subject:(572)

University of Dundee

1. Hukelmann, Jens Ludger. The role of the mammalian target of rapamycin (mTOR) in cytotoxic T lymphocytes.

Degree: PhD, 2014, University of Dundee

URL: http://hdl.handle.net/10588/060165d8-9b47-47aa-9124-c7bb02db9eab

► The serine-threonine kinase mammalian target of rapamycin (mTOR) is an important integrator of nutrient, cytokine and growth factor sensing in T cells and controls transcriptional… (more)

▼ The serine-threonine kinase mammalian target of rapamycin (mTOR) is an important integrator of nutrient, cytokine and growth factor sensing in T cells and controls transcriptional programs that determine CD8+ cytotoxic T cell fate and trafficking. mTORC1 is inhibited by the drug rapamycin which is a powerful immunosuppressant used in the clinic in the context of organ transplantation. However, not much is known about the full extent of the role of mTOR signalling in CTL. We thus utilised high resolution quantitative mass spectrometry to define the mTOR regulated CTL proteome and map the abundance and isoform expression of more than 6700 proteins in CTL. The data provide unbiased analysis of how mTORC1 reprograms the transcriptional and proteome landscape of T cells. The results show that mTORC1 controls expression of approximately 700 proteins with equal numbers of up and down regulated proteins. This illustrates that mTORC1 inhibition does not lead to a general decreases in protein levels but initiates a diverse reprogramming in gene expression and in particular drives selective decreases and increases in the expression of key metabolic regulators, effector molecules, adhesion molecules and adapter proteins. The proteomic approach also allowed us to detect effects caused by mTORC1 inhibition that were not caused by changes in the corresponding transcripts but solely due to posttranscriptional mechanisms. One striking result was the dominance of mTORC1 negative feedback control of the serine/threonine kinase PKB. This prompted detailed analysis of the role of mTOR in the regulation of the phosphatidylinositol (3,4,5)-trisphosphate (PIP3) signalling effector PKB in CTL. A striking observation was that mTOR inhibitors allow T cells to accumulate very high levels of PIP3 and cause T cells to hyperactivate the serine/threonine kinase PKB and moreover reprogram PKB activation. PKB activity was thus uncoupled from mTORC2 activity and explained the similar phenotype of selective mTORC1 vs combined mTORC1/2 inhibition. Collectively these experiments highlight the power of high resolution analysis of proteomes to uncover critical signalling checkpoints that control T cell differentiation and give new insights about how mTORC1 inhibitors control T cell function.

Subjects/Keywords: 572

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APA (6^th Edition):

Hukelmann, J. L. (2014). The role of the mammalian target of rapamycin (mTOR) in cytotoxic T lymphocytes. (Doctoral Dissertation). University of Dundee. Retrieved from http://hdl.handle.net/10588/060165d8-9b47-47aa-9124-c7bb02db9eab

Chicago Manual of Style (16^th Edition):

Hukelmann, Jens Ludger. “The role of the mammalian target of rapamycin (mTOR) in cytotoxic T lymphocytes.” 2014. Doctoral Dissertation, University of Dundee. Accessed February 18, 2020. http://hdl.handle.net/10588/060165d8-9b47-47aa-9124-c7bb02db9eab.

MLA Handbook (7^th Edition):

Hukelmann, Jens Ludger. “The role of the mammalian target of rapamycin (mTOR) in cytotoxic T lymphocytes.” 2014. Web. 18 Feb 2020.

Vancouver:

Hukelmann JL. The role of the mammalian target of rapamycin (mTOR) in cytotoxic T lymphocytes. [Internet] [Doctoral dissertation]. University of Dundee; 2014. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/10588/060165d8-9b47-47aa-9124-c7bb02db9eab.

Council of Science Editors:

Hukelmann JL. The role of the mammalian target of rapamycin (mTOR) in cytotoxic T lymphocytes. [Doctoral Dissertation]. University of Dundee; 2014. Available from: http://hdl.handle.net/10588/060165d8-9b47-47aa-9124-c7bb02db9eab

University of Dundee

2. Schleicher, Katharina. Functional & molecular analysis of Bod1 in mitotic chromosome segregation.

Degree: PhD, 2014, University of Dundee

URL: http://hdl.handle.net/10588/e2e9f122-92c3-44e0-91bc-1ebf4e709d00

► Preservation of genetic integrity during mitotic chromosome segregation requires the attachment of sister chromatids to microtubules emanating from opposite poles of the mitotic spindle. Successful… (more)

Subjects/Keywords: 572

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APA (6^th Edition):

Schleicher, K. (2014). Functional & molecular analysis of Bod1 in mitotic chromosome segregation. (Doctoral Dissertation). University of Dundee. Retrieved from http://hdl.handle.net/10588/e2e9f122-92c3-44e0-91bc-1ebf4e709d00

Chicago Manual of Style (16^th Edition):

Schleicher, Katharina. “Functional & molecular analysis of Bod1 in mitotic chromosome segregation.” 2014. Doctoral Dissertation, University of Dundee. Accessed February 18, 2020. http://hdl.handle.net/10588/e2e9f122-92c3-44e0-91bc-1ebf4e709d00.

MLA Handbook (7^th Edition):

Schleicher, Katharina. “Functional & molecular analysis of Bod1 in mitotic chromosome segregation.” 2014. Web. 18 Feb 2020.

Vancouver:

Schleicher K. Functional & molecular analysis of Bod1 in mitotic chromosome segregation. [Internet] [Doctoral dissertation]. University of Dundee; 2014. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/10588/e2e9f122-92c3-44e0-91bc-1ebf4e709d00.

Council of Science Editors:

Schleicher K. Functional & molecular analysis of Bod1 in mitotic chromosome segregation. [Doctoral Dissertation]. University of Dundee; 2014. Available from: http://hdl.handle.net/10588/e2e9f122-92c3-44e0-91bc-1ebf4e709d00

University of Dundee

3. Gowans, Graeme J. Regulation and role of the LKB1-AMPK pathway.

Degree: PhD, 2014, University of Dundee

URL: http://hdl.handle.net/10588/4d96c32e-75f5-4012-97f8-1e15dd0d3246

► The AMP-activated protein kinase (AMPK) is a sensor of cellular energy that is activated by increases in the intracellular AMP:ATP or ADP:ATP ratios. Once active,… (more)

▼ The AMP-activated protein kinase (AMPK) is a sensor of cellular energy that is activated by increases in the intracellular AMP:ATP or ADP:ATP ratios. Once active, AMPK activates catabolic pathways and inhibits anabolic pathways to restore cellular energy homeostasis. AMPK activity can be increased by phosphorylation of Thr172, a conserved residue in the activation loop of the kinase domain, by either LKB1 or CaMKKß. Increases in Thr172 phosphorylation can be mediated by either protecting this site against dephosphorylation by protein phosphatases, or by promoting its phosphorylation by upstream kinases, both of which were proposed to be mediated by binding of AMP or ADP to the ?-subunit of AMPK. AMPK is also allosterically activated by binding of AMP, but not ADP. Recently, ADP has been proposed as the major regulator of AMPK and the role of AMP has been questioned. The reasons for this are: (i) while both AMP and ADP can increase phosphorylation of Thr172, ADP is present at higher concentrations in the cell and it was proposed that AMP would be unable to compete with ADP for binding at the ?-subunit; (ii) allosteric activation by AMP was reported to increase AMPK activity by less than 2-fold, whereas changes in the phosphorylation of Thr172 can increase AMPK activity by 100-fold. Using cell-free systems and intact cells, the regulation of Thr172 phosphorylation and AMPK activity by AMP, ADP and ATP was re-investigated. AMP promoted Thr172 phosphorylation by LKB1 but not by CaMKKß, while ADP had no effect on Thr172 phosphorylation by either upstream kinase. Additionally, while both AMP and ADP could protect Thr172 against dephosphorylation, AMP was more potent. The allosteric activation of AMPK by AMP was demonstrated to be a significant component of the overall activation mechanism. Using phosphorylation of ACC (a downstream target of AMPK) as a marker, allosteric activation of AMPK was observed under conditions where there were no changes in Thr172 phosphorylation. The changes in Thr172 phosphorylation and AMPK activity observed in intact cells in response to AMPK activators were also examined, and demonstrated to be far lower than the maximal effects observed in cell-free systems. Taken together, these results demonstrate that AMP is a true physiological regulator of AMPK activity. The regulation of LKB1 has also been the subject of much interest. Whilst it appears that LKB1 is constitutively active, several groups have reported that LKB1 activity is regulated by post-translational modifications, particularly phosphorylation. The role of phosphorylation of LKB1 on Ser31 by AMPK was investigated. While AMPK could phosphorylate LKB1 in a cell-free system, an effect lost in an LKB1 [S31A] mutant, this phosphorylation was not observed in intact cells. Additionally, mutation of Ser31 did not appear to alter LKB1 activity. These results suggest that phosphorylation of Ser31 of LKB1 by AMPK, whilst possible, may not be a physiologically relevant event. The mTOR pathway is a key regulator of cell growth and protein…

Subjects/Keywords: 572

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Gowans, G. J. (2014). Regulation and role of the LKB1-AMPK pathway. (Doctoral Dissertation). University of Dundee. Retrieved from http://hdl.handle.net/10588/4d96c32e-75f5-4012-97f8-1e15dd0d3246

Chicago Manual of Style (16^th Edition):

Gowans, Graeme J. “Regulation and role of the LKB1-AMPK pathway.” 2014. Doctoral Dissertation, University of Dundee. Accessed February 18, 2020. http://hdl.handle.net/10588/4d96c32e-75f5-4012-97f8-1e15dd0d3246.

MLA Handbook (7^th Edition):

Gowans, Graeme J. “Regulation and role of the LKB1-AMPK pathway.” 2014. Web. 18 Feb 2020.

Vancouver:

Gowans GJ. Regulation and role of the LKB1-AMPK pathway. [Internet] [Doctoral dissertation]. University of Dundee; 2014. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/10588/4d96c32e-75f5-4012-97f8-1e15dd0d3246.

