Endometriosis is an invasive disease, and a leading cause of pain, infertility and disability among women, with an incidence 10 fold that of cancer. A more complete understanding of disease pathogenesis is essential for the development of non-surgical diagnostic assays and non-hormonal therapeutics. Avoidance of immune clearance and implantation of endometrial tissue on peritoneal surfaces are features of endometriosis lesion formation that overlap with cancer metastasis. Connexins, and the gap junctions they form, have been implicated in cancer progression, and may be associated endometriosis pathophysiology. Single cell transcriptomic profiling of endometrial epithelial and stromal cells from women with endometriosis reveals a striking and progressive shift in expression of connexins and related regulatory and junctional genes. We demonstrate that gap junction coupling between endometrial cells and the peritoneal mesothelium is dramatically induced, specifically in endometriosis patients, and is required for invasion by inducing breakdown of the mesothelial barrier function.

Chromothripsis is an event of genomic instability leading to complex chromosomal alterations in cancer. Frequent long-range chromatin interactions between transcription factors (TFs) and targets may promote extensive translocations and copy-number alterations in proximal contact regions through inappropriate DNA stitching. Although studies have proposed models to explain the initiation of chromothripsis, few discussed how TFs influence this process for tumor progression. This study focused on genomic alterations in amplification associated regions within chromosome 17. Inter−/intra-chromosomal rearrangements were analyzed using whole genome sequencing data of breast tumors in the Cancer Genome Atlas (TCGA) cohort. Common ERα binding sites were defined based on MCF-7, T47D, and MDA-MB-134 breast cancer cell lines using univariate K-means clustering methods. Nanopore sequencing technology was applied to validate frequent rearrangements detected between ATC loci on 17q23 and an ERα hub on 20q13. The efficacy of pharmacological inhibition of a potentially druggable target gene on 17q23 was evaluated using breast cancer cell lines and patient-derived circulating breast tumor cells. There are five adjoining regions from 17q11.1 to 17q24.1 being hotspots of chromothripsis. Inter−/intra-chromosomal rearrangements of these regions occurred more frequently in ERα-positive tumors than in ERα-negative tumors. In addition, the locations of the rearrangements were often mapped within or close to dense ERα binding sites localized on these five 17q regions or other chromosomes. This chromothriptic event was linked to concordant upregulation of 96 loci that predominantly regulate cell-cycle machineries in advanced luminal tumors. Genome-editing analysis confirmed that an ERα hub localized on 20q13 coordinately regulates a subset of these loci localized on 17q23 through long-range chromosome interactions. One of these loci, Tousled Like Kinase 2 (TLK2) known to participate in DNA damage checkpoint control, is an actionable target using phenothiazine antipsychotics (PTZs). The antiproliferative effect of PTZs was prominent in high TLK2-expressing cells, compared to low expressing cells. This study demonstrates a new approach for identifying tumorigenic drivers from genomic regions highly susceptible to ERα-related chromothripsis. We found a group of luminal breast tumors displaying 17q-related chromothripsis for which antipsychotics can be repurposed as treatment adjuncts.

Emerging evidence indicates that adipose stromal cells (ASC) are recruited to enhance cancer development. In this study, we examined the role these adipocyte progenitors play relating to intercellular communication in obesity-associated endometrial cancer. This is particularly relevant given that gap junctions have been implicated in tumor suppression. Examining the effects of ASCs on the transcriptome of endometrial epithelial cells (EEC) in an in vitro coculture system revealed transcriptional repression of GJA1 (encoding the gap junction protein Cx43) and other genes related to intercellular communication. This repression was recapitulated in an obesity mouse model of endometrial cancer. Furthermore, inhibition of plasminogen activator inhibitor 1 (PAI-1), which was the most abundant ASC adipokine, led to reversal of cellular distribution associated with the GJA1 repression profile, suggesting that PAI-1 may mediate actions of ASC on transcriptional regulation in EEC. In an endometrial cancer cohort (n = 141), DNA hypermethylation of GJA1 and related loci TJP2 and PRKCA was observed in primary endometrial endometrioid tumors and was associated with obesity. Pharmacologic reversal of DNA methylation enhanced gap-junction intercellular communication and cell-cell interactions in vitro. Restoring Cx43 expression in endometrial cancer cells reduced cellular migration; conversely, depletion of Cx43 increased cell migration in immortalized normal EEC. Our data suggest that persistent repression by ASC adipokines leads to promoter hypermethylation of GJA1 and related genes in the endometrium, triggering long-term silencing of these loci in endometrial tumors of obese patients. SIGNIFICANCE: Studies reveal that adipose-derived stem cells in endometrial cancer pathogenesis influence epigenetic repression of gap junction loci, which suggests targeting of gap junction activity as a preventive strategy for obesity-associated endometrial cancer.

