Fragile X Mental Retardation Protein is Involved in Protein Synthesis-Dependent Collapse of Growth Cones Induced by Semaphorin-3A

Fragile X syndrome, the most frequent form of familial mental retardation, is caused by mutation of the Fmr1 gene. Fmr1 encodes the fragile X mental retardation protein (FMRP), an mRNA binding protein regulating local, postsynaptic mRNA translation along dendrites necessary for long-term synaptic plasticity. However, recent studies on FMRP localization in axons and growth cones suggest a possible function in the regulation of local protein synthesis needed for axon guidance. Here, we have demonstrated that FMRP is involved in axonal and growth cone responses induced by the axon guidance factor, Semaphorin-3A (Sema3A). In cultured hippocampal neurons from wild type mice, Sema3A-induced growth cone collapse was protein synthesis-dependent. In contrast, Sema3A-induced growth cone collapse was attenuated in Fmr1 knock-out (KO) neurons and insensitive to protein synthesis inhibitors, suggesting that FMRP is involved in protein synthesis-dependent growth cone collapse. Sema3A increased phosphorylation of eukaryotic initiation factor 4E (eIF4E), an indicator of local translation, in distal axons and growth cones of wild type, but not Fmr1 KO neurons. Furthermore, Sema3A rapidly induced a protein synthesis-dependent increase in levels of microtubule associated protein 1B (MAP1B) in distal axons of wild type neurons, but this response was attenuated in Fmr1 KO neurons. These results suggest a possible role of FMRP to regulate local translation and axonal protein localization in response to Sema3A. This study reveals a new link between FMRP and semaphorin signaling in vitro, and raises the possibility that FMRP may have a critical role in semaphorin signaling in axon guidance during brain development

This Is The Story of a Man Named Stanley : Narratology, Authorship and Agency in The Stanley Parable

From Aristotle’s Rhetoric to Kenneth Burke’s more contemporary Grammar of Motives, classical rhetorical scholarship has traditionally focused on examining the strategies, styles, and best methods of persuasion through a focus on the individual rhetor, and how they can achieve their desired end. The rise of interactive technologies in the past twenty years have provided contemporary challenges to these classical theories. A player’s ability to interact with a game, translating into an ability to participate in the construction of a text, has now allowed the argument-building process to become co-creative...or so it would appear. This thesis examines the struggle for authorial control in the digital age by examining The Stanley Parable, a game that pits the player in direct opposition to the narrator. The symbolic struggle in The Stanley Parable not only questions the ways in which game stories are told, but the significance of the player interacting with a game’s story. It also raises questions about the significance of these choices and their role in constructing meaning. I argue that the introduction of such technologies creates new rhetorical opportunities that expose the importance of narrative structure, authorship and agency within persuasion. In doing so, I draw from the works of Michel Foucault, Roland Barthes, and Ian Bogost, to examine why “ownership” in storytelling is significant, and why The Stanley Parable serves as a significant, postmodern addition to the ways persuasion can occur

Development and Application of Ultrastructural in Situ Hybridization to Visualize the Spatial Organization of mRNA: a Dissertation

It has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this thesis was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems and structures involved. This work required the development of novel in situ hybridization methods for use with electron microscopy. This allowed resolution to visualize single mRNA molecules and individual filaments. The development of a silver enhancement methodology for both the light and electron microscopic detection of biotinated oligo-dT probes permitted a synoptic view of the intracellular distribution of poly(A) mRNA. At the light microscope, the distribution of poly(A) mRNA did not resemble the individual distribution patterns of microfilaments, intermediate filaments or microtubules. Ultrastructural examination revealed that poly(A) mRNA was not uniformly distributed along cytoskeletal filaments, but clustered at their intersections. The composition of these mRNA containing structures was investigated by both morphologic and in situ hybridization analysis using antibodies to cytoskeletal proteins. In thin sections, polysomes were observed attached to both microfilaments and intermediate filaments. To permit the simultaneous detection of oligo-dT hybridization and specific cytoskeletal proteins, a double labelling method using colloidal gold conjugated antibodies was developed. The majority of poly(A) mRNA was associated with the actin cytoskeleton, with 72% of the hybridization localized within 5nm of a labelled microfilament. Within the actin cytoskeleton, poly(A) mRNA was localized to intersections of orthogonal networks. Greater than 50% of poly(A) colocalized with the actin crosslinking proteins, filamin and α-actinin, but not vinculin. A significant amount of poly(A) mRNA was found to be associated with intermediate filaments. The double label gold analysis demonstrated that 33% of the hybridization signal localized within 5nm of labelled vimentin filaments. Prior disorganization of the actin cytoskeleton using cytochalasin did not disrupt the association of mRNA with vimentin. These observations are consistent with our morphologic results of polysome-intermediate filament associations, and indicate that microfilaments are not the only filament system to which mRNA is bound. Furthermore, a small amount of hybridization signal (12%) consistently was observed along microtubules, providing an additional cytoskeletal network to distribute mRNA. To further characterize the spatial organization of mRNA within the cytoskeleton, ultrastructural methods were developed to directly visualize individual mRNA molecules. First, oligonucleotide probes chemically modified with a single hapten and directly conjugated primary reagents were used to permit detection of an individual hybridized probe molecule by a single gold particle. Second, biotin and digoxigenin labelled oligonucleotide probes were used to simultaneously visualize the intermolecular and intramolecular relationships of two nucleic acid sequences. Third, reverse transcriptase was used to extend hybridized primers in situ which permitted visualization of the poly(A) sequence concomittant with the conformation of an mRNA molecule. These methods have permitted analysis of how single mRNA molecules may be positioned with respect to each other within the cytoskeleton. The ultrastructural visualization of mRNA within its structural environment has demonstrated heterogeneous interactions with the cytoskeleton. Future work will be needed to further characterize the mechanism of mRNA attachment. The proteins which bridge nucleic acid sequences to specific intersections can be identified. It will be interesting to learn how the identified mRNA-cytoskeletal interactions might be involved in the regulation of both mRNA translation and intracellular location. Lastly, and perhaps the most challenging goal, is to investigate whether the identified mRNA-cytoskeletal interactions are used by the cell to influence its own shape, polarity and architecture