Council of Science Editors:

Gowans GJ. Regulation and role of the LKB1-AMPK pathway. [Doctoral Dissertation]. University of Dundee; 2014. Available from: http://hdl.handle.net/10588/4d96c32e-75f5-4012-97f8-1e15dd0d3246

University of Dundee

4. Kirkwood, Kathryn J. Analysis of human protein complexes by quantitative mass spectrometry.

Degree: PhD, 2014, University of Dundee

URL: http://hdl.handle.net/10588/96892fa1-7861-4d01-87a7-d91e7ef21527

► Proteins are the fundamental building blocks of all living cells and they carry out nearly all cell functions. Proteins predominantly act as part of multiprotein… (more)

▼ Proteins are the fundamental building blocks of all living cells and they carry out nearly all cell functions. Proteins predominantly act as part of multiprotein complexes to carry out these functions. The association of protein isoforms and post-translationally modified forms in protein complexes can influence their subcellular location, activity and substrate specificity. It is therefore crucial to characterise protein complexes at the level of the protein isoforms and post-translationally modified forms they contain to fully decipher the network of signalling and regulatory pathways within cells. The aim of my work has been to develop a technique to study protein complexes through the use of size exclusion chromatography (SEC) combined with tandem mass spectrometry. Combining these approaches has enabled an in-depth analysis of protein complexes in U2OS cells, including those involving post-translationally modified proteins and protein isoforms. The data presented in this thesis provide a proof of concept, together with forming a useful resource, which can be used alongside pull-down analyses to differentiate the interaction partners involved in different protein complexes. This combined approach minimises the need for multiple IP analyses and facilitates a more targeted approach in dissecting the components of individual protein complexes. I developed this technique further by utilising in vivo crosslinking prior to denaturing SEC. This approach enabled more efficient recovery and detection of proteins previously underrepresented using native SEC analysis, including many membrane complexes, thereby providing a more complete picture of endogenous protein complexes. I have applied the native SEC/MS approach in a study of the interactions between the MRFAP1-MORF4L1 proteins. I demonstrated that this complex is distinct from the larger complex involving interactions between the MRGBP-MORF4L1 proteins. In addition, I also demonstrate that the native SEC/MS technique can be extended to assess the effect of drugs on protein-protein interactions. Overall, the methods I present in this thesis enable the rapid, proteome-wide analysis of endogenous protein complexes, which will advance the future study of protein complexes in biology.

Subjects/Keywords: 572

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Kirkwood, K. J. (2014). Analysis of human protein complexes by quantitative mass spectrometry. (Doctoral Dissertation). University of Dundee. Retrieved from http://hdl.handle.net/10588/96892fa1-7861-4d01-87a7-d91e7ef21527

Chicago Manual of Style (16^th Edition):

Kirkwood, Kathryn J. “Analysis of human protein complexes by quantitative mass spectrometry.” 2014. Doctoral Dissertation, University of Dundee. Accessed February 18, 2020. http://hdl.handle.net/10588/96892fa1-7861-4d01-87a7-d91e7ef21527.

MLA Handbook (7^th Edition):

Kirkwood, Kathryn J. “Analysis of human protein complexes by quantitative mass spectrometry.” 2014. Web. 18 Feb 2020.

Vancouver:

Kirkwood KJ. Analysis of human protein complexes by quantitative mass spectrometry. [Internet] [Doctoral dissertation]. University of Dundee; 2014. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/10588/96892fa1-7861-4d01-87a7-d91e7ef21527.

Council of Science Editors:

Kirkwood KJ. Analysis of human protein complexes by quantitative mass spectrometry. [Doctoral Dissertation]. University of Dundee; 2014. Available from: http://hdl.handle.net/10588/96892fa1-7861-4d01-87a7-d91e7ef21527

University of Dundee

5. Munson, Michael. Regulation of the lipid kinase VPS34 by mTOR-mediated UVRAG phosphorylation.

Degree: PhD, 2014, University of Dundee

URL: http://hdl.handle.net/10588/5b823794-dbf9-42a1-a2f7-0a081354fe64

► The lipid kinase VPS34 is an essential mediator of multiple aspects of intracellular trafficking via the formation of phosphoinositide-3-phosphate (PI(3)P) on membranes, this is critical… (more)

▼ The lipid kinase VPS34 is an essential mediator of multiple aspects of intracellular trafficking via the formation of phosphoinositide-3-phosphate (PI(3)P) on membranes, this is critical to mediate efficient trafficking of cargo by endocytosis. In addition, VPS34 kinase activity is essential for inducing autophagy in combination with the protein kinase ULK1. Autophagy acts as a catabolic pathway that is up regulated in response to stress, but is negatively regulated by the master growth protein kinase mTOR. This study sought to identify novel control mechanisms that may mediate the regulation of VPS34 between the pathways of endocytosis and autophagy. During nutrient rich conditions a binding protein of VPS34, UVRAG, was identified to be phosphorylated. Further analysis has identified that phosphorylation is mediated by mTOR and that this occurs at two sites, S550 and S571. Multiple lines of evidence suggest that phosphorylation does not alter the stoichiometry or localisation of the complex nor does it mediate recruitment of additional factors. Phosphorylation of UVRAG acts to increase lipid kinase activity in vitro and cellular PI(3)P levels by ~ 2 fold, mutation of S550 and S571 to alanine residues abrogate this increase in activity. Examination of autophagy, receptor mediated endocytosis and recycling have demonstrated no effect of UVRAG phosphorylation upon their rate of trafficking. Mutation of UVRAG phosphorylation sites however leads to a significant lysosome abnormality that is demonstrated by a dispersed phenotype. Preliminary analysis suggests that this may occur due to abnormalities in the process of autophagic lysosome reformation, a process that is dependent upon the activity of mTOR. This suggests a previously uncharacterised role of PI(3)P in lysosomal regulation and adds to current understanding of regulation between VPS34 and mTOR. Additionally data presented here examines a novel PI3KC3 inhibitor that demonstrates profound selectivity over other PI3K isoforms and lipid kinases. This will be important to further examine the functional role of VPS34 and UVRAG.

Subjects/Keywords: 572

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APA (6^th Edition):

Munson, M. (2014). Regulation of the lipid kinase VPS34 by mTOR-mediated UVRAG phosphorylation. (Doctoral Dissertation). University of Dundee. Retrieved from http://hdl.handle.net/10588/5b823794-dbf9-42a1-a2f7-0a081354fe64

Chicago Manual of Style (16^th Edition):

Munson, Michael. “Regulation of the lipid kinase VPS34 by mTOR-mediated UVRAG phosphorylation.” 2014. Doctoral Dissertation, University of Dundee. Accessed February 18, 2020. http://hdl.handle.net/10588/5b823794-dbf9-42a1-a2f7-0a081354fe64.

MLA Handbook (7^th Edition):

Munson, Michael. “Regulation of the lipid kinase VPS34 by mTOR-mediated UVRAG phosphorylation.” 2014. Web. 18 Feb 2020.

Vancouver:

Munson M. Regulation of the lipid kinase VPS34 by mTOR-mediated UVRAG phosphorylation. [Internet] [Doctoral dissertation]. University of Dundee; 2014. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/10588/5b823794-dbf9-42a1-a2f7-0a081354fe64.

Council of Science Editors:

Munson M. Regulation of the lipid kinase VPS34 by mTOR-mediated UVRAG phosphorylation. [Doctoral Dissertation]. University of Dundee; 2014. Available from: http://hdl.handle.net/10588/5b823794-dbf9-42a1-a2f7-0a081354fe64

Cardiff University

6. Cayzac, Sebastien H. Role of polyamines in the carotid body.

Degree: PhD, 2008, Cardiff University

URL: http://orca.cf.ac.uk/54781/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584371

► Polyamines are small organic molecules which modulate many physiological processes. Here, an inhibitory effect of spermine on rat carotid body chemoreception is reported. Spermine inhibits… (more)

Subjects/Keywords: 572

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Cayzac, S. H. (2008). Role of polyamines in the carotid body. (Doctoral Dissertation). Cardiff University. Retrieved from http://orca.cf.ac.uk/54781/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584371

Chicago Manual of Style (16^th Edition):

Cayzac, Sebastien H. “Role of polyamines in the carotid body.” 2008. Doctoral Dissertation, Cardiff University. Accessed February 18, 2020. http://orca.cf.ac.uk/54781/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584371.

MLA Handbook (7^th Edition):

Cayzac, Sebastien H. “Role of polyamines in the carotid body.” 2008. Web. 18 Feb 2020.

Vancouver:

Cayzac SH. Role of polyamines in the carotid body. [Internet] [Doctoral dissertation]. Cardiff University; 2008. [cited 2020 Feb 18]. Available from: http://orca.cf.ac.uk/54781/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584371.

Council of Science Editors:

Cayzac SH. Role of polyamines in the carotid body. [Doctoral Dissertation]. Cardiff University; 2008. Available from: http://orca.cf.ac.uk/54781/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584371

Cardiff University

7. Forde, Josephine E. Investigation into the role of glycogen synthase kinase 3 (GSK-3) in Dictyostelium discoideum and the role of GSK-3 binding partners in Dictyostelium discoideum and mammalian cells.

Degree: PhD, 2009, Cardiff University

URL: http://orca.cf.ac.uk/54971/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584667

► The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified as a key regulator of insulin-dependent glycogen synthesis. GSK-3 was subsequently shown to function in… (more)

Subjects/Keywords: 572

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APA (6^th Edition):

Forde, J. E. (2009). Investigation into the role of glycogen synthase kinase 3 (GSK-3) in Dictyostelium discoideum and the role of GSK-3 binding partners in Dictyostelium discoideum and mammalian cells. (Doctoral Dissertation). Cardiff University. Retrieved from http://orca.cf.ac.uk/54971/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584667

Chicago Manual of Style (16^th Edition):

Forde, Josephine E. “Investigation into the role of glycogen synthase kinase 3 (GSK-3) in Dictyostelium discoideum and the role of GSK-3 binding partners in Dictyostelium discoideum and mammalian cells.” 2009. Doctoral Dissertation, Cardiff University. Accessed February 18, 2020. http://orca.cf.ac.uk/54971/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584667.

MLA Handbook (7^th Edition):

Forde, Josephine E. “Investigation into the role of glycogen synthase kinase 3 (GSK-3) in Dictyostelium discoideum and the role of GSK-3 binding partners in Dictyostelium discoideum and mammalian cells.” 2009. Web. 18 Feb 2020.

Vancouver:

Forde JE. Investigation into the role of glycogen synthase kinase 3 (GSK-3) in Dictyostelium discoideum and the role of GSK-3 binding partners in Dictyostelium discoideum and mammalian cells. [Internet] [Doctoral dissertation]. Cardiff University; 2009. [cited 2020 Feb 18]. Available from: http://orca.cf.ac.uk/54971/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584667.

Council of Science Editors:

Forde JE. Investigation into the role of glycogen synthase kinase 3 (GSK-3) in Dictyostelium discoideum and the role of GSK-3 binding partners in Dictyostelium discoideum and mammalian cells. [Doctoral Dissertation]. Cardiff University; 2009. Available from: http://orca.cf.ac.uk/54971/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584667

University of Dundee

8. Pavic, Karolina. The development of photo-crosslinkable trapping mutants as tools to investigate the interactions of protein tyrosine phosphatases.

