Phosphatidylinositol (4,5)-bisphosphate turnover by INP51 regulates the cell wall integrity pathway in "Saccharomyces cerevisiae"

Signal transduction pathways are important for the cell to transduce external or internal stimuli where second messengers play an important role as mediators of the stimuli. One important group of second messengers are the phosphoinositide family present in organisms ranging from yeast to mammals. The dephosphorylation and phosphorylation cycle of the phosphatidylinositol species are thought to be important in signaling for recruitment or activation of proteins involved in vesicular transport and/or to control the organization of the actin cytoskeleton. In mammals, phosphatidylinositol (4,5)bisphosphate (PI(4,5)P2) signaling is essential and regulated by various kinases and phosphatases. In the model organism Saccharomyces cerevisiae PI(4,5)P2 signaling is also essential but the regulation remains unclear. My dissertation focuses on the regulation of PI(4,5)P2 signaling in Saccharomyces cerevisiae. The organization of the actin cytoskeleton in Saccharomyces cerevisiae is regulated by different proteins such as calmodulin, CMD1, and here I present data that CMD1 plays a role in the regulation of the only phosphatidylinositol 4-phosphate 5-kinase, MSS4, in Saccharomyces cerevisiae. CMD1 regulates MSS4 activity through an unknown mechanism and thereby controls the organization of the actin cytoskeleton. MSS4 and CMD1 do not physically interact but MSS4 seems to be part of a large molecular weight complex as shown by gel filtration chromatography. This complex could contain regulators of the MSS4 activity. The complex is not caused by dimerization of MSS4 since MSS4 does not interact with itself. Two pathways, the cell wall integrity pathway and TORC2 (target of rapamycin complex 2) signaling cascade are important for the organization of the actin cytoskeleton. Loss of TOR2 function results in a growth defect that can be suppressed by MSS4 overexpression. To further characterize the link between MSS4 and the TORC2 signaling pathway and the cell wall integrity pathway we looked for targets of PI(4,5)P2. The TORC2 pathway and the cell wall integrity pathway signal to the GEF ROM2, an activator of the small GTPase RHO1. In our study we identified ROM2 as a target of PI(4,5)P2 signaling. We observed that the ROM2 localization changes in an mss4 conditional mutant. This suggests that the proper localization needs PI(4,5)P2. This could be mediated by the putative PI(4,5)P2 binding pleckstrin homology (PH) domain of ROM2. To better understand the regulation of PI(4,5)P2 levels in Saccharomyces cerevisiae we focused on one of the PI(4,5)P2 5-phosphatases, INP51. Here we present evidence that INP51 is a new negative regulator of the cell wall integrity pathway as well as the TORC2 pathway. INP51 probably regulates these two pathways by the turnover of PI(4,5)P2 thereby inactivating the effector/s. The deletion of INP51 does not result in any phenotype, but when combined with mutations of the cell wall integrity pathway we observe synthetic interaction. INP51 together with the GTPase activating protein (GAP) SAC7, responsible for the negative regulation of RHO1, negatively regulates the cell wall integrity pathway during vegetative growth. One of the targets of cell wall integrity pathway, the cell wall component chitin, which is normally deposited at the bud end, bud neck and forms bud scars, is delocalized in the mother cell in the sac7 inp51 double deletion mutant. In addition, another downstream component of the cell wall integrity pathway, the MAP kinase MPK1, has increased phosphorylation and protein level in the sac7 inp51 double deletion mutant. This suggests that INP51 is important for the negative regulation of the cell wall integrity pathway. Furthermore, we show evidence that INP51 forms a complex with TAX4 or IRS4, with two EH-domain containing proteins, that positively regulates the activity of INP51 and in this manner negatively regulate the cell wall integrity pathway. The EH-domain is known to bind the NPF-motif. This motif is present in INP51 and is important for INP51 interaction with TAX4 or IRS4. The EH-NPF interaction is a conserved mechanism to build up protein networks. The interaction between an EH-domain containing protein and a PI(4,5)P2 5-phosphatase is conserved. This is demonstrated by the epidermal growth factor substrate EPS15 (EH) interaction with the PI(4,5)P2 5-phosphatase synaptojanin the mammalian orthologue of the Saccharomyces cerevisiae INP proteins. In summary, INP51 together with TAX4 and IRS4, forms complexes important for regulation of PI(4,5)P2 levels. The complexes are linked to the TORC2 signaling pathway and the cell wall integrity pathway, specifically regulating MPK1 activation and chitin biosynthesis. The work presented in this dissertation facilitates the development of a model of the complex regulation of PI(4,5)P2 signaling in Saccharomyces cerevisiae

