Petri Net Siphon Analysis and Network Centrality Measures for Identifying Combination Therapies in Signaling Pathways

Aberrant behavior of biological signaling pathways has been implicated in diseases such as cancers. Therapies have been developed to target proteins in these networks in the hope of curing the illness or bringing about remission. However, identifying targets for drug inhibition that exhibit good therapeutic index has proven to be challenging since signaling pathways have a large number of components and many interconnections such as feedback, crosstalk, and divergence. Unfortunately, some characteristics of these pathways such as redundancy, feedback, and drug resistance reduce the efficacy of single drug target therapy and necessitate the employment of more than one drug to target multiple nodes in the system. However, choosing multiple targets with high therapeutic index poses more challenges since the combinatorial search space could be huge. To cope with the complexity of these systems, computational tools such as ordinary differential equations have been used to successfully model some of these pathways. Regrettably, for building these models, experimentally-measured initial concentrations of the components and rates of reactions are needed which are difficult to obtain, and in very large networks, they may not be available at the moment. Fortunately, there exist other modeling tools, though not as powerful as ordinary differential equations, which do not need the rates and initial conditions to model signaling pathways. Petri net and graph theory are among these tools. In this thesis, we introduce a methodology based on Petri net siphon analysis and graph network centrality measures for identifying prospective targets for single and multiple drug therapies. In this methodology, first, potential targets are identified in the Petri net model of a signaling pathway using siphon analysis. Then, the graph-theoretic centrality measures are employed to prioritize the candidate targets. Also, an algorithm is developed to check whether the candidate targets are able to disable the intended outputs in the graph model of the system or not. We implement structural and dynamical models of ErbB1-Ras-MAPK pathways and use them to assess and evaluate this methodology. The identified drug-targets, single and multiple, correspond to clinically relevant drugs. Overall, the results suggest that this methodology, using siphons and centrality measures, shows promise in identifying and ranking drugs. Since this methodology only uses the structural information of the signaling pathways and does not need initial conditions and dynamical rates, it can be utilized in larger networks.

Evaluation of distributed control signaling protocols in GMPLS

GMPLS is a generalized form of MPLS (MultiProtocol Label Switching). MPLS is IP packet based and it uses MPLS-TE for Packet Traffic Engineering. GMPLS is extension to MPLS capabilities. It provides separation between transmission, control and management plane and network management. Control plane allows various applications like traffic engineering, service provisioning, and differentiated services. GMPLS control plane architecture includes signaling (RSVP-TE, CR-LDP) and routing (OSPF-TE, ISIS-TE) protocols. This paper provides an overview of the signaling protocols, describes their main functionalities, and provides a general evaluation of both the protocols.

A control theoretic paradigm for cell signaling networks : a simple complexity for a sensitive robustness

The fields of molecular biology and cell biology are being flooded with complex genomic and proteomic datasets of large dimensions. We now recognize that each molecule in the cell and tissue can no longer be viewed as an isolated entity. Instead, each molecule must be considered as one member of an interacting network. Consequently, there is an urgent need for mathematical models to understand the behavior of cell signaling networks in health and in disease.

Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma

Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient's metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge.

