Vanishing Point: An examination of some consequences of globalization for contemporary Irish film

In the following article, some films produced with the support of Bord Scannán na hÉireann (The Irish Film Board) since its reconstitution in 1993 are examined in light of the work of global anthropologist Arjun Appadurai and his theory of global cultural flows. I suggest that cinema, primarily of Hollywood origin, has had a notable influence on the development of Irish society and Irish film. Contemporary Irish film itself also reflects the failure of Irish history to excite the imagination of Ireland’s youth as effectively as the seductive depictions of America’s past as mediated through the Western and gangster films. Indeed, films made in Ireland today reflect the influence of both these genres. However, as the key to the Hollywood continuity style of film-making is its own self-effacement, this has sometimes been reflected in the effacement of people, politics and place in contemporary Irish film as film-makers endeavor to attract a global audience for their work

Hormonal regulation and metabolic roles of CCAAT/enhancer-binding proteins

The CCAAT/Enhancer-Binding proteins (C/EBPs) are liver-enriched transcription factors which are known to 'trans'-activate a number of metabolically important genes. The goal of this thesis work has been to advance areas of study on C/EBP isoform regulation and metabolic roles which have not been fully addressed in the current literature. The initial undertaking of this work involved the examination of the effects of hormones and diabetes on C/EBP isoform expression in rat H4IIE hepatoma cells and in rat liver. Treatment of cells with dexamethasone was observed to produce increases in C/EBPá and C/EBPâ mRNA and protein levels. Insulin was observed to produce an interesting bi-phasic response on C/EBPá expression. Treatment of H4IIE cells with 8-chlorophenylthio-cAMP produced greater inductive effects upon C/EBPâ expression than on C/EBPá expression. We observed an inhibition of C/EBPá gene expression in streptozotocin-diabetic rat liver which was reflected by decreases in both its mRNA and protein. However, an interesting alteration in the ratio of alternate C/EBPá translation forms was observed in the streptozotocin-diabetic livers suggesting a potential alteration in the 'trans'-activational activity of C/EBPá. These results suggest that hepatic C/EBP isoforms are under complex control by both hormonal and metabolic signals, which correlates well with their known role as 'trans'-activators of metabolically vital genes. Previous work has demonstrated a role for C/EBPá in mediating the cAMP responsiveness of synthetic phosphoenolpyruvate carboxykinase (PEPCK) promoter constructs within a transiently transfected cell culture system. In order to address the C/EBP isoform requirements for endogenous PEPCK gene expression and regulation, we have produced stable transfected hepatoma cells expressing antisense constructs for the two major C/EBP isoforms in liver. We demonstrate that targeted inhibition of C/EBPá but not C/EBPâ in rat hepatoma H4IIE cells significantly reduces the cAMP responsiveness of the endogenous PEPCK promoter. Cells expressing C/EBPá antisense were characterized by decreases in the levels C/EBPá mRNA and C/EBPá protein levels. The response of PEPCK to cAMP was marginal in C/EBPá antisense expressing cells, compared with a 3-fold induction of PEPCK expression by cAMP observed in wild-type H4IIE cells. The cAMP signaling pathway of C/EBPá antisense expressing cells was intact; in that the cAMP induction of the C/EBPâ gene was similar to that of normal H4IIE cells. Furthermore, the cAMP responsiveness of PEPCK in C/EBPâ antisense expressing cells was nearly identical to that of wild-type H4IIE cells. These data suggest that the á-isoform of C/EBP is specifically required for mediation of the cAMP response of endogenous PEPCK in rat hepatoma cells and cannot be functionally substituted for by C/EBPâ in this context

Phase resetting reveals network dynamics underlying a bacterial cell cycle

Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS)

Single-gene tuning of Caulobacter cell cycle period and noise, swarming motility, and surface adhesion

We established that the sensor histidine kinase DivJ has an important role in the regulation of C. crescentus cell cycle period and noise. This was accomplished by designing and conducting single-cell experiments to probe the dependence of cell cycle noise on divJ expression and constructing a simplified cell cycle model that captures the dependence of cell cycle noise on DivJ with molecular details.In addition to its role in regulating the cell cycle, DivJ also affects polar cell development in C. crescentus, regulating swarming motility and surface adhesion. We propose that pleiotropic control of polar cell development by the DivJ–DivK–PleC signaling pathway underlies divJ-dependent tuning of cell swarming and adhesion behaviors.We have integrated the study of single-cell fluorescence dynamics with a kinetic model simulation to provide direct quantitative evidence that the DivJ histidine kinase is localized to the cell pole through a dynamic diffusion-and-capture mechanism during the C. crescentus cell cycle

Model-Based Deconvolution of Cell Cycle Time-Series Data Reveals Gene Expression Details at High Resolution

In both prokaryotic and eukaryotic cells, gene expression is regulated across the cell cycle to ensure “just-in-time” assembly of select cellular structures and molecular machines. However, present in all time-series gene expression measurements is variability that arises from both systematic error in the cell synchrony process and variance in the timing of cell division at the level of the single cell. Thus, gene or protein expression data collected from a population of synchronized cells is an inaccurate measure of what occurs in the average single-cell across a cell cycle. Here, we present a general computational method to extract “single-cell”-like information from population-level time-series expression data. This method removes the effects of 1) variance in growth rate and 2) variance in the physiological and developmental state of the cell. Moreover, this method represents an advance in the deconvolution of molecular expression data in its flexibility, minimal assumptions, and the use of a cross-validation analysis to determine the appropriate level of regularization. Applying our deconvolution algorithm to cell cycle gene expression data from the dimorphic bacterium Caulobacter crescentus, we recovered critical features of cell cycle regulation in essential genes, including ctrA and ftsZ, that were obscured in population-based measurements. In doing so, we highlight the problem with using population data alone to decipher cellular regulatory mechanisms and demonstrate how our deconvolution algorithm can be applied to produce a more realistic picture of temporal regulation in a cell.</p

Regulation of bacterial surface attachment by a network of sensory transduction proteins

Bacteria are often attached to surfaces in natural ecosystems. A surface-associated lifestyle can have advantages, but shifts in the physiochemical state of the environment may result in conditions in which attachment has a negative fitness impact. Therefore, bacteria employ numerous mechanisms to control the transition from an unattached to a sessile state. The Caulobacter crescentus protein HfiA is a potent developmental inhibitor of the secreted polysaccharide adhesin known as the holdfast, which enables permanent attachment to surfaces. Multiple environmental cues influence expression of hfiA, but mechanisms of hfiA regulation remain largely undefined. Through a forward genetic selection, we have discovered a multi-gene network encoding a suite of two-component system (TCS) proteins and transcription factors that coordinately control hfiA transcription, holdfast development and surface adhesion. The hybrid HWE-family histidine kinase, SkaH, is central among these regulators and forms heteromeric complexes with the kinases, LovK and SpdS. The response regulator SpdR indirectly inhibits hfiA expression by activating two XRE-family transcription factors that directly bind the hfiA promoter to repress its transcription. This study provides evidence for a model in which a consortium of environmental sensors and transcriptional regulators integrate environmental cues at the hfiA promoter to control the attachment decision

Structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen

We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT–CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi

Phase Resetting Reveals Network Dynamics Underlying a Bacterial Cell Cycle

Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS).</p