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Genetic manipulation of folate in riceAnukul, Nampeung January 2010 (has links)
Folate malnutrition is a major problem in many countries around the world especially in Asia and Africa. Stable foods such as rice contain very low amounts of folate. White rice which is the most popular form for human consumption contains less than 80 µg folate per 100 g. Given that it forms a major part of the South East Asian diet, rice represents an important target for enhancing folate levels. The objectives of this thesis is study the mechanisms that regulate total folate levels in rice grains and attempt to enhance and stabilise folate in rice endosperm. Three main strategies were adopted. First, the natural variation of folate biosynthesis gene expression was probed using RT- qPCR. Second, functional genomic approaches were used to manipulate the activity of rice folylpolyglutamate synthetase (FPGS), the enzyme which adds glutamate residues to folate. Third, genetic engineering was used to express FPGS enzymes and mammalian folate binding proteins in rice endosperm. RT-qPCR revealed that the variation in folate biosynthesis transcript abundance was closely correlated with total folate levels among rice varieties. High transcript abundance of all folate biosynthesis genes was associated with high total folate levels in Moroberekan rice mature seed. Comparative genomic studies revealed that rice FPGS is encoded by two distinct genes, FPGS Os03g02030 and FPGS Os10g35940. Transcript abundance of FPGS Os03g02030 appeared higher than that of FPGS Os10g35940 in seed, whilst, transcript abundance of FPGS Os10g35940 was higher in leaf. To determine the function of the FPGS Os03g02030 gene in rice seed, a TDNA knock out line was characterised. Disrupting Os03g02030 gene expression resulted in delayed seed maturation and decreased mono- and polyglutamylated folate pools in mutant seed. RT-qPCR detected an increase in the transcript abundance of folate biosynthesis genes in seed of the knock out plant, whereas the folate deglutamylating enzyme y-glutamyl hydrolase (GGH) mRNA level was reduced. A potential feedback mechanism to maintain folate abundance during rice development was uncovered through the alternative functional FPGS Os10g35940 activity and reduction of folate breakdown. Protein-bound folate forms are better protected from oxidative degradation resulting in greater folate stability (Suh et al. 2001). Two rice FPGS and mammalian folate binding proteins was successfully introduced into rice endosperm using Agrobacterium based transformation in an attempt to retain and stabilise folate pool within rice endosperm. Analysis in terms of folate abundance and bioavailability will form part of future studies.
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Biological characterisation of HER2 amplified breast cancerBarros, Fabrício Félix Tabuada January 2013 (has links)
Breast carcinoma is the most frequent type of cancer affecting women. Among the recently described molecular and phenotypic classes of breast cancer, human epidermal growth factor receptor 2 (HER2)-positive tumours are associated with a poor prognosis. HER2 status is currently assessed in routine breast cancer reporting using immunohistochemistry (IHC) in addition to in situ hybridisation (ISH) in borderline cases. The ability of HER2 gene status to predict response to targeted therapy (Trastuzumab) is well documented. However, prognostic information provided by IHC expression categories and prognostic value added by using ISH in borderline cases remains unclear. HER2 plays an important role in cancer progression being targeted to provide predictive and prognostic information. Moreover, HER2 is related to cancer resistance against a variety of therapies; however, trastuzumab has proved successful in treatment of this subgroup. Nevertheless, patients may acquire resistance to this drug after a period of treatment, which indicates that other molecular mechanisms might influence success of this therapy. Dimerisation between members of the HER family may contribute to resistance against treatments due to different combinations that trigger different downstream pathways. This is promoted by ligands, which are expressed as transmembrane precursor protein molecules and have a conserved epidermal growth factor-like domain. Through resistance to trastuzumab, other drugs are being developed to interact in different domains of HER2 protein. The study of interaction between receptors/ligands will characterise specifically their signalling pathway and understand which strategy to acquire. The main aim of this thesis was to assesses the status of HER2 protein (IHC), HER2 gene (chromogenic ISH) and HER heterodimers (in situ proximity ligation assay (PLA)), including HER2/EGFR, HER2/HER3 and HER2/HER4, in two BC series prepared in tissue microarray format; a series of consecutive primary operable BC cases (n = 1858) including HER2+ trastuzumab naïve cases (TrN, n = 221), the second series of HER2+ trastuzumab adjuvant treated cases (TrT, n = 143). Therefore determining the biological characterisation