Abstract Total osteopontin (OPN) is overexpressed in endometrium carcinoma (EC) and modifications at TP53 and PTEN genes correspond to major genetic alterations in these tumors. Althought total OPN expression has been correlated to p53 and PTEN expression, no data is available regarding OPN splice variants and their association to p53 and PTEN isoforms expression. OPN has three splicing isoforms (OPN-SI), named OPNa, OPNb and OPNc, while p53 has at least 12 variants, such as p53 (full lenght), p53β, p53γ, Δ40p53, Δ133p53 and Δ160p53. Moreover, PTEN splicing isoforms (PTEN-SI) are named vs-fl (full lenght variant), vs-3a-3c, vs-5a-5d, and vs-D6. We aimed to evaluate the expression profile of OPN, p53 and PTEN isoforms as a first approach to establish their presumptive associations and functional interactions in endometrial tumoral and non tumoral cells. Total RNA has been extracted from endometrial non-tumoral (E6/E7 and EM42) and tumoral (Ishikawa, RL95-2, AN3CA and KLE) cell lines, followed by cDNA synthesis. Transcriptional and protein expression patterns have been evaluated for OPN, p53 or PTEN isoforms, using quantitative real time PCR, immunoblot or immunofluorescence. OPNa is overexpressed in tumoral and non-tumoral cell lines in relation to OPNb and OPNc. Regarding p53, the full lengh isoform is overexpressed, followed by Δ40p53, in relation to other variants in EC cells. Conversely, Δ40p53 it is the major expressed transcript in both EM42 and E6/E7 non-tumoral cells. Otherwise, in EC and endometrial non-tumoral cells, PTEN vs-fl isoform is overexpressed in relation to the other splice variants. Specifically in E6/E7 cells, all PTEN-SI, except vs-3b e vs-D6, are overexpressed in relation to EC cell lines. At the protein level, both p53 and PTEN isoforms are overexpressed in E6/E7 cells, when compared to EC cells. Of note, distinct isoforms for most of these variant transcripts have not been detected at the protein level. Our data demonstrate that full lenght OPN, p53 and PTEN are the major expressed variants in EC tumor cell lines. However, most of these transcript variants display differential expression between endometrial tumor and non-tumoral cells and also among distinct EC cell lines. These data provide early evidence that these variants can differently modulate the expression and functional roles of their full length couterparts. Further work should investigate the impact of OPNa overerexpression on p53 and PTEN isoforms and their putative cellular and molecular roles on modulating p53 and PTEN tumor supressive roles. Citation Format: Vanessa F. Franco, Nataly dos Santos Melo, Iaci N. Soares, Ya-Ting Hsu, Ya-Ting, Tim H. Huang, Wallace M. Araujo, José A. Morgado-Diaz, Jerson L. Silva, Etel Rodrigues Pereira Gimba. Osteopontin, p53 and PTEN isoforms expression patterns in endometrium carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4482. doi:10.1158/1538-7445.AM2017-4482

Hsu and colleagues reported on EGF/EGFR–triggered activation of regulated intramembrane proteolysis (RIP) of EpCAM that induces an EMT phenotype ([1][1]). This finding is of utmost interest, as it might explain contradicting effects of EpCAM on adhesion, proliferation, and invasion and shed light

