2024-03-29T02:34:49Zhttps://research-repository.st-andrews.ac.uk/oai/requestoai:research-repository.st-andrews.ac.uk:10023/5422019-07-01T10:16:27Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Elliott, Richard Michael
Eifan, Saleh A.
xviii, 225 p.
2008-10-27T16:20:02Z
2008-10-27T16:20:02Z
2008
uk.bl.ethos.552178
http://hdl.handle.net/10023/542
Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae. It has a
tripartite genome consisting of negative sense RNA segments called large (L),
medium (M) and small (S). The S segment encodes the nucleocapsid protein (N) of
233 amino acids. The N protein encapsidates all three segments to form
transcriptionally active ribonucleoproteins (RNPs). The aim of this project was to
determine the domain map of BUNV N protein.
To investigate residues in BUNV N crucial for its functionality, random and site-
specific mutagenesis were performed on a cDNA clone encoding the BUNV N
protein. In total, 102 single amino acid substitutions were generated in the BUNV N
protein sequence.
All mutant N proteins were used in a BUNV minigenome system to compare their
activity to wt BUNV N. The mutant proteins displayed a wide-range of activity, from
parental-like to essentially inactive. The most disruptive mutations were R94A,
I118N, W134A, Y141C, L177A, K179I and W193A.
Sixty-four clones carrying single substitutions in the BUNV N protein were used in the
BUNV rescue system in an attempt to recover viable mutant viruses. Fifty
recombinant mutant viruses were rescued and 14 N genes were nonrescuable.
The 50 mutant viruses were characterized by: titration, protein labelling, western
blotting, temperature sensitivity and host-restriction. Mutant viruses displayed a wide range of titers between 10³
-10⁸ pfu/ml, and three different plaque sizes large,
medium and small. Protein labelling and western blotting showed that mutations in
the N gene did not affect expression of the other viral genes as much as affecting N
protein expression. It was demonstrated that single amino acid substitutions could
alter N protein electrophoretic mobility in SDS- PAGE (e.g. P19Q and L53F).
Temperature sensitivity tests showed that recombinant viruses N74S, S96S, K228T
and G230R were ts, growing at 33˚C but not at 37˚C or 38˚C, while the parental virus grew at all temperatures. Using the northern blotting technique, mutant viruses N74S
and S96G were shown to have a ts defect in genome-synthesis (late replication
step), while mutant viruses K228T and G230R had a ts defect in antigenome-
synthesis (early replication step).
Host-restriction experiments were performed using 5 different cell lines (Vero-E6,
BHK-21, 2FTGH-V, A549-V and 293-V). Overall, the parental virus grew similarly in
all cell lines. Likewise, the majority of mutant viruses follow this pattern except mutant
virus Y23A. It showed a 100-fold reduction in titer in 2FTGH-V cells. Comparing the
ratios of intracellular and extracellular particles revealed that only 15% of the total
virus particles of mutant Y23A was released as extracellular particles compared to
30% of the parental virus.
Fourteen N genes were nonrescuable. They were characterized by (i) their activity in
the BUNV minigenome system, (ii) their activity in BUNV packaging assay, (iii) their
ability to form multimers, (iv) their ability to interact with L protein, and (v) their impact
on RNA synthesis.
In summary, BUNV N protein was shown to be multi-functional and involved in the
regulation of virus transcription and replication, RNA synthesis and assembly, via
interactions with the viral L polymerase, RNA backbone, itself or the viral
glycoproteins.
15437923 bytes
application/pdf
en
University of St Andrews
The University of St Andrews
Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
http://creativecommons.org/licenses/by-nc-nd/3.0/
QR398.7E5
Bunyaviruses
Viral proteins
Nucleoproteins
Functional analysis of the orthobunyavirus nucleocapsid (N) protein
Thesis
Doctoral
PhD Doctor of Philosophy
THUMBNAIL
Saleh A Eifan PhD thesis.pdf.jpg
Saleh A Eifan PhD thesis.pdf.jpg
Generated Thumbnail
image/jpeg
3717
https://research-repository.st-andrews.ac.uk/bitstream/10023/542/8/Saleh%20A%20Eifan%20PhD%20thesis.pdf.jpg
a09af4a2a19c4354fa903d7ccf62923f
MD5
8
ORIGINAL
Saleh A Eifan PhD thesis.pdf
Saleh A Eifan PhD thesis.pdf
application/pdf
15437923
https://research-repository.st-andrews.ac.uk/bitstream/10023/542/6/Saleh%20A%20Eifan%20PhD%20thesis.pdf
18347dfc3e2507d6bb16bc69c19fb6cd
MD5
6
CC-LICENSE
license_url
license_url
text/plain
49
https://research-repository.st-andrews.ac.uk/bitstream/10023/542/2/license_url
fd26723f8d7edacdb29e3f03465c3b03
MD5
2
license_text
license_text
application/octet-stream
14174
https://research-repository.st-andrews.ac.uk/bitstream/10023/542/3/license_text
fd660d22c2bc09ce4c89722f3e2ca09b
MD5
3
license_rdf
license_rdf
application/octet-stream
5225
https://research-repository.st-andrews.ac.uk/bitstream/10023/542/4/license_rdf
4835cfbe1709c07d040ddb762700f69f
MD5
4
LICENSE
license.txt
license.txt
text/plain
2229
https://research-repository.st-andrews.ac.uk/bitstream/10023/542/5/license.txt
63a121c3cdf69ca6e62674ae948e1355
MD5
5
TEXT
Saleh A Eifan PhD thesis.pdf.txt
Saleh A Eifan PhD thesis.pdf.txt
Extracted text
text/plain
363373
https://research-repository.st-andrews.ac.uk/bitstream/10023/542/7/Saleh%20A%20Eifan%20PhD%20thesis.pdf.txt
cb98eb2ee269ee6dc08caff4ad17ba1d
MD5
7
10023/542
oai:research-repository.st-andrews.ac.uk:10023/542
2019-07-01 11:16:27.861
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/39652019-03-29T11:19:48Zcom_10023_175com_10023_39com_10023_51com_10023_18col_10023_177col_10023_53
Naismith, Jim
Reeks, Judith
330
2013-08-16T11:22:42Z
2013-08-16T11:22:42Z
2013-11-29
uk.bl.ethos.577295
http://hdl.handle.net/10023/3965
http://hdl.handle.net/10023/3954
http://hdl.handle.net/10023/3863
http://hdl.handle.net/10023/3550
http://hdl.handle.net/10023/5991
Clustered regularly interspaced short palindromic repeats (CRISPRs) act to prevent viral infection and horizontal gene transfer in prokaryotes. The genomic CRISPR array contains short sequences (“spacers”) that are derived from foreign genetic elements. The CRISPR array is transcribed and processed into CRISPR RNAs (crRNAs) used in the sequence-specific degradation of foreign nucleic acids. This process is called interference and is mediated by CRISPR-associated (Cas) proteins.
This thesis has focused on the structural and functional characterisation of four Cas proteins from the CRISPR/Cas system of Sulfolobus solfataricus. The crystal structure of Cmr7 (Sso1725), a Sulfolobales-specific subunit of the ssRNA-degrading CMR complex, allowed for the identification of a putative protein-binding site, though no specific function could be ascribed to the protein. Cas6 (Sso1437) is the enzyme responsible for crRNA maturation and the characterisation of this protein allowed for the molecular rationalisation of its atypical RNA cleavage mechanism. Csa5 and Cas8a2 are subunits of the aCascade complex that targets dsDNA. Csa5 (Sso1398) was shown to have a putative role in R-loop stabilisation during interference while the role of Cas8a2 (Sso1401) was not determined. The structures of these two proteins were used to define relationships between the subunits of interference complexes from various CRISPR/Cas systems.
A second aspect of this work has been the expression and purification of eukaryotic ion channels for structural studies. The acid sensing ion channel (ASIC) and FMRFamide-gated sodium channel (FaNaC) are gated ion channels with unknown mechanisms of channel activation. These ion channels must be expressed in eukaryotic systems and so human embryonic kidney (HEK) cells and baculovirus-insect cell expression systems were developed to express ASIC and FaNaC constructs. The expression and purification protocols have been optimised to allow for the preparation of soluble protein that will in future be used for crystallography and electron paramagnetic resonance (EPR) studies.
