Dominant-negative p53 mutations selected in yeast hit cancer hot spots.

Clinically important mutant p53 proteins may be tumorigenic through a dominant-negative mechanism or due to a gain-of-function. Examples for both hypotheses have been described; however, it remains unclear to what extent they apply to TP53 mutations in general. Here it is shown that the mutational spectrum of dominant-negative p53 mutants selected in a novel yeast assay correlates tightly with p53 mutations in cancer. Two classes of dominant-negative mutations are described; the more dominant one affects codons that are essential for the stabilization of the DNA-binding surface of the p53 core domain and for the direct interaction of p53 with its DNA binding sites. These results predict that the vast majority of TP53 mutations leading to cancer do so in a dominant-negative fashion.

Dominant-negative mutant thyroid hormone receptors prevent transcription from Xenopus thyroid hormone receptor beta gene promoter in response to thyroid hormone in Xenopus tadpoles in vivo.

We describe a dominant-negative approach in vivo to assess the strong, early upregulation of thyroid hormone receptor beta (TR beta) gene in response to thyroid hormone, characteristic of the onset of natural and thyroid hormone-induced amphibian metamorphosis, 3,3',5-Triiodo-thyronine (T3) treatment of organ cultures of premetamorphic Xenopus tadpole tails coinjected in vivo with the wild-type Xenopus TR beta (wt-xTR beta) and three different thyroid responsive element chloramphenicol acetyltransferase (TRE-CAT) reporter constructs, including a direct repeat +4 (DR +4) element in the -200/+87 fragment of the xTR beta promoter, resulted in a 4- to 8-fold enhancement of CAT activity. Two human C-terminal TR beta 1 mutants (delta-hTR beta 1 and Ts-hTR beta 1), an artificial Xenopus C-terminal deletion mutant (mt-xTR beta), and the oncogenic viral homology v-erbA, none of which binds T3, inhibited this T3 response of the endogenous wt-xTR in Xenopus XTC-2 cells cotransfected with the -1600/+87 xTR beta promoter-CAT construct, the potency of the dominant-negative effect of these mutant TRs being a function of the strength of their heterodimerization with Xenopus retinoid X receptor gamma. Coinjection of the dominant-negative Xenopus and human mutant TR beta s into Xenopus tadpole tails totally abolished the T3 responsiveness of the wt-xTR beta with different TREs, including the natural DR +4 TRE of the xTR beta promoter.

Inhibition of human immunodeficiency virus type 1 and vaccinia virus infection by a dominant negative factor of the interferon regulatory factor family expressed in monocytic cells.

ICSBP is a member of the interferon (IFN) regulatory factor (IRF) family that regulates expression of type I interferon (IFN) and IFN-regulated genes. To study the role of the IRF family in viral infection, a cDNA for the DNA-binding domain (DBD) of ICSBP was stably transfected into U937 human monocytic cells. Clones that expressed DBD exhibited a dominant negative phenotype and did not elicit antiviral activity against vesicular stomatitis virus (VSV) infection upon IFN treatment. Most notably, cells expressing DBD were refractory to infection by vaccinia virus (VV) and human immunodeficiency virus type 1 (HIV-1). The inhibition of VV infection was attributed to defective virion assembly, and that of HIV-1 to low CD4 expression and inhibition of viral transcription in DBD clones. HIV-1 and VV were found to have sequences in their regulatory regions similar to the IFN-stimulated response element (ISRE) to which IRF family proteins bind. Accordingly, these viral sequences and a cellular ISRE bound a shared factor(s) expressed in U937 cells. These observations suggest a novel host-virus relationship in which the productive infection of some viruses is regulated by the IRF-dependent transcription pathway through the ISRE.

Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

Conventional myosin II is an essential protein for cytokinesis, capping of cell surface receptors, and development of Dictyostelium cells. Myosin II also plays an important role in the polarization and movement of cells. All conventional myosins are double-headed molecules but the significance of this structure is not understood since single-headed myosin II can produce movement and force in vitro. We found that expression of the tail portion of myosin II in Dictyostelium led to the formation of single-headed myosin II in vivo. The resultant cells contain an approximately equal ratio of double- and single-headed myosin II molecules. Surprisingly, these cells were completely blocked in cytokinesis and capping of concanavalin A receptors although development into fruiting bodies was not impaired. We found that this phenotype is not due to defects in myosin light chain phosphorylation. These results show that single-headed myosin II cannot function properly in vivo and that it acts as a dominant negative mutation for myosin II function. These results suggest the possibility that cooperativity of myosin II heads is critical for force production in vivo.

Lymphocytes selected in allogeneic thymic epithelium mediate dominant tolerance toward tissue grafts of the thymic epithelium haplotype.