Abstract Overexpression of epithelial cell adhesion molecule (EpCAM) has been implicated in advanced endometrial cancer, but its roles in this progression remain to be elucidated. In addition to its structural role in modulating cell-surface adhesion, here we demonstrate that EpCAM is a regulatory molecule in which its internalization into the nucleus turns on a transcription program. Activation of EGF/EGFR signal transduction triggered cell-surface cleavage of EpCAM, leading to nuclear internalization of its cytoplasmic domain EpICD. ChIP-seq analysis identified target genes that are coregulated by EpICD and its transcription partner, LEF-1. Network enrichment analysis further uncovered a group of 105 genes encoding functions for tight junction, adherent, and cell migration. Furthermore, nanomechanical analysis by atomic force microscopy revealed increased softness and decreased adhesiveness of EGF-stimulated cancer cells, implicating acquisition of an epithelial–mesenchymal transition (EMT) phenotype. Thus, genome editing of EpCAM could be associated with altering these nanomechanical properties towards a less aggressive phenotype. Using this integrative genomic–biophysical approach, we demonstrate for the first time an intricate relationship between EpCAM-regulated transcription and altered biophysical properties of cells that promote EMT in advanced endometrial cancer. Cancer Res; 76(21); 6171–82. ©2016 AACR.

Abstract Epithelial cell adhesion molecule (EpCAM), a membrane protein known to modulate cell-cell adhesion, is also a regulatory molecule internalized into the nucleus for transcriptional control of gene expression. Here we demonstrate that activated EGF/EGFR is a signaling factor to drive the cleavage of the extracellular fragment EpEX culminating in removal of cell-surface EpCAM as monitored with recognition atomic force microscopy (AFM). As a result, internalization of the cytoplasmic domain EpICD leads to formation of transcription factor complexes with LEF1 that regulate gene transcription for enhancing mobility functions of cancer cells. Comprehensive probing with AFM further reveals increased elasticity and decreased adhesiveness of these cells, implicating acquisition of an epithelial-mesenchymal transition phenotype. While EpCAM cleavage contributes to the loss of cell-surface adhesiveness, its internalized EpICD additionally regulates targets for promoting cell migration. Thus, this EGF/EGFR-modulated action on structural EpCAM and regulatory EpICD can enhance invasion potential of transformed cells. Citation Format: Ya-Ting Hsu, Pawel A. Osmulski, Yao Wang, Yi-Wen Huang, Lu Liu, Jianhua Ruan, Victor X. Jin, Nameer B. Kirma, Maria E. Gaczynska, Tim H.M. Huang. Dual role of EpCAM cleavage in adhesion attenuation and transcription enhancement for cell migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2871.

To develop accurate prognostic models is one of the biggest challenges in "omics"-based cancer research. Here, we propose a novel computational method for identifying dysregulated gene subnetworks as biomarkers to predict cancer recurrence. Applying our method to the DNA methylome of endometrial cancer patients, we identified a subnetwork consisting of differentially methylated (DM) genes, and non-differentially methylated genes, termed Epigenetic Connectors (EC), that are topologically important for connecting the DM genes in a protein-protein interaction network. The ECs are statistically significantly enriched in well-known tumorgenesis and metastasis pathways, and include known epigenetic regulators. Importantly, combining the DMs and ECs as features using a novel random walk procedure, we constructed a support vector machine classifier that significantly improved the prediction accuracy of cancer recurrence and outperformed several alternative methods, demonstrating the effectiveness of our network-based approach.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Abstract Purpose: Endometrial cancer is a common gynecologic cancer whose incidence is increasing annually worldwide. Current methods to detect endometrial cancer are unreliable and biomarkers are unsatisfactory for screening. Cervical scrapings were reported as a potential source of material for molecular testing. DNA methylation is a promising cancer biomarker, but limited use for detecting endometrial cancer. Experimental Design: We analyzed two methylomics databases of endometrioid-type endometrial cancer. Using nonnegative matrix factorization algorithm clustered the methylation pattern and reduced the candidate genes. We verified in pools DNA from endometrial cancer tissues and cervical scrapings, and validated in 146 cervical scrapings from patients with endometrioid-type endometrial cancer (n = 50), uterine myoma (n = 40), and healthy controls (n = 56) using quantitative methylation–specific PCR (QMSP). The logistic regression was used to evaluate the performance of methylation signal and gene combination. Results: We filtered out 180 methylated genes, which constituted four consensus clusters. Serial testing of tissues and cervical scrapings detected 14 genes that are hypermethylated in endometrial cancer. Three genes, BHLHE22, CDO1, and CELF4, had the best performance. Individual genes were sensitivity of 83.7%–96.0% and specificity of 78.7%–96.0%. A panel comprising any two of the three hypermethylated genes reached a sensitivity of 91.8%, specificity of 95.5%, and odds ratio of 236.3 (95% confidence interval, 56.4–989.6). These markers were also applied to cervical scrapings of type II endometrial cancer patients, and detected in 13 of 14 patients. Conclusions: This study demonstrates the potential use of methylated BHLHE22/CDO1/CELF4 panel for endometrial cancer screening of cervical scrapings. Clin Cancer Res; 23(1); 263–72. ©2016 AACR.

