Transcriptional activation by Sp1 and post-transcriptional repression by muscle-specific microRNA miR-133 of expression of human ERG1 and KCNQ1 genes and potential implication in arrhythmogenesis

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Targeted Gene Repression Technologies for Regenerative Medicine, Genomics, and Gene Therapy

Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.

Transcriptional activation by Sp1 and post-transcriptional repression by muscle-specific microRNA miR-133 of expression of human ERG1 and KCNQ1 genes and potential implication in arrhythmogenesis

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

I got drunk and lost my virginity : young women's magazines and the management of sexual identity

It has often been argued that young woman’s magazine’s, like Cosmopolitan, Cleo Dolly and Seventeen, constitute a significant instrument in the patriarchal repression of young women - their hegemonic success lying in the fact that they appear to be sites wherein young women are ‘free’ from the elements of coercion so obviously in evidence within other terrains, such as the school and the family. This paper will suggest an alternative approach to these magazines. Rather than locating such texts within an overall model of repression and patriarchal domination, it will be argued here that they can be regarded as practical manuals which enrol young women to do specific kinds of work on themselves. In doing so, they form an effective link between the governmental imperatives aimed at constructing particular personas (such as, for example, ‘the sexually responsible young woman’), and the actual practices whereby these imperatives are operationalised. These manuals do not prevent young women from learning to ‘project a unique self’, they constitute a significant source of practices and techniques through which particular types of self are shaped.

Asia

Reporters sans frontiéres (RSF) has repeatedly declared Asia to be the most demanding continent for journalists and their news organizations to operate in, and in some countries, even simply to survive in. The many reports issued by RSF and other global agencies regularly show Asia to be the region in which the largest number of murders of journalists occur per year, even when Asian–Arabic states and Central Asia are not included in the definition of ‘Asia’. The reports describe numerous physical, legal and economic threats as well as serious political repression and restrictions that journalists face as they attempt to function as watch-dogs, agenda-setters and gate-keepers for their societies. The statistics and examples provided within these reports, however, do not provide the full picture. Most Asian nations also host vibrant media cultures in which journalists play an important role in supporting social and democratic processes and activities. This chapter outlines the political and economic influences on Asian journalism; the impact of new technologies; the debates about philosophies such as 'development journalism', 'peace journalism' and 'Asian values'; and the influence of the so-called 'envelope culture' or practices of gift-giving and bribery that pervade journalism in some countries. To illustrate how these principles affect journalists' practice, the chapter presents a comparison of the starkly contrasting situations in India versus North Korea (Democratic People's Republic of Korea). The chapter also describes issues affecting countries as far afield as China to Kazakhstan, including a short case study of journalism during the so-called Saffron Revolution in Burma in 2007. The chapter concludes with suggestions about how training and aid for the Asian should be contextualized to take into account the specific cultural, economic and political factors that shape and limit the media’s performance, and how journalists might be best placed to negotiate around them. Such training needs to be sensitive to valid variations in perceptions of what kind of governance and journalism best serves development, without serving politically motivated rhetoric.

Androgen-regulated genes differentially modulated by the androgen receptor coactivator L-dopa decarboxylase in human prostate cancer cells

Background The androgen receptor is a ligand-induced transcriptional factor, which plays an important role in normal development of the prostate as well as in the progression of prostate cancer to a hormone refractory state. We previously reported the identification of a novel AR coactivator protein, L-dopa decarboxylase (DDC), which can act at the cytoplasmic level to enhance AR activity. We have also shown that DDC is a neuroendocrine (NE) marker of prostate cancer and that its expression is increased after hormone-ablation therapy and progression to androgen independence. In the present study, we generated tetracycline-inducible LNCaP-DDC prostate cancer stable cells to identify DDC downstream target genes by oligonucleotide microarray analysis. Results Comparison of induced DDC overexpressing cells versus non-induced control cell lines revealed a number of changes in the expression of androgen-regulated transcripts encoding proteins with a variety of molecular functions, including signal transduction, binding and catalytic activities. There were a total of 35 differentially expressed genes, 25 up-regulated and 10 down-regulated, in the DDC overexpressing cell line. In particular, we found a well-known androgen induced gene, TMEPAI, which wasup-regulated in DDC overexpressing cells, supporting its known co-activation function. In addition, DDC also further augmented the transcriptional repression function of AR for a subset of androgen-repressed genes. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time RT-PCR. Conclusion Taken together, our results provide evidence for linking DDC action with AR signaling, which may be important for orchestrating molecular changes responsible for prostate cancer progression.