Athymic mice grafted at birth with allogeneic thymic epithelium (TE) from day 10 embryos before hematopoietic cell colonization reconstitute normal numbers of T cells and exhibit full life-long tolerance to skin grafts of the TE haplotype. Intravenous transfers of splenic cells, from these animals to adult syngeneic athymic recipients, reconstitute T-cell compartments and the ability to reject third-party skin grafts. The transfer of specific tolerance to skin grafts of the TE donor strain, however, is not observed in all reconstituted recipients, and the fraction of nontolerant recipients increases with decreasing numbers of cells transferred. Furthermore, transfers of high numbers of total or CD4+ T cells from TE chimeras to T-cell receptor-anti-H-Y antigen transgenic immunocompetent syngeneic hosts specifically hinder the rejection of skin grafts of the TE haplotype that normally occurs in such recipients. These observations demonstrate (i) that mice tolerized by allogeneic TE and bearing healthy skin grafts harbor peripheral immunocompetent T cells capable of rejecting this very same graft; and (ii) that TE selects for regulatory T cells that can inhibit effector activities of graft-reactive cells.

Multiple loci govern the bone marrow-derived immunoregulatory mechanism controlling dominant resistance to autoimmune orchitis.

The existence of immunoregulatory genes conferring dominant resistance to autoimmunity is well documented. In an effort to better understand the nature and mechanisms of action of these genes, we utilized the murine model of autoimmune orchitis as a prototype. When the orchitis-resistant strain DBA/2J is crossed with the orchitis-susceptible strain BALB/cByJ, the F1 hybrid is completely resistant to the disease. By using reciprocal radiation bone marrow chimeras, the functional component mediating this resistance was mapped to the bone marrow-derived compartment. Resistance is not a function of either low-dose irradiation- or cyclophosphamide (20 mg/kg)-sensitive immunoregulatory cells, but can be adoptively transferred by primed splenocytes. Genome exclusion mapping identified three loci controlling the resistant phenotype. Orch3 maps to chromosome 11, whereas Orch4 and Orch5 map to the telomeric and centromeric regions of chromosome 1, respectively. All three genes are linked to a number of immunologically relevant candidate loci. Most significant, however, is the linkage of Orch3 to Idd4 and Orch5 to Idd5, two susceptibility genes which play a role in autoimmune insulin-dependent type 1 diabetes mellitus in the nonobese diabetic mouse.

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

Dominant-negative action of the jimpy mutation in mice complemented with an autosomal transgene for myelin proteolipid protein.

Mutations in genes encoding membrane proteins have been associated with cell death of unknown cause from invertebrate development to human degenerative diseases. A point mutation in the gene for myelin proteolipid protein (PLP) underlies oligodendrocyte death and dysmyelination in jimpy mice, an accurate model for Pelizaeus-Merzbacher disease. To distinguish the loss of PLP function from other effects of the misfolded protein, we took advantage of the X chromosomal linkage of the gene and have complemented jimpy with a wild-type PLP transgene. In this artificial heterozygous situation, the jimpy mutation emerged as genetically dominant. At the cellular level oligodendrocytes showed little increase in survival although endogenous PLP gene and autosomal transgene were truly coexpressed. In surviving oligodendrocytes, wild-type PLP was functional and immunodetectable in myelin. Moreover, compacted myelin sheaths regained their normal periodicity. This strongly suggests that, despite the presence of functional wild-type PLP, misfolded jimpy PLP is by itself the primary cause of abnormal oligodendrocyte death.

Discursive constructions of social and environmental accounting in Brazil: the case of Petrobras.