Abstract Endometrial cancer (EC) is the most common gynecologic malignancy, with approximately 20% of EC patients developing advanced stage recurrent tumors and frequent metastasis. Our goal was to investigate whether DNA methylation signatures associated with low promoter methylation (hypomethylation) and specific oncogenic signaling delineate predictive markers of endometrial cancer recurrence. Global screening by Methyl-CpG-capture sequencing revealed aberrant DNA methylation in our endometrial cancer cohort and identified a subset of bone morphogenetic protein family (BMP1, 2, 3, 4, and 7) exhibiting frequent hypomethylation in primary tumors with subsequent recurrence compared with non-recurrent tumors. This epigenetic signature correlated with poor survival and was validated in The Cancer Genome Atlas endometrial cancer cohort. Our functional studies also implicated epidermal growth factor receptor (EGFR) pathway in the transcriptional activation of these BMPs and inducing epithelial-mesenchymal transition (EMT). In addition to AKT and MAPK, the epithelial cell adhesion molecule (EpCAM) mediated these actions by EGFR. EpCAM involvement in cancer progression includes nuclear co-translocation of its intracellular domain EpICD with Lef-1 complexes and targeting oncogene promoter activation. In this study, EGF stimulated EpICD-Lef-1 binding on BMP genes accompanied with histone active modification marks. EpICD knockdown resulted in increased repressive histone marks and DNA methylation at these loci, suggesting that EpICD occupancy is involved in their epigenetic modification to an open transcriptional conformation. Knockdown of candidate BMPs led to decreased endometrial cancer cell invasiveness, implicating them in aggressive growth. Extending our studies, we performed ChIP-Seq analysis to identify global regulation by the EpICD-Lef-1 complexes in endometrial cancer. Interestingly, under basal levels only about 28% of loci targeted by either EpICD or Lef-1 were commonly targeted by EpICD-Lef-1, with common targets increasing to about 50% in 24 hrs and 73% in 48 hrs post EGF treatment. This indicates that EGFR signaling stimulates a time-course dependent enrichment of EpICD-Lef-1 convergence on target loci. Our initial studies of these EpICD target pathways included genes with cell adhesion functions. Future studies on the regulation of the EpICD-Lef-1 regulated genes and their mechanisms of action in aggressive endometrial cancer will provide a better understanding of this gene network in endometrial cancer. Hypomethylation signatures of candidate loci in this regulatory network may present putative predictive markers of poor survival and which may be used to tailor individualized therapy. Citation Format: Ya-Ting Hsu, Fei Gu, Yi-Wen Huang, Joseph Liu, Jianhua Ruan, Rui-Lan Huang, Chiou-Miin Wang, Chun-Liang Chen, Rohit R. Jadhav, Yao Wang, Victor X. Jin, Hung-Cheng Lai, David G. Mutch, Paul J. Goodfellow, Ian M. Thompson, Nameer B. Kirma, Tim H.M. Huang. EpCAM-mediated hypomethylation of BMP and cell adhesion genes is associated with advanced endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1376. doi:10.1158/1538-7445.AM2014-1376