Degree: PhD, 2013, University of Dundee

URL: http://hdl.handle.net/10588/39cd7ecc-d09b-4295-933e-725538437046

► Abnormalities in the coordinated activities of protein phosphatases (PPs) and protein kinases (PKs) contribute to the development of many diseases. Phosphatase of regenerating liver (PRL)-3… (more)

▼ Abnormalities in the coordinated activities of protein phosphatases (PPs) and protein kinases (PKs) contribute to the development of many diseases. Phosphatase of regenerating liver (PRL)-3 and Vaccinia H1-Related (VHR) are two members of the protein tyrosine phosphatase (PTP) family shown to be involved in cancer. PRL-3 is a member of the PRL phosphatases containing a unique post-translationally modifiable prenylation CAAX motif at the carboxy (C)- terminal end. There is an immense body of evidence to support a role for PRL-3 in the development of various types of cancer and in progression to metastasic disease. However, many questions are still pending, especially with respect to the identity of physiological substrates, and interacting partners in general, of PRL-3. VHR is a model for a group of atypical dual specificity protein phosphatases (DUSPs) with a role in cell cycle progression. Only a few of VHR’s physiological substrates have been reported to date and there are very few studies addressing its regulation and physiological role(s). Generally, in order to isolate and identify transient phosphatase-substrate interactions, substrate-trapping mutants of PTPs are employed. Mutants which can function as substrate traps have the ability to recognise and bind substrates, yet they lack functionality of the key catalytic residues and cannot efficiently process the hydrolysis of the substrate. However, it is acknowledged that the efficiency of such standard substrate-trapping mutants of PTPs is low. In this work, the expanded genetic code approach was applied to develop more efficient substrate trapping variants of the PTPs by incorporating the photocross-linkable amino acid para-benzoylphenylalanine (pBPA). The concept was optimised for PRL-3 and VHR, and for both proteins, pBPA-containing variantswere expressed at excellent yields and were highly purified. By utilizing the photo-cross-linkable F68pBPA variant of VHR, dimerisation of VHR was detected in an in vitro ultraviolet (UV) exposure-mediated crosslinking assay. VHR dimerisation was further demonstrated to be a potential novel regulatory mechanism for VHR, having a negative effect on the catalytic activity of the protein. A specific region in VHR known as the variable insert segment was pinpointed as a region in the protein, which is either at the dimer interface or heavily contributing to dimeric association. Furthermore, the intrinsic ability of VHR to self-associate was also demonstrated by complementary methods. For PRL-3 it was demonstrated that its D72pBPA variant could recognise and bind to lipids, with a stronger signal detected in the UV-exposed sample, and without altering the lipid binding profile with respect to the native protein. Lastly, the potential of exploiting photo-cross-linkable variants of the PTPs was also demonstrated by incubating PRL-3 variants, with or without selectively introduced pBPA, with mammalian cell lysates, followed by UV exposure. Western blot analysis detected new bands corresponding to covalently crosslinked…

Subjects/Keywords: 572

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Pavic, K. (2013). The development of photo-crosslinkable trapping mutants as tools to investigate the interactions of protein tyrosine phosphatases. (Doctoral Dissertation). University of Dundee. Retrieved from http://hdl.handle.net/10588/39cd7ecc-d09b-4295-933e-725538437046

Chicago Manual of Style (16^th Edition):

Pavic, Karolina. “The development of photo-crosslinkable trapping mutants as tools to investigate the interactions of protein tyrosine phosphatases.” 2013. Doctoral Dissertation, University of Dundee. Accessed February 18, 2020. http://hdl.handle.net/10588/39cd7ecc-d09b-4295-933e-725538437046.

MLA Handbook (7^th Edition):

Pavic, Karolina. “The development of photo-crosslinkable trapping mutants as tools to investigate the interactions of protein tyrosine phosphatases.” 2013. Web. 18 Feb 2020.

Vancouver:

Pavic K. The development of photo-crosslinkable trapping mutants as tools to investigate the interactions of protein tyrosine phosphatases. [Internet] [Doctoral dissertation]. University of Dundee; 2013. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/10588/39cd7ecc-d09b-4295-933e-725538437046.

Council of Science Editors:

Pavic K. The development of photo-crosslinkable trapping mutants as tools to investigate the interactions of protein tyrosine phosphatases. [Doctoral Dissertation]. University of Dundee; 2013. Available from: http://hdl.handle.net/10588/39cd7ecc-d09b-4295-933e-725538437046

University of Newcastle Upon Tyne

9. Ghiasy, Dena. Control and operation of a spinning disc reactor.

Degree: PhD, 2013, University of Newcastle Upon Tyne

URL: http://hdl.handle.net/10443/1848

► The aim of the present research is to assess the control and operation of a Spinning Disc Reactor (SDR), carried out via four separate investigations.… (more)

▼ The aim of the present research is to assess the control and operation of a Spinning Disc Reactor (SDR), carried out via four separate investigations. Firstly, the effect of equipment size reduction on control is studied by comparing the performance of a PID controller applied to simulated intensified and conventional processes. It was found that superior control performance in terms of Integral of Absolute Error (IAE) is achieved for the simulated intensified system. However, the results showed that intensified systems are more susceptible to disturbances and the controlled variable exhibits larger overshoots. Furthermore, the frequency response analysis of the two systems showed that the simulated intensified system has reduced stability margins. The second part of the research investigates the task of pH control in a SDR using a PID controller by means of simulation and experimental studies. The effectiveness of a disturbance observer (DO) and a pH characteriser to compensate for the severe pH system nonlinearity is also explored in detail. The experimental studies showed that a PID controller provides adequate setpoint tracking and disturbance rejection performances. However, sluggish transient responses prevailed and the effluent pH limit cycled around the setpoint. There were indications of unstable behaviour at lower flowrates, which implied more advanced control schemas may be required to adapt to various operating regions dictated by the complex thin film hydrodynamics. The addition of the DO scheme improved the control performance by reducing the limit cycles. In the third segment of the investigations, the potential of exploiting the disc rotational speed as a manipulated variable is assessed for the process of barium sulphate precipitation. A PI controller is successfully used to regulate the conductivity of the effluent stream by adjusting the disc rotational speed. The results are immensely encouraging and show that the disc speed may be used as an extra degree of freedom in control system design. Finally, the flow regimes and wave characteristics of thin liquid films produced in a SDR are investigated by means of a thermal imaging camera. The film hydrodynamics strongly affect the heat and mass transfer processes within the processing films, and thus the intensification aspects of SDRs. Therefore, effective control and operation of such units is significantly dependent on the knowledge of film hydrodynamics and the underlying impact of the operating parameters and the manipulated variables on a given process. The results provided an interesting insight and unveiled promising potentials for characterisation of thin liquid film flow and temperature profiles across the disc by means of thermographic techniques. The present study reveals both challenges and opportunities regarding the control aspects of SDRs. It is recommended that equipment design and process control need to be considered simultaneously during the early stages of the future developments. Furthermore, intensified sensors and advanced…

Subjects/Keywords: 572

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APA (6^th Edition):

Ghiasy, D. (2013). Control and operation of a spinning disc reactor. (Doctoral Dissertation). University of Newcastle Upon Tyne. Retrieved from http://hdl.handle.net/10443/1848

Chicago Manual of Style (16^th Edition):

Ghiasy, Dena. “Control and operation of a spinning disc reactor.” 2013. Doctoral Dissertation, University of Newcastle Upon Tyne. Accessed February 18, 2020. http://hdl.handle.net/10443/1848.

MLA Handbook (7^th Edition):

Ghiasy, Dena. “Control and operation of a spinning disc reactor.” 2013. Web. 18 Feb 2020.

Vancouver:

Ghiasy D. Control and operation of a spinning disc reactor. [Internet] [Doctoral dissertation]. University of Newcastle Upon Tyne; 2013. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/10443/1848.

Council of Science Editors:

Ghiasy D. Control and operation of a spinning disc reactor. [Doctoral Dissertation]. University of Newcastle Upon Tyne; 2013. Available from: http://hdl.handle.net/10443/1848

University of Leicester

10. Furze, Christopher Michael. Unravelling the mechanism of complement activation via the lectin pathway.

Degree: PhD, 2013, University of Leicester

URL: http://hdl.handle.net/2381/27805

► Activation of complement is involved in the clearance of foreign pathogens, altered-self and apoptotic cells. It proceeds through the Classical, Lectin or Alternative pathways and… (more)

▼ Activation of complement is involved in the clearance of foreign pathogens, altered-self and apoptotic cells. It proceeds through the Classical, Lectin or Alternative pathways and ultimately results in destruction of the cell through lysis by the membrane attack complex. Activation of the Lectin pathway is triggered by the recognition of carbohydrate targets by families of collagenous proteins, called mannose-binding lectins (MBLs) and ficolins. These recognition proteins activate associated proteases called MBL-associated serine proteases (MASPs). Once activated, the MASPs cleave downstream targets to initiate the complement cascade. MASP-2 circulates in a complex with MBL and activates when it recognises mannose-type sugars. The exact molecular mechanisms of the activation of MASP-2 by MBL remains unclear and this is the focus of the current work. In this thesis I demonstrate that MASP binding can be introduced into pulmonary surfactant protein A (SP-A), a protein with a similar architecture to MBL, but which cannot bind MASPs to activate complement, through a series of substitutions to the collagenous domain. Surprisingly, introduction of the MASP-binding site results in constitutive activation of MASP-2, even in the absence of a carbohydrate target, thus lacking the control present in MBL. I then investigated the basis for this control by producing chimeric proteins of MBL and SP-A. The chimeras demonstrate that target-specific activation originates from a portion of MBL comprising the carbohydrate-recognition domains (CRDs) and neck region comprising an α-helical coiled coil. Additional work using a modified MBL that recognises galactose, subsequently demonstrates that the specific activity does not originate from the CRDs themselves, suggesting the neck domain plays an important role in the activation of MASPs. Further work in this thesis revolves around a newly discovered collectin, CL-K1 which is probably involved in complement activation and also important developmental processes. Mutations within the CRD of CL-K1 result in developmental disorders known as 3MC syndrome. The work in this thesis shows that one of these mutants is destabilized and defective in sugar binding hence revealing the likely molecular basis of disease. In addition, the structure of the CRD has been solved by X-ray crystallography enabling the key residues to be visualised.

Subjects/Keywords: 572

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APA (6^th Edition):

Furze, C. M. (2013). Unravelling the mechanism of complement activation via the lectin pathway. (Doctoral Dissertation). University of Leicester. Retrieved from http://hdl.handle.net/2381/27805

Chicago Manual of Style (16^th Edition):

Furze, Christopher Michael. “Unravelling the mechanism of complement activation via the lectin pathway.” 2013. Doctoral Dissertation, University of Leicester. Accessed February 18, 2020. http://hdl.handle.net/2381/27805.

MLA Handbook (7^th Edition):

Furze, Christopher Michael. “Unravelling the mechanism of complement activation via the lectin pathway.” 2013. Web. 18 Feb 2020.

Vancouver:

Furze CM. Unravelling the mechanism of complement activation via the lectin pathway. [Internet] [Doctoral dissertation]. University of Leicester; 2013. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/2381/27805.