VEGFR-3 and Tie pathways in vascular network formation

The blood and lymphatic vascular systems are essential for life, but they may become harnessed for sinister purposes in pathological conditions. For example, tumors learn to grow a network of blood vessels (angiogenesis), securing a source of oxygen and nutrients for sustained growth. On the other hand, damage to the lymph nodes and the collecting lymphatic vessels may lead to lymphedema, a debilitating condition characterized by peripheral edema and susceptibility to infections. Promoting the growth of new lymphatic vessels (lymphangiogenesis) is an attractive approach to treat lymphedema patients. Angiopoietin-1 (Ang1), a ligand for the endothelial receptor tyrosine kinases Tie1 and Tie2. The Ang1/Tie2 pathway has previously been implicated in promoting endothelial stability and integrity of EC monolayers. The studies presented here elucidate a novel function for Ang1 as a lymphangiogenic factor. Ang1 is known to decrease the permeability of blood vessels, and could thus act as a more global antagonist of plasma leakage and tissue edema by promoting growth of lymphatic vessels and thereby facilitating removal of excess fluid and other plasma components from the interstitium. These findings reinforce the idea that Ang1 may have therapeutic value in conditions of tissue edema. VEGFR-3 is present on all endothelia during development, but in the adult its expression becomes restricted to the lymphatic endothelium. VEGF-C and VEGF-D are ligands for VEGFR-3, and potently promote lymphangiogenesis in adult tissues, with direct and remarkably specific effects on the lymphatic endothelium in adult tissues. The data presented here show that VEGF-C and VEGF-D therapy can restore collecting lymphatic vessels in a novel orthotopic model of breast cancer-related lymphedema. Furthermore, the study introduces a novel approach to improve VEGF-C/VEGF-D therapy by using engineered heparin-binding forms of VEGF-C, which induced the rapid formation of organized lymphatic vessels. Importantly, VEGF-C therapy also greatly improved the survival and integration of lymph node transplants. The combination of lymph node transplantation and VEGF-C therapy provides a basis for future therapy of lymphedema. In adults, VEGFR-3 expression is restricted to the lymphatic endothelium and the fenestrated endothelia of certain endocrine organs. These results show that VEGFR-3 is induced at the onset of angiogenesis in the tip cells that lead the formation of new vessel sprouts, providing a tumor-specific vascular target. VEGFR-3 acts downstream of VEGF/VEGFR-2 signals, but, once induced, can sustain angiogenesis when VEGFR-2 signaling is inhibited. The data presented here implicate VEGFR-3 as a novel regulator of sprouting angiogenesis along with its role in regulating lymphatic vessel growth. Targeting VEGFR-3 may provide added efficacy to currently available anti-angiogenic therapeutics, which typically target the VEGF/VEGFR-2 pathway.

Allosteric Communication in Cysteinyl tRNA Synthetase A NETWORK OF DIRECT AND INDIRECT READOUT

Protein structure networks are constructed for the identification of long-range signaling pathways in cysteinyl tRNA synthetase (CysRS). Molecular dynamics simulation trajectory of CysRS-ligand complexes were used to determine conformational ensembles in order to gain insight into the allosteric signaling paths. Communication paths between the anticodon binding region and the aminoacylation region have been identified. Extensive interaction between the helix bundle domain and the anticodon binding domain, resulting in structural rigidity in the presence of tRNA, has been detected. Based on the predicted model, six residues along the communication paths have been examined by mutations (single and double) and shown to mediate a coordinated coupling between anticodon recognition and activation of amino acid at the active site. This study on CysRS clearly shows that specific key residues, which are involved in communication between distal sites in allosteric proteins but may be elusive in direct structure analysis, can be identified from dynamics of protein structure networks.

Performance Evaluation of OBS Networks with INI Signaling Technique

In this paper, we evaluate the performance of a burst retransmission method for an optical burst switched network with intermediate-node-initiation (INI) signaling technique. The proposed method tries to reduce the burst contention probability at the intermediate core nodes. We develop an analytical model to get the burst contention probability and burst loss probability for an optical burst switched network with intermediate-node-initiation signaling technique. The proposed method uses the optical burst retransmission method. We simulate the performance of the optical burst retransmission. Simulation results show that at low traffic loads the loss probability is low compared to the conventional burst retransmission in the OBS network. Result also show that the retransmission method for OBS network with intermediate-node-initiation signaling technique significantly reduces the burst loss probability.