Abstract The endometrium is the major target of oncogenesis in the uterus. Endometrial tissue undergoes several cycles of disintegration and repair during women's reproductive years. The involvement of repair mechanisms that include transforming growth factor beta (TGF-beta) pathway may entail epithelial msenchymal transition (EMT) known to be involved in tissue repair. This suggests that the EMT program is inherent in endometrial epithelial cells and may be subverted during the development of invasive disease, whether benign such as endometriosis or malignant such as endometroid endometrial carcinoma. We therefore examined candidate mechanistic modulators of EMT in normal and malignant endometrial epithelial cells as well as endometrial carcinoma samples using cell signaling, gene expression and epigenetic regulation methodologies. Our evidence strikingly show that although both TGF-beta and epidermal growth factor (EGF) stimulated primary normal endometrial epithelial cell invasiveness, EGF was the main inducer of EMT in these cells. EGF also strongly induced EMT in endometrial cancer cells. In normal and malignant cells, Raf-1/MAPK mediated EGF actions. Interestingly, EGF stimulated epithelial adhesion molecule (EpCAM) cleavage and internalization of its intracellular domain EpICD into the nucleus to activate EMT-related genes such as mesenchymal cadherins. This was associated with loss of the epithelial marker E-cadherin and upregulation of its negative regulator Snail1. The involvement of EMT in aggressive endometrial malignancy was further confirmed in a permissive transcriptional epigenetic profile of mesenchymal cadherins in endometrial cancers with poor survival compared to a silenced epigenetic signature in less aggressive tumors. This epigenetic regulation including DNA methylation and histone modification was mediated by EpCAM, as shown in EpCAM-knockdown endometrial cancer cells, suggesting that EpCAM binding to target promoters maintains an open transcriptional conformation. Combined, our data suggest that while plasticity is required for endometrial regeneration during the menstrual cycles, committed epigenetic signatures may alter the path of normal tissue repair to aberrant growth associated with benign tumors or malignant growth. In concert, activation by cell signaling such as EGF/EpCAM may determine the extent of invasiveness and disease progression. Citation Format: Ya-Ting Hsu, Joseph Liu, Peter A. Binkley, Robert S. Schenken, Rajshwar R. Tekmal, Tim H.-M. Huang, Nameer B. Kirma. Parallel EMT pathways mediated by epidermal growth factor, EpCAM and mesenchymal cadherins in benign endometriotic lesions and endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3326. doi:10.1158/1538-7445.AM2014-3326

DNA hypermethylation of promoter CpG islands is associated with epigenetic silencing of tumor suppressor genes in oral squamous cell carcinomas (OSCCs). We used a methyl-CpG-binding domain protein capture method coupled with next-generation sequencing (MBDCap-seq) to survey global DNA methylation patterns in OSCCs with and without nodal metastasis and normal mucosa (total n 58). Of 1462 differentially methylated CpG islands identified in OSCCs relative to normal controls, MBDCap-seq profiling uncovered 359 loci linked to lymph node metastasis. Interactive network analysis revealed a subset of these loci (n 23), including the anaplastic lymphoma kinase (ALK) gene, are potential regulators and effectors of invasiveness and metastatic progression. Promoter methylation of ALK was preferentially observed in OSCCs without node metastasis, whereas relatively lower methylation levels were present in metastatic tumors, implicating an active state of ALK transcription in the latter group. The OSCC cell line, SCC4, displayed reduced ALK expression that corresponded to extensive promoter CpG island methylation. SCC4 treatment with demethylating agents induced ALK expression and increased invasion and migration characteristics. Inhibition of ALK activity in OSCC cells with high ALK expression (CAL27, HSC3 and SCC25), decreased cell growth and resulted in changes in invasive potential and mesenchymal marker expression that were cell-line dependent. Although ALK is susceptible to epigenetic silencing during oral tumorigenesis, overwriting this default state may be necessary for modulating invasive processes involved in nodal metastases. Given the complex response of OSCC cells to ALK inhibition, future studies are required to assess the feasibility of targeting ALK to treat invasive OSCCs.

Abstract Purpose: Epigenetic regulation by promoter methylation plays a key role in tumorigenesis. Our goal was to investigate whether altered DNA methylation signatures associated with oncogenic signaling delineate biomarkers predictive of endometrial cancer recurrence. Experimental Design: Methyl-CpG-capture sequencing was used for global screening of aberrant DNA methylation in our endometrial cancer cohort, followed by validation in an independent The Cancer Genome Atlas (TCGA) cohort. Bioinformatics as well as functional analyses in vitro, using RNA interference (RNAi) knockdown, were performed to examine regulatory mechanisms of candidate gene expression and contribution to aggressive phenotype, such as epithelial–mesenchymal transition (EMT). Results: We identified 2,302 hypermethylated loci in endometrial tumors compared with control samples. Bone morphogenetic protein (BMP) family genes, including BMP1, 2, 3, 4, and 7, were among the frequently hypermethylated loci. Interestingly, BMP2, 3, 4, and 7 were less methylated in primary tumors with subsequent recurrence and in patients with shorter disease-free interval compared with nonrecurrent tumors, which was validated and associated with poor survival in the TCGA cohort (BMP4, P = 0.009; BMP7, P = 0.007). Stimulation of endometrial cancer cells with epidermal growth factor (EGF) induced EMT and transcriptional activation of these genes, which was mediated by the epithelial cell adhesion molecule (EpCAM). EGF signaling was implicated in maintaining the promoters of candidate BMP genes in an active chromatin configuration and thus subject to transcriptional activation. Conclusions: Hypomethylation signatures of candidate BMP genes associated with EpCAM-mediated expression present putative biomarkers predictive of poor survival in endometrial cancer. Clin Cancer Res; 19(22); 6272–85. ©2013 AACR.

