No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

FtsZ is a tubulin-like protein that polymerizes into a ring like structure at midcell, which is the first step in septum formation. The dynamics of FtsZ polymerization is regulated by a set of proteins, one of which is ZapA. ZapA is a non-essential positive regulator of FtsZ polymerization. In this study we have performed a screen for mutations in Bacillus subtilis that result in a cell division defect when combined with ΔzapA. Three such mutations were found in the yvcL gene. Since this gene is homologous to whiA from Streptomyces coelicolor, and the lack of both proteins imposes in some instances similar phenotypes, we proposed to rename the gene whiA. Mutation of whiA alone had only a mild effect on cells, which became 20-60% longer. However, the double mutant ΔwhiA ΔzapA is filamentous and sick. Evidence is provided that the filamentation is caused by delocalization of FtsZ, and that WhiA is implemented in the early stage of cell division. Interestingly, WhiA localizes to the nucleoid and is important in cells that overinitiate replication. We also found that this protein is essential for survival after UV-induced DNA damage. Its binding sites on DNA were identified using a ChIP-on-chip method and the dif site, which is important for chromosome dimer resolution, was found to be a possible target of WhiA. A transcriptome analysis using whole genome microarray showed that WhiA does not function as transcriptional regulator. We conclude that WhiA is involved in both cell division and chromosome dynamics.

Serum/glucocorticoid regulated kinase 1 (SGK1) is a key component of the pathway that leads to activation of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN). Regulation of ENaC is a major determinant of renal Na+ absorption and overall body fluid homeostasis and blood pressure. Studies from our laboratory have shown that human skin cells express multiple SGK1 isoforms (A-F) that arise from alternative transcriptional start sites and RNA splicing at the SGK1 locus. The aim of this study was to investigate if SGK1 isoforms are also expressed in the ASDN and to assess their potential role in regulating Na+ transport. For these studies the mouse cortical collecting duct cell line mpkCCDcl4, was used as an in vitro model of the ASDN. Comparison of mouse Sgk1 expressed sequence tags (ESTs) with genomic DNA, identified four potential Sgk1 isoforms (Sgk1a-1d). Each isoform has a unique amino terminus of varying size, but otherwise an identical sequence. Using sequence specific primers, mRNA expression of all four isoforms was confirmed by RT-PCR from purified mpkCCDcl4 cell and mouse renal tissue RNA. mpkCCDcl4 cells exposed to aldosterone (Aldo) or Aldo plus insulin (Ins) showed a time-dependent increase in the endogenous expression levels of multiple Sgk1 bands within 1 hour of treatment. These Aldo and Aldo + Ins-induced endogenous Sgk1 bands co-migrated with overexpressed Sgk1 a-d isoform bands. Aldosterone also produced a significant increase in amiloride-sensitive (ENaCmediated) equivalent short circuit current within 2 hours of exposure, peaking after 4 hours. Insulin potentiated the Aldo response, but had no effect alone. Specific inhibitors showed that the hormonal response involved both PI3Kinase and mTOR-dependent and independent pathways. Immunofluorescence studies utilising cloned and tagged SGK1 isoforms in transfected HEK293T cells revealed cytoplasmic network-like staining for all SGK1 isoforms except for SGK1D, which produced plasma membrane staining. In mouse renal tissue, endogenous Sgk1d localised to the basolateral membrane of collecting duct epithelial cells. Furthermore co-immunoprecipitation of cloned human SGK1 and mouse Sgk1 proteins with the Aldo induced regulatory protein, glucocorticoid-induced leucine zipper protein 1 (GILZ1), showed isoform-specific interactions. Collectively, these results build upon our understanding of SGK1 gene expression, protein localisation and function in the ASDN.

