Dissertations / Theses: 'Fields of Research – 270000 Biological Sciences – 270100 Biochemistry and Cell Biology'

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Cooney, Terrence Patrick. "Studies on the biosynthesis of indole-3-acetic acid in tomato shoots." Thesis, University of Auckland, 1989. http://hdl.handle.net/2292/2071.

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The relative contributions of the three main intermediates of indole-3-acetic acid (IAA) biosynthesis from L-tryptophan (L-Trp); indole-3-pyruvate (IPyA), tryptamine (TNH2) and indole-3-acetaldoxime (IAOX), were investigated in vivo in tomato shoots. Initially, L-Trp, D-Trp, IPyA, TNH2 and IAA were purified from shoots, identified by full-scan mass spectrometry and their concentrations measured using gas chromatography with an electron capture detector. High specific activity [5-3H]IAOX and [5-3H]IPyA were synthesized from L-[5-3H]Trp and used as internal standards. Purification of endogenous IPyA was enabled by forming a stable pentafluorobenzyl oxime derivative in the crude plant extract. The respective endogenous concentrations of L-Trp, D-Trp, TNH2, IPyA and IAA were found to be 2,520, 103, 146.3, 5.9 and 8.5 ng g-1 f. wt. However, IAOX could not be identified as a natural constituent of tomato shoots by full-scan GC-MS. Secondly, incubation of tomato shoots for 6, 10 and 21 h in 30% 2H2O was used as a means of labelling IAA and its putative precursors in vivo. L-Trp, D-Trp, TNH2, IPyA and IAA were then extracted and purified and the 2H content measured by combined gas chromatography-mass spectrometry. These indole compounds were labelled rapidly with up to four 2H atoms. Direct comparison of the number and the amount of 2H atoms incorporated (pattern) was obtained from the mass spectral data on the common m/z 130 ion and its isotope peaks. IAA and L-Trp demonstrated an increase in 2H label with up to 17% and 21% of their molecules labelled at 10 h respectively. This was followed by a significant decrease in 2H label at 21 h to 12% for both L-Trp and IAA. This decrease in 2H label was attributed to an increase in protein catabolism, following shoot excision, resulting in the dilution of free L-Trp pool(s) with unlabelled L-Trp from which IAA is biosynthesized. This is reflected in the observed 1.6 to 1.8 fold increase of free L-Trp from 10 to 21 h. In contrast, tryptamine demonstrated a continual increase in 2H label with an average of 8, 20 and 28% of the molecules labelled at 6, 10 and 21 h respectively, suggesting that TNH2 and IAA were synthesized from separate Trp pools. In addition, the relatively slow rate at which 2H is incorporated into tryptamine would not be sufficient to account for the rate at which IAA becomes labelled. However, IPyA demonstrated a rapid increase in 2H with 22% and 37% of its molecules labelled at 6 and 10 h respectively. From the rate at which IPyA was labelled with 2H and the concentration of IPyA in tomato shoots a rate of synthesis for IPyA in tomato shoots was estimated which was sufficient to provide most of the shoot IAA requirements. Furthermore, the extent to which IAA and IPyA were labelled relative to that of total L-Trp would imply that a smaller more rapidly metabolised pool(s) of L-Trp was the precursor of these compounds. The rate and extent that D-Trp was labelled was consistently less than that of IAA precluding it as a possible precursor of IAA. These results indicate that in tomato shoots IAA is biosynthesized from a rapidly metabolized sub-pool(s) of L-trptophan predominantly via IPyA.

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Snowden, Kimberley Cathryn. "The molecular response of wheat roots to aluminium stress." Thesis, University of Auckland, 1994. http://hdl.handle.net/2292/1967.

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Aluminium (Al) toxicity to plants is a significant problem, limiting agricultural production in up to 40% of the world's arable soils. In spite of a large amount of research, there is still no consensus on the physiological mechanisms of Al toxicity in plants. In addition, very little is known about the molecular response of plants to Al stress. This body of research was aimed at identifying the changes in gene expression that occurred in the root tips of plants that had been stressed with Al. A cDNA library made from the root tips of Al-treated wheat (Triticum aestivum L., cultivar Warigal) plants was differentially screened to identify clones whose expression was induced by Al stress. Seven cDNA clones, representing five different genes were identified as being induced in the presence of Al. Initial sequencing and northern analysis revealed that none of the clones isolated were full-length, and that some contained multiple cloning adaptors at their 5' ends. A new cDNA library was then constructed from the root tips of Al-treated Warigal plants, and homologues to each of the original five genes were isolated. These five clones were named wali1 to wali5 (for wheat aluminium induced). Northern analysis showed that wali1, -3 and -5 were induced 24 to 96 h after Al treatment, and their expression declined when the Al was removed. wali4 had a similar pattern of expression with a transient increase in expression also observed after 0.5 h of Al stress. Each of these four genes was induced by inhibitory concentrations of Al in two wheat cultivars - Warigal, an Al-sensitive cultivar, and Waalt, an Al-tolerant cultivar, - and also in two inbred lines of wheat, RR (Al-tolerant) and SS (Al-sensitive). The fifth gene (wali2) had a bimodal pattern of induction, and was induced by Al only in the Al-sensitive Warigal and the Al-tolerant RR. The nucleotide sequence of each of the wali clones was determined, and the databases were searched for homologous sequences. Wali1 was found to be homologous to a group of metallothionein-like proteins (MLPs) from plants, and wali4 was homologous to phenylalanine ammonia-lyase (PAL). wali3 and wali5 encode related, cysteine-rich proteins with homology to Bowman-Birk proteinase inhibitors, and wali2 encodes a novel protein with a repeating motif of cysteine amino acids. The induction of the wali genes was investigated in response to a number of other stresses through northern analysis. The expression of wali1, -3, -4 and -5 was induced in root tips of wheat after 2 d treatments with toxic levels of all other metals tested (Cd, Fe, Zn, Cu, Ga, In and La). The expression levels of wali1, -3, -4, and -5 also increased in the root tips of plants grown in the presence of low levels of Ca (10μM). The transcript levels of wali1, -3 and -5 increased in wounded leaf and root tissue, whereas the transcript levels of wali4 increased only in wounded leaves. The expression of wali2 was greatly reduced by low concentrations of Ca, and showed no induction, or a variable response with most of the other treatments. The site of expression of wali1, -2, -3 and -5 in root tips (and wali1 also in leaf tissue) was identified using in situ hybridisation. Wali1 was expressed predominantly in the meristematic tissue of the root tip, while wali3 and wali5 were expressed predominantly in the cortical tissue of the root. wali2 expression was detected primarily in the epidermis and root cap. Some changes in the site of expression of these genes were evident in the roots of Al-treated plants. In leaf tissue, wali1 expression was found in the mesophyl1 layer of cells. The coding sequences for wali1,-2,-3 and -5 were each cloned into the bacterial expression vector pGEX-2T. The resultant fusion proteins between glutathione S-transferase (GST) and the walis were then successfully purified from E coli. Antibodies were made to the wali1-GST fusion protein and purified by immunoaffinity chromatography. However, when used in western analysis, no specific bands corresponding to the native wali1 protein were identified. The wali2-GST protein was used in a south-western procedure to determine if the protein was capable of binding DNA, but no DNA binding to this protein was detected under the conditions tested. The wali3 and wali5 fusion proteins were tested in proteinase inhibitor assays, where no inhibition of either trypsin or chymotrypsin was detected. It is possible that the native wali3 and wali5 proteins may not function as proteinase inhibitors, or that the lack of activity detected for the fusion proteins may be due to incorrect folding or processing in the bacterial system. This research constitutes the first identification of plant genes whose expression is increased by Al stress. The genes identified are also induced in response to other environmental and nutrient stresses, indicating that they form part of the plant's general response to stress.
Appendix 4 restricted.

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Henry, Stephen Michael. "Further insight into the Lewis histo-blood-group system as revealed from study of Polynesian and Caucasian plasma and erythrocyte glycosphingolipids." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/1975.

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This project involved the study of Lewis and related blood group glycosphingolipid isolated from individuals with normal and aberrant Lewis/secretor phenotypes. The objective was to find a biochemical basis for the unusual expression of Lewis and secretor phenotypes in Polynesians and to use this information to shed light on the "normal" expression of Lewis antigens. By using purified glycolipids, presenting them in the cell free environment of thin layer chromatography to Lewis antibodies and by determining structures by mass spectrometry it has been shown that: l. The Lec epitope is a terminal Galβ1-3Gal sequence, and not an internal branch as proposed by Hanfland (Hanfland et a1.,1986). 2. Lec or H-5-1 are present in Lewis negative phenotypes and their consequent consumption by the Le and ,Se transferases resulting in the known Lea and Leb antigens can be seen in the Lewis positives. 3. Phenotypically Le(a-b-) individuals have small amounts of Lewis antigens. This clearly demonstrates that although the Lewis negative phenotype exists at the crude serological level, this phenotype is not an "all-or-nothing" phenomenon at the chemical level. This also allows it to be postulated that the le gene is probably partially active. 4. Le(a+b+) individuals have both Lea and Leb glycolipids in the erythrocyte membrane and in plasma. Observed phenotyping anomalies appear to be related to there being quantitatively less Leb-6 in the Polynesian Le(a+b+) erythrocyte membrane than in the Le(a-b+) membrane. 5. The Le(a+b-) phenotype of Polynesians is actually the Le(a+b+) phenotype but with serologically undetectable Leb. This allows it to be postulated that the nonsecretor gene (se) is absent in Polynesians. 6. Extended structures are present in most of the Polynesian samples which is in support of a postulated weak secretor gene (Sew). It now appears that the difference between the extended Lewis glycolipids of Caucasians and Polynesians is quantitative. The postulated, Sew transferase appears to be inefficient and allows for increased formation of elongated glycoconjugates (polyglycosylceramides) to result. 7. Reduced fucosyltransferase activity allows increased elongation of the precursor chain to occur, which allows it to be postulated that fucosylation of the precursor prevents, or at least markedly reduces, chain elongation. It is speculated that, as almost everyone is either Lewis and/or secretor positive, perhaps the prevention of chain elongation is a biological reason as to why the Lewis and Secretor polymorphisms exist. 8. Differences in ceramide patterns of Lewis active glycolipids suggests that the small intestinal tract is not the only origin of plasma glycolipids, or there is differential absorption. 9. There is no plasma glycolipid-based reason for there being increased H type 2 antigen reactivity in the Polynesian erythrocyte membrane, nor a reason for the H antigen association with the Le(a+b+) phenotype.

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Hieber, A. David (Andrew David) 1966. "Characterisation of glycoprotein II from bovine adrenal medulla." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/1988.

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Glycoprotein II (GpII) is a glycoprotein isolated from the membranes of chromaffin granules in the adrenal medulla. The chromaffin granules of the adrenal medulla are responsible for the biosynthesis, storage and secretion of catecholamines, neuropeptides and various proteins. The abundance of chromaffin granules makes them an excellent model to further study the organelles specialized in the synthesis and secretion of hormones and neurotransmitters, from both endocrine and synaptic vesicles. When viewed by two-dimensional electrophoresis GpII is a heterogeneoug glycoprotein (80000-100000 daltons) consisting of two components, upper (GpIIa) and lower (GpIIb). Protein and immunological characterisation revealed that the two components of GpII are distinct from each other. Further molecular characterisation of GpIIa showed that it consisted of a 1224 base pair cDNA, encoding a 383 amino acid polypeptide, with a calculated molecular mass of 40500 daltons. This characterisation also revealed 19 potential glycosylation sites, with deglycosylation studies further demonstrating that an estimated 55% of the molecular mass of GpII is asparagine-linked carbohydrate. The presence of poly-N-acetyllactosamine carbohydrate groups on GpII was also demonstrated. The predicted amino acid sequence of GpIIa shares a 72% amino acid identity with the human lamp-l type protein, which belongs to a highly conserved group of lysosomal associated membrane glycoproteins (lamp proteins). As the C-terminal region of GpIIa was identical to the C-terminal region of lamp proteins, a synthetic peptide to the C-terminal region of GpII was used to raise antisera to this region. This antisera was then used to demonstrate that the C-terminal region of GpII wag present on chromaffin granules and facing the cytoplasm. This same C-terminal region on lamp proteins is known to be a cytoplasmic tail that is essential for the intracellular targeting of lamp proteins to the lysosome. A common feature to the pathways of both GpII and lamp proteins is their appearance on the cell surface followed by internalisation via endocytosis. As endocytosis of the chromaffin granule membrane is known to occur, the possibility that GpII was a marker for endocytosis was investigated. The presence of GpII within clathrin coated vesicles was shown, and evidence was presented to demonstrate that the C-terminal region of GpII interacts with adaptor proteins of clathrin coated vesicles. Adaptor proteins are known to mediate between the cytoplasmic domain of proteins internalised by endocytosis, and the outside coat of clathrin. The evidence presented would suggest that the cytoplasmic tail of GpII is a potential marker for endocytosis within the chromaffin granule membrane. The results presented in this study indicate that GpII is the secretory granule and species counterpart to the lamp proteins. This finding however raises some interesting questions regarding intracellular targeting between the lysosomes and secretory granules within the chromaffin cell.

