Cardiomyocytes isolated from rabbit hearts were preconditioned in vitro by 10 min of ischemia or treatment with 100 μM adenosine. Protection was assessed as average integrated mortality following osmotic swelling and determination of viability by trypan blue exclusion over 60-180 min ischemia. Repetitive submaximal stimulations with 1 μM adenosine amplified the protective response. Treatment with adenosine only at the onset of prolonged ischemia afforded a dose-dependent protection. The PKC inhibitor calphostin C (500 nM) blocked preconditioning and, when added during ischemic incubation of non-preconditioned cells, significantly increased injury. The memory of adenosine-induced preconditioning decayed over a 60 min post-incubation period. Light activation of calphostin C initially added to preconditioned ischemic cells in the dark indicated that a 10 min period of PKC activity at the onset of ischemia affords full protection. The reversible PKC inhibitors chelerythrine (5 μM) or staurosporine (100 nM) added only to bracket induction of ischemia, reduced but did not abolish protection. Protection was abolished when either drug was present during induction and a subsequent 30 min post-incubation period. Staurosporine included during initiation and post-incubation but washed out in the final 5 min of post-incubation allowed significant protection to occur. It is concluded that a single adenosine receptor-stimulation induces protection as it preconditions, and PKC activity appears to be required for both induction and protection. Memory may reside in post-receptor amplification of an initial protective response.

This study was designed to test the hypothesis that induction of the preconditioned state results in a sustained translocation of protein kinase C (PKC) which accounts for the memory associated with preconditioning. Isolated rabbit cardiomyocytes were subjected to established preconditioning protocols using either adenosine or transient ischemia. At timed intervals during induction of preconditioning (PC), post-incubation or final sustained ischemia, cells were harvested, subjected to digitonin lysis and separated into cytosolic and particulate fractions. Samples were evaluated by Western blot analysis with monoclonal antibodies to alpha, epsilon, zeta and gamma PKC isozymes, and bands were quantified by densitometry. Internal controls for each experiment included oxygenated cardiomyocytes and cells with PKC translocation evoked by treatment with phorbol 12-myristate 13-acetate (PMA). For control oxygenated cells, the particulate fraction contained about 30% of PKC epsilon, 5-10% of PKC alpha and 60-70% of PKC zeta. Preconditioning with adenosine (100 μM) or 10 min ischemia had no significant effect on these percentages. Furthermore, the relative amounts of the PKC isozymes associated with the particulate fraction of control and preconditioned cells did not differ after a post-incubation in oxygenated buffer or during a final ischemic incubation. PMA and ingenol completely translocated the epsilon and alpha isoforms, while thymeleatoxin totally translocated PKC alpha but only partially (50%) translocated PKC epsilon. The distribution of PKC zeta between fractions was not affected by any drug, The protein phosphatase inhibitor calyculin A protected cells mimicking preconditioning. This protection was blocked by preincubation with the selective PKC inhibitor calphostin C but was largely retained if calphostin C was added only during the final ischemic period. It is concluded that PKC activity is required for preconditioning, but a sustained translocation of PKC above basal levels is not necessary for protection of rabbit cardiomyocytes in vitro.

Calcium tolerant rabbit cardiomyocytes, isolated by collagenase perfusion, were preincubated for varying periods of time followed by resuspension in fresh media and centrifugation into an ischaemic pellet with restricted extracellular fluid. Pellets were incubated for 240 min under oil at 37°C to mimic severe ischaemia. Time to onset of ischaemic contracture (rod to square transformation) and trypan blue permeability following resuspension in 85 mOsm media were monitored at sequential times. The protocol of Series 1 was a 5-10 min pre-incubation, immediately followed by ischaemic pelleting. Preincubation with pinacidil (50 μm) protected cells from ischaemic insult, but pinacidil added only into the ischaemic pellet did not protect. Protection was abolished by the protein kinase (PKC) inhibitors chelerythrine (10 μm) added with pinacidil and calphostin C (200nm) added only into the ischaemic pellet. Neither PKC inhibitor had an effect on injury of untreated ischaemic myocytes (data not shown). Series 2-5 were preconditioning protocols with a 10 min intervention period, followed by a 30 min oxygenated drug-free period, prior to ischaemic pelleting. In series 2 pinacidil protected cells from ischaemic insult and this protection was abolished when glyburide (10 μm) was present during preincubation, or during post-incubation and ischaemia. Glyburide only partially inhibited the protection when glyburide was added only into the ischaemic pellet. In Series 3, 8-sulfophenyltheophyline (SPT)(100 μm) or adenosine deaminase during preincubation, or SPT only added into the ischaemic pellet abolished pinacidil’s protection. In Series 4, cardiomyocytes were ischaemically preconditioned by pelleting for 10 min followed by 30 min reoxygenation. Glyburide during initial ischaemic blocked protection, but when added during post incubation and into the final pellet protection was not reduced. In Series 5 8-cyclopentyl-1,3, dipropylxanthine (DPCPX) (10 μm) added into the final pellet abolished protection by pinacidil, but not protection following ischaemic preconditioning. In contrast to pinacidil, ischaemically preconditioned cells maintain protection in the presence of glyburide, indicating that: (1) pinacidil does not exactly mimic preconditioning and (2) ischaemically preconditioned cells do not require opened K+ATP channels for protection, although they appear to be important during initiation of the preconditioned state. It is hypothesized that pinacidil opening of K+ channels may facilitate induction of preconditioning.