en
University of St Andrews
The University of St Andrews
Reeks , J , Sokolowski , R D , Graham , S , Liu , H , Naismith , J H & White , M F 2013 , ' Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing ' Biochemical Journal , vol 452 , no. 2 , pp. 223-230
Reeks , J A , Naismith , J & White , M F 2013 , ' CRISPR interference : a structural perspective ' Biochemical Journal , vol 453 , no. 2 , pp. 155-166
Reeks , J A , Graham , S , Anderson , L , Liu , H , White , M F & Naismith , J 2013 , ' Structure of the archaeal Cascade subunit Csa5 : Relating the small subunits of CRISPR effector complexes ' RNA Biology , vol 10 , no. 5 , pp. 762-769
Zhang , J , Rouillon , C , Kerou , M , Reeks , J , Brugger , K , Graham , S , Reimann , J , Cannone , G , Liu , H , Albers , S-V , Naismith , J H , Spagnolo , L & White , M F 2012 , ' Structure and mechanism of the CMR complex for CRISPR-Mediated antiviral immunity ' Molecular Cell , vol 45 , no. 3 , pp. 303-313
X-ray crystallography
CRISPR/Cas
S. solfataricus
FaNaC
ASIC
QR92.P75R4
Archaebacteria--Genetic aspects
Bacterial proteins
X-ray crystallography
Ion channels
Structural studies of CRISPR-associated proteins
Thesis
Doctoral
PhD Doctor of Philosophy
THUMBNAIL
JudithReeksPhDThesis.pdf.jpg
JudithReeksPhDThesis.pdf.jpg
Generated Thumbnail
image/jpeg
3603
https://research-repository.st-andrews.ac.uk/bitstream/10023/3965/5/JudithReeksPhDThesis.pdf.jpg
85c3b6b8026bc3ffefb1a03de7ba749d
MD5
5
ORIGINAL
JudithReeksPhDThesis.pdf
JudithReeksPhDThesis.pdf
application/pdf
142655152
https://research-repository.st-andrews.ac.uk/bitstream/10023/3965/3/JudithReeksPhDThesis.pdf
c4c5b57350746984c34e8993164197df
MD5
3
LICENSE
license.txt
license.txt
text/plain
2224
https://research-repository.st-andrews.ac.uk/bitstream/10023/3965/2/license.txt
640efec0e2404cd505c2580e38ee6d69
MD5
2
TEXT
JudithReeksPhDThesis.pdf.txt
JudithReeksPhDThesis.pdf.txt
Extracted text
text/plain
820402
https://research-repository.st-andrews.ac.uk/bitstream/10023/3965/4/JudithReeksPhDThesis.pdf.txt
21303cbd274041c9eaed03492f5f8d82
MD5
4
10023/3965
oai:research-repository.st-andrews.ac.uk:10023/3965
2019-03-29 11:19:48.086
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/103592017-02-24T10:28:06Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Gloster, Tracey
Griffin, Laura Susan
Biotechnology and Biological Sciences Research Council (BBSRC)
278 p.
2017-02-24T10:07:18Z
2017-02-24T10:07:18Z
2017-06-21
http://hdl.handle.net/10023/10359
en
University of St Andrews
The University of St Andrews
Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/
2019-02-03
Thesis restricted in accordance with University regulations. Print and electronic copy restricted until 3rd February 2019
Heparan sulfate
Sulfatases
QP609.S95G8
Sulfatases
Structure and function of heparan sulfate degrading sulfatases
Thesis
Doctoral
PhD Doctor of Philosophy
LICENSE
license.txt
license.txt
text/plain
2188
https://research-repository.st-andrews.ac.uk/bitstream/10023/10359/2/license.txt
a10becd7ef5358e42635fd6aa9fc69b6
MD5
2
CC-LICENSE
license_rdf
license_rdf
application/octet-stream
1223
https://research-repository.st-andrews.ac.uk/bitstream/10023/10359/1/license_rdf
7c9ab7f006165862d8ce9ac5eac01552
MD5
1
10023/10359
oai:research-repository.st-andrews.ac.uk:10023/10359
2017-02-24 10:28:06.034
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
U3QgQW5kcmV3cyBSZXNlYXJjaCBSZXBvc2l0b3J5IC0gREVQT1NJVCBBR1JFRU1FTlQKCkNPVkVSRUQgV09SSwoKSSB3b3VsZCBsaWtlIHRvIGRlcG9zaXQgbXkgbWF0ZXJpYWwgaW4gdGhlIFN0IEFuZHJld3MgUmVzZWFyY2ggUmVwb3NpdG9yeS4gUmVzZWFyY2ggcmVmZXJyZWQgdG8gYmVsb3cgYXMgIldvcmsiIGlzIGNvdmVyZWQgYnkgdGhpcyBhZ3JlZW1lbnQgYW5kIHdoZW4gSSBkZXBvc2l0IG15IFdvcmsgaW4gdGhlIGZ1dHVyZSwgd2hldGhlciBwZXJzb25hbGx5IG9yIHRocm91Z2ggYW4gYXNzaXN0YW50IG9yIG90aGVyIGFnZW50LCBJIGFncmVlIHRvIHRoZSBmb2xsb3dpbmc6CgpOT04tRVhDTFVTSVZFIFJJR0hUUwoKUmlnaHRzIGdyYW50ZWQgdG8gdGhlIGRpZ2l0YWwgcmVwb3NpdG9yeSB0aHJvdWdoIHRoaXMgYWdyZWVtZW50IGFyZSBlbnRpcmVseSBub24tZXhjbHVzaXZlLgpJIGFtIGZyZWUgdG8gcHVibGlzaCB0aGUgV29yayBpbiBpdHMgcHJlc2VudCB2ZXJzaW9uIG9yIGZ1dHVyZSB2ZXJzaW9ucyBlbHNld2hlcmUuIEkgYWdyZWUgdGhhdCB0aGUgVW5pdmVyc2l0eSBvZiBTdCBBbmRyZXdzIG1heSBlbGVjdHJvbmljYWxseSBzdG9yZSwgY29weSBvciB0cmFuc2xhdGUgdGhlIFdvcmsgdG8gYW55IG1lZGl1bSBvciBmb3JtYXQgZm9yIHRoZSBwdXJwb3NlIG9mIGZ1dHVyZSBwcmVzZXJ2YXRpb24gYW5kIGFjY2Vzc2liaWxpdHkuIFRoZSBVbml2ZXJzaXR5IG9mIFN0IEFuZHJld3MgaXMgbm90IHVuZGVyIGFueSBvYmxpZ2F0aW9uIHRvIHJlcHJvZHVjZSBvciBkaXNwbGF5IHRoZSBXb3JrIGluIHRoZSBzYW1lIGZvcm1hdHMgb3IgcmVzb2x1dGlvbnMgaW4gd2hpY2ggaXQgd2FzIG9yaWdpbmFsbHkgZGVwb3NpdGVkLgoKREVQT1NJVCBJTiBTdCBBbmRyZXdzIFJlc2VhcmNoIFJlcG9zaXRvcnkKCkkgdW5kZXJzdGFuZCB0aGF0IHdvcmsgZGVwb3NpdGVkIGluIHRoZSBkaWdpdGFsIHJlcG9zaXRvcnkgd2lsbCBiZSBhY2Nlc3NpYmxlIHRvIGEgd2lkZSB2YXJpZXR5IG9mIHBlb3BsZSBhbmQgaW5zdGl0dXRpb25zIC0gaW5jbHVkaW5nIGF1dG9tYXRlZCBhZ2VudHMgLSB2aWEgdGhlIFdvcmxkIFdpZGUgV2ViLgpBbiBlbGVjdHJvbmljIGNvcHkgb2YgbXkgdGhlc2lzIG1heSBhbHNvIGJlIGluY2x1ZGVkIGluIHRoZSBCcml0aXNoIExpYnJhcnkgRWxlY3Ryb25pYyBUaGVzZXMgT24tbGluZSBTeXN0ZW0gKEVUaE9TKS4KCkkgdW5kZXJzdGFuZCB0aGF0IG9uY2UgdGhlIFdvcmsgaXMgZGVwb3NpdGVkLCBtZXRhZGF0YSB3aWxsIGJlIGluY29ycG9yYXRlZCBpbnRvIHB1YmxpYyBhY2Nlc3MgY2F0YWxvZ3VlcyBhbmQgYSBjaXRhdGlvbiB0byB0aGUgV29yayB3aWxsIGFsd2F5cyByZW1haW4gdmlzaWJsZSwgYWx0aG91Z2ggdGhlIGF1dGhvciByZXRhaW5zIHRoZSByaWdodCB0byB1cGRhdGUgdGhlIFdvcmsuIFJlbW92YWwgb2YgdGhlIGl0ZW0gY2FuIGJlIG1hZGUgYWZ0ZXIgZGlzY3Vzc2lvbiB3aXRoIHRoZSBkaWdpdGFsIHJlcG9zaXRvcnkgYWRtaW5pc3RyYXRvcnMuCgoKSSBBR1JFRSBBUyBGT0xMT1dTOgoKLSBUaGF0IEkgaGF2ZSB0aGUgYXV0aG9yaXR5IG9mIHRoZSBhdXRob3JzIHRvIG1ha2UgdGhpcyBhZ3JlZW1lbnQsIGFuZCB0byBoZXJlYnkgZ2l2ZSB0aGUgVW5pdmVyc2l0eSBvZiBTdCBBbmRyZXdzIHRoZSByaWdodCB0byBtYWtlIGF2YWlsYWJsZSB0aGUgV29yayBpbiB0aGUgd2F5IGRlc2NyaWJlZCBhYm92ZS4KCi0gVGhhdCBJIGhhdmUgZXhlcmNpc2VkIHJlYXNvbmFibGUgY2FyZSB0byBlbnN1cmUgdGhhdCB0aGUgV29yayBpcyBvcmlnaW5hbCwgYW5kIGRvZXMgbm90IHRvIHRoZSBiZXN0IG9mIG15IGtub3dsZWRnZSBicmVhayBhbnkgVUsgbGF3IG9yIGluZnJpbmdlIGFueSB0aGlyZCBwYXJ0eSdzIGNvcHlyaWdodCBvciBvdGhlciBJbnRlbGxlY3R1YWwgUHJvcGVydHkgUmlnaHRzLgoKLSBTdCBBbmRyZXdzIHJlcG9zaXRvcnkgYWRtaW5pc3RyYXRvcnMgZG8gbm90IGhvbGQgYW55IG9ibGlnYXRpb24gdG8gdGFrZSBsZWdhbCBhY3Rpb24gb24gYmVoYWxmIG9mIHRoZSBEZXBvc2l0b3IsIG9yIG90aGVyIHJpZ2h0cyBob2xkZXJzLCBpbiB0aGUgZXZlbnQgb2YgYnJlYWNoIG9mIGludGVsbGVjdHVhbCBwcm9wZXJ0eSByaWdodHMsIG9yIGFueSBvdGhlciByaWdodCwgaW4gdGhlIG1hdGVyaWFsIGRlcG9zaXRlZC4KCg==
oai:research-repository.st-andrews.ac.uk:10023/63192019-03-29T10:30:05Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Jackson, David
Meyer, Bjoern
Medical Research Council (MRC)
vi, 317 p.