In the 29 years following \"Our Common Future\" by the United Nations, there is considerable debate among governments, civil society, interest groups and business organisations about what constitutes sustainable development, which constitutes evidence for a contested discourse concerning sustainability. The purpose of this study is to understand this debate in the developing economic context of Brazil, and in particular, to understand and critique the social and environmental accounting [SEA] discursive constructions relating to the State-owned, Petrobras as well as to understand the Brazilian literature on SEA. The discourse theory [DT]-based analysis employs rhetorical redescription to analyse twenty-two reports from Petrobras from 2004-2013. I investigate the political notions by employing the methodological framework of the Logics of Critical Explanation [LCE]. LCE engenders five methodological steps: problematisation, retroduction, logics (social, political and fantasmatic), articulation and critique. The empirical discussion suggests that the hegemony of economic development operates to obfuscate, rhetorically, the development of sustainability, so as to maintain the core business of Petrobras conceived as capital accumulation. Equally, these articulations also illustrate how the constructions of SEA operate to serve the company\'s purpose with few (none) profound changes in integration of sustainability. The Brazilian literature on SEA sustains the status quo of neo-liberal market policies that operate to protect the dominant business case approach to maintain an agenda of wealth-creation in relation to social and environmental needs. The articulations of the case manifested in policies regarding, for example, corruption, which involved over-payments for contracts and unsustainable practices relating to the use of fossil fuels and demonstrated that there was antagonism between action and disclosure. The corruption scandal that emerged after SEA disclosures highlighted the rhetorical nature of disclosure when financial resources were subtracted from the company for political parties and engineering contractors hid facts through incomplete disclosures. The articulations of SEA misrepresent a broader context of the meanings associated with sustainability, which restricted the constructions of SEA to principally serve and represent the intention of the most powerful groups. The significance of SEA, then is narrowed to represent particular interests. The study argues for more critical studies as limited Brazilian literature concerning SEA kept a \'safe distance\' from substantively critiquing the constructions of SEA and its articulations in the Brazilian context. The literature review and the Petrobras\' case illustrate a variety of naming, instituting and articulatory practices that endeavour to maintain the current hegemony of development in an emerging economy, which allows Petrobras to continue to exercise significant profit at the expense of the social and environmental. The constructed idea of development in Petrobras\' discourses emphasises a rhetoric of wider development, but, in reality, these discourses were the antithesis of political, social and ethical developmental issues. These constructions aim to hide struggles between social inequalities and exploitation of natural resources and constitute excuses about a fanciful notion of rhetorical and hegemonic neo-liberal development. In summary, this thesis contributes to the prior literature in five ways: (i) the addition of DT to the analysis of SEA enhances the discussion of political elements such as hegemony, antagonism, logic of equivalence/difference, ideology and articulation; (ii) the analysis of an emerging economy such as Brazil incorporates a new perspective of the discussion of the discourses of SEA and development; (iii) this thesis includes a focus on rhetoric to discuss the maintenance of the status quo; (iv) the holistic structure of the LCE approach enlarges the understanding of the social, political and fantasmatic logics of SEA studies and; (v) this thesis combines an analysis of the literature and the case of Petrobras to characterise and critique the state of the Brazilian academy and its impacts and reflections on the significance of SEA. This thesis, therefore, argues for more critical studies in the Brazilian academy due to the persistence of idea of SEA and development that takes-for-granted deep exclusions and contradictions and provide little space for critiques.

The Affective Dimensions of Social Controversy

Social controversy is a sustained, mediated debate between at least two oppositional parties which is more than just a difference of opinion; rather it is a persistent conflict over the political and cultural implications that dominant forms of communicative reasoning, practices, and norms have for a public. Simply put, during social controversies the norms guiding public life can be negotiated, reaffirmed, negated, and/or transformed. This can lead to progressive political, cultural, and/or social change in some instances, while establishing or reifying conservative and even oppressive norms, practices, and laws in others. Building upon Olson and Goodnight's (1994) theoretical and methodological framework of social controversy, this dissertation argues that scholars should analyze the role affect plays in this type of conflict as a means to address the regulation of public conduct as well as public discourse. The rhetorical and argumentative significance of the affective dimensions of social controversy have been conceptualized and analyzed via an examination of emotion-based claims and affective states that have become salient, discernable and/or apprehendable during specific public disagreements. Such a conceptualization demonstrates that critical insights regarding the norms that guide public conduct, the role risk and vulnerability play in the regulation of individuals' public behavior, and the relationship between affect and citizenship can be gained by focusing on a controversy's affective dimensions. To highlight the importance of the study of affect in social controversy as well as better understand the larger critical significance affect theory has for rhetorical and argumentation studies, this dissertation has analyzed the affective dimensions of three conflicts. They are: the Abu Ghraib prisoner abuse social controversy, the International Freedom Center social controversy, and the controversy over the 2004 French ban on conspicuous religious attire in public schools. The findings from this dissertation that have specific and general implications for future work in the field of controversy as well as rhetoric and argumentation, respectively.

The Vernacular Discourse of the "Arab Spring:" An Analysis of the Visual, the Embodied, and the Textual Rhetorics of the Karama Revolution

The Vernacular Discourse of the "Arab Spring" is a project that bridges the divide between the East and the West by offering new readings to Arab subjectivities. Through an analysis of the "Arab Spring" through the lens of vernacular discourse, it challenges the Euro-Americo-centric legacies of Orientalism in Western academia and the new wave of extremism in the Arab world by offering alternative representations of Arab bodies and subjectivities. To offer this new reading of the "Arab Spring," it explores the foundations of critical rhetoric as a theory and a practice and argues for a turn towards a critical vernacular discourse. The turn towards critical vernacular discourse is important as it urges the analyses of different artifacts produced by marginalized groups in order to understand their perspectives that have largely been foreclosed in traditional cultural studies research. Building on embodied/performative critical rhetoric, the vernacular discourses of the Arab revolutionary body examines other forms of knowledge productions that are not merely textual; more specifically, through data gathered in the Lhbib Bourguiba, Tunisia. This analysis of the political revolutionary body unveils the complexity underlining the discussion around issues of identity, agency and representation in the Middle East and North Africa, and calls for a critical study towards these issues in the region beyond the binary approach that has been practiced and applied by academics and media analysts. Hence, by analyzing vernacular discourse, this research locates a method of examining and theorizing the dialectic between agency, citizenry, and subjectivity through the study of how power structure is recreated and challenged through the use of the vernacular in revolutionary movements, as well as how marginalized groups construct their own subjectivities through the use of vernacular discourse. Therefore, highlighting the political prominence of evaluating the Arab Spring as a vernacular discourse is important in creating new ways of understanding communication in postcolonial/neocolonial settings.