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Hiyama, Jun. "Isolation and characterisation of N-glycans of ovine and human luteinizing hormones." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/1989.

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Gonadotrophic hormones are heterodimeric glycoproteins and their N-glycans attached to specific amino acid residues are currently thought to play important roles in hormonal biosynthesis, secretion and function. The studies reported in this thesis aimed at isolation and characterisation of structural properties of the N-glycans on ovine and human luteinizing hormones. Initially, chromatographic methods were developed using reverse-phase HPLC for the analytical separation of the three human pituitary glycoprotein hormones and their subunits. Separation of intact oLH and its subunits was also effected by a single HPLC step. A preparative procedure was developed for the efficient purification of hLH and hTSH from crude human pituitary extracts using hydrophobic chromatography which gave highly purified hormones in good yields and with high biological activities. This method did not significantly influence the hormones' extensive charge heterogeneity and it offered potential advantages in the characterisation of their carbohydrate structures. A preparative scheme was developed for the isolation of the N-linked oligosaccharides from each glycosylation site of o- and hLH. Charge heterogeneity of oligosaccharides, which were released by hydrazinolysis from subunits and glycopeptides, was characterised by anion-exchange HPLC. 1H-NMR analysis showed that the structures of all three N-glycans on hLH were highly heterogeneous but mainly diantennary complex-type, with site-specific patterns of terminal sialylation and sulphation as well as core-fucosylation. Sulphated/sialylated and/or disialylated oligosaccharides were the major components at each site. A set of new mono- and disialylated oligosaccharides with the terminal sequence NeuAcα2-6GalNAcβ1-4G1-cNAcβ1-2Manα1-3 was identified. This finding suggested unique site-specific terminal sialylation of oligosaccharides at Asn 78 (hLHα) by an unknown α2-6 sialyltransferase(s) in the human pituitary gonadotroph cell. Each glycosylation site in oLH had a distinct set of oligosaccharides ranging from mainly monosulphated hybrid-type at the two sites of oLHα to predominantly disulphated diantennary complex-type on oLHβ. Core-fucosylation also differed at each site. These results suggested that processing of the oligosaccharides of the α- and β-subunits by α-mannosidase II and α1-6 fucosyltransferase was differently regulated by protein structure in oLH. Whereas hCG, hLH and oLH share similar biological activities, no apparent relationship between their N-glycan structures was found, which suggested that specific branching and peripheral structures of N-glycans on LH and hCG may not be essential for biological function, although the N-glycan nearer the N-terminus of the α-subunit of hCG has been implicated in hormonal activity.

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Podivinsky, Ellen. "Molecular studies on actinidin, a cysteine protease from kiwifruit." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/2001.

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Research in this thesis describes the characterisation of mRNA sequences coding for actinidin, a cysteine protease found in abundance in the fruit of kiwifruit (Actinidia deliciosa). The first step in the characterisation required the isolation of mRNA from ripe kiwifruit tissue. The suitability of a number of RNA extraction procedures was investigated. The method finally adopted differed from that used for unripe fruit tissue, and was chosen as a result of the nature of the polysaccharide that contaminated nucleic acids prepared from extracts of kiwifruit fruit tissue. RNA extracted from ripe fruit was used to synthesis a partial cDNA library and clones for actinidin were isolated. A number of these cDNA clones were sequenced; three clones were almost full-length. The actinidin cDNA clones obtained fall into two broad sequence classes. The majority of them encode acidic proteins (pI˜4.7), with 97% homology to the published amino acid sequence of actinidin. The second class encode basic proteins (pI˜8.1), with 83% homology to the published amino acid sequence of actinidin. Both classes of actinidin cDNA sequence encode zymogens, which contain N- and C-terminal extensions not present in the mature form of the enzyme. The N-terminal extension of both sequence classes includes a putative signal peptide. Northern hybridization analysis was used to investigate the tissue specificity of actinidin mRNA expression, and the expression of mRNA for the two actinidin sequence classes during fruit ripening. Both actinidin sequence classes were expressed differentially during the latter stages of kiwifruit fruit development and through post-harvest fruit ripening. The expression of both sequence classes increased from just prior to fruit maturity through ripening and reached a maximum as fruit attained the stage of 'eating' ripeness. The level of expression of the sequences encoding acidic actinidin reached a plateau at this point, while the expression of the sequence encoding basic actinidin appeared to decrease slightly as fruit continued to ripen. The sequences encoding acidic actinidin were expressed during ripening at a much higher level than those encoding basic actinidin. No actinidin mRNA was detected in other tissues except for very low levels of the acidic form in kiwifruit leaf, and low levels of the basic form in senescing petals. A full-length, acidic, actinidin cDNA sequence was introduced into tobacco (Nicotiana tabacum) plants via Agrobacterium tumefaciens-mediated transformation. Using the binary vector pGA643, the sequence was introduced in both the sense and antisense orientation relative to the cauliflower mosaic virus 35S promoter and transgenic plants were obtained for both sequence orientations. The presence of the T-DNA cassette (containing the actinidin sequence) in the plant genomes was determined using PCR analysis, and confirmed by Southern hybridization. A number of the transgenic plants contained multiple insertions of the actinidin sequence, and most plants contained at least one intact copy of the T-DNA cassette. The transcription of the introduced actinidin sequence was investigated by Northern hybridization analysis. All of the plants containing actinidin in the sense orientation, and some of those incorporating the antisense construct, transcribed the actinidin sequence. Attempts to detect actinidin protein in the transgenic plants were unsuccessful. Acidic actinidin was identified as one of the most abundant bands in the total protein profile from ripe kiwifruit fruit tissue. The identity of the protein was confirmed by N-terminal sequence analysis. The electrophoretic mobility of actinidin, both in the total cell homogenate and when partially purified, suggested that the first step in post-translational processing of the zymogen may be the removal of the N-terminal extension. Actinidin was also partially purified and used to raise antibodies. Poor specificity of the antibody for actinidin led to preliminary evidence for the glycosylation of actinidin.

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Spiers, Andrew J. (Andrew Julian). "Molecular and genetic analysis of RepA from the P307 RepFIB replicon." Thesis, University of Auckland, 1992. http://hdl.handle.net/2292/2044.

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The work in this Thesis concerns the replication control system of the P307 plasmid RepFIB replicon. The basic replicon occupies ≈ 1.6kb of DNA and contains a single large open reading frame (repA) flanked on either side by a series DNA repeat elements. The organisational structure of fie replicon has placed RepFIB into the Step function class of replicons. The placement of RepFIB within this group, as well as a strong homology between RepFIB and mini-P1, has resulted in a series of predictions concerning the control elements of RepFIB replication. The aim of this work was to test some of these predictions and to characterise the fundamental control elements utilised by the replicon. This Thesis describes three different active promoter elements found embedded within the repeat elements flanking repA. Although the functional significance of two of the promoter is unknown (orip and EFp), the third is responsible for the expression of RepA and has been designated 'repAp'. All three promoters are sensitive to RepA in trans, demonstrating that repA is autoregulated and that RepA is a DNA-binding protein capable of recognising copies of the repeat elements. RepA DNA-binding has also been demonstrated in vitro using a modification of the Western analysis technique (referred to a 'Western-DNA') in order to complement the in vivo experimental results. Although the coding region of repA had been determined in earlier work, the identification of the translational start codon was uncertain. This uncertainty has been resolved by limited N-terminal sequence analysis of a RepA:β-galactosidase fusion protein which has demonstrated that translation begins from a CTG codon located upstream of the predicted start sites. Finally, a series of genetic experiments have been used to determine the functional significance of RepA binding to the repeat elements. The repeat group upstream of repA are involved in autoregulation and also form part of the origin of replication, whilst the downstream repeats appear to be involved in the sensing and setting of plasmid copy number. Although the work presented in this Thesis does not directly test the applicability for RepFIB of various control models proposed to explain the behaviour of Step function replicons, the nature and type of control elements identified in RepFIB support the placement of RepFIB within the Step function class. As a result of this work, it is clear that RepFIB is confronted by the same kind of control paradox faced by replicons such as mini-P1 and mini-F. All three replicons use autoregulation and titration to control the supply of initiator protein required for replication. However, concurrent autoregulation and titration appear to be incompatible in current control models, and the identification of both mechanisms in these replicons has lead to a control paradox. Some of the results presented here suggest potentially valuable avenues of future research which may help resolve the paradox faced by RepFIB and other Step function replicons.

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Daja, Mirella Maria. "Enzyme activities associated with gonadotropic hormones." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/2311.

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A structural relationship between gonadotropic hormones and certain types of enzymes has been suggested in previous studies and an investigation into the possibility of enzymatic activity associated with the gonadotropic hormones has been the primary focus of the research presented in this thesis. Partial sequence homology between human chorionic gonadotropin (hCG) and α-chymotrypsin prompted the recent proposal of a tertiary structure of hCG using α-chymotrypsin as a folding template, which suggested the possibility of intrinsic peptidase activity associated with hCG. Highly purified hCG (CR127) was assayed for enzymatic activity against a range of synthetic peptide substrates and was found to exhibit Arg-specific peptidase activity. This activity was almost completely inhibited by diisopropylfluorophosphate (DFP), soybean trypsin inhibitor (STI), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and to a lesser extent by N-α-p-tosyl-L-lysine chloromethyl ketone (TLCK), which indicated that the observed protease activity was serine protease-like. To establish whether this activity was intrinsic to the hormone or due to contaminants, extensive purification procedures were carried out. Hydrophobic interaction chromatography (HIC) and soybean trypsin inhibitor-affinity chromatography were found to effectively separate the protease activity from the hormone, indicating the presence of exogenous protease contaminants in the highly purified preparation of hCG. Further analysis by [3H]-DFP labelling of hCG and SDS-PAGE of the isolated contaminants revealed the presence of possible serine proteases with apparent molecular masses of 60 and 20 kD. Because serine proteases are known to stimulate cAMP production in the same target cells, it was necessary to determine the effects of the contaminating proteases on the receptor binding of hCG and cAMP production. The presence of these contaminants was found to have no apparent effect on the receptor binding capability of hCG, however the in vitro biological activity of hCG as determined by maximal cAMP production was decreased after HIC-HPLC purification of the hormone. These observations suggested that the serine protease-like contaminants contributed to the total cAMP production, thereby introducing significant error in biological assays that use hCG (CR127). The possible intrinsic enzymatic activity of hCG against its receptor as a natural substrate was further investigated. A membrane-bound receptor preparation was isolated from porcine ovaries and a receptor binding assay successfully established. The effects of hCG binding upon the membrane-bound receptor were studied and receptor proteolysis was observed. However, this proteolysis could not be definitively attributed to the actions of hCG. A purified receptor was subsequently prepared by hCG-affinity chromatography and analysed by SDS-PAGE with detection by autoradiography and silver staining. The purified receptor was found to have undergone proteolysis during the purification procedure, presumably following incubation with the hCG affinity matrix. Recent reports of the presence of homologous amino acid sequences in the active site of thioredoxin and the β-subunit of the gonadotropic hormones luteinizing hormone (LH) and follicle stimulating hormone (FSH), and subsequent demonstration of thioredoxin-like activity associated with these hormones, prompted an investigation into the possibility of thioredoxin-like activity associated with hCG. LH, FSH and hCG were all assayed for their ability to promote reactivation of reduced and denatured RNase. Although LH was shown to be capable of reactivating reduced RNase, the level of activity detected was significantly lower than that previously reported, whereas FSH and hCG were not found to be capable of this thioredoxin-like activity. These results suggested that the previously reported thioredoxin-like activity may be due to contamination of the hormone preparation, by the ubiquitous enzyme thioredoxin. The possibility of LH possessing intrinsic dithiol-disulphide interchange activity was investigated further using [3H]-iodoacetic acid. RNase/LH were incubated in an attempt to quench a dithiol intermediate. Preliminary results suggested that the presence of LH in this reaction increased the amount of protein radiolabelled, however, the isolation of a radiolabelled dithiol intermediate which could be conclusively identified as LH was not forthcoming. Furthermore the lack of RNase reactivation activity in hCG, suggests that the putative thioredoxin-like activity of LH, if intrinsic, may not be involved in receptor activation and/or signal transduction, as hCG and LH share the same receptor and should therefore have a similar mechanism of activation.