2015-03-25T12:48:57Z
2015-03-25T12:48:57Z
2014
http://hdl.handle.net/10023/6319
Arenaviruses cause an estimated 300,000 – 500,000 infections annually. Currently there is no arenavirus-specific antiviral drug available to treat these infections. This study sought to use the non-pathogenic New World arenavirus Tacaribe virus (TCRV) as a model for the pathogenic Junin virus (JUNV) and Machupo virus (MACV) that cause haemorrhagic fevers in South America. TCRV was used to explore three different approaches in the search for an antiviral drug against arenavirus infection targeted specifically against the viral nucleoprotein (NP). Of the four expressed arenaviral proteins, NP is the most abundant and is thought to be of multifunctional nature involved in viral replication, suppression of the innate immune system and viral egress. The approaches to find targets for broad-spectrum anti-arenaviral drugs were high throughput screens (HTS) with purified NP using thermal shift assays, exploring the virus interactions with the innate immune system and identifying virus-host protein-protein interactions. HTS resulted in the identification of two small-molecule compounds, [5-(2-Furyl)thien-2-yl]methanol and cyclosporine A (CsA), showing broad-spectrum activity against arenaviruses. Interferon-stimulated genes (ISGs), such as IFIT3, were identified to reduce viral titres and potential 202 protein-interactions between NP and host cell proteins were identified, of which the interaction with apoptosis-inducing factor 1 (AIF1) was described further. To characterise the importance of these interactions as potential drug targets further, a TCRV reverse genetics system was constructed.
en
University of St Andrews
The University of St Andrews
Virology
Virus
Arenavirus
Tacaribe virus
Innate immunity
High throughput screen
Reverse genetics
Interferon
Protein-protein interaction
QR201.A74M4
Arenaviruses
Viral proteins
Nucleoproteins
Protein-protein interactions
Antiviral agents
Molecular studies of the Tacaribe virus nucleoprotein (NP): identification and characterisation of virus-host interactions as novel anti-arenavirus drug targets
Thesis
Doctoral
PhD Doctor of Philosophy
THUMBNAIL
BjoernMeyerPhDThesis.pdf.jpg
BjoernMeyerPhDThesis.pdf.jpg
Generated Thumbnail
image/jpeg
3954
https://research-repository.st-andrews.ac.uk/bitstream/10023/6319/5/BjoernMeyerPhDThesis.pdf.jpg
e471f6fa3d58c4fd6a78d57b857f308d
MD5
5
LICENSE
license.txt
license.txt
text/plain
2224
https://research-repository.st-andrews.ac.uk/bitstream/10023/6319/2/license.txt
640efec0e2404cd505c2580e38ee6d69
MD5
2
ORIGINAL
BjoernMeyerPhDThesis.pdf
BjoernMeyerPhDThesis.pdf
application/pdf
66877239
https://research-repository.st-andrews.ac.uk/bitstream/10023/6319/3/BjoernMeyerPhDThesis.pdf
4bba31b49191fe1eaae8db720aa4c60d
MD5
3
TEXT
BjoernMeyerPhDThesis.pdf.txt
BjoernMeyerPhDThesis.pdf.txt
Extracted text
text/plain
672282
https://research-repository.st-andrews.ac.uk/bitstream/10023/6319/4/BjoernMeyerPhDThesis.pdf.txt
660be5b7618f5518c08449160bffe81c
MD5
4
10023/6319
oai:research-repository.st-andrews.ac.uk:10023/6319
2019-03-29 10:30:05.192
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/8812019-07-01T10:12:46Zcom_10023_175com_10023_39col_10023_177
Coote, Peter John
Harris, Mark R.
Biotechnology and Biological Sciences Research Council (BBSRC)
266
2010-05-26T08:25:22Z
2010-05-26T08:25:22Z
2010-06-23
http://hdl.handle.net/10023/881
Electronic version does not contain associated previously published material
Antimicrobial peptides (AMPs) are found throughout the animal kingdom and act as a natural defence against a broad spectrum of pathogens. These peptides are toxic to invading organisms without acting on host cells, so are of interest for their potential to act as potent new drugs against pathogenic organisms. AMPs traverse the cell wall and predominantly target the plasma membrane, resulting in destabilisation, leakage of intracellular components and cell death. In this thesis the mode of action of several AMPs was investigated. The role of the cell wall was studied and found to mediate peptide binding, the inhibition of certain cell wall components also increased peptide action, subsequent internalisation events were observed with varying localisation patterns and the effect of several genes that alter cell susceptibility to AMP were examined.
Several Candida albicans mutants, each deficient in cell wall protein mannosylation, were tested in relation to their susceptibility to AMPs. It was discovered that cells lacking or deficient in the phosphomannan fraction, with a concomitant reduction in surface negative charge, correlated with reduced susceptibility to AMP action. To ascertain whether peptide binds to negatively charged phosphate, the effect of exogenous glucosamine 6-phosphate (but not glucosamine hydrochloride) was studied demonstrating that peptide efficacy was reduced due to the presence of exogenous phosphate. More specifically, sequestration of the truncated cationic AMP dermaseptin S3 (DsS3(1-16)) was reduced in these phosphomannan deficient mutants. Microscopy analysis of fluorescein tagged DsS3(1-16) also revealed the differential localisation patterns of this AMP: transiently binding to the plasma membrane, localisation to the vacuole or diffuse distribution throughout the cytoplasm. It is proposed that for these cationic AMPs to exert their full antifungal action they must first bind to the negatively charged phosphate.
The echinocandins are a relatively new class of antifungal that function by inhibiting 1,3-β glucan synthase resulting in reduced 1,3-β glucan in the cell wall. As AMPs have to traverse the cell wall it was postulated that cells lacking this fraction would display increased AMP binding to the membrane. Clinical isolate strains of Candida and Cryptococcus spp. were acquired to test their susceptibility to AMP and echinocandin combinations. Comparing the fractional inhibitory concentration index (FICI) (supported by viable cell counts and on a solid surface using disc diffusion assays) synergy was observed between caspofungin, anidulafungin and several AMPs in vitro. In vitro toxicity assays revealed no increase in haemolytic or cytotoxic action on combination. These synergistic combinations could provide a novel treatment against fungal pathogens.
The final area of study was based upon work that identified genes whose expression altered cell susceptibility to AMPs. Three genes were selected for investigation that upon deletion increased the action of DsS3(1-16) or magainin 2 on S. cerevisiae. Results from growth analysis, peptide sequestration and cell viability counts confirmed that deletion of HAL5, LDB7 or IMP2’ did increase susceptibility. Additionally, deletion of HAL5 increased the probability of cell depolarisation upon peptide exposure. Expression of GFP-tagged Imp2’ also increased when cells were exposed to DsS3(1-16). It was concluded that deletion of HAL5 increases depolarisation due to insufficient potassium efflux, leading to ion leakage and cell death facilitated by AMP action. Double strand break repair and DNA protection are probably compromised upon deletion of LDB7 and IMP2’, increasing the inhibitory action of DsS3(1-16) that has previously been shown to bind to DNA.