Council of Science Editors:

Furze CM. Unravelling the mechanism of complement activation via the lectin pathway. [Doctoral Dissertation]. University of Leicester; 2013. Available from: http://hdl.handle.net/2381/27805

Durham University

11. Hermosilla, Belén Solano. A microgripper for single cell manipulation.

Degree: PhD, 2008, Durham University

URL: http://etheses.dur.ac.uk/2920/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.539318

► This thesis presents the development of an electrothermally actuated microgripper for the manipulation of cells and other biological particles. The microgripper has been fabricated using… (more)

▼ This thesis presents the development of an electrothermally actuated microgripper for the manipulation of cells and other biological particles. The microgripper has been fabricated using a combination of surface and bulk micromachining techniques in a three mask process. All of the fabrication details have been chosen to enable a tri-layer, polymer (SU8) - metal (Au) - polymer (SU8), membrane to be released from the substrate stress free and without the need for sacrificial layers. An actuator design, which completely eliminates the parasitic resistance of the cold arm, is presented. When compared to standard U-shaped actuators, it improves the thermal efficiency threefold. This enables larger displacements at lower voltages and temperatures. The microgripper is demonstrated in three different configurations: normally open mode, normally closed mode, and normally open/closed mode. It has-been modelled using two coupled analytical models - electrothermal and thermomechanical - which have been custom developed for this application. Unlike previously reported models, the electrothermal model presented here includes the heat exchange between hot and cold arms of the actuators that are separated by a small air gap. A detailed electrothermomechanical characterisation of selected devices has permitted the validation of the models (also performed using finite element analysis) and the assessment of device performance. The device testing includes electrical, deflection, and temperature measurements using infrared (IR) thermography, its use in polymeric actuators reported here for the first time. Successful manipulation experiments have been conducted in both air and liquid environments. Manipulation of live cells (mice oocytes) in a standard biomanipulation station has validated the microgripper as a complementary and unique tool for the single cell experiments that are to be conducted by future generations of biologists in the areas of human reproduction and stem cell research.

Subjects/Keywords: 572

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APA (6^th Edition):

Hermosilla, B. S. (2008). A microgripper for single cell manipulation. (Doctoral Dissertation). Durham University. Retrieved from http://etheses.dur.ac.uk/2920/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.539318

Chicago Manual of Style (16^th Edition):

Hermosilla, Belén Solano. “A microgripper for single cell manipulation.” 2008. Doctoral Dissertation, Durham University. Accessed February 18, 2020. http://etheses.dur.ac.uk/2920/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.539318.

MLA Handbook (7^th Edition):

Hermosilla, Belén Solano. “A microgripper for single cell manipulation.” 2008. Web. 18 Feb 2020.

Vancouver:

Hermosilla BS. A microgripper for single cell manipulation. [Internet] [Doctoral dissertation]. Durham University; 2008. [cited 2020 Feb 18]. Available from: http://etheses.dur.ac.uk/2920/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.539318.

Council of Science Editors:

Hermosilla BS. A microgripper for single cell manipulation. [Doctoral Dissertation]. Durham University; 2008. Available from: http://etheses.dur.ac.uk/2920/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.539318

University of Leicester

12. Gumiero, Andrea. New insights into heme peroxisases : internediates and mechanisms.

Degree: PhD, 2011, University of Leicester

URL: http://hdl.handle.net/2381/9531

► Heme peroxidises catalyse the H2O2-dependent oxidation of substrates in a two-step process, through formation of two oxy-ferryl intermediates known as Compound I and Compound II.… (more)

▼ Heme peroxidises catalyse the H2O2-dependent oxidation of substrates in a two-step process, through formation of two oxy-ferryl intermediates known as Compound I and Compound II. Despite the considerable effort worldwide, important aspects about the reactivity of these enzymes are still to be clarified. Amongst all, the determination of the nature of the Fe-O bond in the oxy-ferryl intermediates, as well as the mechanism by which protons are delivered to the oxy-ferryl species during turnover, are of highest relevance. In this thesis, high resolution crystal structures of both Compound I and Compound II intermediates in two heme peroxidases, cytochrome c peroxidise (CcP) and ascorbate peroxidise (APX), are presented. In order to rule out the photoreduction arising from X-ray exposure during data collection, which causes alteration of ferryl intermediate structures, a multicrystal method has been employed. Results indicate that Compound I, with an Fe-O distance of 1.63 Å for CcP and 1.73 Å for APX, is consistent with an unprotonated oxy-ferryl species (FeIV=O), whereas Compound II, with an Fe-O bond length of 1.83 Å and 1.84 Å for CcP and APX respectively, is consistent with a protonated oxy-ferryl species (FeIV-OH). Also presented in this thesis is the 2.40 Å structure of resting ferric CcP at room temperature obtained, for the first time, by neutron crystallography. This study allowed to establish the location of individual, exchangeable hydrogen atoms thus revealing the protonation states of several key active site residues in the distal (Arg48, Trp51, His52) and proximal (His163, Trp191, Asp235) heme regions. This information was used to revise the reaction mechanism of heme peroxidises and also to infer a possible delivery pathway of protons during turnover. All together, these data not only clarify long-standing inconsistencies on the nature of the oxy-ferryl species, but they also provide new insights into The reaction mechanism of heme peroxidises and provide important information which May apply to other categories of heme enzymes such as the cytochromes P450 and NO synthases.

Subjects/Keywords: 572

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APA (6^th Edition):

Gumiero, A. (2011). New insights into heme peroxisases : internediates and mechanisms. (Doctoral Dissertation). University of Leicester. Retrieved from http://hdl.handle.net/2381/9531

Chicago Manual of Style (16^th Edition):

Gumiero, Andrea. “New insights into heme peroxisases : internediates and mechanisms.” 2011. Doctoral Dissertation, University of Leicester. Accessed February 18, 2020. http://hdl.handle.net/2381/9531.

MLA Handbook (7^th Edition):

Gumiero, Andrea. “New insights into heme peroxisases : internediates and mechanisms.” 2011. Web. 18 Feb 2020.

Vancouver:

Gumiero A. New insights into heme peroxisases : internediates and mechanisms. [Internet] [Doctoral dissertation]. University of Leicester; 2011. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/2381/9531.

Council of Science Editors:

Gumiero A. New insights into heme peroxisases : internediates and mechanisms. [Doctoral Dissertation]. University of Leicester; 2011. Available from: http://hdl.handle.net/2381/9531

University of Leicester

13. Bavan, Selvan. P2x receptor function in Lymnaea stagnalis and other lower organisms.

Degree: PhD, 2011, University of Leicester

URL: http://hdl.handle.net/2381/10076

► The mollusc Lymnaea stagnalis has a relatively simple central nervous system (CNS) consisting of large and easily identifiable neurons. This feature together with well characterized… (more)

▼ The mollusc Lymnaea stagnalis has a relatively simple central nervous system (CNS) consisting of large and easily identifiable neurons. This feature together with well characterized neuronal circuitry for important physiological processes and an established adenosine 5’-triphosphate (ATP) release system puts Lymnaea forward as an attractive model to examine the CNS function of P2X receptors. Furthermore, lower organism P2X receptors may provide structure function insights by virtue of conservation of functionally important domains. Following the identification of a P2X receptor-like sequence (LymP2X) in the Lymnaea CNS, the cloned complementary deoxyribonucleic acid encoding LymP2X when heterologously expressed in Xenopus oocytes exhibited ATP-evoked inward currents (EC50: 6.2 µM) with slow desensitisation. 2’, 3’-O-(4-Benzoylbenzoyl) adenosine 5’-triphosphate (BzATP) was a partial agonist (EC50: 2.4 µM), whilst pyridoxalphosphate-6-azophenyl-2', 5'-disulphonic acid (PPADS) and suramin were antagonists (IC50: 8.1 and 27.4 µM respectively). A P2X receptor from another invertebrate Hypsibius Dujardini (HdP2X) was also characterised in a comparative study. This tardigrade receptor displayed ATP-evoked currents (EC50: 28.5 µM) with extremely rapid activation and desensitization kinetics, which were concentration-dependently inhibited by copper and zinc. Histidine 306 in HdP2X was found to have a minor role in copper inhibition. Quantitative reverse transcription-polymerase chain reaction and in situ hybridization detected LymP2X expression in all CNS ganglia, including buccal suggesting a possible role in the feeding network. Intracellular recording of motoneuron activity demonstrated that application of ATP (1 mM) to buccal ganglia increased the rate of fictive feeding whereas PPADS abolished this response. The application of BzATP, in an attempt to selectively activate P2X receptors, did not trigger fictive feeding, suggesting that LymP2X activation alone does not initiate the feeding rhythm. BzATP however did cause a significant hyperpolarisation of a key feeding motoneuron, suggesting that LymP2X activation may be required for the latter phase in the ATP-evoked feeding cycle.

Subjects/Keywords: 572

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Bavan, S. (2011). P2x receptor function in Lymnaea stagnalis and other lower organisms. (Doctoral Dissertation). University of Leicester. Retrieved from http://hdl.handle.net/2381/10076

Chicago Manual of Style (16^th Edition):

Bavan, Selvan. “P2x receptor function in Lymnaea stagnalis and other lower organisms.” 2011. Doctoral Dissertation, University of Leicester. Accessed February 18, 2020. http://hdl.handle.net/2381/10076.

MLA Handbook (7^th Edition):

Bavan, Selvan. “P2x receptor function in Lymnaea stagnalis and other lower organisms.” 2011. Web. 18 Feb 2020.

Vancouver:

Bavan S. P2x receptor function in Lymnaea stagnalis and other lower organisms. [Internet] [Doctoral dissertation]. University of Leicester; 2011. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/2381/10076.

Council of Science Editors:

Bavan S. P2x receptor function in Lymnaea stagnalis and other lower organisms. [Doctoral Dissertation]. University of Leicester; 2011. Available from: http://hdl.handle.net/2381/10076

University of Leicester

14. Alhosaini, Khaled A. M. Signalling, desensitization and resensitization of neuromedin U receptors.

Degree: PhD, 2011, University of Leicester

URL: http://hdl.handle.net/2381/10166

► The neuropeptides neuromedin U (NmU) and neuromedin S (NmS) show a high degree of conservation across species, primarily at the amidated C-terminus. NmU in particular… (more)

▼ The neuropeptides neuromedin U (NmU) and neuromedin S (NmS) show a high degree of conservation across species, primarily at the amidated C-terminus. NmU in particular is widely distributed in both the central nervous system and periphery and is involved in a plethora of physiological and pathological events. NmU and NmS mediate their actions via two family A, G protein-coupled receptors, NMU1 and NMU2, which share ~50% homology. The present study confirmed receptor coupling to Gαq/11, leading to increases in intracellular [Ca2+] and activation of extracellular signal-regulated protein kinase (ERK), as well as coupling to Gαi/o, leading to pertussis toxin-sensitive inhibition of adenylyl cyclase activity. This study also confirmed that different NmU analogues bind pseudo-irreversibly to recombinantly expressed NMUs and has shown receptor-dependent internalization of a fluorescently-tagged version of NmU. C-terminal eGFP-tagged NMUs showed co-internalization of ligand and receptor within ~2.5 min of ligand exposure. Cell-surface, receptor-bound ligand could be removed by a rapid (20 s), pH 2.0 washing without detrimental effect on signal transduction or cell viability, allowing examination of desensitization and resensitization in the absence of cell-surface, ligand-bound receptors. Desensitization of NMU2-mediated Ca2+ responses (that was independent of continued ligand binding) occurred within minutes of exposure to human (h) NmU-25. Acute exposure (5 min) to a maximum concentration of hNmU-25 followed by recovery in the absence (pH 2.0 wash) or the presence (pH 7.4 wash) of cell-surface, receptor-bound hNmU-25 showed that the continued presence of ligand markedly delayed receptor resensitization. Receptor internalization via a dynamin-dependent pathway was crucial for resensitization of NMU1 and NMU2. Further, resensitization was dependent on endosomal acidification, recycling and endothelin-converting enzyme-1 (ECE-1) activity, but not de novo protein synthesis. This suggests that processing of hNmU-25 by ECE-1 in acidified endosomes is critical for resensitization. Inhibition of ECE-1 also prolonged NMU-mediated ERK activation, suggesting G protein-independent signalling by a ligand-receptor dependent complex within endosomes. Although no significant differences were demonstrated in potency and signalling between the NMU subtypes or their ligands, resensitization (and potentially therefore G protein-dependent/independent signalling) was influenced by both the ligand (nature and length of the N-terminus) and the receptor (NMU1 versus NMU2).