DNA methylation, a well-studied mechanism of epigenetic regulation, plays important roles in cancer. Increased levels of global DNA methylation is observed in primary solid tumors including endometrial carcinomas and is generally associated with silencing of tumor suppressor genes. The role of DNA methylation in cancer recurrence after therapeutic intervention is not clear. Here, we developed a novel computational method to analyze whole-genome DNA methylation data for endometrial tumors within the context of a human protein-protein interaction (PPI) network, in order to identify subnetworks as potential epigenetic biomarkers for predicting tumor recurrence. Our method consists of the following steps. First, differentially methylated (DM) genes between recurrent and non-recurrent tumors are identified and mapped onto a human PPI network. Then, a PPI subnetwork consisting of DM genes and genes that are topologically important for connecting the DMs on the PPI network, termed epigenetic connectors (ECs), are extracted using a Steiner-tree based algorithm. Finally, a random-walk based machine learning method is used to propagate the DNA methylation scores from the DMs to the ECs, which enables the ECs to be used as features in a support vector machine classifier for predicting recurrence. Remarkably, we found that while the DMs are not enriched in any cancer-related pathways, the ECs are enriched in many well-known tumorgenesis and metastasis pathways and include known epigenetic regulators. Moreover, combining the DMs and ECs significantly improves the prediction accuracy of cancer recurrence and outperforms several alternative methods. Therefore, the network-based method is effective in identifying gene subnetworks that are crucial both for the understanding and prediction of tumor recurrence.

In 2009 UnrealIRCd 3.2.8.1, an IRC (Internet Relay Chat) server, was replaced by a version with a backdoor at its mirror sites. It was not detected until seven months later and it had caused irrevocable damages in IRC services. It is of vital importance and also a challenge to detect implanted malicious code in newly developed systems before their deployment. We apply machine learning to uncover a system implementation structure that includes its normal functions from the design, as well as the hidden malicious behaviors. Published works with machine learning often assume that systems are completely specified. Unfortunately, practical system implementations are usually incompletely specified and the prevalent algorithms do not apply. We design generalized and efficient machine learning algorithms for incompletely specified protocol system implementations for detecting implanted malicious code. We further extend the results where machine learning starts from an approximate model instead of an empty conjecture - a usual approach of machine learning algorithms, and our approach learns an implementation structure more efficiently than the known algorithms. We implement and apply our method to two case studies: an IRC server with backdoor and an MSN client with message flooder. Experiments show that our procedures successfully and efficiently detect the implanted malicious behaviors.

