Gonadotropin-induced ovarian cancer cell migration and proliferation require extracellular signal-regulated kinase 1/2 activation regulated by calcium and protein kinase Cδ

The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCδ inhibitor rottlerin, and downregulation of PKCδ was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCδ in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCδ siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCδ-dependent manner.

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

Stochastic chemical kinetics and the quasi-steady-state assumption : application to the stochastic simulation algorithm and chemical master equation

Recently the application of the quasi-steady-state approximation (QSSA) to the stochastic simulation algorithm (SSA) was suggested for the purpose of speeding up stochastic simulations of chemical systems that involve both relatively fast and slow chemical reactions [Rao and Arkin, J. Chem. Phys. 118, 4999 (2003)] and further work has led to the nested and slow-scale SSA. Improved numerical efficiency is obtained by respecting the vastly different time scales characterizing the system and then by advancing only the slow reactions exactly, based on a suitable approximation to the fast reactions. We considerably extend these works by applying the QSSA to numerical methods for the direct solution of the chemical master equation (CME) and, in particular, to the finite state projection algorithm [Munsky and Khammash, J. Chem. Phys. 124, 044104 (2006)], in conjunction with Krylov methods. In addition, we point out some important connections to the literature on the (deterministic) total QSSA (tQSSA) and place the stochastic analogue of the QSSA within the more general framework of aggregation of Markov processes. We demonstrate the new methods on four examples: Michaelis–Menten enzyme kinetics, double phosphorylation, the Goldbeter–Koshland switch, and the mitogen activated protein kinase cascade. Overall, we report dramatic improvements by applying the tQSSA to the CME solver.

Parallel implementation of stochastic simulation for large scale cellular processes

Experimental and theoretical studies have shown the importance of stochastic processes in genetic regulatory networks and cellular processes. Cellular networks and genetic circuits often involve small numbers of key proteins such as transcriptional factors and signaling proteins. In recent years stochastic models have been used successfully for studying noise in biological pathways, and stochastic modelling of biological systems has become a very important research field in computational biology. One of the challenge problems in this field is the reduction of the huge computing time in stochastic simulations. Based on the system of the mitogen-activated protein kinase cascade that is activated by epidermal growth factor, this work give a parallel implementation by using OpenMP and parallelism across the simulation. Special attention is paid to the independence of the generated random numbers in parallel computing, that is a key criterion for the success of stochastic simulations. Numerical results indicate that parallel computers can be used as an efficient tool for simulating the dynamics of large-scale genetic regulatory networks and cellular processes