A novel corepressor, BCoR-L1, represses transcription through an interaction with CtBP

Corepressors play a crucial role in negative gene regulation and are defective in several diseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe and functionally characterize BCoR-L1, a homolog of BCoR. When tethered to a heterologous promoter, BCoR-L1 is capable of strong repression. Like other corepressors, BCoR-L1 associates with histone deacetylase (HDAC) activity. Specifically, BCoR-L1 coprecipitates with the Class II HDACs, HDAC4, HDAC5, and HDAC7, suggesting that they are involved in its role as a transcriptional repressor. BCoR-L1 also interacts with the CtBP corepressor through a CtBP-interacting motif in its amino terminus. Abrogation of the CtBP binding site within BCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore, BCoR-L1 is located on the E-cadherin promoter, a known CtBP-regulated promoter, and represses the E-cadherin promoter activity in a reporter assay. The inhibition of BCoR-L1 expression by RNA-mediated interference results in derepression of E-cadherin in cells that do not normally express E-cadherin, indicating that BCoR-L1 contributes to the repression of an authentic endogenous CtBP target.

Physical and functional interaction of the archaeal single-stranded DNA-binding protein SSB with RNA polymerase

Archaeal transcription utilizes a complex multisubunit RNA polymerase and the basal transcription factors TBP and TF(II)B, closely resembling its eukaryal counterpart. We have uncovered a tight physical and functional interaction between RNA polymerase and the single-stranded DNA-binding protein SSB in Sulfolobus solfataricus. SSB stimulates transcription from promoters in vitro under TBP-limiting conditions and supports transcription in the absence of TBP. SSB also rescues transcription from repression by reconstituted chromatin. We demonstrate the potential for promoter melting by SSB, suggesting a plausible basis for the stimulation of transcription. This stimulation requires both the single-stranded DNA-binding domain and the acidic C-terminal tail of the SSB. The tail forms a stable interaction with RNA polymerase. These data reveal an unexpected role for single-stranded DNA-binding proteins in transcription in archaea.

Diversity in action : minority group media and social change

My thesis consists of a creative work plus an exegesis. This exegesis uses case study research to investigate three Brisbane-based media organisations and the role they play in encouraging social inclusion and other positive social change for specific disadvantaged and stigmatised minority groups. Bailey, Cammaerts and Carpentier’s theoretical approach to alternative media forms the basis of this research. Bailey et al. (2008, p. 156) view alternative media organisations as having four important roles, two media-centred and two society-centred, which must all be considered to best understand them: • serving their communities • acting as an alternative to mainstream media discourses • promoting and advocating democratisation in the media and through the media in society • functioning as a crossroads in civil society. The first case study, about community radio station 4RPH (Radio for the Print Handicapped), centres on promoting social inclusion for people with a print disability through access to printed materials (primarily mainstream print media) in an audio format. The station also provides important opportunities for members of this group to produce media and, to a lesser extent, provides disability-specific information and discussions. The second case study, about gay print and online magazine Queensland Pride, focuses on promoting social inclusion and combating the discrimination and repression of people who identify as lesbian, gay, bisexual or transgender. Central issues include the representation (including sexualised representation) of a subculture and niche target market, and the impact of commercialisation on this free publication. The third case study, about community radio station 98.9FM, explores the promotion of social inclusion for peoples whose identity, cultures, issues, politics and contributions are often absent or misrepresented in the mainstream media. This radio station provides “a first level of service” (Meadows & van Vuuren, 1998, p. 104) to these people, but also informs and entertains those in the majority society. The findings of this research suggest that there are two key mechanisms that help these media organisations to effect social change: first, strengthening the minority community and serving its needs, and second, fostering connections with the broader society.

Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex

Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G1-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G1-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G 1 into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.

Contemporary State Terrorism: Theory and Practice

This volume aims to 'bring the state back into terrorism studies' and fill the notable gap that currently exists in our understanding of the ways in which states employ terrorism as a political strategy of internal governance or foreign policy. Within this broader context, the volume has a number of specific aims. First, it aims to make the argument that state terrorism is a valid and analytically useful concept which can do much to illuminate our understanding of state repression and governance, and illustrate the varieties of actors, modalities, aims, forms, and outcomes of this form of contemporary political violence. Secondly, by discussing a rich and diverse set of empirical case studies of contemporary state terrorism this volume explores and tests theoretical notions, generates new questions and provides a resource for further research. Thirdly, it contributes to a critical-normative approach to the study of terrorism more broadly and challenges dominant approaches and perspectives which assume that states, particularly Western states, are primarily victims and not perpetrators of terrorism. Given the scarceness of current and past research on state terrorism, this volume will make a genuine contribution to the wider field, particularly in terms of ongoing efforts to generate more critical approaches to the study of political terrorism. This book will be of much interest to students of critical terrorism studies, critical security studies, terrorism and political violence and political theory in general.

Relative abundance of full-length and truncated FOXP1 isoforms is associated with differential NFκB activity in Follicular Lymphoma

FOXP1 is a transcriptional repressor that has been proposed to repress the expression of some NFκB-responsive genes. Furthermore, truncated forms of FOXP1 have been associated with a subtype of Diffuse Large B-cell Lymphoma characterised by constitutive NFκB activity, indicating that they may inhibit this repression. We have shown that FL tumors have increased relative abundance of truncated FOXP1 isoforms and this is associated with increased expression of NFκB-associated genes. Our results provide strong evidence that relative FOXP1 isoform abundance is associated with NFκB activity in FL, and could potentially be used as a marker for this gene signature.