Visualising ribosome profiling and using it for reading frame detection and exploration of eukaryotic translation initiation

Ribosome profiling (ribo-seq) is a recently developed technique that provides genomewide information on protein synthesis (GWIPS) in vivo. The high resolution of ribo-seq is one of the exciting properties of this technique. In Chapter 2, I present a computational method that utilises the sub-codon precision and triplet periodicity of ribosome profiling data to detect transitions in the translated reading frame. Application of this method to ribosome profiling data generated for human HeLa cells allowed us to detect several human genes where the same genomic segment is translated in more than one reading frame. Since the initial publication of the ribosome profiling technique in 2009, there has been a proliferation of studies that have used the technique to explore various questions with respect to translation. A review of the many uses and adaptations of the technique is provided in Chapter 1. Indeed, owing to the increasing popularity of the technique and the growing number of published ribosome profiling datasets, we have developed GWIPS-viz (http://gwips.ucc.ie), a ribo-seq dedicated genome browser. Details on the development of the browser and its usage are provided in Chapter 3. One of the surprising findings of ribosome profiling of initiating ribosomes carried out in 3 independent studies, was the widespread use of non-AUG codons as translation initiation start sites in mammals. Although initiation at non-AUG codons in mammals has been documented for some time, the extent of non-AUG initiation reported by these ribo-seq studies was unexpected. In Chapter 4, I present an approach for estimating the strength of initiating codons based on the leaky scanning model of translation initiation. Application of this approach to ribo-seq data illustrates that initiation at non-AUG codons is inefficient compared to initiation at AUG codons. In addition, our approach provides a probability of initiation score for each start site that allows its strength of initiation to be evaluated.

Form-focused instruction in Irish-medium immersion education: a critical examination of teachers' perspectives and practices. A small-scale qualitative case study

It has been suggested that the less than optimal levels of students’ immersion language “persist in part because immersion teachers lack systematic approaches for integrating language into their content instruction” (Tedick, Christian and Fortune, 2011, p.7). I argue that our current lack of knowledge regarding what immersion teachers think, know and believe and what immersion teachers’ actual ‘lived’ experiences are in relation to form-focused instruction (FFI) prevents us from fully understanding the key issues at the core of experiential immersion pedagogy and form-focused integration. FFI refers to “any planned or incidental instructional activity that is intended to induce language learners to pay attention to linguistic form” (Ellis, 2001b, p.1). The central aim of this research study is to critically examine the perspectives and practices of Irish-medium immersion (IMI) teachers in relation to FFI. The study ‘taps’ into the lived experiences of three IMI teachers in three different IMI school contexts and explores FFI from a classroom-based, teacher-informed perspective. Philosophical underpinnings of the interpretive paradigm and critical hermeneutical principles inform and guide the study. A multi-case study approach was adopted and data was gathered through classroom observation, video-stimulated recall and semistructured interviews. Findings revealed that the journey of ‘becoming’ an IMI teacher is shaped by a vast array of intricate variables. IMI teacher identity, implicit theories, stated beliefs, educational biographies and experiences, IMI school cultures and contexts as well as teacher knowledge and competence impacted on IMI teachers’ FFI perspectives and practices. An IMI content teacher identity reflected the teachers’ priorities as shaped by pedagogical challenges and their educational backgrounds. While research participants had clearly defined instructional beliefs and goals, their roadmap of how to actually accomplish these goals was far from clear. IMI teachers described the multitude of choices and pedagogical dilemmas they faced in integrating FFI into experiential pedagogy. Significant gaps in IMI teachers’ declarative knowledge about and competence in the immersion language were also reported. This research study increases our understanding of the complexity of the processes underlying and shaping FFI pedagogy in IMI education. Innovative FFI opportunities for professional development across the continuum of teacher education are outlined, a comprehensive evaluation of IMI is called for and areas for further research are delineated.

Scientific writing: a randomized controlled trial comparing standard and on-line instruction.