Flexible high voltage pulsed power supply for plasma applications

Demands for delivering high instantaneous power in a compressed form (pulse shape) have widely increased during recent decades. The flexible shapes with variable pulse specifications offered by pulsed power have made it a practical and effective supply method for an extensive range of applications. In particular, the release of basic subatomic particles (i.e. electron, proton and neutron) in an atom (ionization process) and the synthesizing of molecules to form ions or other molecules are among those reactions that necessitate large amount of instantaneous power. In addition to the decomposition process, there have recently been requests for pulsed power in other areas such as in the combination of molecules (i.e. fusion, material joining), gessoes radiations (i.e. electron beams, laser, and radar), explosions (i.e. concrete recycling), wastewater, exhausted gas, and material surface treatments. These pulses are widely employed in the silent discharge process in all types of materials (including gas, fluid and solid); in some cases, to form the plasma and consequently accelerate the associated process. Due to this fast growing demand for pulsed power in industrial and environmental applications, the exigency of having more efficient and flexible pulse modulators is now receiving greater consideration. Sensitive applications, such as plasma fusion and laser guns also require more precisely produced repetitive pulses with a higher quality. Many research studies are being conducted in different areas that need a flexible pulse modulator to vary pulse features to investigate the influence of these variations on the application. In addition, there is the need to prevent the waste of a considerable amount of energy caused by the arc phenomena that frequently occur after the plasma process. The control over power flow during the supply process is a critical skill that enables the pulse supply to halt the supply process at any stage. Different pulse modulators which utilise different accumulation techniques including Marx Generators (MG), Magnetic Pulse Compressors (MPC), Pulse Forming Networks (PFN) and Multistage Blumlein Lines (MBL) are currently employed to supply a wide range of applications. Gas/Magnetic switching technologies (such as spark gap and hydrogen thyratron) have conventionally been used as switching devices in pulse modulator structures because of their high voltage ratings and considerably low rising times. However, they also suffer from serious drawbacks such as, their low efficiency, reliability and repetition rate, and also their short life span. Being bulky, heavy and expensive are the other disadvantages associated with these devices. Recently developed solid-state switching technology is an appropriate substitution for these switching devices due to the benefits they bring to the pulse supplies. Besides being compact, efficient, reasonable and reliable, and having a long life span, their high frequency switching skill allows repetitive operation of pulsed power supply. The main concerns in using solid-state transistors are the voltage rating and the rising time of available switches that, in some cases, cannot satisfy the application’s requirements. However, there are several power electronics configurations and techniques that make solid-state utilisation feasible for high voltage pulse generation. Therefore, the design and development of novel methods and topologies with higher efficiency and flexibility for pulsed power generators have been considered as the main scope of this research work. This aim is pursued through several innovative proposals that can be classified under the following two principal objectives. • To innovate and develop novel solid-state based topologies for pulsed power generation • To improve available technologies that have the potential to accommodate solid-state technology by revising, reconfiguring and adjusting their structure and control algorithms. The quest to distinguish novel topologies for a proper pulsed power production was begun with a deep and through review of conventional pulse generators and useful power electronics topologies. As a result of this study, it appears that efficiency and flexibility are the most significant demands of plasma applications that have not been met by state-of-the-art methods. Many solid-state based configurations were considered and simulated in order to evaluate their potential to be utilised in the pulsed power area. Parts of this literature review are documented in Chapter 1 of this thesis. Current source topologies demonstrate valuable advantages in supplying the loads with capacitive characteristics such as plasma applications. To investigate the influence of switching transients associated with solid-state devices on rise time of pulses, simulation based studies have been undertaken. A variable current source is considered to pump different current levels to a capacitive load, and it was evident that dissimilar dv/dts are produced at the output. Thereby, transient effects on pulse rising time are denied regarding the evidence acquired from this examination. A detailed report of this study is given in Chapter 6 of this thesis. This study inspired the design of a solid-state based topology that take advantage of both current and voltage sources. A series of switch-resistor-capacitor units at the output splits the produced voltage to lower levels, so it can be shared by the switches. A smart but complicated switching strategy is also designed to discharge the residual energy after each supply cycle. To prevent reverse power flow and to reduce the complexity of the control algorithm in this system, the resistors in common paths of units are substituted with diode rectifiers (switch-diode-capacitor). This modification not only gives the feasibility of stopping the load supply process to the supplier at any stage (and consequently saving energy), but also enables the converter to operate in a two-stroke mode with asymmetrical capacitors. The components’ determination and exchanging energy calculations are accomplished with respect to application specifications and demands. Both topologies were simply modelled and simulation studies have been carried out with the simplified models. Experimental assessments were also executed on implemented hardware and the approaches verified the initial analysis. Reports on details of both converters are thoroughly discussed in Chapters 2 and 3 of the thesis. Conventional MGs have been recently modified to use solid-state transistors (i.e. Insulated gate bipolar transistors) instead of magnetic/gas switching devices. Resistive insulators previously used in their structures are substituted by diode rectifiers to adjust MGs for a proper voltage sharing. However, despite utilizing solid-state technology in MGs configurations, further design and control amendments can still be made to achieve an improved performance with fewer components. Considering a number of charging techniques, resonant phenomenon is adopted in a proposal to charge the capacitors. In addition to charging the capacitors at twice the input voltage, triggering switches at the moment at which the conducted current through switches is zero significantly reduces the switching losses. Another configuration is also introduced in this research for Marx topology based on commutation circuits that use a current source to charge the capacitors. According to this design, diode-capacitor units, each including two Marx stages, are connected in cascade through solid-state devices and aggregate the voltages across the capacitors to produce a high voltage pulse. The polarity of voltage across one capacitor in each unit is reversed in an intermediate mode by connecting the commutation circuit to the capacitor. The insulation of input side from load side is provided in this topology by disconnecting the load from the current source during the supply process. Furthermore, the number of required fast switching devices in both designs is reduced to half of the number used in a conventional MG; they are replaced with slower switches (such as Thyristors) that need simpler driving modules. In addition, the contributing switches in discharging paths are decreased to half; this decrease leads to a reduction in conduction losses. Associated models are simulated, and hardware tests are performed to verify the validity of proposed topologies. Chapters 4, 5 and 7 of the thesis present all relevant analysis and approaches according to these topologies.

