[Delta] IDDQ testing of a CMOS 12-bit charge scaling digital-to-analog converter

This work presents design, implementation and test of a built-in current sensor (BICS) for ∆IDDQ testing of a CMOS 12-bit charge scaling digital-to-analog converter (DAC). The sensor uses power discharge method for the fault detection. The sensor operates in two modes, the test mode and the normal mode. In the test mode, the BICS is connected to the circuit under test (CUT) which is DAC and detects abnormal currents caused by manufacturing defects. In the normal mode, BICS is isolated from the CUT. The BICS is integrated with the DAC and is implemented in a 0.5 μm n-well CMOS technology. The DAC uses charge scaling method for the design and a low voltage (0 to 2.5 V) folded cascode op-amp. The built-in current sensor (BICS) has a resolution of 0.5 μA. Faults have been introduced into DAC using fault injection transistors (FITs). The method of ∆IDDQ testing has been verified both from simulation and experimental measurements

Exploring the effects of Cadherin-derived Peptides on Platelet and Endothelial Cell Function

The primary role of platelets is hemostasis, the prevention of excessive bleeding following vascular injury. However, platelets also play a role in the pathology of cardiovascular disease. Platelet mediated thrombosis on ruptured atherosclerotic plaques underlies the acute complications of cardiovascular disease including myocardial infarction and ischemic stroke. Arterial thrombus formation is a complex process involving various cell adhesion molecules and secondary mediators, such as ADP, secreted from activated platelets, which help to recruit platelets. There is an ongoing search for anti-platelet agents that can prevent thrombotic events whilst minimizing bleeding. Previous work in our laboratory identified a palmitoylated peptide derived from Kidney Cadherin (K-Cadherin) as a significant inhibitor of platelet function. Based on this observation, the anti-platelet effects of other cadherin-derived peptides were investigated. Firstly, the effect of peptides derived from membrane-adjacent cytoplasmic regions of K-, Epithelial Cadherin (E-Cadherin) and Neural Cadherin (N-Cadherin) sequences were assessed in assays of platelet function. Although many of these peptides were shown to inhibit platelet secretion and aggregation responses, it emerged that the corresponding control peptide sequences showed a similar ability to inhibit platelet function. Thus it seemed that many of the observed effects were non-specific. Secondly, I identified a novel junctional protein, Vascular Endothelial Cadherin (VE-Cadherin) and its associated protein, P120-catenin in human platelets and demonstrated, for the first time, that VE-Cadherin could form a heterophilic interaction with platelet integrin allb(33. This interaction was Arginine-Glycine-Aspartic acid (RGD) dependent. Peptides derived from juxtamembrane domain (JMD) of VE-Cadherin, such as those studied in the first results chapter, had only non-specific effects on platelet function. Thirdly, the effect of the VE-Cadherin-derived peptides on endothelial cell function and angiogenesis were investigated. Non-specific activity was carefully monitored using matched control peptides and lower peptide doses than in previous studies. This study demonstrates that certain VECadherin peptides specifically inhibited endothelial cell functions, including angiogenesis, migration and proliferation. These results highlight an important role played by the JMD in VE-Cadherin and identified potential inhibitors of cadherin function that may have therapeutic relevance. In the final chapter of this thesis, I investigated which parameters of cadherin-derived peptides, and their control peptides, were associated with the anti-platelet effect. It was found that water-soluble peptides with positively charged amino acids at their N-terminus are associated with anti-platelet activity. In addition, this prediction was also experimentally verified by using peptides with positively charged amino acids. Taken together, this thesis explores the cellular effects of cell-permeable peptides derived from target proteins. It clearly identifies a need for appropriate control peptides in all such experiments to control for sequence, dose and toxicity. Within these constrains, novel peptides were identified that inhibit VE-Cadherin function in endothelial cells

Modified and updated data of Peptides derived from cadherin juxtamembrane region inhibit platelet function

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Data from: Peptides derived from cadherin juxtamembrane region inhibit platelet function