Subjects/Keywords: 572

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APA (6^th Edition):

Alhosaini, K. A. M. (2011). Signalling, desensitization and resensitization of neuromedin U receptors. (Doctoral Dissertation). University of Leicester. Retrieved from http://hdl.handle.net/2381/10166

Chicago Manual of Style (16^th Edition):

Alhosaini, Khaled A M. “Signalling, desensitization and resensitization of neuromedin U receptors.” 2011. Doctoral Dissertation, University of Leicester. Accessed February 18, 2020. http://hdl.handle.net/2381/10166.

MLA Handbook (7^th Edition):

Alhosaini, Khaled A M. “Signalling, desensitization and resensitization of neuromedin U receptors.” 2011. Web. 18 Feb 2020.

Vancouver:

Alhosaini KAM. Signalling, desensitization and resensitization of neuromedin U receptors. [Internet] [Doctoral dissertation]. University of Leicester; 2011. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/2381/10166.

Council of Science Editors:

Alhosaini KAM. Signalling, desensitization and resensitization of neuromedin U receptors. [Doctoral Dissertation]. University of Leicester; 2011. Available from: http://hdl.handle.net/2381/10166

University of Leicester

15. Chadburn, Andrew James. A comparison of the pore-forming subunits of the ATP-sensitive potassium channel.

Degree: PhD, 2010, University of Leicester

URL: http://hdl.handle.net/2381/9957

► Kir6.1 and Kir6.2 display several key differences in characteristics e.g. Kir6.2 can open spontaneously upon removal of inhibitory nucleotides while Kir6.1 cannot. Residues responsible for… (more)

Subjects/Keywords: 572

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APA (6^th Edition):

Chadburn, A. J. (2010). A comparison of the pore-forming subunits of the ATP-sensitive potassium channel. (Doctoral Dissertation). University of Leicester. Retrieved from http://hdl.handle.net/2381/9957

Chicago Manual of Style (16^th Edition):

Chadburn, Andrew James. “A comparison of the pore-forming subunits of the ATP-sensitive potassium channel.” 2010. Doctoral Dissertation, University of Leicester. Accessed February 18, 2020. http://hdl.handle.net/2381/9957.

MLA Handbook (7^th Edition):

Chadburn, Andrew James. “A comparison of the pore-forming subunits of the ATP-sensitive potassium channel.” 2010. Web. 18 Feb 2020.

Vancouver:

Chadburn AJ. A comparison of the pore-forming subunits of the ATP-sensitive potassium channel. [Internet] [Doctoral dissertation]. University of Leicester; 2010. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/2381/9957.

Council of Science Editors:

Chadburn AJ. A comparison of the pore-forming subunits of the ATP-sensitive potassium channel. [Doctoral Dissertation]. University of Leicester; 2010. Available from: http://hdl.handle.net/2381/9957

University of Leicester

16. Bourgognon, Julie-Myrtille. The role of protease-activated receptor-1 in stress-induced neuronal activity and behavioural responses.

Degree: PhD, 2012, University of Leicester

URL: http://hdl.handle.net/2381/11063

► Serine proteases and their target molecules have been implicated in numerous aspects of CNS function, inducing various forms of learning, memory and emotional responses. This… (more)

▼ Serine proteases and their target molecules have been implicated in numerous aspects of CNS function, inducing various forms of learning, memory and emotional responses. This work sought to elucidate the role of the protease-activated receptor-1 (PAR-1) in stress-induced neuronal plasticity and behavioural responses. PAR-1 gene and protein are present in most regions of the mouse brain with high levels in the thalamus, the hippocampus and the amygdala. Due to its localization in limbic areas, PAR-1 regulation in the amygdala was examined following restraint stress or fear conditioning (FC). The finding that PAR-1 protein but not gene levels are downregulated led to the hypothesis that PAR-1 must be cleaved by stress-related proteases. Indeed my results indicate that a limbic-serine protease neuropsin cleaves the receptor in an indirect manner. This led us to investigate behavioural phenotypes of PAR-1־/־ and neuropsin־/־ mice during fear, anxiety, and learning-related tests. PAR-1־/־ and neuropsin־/־ mice are characterised by an anxiogenic and an anxiolytic behaviour, respectively, both profiles being related to amygdala dysfunction. Indeed amygdala-related neuronal activity following FC appeared elevated based on abnormally elevated c-Fos and P-CREB levels in fear conditioned PAR-1־/־ mice. This may underpin a higher susceptibility of these mice towards anxiety and suggest an inhibitory role of PAR-1־/־ during fear learning. Further electrophysiology data suggest that neuronal excitability and plasticity after FC are affected by PAR-1 through modulation of AMPA receptors. A role of the transmembrane AMPA receptor regulatoty protein Stargazin in this process is excluded as its gene levels are not affected by FC. Following fear learning PAR-1־/־ mice display differential phosphorylation levels ofGluR2 subunit. Furthermore dissimilar regulation of AMPAR splice variant between PAR-1־/־ and wild-type mice could account for PAR-I-related behaviour and electrophysiological characteristics. The results presented here reveal a previous uncharacterized inhibitory role of PAR-1 in the central nervous system.

Subjects/Keywords: 572

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APA (6^th Edition):

Bourgognon, J. (2012). The role of protease-activated receptor-1 in stress-induced neuronal activity and behavioural responses. (Doctoral Dissertation). University of Leicester. Retrieved from http://hdl.handle.net/2381/11063

Chicago Manual of Style (16^th Edition):

Bourgognon, Julie-Myrtille. “The role of protease-activated receptor-1 in stress-induced neuronal activity and behavioural responses.” 2012. Doctoral Dissertation, University of Leicester. Accessed February 18, 2020. http://hdl.handle.net/2381/11063.

MLA Handbook (7^th Edition):

Bourgognon, Julie-Myrtille. “The role of protease-activated receptor-1 in stress-induced neuronal activity and behavioural responses.” 2012. Web. 18 Feb 2020.

Vancouver:

Bourgognon J. The role of protease-activated receptor-1 in stress-induced neuronal activity and behavioural responses. [Internet] [Doctoral dissertation]. University of Leicester; 2012. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/2381/11063.

Council of Science Editors:

Bourgognon J. The role of protease-activated receptor-1 in stress-induced neuronal activity and behavioural responses. [Doctoral Dissertation]. University of Leicester; 2012. Available from: http://hdl.handle.net/2381/11063

Heriot-Watt University

17. Foster, Alexander. The heterogeneity of amine oxidase with Rhodoccus opacus.

Degree: PhD, 2013, Heriot-Watt University

URL: http://hdl.handle.net/10399/2703

► Four native amine oxidases have been identified from Rhodococcus opacus to reveal phenotypic plasticity and catalytic activity with respect to structurally diverse natural and synthetic… (more)

Subjects/Keywords: 572

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APA (6^th Edition):

Foster, A. (2013). The heterogeneity of amine oxidase with Rhodoccus opacus. (Doctoral Dissertation). Heriot-Watt University. Retrieved from http://hdl.handle.net/10399/2703

Chicago Manual of Style (16^th Edition):

Foster, Alexander. “The heterogeneity of amine oxidase with Rhodoccus opacus.” 2013. Doctoral Dissertation, Heriot-Watt University. Accessed February 18, 2020. http://hdl.handle.net/10399/2703.

MLA Handbook (7^th Edition):

Foster, Alexander. “The heterogeneity of amine oxidase with Rhodoccus opacus.” 2013. Web. 18 Feb 2020.

Vancouver:

Foster A. The heterogeneity of amine oxidase with Rhodoccus opacus. [Internet] [Doctoral dissertation]. Heriot-Watt University; 2013. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/10399/2703.

Council of Science Editors:

Foster A. The heterogeneity of amine oxidase with Rhodoccus opacus. [Doctoral Dissertation]. Heriot-Watt University; 2013. Available from: http://hdl.handle.net/10399/2703

18. Muñoz, D. Effects of organophosphates on neural and purified liver tissue transglutaminase.

Degree: PhD, 2010, Nottingham Trent University

URL: http://irep.ntu.ac.uk/id/eprint/253/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629185

► Transglutaminase 2 (TGase 2) is a multifunctional calcium dependent enzyme that catalyzes protein modifications. TGase 2 is essential in neuronal cell differentiation and it has… (more)

▼ Transglutaminase 2 (TGase 2) is a multifunctional calcium dependent enzyme that catalyzes protein modifications. TGase 2 is essential in neuronal cell differentiation and it has been reported that certain organophosphates are able to inhibit this process, and the organophosphate phenyl saligenin compound also disrupts TGase 2 activity. It has also been shown that the organophosphates chlorpyrifos (CPF) and chlorpyrifos oxon (CPFO), which cause developmental neurotoxicity, provoke several changes in differentiating rat C6 glioma cells at different levels. The aims of this thesis were to analyse the effects of CPF and CPFO on the TGases present in differentiating rat C6 glioma cells, to develop a new method for the purification of TGase 2 from guinea pig liver, to study possible direct interactions between TGase 2 and esterase inhibitors and to analyze a possible pathway for the externalisation of TGase 2. In the presence of sodium butyrate, rat C6 glial cells differentiated into an astrocyte phenotype. Differentiation of the cells was associated with an increase in the activity, protein levels and gene expression of TGase 2. Differentiation in the presence of CPF or CPFO generated an increase in the activity of TGase 2, a decrease in its levels of gene expression but had no effect on the protein levels. These effects could be associated with a direct interaction between the organophosphates and TGase 2. Chromatographic methods were developed to purify TGase 2 from guinea pig liver and the most effective one was a combination of ion exchange chromatography, protamine sulfate precipitation and hydrophobic interaction chromatography (HIC). The level of purity and yield obtained were superior to that of previously published methods. Furthermore, the final step of HIC could be applied directly to commercially available TGase 2 for the production of a highly purified TGase 2 sample. When TGase 2 purified in this manner was assayed in the presence of CPF and CPFO, enzyme activity was observed to increase significantly, suggesting a direct interaction with TGase 2. By contrast, phenyl saligenin phosphate was found to inhibit TGase activity in vitro, which suggests a direct effect that may involve a different binding site and/or mechanism to CPF or CPFO. The aspartyl protease inhibitor pepstatin A was also able to inhibit directly TGase activity in vitro. The final part of the project involved a short study of the potential association of TGase 2 with exosomes, in order to determine whether the latter might present a means of externalization of this enzyme. Exosomes purified from mouse N2a neuroblastoma cells were found to contain TGase 2, but its localization within the vesicles remains unclear.