of these biomarkers by associating against clinicopathological parameters, survival outcome and understand the trastuzumab therapy value. There was excellent overall concordance between HercepTest negative (scores 0/1+) and positive (3+) with CISH positive/negative (defined as HER2/Chr17 copy number ratio of ≥ 2; p < 0.001). Kaplan-Meier analysis for breast cancer specific survival (BCSS) and disease free interval (DFI) revealed statistically significant differences between HER2 positive and negative cases detected by HercepTest and CISH (p < 0.001). Interestingly, it was identified that HercepTest 2+ non-amplified cases were not significantly different with those amplified 2+ or 3+ cases with respect to their behaviour (BCSS and DFI). The results revealed an inverse association between the HER heterodimerisation status and hormone receptor status (p < 0.001), and a significantly worse outcome amongst cases revealing high levels of all heterodimers (p < 0.001). Among ER+ cases, the heterodimers high levels were significantly associated with worse prognosis (p < 0.001) overall. However amongst the two HER2+ populations dimerisation status did not show an association with patient outcome. The overall concordance between HercepTest and CISH analysis for HER2 status was excellent. All HercepTest 2+ cases identified were observed to have poor outcomes similar to those HercepTest 3+ cases regardless of gene amplification status. In the current clinical environment cases exhibiting IHC 2+ and non-amplified gene HER2 status will not be offered targeted HER2 therapy but do exhibit aggressive clinical behavioural characteristics. Even though those patients with high levels of the HER2 truncated form, p95HER2, have shown poor outcome, this biomarker does not reveal any extra findings comparing with the HER2 expression results. Beside HER2, EGFR is the only monomer that reveals prognostic value amongst the breast cancer patients. Tumours exhibiting high levels of HER heterodimerisation have an adverse prognosis, however in the context of HER2+ breast cancer no association with clinical outcome was observed regardless of use of trastuzumab treatment. HER2/HER3 heterodimerisation status assessed in multivariate analyses has shown that this protein-protein interaction is associated with a poor prognostic outcome, which needs further investigation and assessment of clinical utility to use in the future in breast cancer treatment decision. Further quantification analysis of dimer/ligand complex using PLA of other HER family members may be useful to identify subset of patients associated with distinct outcome, response to treatment and relationships with HER signalling related biomarkers.
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Cytosine methylation and hydroxymethylation at the leptin promoterAl-Azzawi, Haneen January 2013 (has links)
Leptin is an important hormone well known for its role in regulating energy intake and expenditure. DNA methylation levels at the leptin promoter in adult tissues appear to correlate with environmental stresses experienced during early life. This suggests that, once established in early life, DNA methylation is stably transmitted over successive cell generations. The aim of the work presented in this thesis was to determine factors that contribute to the establishment and maintenance of this epigenetic mark at the leptin promoter and to investigate the individual roles of cytosine methylation and cytosine hydroxymethylation at this genomic locus. No effect of a high fat prenatal diet was observed on leptin promoter DNA methylation levels in the adipose tissue of pigs. However, this genomic region exhibited intermediate levels of DNA methylation, which is usually associated with gene silencing, even though adipose tissue is the primary site of leptin expression. Double stranded methylation data obtained from DNA methyltransferase (DNMT) mutant mouse embryonic stem cells (mESCs) was used to investigate the contributions of the three catalytically active DNMT enzymes to leptin promoter DNA methylation patterns. Depletion of DNMT3b resulted in increased methylation levels at the leptin promoter, consistent with preliminary data from mutant DNMT3b mouse tissues where similar increases in methylation levels were observed at specific CpG dinucleotides. Two mESC lines, either hypomethylated or hypermethylated at the leptin promoter, were tested for leptin mRNA expression and neither cell line expressed leptin mRNA, suggesting that some form of methylation may be required for leptin expression. To further investigate the relationship between DNA methylation and leptin expression, in vitro differentiated adipocytes were analysed. 3T3-L1 preadipocytes, which do not express leptin, exhibit high levels of DNA methylation and these high methylation levels are maintained after the cells differentiate into leptin-expressing adipocytes. Induction of cytosine hydroxymethylation at the leptin promoter was detected in differentiating and mature adipocytes and evidence is presented to suggest that cytosine hydroxymethylation at the leptin promoter correlates with leptin expression.