en
University of St Andrews
The University of St Andrews
Creative Commons Attribution 3.0 Unported
http://creativecommons.org/licenses/by/3.0/
Antimicrobial peptide
Echinocandin
Candida
Saccharomyces
RS431.P37H28
Candida
Saccharomyces
Peptide antibiotics
Antifungal agents--Testing
Effects of cationic antimicrobial peptides on Candida and Saccharomyces species
Thesis
Doctoral
PhD Doctor of Philosophy
THUMBNAIL
Mark R Harris PhD thesis.PDF.jpg
Mark R Harris PhD thesis.PDF.jpg
Generated Thumbnail
image/jpeg
3767
https://research-repository.st-andrews.ac.uk/bitstream/10023/881/8/Mark%20R%20Harris%20PhD%20thesis.PDF.jpg
0ca32ab1e331ef56e93f0dde637415d7
MD5
8
ORIGINAL
Mark R Harris PhD thesis.PDF
Mark R Harris PhD thesis.PDF
application/pdf
31883983
https://research-repository.st-andrews.ac.uk/bitstream/10023/881/6/Mark%20R%20Harris%20PhD%20thesis.PDF
84f70a57f303b8ffdada96a5b6ddfd3e
MD5
6
CC-LICENSE
license_url
license_url
text/plain
43
https://research-repository.st-andrews.ac.uk/bitstream/10023/881/2/license_url
5134edb0a80711cbbaa5247c36025151
MD5
2
license_text
license_text
application/octet-stream
19479
https://research-repository.st-andrews.ac.uk/bitstream/10023/881/3/license_text
b283acf571e1ada8a2fa2d9f20807987
MD5
3
license_rdf
license_rdf
application/octet-stream
24437
https://research-repository.st-andrews.ac.uk/bitstream/10023/881/4/license_rdf
126f0814f0f28c509646a12115b4829a
MD5
4
LICENSE
license.txt
license.txt
text/plain
2905
https://research-repository.st-andrews.ac.uk/bitstream/10023/881/5/license.txt
18a17056539dc62c564abf09ab5710e9
MD5
5
TEXT
Mark R Harris PhD thesis.PDF.txt
Mark R Harris PhD thesis.PDF.txt
Extracted text
text/plain
321394
https://research-repository.st-andrews.ac.uk/bitstream/10023/881/7/Mark%20R%20Harris%20PhD%20thesis.PDF.txt
3115f0dd3988cf91ef639bf1e384b35a
MD5
7
10023/881
oai:research-repository.st-andrews.ac.uk:10023/881
2019-07-01 11:12:46.338
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/169832019-07-01T10:15:15Zcom_10023_175com_10023_39com_10023_51com_10023_18col_10023_177col_10023_53
Smith, Terry K.
Roberts, Matthew D.
Wellcome Trust
xxxi, 331 p.
2019-02-01T10:16:43Z
2019-02-01T10:16:43Z
2017-06-21
http://hdl.handle.net/10023/16983
This work expands the knowledge on phospholipid metabolism in the kinetoplastid
parasites: T. brucei, T. cruzi, Leishmania spp. that cause neglected tropical diseases
and the related non-human pathogenic Crithidia fasiculata.
As a close relative of parasitic kinetoplasts, specifically Leishmania, it is hypothesised
that Crithidia fasiculata possesses a similar lipid biosynthetic capability and therefore
represent an attractive model organism. Database mining the Crithidia genome
revealed the ability to biosynthesise all of the main phospholipid species. Utilising
various lipidomic techniques, a high level of an ω-6 18:3 fatty acid was observed,
alongside an uncommon Δ19:0 fatty acid that was later identified to be exclusive
attributed to PE species. Sphingolipid metabolism was shown to resemble that of
Leishmania and T. cruzi, given the exclusive production of inositol-phosphoceramide
species and no sphingomyelin species being observed. Using labelled precursors,
Crithidia were seen to uptake and incorporate extracellular inositol into both
phosphatidylinositol and inositol-phosphoceramide species. Crithidia were also
shown to utilise both the Kennedy pathway and methylation of
phosphatidylethanolamine to form phosphatidylcholine.
The phospholipidome of T. cruzi revealed several phosphatidylserine species for the
first time, suggesting a functional phosphatidylserine synthase. Current knowledge
of T.cruzi sphingolipid biosynthesis was also confirmed as only inositol
xxxi
phosphoceramide species were observed. The identification and subsequent
characterisation of novel phosphonolipid species are reported for the first time.
Utilising lipidomic methodologies and labelled precursors, the relative contribution
of the intracellular inositol pools within bloodstream and procyclic T. brucei towards
PI biosynthesis was examined. This highlighted that the synthesis/turnover rates for
specific phosphatidylinositol and inositol-phosphoceramide species are unequal.
Efforts to optimise media conditions highlighted that under reduced levels of
serum/glucose/inositol, bloodstream T. brucei unexpectedly adjusts its inositol
metabolism. The procyclic parasite exemplifies this fact, as under inositol/glucose
deficient media conditions they appear to have adapted to utilising glucogenesis and
inositol de-novo synthesis.
This work highlights that these parasites are rapidly dividing, their unique features of
lipid metabolism may be exploitable for drug discovery purposes.
en
University of St Andrews
The University of St Andrews
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/
2020-04-05
Thesis restricted in accordance with University regulations. Electronic copy restricted until 5th April 2020
Lipidomic
Mass spectrometry
Typanosoma brucei
Leishmania
Crithidia fasiculata
Phospholipid
Fatty acid
Phosphonolipid
Cyclopropyl fatty acid
Kinetoplastid
Parasite
Model organism
Metabolism
Lipid
Typanosoma cruzi
QP752.P53R7
Phospholipids--Metabolism
Trypanosomatidae
Trypanosoma brucei
Trypanosoma cruzi
Lipidomic investigations into the phospholipid content and metabolism of various kinetoplastids
Thesis
Doctoral
PhD Doctor of Philosophy
CC-LICENSE
license_rdf
license_rdf
application/octet-stream
1223
https://research-repository.st-andrews.ac.uk/bitstream/10023/16983/1/license_rdf
7c9ab7f006165862d8ce9ac5eac01552
MD5
1
LICENSE
license.txt
license.txt
text/plain
2188
https://research-repository.st-andrews.ac.uk/bitstream/10023/16983/2/license.txt
a10becd7ef5358e42635fd6aa9fc69b6
MD5
2
10023/16983
oai:research-repository.st-andrews.ac.uk:10023/16983
2019-07-01 11:15:15.734
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/122732017-12-06T14:36:30Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Taylor, Garry L.
Jameison, Stephen A.
Biotechnology and Biological Sciences Research Council (BBSRC)
University of St Andrews. School of Biology
xxxix, 552 p.
2017-12-06T14:04:53Z
2017-12-06T14:04:53Z
2017-12-07
http://hdl.handle.net/10023/12273
en
University of St Andrews
The University of St Andrews
Biomedical Sciences Research Complex (BSRC)
QR460.J2
Title redacted
Thesis
Doctoral
PhD Doctor of Philosophy
2019-11-22
Thesis restricted in accordance with University regulations. Print and electronic copy restricted until 22nd November 2019
LICENSE
license.txt
license.txt
text/plain
2188
https://research-repository.st-andrews.ac.uk/bitstream/10023/12273/1/license.txt
a10becd7ef5358e42635fd6aa9fc69b6
MD5
1
10023/12273
oai:research-repository.st-andrews.ac.uk:10023/12273
2017-12-06 14:36:30.773
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/4682019-07-01T10:18:35Zcom_10023_175com_10023_39col_10023_177
White, Malcolm F.
Taylor, Garry L.
Mackay, Dale Tara
Biotechnology and Biological Sciences Research Council (BBSRC)
209
2008-04-23T14:13:19Z
2008-04-23T14:13:19Z
2008-06-27
http://hdl.handle.net/10023/468
Compaction of DNA into chromatin is an important feature of every living cell. This compaction phenomenon is brought about and maintained by a variety of DNA binding proteins, which have evolved to suit the specific needs of the different cell types spanning the three kingdoms of life; the eukaryotes, prokaryotes and archaea. Sulfolobus solfataricus, a member of the crenarchaeal subdivision of the archaea, has two prominent DNA binding proteins known as Alba (1&2) and Sso7d. Alba1 is acetylated in vivo at two positions and this modification lowers its’ affinity for binding DNA. Acetylation levels impact many cellular processes and in higher organisms play a critical role in the development of many cancers and other diseases.
This thesis documents the finding and characterisation of the N-terminal acetyltransferase (ssArd1) of SsoAlba1, based on its’ sequence homology to the catalytic subunits Ard1, Nat3 and Mak3 belonging to the larger eukaryal Nat complexes NatA, NatB and NatC, respectively. Mutagenesis studies revealed that ssArd1 preferentially acetylates N-termini bearing a serine or alanine residue at position 1 (after methionine cleavage). It is also capable of acetylating other proteins with very different physical structures. These findings allow classification of ssArd1 as a promiscuous acetyltransferase belonging to the Gcn5-N-acetyltransferase (GNAT) superfamily. The active site of the enzyme was examined through mutagenesis studies, revealing that the mechanism of acetylation is likely to proceed through a direct acetyl transfer involving a tetrahedral intermediate. Structural studies provided some insight into the molecular structure of ssArd1.