Subjects/Keywords: 572

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APA (6^th Edition):

Muñoz, D. (2010). Effects of organophosphates on neural and purified liver tissue transglutaminase. (Doctoral Dissertation). Nottingham Trent University. Retrieved from http://irep.ntu.ac.uk/id/eprint/253/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629185

Chicago Manual of Style (16^th Edition):

Muñoz, D. “Effects of organophosphates on neural and purified liver tissue transglutaminase.” 2010. Doctoral Dissertation, Nottingham Trent University. Accessed February 18, 2020. http://irep.ntu.ac.uk/id/eprint/253/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629185.

MLA Handbook (7^th Edition):

Muñoz, D. “Effects of organophosphates on neural and purified liver tissue transglutaminase.” 2010. Web. 18 Feb 2020.

Vancouver:

Muñoz D. Effects of organophosphates on neural and purified liver tissue transglutaminase. [Internet] [Doctoral dissertation]. Nottingham Trent University; 2010. [cited 2020 Feb 18]. Available from: http://irep.ntu.ac.uk/id/eprint/253/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629185.

Council of Science Editors:

Muñoz D. Effects of organophosphates on neural and purified liver tissue transglutaminase. [Doctoral Dissertation]. Nottingham Trent University; 2010. Available from: http://irep.ntu.ac.uk/id/eprint/253/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629185

19. Smida, F. A. Photochemical harpoons : covalent labels for multi-protein complexes.

Degree: PhD, 2013, Nottingham Trent University

URL: http://irep.ntu.ac.uk/id/eprint/69/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629292

► The identification of the biomolecular interaction partners of small bioactive molecules is a fundamental problem in drug discovery and cell biology. This thesis describes the… (more)

▼ The identification of the biomolecular interaction partners of small bioactive molecules is a fundamental problem in drug discovery and cell biology. This thesis describes the development of fluorescent chemical probes to identify the biomolecular targets of the known organophosphate toxin, phenyl saligenin phosphate (PSP), and the cardioprotective agent diazoxide. PSP is an organophosphate toxin that irreversibly inhibits hydrolase enzymes such as trypsin and chymotrypsin along with the common organophosphate target acetylcholine esterase. PSP is also suspected of affecting many other cell functions and may interact with a large number of cellular proteins. In this work phenyl saligenin phosphate has been synthesised and its inhibitory effect on the action of transglutaminase 2 (TGase2) demonstrated. Analogues of PSP containing an attached dansyl amide fluorescent group have been prepared and incubated with purified enzymes trypsin, chymotrypsin and TGase2. SDS-PAGE analysis demonstrates effective fluorescent labelling and a covalent interaction between the toxin analogue and the enzymes. The KATP channel opener, diazoxide displays marked cardioprotective effects and is reported to bind to mitochondrial KATP channels. However, the molecular structure of these channels is still largely unknown. This thesis describes the design and the synthesis of a chemical tool to covalently attach fluorescently labels to the proteins which will bind diazoxide. Chemical tools for fluorescent labelling of diazoxide targeted proteins have been prepared. Each consists of a photochemically activated reactive ‘barb’ and coupled fluorescent component linked to modified diazoxide bait. In developing these molecules, a range of functionalised diazoxide bait components were prepared and tested for retained biological activity compared to the parent compound. Two active analogues were linked to either benzophenone or diazirine (photoreactive) and dansyl amide (fluorescent) components. The non-specific photochemical reactivity of these labelling compounds with bovine serine albumin was established. The incubation and photolysis with mitochondrial extracts showed selective photo labelling of only three biomolecular components. The identification of these biomolecules is the subject of on-going investigation.

Subjects/Keywords: 572

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APA (6^th Edition):

Smida, F. A. (2013). Photochemical harpoons : covalent labels for multi-protein complexes. (Doctoral Dissertation). Nottingham Trent University. Retrieved from http://irep.ntu.ac.uk/id/eprint/69/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629292

Chicago Manual of Style (16^th Edition):

Smida, F A. “Photochemical harpoons : covalent labels for multi-protein complexes.” 2013. Doctoral Dissertation, Nottingham Trent University. Accessed February 18, 2020. http://irep.ntu.ac.uk/id/eprint/69/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629292.

MLA Handbook (7^th Edition):

Smida, F A. “Photochemical harpoons : covalent labels for multi-protein complexes.” 2013. Web. 18 Feb 2020.

Vancouver:

Smida FA. Photochemical harpoons : covalent labels for multi-protein complexes. [Internet] [Doctoral dissertation]. Nottingham Trent University; 2013. [cited 2020 Feb 18]. Available from: http://irep.ntu.ac.uk/id/eprint/69/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629292.

Council of Science Editors:

Smida FA. Photochemical harpoons : covalent labels for multi-protein complexes. [Doctoral Dissertation]. Nottingham Trent University; 2013. Available from: http://irep.ntu.ac.uk/id/eprint/69/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629292

20. Rajakaruna, L. K. Proteomics as a tool for the characterisation of nosocomial pathogens.

Degree: PhD, 2010, Nottingham Trent University

URL: http://irep.ntu.ac.uk/id/eprint/32/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629158

► The aim of the present investigation was to develop and assess the potential of various proteomic approaches for the characterisation of two major nosocomial pathogens,… (more)

Subjects/Keywords: 572

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APA (6^th Edition):

Rajakaruna, L. K. (2010). Proteomics as a tool for the characterisation of nosocomial pathogens. (Doctoral Dissertation). Nottingham Trent University. Retrieved from http://irep.ntu.ac.uk/id/eprint/32/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629158

Chicago Manual of Style (16^th Edition):

Rajakaruna, L K. “Proteomics as a tool for the characterisation of nosocomial pathogens.” 2010. Doctoral Dissertation, Nottingham Trent University. Accessed February 18, 2020. http://irep.ntu.ac.uk/id/eprint/32/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629158.

MLA Handbook (7^th Edition):

Rajakaruna, L K. “Proteomics as a tool for the characterisation of nosocomial pathogens.” 2010. Web. 18 Feb 2020.

Vancouver:

Rajakaruna LK. Proteomics as a tool for the characterisation of nosocomial pathogens. [Internet] [Doctoral dissertation]. Nottingham Trent University; 2010. [cited 2020 Feb 18]. Available from: http://irep.ntu.ac.uk/id/eprint/32/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629158.

Council of Science Editors:

Rajakaruna LK. Proteomics as a tool for the characterisation of nosocomial pathogens. [Doctoral Dissertation]. Nottingham Trent University; 2010. Available from: http://irep.ntu.ac.uk/id/eprint/32/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629158

21. Croft, S. M. Mammalian-wide interspersed repeats (MIRs) and their role in mammalian gene function and evolution.

Degree: PhD, 2009, Nottingham Trent University

URL: http://irep.ntu.ac.uk/id/eprint/104/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629132

► Transposable elements (TEs) are ubiquitous components of plant and animal genomes and constitute more than ~45% of the human genome. Though originally considered as 'parasitic'… (more)

▼ Transposable elements (TEs) are ubiquitous components of plant and animal genomes and constitute more than ~45% of the human genome. Though originally considered as 'parasitic' or 'junk' DNA, TEs are now thought to have played a role in shaping genomes during evolution, contributing to genome plasticity and diversity. All classes of retrotransposons accumulate in the genome via a process termed retrotransposition, wherein the elements are reverse transcribed into RNA and inserted into the genome as DNA. Exaptation of these elements can provide additional or novel function for endogenous genes. Mammalian-wide interspersed repeats (MIRs) are short interspersed nuclear elements (SINEs), belonging to the non-autonomous class of retroelements and are found in all mammals. The recruitment of an MIR element by a gene may provide insight into mammalian evolution and gene function. The human genome was screened for genes that have exaptated MIR elements and the compiled dataset was analysed to determine any commonality which may suggest conserved function(s). Subsequently 1359 genes were identified that have exaptated MIR elements, constituting 5% of the total genes in the human genome. MIR elements may be multifunctional, as 1% of the total human genes contain MIRs that are spliced and/or are contributing to protein coding sequences. Subsequently sequence motifs were identified in the MIR consensus sequences which resemble canonical mammalian splice sites; therefore MIR elements recruited in the 5'-UTR and coding sequence may be a result of the exonisation of intronic elements. The MIR-containing transcripts are frequently expressed in neurological tissue, suggesting a role in neuronal function. Moreover a number of MIR-containing mRNA transcripts are known to be localised to the dendritic compartment of the neurone, and ciliated region of photoreceptors. Some of the localised mRNAs contain putative microRNA binding sites within the MIR sequence, and possible dsRNA structures were noted between MIR elements. It is proposed that exaptated MIR elements may be a source of cis-acting regulatory elements, involved in post-transcriptional control of gene expression, including localisation of mRNA to distinct intracellular compartments.

Subjects/Keywords: 572

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APA (6^th Edition):

Croft, S. M. (2009). Mammalian-wide interspersed repeats (MIRs) and their role in mammalian gene function and evolution. (Doctoral Dissertation). Nottingham Trent University. Retrieved from http://irep.ntu.ac.uk/id/eprint/104/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629132

Chicago Manual of Style (16^th Edition):

Croft, S M. “Mammalian-wide interspersed repeats (MIRs) and their role in mammalian gene function and evolution.” 2009. Doctoral Dissertation, Nottingham Trent University. Accessed February 18, 2020. http://irep.ntu.ac.uk/id/eprint/104/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629132.

MLA Handbook (7^th Edition):

Croft, S M. “Mammalian-wide interspersed repeats (MIRs) and their role in mammalian gene function and evolution.” 2009. Web. 18 Feb 2020.

Vancouver:

Croft SM. Mammalian-wide interspersed repeats (MIRs) and their role in mammalian gene function and evolution. [Internet] [Doctoral dissertation]. Nottingham Trent University; 2009. [cited 2020 Feb 18]. Available from: http://irep.ntu.ac.uk/id/eprint/104/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629132.