As computer networks turn into an indispensable part of technology and entertainment of human life today, security and reliability become the forefront issue of network protocol design and implementation. Any security or reliability flaws in network protocol operation can lead to great loss in private data, business opportunity and reputation. As a result, methods and tools for preventing and detecting such security breach in network protocol design and implementation are urgently needed. Formal methods have proven to be successful in system verification and testing. This thesis is devoted to formally design algorithm and tools to efficiently and effectively investigate security of both protocol specification and implementation.For protocol specification, we first model a protocol as a state machine. To verify certain security property against the state machine, we need to explore all reachable states in the state machine. However, traditional reachability problem suffers from the classic state space explosion problem. We propose two techniques to tackle this problem: trace inclusion transformation and online minimization. We first transform the original state machine to a simplified machine which is a trace inclusion transformation of the original machine by removing certain system parameters. We apply online minimization to construct a minimal reachable graph of manageable size that is bisimulation equivalent to the reachability graph of the simplified machine. Then we can verify the desired security property against the minimal reachable graph. However, we may introduce false positive and false negative since we have simplified the original machine. In the second phase of analysis, we show that there is no false negative if the simplified machine is a trace inclusion transformation of the original machine. We also design algorithm to eliminate false positive. Then we can conclude if the state machine (the protocol design) is security against the checked security property.However, even with perfect design, fault can still be introduced during the implementation and integration phase. Due to the black-box nature of protocol implementation and often unavailability of specification, traditional verification techniques do not apply here. In this thesis, we first automatically synthesize a formal model to represent the behavior of the implementation and then use the synthesized model to assist different security flaw detection applications. We study both active and passive synthesize approaches and compare their advantages and disadvantages. We incorporate the formal model to two different security flaw detection applications. In the first application, we build a model based fuzz testing framework in which the formal model is used to guide test input selection and served as test coverage criteria. Our framework significantly improves existing black-box protocol security testing techniques. In the second application, we examine the synthesized model to determine if there is any malicious function hidden inside a protocol implementation and determine the behavior of the hidden function.We show the proposed approaches are effective with extensive case studies for various scenarios and applications. Nevertheless, the techniques and framework proposed in this thesis is generic and could be easily adapted for different problem domains and applications.

A major hurdle of formal analysis of protocol security properties is the well-known state explosion - a protocol system usually contains infinitely many or a formidable number of states. As a result, most of the analysis resorts to heuristics, such as state space pruning. Given the temporal property of authentication and authorization protocols, we introduce trace inclusion transformation of protocol specification to reduce significantly the state space. We further cut down the number of states by online minimization for obtaining a model of a manageable size for a formal and rigorous analysis. However, the two state space reduction procedures may result in false negative and false positives. We show that our trace inclusion transformation and online minimization do not introduce any false negative. On the other hand, we design an efficient algorithm for ruling out all the possible false positives. Therefore, our analysis is sound and complete. For a case study, we analyze OAuth, a standardization of API authentication protocols. Our automated analysis identifies a number of attacks in the original specification, including the one that has been detected. We also analyze the second version of OAuth and prove it is secure if the API interface is secure.

In geophysical tomography, a proper model parameterization scheme for forward modeling is not necessarily a suitable one for the inversion stage, and vice versa. To take full advantage of the merits of parameterization in both stages, we propose a two‐step model parameterization approach, in which different model bases for forward computation and inversion are adopted and the basis change is achieved by applying a spatial projection directly to the sensitivity matrix. We demonstrate this approach through an experimental study of waveform tomography for the Pacific upper mantle shear wave structure using first‐orbit long‐period Rayleigh waves. In the forward modeling, a normal‐mode‐based nonlinear asymptotic coupling theory is used for the computation of the synthetics and sensitivity matrix, and the model is parameterized in terms of spherical harmonics which provide efficient analytical solutions for path integrals in the forward modeling. Prior to the inversion, the model basis of the sensitivity matrix is transformed to local functions within the study region. After mapping, only local bases around the data sampling path receive effective sensitivities. Accordingly, the computation cost in the inversion is significantly reduced. Furthermore, the two‐step model parameterization also adds flexibility to the inversion schemes. In particular, a wavelet‐based multiscale inversion is implemented, and its results are compared to simple damping solutions. The general concept and applications of the two‐step model parameterization are not restricted to the forwarding modeling technique or model parameterization schemes employed in this experimental study. This approach benefits any inverse problems wherever transformation of model bases helps to better constrain the results.

Aminopropyl-functionalized mesoporous KIT-5 materials have been synthesized by co-condensation using carboxyl-terminated Pluronic F127 as a structure-directing agent. The KIT-5 materials synthesized with carboxyl- or hydroxyl-terminated copolymers had distinct physicochemical properties. The carboxyl end groups were crucial for forming ordered materials with high aminopropyl content, but the structural ordering decreased with increasing the loading of functional groups. While analyses suggested that significant amount of the aminopropyl groups were embedded in the silica matrix, part of them could still contribute to the chemisorption of carbon dioxide. The aminopropyl-functionalized mesoporous KIT-5 materials were applied to deposit gold, resulting in small (2−3 nm) nanoparticles uniformly dispersed inside the interconnected cagelike mesopores.