11908437 bytes
application/pdf
en
University of St Andrews
The University of St Andrews
Creative Commons Attribution 3.0 Unported
http://creativecommons.org/licenses/by/3.0/
Sulfolobus
Acetylation
QR82.A69M6
Acetyltransferases
Archaebacteria
Thermophilic bacteria
Sulfur bacteria
Characterisation of Sulfolobus solfataricus Ard1, a promiscuous N-acetyltransferase
Thesis
Doctoral
PhD Doctor of Philosophy
THUMBNAIL
DT Mackay thesis.pdf.jpg
DT Mackay thesis.pdf.jpg
Generated Thumbnail
image/jpeg
3784
https://research-repository.st-andrews.ac.uk/bitstream/10023/468/8/DT%20Mackay%20thesis.pdf.jpg
fb7f30770a628631f9c56e7a8738b0ad
MD5
8
CC-LICENSE
license_url
license_url
text/plain
43
https://research-repository.st-andrews.ac.uk/bitstream/10023/468/2/license_url
5134edb0a80711cbbaa5247c36025151
MD5
2
license_text
license_text
application/octet-stream
11921
https://research-repository.st-andrews.ac.uk/bitstream/10023/468/3/license_text
cc99368e9cd6bba1f0e066652c679c15
MD5
3
license_rdf
license_rdf
application/octet-stream
11682
https://research-repository.st-andrews.ac.uk/bitstream/10023/468/4/license_rdf
639a46cae0c97a2b05fbeb6ae0d1a818
MD5
4
LICENSE
license.txt
license.txt
text/plain
2235
https://research-repository.st-andrews.ac.uk/bitstream/10023/468/5/license.txt
9063452bad17e5a68b1bf2a46cf9b5aa
MD5
5
ORIGINAL
DT Mackay thesis.pdf
DT Mackay thesis.pdf
application/pdf
11908437
https://research-repository.st-andrews.ac.uk/bitstream/10023/468/6/DT%20Mackay%20thesis.pdf
525cf7a0d948e7f940dc01a963cf12ab
MD5
6
TEXT
DT Mackay thesis.pdf.txt
DT Mackay thesis.pdf.txt
Extracted text
text/plain
340005
https://research-repository.st-andrews.ac.uk/bitstream/10023/468/7/DT%20Mackay%20thesis.pdf.txt
7b7ae5088e0fc1ab72ba327a6128b2c2
MD5
7
10023/468
oai:research-repository.st-andrews.ac.uk:10023/468
2019-07-01 11:18:35.174
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/44222019-07-01T10:19:02Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
MacNeill, Stuart
Schnick, Jasmin
xi, 267 p.
2014-01-28T08:40:10Z
2014-01-28T08:40:10Z
2014-06
uk.bl.ethos.588948
http://hdl.handle.net/10023/4422
The MCM helicase plays an important role in eukaryotic DNA replication, unwinding double stranded DNA ahead of the replication fork. MCM is a hetero-hexamer consisting of the six related proteins, Mcm2-Mcm7. The distantly related MCM-binding protein (MCM-BP) was first identified in a screen for proteins interacting with MCM2-7 in human cells and was found to specifically interact with Mcm3-7 but not Mcm2. It is conserved in most eukaryotes and seems to play an important role in DNA replication but its exact function is not clear yet.
This study contributes to the understanding of the fission yeast homologue of MCM-BP, named Mcb1, but also of MCM-BP in general. Results presented in this thesis document the initial biochemical characterisation of the complex Mcb1 forms with Mcm proteins, the MCMᴹᶜᵇ¹ complex. Interactions of Mcb1 with Mcm proteins, potential interaction sites between the proteins and the size of the complex were analysed using a variety of methods, including tandem affinity purification, co-immunoprecipitation, sucrose gradients and in vitro pull-down assays. Sequence analysis and structure prediction were utilised to gain some insight into Mcb1 and MCM-BP ancestry and structure. Results presented here indicate that fission yeast Mcb1 shares homology with Mcm proteins and forms a complex with Mcm3-Mcm7 but not Mcm2 and thus replaces the latter in an alternative high molecular weight complex that is likely to have an MCM-like appearance.
Deletion of mcb1⁺ showed that Mcb1 is essential in fission yeast. To examine the cellular function of the protein, temperature-sensitive mutants were generated. Inactivation of Mcb1 leads to an increase in DNA damage and cell cycle arrest in G2-phase depending on the activation of the Chk1 dependent DNA damage checkpoint. Similar observations were made when Mcb1 was overexpressed, indicating that certain levels of the protein are important for accurate DNA replication. Construction of truncated versions of Mcb1 suggested that almost the full-length protein is needed for proper function.
en
University of St Andrews
The University of St Andrews
Creative Commons Attribution-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-sa/4.0/
DNA replication
Minichromosome maintenance binding protein
Schizosaccharomyces pombe
Mcm2-7 complex
Protein interaction
DNA damage checkpoint
Temperature-sensitive mutants
QP616.D54S6
DNA helicases
DNA replication
Structural and functional characterisation of Mcb1 and the MCMᴹᶜᵇ¹ complex in Schizosaccharomyces pombe
Thesis
Doctoral
PhD Doctor of Philosophy
Schizosaccharomyces pombe
THUMBNAIL
JasminSchnickPhDThesis.pdf.jpg
JasminSchnickPhDThesis.pdf.jpg
Generated Thumbnail
image/jpeg
3969
https://research-repository.st-andrews.ac.uk/bitstream/10023/4422/8/JasminSchnickPhDThesis.pdf.jpg
bdcfdd9a54f8144f7b8a1d32f72bad13
MD5
8
ORIGINAL
JasminSchnickPhDThesis.pdf
JasminSchnickPhDThesis.pdf
application/pdf
108162901
https://research-repository.st-andrews.ac.uk/bitstream/10023/4422/6/JasminSchnickPhDThesis.pdf
f492ec87ebd095db6808a01a8d86f484
MD5
6
CC-LICENSE
license_text
license_text
application/octet-stream
20896
https://research-repository.st-andrews.ac.uk/bitstream/10023/4422/3/license_text
531c023614f8d3178286f9e9f12bf880
MD5
3
license_rdf
license_rdf
application/octet-stream
21889
https://research-repository.st-andrews.ac.uk/bitstream/10023/4422/4/license_rdf
5f21d45308ffc58e8d263280cb61c64d
MD5
4
license_url
license_url
text/plain
46
https://research-repository.st-andrews.ac.uk/bitstream/10023/4422/2/license_url
6f1da3ff281999354d4abd56d1551468
MD5
2
LICENSE
license.txt
license.txt
text/plain
2224
https://research-repository.st-andrews.ac.uk/bitstream/10023/4422/5/license.txt
640efec0e2404cd505c2580e38ee6d69
MD5
5
TEXT
JasminSchnickPhDThesis.pdf.txt
JasminSchnickPhDThesis.pdf.txt
Extracted text
text/plain
552839
https://research-repository.st-andrews.ac.uk/bitstream/10023/4422/7/JasminSchnickPhDThesis.pdf.txt
fa596885fb3dc44de1976ce45b85ae71
MD5
7
10023/4422
oai:research-repository.st-andrews.ac.uk:10023/4422
2019-07-01 11:19:02.426
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/45062019-03-29T10:37:03Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Taylor, Garry L.
Yang, Lei
xvii, 171 p.
2014-03-11T10:53:50Z
2014-03-11T10:53:50Z
2013
uk.bl.ethos.595627
http://hdl.handle.net/10023/4506
A number of dihydroxyacetone kinases (DAK) have been described that can utilize
either ATP (in animals, plants and some bacteria) or phosphoenolpyruvate (PEP) (in
most bacteria) as the energy source to convert dihydroxyacetone (Dha) to
dihydroxyacetone phosphate (DhaP), which plays critical roles in many metabolic
pathways. It has also been described that Homo sapiens DAK is able to regulate the
innate immune system via its interaction with Melanoma differentiation-associated
protein 5 (MDA5), which is recognized as an RNA sensor during virus infection.
These findings make H. sapiens DAK a very noteworthy research target due to its
multiple functions in many fields. Therefore, structural studies of DAK from H.
sapiens are presented.
The initial structure of the wild type (WT) H. sapiens DAK was determined to 2.5Å
resolution and solved by molecular replacement. The structure forms a homodimer
and four dimers were shown to be present in each asymmetric unit. However, within
each monomer, most regions of the C-terminal domain were disordered, and therefore
in order to improve structure quality, multiple site-directed mutagenesis was used. The
mutated protein was then crystallized and the structure was determined to 1.4Å. The
N-terminal Dha binding domain consists of two α/β regions and the C-terminal ATP
binding domain is comprised of eight anti-parallel α-helices, which forms a deep
pocket and is filled with a phospholipid molecule. In addition, the structures of
mutated DAK in complex with ATP analogues and Dha are also described in the
current study.
The second part of the project concerned sialidases, which are glycoside hydrolases that specifically hydrolyse terminal sialic acid from various glycans. Streptococcus
pneumoniae is one of the most common pathogenic bacteria of humans, and is
reported to encode three sialidases that act as virulence factors in bacterial
colonization and infection. One of these sialidases, NanA, was reported to be present
in all clinical strains and plays a vital role during the bacterial infection.
Consequently, the structure of N-terminal Carbohydrate-binding module (CBM)
domain of NanA has been determined to 1.8Å, and reveals a β-sandwich fold. The apo
form of NanA-CBM is present as a dimer in the asymmetric unit, whereas a monomer
was detected when it is bound to sialic acid or its derivatives. Structural comparisons
between NanA-CBM and other structures of the CBM40 family were also performed.