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Janssen, Bart-Jan. "Agrobacterium-mediated gene transfer into kiwifruit." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/2313.

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A system has been developed to aid in the establishment of Agrobacterium-mediated transformation for new plant species. A series of binary vectors have been constructed that express a chimaeric β-D-glucuronidase (GUS) gene in plants cells but not in bacterial cells. This feature allows GUS activity from transformed plant cells to be assayed in the presence of Agrobacterium. Preliminary experiments examined the expression of these chimaeric GUS genes in transformed petunia leaf discs. GUS expression was detectable 2 days after inoculation, peaked at 3 – 4 days and then declined; if selection was imposed expression increased again after 10 - 14 days. The amount of expression observed 4 days after inoculation correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3 - 4 days after inoculation. Surprisingly, GUS expression was concentrated in localized zones on the circumference of the disc; within these zones essentially all the cells appeared to be expressing GUS. These results suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of the petunia leaf explants, but that the frequency of stable integration is several orders of magnitude lower. A reliable Agrobacterium-mediated transformation system has been established for kiwifruit (Actinidia deliciosa var. deliciosa cv. Hayward) by using transient expression of GUS to monitor gene transfer frequencies. In vitro culture of kiwifruit plants and conditions for regeneration of plants from leaf discs have been established. Several factors were found to improve gene transfer frequencies in kiwifruit: (i) healthy actively growing source tissue; (ii) the use of Agrobacterium strain A281; (iii) the presence of a layer of moistened filter paper between the leaf explants and the cocultivation media; and (iv) the presence of 20 μM acetosyringone in both the bacterial culture media and in the cocultivation media. Pre-culture of leaf explants significantly inhibited gene transfer, particularly at the cut edge of the explants. Using the optimized transformation system, at least one transgenic plant can be regenerated from each leaf inoculated. Stable transformation frequencies have been shown to vary significantly between different binary vectors. Phenotypic, PCR, and Southern analysis has confirmed the presence of stably integrated T-DNA in several transgenic kiwifruit plants.

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Merriman-Smith, B. Rachelle. "Glucose Transporters in Diabetic Complications of the Lens." Thesis, University of Auckland, 2001. http://hdl.handle.net/2292/2338.

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Lens transparency is primarily maintained by the anaerobic metabolism of glucose. Glucose is transported from the aqueous humuor to the lens epithelial cells however, it has not yet been established how glucose penetrates to the inner part of the lens. The core of the lens is acidic, approximately pH 6.5, as an effect of the accumulation of lactate, the end-product of glycolysis. This confirms that glucose is drawn deep into the core of the lens. Until recently it was assumed that glucose was transported to the core via a gap junction-mediated route by cell-cell diffusion. However, passive diffusion is limited in capacity and is unlikely to be sufficient for nutrient transport especially for larger lenses. Instead, an active circulation system has been proposed that has the potential to transport glucose deep into the lens via an extracellular route. This would imply that fibre cells may have evolved their own glucose uptake system, yet no direct evidence to this effect has been available. My thesis describes new molecular evidence that both epithelial and fibre cells have evolved their own glucose uptake system. The rat lens expresses the facilitative glucose transporters GLUTI and GLUT3 differentially. GLUTI is predominantly expressed in the epithelium while GLUT3 is predominantly expressed in the fibre cells of the lens. In the normal lens, this makes good physiological sense. GLUTI has a high Km suitable for situations of high glucose concentrations, as is the case for the epithelium where the aqueous humour mirrors glucose concentrations found in blood. GLUT3 has a lower Km and is particularly suitable for the fibre cells, where the supply of glucose from the tortuous extracellular space is limited. The discovery of GLUT3 in the fibre cells lends strong support for the existence of an active circulation system in the lens and makes it seem unlikely that glucose is transported into the core via gap junction-mediated diffusion. In the diabetic state, sorbitol - a product of glucose metabolism, occurs at elevated levels of about 30 times more than that of the normal, suggesting a significant increase in glucose uptake. This imposes an osmotic stress on the lens, which can be countered by regulated cell volume decrease only in the outer but not in the inner cortex. As a consequence inner cortex tissue breaks down and opacities result. My studies of the diabetic rat lens shows that the situation is made worse by an apparent up-regulation of GLUT3 in the fibre cells. Quantitative RT-PCR shows that the GLUT3 transcript is up-regulated six-fold during the initial weeks of diabetic insult in the streptozotocin rat model. The up-regulated GLUT3 protein is detected in the region where maximum tissue damage occurs. These results suggest a new mechanism for the initial tissue damage in the diabetic lens, whereby increased uptake of glucose leads to an over-production of sorbitol which causes osmotic stress on the fibre cells that is beyond their defense capability of regulated cell volume decrease. While my results described above have revolutionized our view of nutrient transport in the lens and its potential role in the early stages of diabetic cataract, the picture is only complete when the functionality of GLUT3 can be demonstrated. For this purpose, vesicles were prepared from isolated lens fibre cells and subjected to quantitative uptake studies using a fluorescent glucose derivative. Uptake was greatly reduced using the GLUT-specific inhibitor phloretin demonstrating that GLUT3 of the rat lens fibre cells is indeed fully functional. In summary, my results add a new dimension to our understanding of how the lens maintains homeostasis and tissue transparency. The lens has evolved an ingenious system to supply, nutrients to the core region to compensate for the absence of a vasculature. However, by achieving this, it has rendered itself unable to defend itself against the adverse effects of high glucose. My results also contribute towards developing new strategies of rational drug design to prevent or delay cataract.

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Simpson, Robert Malcolm. "The biosynthesis and control of indoleacetic acid." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/2344.

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Attempts were made to form indoleacetic acid in cellfree extracts of mung bean (Vigna radiata) shoots. The extracts were incubated with radiolabelled tryptophan and other substrates and cofactors thought to be involved in indoleacetic acid biosynthesis. After incubation indolepyruvate and indoleacetic acid were separated and quantified by HPLC. There was no significant difference in the conversion of tryptophan to indolepyruvate and indoleacetic acid between the incubations and control incubations using boiled extract. The concentrations of indolepyruvate and indoleacetic acid in mung bean hypocotyl suspension cultures were measured using GC-MS SIM over the growth of the culture, a period of 29 days. Indoleacetic acid concentrations, although scattered, mostly remained at constant low levels in the range of 6 to 9ng/g fwt of culture. The indolepyruvate levels steadily increased to a maximum level after 14 days, then remained at this level, 10 to 12 ng/g fwt, for the remainder of the culture period. This plateau in indolepyruvate concentration matched the period that the suspension culture was in the logarithmic phase of growth. An aromatic amino acid aminotransferase was purified over 33,000 fold from the shoots and primary leaves of mung beans, as determined using a tryptophan aminotransferase activity assay. The enzyme was a monomer, with a molecular weight of about 58kDa. The pH optimum was broad, with a maximum at about 8.6. The relative activities of the aromatic amino acids were: tryptophan 100, tyrosine 83 and phenylalanine 75, and the Kms were 0.095, 0.08 and 0.07mM respectively. The enzyme was able to use 2-oxoglutarate, oxaloacetate and pyruvate as the oxo acid substrate at relative activities 100, 128 and 116 and Kms 0.65, 0.25 and 0.24mM respectively In addition to the aromatic amino acids the enzyme was able to transaminate alanine, arginine, leucine and lysine to a lesser extent, and showed slight activity with asparagine, aspartate, histidine, valine and D-tryptophan and tyrosine. Inhibition studies showed that the alanine, aspartate and histidine activities were part of the aromatic amino acid aminotransferase activity. The enzyme was not inhibited by indoleacetic acid, although naphthaleneacetic acid did inhibit slightly. There was evidence of substrate inhibition by hydroxyphenylpyruvate at high concentrations. Addition of the cofactor pyridoxal phosphate only slightly increased the activity of the enzyme. The enzyme was blotted onto a PVDF membrane cleaved by in situ trypsin digest. Three of the tryptic fragments were sequenced. These fragments had approximately 60% sequence similarity with plant aspartate aminotransferases and tyrosine aminotransferases.

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12

Taylor, Jacqueline Ann, and Jackie (name change) O'Flaherty. "Factors affecting the metabolic control of cytosolic and lysosomal glycogen levels in the liver." Thesis, University of Auckland, 1985. http://hdl.handle.net/2292/2380.

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Although glycogen is a chemically homogeneous material it is polydisperse, exhibiting a broad molecular weight spectrum and a metabolic lability that is molecular weight dependent. The lower molecular weight (β-particle) glycogen was found to be extremely labile, while the higher molecular weight (α-particle) exhibited a far lower metabolic activity, indicating that it may act as a glycogen store for mobilisation in stress situations. These observations, coupled to the existence of Pompe’s Disease, a glycogen storage disease involving the lysosomal system, supports the hypothesis that α - and β -particulate glycogen may be partially separated from one another within the cell i.e. compartmentalised. By the use of a rapid differential centrifugation technique it was possible to show, both physiochemically and ultrastructurally, the existence of glycogen of a very large molecular size associated with the lysosomal fraction. This glycogen exhibited a different molecular weight distribution from that isolate from the liver as a whole i.e. cytosol + lysosomal. It is suggested that appreciably more than 10% of cellular glycogen is located within the lysosomes and that this is of predominantly high molecular weight. The size-distribution of liver glycogen was shown to be distinctly affected by the anti-inflammatory drugs, salicylate and Indomethacin. By measurement of the incorporation of radioactive glucose into glycogen, salicylate was shown to have a depressing effect on overall liver glycogen metabolism. These effects appear to arise from a stabilisation of the lysosomal membrane by the drugs. The incorporation, via liposomes, of purified anti-1,4-α-glucosidase antibodies into the liver lysosomes of normal Wistar rats and rats with a genetic deficiency of phosphorylase kinase, caused a distinct decrease in 1,4-α-glucosidase activity and in the content of high molecular weight glycogen. These changes were enhanced by prolonged liposomal-antibody treatment and suggested that a possible feedback control mechanism operates in the incorporation of glycogen into lysosomes. The 1-4- α -glucosidase inhibitor, Acarbose, when injected intraperitoneally into normal and phosphorylase kinase-deficient rats similarly disturbed liver lysosomal metabolism, causing distinct and persistent inhibition of enzymes and acute disturbances of lysosomal glycogen metabolism. Again a feedback control mechanism appears to operate, which effects cytosolic carbohydrate metabolism. The biochemical effects closely resembled those occurring in Pompe’s disease and were confirmed by electron microscopy. A model for the adult form of the lysosomal storage disease has been suggested.

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13

Powell, Kevin F. H. (Kevin Frederick Herbert). "Gene sequencing and in vitro synthesis of the rotavirus non-structural glycoprotein." Thesis, University of Auckland, 1986. http://hdl.handle.net/2292/2385.

