Integrated Genomic Studies to Discern Tumor Heterogeneity and Prognostic Subgroups in Hepatocellular Carcinoma
Globally, hepatocellular carcinoma (HCC) accounts for 70-85% of primary liver cancers and is the second leading cause of male cancer death. Among patients there is widespread tumor heterogeneity, yet only a single FDA-approved therapeutic, sorafenib, for standard of care in advanced HCC. Identifying homogeneous subgroups of patients will allow development of effective treatments targeted to specific pathways that drive poor survival. In this study, I took both a specific and global approach to characterize HCC tumors with poor prognosis. In the specific approach, I focused on serum alpha-fetoprotein (AFP), a diagnostic biomarker for HCC. AFP, normally only expressed in the liver during fetal development, is reactivated in 60% of HCC tumors and associated with poor outcome. Therefore, I hypothesized that AFP+ and AFP- tumors differ biologically. Using microRNA and mRNA microarray-profiling in predominantly Hepatitis B+ HCC cases from Zhongshan Hospital, I found that miR-29 family members were the most significantly down-regulated miRNAs in AFP+ HCC. Moreover, global DNA methylation profiling revealed that increased methylation was associated with AFP+ HCC. Experimentally, I demonstrated that AFP inhibited transcription of the miR-29a/b-1 locus and this effect is mediated through the c-MYC protein. These findings suggest that AFP is a functional antagonist of miR-29, which may contribute to epigenetic alterations and poor prognosis in AFP+ HCC. In the more global approach to clarify tumor heterogeneity, I aimed to integrate data from multiple platforms to define prognostic tumor subgroups. First, we identified the tumor-specific genes in two complementary platforms: Illumina 27k methylation and Affymetrix gene expression. An epigenetic-driven signature was derived from the tumor-specific genes that inversely correlated with methylation. Employing the epigenetic-driven gene signature along with a somatic copy number alteration (SCNA)-driven signature developed by the lab, we found that epigenetic changes drive poor outcome in one subset of patients while SCNA-related changes drive poor outcome in a different group. In summary, I found that HCC tumors could be separated into prognostic subgroups based on AFP level, as well as epigenetic-driven or SCNA-driven genetic changes. Future work will elucidate key signaling pathways activated in each subgroup to better characterize the differences in HCC tumors.
ERRβ Splice Variants Differentially Regulate Cell Cycle Progression
Orphan receptors comprise nearly half of all members of the nuclear receptor superfamily. Despite having broad structural similarities to the classical estrogen receptors, estrogen-related receptors (ERRs) have their own unique DNA response elements and functions. In this study, we focus on two ERRβ splice variants, short form ERRβ (ERRβsf) and ERRβ2, and identify their differing roles in cell cycle regulation. Using the acyl hydrazone DY131 (a synthetic agonist of ERRβ) as a tool to modulate endogenous ERRβ function, splice-variant selective shRNA, and exogenous ERRβsf and ERRβ2 cDNAs, we differentiate between splice variant function in cell cycle regulation. We demonstrate the role of ERRβsf in mediating the G1/S checkpoint through p21. We also show ERRβsf is required for DY131-induced cellular senescence. A key novel finding of this study is that ERRβ2 can mediate a G2/M arrest. Furthermore, in the absence of ERRβ2, the G2/M arrest is reversed and ERRβsf now induces p21 and initiates a G1/S arrest. These data imply a potential dominant inhibitory role for ERRβ2 on ERRβsf. Furthermore, we demonstrate DY131 cytotoxic sensitivity is dependent on p53 status. In the absence of wild-type p53 (null or mutated), activated ERRβ2 initiates an apoptotic response. In wild-type p53, ERRβsf induces p21 in a cytoprotective response and the magnitude of cell death is lessened. This study illustrates both novel functions for ERRβ splice variants as well as evidence for splice variant interaction.
