systems is expressed in terms of a mapping from continuous action systems to action systems. First, we present a new mapping from continuous act ion systems to action systems, such that Back's definition of trace refinement is correct with respect to it. Second, we present a stream semantics that is compatible with the trace semantics, but is preferable to it because it is more general. Although action system trace refinement rules are applicable to continuous action systems with a stream semantics, they are not complete. Finally, we introduce a new data refinement rule that is valid with respect to the stream semantics and can be used to prove refinements that are not possible in the trace semantics, and we analyse the completeness of our new rule in conjunction with the existing trace refinement rules.

Bifurcation analysis of a dynamic power system with SVC devices

Bifurcation analysis is a very useful tool for power system stability assessment. In this paper, detailed investigation of power system bifurcation behaviour is presented. One and two parameter bifurcation analysis are conducted on a 3-bus power system. We also examined the impact of FACTS devices on power system stability through Hopf bifurcation analysis by taking static Var compensator (SVC) as an example. A simplified first-order model of the SVC device is included in the 3-bus sample system. Real and reactive powers are used as bifurcation parameter in the analysis to compare the system oscillatory properties with and without SVC. The simulation results indicate that the linearized system model with SVC enlarge the voltage stability boundary by moving Hopf bifurcation point to higher level of loading conditions. The installation of SVC increases the dynamic stability range of the system, however complicates the Hopf bifurcation behavior of the system

Developing relational navigation to effectively understand software

Effective comprehension of complex software systems requires understanding of both the individual documents that represent software and the complex relationships that exist within and between documents. Relationships of all kinds play a vital role in a software engineer's comprehension of, and navigation within and between, software documents. User-determined relationships have the additional role of enabling the engineer to create and maintain relational documentation that cannot be generated by tools or derived from other relationships. We argue that for a software development environment to effectively support the understanding of complex software systems, relational navigation must be supported at both the document-focused (intra-document) and relation-focused (inter-document) levels. The need for a relation-focused approach is highlighted by an evaluation of an existing document-focused relational interface. We conclude with the requirements for a relation-focused approach to relational navigation. These requirements focus on the user's perspective when interacting with a collection of related documents. We define the requirements for a software development environment that effectively supports the understanding of the software documents and relationships that define a complex software system.

Designing a Computer Vision System for Underwater Robotic Interventions

This thesis deals with the challenging problem of designing systems able to perceive objects in underwater environments. In the last few decades research activities in robotics have advanced the state of art regarding intervention capabilities of autonomous systems. State of art in fields such as localization and navigation, real time perception and cognition, safe action and manipulation capabilities, applied to ground environments (both indoor and outdoor) has now reached such a readiness level that it allows high level autonomous operations. On the opposite side, the underwater environment remains a very difficult one for autonomous robots. Water influences the mechanical and electrical design of systems, interferes with sensors by limiting their capabilities, heavily impacts on data transmissions, and generally requires systems with low power consumption in order to enable reasonable mission duration. Interest in underwater applications is driven by needs of exploring and intervening in environments in which human capabilities are very limited. Nowadays, most underwater field operations are carried out by manned or remotely operated vehicles, deployed for explorations and limited intervention missions. Manned vehicles, directly on-board controlled, expose human operators to risks related to the stay in field of the mission, within a hostile environment. Remotely Operated Vehicles (ROV) currently represent the most advanced technology for underwater intervention services available on the market. These vehicles can be remotely operated for long time but they need support from an oceanographic vessel with multiple teams of highly specialized pilots. Vehicles equipped with multiple state-of-art sensors and capable to autonomously plan missions have been deployed in the last ten years and exploited as observers for underwater fauna, seabed, ship wrecks, and so on. On the other hand, underwater operations like object recovery and equipment maintenance are still challenging tasks to be conducted without human supervision since they require object perception and localization with much higher accuracy and robustness, to a degree seldom available in Autonomous Underwater Vehicles (AUV). This thesis reports the study, from design to deployment and evaluation, of a general purpose and configurable platform dedicated to stereo-vision perception in underwater environments. Several aspects related to the peculiar environment characteristics have been taken into account during all stages of system design and evaluation: depth of operation and light conditions, together with water turbidity and external weather, heavily impact on perception capabilities. The vision platform proposed in this work is a modular system comprising off-the-shelf components for both the imaging sensors and the computational unit, linked by a high performance ethernet network bus. The adopted design philosophy aims at achieving high flexibility in terms of feasible perception applications, that should not be as limited as in case of a special-purpose and dedicated hardware. Flexibility is required by the variability of underwater environments, with water conditions ranging from clear to turbid, light backscattering varying with daylight and depth, strong color distortion, and other environmental factors. Furthermore, the proposed modular design ensures an easier maintenance and update of the system over time. Performance of the proposed system, in terms of perception capabilities, has been evaluated in several underwater contexts taking advantage of the opportunity offered by the MARIS national project. Design issues like energy power consumption, heat dissipation and network capabilities have been evaluated in different scenarios. Finally, real-world experiments, conducted in multiple and variable underwater contexts, including open sea waters, have led to the collection of several datasets that have been publicly released to the scientific community. The vision system has been integrated in a state of the art AUV equipped with a robotic arm and gripper, and has been exploited in the robot control loop to successfully perform underwater grasping operations.