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1. Recombinant DNA techniques have been applied to the dsRNA genome of the bovine rotavirus Nebraska Calf Scours Diarrhoea virus (NCDV). The sequence of a full - length cloned copy of genomic segment 10 of NCDV has been determined using the Sanger dideoxynucleoside sequencing technique by subcloning cDNA into M13 vectors. 2. Genomic segment 10 codes for the non-structural protein NCVP5, a protein which appears to be involved in virus maturation (Estes et al., 1983). Determination of the nucleic acid sequence of the gene has enabled the amino acid sequence o f the bovine NCVPS protein to be inferred. Comparison of the inferred amino acid sequence with homologous sequences derived from other virus strains (Both et al., 1983c; Baybutt and McCrae, 1984; Okada et al., 1984; Ward et al., 1985) has enabled conserved regions of the molecule to be identified. A small region of the NCVPS protein has been identified (residues 131-161) which exhibits considerable variability between rotavirus strains. 3. A computer-based algorithm has been utilised to predict the folding pattern of NCDV gene 10 mRNA. This reveals a 'panhandle ' structure which differs from that proposed for the related gene of strain Wa rotavirus (Okada et al., 1984) but both molecules possess a common feature in that the initiation codon falls within a potentially-stable duplex formed with a portion of the 3 untranslated region. 4. A series of four site-directed deletion mutants of the cloned gene were constructed in order to investigate the functional significance of the three N-terminal hydrophobic regions of the NCVP5 protein. Two mutants were constructed using conveniently-located HindIII and BamHI restriction enzyme sites. The other two mutants were generated using M13 vectors and synthetic oligonucleotides. These modifications yielded genes coding for proteins in which portions of the first and second hydrophobic regions had been deleted. 5. DNA corresponding to the 'wild-type' coding region was inserted into an SP6 transcription vector to enable mRNA to be produced in vitro. This mRNA, when incubated in a reticulocyte lysate, directed the synthesis of a protein of the correct size (20 K). The addition of dog pancreatic microsomes to the reaction yielded a protein product (29 K) of a size consistent with the glycosylated ('wild-type') form of the NCVP5 protein. 6. The four variant forms of the NCVP5 gene were also inserted into SP6 transcription vectors and the protein products synthesised by the resulting mRNAs studied. All four mRNAs directed synthesis of variant protein products of the anticipated size. 7. The ability of the four variant proteins to become glycosylated and to associate with membranes was investigated. The topology of the proteins in the membrane was examined by digestion with proteolytic enzymes. Variant proteins altered in the first or second hydrophobic regions retained their ability to associate with membranes, suggesting that the third hydrophobic region, which was not altered, might play a role in membrane association. 8. A model for the disposition of NCVPS in the endoplasmic reticulum is proposed in which the first hydrophobic region is located within the lumen of the endoplasmic reticulum, the second hydrophobic region spans the membrane and the third hydrophobic sequence associates independently with the membrane from the cytoplasmic side leaving the C-terminus of the molecule exposed to the cytoplasm. The model proposed accounts for the experimental observations but is in conflict with current mechanisms proposed for the insertion of proteins into membranes. (Wickner and Lodish, 1985).
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14

Beuning, Lesley L. "Cytokinins and the division or expansion of plant cells." Thesis, University of Auckland, 1988. http://hdl.handle.net/2292/2406.

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The effect of cytokinins was studied in three systems: the alga Chlorella, callus cultures and etiolated cucumber cotyledons. In Chlorella cultures: 1) A range of concentrations of 6BA and IP had no effect on growth; 2) Low concentrations of an anticytokinin had no effect on growth, whereas higher concentrations appeared to be inhibitory. 3) Characterisation of the Chlorella species suggested that it was surrounded by an impermeable sporopollenin layer which hindered the uptake of cytokinin. 4) The uptake of radioactive adenine occurred readily, whereas the uptake of radioactive 6BA was very slow in both growing and saturated cultures of Chlorella. 5) Extracts isolated from Chlorella and the medium in which Chlorella was growing contained cytokinin-like activity in two bioassays. 6) HPLC analyses of these extracts showed that there were fractions which eluted at the positions of IP and IPA. In callus cultures: A.1) A carrot callus was grown from the secondary phloem of the storage root of carrot. 2) This callus, which was grown on 2,4-D and kinetin, produced roots and shoots when subcultured onto IAA and kinetin. 3) Growth on 2,4-D alone was independent of the presence of kinetin. 4) Growth was inhibited in the presence of an anticytokinin, suggesting that the callus produced a cytokinin. B.1) A tobacco callus was grown from a young leaf of tobacco. 2) This callus habituated to cytokinin independence following subculture onto lower concentrations of kinetin. 3) Subculture of the habituated callus onto a higher concentration of kinetin resulted in the production of roots and shoots. C.1) Cytokinin-dependent soybean and tobacco callus cultures were obtained from the Botany Department, University of Otago. 2) Analysis of the total proteins from suspension and callus cultures of soybean by 1-D polyacrylamide gel electrophoresis showed one small 6BA-induced change in the proteins from the suspension cultures. In etiolated cucumber cotyledons: 1) 6BA caused the expansion of excised etiolated cucumber cotyledons after a 24h-hour incubation in the dark in a solution containing 6BA, in comparison to cotyledons incubated in water only. 2) The cotyledons curved upwards and in the light microscope the cells of the vascular bundles and the lower epidermis exhibited greater expansion than the upper epidermis. 3) Electron microscopic examination showed that the central vacuole of palisade cells from cotyledons treated with 6BA had expanded and that the cytoplasm had probably lost water and was compressed by the vacuole against the cell wall. 4) In contrast to other research, there was no apparent increase in polysome formation in 6BA-treated cotyledons in comparison to untreated cotyledons examined in the electron microscope. 5) A number of protein extraction methods were tried before a method was found which produced a protein extract suitable for both 1-D and 2-D polyacrylamide gel electrophoresis analyses. 6) 1-D and 2-D polyacrylamide gel electrophoresis showed that a number of proteins either increased or decreased following the treatment of cotyledons with 6BA. 7) A number of RNA extraction methods were tried, to obtain RNA suitable for translation in vitro. Only one method produced RNA which appeared to be free of contaminating substances. Weak translation of this RNA was obtained in vitro and it might be possible to develop conditions for optimal translation of the RNA given an adequate supply of an in vitro translation system.

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15

Kennedy, Martin A. "Transcriptional promoters in a replication region of F plasmid." Thesis, University of Auckland, 1986. http://hdl.handle.net/2292/2482.

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This thesis describes aspects of genetic regulation within and near a replication origin (ori-1) of the F plasmid. A number of transcriptional promoters were isolated, precisely mapped, and characterized with respect to their strengths and modes of regulation. The principal techniques employed in these investigations were: "shotgun" molecular cloning of restriction fragments into a galactokinase-based promoter selection vector, assays for galactokinase activities, DNA sequencing and S1 nuclease mapping of transcripts. Major findings from this study can be summarized as follows: 1). Promoters for the essential replication genes pifC and E were cloned and shown to be autoregulated at the transcriptional level. 2). An E.coli protein, integration host factor (IHF), was found to modulate the activity of the pif operon promoter. 13). Two promoters which direct transcription in opposite directions from within the minimal ori-1 region were discovered. 4). Transcription from both ori-1 promoters was shown to be repressed by the mini-F encoded D protein. 5). Precise transcriptional startsites of the pifC gene and the two ori-1 promoters were determined. 6). A mini-F protein (D) was shown to resolve dimers of a plasmid which contains a site-specific recombination locus from near ori-1, and a facile assay system for this function was developed.

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16

Hole, Rebecca. "Mammalian ADP-dependent glucokinase : a thesis presented in partial fulfilment of the requirement for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand." Massey University, 2009. http://hdl.handle.net/10179/1154.

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The mammalian ADP-dependent glucokinase is the most recent mammalian glucokinase to have been discovered, and is unique in its ability to catalyse the phosphorylation of glucose to glucose-6-phosphate using ADP as the phosphoryl donor. Up until the discovery of this enzyme, the traditional biochemical view was that the first step of glycolysis was solely catalysed by ATP-dependent hexokinases, types I-IV. The particular role played by ADP-GK in the mammalian cell and the significance of this role has not yet been determined, although it is hypothesised that the ADP-dependent glucokinase could be potentially significant in contributing to the survival of cells under low energy and hypoxic or ischemic conditions. By using ADP as the energy investment in phase one of the glycolytic cycle instead of ATP, it is predicted that glycolysis could be sustained for longer during lower energy conditions (conditions of high ADP:ATP ratios). Since the phosphorylation of glucose by ADP-GK results in the production of AMP, it may also be possible that this has a direct effect on the energy charge of the cell. The AMP produced could lead to the regulation of cellular metabolism during hypoxia and/or ischemia via the activation of the cell-energy regulator AMPK. The study of mammalian ADP-dependent glucokinase is a very new area, and prior to this no investigation of the human ADP-GK enzyme had been undertaken. The main objective of this project was to clone, express and purify the recombinant ADP-GK so it could be kinetically characterised and directly compared with the recombinant mouse kinetic characteristics, the only other mammalian ADP-GK to have been studied. Unfortunately, due to complications in the expression and purification of soluble recombinant human ADP-GK, the project did not incorporate the kinetic characterisation of the enzyme. Acquiring data on the kinetic characteristics of the human ADP-GK will, in the long term, assist in the elucidation of the metabolic role of this enzyme, so the continuation of this project would be worthwhile.

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17

MacAskill, Ursula Kate. "A structural investigation of squash aspartic peptidase inhibitor (SQAPI) using Nuclear Magnetic Resonance spectroscopy (NMR) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand." Massey University, 2007. http://hdl.handle.net/10179/985.

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Peptidases are enzymes that hydrolyse peptide bonds. This potentially dangerous activity is regulated by post translational modification and peptidase inhibitors. The best characterized of the peptidase inhibitors are the serpins whilst the aspartic peptidase inhibitors are the least characterized. Aspartic peptidase inhibitors are rare with only nine known sources. However, they are of great interest because they play an important part in several human diseases such as metastasis of breast cancer cells, Candida albicans infections and HIV. The aims of this research project were to investigate the structure of Squash Aspartic peptidase inhibitor (SQAPI), using nuclear magnetic resonance spectroscopy (NMR). This required large amounts of relatively pure and isotopically labeled protein, which was achieved by heterologously expressing His-tagged rSQAPI fusion protein in Escherichia coli using a rich to minimal media transfer method. The fusion protein was purified with a nickel column and the N-terminal extension containing the His6-tag was removed by cleavage of the fusion protein with enterokinase followed by nickel column purification. Preliminary 1 dimensional NMR spectra indicated that SQAPI was folded in solution at pH 3. This was confirmed from the results of a preliminary 15N-edited HSQC. These results combined justified the production of a 15N 13C labeled SQAPI sample for the collection of further NMR spectra. From the spectra produced with double labeled protein the backbone and the side-chain atoms of SQAPI were assigned. The chemical shifts are currently 88.89% complete and have been submitted to the biological magnetic resonance bank (BMRB). A preliminary estimate of the secondary structure of SQAPI has been calculated from the HNHA spectrum suggesting that the SQAPI structure has some similarity to the previously proposed model of the inhibitor’s structure. Furthermore, the region corresponding to the putative binding loop on the model of SQAPI was found to be mobile and deuterium exchange experiments indicate that the SQAPI structure is more globular than open.

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18

Grievink, Hilbert. "Malignant hyperthermia: allele specific expression and mutation screening of the ryanodine receptor 1 : a dissertation presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry." Massey University, 2009. http://hdl.handle.net/10179/1051.

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Malignant hyperthermia (MH) is a dominant skeletal muscle disorder caused by mutations in the ryanodine receptor skeletal muscle calcium release channel (RyR1). Allele-specific differences in RyR1 expression levels might provide insight into the observed incomplete penetrance and variations in MH phenotypes between individuals. Firstly, an H4833Y allele-specific PCR (AS-PCR) assay was designed that allowed for the relative quantification of the two RYR1 mRNA alleles in heterozygous samples. In four MHS skeletal muscle samples and two lymphoblastoid cell lines (LCLs), the wild type allele was found to be expressed at higher levels than the mutant RyR1 allele. These differences were not caused by variations in RYR1 mRNA stabilities. Secondly, high-throughput amplicon sequencing was employed for the quantification of both the T4826I and H4833Y causative MH mutations in heterozygous MHS samples. With the exception of one, all detected H4833Y and T4826I mutation frequencies were about 50%. This included a control, which was constructed and proven to have a 3:1 ratio of the wild type (H4833) versus the mutant (Y4833) RYR1 allele. This suggested that that the high-throughput amplicon sequencing approach as used here, was not suitable for accurate quantification of the two RyR1 alleles in heterozygous H4833Y MHS samples. To detect possible variations in RyR1 alleles at the protein level, the RyR1 was to be isolated from microsomes prepared from a H4833Y MHS frozen skeletal muscle tissue. Microsomes isolated from MHS skeletal muscle tissues lacked the immunoreactive band that was believed to be the full length RyR1. Poor muscle quality, due to long term storage was believed to be the main cause of RyR1 depletion. Faster and less expensive screening methodologies are required for the identification of genetic variants in MH research. Thus, in an additional project inexpensive and high-throughput high-resolution melting (HRM) assays were developed to allow screening of the RYR1 gene, for mutations associated with MH and/or central core disease (CCD).