Social Isolation Stress, Obesity, and Breast Cancer Risk in Mice
Social isolation is a potent psychosocial stressor and is associated with obesity and increased breast cancer risk and mortality. In this thesis, I investigated the combined effects of social isolation stress and obesity on insulin resistance, mammary tumorigenesis, and the mechanisms involved.; First, I studied the effects of social isolation stress in combination with an obesity inducing diet (OID) on mammary tumorigenesis induced by medroxyprogesterone acetate (MPA) and 7,12-dimethylbenz(a)anthracene (DMBA) in female C57BL/6 mice. Social isolation, with or without OID, increased food intake and body weight, and caused impaired insulin and glucose tolerance. Serum NPY levels were increased only in socially isolated OID fed mice, whilst adiponectin levels were reduced both by OID and social isolation. These changes may have contributed to insulin resistance. Mammary tumorigenesis was significantly higher in the socially isolated OID fed mice than in group-housed obese or control mice. Consistent with a previous study indicating that social isolation does not increase mammary tumorigenesis in p53 knockout mice, we found an increase in p53 mRNA expression in socially-isolated mice that exhibited increased mammary cancer risk. Apoptosis was not altered in these mice, but cell proliferation was significantly increased. Social isolation increased the expression of p53-induced autophagy-linked genes and other genes indicative of autophagy in the mammary gland. In summary, we found that obesity potentiates the effects of social isolation on mammary carcinogenesis, but primarily social isolation induced autophagy and cell proliferation but not apoptosis.; Next, histone deacetylase inhibitor (HDACi) and DNA methyltransferase inhibitor (DNMTi) were administered in drinking water to group-housed or socially-isolated OID fed mice. Insulin resistance and body weight gain were reversed with these inhibitors. However, HDACi/DNMTi treatment did not reverse mammary tumorigenesis and neither did they reverse up-regulation of p53 or alter the expression of cell cycle and cell death genes. Taken together, these data suggest that insulin resistance in obese mice may result from epigenetically-induced changes and therefore DNMTi/HDACi drugs may be useful in preventing weight gain and improving insulin sensitivity, but the increase in mammary tumorigenesis in socially-isolated obese mice is not caused by an increase in DNMT and HDAC activity.
In Vivo DNA Repair Mechanisms and Carcinogenesis
The maintenance of the genome is an important barrier to carcinogenesis, and one of the major cellular pathways involved in genome maintenance is DNA repair. There are several types of DNA repair pathways, but one type, the base excision repair (BER) pathway repairs the majority of endogenous DNA. Understanding how repair deficiencies predispose cells to damage accumulation and mutation is particularly important to understanding the carcinogenic process. In the first theme (Theme I) of this work, two types of BER deficiencies are explored. The first study involves inflammation-mediated carcinogenesis and the decrease of BER capacity under inflammatory conditions observed in some studies (Theme IA). The types of lesions repaired by the BER pathway are often the result of inflammatory processes, and, in fact, chronic inflammatory diseases are often pre-malignant conditions and confer high risk for cancer development. This highlights the importance of BER in inflammatory diseases as a critical "tumor suppressor" pathway. In an animal model of inflammation-mediated hepatocarcinogenesis, the Long Evans Cinnamon (LEC) rat, we observed a significant decrease in BER activity in liver tissues during acute hepatitis and determined through extensive gene expression microarray analysis that early carcinogenic changes occurred during acute hepatitis. We hypothesized that decreased BER during acute hepatitis induced oncogenic mutations and pursued the development of an appropriate cell culture model to test this hypothesis. We developed a hepatocyte cell line from the LEC rat during acute hepatitis (LEC-AH) and a tumor cell line from a LEC rat with HCC (LEC-T). The LEC-AH cell line exhibited decreased BER and increased DNA damage, which was consistent with the observations in LEC tissues. Furthermore, we determined that the LEC-T cell line harbored an activating mutation in codon 12 of the K-ras oncogene which was characteristic of deficient BER. The LEC-AH cell line characterization and the mutation observed in LEC-T cells provide an appropriate model in which to perform lesion mapping and establish a link between deficient BER during acute hepatitis and oncogenic mutation. The second study examines the role of mutation site-specific deficiencies in repair of an adduct, 1, N6-ethenoadenine (eA), associated with pre-malignant inflammatory disease and the relationship of sequence-dependent deficiencies to the location and pattern of mutation sites in the tumor suppressor gene p53 (Theme IB). We determined that eA at frequently mutated codons in p53 was dramatically impaired compared to eA at non-mutation sites. We determined the mechanism of slow repair at mutation sites was codon-specific slow turnover of the BER enzyme, N-methylpurine DNA glycosylase (MPG), the enzyme that initates repair of eA. Finally, one of the disadvantages inherent in the study of BER mechanisms is the lack of appropriate in vivo methodology to study the pathway in a physiologically relevant manner. Therefore, the second theme (Theme II) describes the development of a fluorescence microscopy-based technique to monitor interactions between DNA repair proteins and DNA adducts in vivo. We utilized the interaction of a well-known DNA repair protein, human apurinic apyrimidinic endonuclease 1 (APE1) with its substrate, the abasic (AP) site, as a model. The method developed borrowed from techniques generally used to detect protein-protein interactions, such as co-localization and fluorescence resonance energy transfer (FRET), and was able to reliably monitor functional APE1 interaction with AP-site DNA. Importantly, the method was also able to distinguish specific and non-specific interaction over time.