The substrate binding sites of NanA-CBM forms a cavity that is able to accommodate
the substrates. A potential molecular binding site located beside the sialic acid binding
site was revealed, and is occupied by the side chain of a lysine from a symmetry-
related molecule. Heteronuclear single quantum coherence (HSQC) NMR
spectroscopy and fluorescent-based thermal shift assays were also carried out to
further characterise the protein.
The current results reveal the structure of both DAK from H. sapiens and NanA-CBM
from S. pneumoniae, which may contribute to a better understanding towards cell
metabolism and bacterial colonization.
en
University of St Andrews
The University of St Andrews
QP601.Y2
Enzymes--Structure
Structural studies on human dihydroxyacetone kinase (DAK) and the carbohydrate-binding domain of Streptococcus pneumoniae NanA
Thesis
Doctoral
PhD Doctor of Philosophy
THUMBNAIL
LeiYang_PhDThesis.pdf.jpg
LeiYang_PhDThesis.pdf.jpg
Generated Thumbnail
image/jpeg
3207
https://research-repository.st-andrews.ac.uk/bitstream/10023/4506/5/LeiYang_PhDThesis.pdf.jpg
f2c09281a805a26c8b7d4f000f9ca166
MD5
5
LICENSE
license.txt
license.txt
text/plain
2224
https://research-repository.st-andrews.ac.uk/bitstream/10023/4506/2/license.txt
640efec0e2404cd505c2580e38ee6d69
MD5
2
ORIGINAL
LeiYang_PhDThesis.pdf
LeiYang_PhDThesis.pdf
application/pdf
226546770
https://research-repository.st-andrews.ac.uk/bitstream/10023/4506/3/LeiYang_PhDThesis.pdf
35b6ae6636e2ac8920c0e2f9b53b5e45
MD5
3
TEXT
LeiYang_PhDThesis.pdf.txt
LeiYang_PhDThesis.pdf.txt
Extracted text
text/plain
345064
https://research-repository.st-andrews.ac.uk/bitstream/10023/4506/4/LeiYang_PhDThesis.pdf.txt
08818eceb0ed784e0ab810b3c69c0b25
MD5
4
10023/4506
oai:research-repository.st-andrews.ac.uk:10023/4506
2019-03-29 10:37:03.716
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
UmVzZWFyY2hAU3RBbmRyZXdzOkZ1bGxUZXh0IC0gREVQT1NJVCBBR1JFRU1FTlQKCkNPVkVSRUQgV09SSwoKSSB3b3VsZCBsaWtlIHRvIGRlcG9zaXQgbXkgbWF0ZXJpYWwgaW4gdGhlIFVuaXZlcnNpdHkgb2YgU3QgQW5kcmV3cyBEaWdpdGFsIFJlcG9zaXRvcnksClJlc2VhcmNoQFN0QW5kcmV3czpGdWxsVGV4dC4gUmVzZWFyY2ggcmVmZXJyZWQgdG8gYmVsb3cgYXMgIldvcmsiIGlzIGNvdmVyZWQgYnkgdGhpcwphZ3JlZW1lbnQgYW5kIHdoZW4gSSBkZXBvc2l0IG15IFdvcmsgaW4gdGhlIGZ1dHVyZSwgd2hldGhlciBwZXJzb25hbGx5IG9yIHRocm91Z2ggYW4KYXNzaXN0YW50IG9yIG90aGVyIGFnZW50LCBJIGFncmVlIHRvIHRoZSBmb2xsb3dpbmc6CgpOT04tRVhDTFVTSVZFIFJJR0hUUwoKUmlnaHRzIGdyYW50ZWQgdG8gdGhlIGRpZ2l0YWwgcmVwb3NpdG9yeSB0aHJvdWdoIHRoaXMgYWdyZWVtZW50IGFyZSBlbnRpcmVseSBub24tZXhjbHVzaXZlLgpJIGFtIGZyZWUgdG8gcHVibGlzaCB0aGUgV29yayBpbiBpdHMgcHJlc2VudCB2ZXJzaW9uIG9yIGZ1dHVyZSB2ZXJzaW9ucyBlbHNld2hlcmUuIEkgYWdyZWUKdGhhdCB0aGUgVW5pdmVyc2l0eSBvZiBTdCBBbmRyZXdzIG1heSBlbGVjdHJvbmljYWxseSBzdG9yZSwgY29weSBvciB0cmFuc2xhdGUgdGhlIFdvcmsgdG8KYW55IG1lZGl1bSBvciBmb3JtYXQgZm9yIHRoZSBwdXJwb3NlIG9mIGZ1dHVyZSBwcmVzZXJ2YXRpb24gYW5kIGFjY2Vzc2liaWxpdHkuIFRoZQpVbml2ZXJzaXR5IG9mIFN0IEFuZHJld3MgaXMgbm90IHVuZGVyIGFueSBvYmxpZ2F0aW9uIHRvIHJlcHJvZHVjZSBvciBkaXNwbGF5IHRoZSBXb3JrIGluCnRoZSBzYW1lIGZvcm1hdHMgb3IgcmVzb2x1dGlvbnMgaW4gd2hpY2ggaXQgd2FzIG9yaWdpbmFsbHkgZGVwb3NpdGVkLgoKREVQT1NJVCBJTiBSZXNlYXJjaEBTdEFuZHJld3M6RnVsbFRleHQKCkkgdW5kZXJzdGFuZCB0aGF0IHdvcmsgZGVwb3NpdGVkIGluIHRoZSBkaWdpdGFsIHJlcG9zaXRvcnkgd2lsbCBiZSBhY2Nlc3NpYmxlIHRvIGEgd2lkZQp2YXJpZXR5IG9mIHBlb3BsZSBhbmQgaW5zdGl0dXRpb25zIC0gaW5jbHVkaW5nIGF1dG9tYXRlZCBhZ2VudHMgLSB2aWEgdGhlIFdvcmxkIFdpZGUgV2ViLgpBbiBlbGVjdHJvbmljIGNvcHkgb2YgbXkgdGhlc2lzIG1heSBhbHNvIGJlIGluY2x1ZGVkIGluIHRoZSBCcml0aXNoIExpYnJhcnkgRWxlY3Ryb25pYwpUaGVzZXMgT24tbGluZSBTeXN0ZW0gKEVUaE9TKS4KCkkgdW5kZXJzdGFuZCB0aGF0IG9uY2UgdGhlIFdvcmsgaXMgZGVwb3NpdGVkLCBtZXRhZGF0YSB3aWxsIGJlIGluY29ycG9yYXRlZCBpbnRvIHB1YmxpYwphY2Nlc3MgY2F0YWxvZ3VlcyBhbmQgYSBjaXRhdGlvbiB0byB0aGUgV29yayB3aWxsIGFsd2F5cyByZW1haW4gdmlzaWJsZSwgYWx0aG91Z2ggdGhlCmF1dGhvciByZXRhaW5zIHRoZSByaWdodCB0byB1cGRhdGUgdGhlIFdvcmsuIFJlbW92YWwgb2YgdGhlIGl0ZW0gY2FuIGJlIG1hZGUgYWZ0ZXIgZGlzY3Vzc2lvbgp3aXRoIHRoZSBkaWdpdGFsIHJlcG9zaXRvcnkgYWRtaW5pc3RyYXRvcnMuCgoKSSBBR1JFRSBBUyBGT0xMT1dTOgoKLSBUaGF0IEkgaGF2ZSB0aGUgYXV0aG9yaXR5IG9mIHRoZSBhdXRob3JzIHRvIG1ha2UgdGhpcyBhZ3JlZW1lbnQsIGFuZCB0byBoZXJlYnkgZ2l2ZSB0aGUKVW5pdmVyc2l0eSBvZiBTdCBBbmRyZXdzIHRoZSByaWdodCB0byBtYWtlIGF2YWlsYWJsZSB0aGUgV29yayBpbiB0aGUgd2F5IGRlc2NyaWJlZCBhYm92ZS4KCi0gVGhhdCBJIGhhdmUgZXhlcmNpc2VkIHJlYXNvbmFibGUgY2FyZSB0byBlbnN1cmUgdGhhdCB0aGUgV29yayBpcyBvcmlnaW5hbCwgYW5kIGRvZXMgbm90IHRvCnRoZSBiZXN0IG9mIG15IGtub3dsZWRnZSBicmVhayBhbnkgVUsgbGF3IG9yIGluZnJpbmdlIGFueSB0aGlyZCBwYXJ0eSdzIGNvcHlyaWdodCBvciBvdGhlcgpJbnRlbGxlY3R1YWwgUHJvcGVydHkgUmlnaHQuCgotIFN0IEFuZHJld3MgcmVwb3NpdG9yeSBhZG1pbmlzdHJhdG9ycyBkbyBub3QgaG9sZCBhbnkgb2JsaWdhdGlvbiB0byB0YWtlIGxlZ2FsIGFjdGlvbiBvbgpiZWhhbGYgb2YgdGhlIERlcG9zaXRvciwgb3Igb3RoZXIgcmlnaHRzIGhvbGRlcnMsIGluIHRoZSBldmVudCBvZiBicmVhY2ggb2YgaW50ZWxsZWN0dWFsIHByb3BlcnR5CnJpZ2h0cywgb3IgYW55IG90aGVyIHJpZ2h0LCBpbiB0aGUgbWF0ZXJpYWwgZGVwb3NpdGVkLgoKCg==
oai:research-repository.st-andrews.ac.uk:10023/102542017-02-09T10:24:15Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Gloster, Tracey
Pengelly, Robert Joseph
Wellcome Trust
xxiii, 297 p.