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19

Muir, Matthew Stewart. "Proteomics of the ovine cataract." Diss., Lincoln University, 2008. http://hdl.handle.net/10182/792.

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The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.

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20

Clark, Alice Rosemary. "The filamin A actin binding domain structure and function: implications for a gain-of-function mechanism for the otopalatodigital syndrome: a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand [Ph. D] EMBARGOED." Massey University, 2010. http://hdl.handle.net/10179/1185.

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Embargoed until 1 January 2011
The filamin family act as scaffolding proteins associating with actin filmanents, acting through a highly conserved actin binding domain (ABD). The ABD of the filamins is homologous to that found in other F-actin binding proteins such as dystrophin. Mutations in the filamin A gene cause a wide range of disease symptoms in humans reflecting the diversity of the roles that filamin A has in cell structure and signalling pathways. The diseases fall into two separate phenotypic groups. Periventricular nodular heterotopia (PVNH) generally results from the complete loss of filamin A protein, and affects the central nervous system. The clinically separate otopalatodigital disorders (OPD) spectrum disorders are skeletal disorders and were hypothesised to be gain of function phenotype diseases. At the beginning of this work, there was very little structural data available for the human filamins, and none for the crucial highly conserved actin binding domain. This lack of structural data limited the interpretation of the biochemical and genetic data and constrained our understanding of the disease associated mutations that cluster in this domain. These studies aimed to provide insights into the structure and mechanism of actin binding domains, and thus provide a better understanding of the diseases caused when this domain is mutated. A secondary structural analysis and crystal structures of the wildtype and OPD2 associated mutant ABDs were obtained. The overall fold of the three proteins was equivalent as determined by circular dichroism spectroscopy and x-ray crystallography. The ABD from filamin A E254K showed 3.7 fold increased F-actin affinity, accompanied by a reduced thermostability (of 5.6 °C). Western blotting of OPD2, frontometaphyseal dysplasia (FMD) and PVNH patient fibroblast lysates showed similar levels of filamin A compared to the control cells. In addition the OPD and PVNH patient fibroblasts were able to adhere to fibronectin and migrate with an equivalent rate to control cells. Together these results have allowed correlations to be developed between structure, protein stability, actin affinity, cellular phenotype and the overall clinical phenotype. Showing that, at least in one example, OPD2 may be due to an increased actin affinity providing further evidence for a gain of function mechanism of OPD2.

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21

Bulman, S. R. "Testing the effect of in planta RNA silencing on Plasmodiophora brassicae infection." Lincoln University, 2006. http://hdl.handle.net/10182/1856.

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In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

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22

McDermott, Joshua D. "The ovine lens cytoskeleton." Lincoln University, 2007. http://hdl.handle.net/10182/700.

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The lens of the eye is a vital tissue in the visual system, responsible for the collection and focusing of light on to the retina. Comprised of epithelial cells at differing stages of differentiation, the transparency of the lens is dependent on the highly ordered crystalline structure of lens proteins. The lens consists of several proteins including crystallins (α, β, γ) that make up 90% of the soluble protein, and the lens cytoskeletal proteins. Cytoskeletal proteins contribute only a fraction of the total lens protein, but are thought to play an important role in the establishment and maintenance of transparency. Calpain-induced degradation of these proteins may be involved in the development of cataracts. This has been an area of research at Lincoln University where a flock of sheep genetically predisposed to cataract maintained as a cataract development model. The aim of this research was to investigate the distribution of cytoskeletal proteins in the lens, and to examine the effects of calpain proteolysis on these proteins, with the goal of establishing the role of the lens cytoskeletal proteins in the ovine cataract model. A combination of techniques was used including immunohistochemistry, which required the development of a specific protocol for ovine lenses. Cytoskeletal proteins were identified using immunohistochemistry in lens tissue sections and exhibited characteristic distributions. Actin displayed preferential distribution in the short sides of the fibre cells in the cortex of the lens but was absent in the lens nucleus, while spectrin in the cortex and nucleus was associated with the fibre cell membrane. Filensin was observed in the outer cortex of lens sections associated with the fibre cell membrane and cytoplasm, although the pattern of localisation was indistinct due to the abundance of filensin breakdown products. Vimentin displayed membrane and cytoplasmic association in the outer cortex that diminished toward the lens nucleus, with membrane associated vimentin only persisting in the deeper regions of the cortex and nucleus. Additionally, the effect of novel calpain inhibitors (Cat0059 and Cat811) in preventing proteolysis of lens cytoskeletal protein was investigated and compared with calpain inhibitors developed elsewhere (SJA6017). The inhibitors were tested at between 10 and 0.1 μM (100 nM). All inhibitors were effective at 10 μM. SJA6017 provided significant protection to vimentin at 1 μM. Cat0059 was found to protect spectrin and filensin at 1 μM, but not vimentin, while inhibitor Cat811 was found to protect spectrin only. SJA6017 added to assays at 100 nM offered significant protection to spectrin, and Cat0059 was found to protect filensin and spectrin to a significant degree at 100 nM, indicating the novel inhibitors were comparable to those developed elsewhere in terms of their effectiveness. Taken together, the evidence presented in this thesis shows the cytoskeletal proteins as crucial elements in the lens. Their pervasive presence coupled with evidence that lens cytoskeletal proteins are sensitive to calpain-induced proteolysis that is inhibited with novel calpain inhibitors suggests that the lens cytoskeletal proteins may be useful targets in cataract prevention for future research.

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23

Lei, Jie. "The role of antioxidants in the hydrogen peroxide-induced opacification of sheep lens." Master's thesis, Lincoln University. Agriculture and Life Sciences Division, 2006. http://theses.lincoln.ac.nz/public/adt-NZLIU20070517.162145/.

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The lens of the eye needs to be transparent with a high refractive index to focus images on the retina. In cataracts the lens becomes opaque, eventually leading to blindness. There are many possible causes of cataract but a lot of evidence implicates oxidative damage as contributing to opacification. This includes epidemiological studies showing that diets rich in antioxidants lowered the prevalence of cataract. This research tested the hypothesis that if cataracts were at least partially caused by oxidative damage then their progression would be slowed by application of antioxidants. The antioxidants used were two plant compounds found in the diet, resveratrol and quercetin. The system used was sheep lenses cultured in Eagles Minimal Essential Medium (EMEM). Lenses remained transparent for up to 7 days in EMEM but became opaque within 24 h when exposed to 1 mM hydrogen peroxide (H2O2). The lens is exposed to H2O2 in vivo as it is found in the aqueous humor. Prior Lenses pre-treated with quercetin reduced but did not prevent opacification. Lens cell death, as determined by measurement of leakage of lactate dehydrogenase, was found to increase with H2O2 and the increase was prevented by pre-treatment with antioxidants. The role of the endogenous antioxidant glutathione was also investigated. It was found that H2O2 decreased the amount of reduced glutathione in the lens cortex and increased the levels of oxidised glutathione but only at levels of 2 mM and above. Thus the results of this research indicate that H2O2 at low concentration (1 mM) is able to damage lens cells and cause opacification without affecting the reduced glutathione levels and that the exogenous antioxidants have some ability to protect the lens.

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24

Mathews, Antony James. "Studies on chemically modified cytochromes Cl2." 1985. http://hdl.handle.net/2292/1744.

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Tuna and Horse cytochromes c were purified and chemically modified with the water soluble carboxyl group modifying reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using the method of Timkovich (1980). The time-course of modification was followed by visible spectroscopy and by functional measurements. Both methods indicated that disruption of the haem crevice regions of the proteins was largely complete within 10-15 minutes exposure to EDC. The EDC modified Tuna and Horse proteins (THC* and HHc* respectively) showed essentially identical functional properties to those described by Timko-vich (1980) for THc*. These include a typical methionine coordination to the haem iron, as shown by the absence of the 697nm visible absorption band from the spectra of the oxidized derivatives, and a pH dependent high spin - low spin transition for the derivatives in this oxidation state. In the reduced forms, THc* and HHc* show reactivity with carbon monoxide and oxygen indicating the haem crevice regions of these proteins is disrupted compared to those of the native proteins. The tryptic peptides of native Tuna and Horse cytochromes c were mapped by HPLC and identified by amino acid analysis. Examination of the tryptic digests of THc* and HHc* by gel filtration and HPLC revealed the presence of trypsin indigestible material indicating the presence of EDC promoted intramolecular cross-links within the modified proteins. Failure to extract the haem groups from THc* and HHc* after cleavage of the protein-haem thio-ether bonds, indicates cross-linking of the exposed haem propionate groups to the proteins. Labelling of the native and EDC modified proteins with glycine methyl ester showed that THc* and HHc* contain several covalently modified carboxyl groups. The results of Timkovich (1980) for THc* are discussed with respect to these findings. The reduction of THc*3+ and HHc*3+ by L-ascorbic acid and by the strong inorganic reducing agent sodium dithionite, was studied using stopped flow spectrophotometry. The kinetics of reduction of the modified proteins by these reagents could be accounted for by mechanisms similar to those proposed for the reduction of the native proteins by these reagents (Myer et al, 1980; Lambeth and Palmer, 1973). The effect on the reactions, of chemical modification of the proteins, is discussed. The reaction of the reduced derivatives with carbon monoxide was studied by stopped flow spectrophotometry, flash photolysis, and by equilibrium binding measurements. The reduced derivatives have a high affinity for carbon monoxide. Flash photolytic studies indicated very low quantum yields for photo-dissociation of the reduced protein CO complexes. The reduced derivatives formed complexes with oxygen which were unstable, decaying to form the oxidized protein. The rate of conversion to the oxidized protein was dependent on the solution pH. It was found that in the presence of excess reducing agent, THc*2+ and HHc*2+ could catalytically reduce oxygen. Steady state kinetic measurements were carried out to determine the dependence of the rate of oxygen reduction on various parameters. Similarities to the reactions of reduced carboxymethylated cytochrome c with oxygen are discussed.
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25

Stapleton, Patricia M. (Patricia Mary). "Molecular genetics of restriction fragment length polymorphisms linked to the Huntington disease locus." 1988. http://hdl.handle.net/2292/2601.

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New Zealand families segregating the Huntington disease (HD) phenotype were investigated for linkage of the HD locus to the anonymous DNA locus G8, from chromosome 4p16.3. Linkage of the two loci was indicated in the largest family assessed. The results from this family together with those from 10 other smaller families were consistent with the existence of a single locus (that is, genetic homogeneity for HD). One crossover between the HD locus and the G8 marker locus was detected. Overall, the results of the linkage analysis indicate a distance of 4cM separating HD from G8. The usefulness of G8 as a marker in predictive testing for HD was examined in the New Zealand families. In agreement with overseas findings, G8 and DNA contiguous with G8 are useful for predictive testing in some families. The main limitation for predictive testing is the family structure which often results in the unavailability of key individuals for testing and thus prevents prediction in others. Sequence analysis of three sites which produce restriction fragment length polymorphisms (RFLPs) detected by G8 revealed that single point mutations were responsible for the presence or absence of the polymorphic sites *H1, *H2 and *E. The sequences were highly conserved between individuals in the regions investigated. The conservation of sequence provided the potential for the use of the polymerase chain reaction (PCR) to amplify each polymorphic region for more rapid assessment of these RFLPs. The three regions were amplified successfully from genomic DNA. In addition, amplification of the three regions was possible with template DNA which was degraded or crude or isolated from tissue which had been fixed in formalin. However, the results of the subsequent analyses of the amplified products by restriction enzyme digestion showed that there are problems that can render the PCR unreliable. The presence of non-target sequences hindered detection of the genotype in the *H1 and *H2 regions and masked the true genotype of a person in the *E region. Thus, the potential of the PCR for presymptomatic diagnosis of HD remains to be realised. However, when the problems are overcome a very rapid analysis of RFLPs linked with HD using the PCR will be possible, as will be retrospective analyses.

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26

Robertson, Andrew James. "The bovine spliceosomal U1 small nuclear ribonucleoprotein particle : a study of its autoantigenicity and biochemical properties : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand." 2006. http://hdl.handle.net/10179/1504.