Regulation of MicroRNAs Targeting the Angiogenic Switch Molecule Fibroblast Growth Factor Binding Protein 1 by Retinoic Acid Receptor Activation
This dissertation examines the role of retinoic acid receptor activation in the post-transcriptional regulation of a fibroblast growth factor binding protein. Previous work showed that all-trans retinoic acid (ATRA) reduces mRNA expression of the angiogenic switch molecule, Fibroblast Growth Factor Binding Protein 1 (FGFBP1 or FGF-BP), independent of an effect on transcription of the FGFBP1 mRNA. I hypothesized that a retinoid-induced microRNA was involved in FGFBP1 mRNA loss. MicroRNAs (miRs) are 19-22 nucleotide (nt) single stranded non-coding RNAs that post-transcriptionally repress mature mRNA function, thereby reducing expression of their target proteins. The current dogma suggests that miRs canonically bind to the 3' untranslated region (UTR) of mRNA through a 7-nt seed-matched site. However, recent data indicate that miRs may also bind the open reading frame (ORF) of mRNAs. In this dissertation, I show that miR-27b-3p and miR-125a-5p are induced by ATRA and target FGFBP1. Overexpression of miR-27b-3p and miR-125a-5p rapidly reduced FGFBP1 mRNA levels through a target site in the open reading frame of the FGFBP1 mRNA. Both microRNAs showed specificity for regions within the ORF of FGFBP1, suggesting that these microRNAs may also be involved in inhibiting translation of the FGFBP1 protein. Next generation sequencing data from The Cancer Genome Atlas shows that loss of these microRNAs is characteristic of several epithelial cancers, including head and neck, lung, and cervical squamous cell carcinomas, suggesting a tumor suppressor role for miRs 27a-3p and 125a-5p. In total, these data suggest an important regulatory role for miRs 27b-3p and 125a-5p in the oncogenesis of squamous cell carcinomas, through modulation of FGFBP1 expression.
Molecular Modulation of Estrogen-Induced Apoptosis in Long-Term Estrogen-Deprived Breast Cancer Cells
Estrogen receptor (ER)-positive breast cancer cell lines have been instrumental in modeling breast cancer and providing an opportunity to document the development and evolution of acquired anti-hormone resistance. Models of long-term estrogen-deprived breast cancer cells are utilized in the laboratory to mimic clinical aromatase inhibitor-resistant breast cancer and serve as a tool to discover new therapeutic strategies. The MCF-7:5C and MCF-7:2A subclones were generated through long-term estrogen deprivation of ER-positive MCF-7 cells, and represent anti-hormone resistant breast cancer. MCF-7:5C cells paradoxically undergo estrogen-induced apoptosis within seven days of estrogen (estradiol, E2) treatment; MCF-7:2A cells also experience E2-induced apoptosis but evade dramatic cell death until approximately 14 days of treatment. Our data suggest that MCF-7:2A cells employ stronger antioxidant defense mechanisms than do MCF-7:5C cells, and that oxidative stress is ultimately required for MCF-7:2A cells to die in response to E2 treatment. Tumor necrosis factor (TNF) family member activation also correlates with E2-induced apoptosis in MCF-7:2A cells; up-regulation of TNFα occurs simultaneously with oxidative stress activation. Additionally, increased insulin-like growth factor receptor beta (IGF-1Rβ) confers a mechanism of growth and anti-apoptotic advantage in MCF-7:2A cells.; Hormone replacement therapy (HRT) is widely used to manage menopausal symptoms in women, and can comprise an estrogen alone or an estrogen combined with a progestin. The Women's Health Initiative demonstrated in their randomized trials that estrogen alone HRT decreases the risk of breast cancer in post-menopausal women, while combined estrogen plus a progestin (medroxyprogesterone acetate, MPA) HRT increases this risk. We sought to elucidate the mechanism through which these opposing effects occur. The data suggests that MPA acts as a glucocorticoid which blocks estrogen-induced apoptosis in long-term estrogen-deprived breast cancer cells thereby increasing cancer risk.