BACKGROUND: Writing plays a central role in the communication of scientific ideas and is therefore a key aspect in researcher education, ultimately determining the success and long-term sustainability of their careers. Despite the growing popularity of e-learning, we are not aware of any existing study comparing on-line vs. traditional classroom-based methods for teaching scientific writing. METHODS: Forty eight participants from a medical, nursing and physiotherapy background from US and Brazil were randomly assigned to two groups (n = 24 per group): An on-line writing workshop group (on-line group), in which participants used virtual communication, google docs and standard writing templates, and a standard writing guidance training (standard group) where participants received standard instruction without the aid of virtual communication and writing templates. Two outcomes, manuscript quality was assessed using the scores obtained in Six subgroup analysis scale as the primary outcome measure, and satisfaction scores with Likert scale were evaluated. To control for observer variability, inter-observer reliability was assessed using Fleiss's kappa. A post-hoc analysis comparing rates of communication between mentors and participants was performed. Nonparametric tests were used to assess intervention efficacy. RESULTS: Excellent inter-observer reliability among three reviewers was found, with an Intraclass Correlation Coefficient (ICC) agreement = 0.931882 and ICC consistency = 0.932485. On-line group had better overall manuscript quality (p = 0.0017, SSQSavg score 75.3 +/- 14.21, ranging from 37 to 94) compared to the standard group (47.27 +/- 14.64, ranging from 20 to 72). Participant satisfaction was higher in the on-line group (4.3 +/- 0.73) compared to the standard group (3.09 +/- 1.11) (p = 0.001). The standard group also had fewer communication events compared to the on-line group (0.91 +/- 0.81 vs. 2.05 +/- 1.23; p = 0.0219). CONCLUSION: Our protocol for on-line scientific writing instruction is better than standard face-to-face instruction in terms of writing quality and student satisfaction. Future studies should evaluate the protocol efficacy in larger longitudinal cohorts involving participants from different languages.

The Role of mRNA Translation in the Regulation of Ribosome Trafficking to the Endoplasmic Reticulum

The goal of this research is to identify the trafficking patterns that direct ribosomes to the endoplasmic reticulum (ER). It is widely believed that the SRP pathway is the only mechanism that cells use to localize mRNA and ribosomes to the ER, but this has been found not to be a sufficient explanation for the patterns of RNA localization in cells, namely that non-signal sequence-containing mRNA are translated on the ER and that ribosomes retain their membrane association after translation termination. First, a summary of the history of the field is presented to provide context for the key, unanswered questions in the field. Then, experiments employing [32Pi] pulse-chase labeling of HeLa cells over a time course to follow nascent ribosome trafficking are presented. The purpose of the cell labeling was to track rRNA processing and assembly into nascent ribosomes, followed by their export into the cytoplasm and recruitment into active polysomes. A detergent-based cell fractionation procedure was also utilized to separate the cytosol and ER compartments in order to observe ribosomes on their path as they exit the nucleus and either localize to the ER or cytosolic cellular compartment. Through this method, it was seen that ribosomes appear in both compartments at the same time, suggesting a mechanism may be occurring in addition to SRP-dependent ribosome trafficking. This research provides an understanding toward a mechanism that is not currently known, but will one day more fully explain the patterns of ribosomal localization.

Effects of task instruction on autobiographical memory specificity in young and older adults

Older adults tend to retrieve autobiographical information that is overly general (i.e., not restricted to a single event, termed the overgenerality effect) relative to young adults' specific memories. A vast majority of studies that have reported overgenerality effects explicitly instruct participants to retrieve specific memories, thereby requiring participants to maintain task goals, inhibit inappropriate responses, and control their memory search. Since these processes are impaired in healthy ageing, it is important to determine whether such task instructions influence the magnitude of the overgenerality effect in older adults. In the current study participants retrieved autobiographical memories during presentation of musical clips. Task instructions were manipulated to separate age-related differences in the specificity of underlying memory representations from age-related differences in following task instructions. Whereas young adults modulated memory specificity based on task demands, older adults did not. These findings suggest that reported rates of overgenerality in older adults' memories might include age-related differences in memory representation, as well as differences in task compliance. Such findings provide a better understanding of the underlying cognitive mechanisms involved in age-related changes in autobiographical memory and may also be valuable for future research examining effects of overgeneral memory on general well-being. © 2013 Taylor & Francis.