The juxtamembrane domains (JMD) of transmembrane proteins are rich in critical peptide sequences that participate in dynamic cell signaling events. Synthetic JMD peptides derived from cadherin cell adhesion proteins have previously been shown to modulate platelet function. In this study, we aimed to develop functional bioactive agents from bioinformatically-identified critical peptide sequences. We synthesized overlapping 12-15 amino acids peptides from E- and N-cadherin JMD and assessed their effect on platelet aggregation and platelet ATP secretion. Peptides derived from close to the membrane proximal region inhibit platelet function. Sequential deletion of amino acids from the N- and C-termini of the inhibitory E-cadherin peptides identified the short K756EPLLP763 motif as a critical bioactive sequence. Alanine scanning studies further identified that the dileucine (LL) motif and positively charged lysine (K) are crucial for peptide activity. Moreover, scrambled peptides failed to show any effect on platelet activity. We conclude that peptides derived from JMD of E-cadherin provide potential lead peptides for the development of anti-thrombotic agents and to enable further understanding of the role of cadherins in platelet function

Discovering Anti-platelet Drug Combinations with an Integrated Model of Activator-Inhibitor Relationships, Activator-Activator Synergies and Inhibitor-Inhibitor Synergies

<div><p>Identifying effective therapeutic drug combinations that modulate complex signaling pathways in platelets is central to the advancement of effective anti-thrombotic therapies. However, there is no systems model of the platelet that predicts responses to different inhibitor combinations. We developed an approach which goes beyond current inhibitor-inhibitor combination screening to efficiently consider other signaling aspects that may give insights into the behaviour of the platelet as a system. We investigated combinations of platelet inhibitors and activators. We evaluated three distinct strands of information, namely: activator-inhibitor combination screens (testing a panel of inhibitors against a panel of activators); inhibitor-inhibitor synergy screens; and activator-activator synergy screens. We demonstrated how these analyses may be efficiently performed, both experimentally and computationally, to identify particular combinations of most interest. Robust tests of activator-activator synergy and of inhibitor-inhibitor synergy required combinations to show significant excesses over the double doses of each component. Modeling identified multiple effects of an inhibitor of the P2Y12 ADP receptor, and complementarity between inhibitor-inhibitor synergy effects and activator-inhibitor combination effects. This approach accelerates the mapping of combination effects of compounds to develop combinations that may be therapeutically beneficial. We integrated the three information sources into a unified model that predicted the benefits of a triple drug combination targeting ADP, thromboxane and thrombin signaling.</p></div

Identification of activator-activator synergy, inhibitor-inhibitor synergy, and activator-inhibitor combination effects.

<p>Combination experiments of activators and inhibitors. <b>(A)</b> Mean log₁₀ ADP release across platelets from 10 blood donors are shown, with green indicating platelet activation. Combinations of activator and inhibitors. Single and double doses (concentrations) of each activator alone are shown at the bottom; single and double doses of each inhibitor in the presence of a cocktail of all five activators are shown to the right; resting and cocktail are shown bottom right <b>(B)</b> Activator-activator combinations and inhibitor-inhibitor combinations, log(AAU) ADP release. Inhibitor-inhibitor data represents the inhibition of a cocktail of all five agonists. <b>(C)</b> To more easily visualize the data allowing for the differences in levels of activation among the five activators, a simple correction of the data is shown, with the values in panel A subtracted by the value of the single dose activator alone (thus, for CaXi the value is 4.90–4.97 = −0.07). Four significant activator-inhibitor combinations identified by statistical modeling (see text) are highlighted within a white box. Two of these lie on the diagonal, as expected <i>a priori</i>. <b>(D)</b> As for panel B, but calculated to display the difference of the activation or inhibition from the most effective double dose of either the first or the second agent within the combination (thus, for PiTi the value is 5.29–5.13 = 0.16). Positive synergy corresponds to more combined stimulation for the activator-activator pairs, indicated in magenta, and also to less combined stimulation for the inhibitor-inhibitor pairs, which are also indicated in magenta (i.e. magenta implies strong positive synergy of either activation, or of inhibition).</p

Interpreting the integrated model in the context of platelet signaling pathways.