Roberts, Jenifer, 1943- author
Lambert, Arnaud F., author
Authored By: Derek Featherstone
Released On: 8/15/2014
The Internet has removed many obstacles to communication and interaction. However, when websites, technologies, or tools are poorly designed, they can create barriers that exclude people with disabilities from using the web. In this course, you'll learn how accessibility lowers the barrier to entry, by providing equal access and opportunity to people with a diverse range of hearing, movement, sight, and cognitive abilities. Derek Featherstone introduces the core concepts of accessibility as they apply to UX design, including an overview of the assistive technology visitors may use to access your projects. He includes practical examples, from re-creating visual interactions to writing great alternative text; introduces different personas and their unique challenges navigating the web; and shows how to make accessibility a regular part of your design workflow. Once you learn the foundations, you'll find that the benefits of accessibility—enhanced search, improved usability, and increased audience reach—aid your designs almost as much as they aid your users.
Authored By: Todd Howard
Released On: 8/15/2014
Songwriting in Logic Pro is an in-depth course geared toward musicians and songwriters who are starting to use Logic Pro to write, record, and mix their songs. There are two groups of techniques: one to get your demo ideas down quickly, and another to polish rough ideas into songs worth presenting to your band mates, producer, publisher, or label. Author Todd Howard takes you through his creative process in real time, starting with an empty project based on one of Logic's prebuilt templates. Then he records the initial song idea and creates a groove with Drummer. He works on the lyric and then lays down additional tracks—a driving bassline, acoustic guitar, and some scratch vocals—and builds out the arrangement with layered keyboard parts. After using improvisation to create a bridge section, he adds effects for a layered and nuanced song. Next he shows techniques for editing and mixing the track, using EQ, compression, reverb, and delay. Finally, Todd shows how to bounce down the track and share it with the world.
Want to learn about other tools for songwriting? Check out our Songwriting series, which covers other DAWs like Pro Tools and GarageBand.
Authored By: Brian Thurston Bralczyk
Released On: 8/15/2014
If users really like your website or app, they're not logging in from a single device. They're visiting from their desktop at work, their phone on the train, and their tablet at home. Your design also needs to perform elegantly and consistently on all platforms. In this course, Brian Thurston shows how to design complete experiences that take visitors from one device to another, and back again. You'll leave with a solid understanding of the planning it takes to ensure your site is prepared for success, the importance of flexible content creation and preparation, and special considerations involved in designing for touch screens. Brian also takes a look forward at the impact wearable devices and future tech will have on the UX designs of today.
Authored By: Brad Batesole
Released On: 8/15/2014
Learn how to leverage the most powerful tools in Facebook and manage large ad campaigns across different accounts and pages. Facebook advertising expert Brad Batesole shares a new set of advanced, time-saving techniques, such as bulk uploading ad images, using tags to organize campaigns, making campaign-wide changes quickly, and creating hundreds of custom ads on the spot.
The course also covers conversion tracking with conversion pixels, and retargeting users who have previously interacted with your brand's page or website. Plus, learn how to use the Power Editor to quickly duplicate ads with different targeting or imagery, and move ads between accounts. And last but not least, Brad reveals tips for mimicking A/B testing of ads and generating more leads.
If you need a Facebook advertising refresher before diving into these advanced tactics, check out Facebook Advertising Fundamentals.
Illness as Discourse Medical Welfare of Egyptian Labor in an Age of Liberalism and Progress (1919-1939)
This thesis is about discourses and practices formulated around health issues of workers in Egypt during the interwar period (1919-1939). It locates working-class illness within three major social transformations: the rise of large-scale mechanized production and its impact on human bodies, the ascendance of Egyptian nationalism, and the major economic adjustment from laissez-faireism to state intervention. This thesis concerns the somatic suffering of workers as a result of the development in modern industrial sectors, and raises the question of who spoke for the bodies of workers; why were these discourses designed; and how did the practices of discourses conversely reshape these tortured bodies? To answer these questions, I seek to explore and understand the emergence of working-class illness as a social problem and the ways in which social elites popularized nationalist ideas and constructed working-class health into a national project during years of rapid economic transformation and state building. Using numerous primary and secondary sources, the pages that followed will look at symbolic systems that nationalist elites artificially designed to incorporate workers into anti-colonial struggles. This thesis also uncovers the enforcement of these strategies, the practices of the state to gradually build a medical welfare system as a rescue operation in order to minimize class antagonism, as well as the agency of medical expertise in the making of a modern interventionist state.