Modelling resilience in a water supply system : contrasting conditions of drought and flood

This paper presents an approach to modelling the resilience of a generic (potable) water supply system. The system is contextualized as a meta-system consisting of three subsystems to represent the natural catchment, the water treatment plant and the water distribution infrastructure for urban use. An abstract mathematical model of the meta-system is disaggregated progressively to form a cascade of equations forming a relational matrix of models. This allows the investigation of commonly implicit relationships between various operational components within the meta system, the in-depth understanding of specific system components and influential factors and the incorporation of explicit disturbances to explore system behaviour. Consequently, this will facilitate long-term decision making to achieve sustainable solutions for issues such as, meeting a growing demand or managing supply-side influences in the meta-system under diverse water availability regimes. This approach is based on the hypothesis that the means to achieve resilient supply of water may be better managed by modelling the effects of changes at specific levels that have a direct or in some cases indirect impact on higher-order outcomes. Additionally, the proposed strategy allows the definition of approaches to combine disparate data sets to synthesise previously missing or incomplete higher-order information, a scientifically robust means to define and carry out meta-analyses using knowledge from diverse yet relatable disciplines relevant to different levels of the system and for enhancing the understanding of dependencies and inter-dependencies of variable factors at various levels across the meta-system. The proposed concept introduces an approach for modelling a complex infrastructure system as a meta system which consists of a combination of bio-ecological, technical and socio-technical subsystems.

Prognostic significance of IGF and ECM induced signalling proteins in breast cancer patients

Breast cancer is a leading contributor to the burden of disease in Australia. Fortunately, the recent introduction of diverse therapeutic strategies have improved the survival outcome for many women. Despite this, the clinical management of breast cancer remains problematic as not all approaches are sufficiently sophisticated to take into account the heterogeneity of this disease and are unable to predict disease progression, in particular, metastasis. As such, women with good prognostic outcomes are exposed to the side effects of therapies without added benefit. Furthermore, women with aggressive disease for whom these advanced treatments would deliver benefit cannot be distinguished and opportunities for more intensive or novel treatment are lost. This study is designed to identify novel factors associated with disease progression, and the potential to inform disease prognosis. Frequently overlooked, yet common mediators of disease are the interactions that take place between the insulin-like growth factor (IGF) system and the extracellular matrix (ECM). Our laboratory has previously demonstrated that multiprotein insulin-like growth factor-I (IGF-I): insulin-like growth factor binding protein (IGFBP): vitronectin (VN) complexes stimulate migration of breast cancer cells in vitro, via the cooperative involvement of the insulin-like growth factor type I receptor (IGF-IR) and VN-binding integrins. However, the effects of IGF and ECM protein interactions on the dissemination and progression of breast cancer in vivo are unknown. It was hypothesised that interactions between proteins required for IGF induced signalling events and those within the ECM contribute to breast cancer metastasis and are prognostic and predictive indicators of patient outcome. To address this hypothesis, semiquantitative immunohistochemistry (IHC) analyses were performed to compare the extracellular and subcellular distribution of IGF and ECM induced signalling proteins between matched normal, primary cancer, and metastatic cancer among archival formalin-fixed paraffin-embedded (FFPE) breast tissue samples collected from women attending the Princess Alexandra Hospital, Brisbane. Multivariate Cox proportional hazards (PH) regression survival models in conjunction with a modified „purposeful selection of covariates. method were applied to determine the prognostic potential of these proteins. This study provides the first in-depth, compartmentalised analysis of the distribution of IGF and ECM induced signalling proteins. As protein function and protein localisation are closely correlated, these findings provide novel insights into IGF signalling and ECM protein function during breast cancer development and progression. Distinct IGF signalling and ECM protein immunoreactivity was observed in the stroma and/or in subcellular locations in normal breast, primary cancer and metastatic cancer tissues. Analysis of the presence and location of stratifin (SFN) suggested a causal relationship in ECM remodelling events during breast cancer development and progression. The results of this study have also suggested that fibronectin (FN) and ¥â1 integrin are important for the formation of invadopodia and epithelial-to-mesenchymal transition (EMT) events. Our data also highlighted the importance of the temporal and spatial distribution of IGF induced signalling proteins in breast cancer metastasis; in particular, SFN, enhancer-of-split and hairy-related protein 2 (SHARP-2), total-akt/protein kinase B 1 (Total-AKT1), phosphorylated-akt/protein kinase B (P-AKT), extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (ERK1/2) and phosphorylated-extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (P-ERK1/2). Multivariate survival models were created from the immunohistochemical data. These models were found to fit well with these data with very high statistical confidence. Numerous prognostic confounding effects and effect modifications were identified among elements of the ECM and IGF signalling cascade and corroborate the survival models. This finding provides further evidence for the prognostic potential of IGF and ECM induced signalling proteins. In addition, the adjusted measures of associations obtained in this study have strengthened the validity and utility of the resulting models. The findings from this study provide insights into the biological interactions that occur during the development of breast tissue and contribute to disease progression. Importantly, these multivariate survival models could provide important prognostic and predictive indicators that assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy. The outcomes of this study further inform the development of new therapeutics to aid patient recovery. The findings from this study have widespread clinical application in the diagnosis of disease and prognosis of disease progression, and inform the most appropriate clinical management of individuals with breast cancer.

