Alterations in mRNA and protein profiles in eIF4E-transfected human lung carcinoma cells

Previous work has shown that treatment of the lung cell carcinoma cell line DLKP with the differentiation modulating agent bromodeoxyuridine (BrdU) causes posttranscriptionally regulated changes in the expression of growth and differentiation related genes. These changes in gene expression were found to coincide with an increase in the level of expression and phosphorylation of the translation initiation factor eIF-4E. In this study we have overexpressed eIF4E in DLKP cells to determine its role in mediating the changes seen in BrdU treatment and what effects it may have on the growth of lung cancer cells in general as studies have shown eIF4E to play a role in regulating gene expression in carcinogenesis We also analysed the overexpression of Ser209 mutated non-phosphorylatable eIF4E in DLKP cells to determine the role of the eIF4E Ser209 (S209) phosphorylation site in regulating translational changes in gene expression and functional changes in DLKP cells. The exact role of eIF4E Ser209 phosphorylation in translation initiation is currently unknown and conflicting views have emerged as to whether it is necessary for regulation of translation by eIF4E. Stable transfections were carried out using wild type (4E), S209 mutant (4E-S209) and HA (hemagluttinm) epitope-tagged human eIF4E constructs Stable transfections were also earned using empty pcDNA plasmid vector as a negative control .Western blot analysis showed that transfected HA-tagged eEF4E protein was effectively overexpressed in DLKP cells. The transfected cells were cloned out by limiting dilution and clones were chosen for further analysis. Two clones expressing wild type HA tagged eIF4E, two clones expressing HA-tagged 4E-S209 phosphorylation site mutant and two pcDNA vector transfected control clones have been analysed in this study. An eDF4E overexpressing clone which expresses a high level of transfected protein showed increased keratin 8 expression. These cells also showed an increase in pi integrm expression which was not seen in other eIF4E overexpressing clones indicating high levels of 4E overexpression may induce expression of this protein Immunocytochemical analysis of alpha integrm subunits showed increased expression of alpha 3 integrm in eIF4E overexpressing cells. Invasion assays were performed on eIF4E overexpressing cells as increased 4E expression has been detected in certain cancers. The eIF4E overexpressing clone which expresses a high level of transfected protein displayed a large increase in invasiveness compared to control transfected cells whereas other eIF4E transfected clones did not display increased mvasiveness eIF4E-S209 mutant transfected cells showed similar levels of mvasiveness compared to controls. Large scale analysis of the effects of eIF4E overexpression on protein expression levels was undertaken using the novel 2D-DIGE (2 dimensional-differential in gel analysis) two dimensional electrophoresis technique Differentially expressed proteins were identified using mass-spectrometry based techniques. Among the proteins identified were proteins involved in mRNA processing, protein degradation and cytoskeletai regulation Of particular interest, were a number of proteins involved in regulating cytoskeletai dynamics whose expression was down regulated in eIF4E S209 mutant overexpressing cells. A common regulatory element in the mRNA of these proteins was identified which led to the development of a hypothesis for localised translation of these proteins. The possible involvement of eIF4E in regulation of localised translation of these proteins represents a novel aspect of translational regulation by eIF4E which may contribute to its role in oncogenesis. Changes seen in the expression of proteins involved in mRNA processing and protein degradation indicate that other post-transcriptional processes apart from translational regulation may play an important role in regulating gene expression in these cells. Oligonucleotide microarray analysis of the mRNA expression levels of genes in eIF4E overexpressing cells has also been conducted to determine the effects of eIF4E overexpression on transcriptional regulation downstream of its effects on translation regulation. Microarray analysis showed that there are changes in the expression of a large number of genes with diverse cellular functions. This indicates changes in transcriptional regulation occur as a result of eEF4E overexpression mRNA expression levels of a large number of genes involved in cytoskeletai regulation were found to be altered in eIF4E and eIF4E-S209 mutant overexpressing cells. Microarray analysis showed that the integrm signalling gene FAK (focal adhesion kinase) was downregulated in both 4E and 4E S209 overexpesssing cells Western blot analysis showed this gene was also downregulated at the protein level Immunofluorescent staining of FAK showed the localisation of this protein was altered in eIF4E and eIF4E-S209 mutant overexpressing cells and may affect the growth and mvasiveness of these cells. A large number of genes and proteins found to be altered in proteomic and microarray anlysis were involved in regulating actin cytoskeletai dynamics. Cells were therefore analysed for expression of actin cytoskeletai structures. Major changes were detected in actin cytoskeletai structures in an eIF4E overexpressing clone which expresses a high level of transfected protein. We have therefore conducted an in depth analysis into the regulation of growth and differentiation related gene expression by eIF4E