The People Want to Topple the System: An Alternative Narrative of the Arab Uprisings
Since the toppling of Tunisian President Zine El Abidine Ben Ali, a one-dimensional narrative of the Arab Uprisings has become axiomatic in both foreign and Arab spheres. The mainstream narrative paints the movement as one for Western-style democracy and discursively associated economic neoliberalism. This account of the Uprisings not only obfuscates but also undermines the story that was put forth by activists themselves and that resonated so widely with diverse sectors of Arab populations. My thesis explores a narrative of the Arab Uprisings as put forth by activists. It presents and analyzes three demonstrations from Tunisia and Egypt to illustrate how, through their contentious performances, Arab activists attempt to challenge a system of imperial continuity--a centuries-old pattern of political, economic and social relations between foreign actors and the Arab people, and between Arab political elite and populations as a whole. Given the reach and history of this political system, such resistance is not easy. Nor is it straightforward; resistance to such a layered and variegated power formation must be relational. The Arab Uprisings present a dynamic by which actors revolted against local forms of oppression but also consistently reached for repertoires that were regional (rather than purely local) and drew upon networks and strategies that were Arab (rather than merely national). This indicates our need for a more nuanced narrative of how the Uprisings of 2011 constituted an event of Arab history.
"Building the Earth": Labor Politics, Technopolitics, and Tapline in Lebanon, 1950-1964
This project examines the history of the Aramco-owned Trans-Arabian Pipeline, or Tapline, focusing particularly on the history of its operations in Lebanon and labor activism by its Lebanese employees between 1950 and 1964. It applies the framework Timothy Mitchell develops in Carbon Democracy - entailing the analysis of the "chokepoints" created by energy infrastructure - to the particular case of Tapline in Lebanon, while supplementing Mitchell's "technopolitical" theoretical framework with attention to working conditions at Tapline and the particular the demands they made on their American employers. It therefore engages with literature on moral economy, class-formation, and labor politics in colonial and post-colonial settings.; My research revolves primarily around Tapline's management strategies, the Tapline workplace as experienced by its Lebanese employees, Tapline's vulnerabilities to strike action and workers' mobilization, and Lebanese Tapline employees' unionization and participation in a mass strike in 1964. Borrowing from Frederick Cooper's analysis of labor activism in colonial West Africa, I argue that Lebanese Tapline workers exploited Tapline's "chokepoints" to force the company to live up to its promises of meritocracy and welfare for its employees, and thus tried to improve their position within the status quo of their workplace rather than radically alter it.; This project is based on a diverse body of primary and secondary sources. Primary sources include interviews with the former head of the Tapline Laborers' Syndicate, internal documents from Aramco and Tapline, the Pipeline Periscope, an in-house magazine produced by the company for distribution to employees, coverage of Tapline and its employees' labor activism from the Lebanese press, and declassified US government documents. It aims to bring the literatures on the politics of labor and technopolitics into a productive dialogue. It also seeks to develop the historical literature on Tapline, which has received only fragmentary academic attention.
Developing Renaissance: Nahda Discourse in Jordanian Humanities Textbooks
This analysis addresses the subject of nahda, or renaissance, in Jordanian history, civics, religion, and literature textbooks from the 1970s to 2009. "Nahda" is a keyword in the textbooks signifying both the historical period of Arab modernist thought and nationalism in the late nineteenth and early twentieth centuries and the Jordanian state's present-day national development. The textbooks use the language of nahda to portray the Hashemite king as Jordan's historically qualified and legitimate leader. Nahda operates in nuanced ways across the genres. History and civics textbooks reflect "nahda as national development" in which the Hashemite kings are the primary actors in history and leaders of a comprehensive societal renaissance. Religion and literature textbooks, meanwhile, rely on "nahda as intellectual ideal" in which the state is hesitant to co-opt the ideals of pan-Arab and pan-Islamic unity because they exceed the territorially bounded Jordanian nation-state. These portrayals of nahda in the textbooks are coercive in the sense that they seek to preserve monarchic power by reducing Jordanian citizens to the loyal subjects of a progress-oriented king who not only raises their material well-being, but serves as the model for their civic virtue, tolerance, pluralism, and piety.