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Despite individual autoimmune diseases being relatively rare, collectively these diseases afflict 8 % of the population according to the American Autoimmune Related Diseases Association. With over 75 % of those affected being women, autoimmune disease has been recognised, by the World Health Organisation and the US National Institutes of Health, as a major global women's health issue. One third of autoimmune sufferers have a rheumatological disorder, which commonly affect the joints, muscle, skin, salivary glands and kidneys. Antibodies against nuclear antigens are a serological hallmark of these diseases. Detection of these antibodies is used in the diagnosis and prognosis of the disease. The sensitivity and specificity of the test, of which the antigen is a key component, is pivotal to correct disease diagnosis and management. The relationship between circulating autoantibodies and the target antigen is complex. Improving the effectiveness of a test to assist in diagnosis and prognosis comes from characterisation and understanding these complex relationships. This thesis compares bovine spliceosomal U1 small nuclear ribonucleoprotein particle (U1 snRNP) complex with its human equivalent, and examines the validity of using this bovine derived autoantigen in the diagnosis of the human autoimmune diseases, systemic lupus erythematosus and mixed connective tissue disease. Differences between bovine and human U1 snRNP composition were characterised using a combination of electrophoretic, immunoassay and mass spectrometry techniques. Although the U1C protein could not be identified in bovine U1 snRNP, all other specificities were present. U1A remained intact, whilst the U1 snRNP specific 68K protein was dephosphorylated and a large C-terminal domain was removed, such that 68K migrated as a 30-36 kDa cluster on SDS-PAGE. Bovine SmD proteins, present in U1 and non-U1 snRNPs, were unaffected, whereas, SmB'/B was truncated to a 12 kDa peptide, which interestingly, was no longer reactive with anti-RNP sera in western blot. The recognition of human SmB'/B protein by anti-RNP sera in western blot was further examined. A technique was developed to immunoaffinity purify tryptic digests of SmB'/B which could then be analysed by mass spectrometry. Interestingly, the human replication element protein (HREP) was tentatively identified, rather than SmB'/B as expected. It may be possible, therefore, that anti-RNP sera may be reacting with a protein other than SmB'/B. To examine the contribution of the individual U1 snRNP proteins to anti-RNP and anti-Sm sera reactivities, a method was developed to dissociate bovine U1 snRNP and to purify the individual component antigens. It was demonstrated both empirically and through anecdotal feedback from a commercial diagnostic kit producer that patient sera respond better to purified Sm-free 68K than the recombinant 68K antigen. The effect of commercial processing of bovine thymus, the source for U1 snRNP antigen, was determined. In this study, variables that may be controlled during processing, such as temperature, protease activity and pH, were investigated. Hydrolysis of the intact human 68K protein with the necrotic protease, cathepsin L, produced 38 and 25 kDa fragments, whereas exposure to ambient temperature and low pH produced 32 kDa peptide fragments similar to those observed in purified bovine 68K. It was therefore proposed that 68K protein may undergo autocatalytic hydrolysis during necrotic cell death. Thorough characterisation of the bovine spliceosomal U1 snRNP proteins has not only validated their use as diagnostic reagents in autoimmune disease but also provided some insight into the inactivation of U1 snRNP function during early cell death.

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Webby, Celia Jane. "Structural & functional characterization of 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase from Helicobacter pylori & Mycobacterium tuberculosis : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Turitea, Palmerston North, New Zealand." 2006. http://hdl.handle.net/10179/1584.

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Content removed due to copyright restrictions: Webby, C.J., Patchett, M.L. & Parker, E.J. (2005) Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori. Biochemical Journal 390, 223-230 Webby C.J., Lott J.S., Baker H.M., Baker E.N., & Parker E.J. (2005) Crystallization and preliminary X-ray crystallographic analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Mycobacterium tuberculosis. Acta Crystallographica Section F - Sturctural Biology and Crystallization Communications 61(4) 403-406. Webby C.J., Baker H.M., Lott J.S., Baker E.N. & Parker E.J. (2005) The structure of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Mycobacterium tuberculosis reveals a common catalytic scaffold and ancestry for type I and type II enzymes. Journal of Molecular Biology 354(4), 927-939
The shikimate pathway, responsible for the biosynthesis of aromatic compounds, is found in microorganisms and plants but absent in higher organisms. This makes the enzymes of this pathway attractive as targets for the development of antibiotics and herbicides. Recent gene disruption studies have shown that the operation of the shikimate pathway is essential for the viability of M. tuberculosis, validating the choice of enzymes from this pathway as targets for the development of novel anti-TB drugs. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first committed step of the shikimate pathway. Two distinct classes of DAH7PS have been defined based on sequence similarity. The type I DAH7PSs are well characterized, however prior to this project there was limited mechanistic and no structural information about type II enzymes. Sequence identity between type I and type II enzymes is less than 10% raising the possibility that they represent distinct protein families, unrelated by evolution. We have functionally characterized the type II enzyme from Helicobacter pylori, and have shown that type I and type II enzymes catalyze a metal-dependent ordered sequential reaction following the same stereochemical course. We have solved the structure of the type II DAH7PS from M. tuberculosis using single-wavelength anomalous diffraction (SAD) methods and the structure reveals a tightly associated dimer of (β/α)8 TIM barrels. The monomer fold, the arrangement of key residues in the active site, and the binding modes of PEP and Mn2+, all match those of the type I enzymes. This similarity of protein fold and catalytic architecture makes it unequivocal that type I and type II enzymes are related by divergent evolution from a common ancestor. Interestingly, there are significant differences in the additional structural elements that extend from the core (β/α)8 barrel and in the quaternary structure. Further structural and functional analysis of M. tuberculosis DAH7PS revealed that the two major additions decorating the barrel are involved in the binding of the aromatic amino acids. Two distinct inhibitory binding sites for Trp and Phe have been identified providing an explanation for the synergistic inhibition displayed with Trp and Phe. The role of several active site residues of Mt-DAH7PS in enzyme catalysis has also been investigated.

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Bennett, Matthew David. "The structure and function of esterases from lactic acid bacteria : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosphy in the Institute of Molecular BioSciences, Massey University, New Zealand." 2007. http://hdl.handle.net/10179/810.

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Compounds derived from the breakdown of glyceride esters of milk fat, such as free fatty acids and short chain esters, are recognised as playing an important role in the flavour of a range of fermented foods. Esterases, capable of hydrolysing ester bonds, and in some cases, synthesising them via an acyltransferase mechanism, typically enter the fermentation from the starter and adjunct lactic acid bacteria that are used to inoculate milk to initiate the fermentation process. With such an important role in the development of both desirable and undesirable flavours, understanding how these enzymes operate is essential for product control. In this study, the crystal structures of three lactic acid bacterial esterases were solved: EstA from Lactococcus lactis, and AA7 from Lactobacillus rhamnosus which are both capable of hydrolysis of short chain triglycerides as well as synthesising esters via a transferase mechanism, and AZ4, an esterase from L. rhamnosus which appears to be limited to hydrolysis reactions. Whilst all three were found to be members of the hydrolase family, unique features were found for each enzyme, reflecting the large differences in their primary sequences, substrate specificities and activities. EstA and AA7 were both found to have a shallow substrate binding cleft, bisected by the catalytic machinery. The divided binding cleft suggests that during a transferase reaction the transferred group binds in one pocket, with the donor and acceptor groups (dependant on the stage of catalysis) binding in the other. In contrast, AZ4 was found to have a single deep substrate binding cavity, extending into the enzyme interior, with the catalytic residues located near its entrance. The absence of a second binding site for an acceptor is consistent with AZ4 having only one function – that of a hydrolase. The structures presented in this study are the first three dimensional structures of esterases from lactic acid bacteria to be reported. Their analyses, both in native form, and complexed with a varity of ligands mimicking various stages of the reaction cycle have highlighted how this basic fold can be adapted to efficiently catalyse different reactions. More importantly, in the case of AZ4, these structures have suggested that there is a novel mechanism used by the esterases to promote the enzyme reaction to proceed to completion, by preventing a futile catalytic reaction.

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29

Benny, Athol Graeme. "An integrated process for the recovery of clinically significant trace proteins from human plasma." 1990. http://hdl.handle.net/2292/2157.

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Methods for the preparation of concentrates of factor VIII, factor IX, high purity factor IX, Cl esterase inhibitor, specific immunoglobulin and platelet factor XIII are described. These procedures were developed or modified with the aim of integration into an automated process that would allow sequential recovery of all the clinically significant trace proteins from a single plasma pool. Concomitant recovery of important proteins such as transferrin, alpha-1-antitrypsin and platelet-derived growth factor was considered. A high-purity factor VIII concentrate heat-treated at 80°C for 96 h was prepared by a process that incorporated heparin fractionation. This method was shown to be suitable for assimilation into an existing regional blood processing laboratory. Several ion-exchange procedures for the recovery of factor IX were evaluated and higher purification of a factor IX concentrate was achieved on a new cellulose-based chromatographic medium. A chromatographic procedure for the preparation of a heat-treated high-purity Cl esterase inhibitor concentrate was described and the performance of a new cellulose-based desalting medium was evaluated in comparison with ultrafiltration. A heat-treated specific immunoglobulin concentrate was prepared from side-stream fractions of an automated chromatographic process for the production of albumin concentrate, and a pilot study for the fractionation of outdated platelet concentrates was carried out with the aim preparing components of potential therapeutic value. See summary flow diagrams of fractionation processes included with references in the back of this thesis.

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30

Glyn-Jones, Sarah. "Investigation of diabetic cardiomyopathy and its treatment by copper chelation." 2008. http://hdl.handle.net/2292/2421.

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Diabetes mellitus is estimated to affect approximately 7% of the populations living a western lifestyle. Of the multiple etiologies associated with diabetes, heart failure is the most common cause of death. A specific type of heart disease called diabetic cardiomyopathy is thought to be partially responsible. At this time, no one specific treatment is available for diabetic cardiomyopathy due to the wide variety of possible complex molecular changes, including metabolic disturbances, myocardial fibrosis, LV hypertrophy, and increased ROS production. Abnormal copper metabolism in diabetes has been proposed to form part of the pathway that leads to diabetic cardiomyopathy. Our group have shown that treatment with the copper (CuII) chelator, triethylenetetramine, ameliorates the effects of diabetes on the heart at both the functional and molecular level. This thesis aimed to further these studies by increasing our understanding of the mechanism of triethylenetetramine action on the diabetic heart. This was primarily achieved through the use of microarray technology but included the use of a range of molecular experimental techniques. During this investigation it was determined that the most suitable microarray platform for our studies was the Affymetrix GeneChip® system. Using this system we identified more than 1600 gene changes associated with diabetes in the left ventricle wall. A disproportionate number of significant messenger RNA transcript changes were associated with the mitochondria and further investigation of these genes revealed changes associated with perturbed lipid metabolism and increased oxidative stress. A second study investigated the molecular mechanisms underpinning improved cardiac function in the left ventricle of the heart from diabetic and sham animals treated with triethylenetetramine. There was an observed decrease in diabetic cardiac tissue triglyceride towards normal, possibly through improvement of the structure and stability of the mitochondria. Only a small number of changes in gene expression were detected after triethylenetetramine treatment using microarray technology, and none were detected using real time-quantitative PCR. The final aim of this thesis was to understand the absorption and excretion of triethylenetetramine by both sham and diabetic animals. Our study found differences in the ability of diabetic animals to absorb and metabolise triethylenetetramine compared to sham animals. Also, the length of exposure was found to be an influencing factor in triethylenetetramine metabolism.

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31

Schwalbe, Martin. "Intrinsic disorder and coiled coil formation in prostate apoptosis response factor-4 (Par-4) : submitted in fulfilment of the requirements of the degree of Doctor of Philosphy, Institute of Fundamental Sciences, Massey University, New Zealand." 2010. http://hdl.handle.net/10179/1688.

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Prostate apoptosis response factor-4 (Par-4) is a ubiquitously expressed pro-apoptotic and tumour suppressive protein. Par-4 contains a highly conserved coiled coil (CC) region at the Cterminus, particularly the distal 40 residues fulfil the criteria for a leucine zipper (LZ). This Cterminal domain serves as the primary recognition domain for a large number of binding partners. Par-4 is tightly regulated by the aforementioned binding partners and also by posttranslational modifications. Biophysical data presented here describe Par-4 as primarily an intrinsically disordered protein (IDP). Bioinformatic analysis of the highly conserved Par-4 reveals low sequence complexity and enrichment in polar and charged amino acids. High proteolytic susceptibility and increased hydrodynamic radii are consistent with largely extended structures in solution. Spectroscopic measurements using circular dichroism (CD) and nuclear magnetic resonance (NMR) also reveal characteristic features of intrinsic disorder. Under physiological conditions, data show that Par-4 self-associates via the C-terminal domain possibly through coiled coil formation. Analysis of various constructs comprising the Par-4 LZ domain by NMR, CD, light scattering and other techniques reveals an environment-dependent conformational equilibrium between primarily disordered monomers and predominantly coiled coil dimers. Whereas the disordered monomers are easily observed by NMR, the coiled coil fraction is not amenable to NMR studies possibly due to intermediate exchange processes. Mutational approaches that stabilise the coiled coil fraction result in NMR spectra of lower quality compared to the wild-type form. The high degree of sequence conservation suggest that coiled coil formation and intrinsic disorder are essential for Par-4 to function as an effective regulator of apoptosis.