Seasonal climate forecasts for more effective raingrown grain-cotton production systems

Cropping is a risky business. Our highly variable climate makes it difficult to decide how best to manage crops and cropping systems. What works well one year might not work well the next. To develop better risk management practices, this project uses the APSIM cropping systems model to examine the profitability and sustainability of a range of alternative dryland cotton/grain cropping systems throughout the northern grain region of eastern Australia. It involves working closely with farmer collaborators in Central Queensland, the Darling Downs, the northwest slopes of NSW and the Liverpool Plains

The Madden Julian Oscillation and its relationship with rainfall in Queensland

The Madden Julian Oscillation is a large-scale atmospheric phenomenon that is generated above the tropical Indian Ocean. It is associated with large convective systems that propagate eastward across the Pacific Ocean. Since it is an atmospheric event limited to the equatorial domain, it was believed that it has little effect on non-tropical regions. However, recent research found correlations between the positioning of the active Madden Julian Oscillation phase along the Equator and rainfall events northeast Australia. The correlations were significant throughout Queensland. The phenomenon is subject to a study by climate scientists at four Australian institutions. It aims to develop a simple predictive tool of rainfall events that are linked with the active phase of the Madden Julian Oscillation and that is applicable throughout Queensland and possible beyond. The outcome of this research is to be linked with agricultural production systems model in order to help Queensland farmers to better time planting and harvesting, as well as scheduling of contractors whose operations might be delayed by rain

Bases teóricas e importância da variância da vazão como variável bidimensional no dimensionamento de linhas laterais em microirrigação

Visando a reforçar as bases teóricas e contribuir com a melhoria da capacitação educacional em assuntos relacionados a dimensionamento de sistemas de irrigação, o presente ponto de vista revela uma dedução alternativa para a variância da vazão dos emissores, variável aleatória independente bidimensional. Posteriormente apresenta breve aplicação do modelo aceito para dimensionamento de linhas laterais em sistemas de microirrigação, de acordo com a abordagem estatística. A melhor precisão na fabricação de emssores permite, para uma dada inclinação de terreno, dimensionar laterais mais longas, o que não significa necessariamente que uniformidade de emissão dessas laterais será projetada como mais homogênea.In order to support the theoretical basis and contribute to the improvement of educational capability issues relating to irrigation systems design, this point of view presents an alternative deduction of the variance of the discharge as a bidimensional and independent random variable. Then a subsequent brief application of an existing model is applied for statistical design of laterals in micro-irrigation. The better manufacturing precision of emitters allows lengthening a lateral for a given soil slope, although this does not necessarily mean that the statistical uniformity throughout the lateral will be more homogenous

The 1916 Easter Rising web archive project

The 1916 Easter Rising Web Archive was a collaborative project in 2015/2016 between the Library of Trinity College Dublin (University of Dublin), the Bodleian Libraries (University of Oxford), and the British Library. The project aimed to identify, collect, and preserve online resources that can contribute to an understanding of the causes, course, and consequences of the 1916 Easter Rising, and help to enable critical reflection on the Easter Rising and the diverse ways it was commemorated and engaged with in 2016. The context for the project was the UK legal deposit environment in which the six Legal Deposit Libraries1 (LDLs) work together to help preserve the UK’s knowledge and memory

Using seasonal climate forecasts for more effective grain-cotton production systems

The overall aim of this project is to significantly improve financial profitability, economic efficiency and resource risk management of dryland grain/cotton systems through effective use of seasonal climate forecasts and quantification of climatic variability