Apixaban for the prevention of thromboembolism in adults after elective hip or knee replacement surgery, and in symptomatic venous thromboembolism

The anticoagulant effect of apixaban is due to direct inhibition of FXa in the coagulation cascade. The main advantages apixaban has over the current anti-coagulant drugs is that it is active after oral administration, and its coagulation effect does not require monitoring. Apixaban has been compared to enoxaparin in the prevention of venous thromboembolism associated with knee and hip replacement, where it is as efficacious as enoxaparin, but causes less bleeding. However, apixaban is not the only FXa inhibitor that could replace enoxaparin for this indication, as the FXa inhibitor rivaroxaban is as efficacious and safe as enoxaparin in preventing thromboembolism associated with these surgical procedures. Until the results of the AMPLIFY Phase III trial are known, it is too early to consider apixaban as an alternative to enoxaparin in symptomatic thromboembolism. Apixaban should not be used to prevent thromboembolism in medical immobilised subjects or acute coronary syndromes, as it causes excess bleeding in these conditions without benefit.

Vitronectin – master controller or micromanager?

The concept of the cellular glycoprotein vitronectin acts as a biological ‘glue’ and key controller of mammalian tissue repair and remodelling activity is emerging from nearly 50 years of experimental in vitro and in vivo data. Unexpectedly, the vitronectin-knock-out mouse was found to be viable and to have largely normal phenotype. However, diligent observation revealed that the VN-KO animal exhibits delayed coagulation and poor wound healing. This is interpreted to indicate that vitronectin occupies a role in the earliest events of thrombogenesis and tissue repair. That role is as a foundation upon which the thrombus grows in an organised structure. In addition to closing the wound, the thrombus also serves to protect the underlying tissue from oxidation, is a reservoir of mitogens and tissue repair mediators and provides a provisional scaffold for the repairing tissue. In the absence of vitronectin (e.g. VN-KO animal) this cascade is disrupted before it begins. Our data demonstrates that a wide variety of biologically active species associate with VN. While initial studies were focused on mitogens, other classes of bioactives (e.g. glycosaminoglycans, metalloproteinases) are now also known to specifically interact with VN. Many of these interactions are long-lived, often resulting in multi-protein complexes, while others are stable for prolonged periods. Multiprotein complexes provide several advantages: prolonging molecular interaction; sustaining local concentrations, facilitating co-stimulation of cell surface receptors and thereby enhancing cellular / biological responses. We contend that these, or equivalent, multi-protein complexes mediate vitronectin functionality in vivo. It is also likely that many of the species demonstrated to associate with vitronectin in vitro, also associate with vitronectin in vivo in similar multi-protein complexes. Thus the predominant biological function of vitronectin is that of a master controller of the extracellular environment; informing, and possibly instructing cells ‘where’ to behave, ‘when’ to behave, and ‘how’ to behave (i.e. appropriately for the current circumstance).