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32

Saikia, Sanjay. "Functional analysis of Penicillium paxilli genes required for biosynthesis of paxilline : this thesis is presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Biochemistry at Massey University, Palmerston North, New Zealand." 2006. http://hdl.handle.net/10179/1489.

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Paxilline belongs to a large, structurally and functionally diverse group of indole-diterpenes and is synthesised by the filamentous fungus Penicillium paxilli. A gene cluster for paxilline biosynthesis in P. paxilli has been identified and characterised. However, none of the steps proposed in the biosynthesis of paxilline or paxilline-like indole-diterpenes have been validated. In some diterpene-producing filamentous fungi, including P. paxilli, two distinct copies of geranylgeranyl diphosphate (GGPP) synthase, that catalyses the committed step in diterpene biosynthesis, have been identified. However, the biological significance of the presence of two distinct GGPP synthases is not known. In this study, biochemical analysis of the paxilline gene products in P. paxilli and subcellular localisation of the two P. paxilli GGPP synthases, Ggs1 and PaxG, were carried out. Transfer of constructs containing different combinations of pax genes into a pax cluster negative deletion derivative of P. paxilli identified four Pax proteins that are required for the biosynthesis of a paxilline intermediate, paspaline. These proteins are PaxG, a GGPP synthase, PaxM, a FAD-dependent monooxygenase, PaxB, a putative membrane protein, and PaxC, a prenyltransferase. Using precursor feeding experiments, it was confirmed that the indole-diterpenes paspaline and β-PC-M6 are substrates for the cytochrome P450 monooxygenase, PaxP, and are converted to 13-desoxypaxilline. Further, it was confirmed that the indole-diterpene 13-desoxypaxilline is a substrate for PaxQ, a cytochrome P450 monooxygenase, and is converted to paxilline. Unlike PaxQ, PaxP is specific for indole-diterpene substrates that have a β-stereochemistry. The detection of the indole-diterpene products was related to the expression of the transgene in the pax cluster negative background. Reporter fusion studies of the two P. paxilli GGPP synthases, Ggs1 and PaxG, showed that the Ggs1-EGFP fusion protein was localised to punctuate structures whose identity could not be established, and the EGFP-GRV fusion protein, containing the C-terminal tripeptide GRV of PaxG, was localised to peroxisomes.

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Chen, Chunhong. "Structure function studies on lectin nucleotide phosphohydrolases (LNPs)." 2008. http://hdl.handle.net/2292/3369.

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Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

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Yeoman, Jeffrey Aaron. "Biochemical characterization of metal-dependent 3-deoxy-D-manno-octulosonate 8-phosphate synthases from Chlorobium tepidum & Acidithiobacillus ferrooxidans : a thesis presented in partial fulfillment of the requirements for the degree of Masterate of Science in Biochemistry at Massey University, Turitea, Palmerston North, New Zealand." 2007. http://hdl.handle.net/10179/706.

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3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase is the enzyme responsible for catalyzing the first reaction in the biosynthesis of KDO. KDO is an essential component in the cell wall of Gram-negative bacteria and plants. This compound is not present in mammals; therefore the enzymes responsible for its biosynthesis are potential targets for the development of new antibiotic agents. KDO8P synthase catalyzes the condensation reaction between phosphoenol pyruvate (PEP) and D-arabinose 5-phosphate (A5P) to form KDO8P. Two types of KDO8P synthase have been identified; a metal-dependent type and a non metal-dependent type. KDO8P synthase from the organism Chlorobium tepidum (Cte) has been partially purified and partially characterized. In line with predictions based on sequence alone, the activity of this enzyme is dependent on the presence of a divalent metal ion and is sensitive to the presence of the metal chelating agent EDTA. Cte KDO8P synthase was found to have the highest activity in the presence of Mn2+ or Cd2+. KDO8P synthase from the organism Acidithiobacillus ferrooxidans (Afe) has also been cloned, purified and biochemically characterized. Afe KDO8P synthase was also found to be a metallo enzyme and the catalytic activity is highest in the presence of Mn2+ or Co2+. Afe KDO8P synthase was found to exist as a tetramer in solution and is most active within the pH range of 6.8 to 7.5 and within a temperature range of 35 ºC to 40 ºC. Sequence analysis suggests that this enzyme has characteristics conserved throughout the metallo and the non-metallo KDO8P synthases and is closely related to the metal-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthases. The role of several active-site residues of Afe KDO8P synthase has been investigated. A C21N mutant of Afe KDO8P synthase was found to retain 0.5% of wildtype activity and did not require a divalent metal ion for catalytic activity. This suggests that the metallo and non-metallo KDO8P synthases have similar catalytic mechanisms.

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Poulsen, Raewyn Carol. "Long chain polyunsaturated fatty acids and their possible interaction with phytoestrogens : impact on bone and bone cell function in vivo and in vitro : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand." 2007. http://hdl.handle.net/10179/1595.

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Inflammation is a major contributor to postmenopausal bone loss. Various long chain polyunsaturated fatty acids (LCPUFAs), particularly those of the n-3 family, are known to have anti-inflammatory activity and may have a role in minimising postmenopausal bone loss. The objectives of this thesis were to determine whether some LCPUFAs have greater bone-protective effects than others; to identify some of the mechanisms of action of LCPUFAs in bone and to explore the possibility that combined treatment with LCPUFAs and phytoestrogens offers greater bone-protective effects than either treatment alone. Using the ovariectomised rat model for postmenopausal bone loss, the relative effectiveness of eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and gamma-linolenic acid (GLA, 18:3n-6) in minimising bone loss post-ovariectomy was investigated. GLA exacerbated bone loss post ovariectomy in rats. In vitro, treatment of MC3T3-E1/4 osteoblast-like cells with GLA resulted in greater membrane-bound RANKL expression suggesting a possible stimulatory effect of GLA on osteoclastogenesis and osteoclast activity. EPA had no effect on overall bone mass in vivo. DHA significantly ameliorated ovariectomy-induced bone loss possibly by increasing plasma IGF-1 concentration, modulating vitamin D metabolism and, as observed in a second study, by increasing the concentration of gamma-carboxylated osteocalcin. In vitro both EPA and DHA reduced the prostaglandin E2 (PGE2)-induced increase in membrane-bound RANKL expression in MC3T3-E1/4 osteoblast-like cells. However as RANKL-independent pathways are believed to be largely responsible for the ovariectomy-induced increase in osteoclastogenesis in vivo, inhibition of RANKL expression may not significantly contribute to the prevention of ovariectomy-induced bone loss. In a second study in ovariectomised rats, combined treatment with DHA and 17β-oestradiol was associated with significantly higher femur bone mineral content than either treatment alone. However, no beneficial effects of combined treatment with DHA and either of the phytoestrogens genistein or daidzein, on bone mass were apparent. In vitro, co-treatment of TNF-α - exposed MC3T3-E1/4 cells with DHA and 17β-oestradiol was associated with a higher cell number compared to either treatment alone indicating a protective effect of combined treatment against the cytotoxic and/or anti-proliferative effects of TNF-α. In contrast, combined treatment of MC3T3-E1/4 cells with DHA and genistein, but not daidzein, was associated with significantly lower cell number than either treatment alone. As genistein, but not daidzein, is a tyrosine kinase inhibitor, this may indicate that DHA requires tyrosine kinase activity for its protective effect on cell number in TNF-α - exposed osteoblasts. Whether DHA itself is bioactive in bone cells or whether lipid mediators formed from DHA are responsible for the observed bone-protective effects is unknown. Using lipid mediator lipidomic analysis, the presence of DHA-derived lipid mediators in bone marrow in quantities known to be physiologically significant in other tissues was confirmed. Further research into the effects of these lipid mediators in bone and confirmation of the mechanisms of action of DHA in bone cells is required. This thesis demonstrates that consumption of DHA provides some protection against ovariectomy-induced bone loss in vivo and mitigates the effects of inflammation on RANKL signalling and osteoblast cell number in vitro. The bone-protective effects of DHA are complemented by co-treatment with 17β-oestradiol but may be inhibited by co-treatment with the phytoestrogens daidzein or genistein.

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Sato, Keisaku. "A functional analysis of RYR1 mutations causing malignant hyperthermia : a thesis presented to Massey University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry." 2009. http://hdl.handle.net/10179/1228.

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Malignant hyperthermia (MH) is a rare pharmacogenetic disorder in humans induced by volatile anaesthetics and depolarising muscle relaxants. An MH reaction shows abnormal calcium homeostasis in skeletal muscle leading to a hypermetabolic state and increased muscle contracture. A mutation within the skeletal muscle calcium release channel ryanodine receptor gene (RYR1) is associated with MH and is thought to cause functional defects in the RYR1 channel leading to abnormal calcium release to the sarcoplasm and consequent MH reactions. Mutations within RYR1 are also associated with a rare congenital myopathy, central core disease (CCD). CCD is characterised by muscle weakness and is thought to be caused by insufficient calcium release from the RYR1 channel during excitation-contraction (EC) coupling. To investigate functional effects of RYR1 mutations, the entire coding region of human RYR1 was assembled and cloned into an expression vector. Mutant clones containing RYR1 mutations linked to MH or CCD were also constructed. Wild-type (WT) and mutant RYR1 clones were used for transient transfection of HEK-293 cells. Western blotting was performed after harvesting and expressed WT and mutant RYR1 proteins were successfully detected. Immunofluorescence showed co-localisation of RYR1 proteins and the endoplasmic reticulum in HEK-293 cells. [3H]ryanodine binding assays showed that RYR1 mutants linked to MH were more sensitive to the agonist 4-chloro-m-cresol (4-CmC) and less sensitive to the antagonist Mg2+ compared with WT. Two C-terminal RYR1 mutants T4826I and H4833Y were very significantly hypersensitive to 4-CmC and they may also result in a leaky channel. This hypersensitivity of mutants linked to MH may result in abnormal calcium release through the RYR1 channel induced by triggering agents leading to MH reactions. RYR1 mutants linked to CCD showed no response to 4-CmC showing their hyposensitive characteristics to agonists. This study showed that the human RYR1 proteins could be expressed in HEK-293 cells. Moreover, using the recombinant human RYR1 clone, a single mutation within RYR1 resulted in a functional defect in expressed RYR1 proteins and functions of mutant RYR1 proteins varied from hypersensitive to hyposensitive depending on the mutation and whether it was linked to MH or CCD.

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37

Starck, Carlene Sheree. "The human myostatin precursor protein : structure, function and amyloid formation : implications for the muscle wastage disease sporadic inclusion body myositis : a dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand." 2010. http://hdl.handle.net/10179/1408.

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Myostatin is a major player in the regulation of mammalian muscle growth and development, maintaining the balance between proliferation and differentiation prenatally and the quiescence of satellite cells in adults. An absence or overexpression of myostatin results in double-muscling and cachexia respectively, placing myostatin as a promising target in the treatment of muscle wastage diseases. As a transforming growth factor-β superfamily member, myostatin is produced as a precursor protein, consisting of a propeptide region N-terminal to the growth factor domain. Cleavage of the precursor between the domains forms the myostatin latent complex, an inhibitory structure which is exported from the cell where a second cleavage event releases the active myostatin growth factor. The precursor protein, propeptide, and latent complex play important roles in the regulation of myostatin. However, their structure and function are poorly understood, and a possible role for the myostatin precursor protein in the muscle wastage disease sporadic inclusion body myositis, suggests that pre-growth factor forms of myostatin may be additional important therapeutic targets. This thesis presents an investigation into the structure and function of the myostatin precursor protein, the latent complex, and the propeptide region within these, with comparisons to a mutant form of myostatin responsible for the naturally-occurring double-muscled phenotype of the Piedmontese cattle breed. Results suggest that the diverse functions of the propeptide region are facilitated by regions of intrinsic disorder within a primarily structured domain, and that conformational alterations accompany the precursor to latent complex transition, resulting in a tighter inhibitory structure. Comparative analyses between the wild-type and mutant proteins suggest that the Piedmontese phenotype is due to a reduced capacity for covalent dimerisation and significant structural alterations within the type I receptor-binding domain. Investigation into misfolded myostatin precursor protein found that the precursor is able to form cytotoxic amyloid aggregates and mature fibrils under partially denaturing conditions, suggesting a possible mechanism for the role of the myostatin precursor in sporadic inclusion body myositis. Together, these novel results contribute important information towards an understanding of myostatin structure, function and regulation in both normal and disease scenarios.