Council of Science Editors:

Croft SM. Mammalian-wide interspersed repeats (MIRs) and their role in mammalian gene function and evolution. [Doctoral Dissertation]. Nottingham Trent University; 2009. Available from: http://irep.ntu.ac.uk/id/eprint/104/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629132

22. Dookie, S. Transglutaminase in the life and death of the pancreatic β-cell.

Degree: PhD, 2010, Nottingham Trent University

URL: http://irep.ntu.ac.uk/id/eprint/323/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629178

► Tissue transglutaminase (TG2) is a ubiquitous enzyme that catalyses both the Ca2+-dependent formation of protein cross-links via intermolecular isopeptide bonds, and the Ca2+-independent hydrolysis of… (more)

▼ Tissue transglutaminase (TG2) is a ubiquitous enzyme that catalyses both the Ca2+-dependent formation of protein cross-links via intermolecular isopeptide bonds, and the Ca2+-independent hydrolysis of GTP. The multifunctional nature of the TG2 protein has been reported in numerous intracellular mechanisms, cell-surface associations, and extracellular matrix (ECM) interactions. In the pancreas, the expression of TG may be fundamental to the insulin-secretion function of β-cells, and associated diabetic disorders. The functional roles of TG2 in the pancreas were investigated in the present study using in vitro models of rat pancreatic insulinoma β-cells (BRIN-BD11), and ex vivo islet of Langerhans models from human, rat, TG2(-/-) knockout mice and their wild-type counterparts. The importance of ECM-associated TG2 in the maintenance of β-cell survival and function was also investigated using an in vitro human urinary bladder carcinoma support matrix (5637 cells). Biochemical analysis of both clonal BRIN-BD11 and islet β-cells showed a thiol-dependent TG2 activity mechanism that was reciprocally regulated by Ca2+ and GTP. Intracellular TG2 cross-linking activity was up-regulated in the presence of glucose and retinoic acid, while cell-surface associated TG2 activity was reduced in the presence of membrane proteases. The in vitro application of irreversible active-site directed TG inhibitors (R281, R283 and BOC-peptide) strongly supported a role for TG2 in the glucose-stimulated insulin secretion function of BRIN-BD11 β-cells and rat islets, with a possible survival role for the enzyme under conditions of β-cell oxidative stress. Further characterisation of the pancreatic β-cell TG led to the discovery of a novel short-form (~60-kDa) TG2 protein expressed in BRIN-BD11, human and mouse islets that was immunoreactive to CUB7402 antibody, and showed GTP-binding potential. Analysis of the BRIN-BD11 proteome using 2-D SDS-PAGE and western blotting exhibited possible phosphorylation of the TG2 protein at ~60-kDa (pI 4-6), with an additional 120-kDa and ~35-kDa protein. Molecular screening using northern blot analysis of BRIN-BD11 mRNA confirmed the presence of two short-form TG2 transcripts at ~2.5-kb and ~1.0-kb in addition to the full length 3.5-kb TG2 transcript, while RT-PCR analysis using C-terminal directed primers revealed a short-form BRIN-BD11 product similar to an alternatively spliced shorter TG2 isoform found in rat brain. Modifications to ECM-associated TG2 in an in vitro 3-day 5637 support matrix demonstrated the importance of this protein in cell adhesion, cell spreading and aggregation in the maintenance of BRIN-BD11 survival and function. Preliminary studies produced an up-regulation of β-cell focal adhesion kinase, and actin-stained focal adhesion points, in response to the TG2-rich support matrices. The 5637 pre-conditioned matrix also supported greater β-cell viability in response to diabetic conditions such as hyperglycaemia, oxidative stress, and hyperlipidaemia. These findings demonstrate that the primary…

Subjects/Keywords: 572

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APA (6^th Edition):

Dookie, S. (2010). Transglutaminase in the life and death of the pancreatic β-cell. (Doctoral Dissertation). Nottingham Trent University. Retrieved from http://irep.ntu.ac.uk/id/eprint/323/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629178

Chicago Manual of Style (16^th Edition):

Dookie, S. “Transglutaminase in the life and death of the pancreatic β-cell.” 2010. Doctoral Dissertation, Nottingham Trent University. Accessed February 18, 2020. http://irep.ntu.ac.uk/id/eprint/323/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629178.

MLA Handbook (7^th Edition):

Dookie, S. “Transglutaminase in the life and death of the pancreatic β-cell.” 2010. Web. 18 Feb 2020.

Vancouver:

Dookie S. Transglutaminase in the life and death of the pancreatic β-cell. [Internet] [Doctoral dissertation]. Nottingham Trent University; 2010. [cited 2020 Feb 18]. Available from: http://irep.ntu.ac.uk/id/eprint/323/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629178.

Council of Science Editors:

Dookie S. Transglutaminase in the life and death of the pancreatic β-cell. [Doctoral Dissertation]. Nottingham Trent University; 2010. Available from: http://irep.ntu.ac.uk/id/eprint/323/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629178

University of Newcastle upon Tyne

23. Stroukova, Daria. Recombinant expression and characterisation of the colicin N immunity protein.

Degree: PhD, 2016, University of Newcastle upon Tyne

URL: http://hdl.handle.net/10443/3250

► Some Escherichia coli express plasmid encoded toxins called colicins to eliminate ecological competitors. Colicins are a diverse group of toxins, usually divided into three groups:… (more)

▼ Some Escherichia coli express plasmid encoded toxins called colicins to eliminate ecological competitors. Colicins are a diverse group of toxins, usually divided into three groups: nucleases, pore-formers and peptidoglycan synthesis inhibitors. In addition, each colicinogenic plasmid codes for an immunity protein - a self-protection mechanism against its own toxin. Immunity proteins are highly specific to their cognate toxins but it is not clearly known how immunity proteins to pore-forming colicins achieve protection. This work focuses on the immunity protein for the smallest pore-forming colicin, colicin N. To investigate in vitro how the Colicin N immunity protein (CNI) neutralises the toxin, different overexpression and purification methods were tested. The fusion CNI-3C-HALO7-6His yielded the most protein, when expressed in C41 E. coli cells at 37 °C overnight in Terrific Broth. The fusion protein increases resistance to Colicin N dramatically and is therefore folded and localised correctly. Decyl β-Dmaltopyranoside is the most effective detergent to solubilise and stabilise the protein fusion. To investigate if CNI inactivates ColN by binding to it via hydrophobic α-helical interactions, the protein’s helical regions were defined in silico and swapped with the respective parts of the Colicin A immunity protein, CNI’s closest homologue, which provides immunity to Colicin A. The N-terminal region of CNI, including the first transmembrane helix, is not necessary for specific CNI-Colicin N interaction but the other helices are crucial for protein stability and function. CNI can be expressed in an active form fused to GFP for microscopy. Epifluorescent and TIRF microscopy revealed that CNI is localised to the cell periphery and appears to be very evenly distributed in the cell membrane. Colicin N addition does not affect CNI distribution. Single molecule tracking in TIRF microscopy showed that the diffusion rate of CNI does not change when ColN is added. Future work can pursue either of the three approaches used here to study CNI using in vitro structural analysis, site-directed mutagenesis of the active site or in vivo interaction of CNI with other E. coli inner membrane proteins.

Subjects/Keywords: 572

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APA (6^th Edition):

Stroukova, D. (2016). Recombinant expression and characterisation of the colicin N immunity protein. (Doctoral Dissertation). University of Newcastle upon Tyne. Retrieved from http://hdl.handle.net/10443/3250

Chicago Manual of Style (16^th Edition):

Stroukova, Daria. “Recombinant expression and characterisation of the colicin N immunity protein.” 2016. Doctoral Dissertation, University of Newcastle upon Tyne. Accessed February 18, 2020. http://hdl.handle.net/10443/3250.

MLA Handbook (7^th Edition):

Stroukova, Daria. “Recombinant expression and characterisation of the colicin N immunity protein.” 2016. Web. 18 Feb 2020.

Vancouver:

Stroukova D. Recombinant expression and characterisation of the colicin N immunity protein. [Internet] [Doctoral dissertation]. University of Newcastle upon Tyne; 2016. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/10443/3250.

Council of Science Editors:

Stroukova D. Recombinant expression and characterisation of the colicin N immunity protein. [Doctoral Dissertation]. University of Newcastle upon Tyne; 2016. Available from: http://hdl.handle.net/10443/3250

24. Dowle, Adam Ashley. Leveraging the power of mass spectrometry-based proteomics to reveal novel biological insights.

Degree: PhD, 2016, University of York

URL: http://etheses.whiterose.ac.uk/15722/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701469

► Mass spectrometry-based proteomics has the potential to offer new qualitative and quantitative insights into a wide array of biological questions. The work presented in this… (more)

Subjects/Keywords: 572

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APA (6^th Edition):

Dowle, A. A. (2016). Leveraging the power of mass spectrometry-based proteomics to reveal novel biological insights. (Doctoral Dissertation). University of York. Retrieved from http://etheses.whiterose.ac.uk/15722/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701469

Chicago Manual of Style (16^th Edition):

Dowle, Adam Ashley. “Leveraging the power of mass spectrometry-based proteomics to reveal novel biological insights.” 2016. Doctoral Dissertation, University of York. Accessed February 18, 2020. http://etheses.whiterose.ac.uk/15722/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701469.

MLA Handbook (7^th Edition):

Dowle, Adam Ashley. “Leveraging the power of mass spectrometry-based proteomics to reveal novel biological insights.” 2016. Web. 18 Feb 2020.

Vancouver:

Dowle AA. Leveraging the power of mass spectrometry-based proteomics to reveal novel biological insights. [Internet] [Doctoral dissertation]. University of York; 2016. [cited 2020 Feb 18]. Available from: http://etheses.whiterose.ac.uk/15722/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701469.

Council of Science Editors:

Dowle AA. Leveraging the power of mass spectrometry-based proteomics to reveal novel biological insights. [Doctoral Dissertation]. University of York; 2016. Available from: http://etheses.whiterose.ac.uk/15722/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701469

Durham University

25. Lear, Sam. Total synthesis of bioactive peptides and whole proteins.

Degree: PhD, 2016, Durham University

URL: http://etheses.dur.ac.uk/11946/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702083

► Total chemical synthesis is an essential tool for the validation of natural product structures and the discovery and elaboration of novel therapeutic scaffolds. Chapter 1… (more)

Subjects/Keywords: 572

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APA (6^th Edition):

Lear, S. (2016). Total synthesis of bioactive peptides and whole proteins. (Doctoral Dissertation). Durham University. Retrieved from http://etheses.dur.ac.uk/11946/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702083

Chicago Manual of Style (16^th Edition):

Lear, Sam. “Total synthesis of bioactive peptides and whole proteins.” 2016. Doctoral Dissertation, Durham University. Accessed February 18, 2020. http://etheses.dur.ac.uk/11946/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702083.

MLA Handbook (7^th Edition):

Lear, Sam. “Total synthesis of bioactive peptides and whole proteins.” 2016. Web. 18 Feb 2020.

Vancouver:

Lear S. Total synthesis of bioactive peptides and whole proteins. [Internet] [Doctoral dissertation]. Durham University; 2016. [cited 2020 Feb 18]. Available from: http://etheses.dur.ac.uk/11946/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702083.

