Optimisation of the Schizosaccharomyces pombe urg1 expression system

The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down P urg1 "OFF-levels" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining P urg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair

Identification and Correction of Mechanisms Underlying Inherited Blindness in Human iPSC-Derived Optic Cups

Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290, which causes missplicing and premature termination, but the basis of this sensitivity is unclear. Here, we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups, despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups, explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290, restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention

Contemporary developments in Negro middle-class religion : an overview

The sociology of religion is often placed in a defensive position in which it must define its status as a legitimate area of scientific inquiry. Many doubt that religious phenomena, with a concern for the supernatural and with individual, subjective, non-empirical elements can be studied in any systematic, reliable fashion. But as the sociologist views religion, individual and subjective attributes are only one aspect of religious phenomena. Beyond this, religion is viewed as having significant collective and social elements. Religion is seen as a part of a cultural complex such that there is an interrelation of religion and society which involves mutual influences. The social system and social changes affect religious forms and expressions. At the same time, religion has significant effects on the socio-cultural system

Investigating the North Carolina charter school movement: a critical discourse analysis of editorial-page items from 1995 to 2014

Charter schools remain hotly contested nationally and in North Carolina, where the number of charter schools has grown rapidly from 100 in 2011 to 148 in 2014. Interest shows no signs of abating. North Carolina has approved 11 new charter schools to open in 2015, along with two new virtual charter schools. The state has received 40 applications from charter operators hoping to open new schools in 2016. This dissertation conducts a critical discourse analysis of 114 randomly selected editorial-page items and 60 news articles about charter schools published by 20 North Carolina newspapers from 1995 to 2014. The findings indicate that the strategic use of political discourse, consistent message framing, and editorial board support helped charter school proponents gain more social, economic, and cultural power in North Carolina, despite charter schools' tendency toward hyper-segregation by race and class, limited gains in student achievement, and lingering doubts regarding equity of access, scalability, oversight, and public accountability. Key words: critical discourse analysis (CDA), frames, editorial(s), news, charter schools, race, class, equity, North Carolina, school reform, public policy

Examining the time course of attention in a soccer kick using a dual task paradigm

A dual-task paradigm was implemented to determine the time course of attention demands of a soccer penalty kick. A sample of experienced soccer players was selected to participate in the study. Participants (N= 15) were asked to perform a 12-yard soccer-style penalty kick into a FIFA-regulation soccer goal that was divided into smaller scoring zones. As part of the dual task paradigm, participants were instructed to respond to an audible cue that was administered during one of three probe positions (PP) during the primary task. Tests were performed with both the dominant foot (DFP) and the non-dominant foot (NDFP). The order of presentation of the tasks and of the probe positions was randomized and counterbalanced. I hypothesized that the Verbal Response Reaction Time (VRRT) would follow the pattern observed in previous research conducted with other sport skills. Reaction times would be slowest at the beginning and end of the performance of the motor skill in both the DFP and NDFP situations. I also hypothesized that overall VRRT would be slower in the NDFP than DFP, but that kicking foot would not affect the pattern of attentional demands. Results indicated that VRRT was slowest at PP1 for both the DFP and the NDFP and that VRRT was significantly slower at PP1 for the DFP than for the NDFP. This suggests that soccer players may engage in more complex planning during the preparatory phases when executing a kick with their dominant foot or, alternatively, that the determinant of attentional demands is the support foot rather than the kicking foot. Future research should be designed to further our understanding of foot dominance with regard to kicking and to explore attentional demands of striking tasks

Visual design of Jean Genet's The balcony

The purpose of this thesis is to present in three parts the design production of Jean Genet’s The Balcony. The thesis is divided into three parts: (l) Part I, the playwright, the play, and the design approach, (2) Part II, the technical production, and (3) Part III, an analysis. Part I deals with the historical and stylistic considerations influencing the design approach and a description of the design approach to setting, costumes and makeup, and lighting. Part II, largely illustrative, contains renderings, production photographs, and working plots for sets, costumes, and lighting. Part III discusses the weaknesses of the set, costumes, and lighting as part of the total visual design

Local public sector allocation of scarce emergency assets : an evaluation of the fire service.

The methodology utilized by public sector managers to allocate scarce resources determines the level, efficiency, and effectiveness of service delivery. These decisions are influenced by a myriad of factors, not the least of which is the ideal goal to distribute services in a fair and equitable manner. This ideal becomes problematic if service outcomes are important to public decision-makers, because the level of need for these resources is not spread equally across local jurisdictions. Therefore, when goods and services are located or distributed equally to all “customers,” many do not receive enough assistance and others receive more than they prefer. This causes inefficient service delivery that fails to maximize potential positive outcomes with the available limited resources. This is particularly true with the geographic distribution of fire service resources across most communities in this country. This research effort attempts to model the demographic characteristics that drive emergency service demand and workload across local jurisdictions. Specifically, data about demographic characteristics was collected at the Census block group level and compared to emergency response data collected by the Charlotte Fire Department. The findings from this effort are promising, as the bivariate correlation and multivariate regression analyses indicate that economic and structural factors common to all local communities can be used to confidently predict demand and workload on local public safety systems. Measuring these characteristics at the block group level permitted the opportunity to isolate homogenous groups within the population that have risk characteristics associated with more or less demand for these services. These findings provide a solid base to support the development of an alternative model for locating these critical emergency resources according to demand and workload to better meet the needs of individual communities

The Value Of Using Biracial Young Adult Literature In The Secondary Classroom

This thesis argues for the inclusion of biracial young adult literature in the secondary classroom because of the value it can add to students', teachers', and communites’ lives. As the population of biracial young adults grows, as does the need for them to see themselves in the classroom for the development of their identity. By including biracial young adult literature in the secondary classroom, it provides a tremendous learning experience for students who are biracial and non-biracial. For students who are biracial, it allows them to connect with another who faces similar problems to themselves. For non-biracial students, these novels provide them the opportunity to learn about the issues their classmates may face and also provides a platform for these issues to be discussed. For teachers, it provides the opportunity to teach about issues of social justice, bullying, and advocating for others. By examining multiple biracial young adult novels and the issues discussed within, this thesis defends and asserts the value of using these novels in the classroom

Polymerase δ replicates both strands after homologous recombination-dependent fork restart

To maintain genetic stability DNA must be replicated only once and replication completed even when individual replication forks are inactivated. Because fork inactivation is common, the passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination-dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via Homologous Recombination Restarted Replication (HoRReR) ensures cell survival, but at a cost. One such cost is increased mutagenesis caused by HoRReR being more error prone than canonical replication. This increased error rate implies that the HoRReR mechanism is distinct from that of a canonical fork. Here we exploit the fission yeast Schizosaccharomyces pombe to demonstrate that a DNA sequence duplicated by HoRReR during S phase is replicated semi-conservatively, but that both the leading and lagging strands are synthesised by DNA polymerase delta