Chromosome segregation is a complex, but subsequently error- prone process, who’s accuracy is essential to prevent uneven DNA distribution between mother and daughter cells. Such unequal chromosome segregation can often result in aneuploidy, which is a prevalent phenotype of cancer cells, and so surveillance mechanisms must exist within the cell cycle to detect and correct the cause of such chromosome division errors, before allowing the cell to divide. The Spindle Assembly Checkpoint (SAC) has evolved to monitor the interaction between microtubules, and the point at which they attach sister chromosomes, the kinetochore. By detecting attachment and resulting tension abnormalities, the SAC halts the metaphase to anaphase transition if chromosomes are not aligned correctly at the metaphase plate. By disallowing cell division to occur in the absence of proper chromosome alignment, the SAC minimises the frequency of uneven DNA distribution and the consequent problems this can incur. Silencing of the SAC, and normal cell progression is not promoted until correction mechanisms have achieved proper bioriented chromosome attachments. The target of the SAC is widely accepted to be Cell Division Cycle 20 (Cdc20), which is the activator of the Anaphase Promoting Complex or Cyclosome (APC/C), the E3 ubiquitin ligase that drives cells into anaphase. By inhibiting Cdc20, the activity of the APC/C is halted, and cells are arrested at metaphase. A number of key proteins are believed to be involved in the sequestration of Cdc20, by incorporating it into an inhibitory Mitotic Checkpoint Complex (MCC). This MCC complex is believed to comprise of Cdc20, BubR1, Bub1 and Mad2, although there is speculation as to whether Mad2 is part of the complex, or merely promotes its formation. The proteins involved in the MCC all localise to kinetochores with activation of the SAC, although it remains unclear as to whether the MCC forms at the kinetochore upon localisation of the various components, or can form in part or as a whole, moving to kinetochores upon SAC activation. Sub-complexes of the MCC have been detected outside of mitosis, which provide evidence in favour of a kinetochore-independent MCC formation. However, if this were the case, it could be assumed that modification (such as phosphorylation) to either MCC components or the APC/C itself would need to occur in mitosis or with SAC activation, allowing for APC/C inhibition only with SAC activation, and to prevent IV non-specific inhibition of APC/C by the MCC elsewhere in the cell cycle. These issues still remain unclear. In order to investigate further, the requirement of direct kinetochore localisation of MCC components in the formation of the complex, this thesis aims to provide evidence of the effect of disrupting such kinetochore localisation upon checkpoint function, as well as the impact of removal of Cdc20 modifications on MCC formation. In addition to this, the protein-protein interaction domains between Cdc20 and BubR1, proven essential for SAC function, are investigated within Drosophila melanogaster. Collectively, the data in this thesis provides an insight into the regulation of SAC in Drosophila. The Cdk1/Cyclin B phosphorylation of Fizzy (the Drosophila homologue of Cdc20) is confirmed to have an effect on MCC formation, and can be mapped to three specific sites on the N-terminal of Fizzy, which are conserved across various species. In addition to the effect of Cdk1/Cyclin B phosphorylation on the interaction between Fizzy and other SAC proteins, the importance of the BubR1 KEN box motif on the Fizzy-BubR1-Mad2 interaction is confirmed, implicating another essential domain for MCC formation in Drosophila. With regard to kinetochore localisation of SAC components, a model is achieved in which a dramatic reduction of Mps1, previously shown to disturb kinetochore localisation of Mad1, Mad2 and BubR1 in Xenopus, confirms a role for Mad2 kinetochore localisation in SAC activation, even though Fizzy localisation is unperturbed. Overall, these findings may provide a useful insight into the complex relationships, kinetochore localisation requirements and inter-protein dependencies within the regulation of MCC formation and SAC signalling in Drosophila melanogaster.

Polyunsaturated Fatty Acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are increasingly attracting scientific attention due to their significant health promoting role in the human body. However, the human body lacks the ability to produce them in vivo. The limitations associated with the current sources of ω-3 fatty acids and ω-6 fatty acids from animal and plant sources have led to increased interest in microbial production. Marine bacteria provide a suitable alternative, although the isolation of production strains and the identification of operating conditions must be addressed before manufacturing processes become economically viable. Sea sediment samples were collected from three different environments including Mid Atlantic Ridge, Red Sea and Mediterranean Sea. The isolates were screened for PUFA production using a fast colourimetric method and verified by gas chromatography/mass spectroscopy. The isolated PUFA producers were characterised and identified on the basis of 16S rRNA gene sequencing and analysis. Three different isolates were chosen for this study. These were labelled as 717, 66 and Hus-27. The chosen isolates were subjected to an optimisation study to maximise their productivity. This optimisation strategy included identifying a suitable production medium by applying a statistical design of experiment methodology (Plackett-Burman and Central Composite Design). A chemically defined media was identified for isolates 717 and 66 in order to determine the limiting media components and to study the effect of carbon/nitrogen ratio on the productivity of isolates. As an important step in the process development of the microbial PUFA production, the culture conditions at the bioreactor scale were optimised for isolate 717 using a Response Surface Methodology (RSM) revealing the significant effect of temperature, dissolved oxygen and the interaction between them on the EPA production. Two sets of continuous stirred-tank reactor (CSTR) experiments were also performed to test the effect of growth rates on EPA production and the effect of temperature at constant growth rate as this was identified as the most significant factor affecting EPA production. This optimisation strategy led to a significant increase in the amount of EPA produced by isolates under investigation, where the amount of EPA increased from 9 mg/g biomass, 33 mg/l representing 7.6% of the total fatty acids to 45 mg/g, 350 mg/l representing 25% of the total fatty acids using isolate 717. A significant increase was also achieved using isolate 66 with the amount of EPA increased from 5.5 mg/g, 14 mg/l representing 3.5% of the total fatty acids to 32 mg/g, 285 mg/l representing 15% of the total fatty acids. For isolate Hus-27 the amount of EPA increased from 0.6 mg/g, 3 mg/l representing 2.2% of the total fatty acids to 8 mg/g, 36 mg/l representing 8% of the total fatty acids. The stability of the produced oil and the complete absence of heavy metals in bacterial biomass are considered as an additional benefit of bacterial EPA compared to other sources of PUFA. To our knowledge this is the first report of a bacterial isolate producing EPA with such high yields making large scale manufacture much more economically viable.