Marketing Leadership in a Knowledge Economy

Often the most valuable assets of a marketing driven firm are intangible assets such as a brand name, intellectual capital, and the expertise and knowledge of employees. The new breed of marketing leaders understand that it is important for employees to collaborate and be engaged and that leaders must be agents of change, creative, ethical, and global thinkers who can create learning organizations. The research reveals that organizations that are going to thrive in the knowledge economy are those that have marketing leaders who can build learning organizations, encourage diversity, and ensure employees are engaged in meaningful work

Storytelling And Reflective Pedagogy: Transforming Nursing Education Through Faculty Development

Nurse educators require pedagogical approaches beyond traditional methods to facilitate student learning of new competencies to practice in complex health care environments. However, little direction is available about how to effectively transform education. The purpose of this quality improvement project was to develop and implement steps to initiate change in both systems and processes of teaching and learning; to provide an efficient, sustainable method to incorporate transformative pedagogies through innovative faculty development; and, to collect outcomes of an e-Learning course to support teaching, using Kirkpatrick’s 4-level Model. An innovative course using storytelling and reflective pedagogy was developed to guide faculty into a transformative learning experience to challenge assumptions, gather insights, and raise questions about teaching practices. Pre- and post-course surveys captured data across three levels: satisfaction, knowledge and skill acquisition, and change in behavior. Forty-five participants were initially evaluated, while 31 were eligible for evaluation at three months. Follow-up survey results yielded a 42% response rate. Pre- and post-surveys were analyzed using a two-tailed, dependent t-test. Significant gains were recorded across all three areas (p<0.05), with large to medium effect size noted using Cohen’s d. Follow-up surveys revealed a significant change in knowledge (p<0.05), whereas the skill and attitude effect change were not statistically significant (p<0.05). Results suggest storytelling and reflective pedagogy are effective for faculty to confront and resolve actual and desired teaching practices, and that faculty placed value on reflection to facilitate self-awareness, question assumptions, and nurture ideas about personal and professional growth

High-efficiency transfection of cultured primary motor neurons to study protein localization, trafficking, and function

<p>Abstract</p> <p>Background</p> <p>Cultured spinal motor neurons are a valuable tool to study basic mechanisms of development, axon growth and pathfinding, and, importantly, to analyze the pathomechanisms underlying motor neuron diseases. However, the application of this cell culture model is limited by the lack of efficient gene transfer techniques which are available for other neurons. To address this problem, we have established magnetofection as a novel method for the simple and efficient transfection of mouse embryonic motor neurons. This technique allows for the study of the effects of gene expression and silencing on the development and survival of motor neurons.</p> <p>Results</p> <p>We found that magnetofection, a novel transfection technology based on the delivery of DNA-coated magnetic nanobeads, can be used to transfect primary motor neurons. Therefore, in order to use this method as a new tool for studying the localization and transport of axonal proteins, we optimized conditions and determined parameters for efficient transfection rates of >45% while minimizing toxic effects on survival and morphology. To demonstrate the potential of this method, we have used transfection with plasmids encoding fluorescent fusion-proteins to show for the first time that the spinal muscular atrophy-disease protein Smn is actively transported along axons of live primary motor neurons, supporting an axon-specific role for Smn that is different from its canonical function in mRNA splicing. We were also able to show the suitability of magnetofection for gene knockdown with shRNA-based constructs by significantly reducing Smn levels in both cell bodies and axons, opening new opportunities for the study of the function of axonal proteins in motor neurons.</p> <p>Conclusions</p> <p>In this study we have established an optimized magnetofection protocol as a novel transfection method for primary motor neurons that is simple, efficient and non-toxic. We anticipate that this novel approach will have a broad applicability in the study of motor neuron development, axonal trafficking, and molecular mechanisms of motor neuron diseases.</p

Transforming Leaders Into Stewards Of Teaching Excellence: Building And Sustaining An Academic Culture Through Leadership Immersion

Nursing must transform education and practice to meet the changing healthcare environment; yet, steps to desired change remain unknown. Academic leaders are well-positioned to initiate change and transform the academic landscape. However, many advance to leadership positions with minimal orientation to the role. Moreover, leaders in academic nursing often have expertise as clinicians and administrators, and not as academics. It is incumbent on nurse leaders to acquire needed competencies to fulfill the academic role. The purpose of this quality improvement project was to immerse leaders in an exploration of steps to initiate and sustain change in the teaching and learning process. Self-reported low- and high-level outcomes were analyzed using Kirkpatrick’s Model (1994) to evaluate the effectiveness of the immersion in preparing leaders to build and sustain a quality academic culture. Leadership immersions were implemented to transform leaders into stewards of teaching excellence. Pre- and post-immersion surveys captured data across three levels: satisfaction, knowledge and skill acquisition, and change in behavior. Seventy-three participants were evaluated. Participants for inclusion in the three-month analysis culminated in a 41% response rate. Findings were analyzed using ANOVA and t-tests. Further analysis was performed using Cohen’s d to determine effect size. Three-month follow-up surveys revealed no significant effect change (p&lt;0.05). Results suggest immersion is effective in preparing leaders of academic nursing to build a quality academic culture. Through immersion, leaders established a collective vision of teaching excellence and proficiency in confronting and resolving actual and desired teaching practices, while enriching the life and work of faculty