Effects of task instruction on autobiographical memory specificity in young and older adults.

Older adults tend to retrieve autobiographical information that is overly general (i.e., not restricted to a single event, termed the overgenerality effect) relative to young adults' specific memories. A vast majority of studies that have reported overgenerality effects explicitly instruct participants to retrieve specific memories, thereby requiring participants to maintain task goals, inhibit inappropriate responses, and control their memory search. Since these processes are impaired in healthy ageing, it is important to determine whether such task instructions influence the magnitude of the overgenerality effect in older adults. In the current study participants retrieved autobiographical memories during presentation of musical clips. Task instructions were manipulated to separate age-related differences in the specificity of underlying memory representations from age-related differences in following task instructions. Whereas young adults modulated memory specificity based on task demands, older adults did not. These findings suggest that reported rates of overgenerality in older adults' memories might include age-related differences in memory representation, as well as differences in task compliance. Such findings provide a better understanding of the underlying cognitive mechanisms involved in age-related changes in autobiographical memory and may also be valuable for future research examining effects of overgeneral memory on general well-being.

The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration.

Growth cone guidance and synaptic plasticity involve dynamic local changes in proteins at axons and dendrites. The Dual-Leucine zipper Kinase MAPKKK (DLK) has been previously implicated in synaptogenesis and axon outgrowth in C. elegans and other animals. Here we show that in C. elegans DLK-1 regulates not only proper synapse formation and axon morphology but also axon regeneration by influencing mRNA stability. DLK-1 kinase signals via a MAPKAP kinase, MAK-2, to stabilize the mRNA encoding CEBP-1, a bZip protein related to CCAAT/enhancer-binding proteins, via its 3'UTR. Inappropriate upregulation of cebp-1 in adult neurons disrupts synapses and axon morphology. CEBP-1 and the DLK-1 pathway are essential for axon regeneration after laser axotomy in adult neurons, and axotomy induces translation of CEBP-1 in axons. Our findings identify the DLK-1 pathway as a regulator of mRNA stability in synapse formation and maintenance and also in adult axon regeneration.

Safety-critical medical device development using the UPP2SF model translation tool

Software-based control of life-critical embedded systems has become increasingly complex, and to a large extent has come to determine the safety of the human being. For example, implantable cardiac pacemakers have over 80,000 lines of code which are responsible for maintaining the heart within safe operating limits. As firmware-related recalls accounted for over 41% of the 600,000 devices recalled in the last decade, there is a need for rigorous model-driven design tools to generate verified code from verified software models. To this effect, we have developed the UPP2SF model-translation tool, which facilitates automatic conversion of verified models (in UPPAAL) to models that may be simulated and tested (in Simulink/Stateflow). We describe the translation rules that ensure correct model conversion, applicable to a large class of models. We demonstrate how UPP2SF is used in themodel-driven design of a pacemaker whosemodel is (a) designed and verified in UPPAAL (using timed automata), (b) automatically translated to Stateflow for simulation-based testing, and then (c) automatically generated into modular code for hardware-level integration testing of timing-related errors. In addition, we show how UPP2SF may be used for worst-case execution time estimation early in the design stage. Using UPP2SF, we demonstrate the value of integrated end-to-end modeling, verification, code-generation and testing process for complex software-controlled embedded systems. © 2014 ACM.