The hypothermic patient

Concepts used in this chapter include: Thermoregulation:- Thermoregulation refers to the body’s sophisticated, multi-system regulation of core body temperature. This hierarchical system extends from highly thermo-sensitive neurons in the preoptic region of the brain proximate to the rostral hypothalamus, down to the brain stem and spinal cord. Coupled with receptors in the skin and spine, both central and peripheral information on body temperature is integrated to inform and activate the homeostatic mechanisms which maintain our core temperature at 37oC.1 Body heat is lost through the skin, via respiration and excretions. The skin is perhaps the most important organ in regulating heat loss. Hyporthermia:- Hypothermia is defined as core body temperature less than 350C and is the result of imbalance between the body’s heat production and heat loss mechanisms. Hypothermia may be accidental, or induced for clinical benefit i.e: neurological protection (therapeutic hypothermia). External environmental conditions are the most common cause of accidental hypothermia, but not the only causes of hypothermia in humans. Other causes include metabolic imbalance; trauma; neurological and infectious disease; and exposure to toxins such as organophosphates. Therapeutic Hypothermia:- In some circumstances, hypothermia can be induced to protect neurological functioning as a result of the associated decrease in cerebral metabolism and energy consumption. Reduction in the extent of degenerative processes associated with periods of ischaemia such as excitotoxic cascade; apoptotic and necrotic cell death; microglial activation; oxidative stress and inflammation associated with ischaemia are averted or minimised.2 Mild hypothermia is the only effective treatment confirmed clinically for improving the neurological outcomes of patient’s comatose following cardiac arrest.3

ECMO : the clinician’s view

Background: Extra corporeal membrane oxygenation (ECMO) is a complex rescue therapy used to provide cardiac and/or respiratory support for critically ill patients who have failed maximal conventional medical management. ECMO is based on a modified cardiopulmonary bypass (CPB) circuit, and can provide cardiopulmonary support for up-to several months. It can be used in a veno venous configuration for isolated respiratory failure, (VV-ECMO), or in a veno arterial configuration (VA-ECMO) where support is necessary for cardiac +/- respiratory failure. The ECMO circuit consists of five main components: large bore cannulae (access cannulae) for drainage of the venous system, and return cannulae to either the venous (in VV-ECMO) or arterial (in VA ECMO) system. An oxygenator, with a vast surface area of hollow filaments, allows addition of oxygen and removal of carbon dioxide; a centrifugal blood pump allows propulsion of blood through the circuit at upto 10 L/minute; a control module and a thermoregulatory unit, which allows for exact temperature control of the extra corporeal blood. Methods: The first successful use of ECMO for ARDS in adults occurred in 1972, and its use has become more commonplace over the last 30 years, supported by the improvement in design and biocompatibility of the equipment, which has reduced the morbidity associated with this modality. Whilst the use of ECMO in neonatal population has been supported by numerous studies, the evidence upon which ECMO was integrated into adult practice was substantially less robust. Results: Recent data, including the CESAR study (Conventional Ventilatory Support versus Extra corporeal membrane oxygenation for Severe Respiratory failure) has added a degree of evidence to the role of ECMO in such a patient population. The CESAR study analysed 180 patients, and confirmed that ECMO was associated with an improved rate of survival. More recently, ECMO has been utilized in numerous situations within the critical care area, including support in high-risk percutaneous interventions in cardiac catheter lab; the operating room, emergency department, as well in specialized inter-hospital retrieval services. The increased understanding of the risk:benefit profile of ECMO, along with a reduction in morbidity associated with its use will doubtless lead to a substantial rise in the utilisation of this modality. As with all extra-corporeal circuits, ECMO opposes the basic premises of the mammalian inflammation and coagulation cascade where blood comes into foreign circulation, both these cascades are activated. Anti-coagulation is readily dealt with through use of agents such as heparin, but the inflammatory excess, whilst less macroscopically obvious, continues un-abated. Platelet consumption and neutrophil activation occur rapidly, and the clinician is faced with balancing the need of anticoagulation for the circuit, against haemostasis in an acutely bleeding patient. Alterations in pharmacokinetics may result in inadequate levels of disease modifying therapeutics, such as antibiotics, hence paradoxically delaying recovery from conditions such as pneumonia. Key elements of nutrition and the innate immune system maysimilarly be affected. Summary: This presentation will discuss the basic features of ECMO to the nonspecialist, and review the clinical conundrum faced by the team treating these most complex cases.