2017-02-09T10:04:14Z
2017-02-09T10:04:14Z
2017-06-21
http://hdl.handle.net/10023/10254
en
University of St Andrews
The University of St Andrews
Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/
2019-02-03
Thesis restricted in accordance with University regulations. Print and electronic copy restricted until 3rd February 2019
QP606.G6P4
Glycosyltransferases
Structural and mechanistic studies of specific carbohydrate processing enzymes
Thesis
Doctoral
PhD Doctor of Philosophy
CC-LICENSE
license_rdf
license_rdf
application/octet-stream
1223
https://research-repository.st-andrews.ac.uk/bitstream/10023/10254/1/license_rdf
7c9ab7f006165862d8ce9ac5eac01552
MD5
1
LICENSE
license.txt
license.txt
text/plain
2188
https://research-repository.st-andrews.ac.uk/bitstream/10023/10254/2/license.txt
a10becd7ef5358e42635fd6aa9fc69b6
MD5
2
10023/10254
oai:research-repository.st-andrews.ac.uk:10023/10254
2017-02-09 10:24:15.627
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/32302019-03-29T10:37:43Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Elliott, Richard Michael
Mazel-Sanchez, Beryl
214
2012-10-25T15:18:57Z
2012-10-25T15:18:57Z
2012-11-30
uk.bl.ethos.558151
http://hdl.handle.net/10023/3230
Bunyamwera orthobunyavirus (BUNV) is the prototype for the family Bunyaviridae.
BUNV has a tripartite RNA genome of negative polarity composed of the large(L),medium (M)and small(S)segments. Each segment contains an open reading frame (ORF) flanked by untranslated regions (UTRs). The eleven terminal nucleotides are conserved between the three segments while the internal regions are unique. The UTRs play an important role in the virus life cycle by promoting transcription, replication and encapsidation of the viral genome.The work presented in this thesis explores UTRs plasticity and examines ways to engineere attenuated viruses by modifying only their UTRs. Using reverse genetics, mainly two ways of attenuation were explored: rescue of viruses either carrying deletions within their 3’ and/or 5’ UTRs in all three segments, or of viruses carrying one segment bearing heterologous UTRs. Both approaches resulted in virus attenuation in tissue culture, with viruses producing smaller plaques or even no plaques, and growing to lower titres than wild-type BUNV. Through serial passage, viruses were shown to
regain some level of fitness while the mutations introduced in the UTRs proved to be stable. Thus, to investigate the mechanism behind fitness recovery, the nucleotide sequence of the entire genome of viruses with deletions in their UTRs was determined. Amino acid changes were observed in the viral polymerase (L protein) of most mutant viruses and the vast majority of the amino acid changes occured in the C-terminal region. The function of this domain is unclear to date, however data obtained using a mini-replicon assay suggest that the
C-terminal domain of the L protein might be involved in UTR recognition. Full genome
sequencing also allowed the identification of an amino acid mutation within the polymerase that resulted in a temperature sensitive phenotype when introduced in an otherwise wild-type BUNV. Thus, it was shown that mutations introduced within the UTR regions of the genome were stable through serial passage and resulted in attenuation. Such a strategy could be used in
combination with mutations of the ORF to design live-attenuated vaccines against serious pathogens within the family Bunyaviridae.
en
University of St Andrews
The University of St Andrews
QR398.7M2
Bunyaviruses
Recombinant viruses
Non-coding RNA
Attenuation of bunyavirus replication by modification of genomic untranslated regions
Thesis
Doctoral
PhD Doctor of Philosophy
THUMBNAIL
BerylMazel-SanchezPhDThesis.pdf.jpg
BerylMazel-SanchezPhDThesis.pdf.jpg
Generated Thumbnail
image/jpeg
3578
https://research-repository.st-andrews.ac.uk/bitstream/10023/3230/5/BerylMazel-SanchezPhDThesis.pdf.jpg
b706037db8f7f7609fa92b5b9c0400ff
MD5
5
ORIGINAL
BerylMazel-SanchezPhDThesis.pdf
BerylMazel-SanchezPhDThesis.pdf
application/pdf
53175589
https://research-repository.st-andrews.ac.uk/bitstream/10023/3230/3/BerylMazel-SanchezPhDThesis.pdf
d603a2006498cb09ec35aef7e688caad
MD5
3
LICENSE
license.txt
license.txt
text/plain
2224
https://research-repository.st-andrews.ac.uk/bitstream/10023/3230/2/license.txt
640efec0e2404cd505c2580e38ee6d69
MD5
2
TEXT
BerylMazel-SanchezPhDThesis.pdf.txt
BerylMazel-SanchezPhDThesis.pdf.txt
Extracted text
text/plain
436754
https://research-repository.st-andrews.ac.uk/bitstream/10023/3230/4/BerylMazel-SanchezPhDThesis.pdf.txt
6c7d7fb6ab266831934ed6ff9487acda
MD5
4
10023/3230
oai:research-repository.st-andrews.ac.uk:10023/3230
2019-03-29 10:37:43.995
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/25702019-07-01T10:11:49Zcom_10023_175com_10023_39col_10023_177
Elliott, Richard Michael
Szemiel, Agnieszka M.
University of St Andrews
2012-04-24T15:41:40Z
2012-04-24T15:41:40Z
2011
http://hdl.handle.net/10023/2570
The Bunyaviridae family is one of the largest among RNA viruses, comprising more
than 350 serologically distinct viruses. The family is classified into five genera,
Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus.
Orthobunyaviruses, nairoviruses and phleboviruses are maintained in nature by a
propagative cycle involving blood-feeding arthropods and susceptible vertebrate hosts.
Like most arthropod-borne viruses, bunyavirus replication causes little damage to the
vector, whereas infection of the mammalian host may lead to death. This situation is
mimicked in the laboratory: in cultured mosquito cells no cytopathology is observed
and a persistent infection is established, whereas in cultured mammalian cells
orthobunyavirus infection is lytic and leads to cell death.
Bunyaviruses encode four common structural proteins: an RNA-dependent RNA
polymerase, two glycoproteins (Gc and Gn), and a nucleoprotein N. Some viruses also
code for nonstructural proteins called NSm and NSs. The NSs protein of the prototype
bunyavirus, Bunyamwera virus, seems to be one of the factors responsible for the
different outcomes of infection in mammalian and mosquito cell lines. However, only
limited information is available on the growth of bunyaviruses in cultured mosquito cell
lines other than Aedes albopictus C6/36 cells. Here, I compared the replication of
Bunyamwera virus in two additional Aedes albopictus cell clones, C7-10 and U4.4, and
two Aedes aegypti cell clones, Ae and A20, and investigated the impact of virus
replication on cell function. In addition, whereas the vertebrate innate immune
response to arbovirus infection is well studied, relatively little is known about
mosquitoes’ reaction to these infections. I investigated the immune responses of the
different mosquito cells to Bunyamwera virus infection, in particular antimicrobial
signaling pathways (Toll and IMD) and RNA interference (RNAi). The data obtained in
U4.4 cells suggest that NSs plays an important role in the infection of mosquitoes.
Moreover infection of U4.4 cells more closely resembles infection in Ae and A20 cells
and live Aedes aegypti mosquitoes. My data showed that the investigated cell lines
have various properties, and therefore they can be used to study different aspects of
mosquito-virus interactions.
en
University of St Andrews
The University of St Andrews
Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
http://creativecommons.org/licenses/by-nc-nd/3.0/
QR398.7S8
Bunyaviruses
Mosquitoes--Cytology
Virology--Cultures and culture media
Replication of Bunyamwera virus in mosquito cells
Thesis
Doctoral
PhD Doctor of Philosophy
THUMBNAIL
AgnieszkaSzemielPhDThesis.pdf.jpg
AgnieszkaSzemielPhDThesis.pdf.jpg
Generated Thumbnail
image/jpeg
3858
https://research-repository.st-andrews.ac.uk/bitstream/10023/2570/7/AgnieszkaSzemielPhDThesis.pdf.jpg
57c7cf01aeaa212ad3b14a304d0a5f37
MD5
7
ORIGINAL
AgnieszkaSzemielPhDThesis.pdf
AgnieszkaSzemielPhDThesis.pdf
application/pdf
126169166
https://research-repository.st-andrews.ac.uk/bitstream/10023/2570/6/AgnieszkaSzemielPhDThesis.pdf
af997b710980d99ae02595bf005162b5
MD5
6
CC-LICENSE
license_url
license_url
text/plain
49
https://research-repository.st-andrews.ac.uk/bitstream/10023/2570/2/license_url
fd26723f8d7edacdb29e3f03465c3b03
MD5
2
license_text
license_text
application/octet-stream
20904
https://research-repository.st-andrews.ac.uk/bitstream/10023/2570/3/license_text
e09bf99e64678e4285abf3ef3e05412d
MD5
3
license_rdf
license_rdf
application/octet-stream
22564
https://research-repository.st-andrews.ac.uk/bitstream/10023/2570/4/license_rdf
ff373f1ad387898ef8ba4f8bc9e37073
MD5
4
LICENSE
license.txt
license.txt
text/plain
2318
https://research-repository.st-andrews.ac.uk/bitstream/10023/2570/5/license.txt
f28e290459cb3585e032d74cfdcb2a08
MD5
5
10023/2570
oai:research-repository.st-andrews.ac.uk:10023/2570
2019-07-01 11:11:49.038
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/5712019-07-01T10:18:00Zcom_10023_175com_10023_39com_10023_51com_10023_18col_10023_177col_10023_53
Conway, Stuart J.