MPTP activates ASK1-p38 MAPK signaling pathway through TNF-dependent Trx1 oxidation in parkinsonism mouse model

Activation of apoptosis signal regulating kinase 1 (ASK1)-p38 MAPK death signaling cascade is irn plicated in the death of dopaminergic neurons in substantia nigra in Parkinson's disease (PD). We investigated upstream activators of ASK1 using an MPTP mouse model of parkinsonism and assessed the temporal cascade of death signaling in ventral midbrain (VMB) and striatum (ST). MPTP selectively activated ASK1 and downstream 1)38 MAPK in a time dependent manner in VMB alone. This occurred through selective protein thiol oxidation of the redox-sensitive thiol disulfide oxidoreductase, thiorcdoxin (Trxl), resulting in release of its inhibitory association with ASK1, while glutathione-S-transferase ji 1 (GSTM1) remained in reduced form in association with ASK1. Levels of tumor necrosis factor (TNF), a known activator of ASK1, increased early after MPTP in VMB. Protein ovariation netvvork analysis (PCNA) using protein states as nodes revealed TNF to be an important node regulating the ASK1 signaling cascade. In confirmation, blocking MPTP-mecliated TNF signaling through intrathecal administration of TNFneutralizing antibody prevented Trxl oxidation and downstream ASK1-p38 MAPK activation. Averting an early increase in TNF, which leads to protein thiol oxidation resulting in activation of ASK1-p38 signaling, may be critical for neuroprotection in PD. Importantly, network analysis can help in understanding the cause/effect relationship within protein networks in complex disease states. (C) 2015 Published by Elsevier Inc.

Analytical efficiency evaluation of a network mobility management protocol for Intelligent Transportation Systems

One of the major concerns in an Intelligent Transportation System (ITS) scenario, such as that which may be found on a long-distance train service, is the provision of efficient communication services, satisfying users' expectations, and fulfilling even highly demanding application requirements, such as safety-oriented services. In an ITS scenario, it is common to have a significant amount of onboard devices that comprise a cluster of nodes (a mobile network) that demand connectivity to the outside networks. This demand has to be satisfied without service disruption. Consequently, the mobility of the mobile network has to be managed. Due to the nature of mobile networks, efficient and lightweight protocols are desired in the ITS context to ensure adequate service performance. However, the security is also a key factor in this scenario. Since the management of the mobility is essential for providing communications, the protocol for managing this mobility has to be protected. Furthermore, there are safety-oriented services in this scenario, so user application data should also be protected. Nevertheless, providing security is expensive in terms of efficiency. Based on this considerations, we have developed a solution for managing the network mobility for ITS scenarios: the NeMHIP protocol. This approach provides a secure management of network mobility in an efficient manner. In this article, we present this protocol and the strategy developed to maintain its security and efficiency in satisfactory levels. We also present the developed analytical models to analyze quantitatively the efficiency of the protocol. More specifically, we have developed models for assessing it in terms of signaling cost, which demonstrates that NeMHIP generates up to 73.47% less signaling compared to other relevant approaches. Therefore, the results obtained demonstrate that NeMHIP is the most efficient and secure solution for providing communications in mobile network scenarios such as in an ITS context.

Fluid flow induced calcium response in bone cell network

In our previous work, bone cell networks with controlled spacing and functional intercellular gap junctions had been successfully established by using microcontact printing and self assembled monolayers technologies [Guo, X. E., E. Takai, X. Jiang, Q. Xu, G. M. Whitesides, J. T. Yardley, C. T. Hung, E. M. Chow, T. Hantschel, and K. D. Costa. Mol. Cell. Biomech. 3:95-107, 2006]. The present study investigated the calcium response and the underlying signaling pathways in patterned bone cell networks exposed to a steady fluid flow. The glass slides with cell networks were separated into eight groups for treatment with specific pharmacological agents that inhibit pathways significant in bone cell calcium signaling. The calcium transients of the network were recorded and quantitatively evaluated with a set of network parameters. The results showed that 18 alpha-GA (gap junction blocker), suramin (ATP inhibitor), and thapsigargin (depleting intracellular calcium stores) significantly reduced the occurrence of multiple calcium peaks, which were visually obvious in the untreated group. The number of responsive peaks also decreased slightly yet significantly when either the COX-2/PGE(2) or the NOS/nitric oxide pathway was disrupted. Different from all other groups, cells treated with 18 alpha-GA maintained a high concentration of intracellular calcium following the first peak. In the absence of calcium in the culture medium, the intracellular calcium concentration decreased slowly with fluid flow without any calcium transients observed. These findings have identified important factors in the flow mediated calcium signaling of bone cells within a patterned network.