Isolated adult rat myocytes were subjected to 180 min of metabolic inhibition or incubated in ischaemic pellets, in the presence and absence of 10 μm okadaic acid (OA) or calyculin A (CL-A). Contracture and viability was determined by light microscopic analysis of trypan blue-stained preparations and ATP levels by HPLC. Osmotic fragility was assessed by brief hypotonic swelling of cells in 170 or 85 mOsm media prior to determination of viability. Neither drug significantly affected the relatively rapid rates of contracture of myocytes during metabolic inhibition, and both afforded significant protection from development of trypan blue permeability and osmotic fragility. Both OA and CL-A significantly accelerated the rates of contracture and ATP depletion of myocytes during ischaemic incubations. Despite an enhanced rate of ATP depletion, which would be expected to accelerate development of injury, neither drug accelerated development of loss of viability or development of osmotic fragility as measured by 170 mOsm swelling. Mathematical compensation for different rates of ATP depletion confirmed that a protective effect of the drugs, during ischaemic incubation, was masked by their enhancement of the rate of injury, following swelling at 170 mOsm. When the effects of CL-A on ischaemic cells were examined at 85 mOsm, a more stringent test for osmotic fragility, protection was found without compensation for differing rates of ATP depletion. A dose/response curve for CL-A showed some effect at 100 nm and a nearly full effect during metabolic inhibition at 1 μm concentrations. It is concluded that protein phosphatase inhibitors reduce the rates of development of osmotic fragility of metabolically inhibited cells and reduces the rate of injury relative to the rate of ATP depletion of ischaemic cardiomyocytes. Phosphorylation mechanisms may be important to development of irreversible myocardial cell injury.

The relationship between myocardial cell contracture and injury during total metabolic inhibition (amylobarbital and iodacetic acid) and ischemia was examined, using 5-50 mm butanedione monoxime (BDM) as an inhibitor of contracture. BDM had no apparent effect on control myocytes during 180 min incubations, but inhibited contracture following anoxia or ischemia in a dose-dependent fashion, as directly quantitated by length/width ratios. Cellular ATP levels decreased at a similar rate in the absence or presence of BDM, following metabolic inhibition. BDM-mediated inhibition of contracture was associated with accelerated cell injury, as defined by: the uptake of an extracellular marker (trypan blue) by the cardiomyocytes, by direct analysis of myoglobin released into the supernatant and by ultrastructural demonstration of defects in sarcolemmal membrane integrity. Calcium was not required for BDM's enhancement of injury, in that cells incubated in calcium free-EGTA buffer showed a similar BDM-mediated acceleration of injury. In the presence or absence of calcium, enhancement of injury was more marked in cells osmotically stressed with a brief incubation in hypotonic buffer, than in cells resuspended in isotonic media. It is concluded that BDM enhances development of osmotic fragility of inhibited or ischemic cardiomyocytes and that contracture is not a necessary contributing factor to myocardial cell death.

The challenges imposed on Voltage Regulator Modules (VRM) become difficult to be achieved with the conventional multiphase buck converter commonly used on PC motherboards. For faster data transfer, a decrease in the output voltage is needed. This decrease causes small duty cycle that is accompanied by critical problems which impairs the efficiency. Therefore, these problems need to be addressed. Transformer-based non-isolated topologies are not new approaches to extend the duty cycle and avoid the associated drawbacks. High leakage, several added components and complicated driving and control schemes are some of the trade-offs to expand the duty cycle. The objective of this work is to present a new dc-dc buck-based topology, which extends the duty cycle with minimum drawbacks by adding two transformers that can be integrated to decrease the size and two switches with zero voltage switching (ZVS). Another issue addressed in this thesis is deriving a small signal model for a two-input two-phase buck converter as an introduction to a new evolving field of multi-input converters.