Murray, Ross George
sanofi-aventis
192
2008-12-02T16:26:28Z
2008-12-02T16:26:28Z
2008-06-25
uk.bl.ethos.552124
http://hdl.handle.net/10023/571
The Bucherer-Bergs reaction is a classical multi-component reaction that yields hydantoins, which can be hydrolysed to afford α-amino acids. Hydantoins have many uses in modern organic synthesis, and this moiety has been included in a number of therapeutic agents, which have a wide range of biological activities. Herein, we report a mild synthesis of 5- and 5,5-substituted hydantoins from α-aminonitriles using Hünig’s base and carbon dioxide. This reaction can be performed in excellent yields, using a variety of organic solvents and is applicable to a range of substrates. In an extension to the above methodology, a one-pot Lewis acid-catalysed synthesis of hydantoins from ketones has also been developed and optimised in organic media. This reaction can be performed in excellent yields and is suitable for the synthesis of 5- and 5,5-substituted hydantoins.
2675 bytes
application/pdf
en
University of St Andrews
The University of St Andrews
Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
http://creativecommons.org/licenses/by-nc-nd/3.0/
Hydantoin
Aminonitrile
Synthesis
Lewis acid catalysis
QD401.M8
Hydantoin--Synthesis
The synthesis of 5-substituted hydantoins
Thesis
Doctoral
PhD Doctor of Philosophy
THUMBNAIL
RossMurrayPhDThesis.pdf.jpg
RossMurrayPhDThesis.pdf.jpg
Generated Thumbnail
image/jpeg
3718
https://research-repository.st-andrews.ac.uk/bitstream/10023/571/8/RossMurrayPhDThesis.pdf.jpg
be2bedcfd7ade668d9e92d0a5a7c3b36
MD5
8
ORIGINAL
RossMurrayPhDThesis.pdf
RossMurrayPhDThesis.pdf
application/pdf
5904287
https://research-repository.st-andrews.ac.uk/bitstream/10023/571/6/RossMurrayPhDThesis.pdf
1ef228c8d2124e4cabfad5a4b5956b44
MD5
6
CC-LICENSE
license_url
license_url
text/plain
49
https://research-repository.st-andrews.ac.uk/bitstream/10023/571/2/license_url
fd26723f8d7edacdb29e3f03465c3b03
MD5
2
license_text
license_text
application/octet-stream
13525
https://research-repository.st-andrews.ac.uk/bitstream/10023/571/3/license_text
92179d1159b98a0af9b2d137dbe8d013
MD5
3
license_rdf
license_rdf
application/octet-stream
4935
https://research-repository.st-andrews.ac.uk/bitstream/10023/571/4/license_rdf
55f83bcfdf4d411736c00d6b24771e3a
MD5
4
LICENSE
license.txt
license.txt
text/plain
2227
https://research-repository.st-andrews.ac.uk/bitstream/10023/571/5/license.txt
8c3d9ff55342b336e077d22f2e9996bd
MD5
5
TEXT
RossMurrayPhDThesis.pdf.txt
RossMurrayPhDThesis.pdf.txt
Extracted text
text/plain
347627
https://research-repository.st-andrews.ac.uk/bitstream/10023/571/7/RossMurrayPhDThesis.pdf.txt
2faf4bbd292b15233ad508d29476485d
MD5
7
10023/571
oai:research-repository.st-andrews.ac.uk:10023/571
2019-07-01 11:18:00.588
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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
oai:research-repository.st-andrews.ac.uk:10023/36802019-03-29T10:41:20Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Schiemann, Olav
Sigurdsson, Snorri T.
Reginsson, Gunnar Widtfeldt
372
2013-06-13T08:43:30Z
2013-06-13T08:43:30Z
2013-06-26
uk.bl.ethos.574784
http://hdl.handle.net/10023/3680
The function of biomacromolecules is controlled by their structure and conformational flexibility. Investigating the structure of biologically important macromolecules can, therefore, yield information that could explain their complex biological function. In addition to X ray crystallography and nuclear magnetic resonance (NMR) methods, pulsed electron paramagnetic resonance (EPR) methods, in particular the pulsed electron electron double resonance (PELDOR) technique has, during the last decade, become a valuable tool for structural determination of macromolecules. Long range distance constraints obtained from pulsed EPR measurements, make it possible to carry out structural refinements on structures from NMR and X ray methods. In addition, EPR yields distance distributions that give information about structural flexibility.
The use of EPR for structural studies of biomacromolecules requires in most cases site specific incorporation of paramagnetic centres known as spin labelling. To date, spin labelling nucleic acids has required complex spin labelling chemistry. The first application of a site directed and noncovalent spin labelling method for distance measurements on DNA is described. It is demonstrated that noncovalent spin labelling with a rigid spin label can afford detailed information on internal DNA dynamics using PELDOR. Furthermore, it is shown that noncovalent spin labelling can be used to study DNA protein complexes.
PELDOR can also yield information about spin label orientation. Therefore, spin labels with limited flexibility can be used to measure the relative orientation of the spin labelled sites. Although information on orientation can be obtained from 9.7 GHz PELDOR measurements in selected applications, measurements at 97 GHz or higher, increases orientation selection. It is shown that PELDOR measurements on semi rigid and rigid nitroxide biradicals using a home built high power 97 GHz EPR spectrometer (Hiper) and model based simulations yield quantitative information on spin label orientations and dynamics.
The most widely used spin labels for EPR studies on biomacromolecules are the aminoxyl (nitroxide) radicals. The major drawbacks of nitroxide spin labels include low sensitivity for distance measurements, fast spin spin relaxation in solution and limited stability in reducing environments. Carbon centered triarylmethyl (trityl) radicals have properties that could eliminate some of the limitations of nitroxide spin labels. To evaluate the use of trityl spin labels for nanometer distance measurements, models systems with trityl and nitroxide spin labels were measured using PELDOR and Double Quantum Coherence (DQC). This study shows that trityl spin labels yield reliable information on interlabel distances and dynamics, establishing the trityl radical as a viable spin label for structural studies on biomacromolecules.
en
University of St Andrews
The University of St Andrews
University of Iceland
EPR
PELDOR
DEER
Distance measurements
Free radicals
Spin labels
Nitroxide radicals
Trityl radicals
Biomolecular structure
Nucleic acid structure
QH324.9S62R4
Biomolecules--Structure
Electron paramagnetic resonance spectroscopy
Spin labels
Nitroxides
Triarylmethyls
Distance measurements using pulsed EPR : noncovalently bound nitroxide and trityl spin labels
Thesis
Doctoral
PhD Doctor of Philosophy
THUMBNAIL
GunnarWidtfeldtReginssonPhDThesis.pdf.jpg
GunnarWidtfeldtReginssonPhDThesis.pdf.jpg
Generated Thumbnail
image/jpeg
3800
https://research-repository.st-andrews.ac.uk/bitstream/10023/3680/5/GunnarWidtfeldtReginssonPhDThesis.pdf.jpg
1d1ce160f6bec468bb24e12581c14f9e
MD5
5
ORIGINAL
GunnarWidtfeldtReginssonPhDThesis.pdf
GunnarWidtfeldtReginssonPhDThesis.pdf
application/pdf
24489064
https://research-repository.st-andrews.ac.uk/bitstream/10023/3680/3/GunnarWidtfeldtReginssonPhDThesis.pdf
ad697d1aa7eb2c6911df1cc3f8d6fe84
MD5
3
LICENSE
license.txt
license.txt
text/plain
2224
https://research-repository.st-andrews.ac.uk/bitstream/10023/3680/2/license.txt
640efec0e2404cd505c2580e38ee6d69
MD5
2
TEXT
GunnarWidtfeldtReginssonPhDThesis.pdf.txt
GunnarWidtfeldtReginssonPhDThesis.pdf.txt
Extracted text
text/plain
714563
https://research-repository.st-andrews.ac.uk/bitstream/10023/3680/4/GunnarWidtfeldtReginssonPhDThesis.pdf.txt
92481b6b6d26316112f6bd3fddfb115c
MD5
4
10023/3680
oai:research-repository.st-andrews.ac.uk:10023/3680
2019-03-29 10:41:20.225
St Andrews Research Repository
Digital-Repository@st-andrews.ac.uk
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