<p>As in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004119#pcbi.1004119.g007" target="_blank">Fig. 7</a>, activatory synergies are represented by green lines, inhibitory synergies by red lines. Activators U46619 (Xa), TRAP (Ta), Epinephrine (Ea) ADP (Aa) and CRP (Ca) are indicated extracellularly, acting on their receptors, namely the thromboxane receptor (TXA2R), the thrombin receptor (PAR1), the Epinephrine receptor (α2AR), the ADP receptors (P2Y12, P2Y1 and P2X), and the collagen receptor (GPVI).</p

Computational and experimental analysis of bioactive peptide linear motifs in the integrin adhesome.

Therapeutic modulation of protein interactions is challenging, but short linear motifs (SLiMs) represent potential targets. Focal adhesions play a central role in adhesion by linking cells to the extracellular matrix. Integrins are central to this process, and many other intracellular proteins are components of the integrin adhesome. We applied a peptide network targeting approach to explore the intracellular modulation of integrin function in platelets. Firstly, we computed a platelet-relevant integrin adhesome, inferred via homology of known platelet proteins to adhesome components. We then computationally selected peptides from the set of platelet integrin adhesome cytoplasmic and membrane adjacent protein-protein interfaces. Motifs of interest in the intracellular component of the platelet integrin adhesome were identified using a predictor of SLiMs based on analysis of protein primary amino acid sequences (SLiMPred), a predictor of strongly conserved motifs within disordered protein regions (SLiMPrints), and information from the literature regarding protein interactions in the complex. We then synthesized peptides incorporating these motifs combined with cell penetrating factors (tat peptide and palmitylation for cytoplasmic and membrane proteins respectively). We tested for the platelet activating effects of the peptides, as well as their abilities to inhibit activation. Bioactivity testing revealed a number of peptides that modulated platelet function, including those derived from α-actinin (ACTN1) and syndecan (SDC4), binding to vinculin and syntenin respectively. Both chimeric peptide experiments and peptide combination experiments failed to identify strong effects, perhaps characterizing the adhesome as relatively robust against within-adhesome synergistic perturbation. We investigated in more detail peptides targeting vinculin. Combined experimental and computational evidence suggested a model in which the positively charged tat-derived cell penetrating part of the peptide contributes to bioactivity via stabilizing charge interactions with a region of the ACTN1 negatively charged surface. We conclude that some interactions in the integrin adhesome appear to be capable of modulation by short peptides, and may aid in the identification and characterization of target sites within the complex that may be useful for therapeutic modulation

Integrated modelling and validation of synergy and activator-inhibitor combination effects.

<p><b>(A)</b> a schematic of the integrated model (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004119#pcbi.1004119.s010" target="_blank">S4 Table</a>), investigating the influence of five activators (green dots) and five inhibitors (red dots) on platelet activation. Each solid line (10 black main effects, 4 purple activator-inhibitor combination effects, 3 red inhibitor-inhibitor synergy effects, 2 green activator-activator synergy effects) represents a parameter within the multiple regression model predicting platelet activation. The five receptors and the kinase shown in the model are not explicitly modelled since there is no direct data on their activation states. The predictions of this model were used to assess the impact of all possible three way combinations of inhibitors on platelets activated by a cocktail of five activators (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004119#pcbi.1004119.s011" target="_blank">S5 Table</a>). <b>(B)</b> testing the most strongly predicted inhibitor triple combination. This shows that the most strongly predicted three-way combination of <i>Xi</i>, <i>Ai</i>, <i>Ti</i> had a clearly stronger effect than the alternative <i>Xi</i>, <i>Ai</i>, <i>Pi</i> combination which was ranked more weakly by the predictive model (p = 0.0003).</p