Reactive oxygen species induce antigenic changes in DNA

Reactive oxygen species (ROS) are released at sites of inflammation during the respiratory burst which accompanies the phagocytic process. Using an in vitro system to simulate this process we have shown that ROS induce antigenic changes in DNA. More specifically, results of experiments using ROS scavengers have shown that hydroxyl radicals produced in close proximity to DNA-bound metal ions play a predominant role. ROS-mediated attack resulted in increased binding of anti-DNA antibodies to the denatured DNA. These changes were detected using IgG, IgA and IgM isotype binding to antibodies in systemic lupus erythematosus sera. Of these the IgA isotype was most discriminating in its detection of hydroxyl radical-induced damage.

Fast response static var generator for improving the power system performance of an electric arc furnace (EAF)

Cascaded multilevel inverters-based Static Var Generators (SVGs) are FACTS equipment introduced for active and reactive power flow control. They eliminate the need for zigzag transformers and give a fast response. However, with regard to their application for flicker reduction in using Electric Arc Furnace (EAF), the existing multilevel inverter-based SVGs suffer from the following disadvantages. (1) To control the reactive power, an off-line calculation of Modulation Index (MI) is required to adjust the SVG output voltage. This slows down the transient response to the changes of reactive power; and (2) Random active power exchange may cause unbalance to the voltage of the d.c. link (HBI) capacitor when the reactive power control is done by adjusting the power angle d alone. To resolve these problems, a mathematical model of 11-level cascaded SVG, was developed. A new control strategy involving both MI (modulation index) and power angle (d) is proposed. A selected harmonics elimination method (SHEM) is taken for switching pattern calculations. To shorten the response time and simplify the controls system, feed forward neural networks are used for on-line computation of the switching patterns instead of using look-up tables. The proposed controller updates the MI and switching patterns once each line-cycle according to the sampled reactive power Qs. Meanwhile, the remainder reactive power (compensated by the MI) and the reactive power variations during the line-cycle will be continuously compensated by adjusting the power angles, d. The scheme senses both variables MI and d, and takes action through the inverter switching angle, qi. As a result, the proposed SVG is expected to give a faster and more accurate response than present designs allow. In support of the proposal there is a mathematical model for reactive powered distribution and a sensitivity matrix for voltage regulation assessment, MATLAB simulation results are provided to validate the proposed schemes. The performance with non-linear time varying loads is analysed and refers to a general review of flicker, of methods for measuring flickers due to arc furnace and means for mitigation.

The mechanochemical modification of polyolefins using reactive modifier systems

The principal objective of this work was to improve the mechanical properties of glass fibre reinforced polypropylene (PP) composites by the mechanochemical modification of the PP. The modification of the PP was carried out by reactive processing of the PP with a modifier in a Buss Ko-Kneader. Two main types of modifier were evaluated one type based on N-substituted maleimides the others based on 2-allylamino-4,6-dichloro-1,3,5-triazine (ACCT). The modification of the PP was carried out in two stages. Firstly the PP was reactively processed with the modifier and a free radical initiator. The objective of this stage was to bind the modifier to the PP. In the second stage the modified PP was reactively processed with the glass fibre. The objective in this stage was to form a chemical bond between the bound modifier and the silane coupling agent on the surface of the glass. Two silane coupling agents were evaluated these had a aliphatic chloro group and an aliphatic amino group respectively available for reaction with the modifier. The modifiers synthesised for this work had two main functional groups. The first was a double bond for free radical addition to the PP. The second was an organic group chosen for its potential reactivity to the silane coupling agent. A preliminary investigation was carried out using maleic anhydride (MA) as the modifier, this is reactive to the amino silane coupled glass. Studies of a commercially available system were also carried out for comparison purposes. During the work it was found that the amino silane coupled glass fibres produced, without any modification being made to the PP, mechanical properties comparable to the commercial system. Further any modification added to the amino silane system failed to improve the mechanical performance and in some cases acted in the opposite fashion. This failure was evident even when a chemical bond between glass fibre and PP could be shown. In the case of the chloro silane coupled glass fibres the mechanical properties of the composite without modification were poorer than those of the commercial system. It was found that the mechanical properties of these systems could be enhanced by the modifiers, however, no system tested significantly out performed the commercial system. Of the two modifier systems tested those based on the n-substituted maleimides were more successful at enhancing mechanical properties than those based on ACCT. This was attributed to the Poor chemical binding of the ACCT based modifiers to the PP. During the work it was found that several of the modifiers improved the properties of the PP when no glass fibres were present, particularly the % elongation and impact strength. It is possible that these modifiers could be used to improve the impact performance of PP, this may be of particular interest in recycling. These modifiers have only been tested for improving the properties of glass fibre composites. The N-substituted maleimide based modifiers could be used as compatibleisers for alloys of PP and other polymers. These could function by the formation of the bond with PP via the double bond whilst the group attached to the nitrogen atom could react with the alloying polymer.