Adaptive Polling for Responsive Web Applications

YesThe web environment has been developing remarkably, and much work has been done towards improving web based notification systems, where servers act smartly by notifying and feeding clients with subscribed data. In this paper we have reviewed some of the problems with current solutions to real-time updates of multi user web applications; we introduce a new concept “adaptive polling” based on one AJAX technique “Polling” to reduce the high volume of redundant server connections with reasonable latency, we demonstrated a prototype implementation of the new concept which is then evaluated against the existing one; the positive results clearly indicated more efficiency in terms of client-server bandwidth

Music and Arts in Health Promotion and Death Education: The St Christopher’s Schools Project

The reality of death and dying is rarely discussed openly in modern Western societies, while death sometimes is even considered to be a ‘failure’ in the context of traditional, medically-focused healthcare systems. Similarly, loss and transition are part of the National School Curriculum in the UK, but many schools still find approaching these subjects difficult. In this context St Christopher’s hospice in London has initiated and delivered the ‘Schools Project’ since 2005. The St Christopher’s Schools Project is an innovative community arts programme. It takes the form of short-term collaborative arts projects between terminally ill patients and students from primary and secondary schools, as well as colleges within the hospice’s catchment area. The Schools Project has attracted the interest of many other hospices, as well as other healthcare institutions and inspired the development of similar projects, both nationally and internationally. The aim of the Schools Project is to introduce the hospice and its work to the school communities in a creative and non-threatening way. Within a structured framework students are given the opportunity to interact and engage in music and art making together with terminally ill patients, culminating in an exhibition or performance. Promoting healthier attitudes towards death and dying amongst the students, their teachers, school peers, parents and carers, is at the core of the project. This paper presents the philosophy and aims, as well as the process and outcomes, of the Schools Project. Additionally, an overview of all of the projects that have taken place at St Christopher’s since 2005, as well as some prospects for future development, are given. This will hopefully stimulate a constructive dialogue with regards to the potential role of hospices and the arts in the promotion of health and death education, as well as their potential impact on the development of sustainable healthcare policies and practices not only in palliative care, but also in other health and social care contexts

Database metadata requirements for automated web development : a case study using PHP

The Web has come a long way. It started as a distributed document repository and quickly became the spring board for a new type of application. Propped on top of the original HTML+HTTP architecture, this new application platform shifted the way the architecture was used so that commands and functionality were embedded in the form data of Web requests rather than in the HTTP command conveying the request. This approach enabled Web requests to convey any type of data, not just document operations. This is occurring because the Web provides such a powerful platform on which to create applications. This is occurring because web development methods are still evolving toward the structure and stability required taking on this enormous new role. As the needs of developers change, certain themes that arise more frequently than others become embedded into new environments to support those needs. Until recently, Web application programming has largely been done with a set of keywords and metaphors developed long before the Web became a popular place to program. APIs have been developed to support Web specific features, but they are no replacement for fundamental changes in the programming environment itself. The growth of Web applications requires a new type of programming designed specifically for the needs of the Web. This thesis aims to contribute towards the development of an abstract framework to generate abstract and dynamic Web user interfaces that are not developed to a specific platform. To meet this aim, this thesis suggests a general implementation of a prototype system that uses the information in database metadata in conjunction with PHP. Database metadata is richer in providing the information needed to build dynamic user interfaces. This thesis uses PHP and the abstract library ADOdb to provide us with a generalised database metadata based prototype. PHP does not have any restrictions on accessing and extracting database metadata from numerous database management systems. As a result, PHP and relational database were used to build the proposed framework. Additionally, ADOdb was used to link the two mentioned technologies. The implemented framework in this thesis demonstrates that it is possible to generate different automatic Web entry forms that are not specific at any platform.EThOS - Electronic Theses Online ServiceGBUnited Kingdo