Cell death control : the interplay of apoptosis and autophagy in the pathogenicity of sclerotinia sclerotiorum

Programmed cell death is characterized by a cascade of tightly controlled events that culminate in the orchestrated death of the cell. In multicellular organisms autophagy and apoptosis are recognized as two principal means by which these genetically determined cell deaths occur. During plant-microbe interactions cell death programs can mediate both resistant and susceptible events. Via oxalic acid (OA), the necrotrophic phytopathogen Sclerotinia sclerotiorum hijacks host pathways and induces cell death in host plant tissue resulting in hallmark apoptotic features in a time and dose dependent manner. OA-deficient mutants are non-pathogenic and trigger a restricted cell death phenotype in the host that unexpectedly exhibits markers associated with the plant hypersensitive response including callose deposition and a pronounced oxidative burst, suggesting the plant can recognize and in this case respond, defensively. The details of this plant directed restrictive cell death associated with OA deficient mutants is the focus of this work. Using a combination of electron and fluorescence microscopy, chemical effectors and reverse genetics, we show that this restricted cell death is autophagic. Inhibition of autophagy rescued the non-pathogenic mutant phenotype. These findings indicate that autophagy is a defense response in this necrotrophic fungus/plant interaction and suggest a novel function associated with OA; namely, the suppression of autophagy. These data suggest that not all cell deaths are equivalent, and though programmed cell death occurs in both situations, the outcome is predicated on who is in control of the cell death machinery. Based on our data, we suggest that it is not cell death per se that dictates the outcome of certain plant-microbe interactions, but the manner by which cell death occurs that is crucial.

Humanised xenograft models of bone metastasis revisited : novel insights into species-specific mechanisms of cancer cell osteotropism

The determinants and key mechanisms of cancer cell osteotropism have not been identified, mainly due to the lack of reproducible animal models representing the biological, genetic and clinical features seen in humans. An ideal model should be capable of recapitulating as many steps of the metastatic cascade as possible, thus facilitating the development of prognostic markers and novel therapeutic strategies. Most animal models of bone metastasis still have to be derived experimentally as most syngeneic and transgeneic approaches do not provide a robust skeletal phenotype and do not recapitulate the biological processes seen in humans. The xenotransplantation of human cancer cells or tumour tissue into immunocompromised murine hosts provides the possibility to simulate early and late stages of the human disease. Human bone or tissue-engineered human bone constructs can be implanted into the animal to recapitulate more subtle, species-specific aspects of the mutual interaction between human cancer cells and the human bone microenvironment. Moreover, the replication of the entire "organ" bone makes it possible to analyse the interaction between cancer cells and the haematopoietic niche and to confer at least a partial human immunity to the murine host. This process of humanisation is facilitated by novel immunocompromised mouse strains that allow a high engraftment rate of human cells or tissue. These humanised xenograft models provide an important research tool to study human biological processes of bone metastasis.

Improving ultrasound excitation systems using a flexible power supply with adjustable voltage and frequency to drive piezoelectric transducers