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Koehn, Henning. "Differentially regulated proteins in breast cancer chemotherapy : a thesis presented to Massey University in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Biochemistry." 2005. http://hdl.handle.net/10179/1554.

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Intrinsic or acquired drug resistance of tumours is a major problem for successful therapy of breast cancer patients. The efficacy of doxorubicin, one of the most important and commonly used drugs in chemotherapy, can be severely compromised by a variety of unspecific mechanisms rendering tumours drug resistant. Little is known however, about the specific events taking place in response to doxorubicin treatment, which may repair doxorubicin-induced damage, leading to drug resistance. Doxorubicin is a topoisomerase II poison, which interferes with topoisomerase II enzymes during DNA replication, resulting in DNA double-strand breaks. Topoisomerase II enzymes mediate the passage of DNA strands by introducing transient DNA breaks, and are essential for changes in DNA topology during replication. The DNA lesions induced by the combination of topoisomerase II and doxorubicin can be repaired by either non homologous end-joining or homologous recombination repair, as both pathways are specifically responsible for the repair of DNA double-strand breaks. The DNA-dependent protein kinase catalytic subunit in non homologous end-joining and Rad51 in homologous recombination repair are essential for each of these pathways. If it was possible to specifically target these proteins or other antagonistic mechanisms of doxorubicin-induced cell death, which may be activated in response to doxorubicin treatment, chemosensitivity of tumours could be restored and chemotherapy made more effective. Hence it was the purpose of this study to investigate proteome-wide changes in protein expression in response to drug treatment, as well as specifically analysing alterations in the protein levels of the DNA-dependent protein kinase catalytic subunit and Rad51. Global changes in protein regulation of breast and breast cancer cells were investigated using mass spectrometric and electrophoretic analysis techniques. These experiments however, could not reproducibly identify any genuine drug-induced changes in protein levels, as only proteins of relatively high abundance could be analysed. Immunoblotting results however, showed that Rad51 was differentially regulated in a cell line- and drug dosage-dependent manner, while levels of the DNA-dependent protein kinase catalytic subunit remained largely unchanged. Furthermore, increased levels of topoisomerase II alpha protein were also detected. In addition, immunohistochemical analysis demonstrated that both Rad51 and the DNA-dependent protein kinase catalytic subunit could be independently overexpressed in breast tumours and therefore may represent potential targets for selectively enhancing chemosensitivity of breast cancers.

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39

Magan, Natisha. "Protein interactions at the human topoisomerase II[alpha] promoter : a thesis presented to Massey University in partial fulfilment of the requirement for the degree Doctor of Philosophy in Biochemistry." 2009. http://hdl.handle.net/10179/1292.

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Among women in the 45 to 64 age group, over half of the recorded deaths are from cancer, breast cancer being the most common. Just over 30% off all deaths in New Zealand women is caused by breast cancer. Treatment of cancer is difficult, not only due to the physiological and immunological similarities between a cancer cell and a normal cell, but also due to the high cardiotoxicity of many treatments, and also the problems related with the development of resistance. Approximately 40% of the cancer cells treated with the chemotherapy drug doxorubicin will become resistant to treatment. Drug efficacy is strongly associated with the proliferation status of a cell, as cancer cells divide rapidly, this can often be the defining factor between effective treatments or the development of resistance. Central to this proliferation status is an enzyme known as topoisomerase IIa. This essential enzyme is expressed in all cells and is required to relieve the torsional stress in DNA that is created during normal cellular processes. A number of commonly used anti-cancer drugs have been found to target topoisomerase IIa in cancer cells and significantly, during the development of drug resistance levels of topoisomerase IIa enzyme have been found to be reduced in some cell lines and tumours. There are a number of factors that can modulate the amount of topoisomerase IIa enzyme found in a cell, and one of the ways to understand this is to examine the regulation of the topoisomerase IIa gene, most importantly the proteins that interact with the promoter region to direct transcription. The human topoisomerase IIa promoter has been found to be regulated by a number of transcription factors that can bind to their cognate sequences. The introduction of mutations within specific sequences of the topoisomerase IIa promoter has enabled the identification of a key regulatory region within the promoter, a sequence of DNA that encompasses both the ICB1 and GC1 regulatory elements. Transcription factor NF-Y is found to bind to ICB1 element, whereas transcription factors Sp1 and Sp3 have been found to associate with the GC element. However this region of the promoter was also found to bind a fourth uncharacterised component. This research aims to further define the protein components that are found to bind to this important ICB1/GC1 regulatory region and distinguish the protein-protein and protein-DNA interactions that are important for the regulation of the human topoisomerase IIa promoter.

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40

Nikmatullah, Aluh. "Regulation of ethylene biosynthesis in vegetative tissues of white clover (Trifolium repens L.) during water deficit : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology, Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand." 2009. http://hdl.handle.net/10179/1229.

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The investigation in this thesis is divided into two parts. In the first part, the expression and accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO), the enzyme which catalyses the final step of ethylene biosynthesis in higher plants, is examined during exposure of white clover (Trifolium repens L.) to a water deficit. The second part of this thesis is focused on the identification and characterisation of a water-deficit-associated ACC synthase (ACS), the enzyme which catalyses the production of ACC. In the first part, two white clover varieties with differing sensitivity to water deficit, a drought-tolerant Tienshan ecotype and a drought-sensitive Grasslands Challenge cv. Kopu II cultivar were exposed to two water deficit treatments: one cycle of water deficit (designated non-prestressed; NPS) and a water deficit, a rehydration period and then a second water deficit treatment (designated pre-stressed; PS) in the New Zealand Climate Environment Laboratory (NZCEL). Treatments were terminated when the petiole elongation rate (PER) in the first fully-expanded leaf reached zero. Water relations, growth responses, the expression of the white clover ACO genes, TR-ACO1 TR-ACO2 and TR-ACO3 and the accumulation of two of the corresponding proteins, TR-ACO1 and TR-ACO2, were then examined. The soil water content (SWC) and leaf water potential (LWP) measured in both varieties and in both water deficit treatments declined progressively. The rate of decline in SWC and LWP was slower in the Tienshan ecotype with no difference between the NPS and PS treatments. However, the LWP in the Tienshan ecotype at the point at which the PER ceased was less negative (ca. -1.4 MPa) compared to Kopu (ca. -1.7 MPa). In addition, the decline in the PER differed between NPS- and PS-treated Kopu. In the NPS-treated Kopu, the PER was maintained at a high rate when plants were exposed to SWC above 18%, but declined sharply as the SWC declined further. However, in the PS-treated Kopu, the PER declined more progressively in a similar pattern to that determined for NPS- and PS-treated Tienshan. Expression of TR-ACO1 and accumulation of TR-ACO1 was observed in the apical structure of the stolon. As the water deficit progressed, no significant alteration in TR-ACO1 expression and TR-ACO1 protein accumulation was observed in the apical structures of both the NPS- and PS-treated Tienshan ecotype suggesting some degree of protection of the meristem tissues in this more drought-tolerant variety. However, a discernable decline in expression of TR-ACO1 and accumulation of TR-ACO1 protein was observed in the NPS-treated Kopu suggesting some degree of tissue injury in this more drought-susceptible variety. However, after the pre-stress (PS) treatment, no real changes in TR-ACO1 expression and TR-ACO1 protein accumulation were observed, in common with the observations for the NPS- and PS-treated Tienshan ecotype suggesting that meristem protection may now be occurring. The results suggest further that the pre-stress treatment of the more drought-susceptible Kopu may result in a degree of acclimation to the water deficit. For the first-fully expanded leaves, expression of two transcripts, TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein was monitored as the SWC decreased. The expression of TR-ACO2 and accumulation of TR-ACO2 decreased as the water deficit progressed in both the NPS- and PS-treated Tienshan ecotype and correlated with the decrease in PER. By contrast, in the NPS-treated Kopu, TR-ACO2 expression and TR-ACO2 protein accumulation increased, but again, after a period of pre-stress, TR-ACO2 expression and TR-ACO2 accumulation decreased, in common with the Tienshan ecotype. Again, the pre-stress treatment of the drought-susceptible Kopu may result in a degree of acclimation to the water deficit such that the responses become similar to those observed in the more drought-tolerant Tienshan ecotype. However, in both NPS- and PS-treated Tienshan and Kopu there was no significant alteration in the expression of TR-ACO3 in the first fully-expanded leaf. The expression of TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein were also observed in the second fully-expanded leaves (an older tissue). Again similar patterns in the expression of TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein were observed in both NPS- and PS-treated Tienshan and Kopu. In these leaves, expression of TR-ACO2 and accumulation of TR-ACO2 protein decreased as the water deficit progressed, but expression of TR-ACO3 increased as the water deficit decreased to less than 10%. These results suggest that responses of younger tissues (apical structure; first-fully expanded leaf) maybe the critical determinant for the tolerant (or otherwise) of white clover plants to water deficit. In the second part of this thesis, four ACS genes were identified from the Tienshan ecotype exposed to water deficit and designated TR-ACS-T. Three of these were similar to previously identified TR-ACS genes from Grasslands Challenge genotype 10F while the fourth was a novel gene designated TR-ACS4-T. TR-ACS4-T is 64%, 64% and 63% homologous to TR-ACS1-T, TR-ACS2-T and TR-ACS3-T, respectively in terms of nucleotide sequence. In the GeneBank database, TR-ACS4-T shares highly homology to ACC synthase sequences from a wide range of tissues including seedlings and fruit tissues, in addition to a high homology to ACS genes induced in auxin-, wounding- and ethylene-treated tissues. The pattern of TR-ACS4-T expression observed during leaf development suggests that the gene is expressed initially in the apical structures and in the newly initiated leaves, and then again in the later mature leaves and those at the onset of senescence. Expression decreases again during senescence. TR-ACS4-T expression is not altered by water deficit, but is induced by both ethylene and NAA treatment, but the auxin-induced TR-ACS4-T is mediated by ethylene treatment.

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41

Rutherford, William Ernest. "Epigenetic characterisation of the 06 methyl-guanine DNA-methyltransferase promoter in New Zealand melanoma cell lines : a thesis presented to Massey University in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand." 2010. http://hdl.handle.net/10179/1414.

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New Zealand has the second highest incidence of melanoma skin cancer in the world. Chemotherapy is the standard treatment for melanoma derived tumours which have undergone metastasis and current therapies have limited benefit. There is a great need for new therapies and to increase the efficacy of current therapies. Temozolomide (TMZ) is a chemotherapy agent effective in the treatment of both metastatic melanoma and glioblastoma (brain cancer), although TMZ resistance has been observed in many tumours. The activity of the DNA repair enzyme O6 methyl-guanine methyltransferase (MGMT) is thought to be largely responsible for TMZ resistance. MGMT protects the cell from the effects of TMZ by removing cytotoxic lesions placed on the DNA. Mechanisms of regulation of MGMT expression remain unclear in melanoma. DNA methylation at the MGMT promoter has been linked to MGMT silencing in some cancers and has been associated with specific chromatin modifications. The present study was aimed at investigating the promoter methylation status of MGMT in primary melanoma cell lines using a new technique named methyl DNA immuno-precipitation (MeDIP). Next, the chromatin immuno-precipitation (ChIP) method was used to examine post translational modifications on the surrounding chromatin. The data obtained was correlated with both MGMT transcription levels and TMZ sensitivity. The promoter methylation status of MGMT has been used to predict the clinical responsiveness of glioblastoma patients to TMZ. Establishing the regulatory mechanisms of MGMT expression in melanoma patients would validate a means to predict clinical responsiveness to TMZ. Furthermore, establishing mechanisms of MGMT silencing may provide the basis for future clinical trials of novel therapies for melanoma and glioblastoma.

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Dissertations / Theses on the topic 'Fields of Research – 270000 Biological Sciences – 270100 Biochemistry and Cell Biology'

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