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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

Identification of a novel cell division protein in Bacillus subtilis

Surdová, Katarína January 2012 (has links)
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.

Investigation of the role of novel SGK1 isoforms in regulation of sodium transport in kidney epithelial cells

Daniels, Nigel Allan January 2010 (has links)
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.

Regulation of spindle assembly checkpoint (SAC) by phosphorylation and protein-protein interactions in Drosophila melanogaster

Herriott, Ashleigh Jane January 2012 (has links)
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.

Production of polyunsaturated fatty acids from marine microorganisms

Abd Elrazak, Ahmed Abdo Ahmed January 2012 (has links)
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.

Mechanisms by which glycoside hydrolases recognize plant, bacterial and yeast polysaccharides

Cuskin, Fiona Marie January 2013 (has links)
The deconstruction of complex carbohydrates by glycoside hydrolases requires extensive enzyme consortia in which specificity is often conferred by accessory modules and domains that are distinct from the active site. The diverse mechanisms of substrate recognition were explored in this thesis using selected yeast, bacterial and plant polysaccharides as example substrates. Carbohydrate binding modules (CBM) are non-catalytic modules that enhance the catalytic activity of their glycoside hydrolase counterparts through binding to polysaccharide. Normally CBMs are found attached to glycoside hydrolases that target insoluble recalcitrant substrates resulting in a moderate, 2-5 fold, potentiation in enzyme activity. A CBM, defined herein as CBMX40, is found at the C-terminal of a glycoside hydrolase family (GH) 32 enzyme, SacC, which displays exo-levanase activity. CBMX40 binds the non-reducing end of the levan chain targeting the disaccharide fructose--fructose unit. Removal of CBMX40 results in a >100-fold decrease in catalytic activity against levan, compared to the full length native enzyme. The truncated SacC catalytic domain acts as a non-specific exo-β-fructosidase displaying similar activity on β2,1- (inulin) and β2,6-linked fructose polymers, both polysaccharides and oligosaccharides. When CBMX40 was fused to a non-related exo-β-fructosidase, BT 3082, it conferred exo-levanase specificity on the enzyme. Thus CBMX40 is not only able to enhance catalytic activity but is also able to confer catalytic specificity. This led to the hypothesis that the CBM and the active site of the enzyme bind to different terminal residues of branched fructans such as levan. This results in enhanced affinity through avidity effects leading to the potentiation of catalytic activity. The gut bacterium Bacteroides thetaiotaomicron contributes to the maintenance of a healthy human gut. B. thetaiotaomicron is able to acquire and utilise complex carbohydrates that are not attacked by the intestinal enzymes of the host. B. thetaiotaomicron dedicates a large proportion of its genome to glycan degradation with a large expansion of α-mannan degrading enzymes. The B. thetaiotaomicron genome encodes 23 GH92 α-mannanosidases and 10 GH76 α-mannanases. While GH92 has recently been characterised the activities displayed by GH76 relies on the characterization of a single enzyme in this family. B. thetaiotaomicron organises the genes required to sense, degrade, transport and utilise specific complex glycans into genetic clusters defined as Polysaccharide Utilisation Loci (PULs). Transcriptomics revealed that two PULs are up regulated in response to yeast mannan, PUL 36 and PUL 68. These PULs contain both GH76 enzymes along with GH92 enzymes and other CAZy annotated enzymes. Biochemical analysis of the GH76 enzymes found in the two PULs show they are α1, 6 mannanases capable of hydrolysing the α1, 6 mannan backbone of yeast mannan, with the putative periplasmic enzymes generating small oligosaccharides, while the surface mannanases releasing larger products. The three GH92 enzymes encoded by the two PULs have been shown to remove α1, 2 and α1, 3 linked mannose branches from yeast mannan polysaccharide. In addition PUL 68 also encodes a phosphatase that removes the phosphate from mannose-6-phosphate and glucose-6-phosphate but not from intact mannan. Therefore, this study describes the ability of B. thetaiotaomicron to target and degrade yeast α-mannans. The GH5 enzyme CtXyl5A from Clostridium thermocellum is an arabinoxylan specific xylanase that contains a GH5 catalytic module appended to several CBMs. The apo structure of the GH5 catalytic module appended to a family 6 CBM reveals a large pocket abutted to the -1 subsite of the active site. This pocket was thought to bind the arabinose decoration appended to the O3 of the xylan backbone. Here mutational and structural studies showed that the fulfilment of arabinose is this pocket is the key specificity determinant for the novel arabinoxylanase activity. Significantly the bound arabinose displayed a pyranose conformation, rather than a furanose structure which is the typical conformation adopted by arabinose side chains in arabinoxylans. This structural information suggests that CtXyl5A may be able to exploit side chains other than arabinofuranose residues as substrate specificity determinants.

Studies on the control of cation permeability in skeletal muscle of the laboratory rat

Parkin, A. C. January 1975 (has links)
The present study was undertaken in an attempt to examine the importance of the NaKMgATPase (the 'sodium pump' enzyme) in cation movements in rat skeletal muscle. Though the 'sodium pump' was first postulated in an attempt to explain some of the cation movements recorded in muscle tissue, at the commencement of this study clear evidence of the presence of the NaKMgATPase was still awaited. Several approaches to the problem were made, using physiological and biochemical preparations. Isolated preparations of muscle, maintained in optimum conditions, were subjected to treatments with reagents known to affect the NaKMgATPase and the changes in cation distribution were observed. Similar preparations were studied with respect to their oxygen consumption, to determine whether treatments known to cause changes in NaKMgATPase activity would in turn lead to alterations in oxidative metabolism as had been reported in a variety of other tissues, e.g. kidney, bain. Biochemical approaches consisted largely of attempts to isolate a fraction from skeletal muscle which demonstrated; the properties of the NaKMgATPase that had been isolated from a wide range of tissues, and was especially active in those tissues in which a great deal of active cation movement was known to occur. The studies of cation distribution were largely inconclusive, as though modified cation movements were clearly seen in various conditions known to inhibit NaKMgATPase activity, it was not possible to identify firmly the position of such an enzyme or indeed that it was the sole site of action. Measurements of oxygen uptake failed to reveal any portion of oxidative metabolism which could be ascribed to metabolic activity linked to NaKMgATPase activity. After a variety of isolation techniques had failed to produce a fraction of skeletal muscle containing a clearly demonstrable NaKMgATPase, a procedure involving exposure of the muscle homogenate to 2 M NaI was successful in separating a membrane fraction exhibiting NaKMgATPase activity from rat diaphragm and hind-limb muscle. Whilst isolation of a 'pure' enzyme was not made, the study showed that the properties of the fraction isolated resembled those described for NaKMgATPases isolated from other mammalian tissues.

Potassium Channels in Interstitial Cells of Cajal and Smooth Muscle Cells of the Urinary Bladder

Carson, C. January 2010 (has links)
No description available.

Gating mechanisms of the TRPM* ion channel

Fernandez, Jose A. January 2011 (has links)
No description available.

The chemistry of amphibian skin secretions

Clark, V. C. January 2012 (has links)
No description available.

The Identification, Molecular Cloning, Synthesis and Biological Evaluation of Novel Protease Inhibitors from Chinese Ranid Frogs (Huia Versabillis)

Ganhong, Song January 2010 (has links)
No description available.

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