Metacommunity connectivity, i.e., multi-species dispersal events, is vital to metapopulation persistence in patchy landscapes. Assessments of metacommunity connectivity are not trivial. However, a relationship between trophic rank and the species-area relationship has been found in previous studies, allowing for the use of the predator species-area relationship to act as a surrogate measure of actual metacommunity connectivity of prey species in some systems. For this study, avian species were selected as they are generalist top predators within the study system. Predator species richness within geographically isolated freshwater marshes is influenced by a number of factors. I explore the relative roles of patch area, seasonality, hydroperiod, isolation, and vegetation structure on habitat use in the isolated freshwater marshes embedded within the dry prairie ecosystem of Central Florida. Predator species richness was surveyed in 50 sites for three seasons: fall 2005, winter 2005/06, and spring 2006 and the observed avian assemblage measures were subdivided into foraging guilds for analysis. Wading guild (e.g., egrets, herons, bitterns) species richness was correlated with hydroperiod and vegetation structural variables while perching guild (e.g., blackbirds, sparrows, meadowlarks) species richness was correlated with isolation, hydroperiod, and area annually. Overall predator and all guild species richness measures were also correlated with patch area for all seasons. These results suggest that while a complex mixture of patch area, hydroperiod and isolation influence habitat utilization that varies by season and at the community, guild and individual species level, the underlying predictors that define habitat use in wetlands annually includes hydroperiod, and is not exclusively patch area. Additionally, seasonal differences in predator species richness were found to be significant in some cases indicating that future avian population studies may benefit by sampling outside of the normally studied spring breeding season. Results of this study support the use of predator species richness as a suitable assay of metacommunity connectivity of prey species. Applications and implications of this approach toward future conservation efforts are discussed.

Yes / The single-stage flyback Photovoltaic (PV) micro-inverter is considered as a simple and small in size topology but requires expensive digital microcontrollers such as Field-Programmable Gate Array (FPGA) or Digital Signal Processor (DSP) to increase the system efficiency, this would increase the cost of the overall system. To solve this problem, based on a single-stage flyback structure, this paper proposed a low cost and simple analog-digital control scheme. This control scheme is implemented using a low cost ATMega microcontroller built in the Arduino Uno board and some analog operational amplifiers. First, the single-stage flyback topology is analyzed theoretically and then the design consideration is obtained. Second, a 120 W prototype was developed in the laboratory to validate the proposed control. To prove the effectiveness of this control, we compared the cost price, overall system efficiency, and THD values of the proposed results with the results obtained by the literature. So, a low system component, single power stage, cheap control scheme, and decent efficiency are achieved by the proposed system. Finally, the experimental results present that the proposed system has a maximum efficiency of 91%, with good values of the total harmonic distortion (THD) compared to the results of other authors / This work was supported in-part by Innovate UK GCRF Energy Catalyst PiCREST project under Grant number 41358, in-part by British Academy GCRF COMPENSE project under Grant GCRFNGR3\1541

The objective of this research is to simulate the response of an isolated substructure such that the response of the substructure in isolation would be the same as the substructure within the structure. Generally, the behaviour of an isolated subsystem (substructure) subjected to dynamic loading is different than the behaviour of the same substructure within a system (structure). This is primarily caused by the boundary conditions that are imposed on the substructure from the surrounding subsystem in the entire structure. A new systematic approach (methodology) is developed for performing impact analysis on the isolated substructure. The developed technique is fundamentally based on enforcing the mode shapes around the boundary of the substructure in the full structure to be similar to the mode shapes of the isolated substructure. This is achieved by providing a consistent adjustment to the loading conditions (impact velocity and mass) to account for the loss of restraint at the interface with the full structure. Another important aspect of this research is experimental validation of proposed method. This method allows the experimental testing of an isolated substructure since the testing is performed by impacting the isolated substructure with an appropriate mass and velocity. In the finite element analysis, the structure is analyzed, and then the isolated substructure simulation is performed using the developed technique. The results obtained from the numerical simulations, for both the substructure in situ and the substructure in isolation, are compared and found to be in good agreement. For instance, the effective plastic strains, kinetic and internal energies for the substructure within the structure and the substructure in isolation range from 7% to 12% discrepancies between two analyses. The numerical simulations of a full structure are verified by performing a series of experimental impact tests on the full structure. Finally, the experimental applicability of the technique is studied and its results are validated with FE simulation of substructure in isolation. This problem of experimentally testing an isolated substructure had previously not been addressed. The comparisons of FE simulation and experimental testing are made based on the deformed geometries, out-of-plane deflections and accelerometer readings. For example, the out-of-plane deformations from the FE analysis and the experimental test were determined to be within 7% to 9%. The experimental validation and numerical simulations indicates the technique is reliable, repeatable and can predict dynamic response of the substructures when tested in isolation. / Thesis / Doctor of Philosophy (PhD)

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