[Book review] Instruction Paintings

Tony Mann provides a review of the book: Yoko Ono, Instruction Paintings, Weatherhill Inc., 1996, ISBN 0-8348-0348-8, £12.99

Hermeneutics, accreting receptions, hypermedia: A tool for reference versus a tool for instruction

We provide a select overview of tools supporting traditional Jewish learning. Then we go on to discuss our own HyperJoseph/HyperIsaac project in instructional hypermedia. Its application is to teaching, teacher training, and self-instruction in given Bible passages. The treatment of two narratives has been developed thus far. The tool enables an analysis of the text in several respects: linguistic, narratological, etc. Moreover, the Scriptures' focality throughout the cultural history makes this domain of application particularly challenging, in that there is a requirement for the tool to encompass the accretion of receptions in the cultural repertoire, i.e., several layers of textual traditions—either hermeneutic (i.e., interpretive), or appropriations—related to the given core passage, thus including "secondary" texts (i.e., such that are responding or derivative) from as disparate realms as Roman-age and later homiletics, Medieval and later commentaries or supercommentaries, literary appropriations, references to the arts and modern scholarship, etc. in particular, the Midrash (homiletic expansions) is adept at narrative gap filling, so the narratives mushroom at the interstices where the primary text is silent. The genealogy of the project is rooted in Weiss' index of novelist Agnon's writings, which was eventually upgraded into a hypertextual tool, including Agnon's full-text and ancillary materials. Those early tools being intended primarily for reference and research-support in literary studies, the Agnon hypertext system was initially emulated in the conception of HyperJoseph, which is applied to the Joseph story from Genesis. Then, the transition from a tool for reference to an instructional tool required a thorough reconception in an educational perspective, which led to HyperIsaac, on the sacrifice of Isaac, and to a redesign and upgrade of HyperJoseph as patterned after HyperIsaac.

Research and perspectives on processing instruction

This volume tracks the impact processing instruction has made since its conception. It provides an overview of new research trends on measuring the relative effects of processing instruction. Firstly, the authors explain processing instruction, both its main theoretical underpinnings as well as the guidelines for developing structured input practices. Secondly, they review the empirical research conducted, to date, so that readers have an overview of new research carried out on the effects of processing instruction. The authors finally reflect on the generalizability and limits of the research on processing instruction and offer future directions for processing instruction research.

Delivering processing instruction in classroom and virtual contexts

Processing Instruction (PI) is an approach to grammar instruction for second language learning. It derives its name from the fact that the instruction (both the explicit explanation as well as the practices) attempt to influence, alter, and/or improve the way learners process input. PI contrasts with traditional grammar instruction in many ways, most principally in its focus on input whereas traditional grammar instruction focuses on learners' output. The greatest contribution of PI to both theory and practice is the concept of "structured input", a form of comprehensible input that has been manipulated to maximize learners' benefit of exposure to input. This volume focuses on a new issue for PI, the role of technology in language learning. It examines empirically the differential effects of delivering PI in classrooms with an instructor and students interacting (with each other and with the instructor) versus on computers to students working individually. It also contributes to the growing body of research on the effects of PI on different languages as well as different linguistic items: preterite/imperfect aspectual contrast and negative informal commands in Spanish, the subjunctive of doubt and opinion in Italian, and the subjunctive of doubt in French. Further research contributions are made by comparing PI with other types of instruction, specifically, with meaning-oriented output instruction.

Grammar acquisition and processing instruction: Secondary and cumulative effects

Research on Processing Instruction has so far investigated the primary effects of Processing Instruction. In this book, the results of a series of experimental studies investigating possible secondary and cumulative effects of Processing Instruction on the acquisition of French, Italian and English as a second language will be presented. The results of the three experiments have demonstrated that Processing Instruction not only provides learners the direct or primary benefit of learning to process and produce the morphological form on which they received instruction, but also a secondary benefit in that they transferred that training to processing and producing another morphological form on which they had received no instruction.

Lost in Translation, book review of Urban Landscape Perspectives

In Sofia Coppola's 2003 film Lost in Translation, Bill Murray and Scarlett Johansson's characters find themselves culturally stranded and oddly mismatched as an improvised tourist couple in contemporary Tokyo. This is an urban landscape that they cannot comprehend but only temporarily experience, in a fragmented and surreptitious way that allows no possible understanding and categorizations, but offers physical inclusion, emotional participation and momentary embeddedness.