Everything in di Dark Muss Come to Light: A Postcolonial Investigation of the Practice of Extra Lessons at the Secondary Level in Jamaica's Education System

Despite the vast research examining the evolution of Caribbean education systems, little is chronologically tied to the postcolonial theoretical perspectives of specific island-state systems, such as the Jamaican education system and its relationship with the underground shadow education system. This dissertation study sought to address the gaps in the literature by critically positioning postcolonial theories in education to examine the macro- and micro-level impacts of extra lessons on secondary education in Jamaica. The following postcolonial theoretical (PCT) tenets in education were contextualized from a review of the literature: (a) PCT in education uses colonial discourse analysis to critically deconstruct and decolonize imperialistic and colonial representations of knowledge throughout history; (b) PCT in education uses an anti-colonial discursive framework to re-position indigenous knowledge in schools, colleges, and universities to challenge hegemonic knowledge; (c) PCT in education involves the "unlearning" of dominant, normative ideologies, the use of self-reflexivity, and deconstruction; and (d) PCT in education calls for critical pedagogical approaches that reject the banking concept of education and introduces inclusive pedagogy to facilitate "the passage from naïve to critical transitivity" (Freire, 1973, p. 32). Specifically, using a transformative mixed-methods design, grounded and informed by a postcolonial theoretical lens, I quantitatively uncovered and then qualitatively highlighted how if at all extra lessons can improve educational outcomes for students at the secondary level in Jamaica. Accordingly, the quantitative data was used to test the hypotheses that the practice of extra lessons in schools is related to student academic achievement and the practice of critical-inclusive pedagogy in extra lessons is related to academic achievement. The two-level hierarchical linear model analysis revealed that hours spent in extra lessons, average household monthly income, and critical-inclusive pedagogical tents were the best predictors for academic achievement. Alternatively, the holistic multi-case study explored how extra-lessons produces increased academic achievement. The data revealed new ways of knowledge construction and critical pedagogical approaches to galvanize systemic change in secondary education. Furthermore, the data showed that extra lessons can improve educational outcomes for students at the secondary level if the conditions for learning are met. This study sets the stage for new forms of knowledge construction and implications for policy change.

The orchestration of modes and EFL audio-visual comprehension: A multimodal discourse analysis of vodcasts

This thesis explores the role of multimodality in language learners’ comprehension, and more specifically, the effects on students’ audio-visual comprehension when different orchestrations of modes appear in the visualization of vodcasts. Firstly, I describe the state of the art of its three main areas of concern, namely the evolution of meaning-making, Information and Communication Technology (ICT), and audio-visual comprehension. One of the most important contributions in the theoretical overview is the suggested integrative model of audio-visual comprehension, which attempts to explain how students process information received from different inputs. Secondly, I present a study based on the following research questions: ‘Which modes are orchestrated throughout the vodcasts?’, ‘Are there any multimodal ensembles that are more beneficial for students’ audio-visual comprehension?’, and ‘What are the students’ attitudes towards audio-visual (e.g., vodcasts) compared to traditional audio (e.g., audio tracks) comprehension activities?’. Along with these research questions, I have formulated two hypotheses: Audio-visual comprehension improves when there is a greater number of orchestrated modes, and students have a more positive attitude towards vodcasts than traditional audios when carrying out comprehension activities. The study includes a multimodal discourse analysis, audio-visual comprehension tests, and students’ questionnaires. The multimodal discourse analysis of two British Council’s language learning vodcasts, entitled English is GREAT and Camden Fashion, using ELAN as the multimodal annotation tool, shows that there are a variety of multimodal ensembles of two, three and four modes. The audio-visual comprehension tests were given to 40 Spanish students, learning English as a foreign language, after the visualization of vodcasts. These comprehension tests contain questions related to specific orchestrations of modes appearing in the vodcasts. The statistical analysis of the test results, using repeated-measures ANOVA, reveal that students obtain better audio-visual comprehension results when the multimodal ensembles are constituted by a greater number of orchestrated modes. Finally, the data compiled from the questionnaires, conclude that students have a more positive attitude towards vodcasts in comparison to traditional audio listenings. Results from the audio-visual comprehension tests and questionnaires prove the two hypotheses of this study.