The ability of a piezoelectric transducer in energy conversion is rapidly expanding in several applications. Some of the industrial applications for which a high power ultrasound transducer can be used are surface cleaning, water treatment, plastic welding and food sterilization. Also, a high power ultrasound transducer plays a great role in biomedical applications such as diagnostic and therapeutic applications. An ultrasound transducer is usually applied to convert electrical energy to mechanical energy and vice versa. In some high power ultrasound system, ultrasound transducers are applied as a transmitter, as a receiver or both. As a transmitter, it converts electrical energy to mechanical energy while a receiver converts mechanical energy to electrical energy as a sensor for control system. Once a piezoelectric transducer is excited by electrical signal, piezoelectric material starts to vibrate and generates ultrasound waves. A portion of the ultrasound waves which passes through the medium will be sensed by the receiver and converted to electrical energy. To drive an ultrasound transducer, an excitation signal should be properly designed otherwise undesired signal (low quality) can deteriorate the performance of the transducer (energy conversion) and increase power consumption in the system. For instance, some portion of generated power may be delivered in unwanted frequency which is not acceptable for some applications especially for biomedical applications. To achieve better performance of the transducer, along with the quality of the excitation signal, the characteristics of the high power ultrasound transducer should be taken into consideration as well. In this regard, several simulation and experimental tests are carried out in this research to model high power ultrasound transducers and systems. During these experiments, high power ultrasound transducers are excited by several excitation signals with different amplitudes and frequencies, using a network analyser, a signal generator, a high power amplifier and a multilevel converter. Also, to analyse the behaviour of the ultrasound system, the voltage ratio of the system is measured in different tests. The voltage across transmitter is measured as an input voltage then divided by the output voltage which is measured across receiver. The results of the transducer characteristics and the ultrasound system behaviour are discussed in chapter 4 and 5 of this thesis. Each piezoelectric transducer has several resonance frequencies in which its impedance has lower magnitude as compared to non-resonance frequencies. Among these resonance frequencies, just at one of those frequencies, the magnitude of the impedance is minimum. This resonance frequency is known as the main resonance frequency of the transducer. To attain higher efficiency and deliver more power to the ultrasound system, the transducer is usually excited at the main resonance frequency. Therefore, it is important to find out this frequency and other resonance frequencies. Hereof, a frequency detection method is proposed in this research which is discussed in chapter 2. An extended electrical model of the ultrasound transducer with multiple resonance frequencies consists of several RLC legs in parallel with a capacitor. Each RLC leg represents one of the resonance frequencies of the ultrasound transducer. At resonance frequency the inductor reactance and capacitor reactance cancel out each other and the resistor of this leg represents power conversion of the system at that frequency. This concept is shown in simulation and test results presented in chapter 4. To excite a high power ultrasound transducer, a high power signal is required. Multilevel converters are usually applied to generate a high power signal but the drawback of this signal is low quality in comparison with a sinusoidal signal. In some applications like ultrasound, it is extensively important to generate a high quality signal. Several control and modulation techniques are introduced in different papers to control the output voltage of the multilevel converters. One of those techniques is harmonic elimination technique. In this technique, switching angles are chosen in such way to reduce harmonic contents in the output side. It is undeniable that increasing the number of the switching angles results in more harmonic reduction. But to have more switching angles, more output voltage levels are required which increase the number of components and cost of the converter. To improve the quality of the output voltage signal with no more components, a new harmonic elimination technique is proposed in this research. Based on this new technique, more variables (DC voltage levels and switching angles) are chosen to eliminate more low order harmonics compared to conventional harmonic elimination techniques. In conventional harmonic elimination method, DC voltage levels are same and only switching angles are calculated to eliminate harmonics. Therefore, the number of eliminated harmonic is limited by the number of switching cycles. In the proposed modulation technique, the switching angles and the DC voltage levels are calculated off-line to eliminate more harmonics. Therefore, the DC voltage levels are not equal and should be regulated. To achieve this aim, a DC/DC converter is applied to adjust the DC link voltages with several capacitors. The effect of the new harmonic elimination technique on the output quality of several single phase multilevel converters is explained in chapter 3 and 6 of this thesis. According to the electrical model of high power ultrasound transducer, this device can be modelled as parallel combinations of RLC legs with a main capacitor. The impedance diagram of the transducer in frequency domain shows it has capacitive characteristics in almost all frequencies. Therefore, using a voltage source converter to drive a high power ultrasound transducer can create significant leakage current through the transducer. It happens due to significant voltage stress (dv/dt) across the transducer. To remedy this problem, LC filters are applied in some applications. For some applications such as ultrasound, using a LC filter can deteriorate the performance of the transducer by changing its characteristics and displacing the resonance frequency of the transducer. For such a case a current source converter could be a suitable choice to overcome this problem. In this regard, a current source converter is implemented and applied to excite the high power ultrasound transducer. To control the output current and voltage, a hysteresis control and unipolar modulation are used respectively. The results of this test are explained in chapter 7.

Impact energy dependence of Al13 cluster deposition on Ni(001) surface

In this paper, the influence of the impact energy on the initial fabrication of thin films formed by low energy cluster deposition was investigated by molecular dynamics simulation of All 3 clusters depositing on Ni(0 0 1) substrate. In the case of soft-landing, (0.01 eV/atom), clusters are rearranged from I-h symmetry into fcc-like clusters on the surface. Then they aggregate each other, which result in thin film growing in 3D island mode. While, growth will be in layer-by-layer mode at the impact energy of a few electron volt due to the transient lateral spread of cluster atoms induced by dense collision cascade. This effect has been traced to collision cascade inside the cluster. which is enhanced by collision with a hard Ni substrate. (C) 2002 Elsevier Science B.V. All rights reserved.