Council of Science Editors:

Lear S. Total synthesis of bioactive peptides and whole proteins. [Doctoral Dissertation]. Durham University; 2016. Available from: http://etheses.dur.ac.uk/11946/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702083

University of Sheffield

26. Wacnik, Katarzyna. Dissecting cell division in the human pathogen Staphylococcus aureus.

Degree: PhD, 2016, University of Sheffield

URL: http://etheses.whiterose.ac.uk/14257/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696007

► Bacterial cell division is a fundamental process mediated by a large collection of proteins, collectively called the divisome. The divisome is responsible for the appropriate… (more)

▼ Bacterial cell division is a fundamental process mediated by a large collection of proteins, collectively called the divisome. The divisome is responsible for the appropriate synthesis of new cell wall peptidoglycan to produce the septum, allowing the production of two new daughter cells. Although divisome components have been identified, their precise roles are not well understood. Many cell division proteins interact and coordination of information from the cytoplasm, through the membrane, to the peptidoglycan biosynthesis machinery is required. FtsZ is a key cytoplasmic component, which initiates septum formation resulting in new peptidoglycan synthesis via penicillin binding proteins (PBPs). In Staphylococcus aureus, cell division components, including EzrA, have been identified by a bacterial two hybrid analysis. EzrA, a membrane-associated protein, interacts with both cytoplasmic proteins and those with periplasmic domains. It is therefore proposed to act as an interface between FtsZ in the cytoplasm and PBPs in the membrane forming a scaffold for other cell division components. In this study, a combination of protein labelling and super-resolution microscopy approaches have been used to study the architecture of the cell division process in S. aureus. Conventional wide field fluorescence microscopy shows EzrA and FtsZ as uniform rings at the division site. Super resolution fluorescence microscopy of a strain, in which the only copy of EzrA was tagged with a fluorophore, revealed that EzrA does not form a homogenous ring but it is rather a collection of ‘patches’ distributed at the division site. FtsZ precedes EzrA and also forms heterogeneous structures around the midcell. Unsuccessful attempts were made to investigate the localisation of the essential PBP2 at molecular level using fluorescent proteins or fluorescent derivatives of β-lactams. A model of septum formation in S. aureus, in which a decreasing concentration gradient of cell division components wraps around the septum and forms a framework for peptidoglycan synthesis, has been developed.

Subjects/Keywords: 572

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APA (6^th Edition):

Wacnik, K. (2016). Dissecting cell division in the human pathogen Staphylococcus aureus. (Doctoral Dissertation). University of Sheffield. Retrieved from http://etheses.whiterose.ac.uk/14257/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696007

Chicago Manual of Style (16^th Edition):

Wacnik, Katarzyna. “Dissecting cell division in the human pathogen Staphylococcus aureus.” 2016. Doctoral Dissertation, University of Sheffield. Accessed February 18, 2020. http://etheses.whiterose.ac.uk/14257/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696007.

MLA Handbook (7^th Edition):

Wacnik, Katarzyna. “Dissecting cell division in the human pathogen Staphylococcus aureus.” 2016. Web. 18 Feb 2020.

Vancouver:

Wacnik K. Dissecting cell division in the human pathogen Staphylococcus aureus. [Internet] [Doctoral dissertation]. University of Sheffield; 2016. [cited 2020 Feb 18]. Available from: http://etheses.whiterose.ac.uk/14257/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696007.

Council of Science Editors:

Wacnik K. Dissecting cell division in the human pathogen Staphylococcus aureus. [Doctoral Dissertation]. University of Sheffield; 2016. Available from: http://etheses.whiterose.ac.uk/14257/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696007

University of Leeds

27. Wilson, Michael Christopher. Comparing the thermal, chemical and mechanical stabilities of extremophilic cold shock protein variants.

Degree: PhD, 2016, University of Leeds

URL: http://etheses.whiterose.ac.uk/15982/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701722

► Proteins conduct a vast array of chemical processes and achieve this through a balance of flexibility and function. Proteins from extremophilic organisms have evolved to… (more)

▼ Proteins conduct a vast array of chemical processes and achieve this through a balance of flexibility and function. Proteins from extremophilic organisms have evolved to adjust this balance to function in extreme conditions. Research into hot-adapted proteins has been encouraged through successes in improving enzyme thermostability, though cold-adapted proteins also offer substantial potential as they can function effectively at lower temperatures. Research into cold-adapted proteins remains limited despite cold climates comprising over 80% of Earth’s biosphere. This study compares the stabilities of cold shock protein (Csp) homologues from bacteria growing at vastly different temperatures. The cold-adapted Csps show reduced thermal denaturation mid-points (Tm values) compared to a temperate Csp with PB6 Csp displaying the lowest Tm of 42.8 ⁰C contrasting with 52.3 ⁰C shown by BsCsp. Thermostability projections using the Gibbs-Helmholtz equation show cold-adapted Csps display slightly reduced thermostabilities and remain folded over a narrower range of temperatures. Csp thermostabilities were found to be very similar at around 8 degrees below the optimal growth temperature of the bacteria each Csp was derived from, suggesting thermostabilities evolved relating to Csp operating temperatures. The roles of electrostatics and hydrophobic packing in stabilizing a hyperthermophilic Csp are evaluated using mutants that highlight how a single side chain length reduction dramatically decreases Csp thermostability. Kinetic studies showed that the Csps exhibit similar folding rate constants but different unfolding rate constants. Initial flexibility studies suggest that DNA binding increases Csp rigidity and triggers a conformational change in a psychrotrophic Csp. Csp mechanical stabilities showed the same hierarchy as thermostabilities and similar sensitivity to temperature changes. Energy landscape projections constructed using Monte Carlo simulations showed the mechanical energy barrier height of Csp unfolding was temperature independent. At 5 ⁰C the Csps showed similar mechanical softness however the hyperthermophilic TmCsp shows greater mechanical softness at higher temperatures.

Subjects/Keywords: 572

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APA (6^th Edition):

Wilson, M. C. (2016). Comparing the thermal, chemical and mechanical stabilities of extremophilic cold shock protein variants. (Doctoral Dissertation). University of Leeds. Retrieved from http://etheses.whiterose.ac.uk/15982/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701722

Chicago Manual of Style (16^th Edition):

Wilson, Michael Christopher. “Comparing the thermal, chemical and mechanical stabilities of extremophilic cold shock protein variants.” 2016. Doctoral Dissertation, University of Leeds. Accessed February 18, 2020. http://etheses.whiterose.ac.uk/15982/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701722.

MLA Handbook (7^th Edition):

Wilson, Michael Christopher. “Comparing the thermal, chemical and mechanical stabilities of extremophilic cold shock protein variants.” 2016. Web. 18 Feb 2020.

Vancouver:

Wilson MC. Comparing the thermal, chemical and mechanical stabilities of extremophilic cold shock protein variants. [Internet] [Doctoral dissertation]. University of Leeds; 2016. [cited 2020 Feb 18]. Available from: http://etheses.whiterose.ac.uk/15982/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701722.

Council of Science Editors:

Wilson MC. Comparing the thermal, chemical and mechanical stabilities of extremophilic cold shock protein variants. [Doctoral Dissertation]. University of Leeds; 2016. Available from: http://etheses.whiterose.ac.uk/15982/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701722

Cardiff University

28. Redding, Sadie Jane. Post-transcriptional regulation of ALAS1 expression by haem.

Degree: PhD, 2007, Cardiff University

URL: http://orca.cf.ac.uk/55682/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.575160

► Haem is the prosthetic moiety of numerous haemoproteins critical for the function of all aerobic cells. Its biosynthesis is a tightly controlled process since high… (more)

Subjects/Keywords: 572

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Redding, S. J. (2007). Post-transcriptional regulation of ALAS1 expression by haem. (Doctoral Dissertation). Cardiff University. Retrieved from http://orca.cf.ac.uk/55682/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.575160

Chicago Manual of Style (16^th Edition):

Redding, Sadie Jane. “Post-transcriptional regulation of ALAS1 expression by haem.” 2007. Doctoral Dissertation, Cardiff University. Accessed February 18, 2020. http://orca.cf.ac.uk/55682/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.575160.

MLA Handbook (7^th Edition):

Redding, Sadie Jane. “Post-transcriptional regulation of ALAS1 expression by haem.” 2007. Web. 18 Feb 2020.

Vancouver:

Redding SJ. Post-transcriptional regulation of ALAS1 expression by haem. [Internet] [Doctoral dissertation]. Cardiff University; 2007. [cited 2020 Feb 18]. Available from: http://orca.cf.ac.uk/55682/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.575160.

Council of Science Editors:

Redding SJ. Post-transcriptional regulation of ALAS1 expression by haem. [Doctoral Dissertation]. Cardiff University; 2007. Available from: http://orca.cf.ac.uk/55682/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.575160

Cardiff University

29. Reviriego Santos, Pablo. Engineering aequorin as an indicator of calcium signals near the BK channel.

Degree: PhD, 2009, Cardiff University

URL: http://orca.cf.ac.uk/55830/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584705

► The BK channel is a large conductance calcium-activated voltage- dependent potassium channel. This channel plays a key role as a negative feedback mechanism of membrane… (more)

Subjects/Keywords: 572

Record Details Similar Records Cite « Share

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Reviriego Santos, P. (2009). Engineering aequorin as an indicator of calcium signals near the BK channel. (Doctoral Dissertation). Cardiff University. Retrieved from http://orca.cf.ac.uk/55830/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584705

Chicago Manual of Style (16^th Edition):

Reviriego Santos, Pablo. “Engineering aequorin as an indicator of calcium signals near the BK channel.” 2009. Doctoral Dissertation, Cardiff University. Accessed February 18, 2020. http://orca.cf.ac.uk/55830/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584705.

MLA Handbook (7^th Edition):

Reviriego Santos, Pablo. “Engineering aequorin as an indicator of calcium signals near the BK channel.” 2009. Web. 18 Feb 2020.

Vancouver:

Reviriego Santos P. Engineering aequorin as an indicator of calcium signals near the BK channel. [Internet] [Doctoral dissertation]. Cardiff University; 2009. [cited 2020 Feb 18]. Available from: http://orca.cf.ac.uk/55830/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584705.

Council of Science Editors:

Reviriego Santos P. Engineering aequorin as an indicator of calcium signals near the BK channel. [Doctoral Dissertation]. Cardiff University; 2009. Available from: http://orca.cf.ac.uk/55830/ ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584705

University of Newcastle Upon Tyne

30. Sloan, Katherine. The exosome and human ribosome biogenesis.

Degree: PhD, 2012, University of Newcastle Upon Tyne

URL: http://hdl.handle.net/10443/1462

► Exoribonucleases have many important functions in the cell including RNA processing, turnover and quality control. One of the key 3’-5’ exonucleases is the exosome, a… (more)

Subjects/Keywords: 572

Record Details Similar Records Cite « Share

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Sloan, K. (2012). The exosome and human ribosome biogenesis. (Doctoral Dissertation). University of Newcastle Upon Tyne. Retrieved from http://hdl.handle.net/10443/1462

Chicago Manual of Style (16^th Edition):

Sloan, Katherine. “The exosome and human ribosome biogenesis.” 2012. Doctoral Dissertation, University of Newcastle Upon Tyne. Accessed February 18, 2020. http://hdl.handle.net/10443/1462.

MLA Handbook (7^th Edition):

Sloan, Katherine. “The exosome and human ribosome biogenesis.” 2012. Web. 18 Feb 2020.

Vancouver:

Sloan K. The exosome and human ribosome biogenesis. [Internet] [Doctoral dissertation]. University of Newcastle Upon Tyne; 2012. [cited 2020 Feb 18]. Available from: http://hdl.handle.net/10443/1462.

Council of Science Editors:

Sloan K. The exosome and human ribosome biogenesis. [Doctoral Dissertation]. University of Newcastle Upon Tyne; 2012. Available from: http://hdl.handle.net/10443/1462

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Open Access Theses and Dissertations