Analysis of a transcriptional network involving PU.1, Notch, and Gata3 in the lymphomyeloid lineage decision during early T-cell development

Hematopoiesis is a well-established system used to study developmental choices amongst cells with multiple lineage potentials, as well as the transcription factor network interactions that drive these developmental paths. Multipotent progenitors travel from the bone marrow to the thymus where T-cell development is initiated and these early T-cell precursors retain lineage plasticity even after initiating a T-cell program. The development of these early cells is driven by Notch signaling and the combinatorial expression of many transcription factors, several of which are also involved in the development of other cell lineages. The ETS family transcription factor PU.1 is involved in the development of progenitor, myeloid, and lymphoid cells, and can divert progenitor T-cells from the T-lineage to a myeloid lineage. This diversion of early T-cells by PU.1 can be blocked by Notch signaling. The PU.1 and Notch interaction creates a switch wherein PU.1 in the presence of Notch promotes T-cell identity and PU.1 in the absence of Notch signaling promotes a myeloid identity. Here we characterized an early T-cell cell line, Scid.adh.2c2, as a good model system for studying the myeloid vs. lymphoid developmental choice dependent on PU.1 and Notch signaling. We then used the Scid.adh.2c2 system to identify mechanisms mediating PU.1 and Notch signaling interactions during early T-cell development. We show that the mechanism by which Notch signaling is protecting pro-T cells is neither degradation nor modification of the PU.1 protein. Instead we give evidence that Notch signaling is blocking the PU.1-driven inhibition of a key set of T-regulatory genes including Myb, Tcf7, and Gata3. We show that the protection of Gata3 from PU.1-mediated inhibition, by Notch signaling and Myb, is important for retaining a T-lineage identity. We also discuss a PU.1-driven mechanism involving E-protein inhibition that leads to the inhibition of Notch target genes. This is mechanism may be used as a lockdown mechanism in pro-T-cells that have made the decision to divert to the myeloid pathway.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

Cell types, network homeostasis, and pathological compensation from a biologically plausible ion channel expression model.

How do neurons develop, control, and maintain their electrical signaling properties in spite of ongoing protein turnover and perturbations to activity? From generic assumptions about the molecular biology underlying channel expression, we derive a simple model and show how it encodes an "activity set point" in single neurons. The model generates diverse self-regulating cell types and relates correlations in conductance expression observed in vivo to underlying channel expression rates. Synaptic as well as intrinsic conductances can be regulated to make a self-assembling central pattern generator network; thus, network-level homeostasis can emerge from cell-autonomous regulation rules. Finally, we demonstrate that the outcome of homeostatic regulation depends on the complement of ion channels expressed in cells: in some cases, loss of specific ion channels can be compensated; in others, the homeostatic mechanism itself causes pathological loss of function.

A dual role for ErbB2 signaling in cardiac trabeculation.

Cardiac trabeculation is a crucial morphogenetic process by which clusters of ventricular cardiomyocytes extrude and expand into the cardiac jelly to form sheet-like projections. Although it has been suggested that cardiac trabeculae enhance cardiac contractility and intra-ventricular conduction, their exact function in heart development has not been directly addressed. We found that in zebrafish erbb2 mutants, which we show completely lack cardiac trabeculae, cardiac function is significantly compromised, with mutant hearts exhibiting decreased fractional shortening and an immature conduction pattern. To begin to elucidate the cellular mechanisms of ErbB2 function in cardiac trabeculation, we analyzed erbb2 mutant hearts more closely and found that loss of ErbB2 activity resulted in a complete absence of cardiomyocyte proliferation during trabeculation stages. In addition, based on data obtained from proliferation, lineage tracing and transplantation studies, we propose that cardiac trabeculation is initiated by directional cardiomyocyte migration rather than oriented cell division, and that ErbB2 cell-autonomously regulates this process.

A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.

In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.