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Identification of novel transcripts of CHRNA7 and CHRFAM7A in airway epithelial cellsAhmad, Omar Akram Jerjees January 2013 (has links)
Background: RNA splicing is a crucial process for delivering the appropriate message for protein synthesis. Most genes are affected by alternative splicing, and among these is CHRNA7. This gene encodes for the nicotinic acetylcholine α7 receptor subunit that is involved in the cholinergic anti-inflammatory pathway. This anti-inflammatory pathway is considered an important part of the human body’s defence line against tissue injury or infection and causative mechanism in COPD. Aim: The aim of the present study was to investigate the role of alternative splicing on the nature of the transcripts generated by CHRNA7 gene and its partial duplicate, CHRFAM7A. Methods: Airway epithelial cell lines, A549 and BEAS2B, were mainly used as targets for testing alternative splicing. RT-PCR, TA cloning and gel extraction methods were used for testing CHRNA7 and CHRFAM7A transcripts. Following RT-PCR, the resulting product band intensities were analysed using densitometric analysis tools. This was followed by the use of several bioinformatics analysis tools to predict the protein structure for the resulting transcripts. For one of the detected transcripts, minigene methods were used to test for the source of expression. Results: A novel transcript missing exon 9 is reported for the first time. Both genes showed the expression of full length and the novel transcripts (missing exon 9) at similar ratios (~2:1). These results could be detected in immortalised cell lines from human alveolar and bronchial epithelial cells (A549 and BEAS2B, respectively) and in BE (2)-c cells (neuroblastoma cells with bone marrow metastasis). The same results were shown when primary human peripheral blood monocytes cells (PBMC) were tested. This means that the effect of missing exon 9 is not tissue-specific, and is not only found in cancerous cells, indicating that it could be a common feature of splicing for these two genes. Furthermore, another novel transcript was detected which is inserted exon 9b. The initial RT-PCR experiments seemed to suggest that this was derived from CHRFAM7A only. The use of minigene methods showed that this transcript could be expressed from both genes, CHRNA7 and CHRFAM7A, but a single nucleotide base within the inserted sequence (at position 77 from the 5` end) could play a role in enhancing of exon 9b in the mRNA transcripts. This base is C allele in CHRFAM7A sequence of exon 9b, while its corresponding base in CHRNA7 is G allele that has less prominent effect on exon 9b inclusion. Conclusion: CHRNA7 and CHRFAM7A express novel transcripts in different human cells that are missing exon 9. This could be due to inactive splicing factors that are required for recognition of exon 9 as a constitutive exon. For exon 9b transcripts, these lie within the common sequence of CHRNA7 and CHRFAM7A, and it seems that the presence of C allele at position 77 could enhance the inclusion of exon 9b in CHRFAM7A more than the presence of G allele in CHRNA7 sequences. The results shown in this study implicate a possible regulatory role of the transcripts detected on the control mechanism exerted by CHRFAM7A on CHRNA7. These results help to suggest a possible role of in the development of COPD in the form of inflammatory/anti-inflammatory control imbalance.
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Folate profiling in wild and transgenic riceAbilgos Ramos, Riza January 2010 (has links)
Quantitative profiling of mono- and polyglutamyl folates in rice was achieved using the microbiological assay (MA) and a newly developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method. MA was used to screen 51 rice cultivars for their total folate content and LC-MS/MS was employed to measure naturally occurring mono- and polyglutamated forms of the vitamin in wild type, FPGS Os03g02030 knockout and transgenic lines with overexpressed FPGS genes and with folate binding protein from cow’s milk (cFBP) and rat’s liver (GNMT). Natural variation among rice cultivars in terms of total folate content was measured using MA screening and the validated LC-MS/MS technique of simultaneous profiling of mono- and polyglutamated folates through MeOHAA/PO4 extraction revealed that the naturally-occurring species in wild type rice are 5-CH3-H4PteGlu, 5/10-CHO-H4PteGlu, 5-CH3-H4PteGlu4, 5-CH3-H4PteGlu5 and 5/10-CHO-PteGlu5. There was a general decrease in these folate forms in the FPGS Os03g02030 knockout rice line while a dramatic increase was observed in overexpressed FPGS, cFBP and GNMT compared to Nipponbare in terms of 5-CH3-H4PteGlu4, 5/10-CHO-H4Pteglu5, 5-CH3-H4PteGlu6, and 5/10-CHO-H4Pteglu6 levels, resulting in a 2.5 to 8.8-fold increase in the total folate pool in the unpolished grains of rice. This study looked at the role of the two FPGS genes (Os03g02030 and Os10g35940) found in rice and the possible effect of introducing folate binding proteins (cFBP and GNMT) in terms of the overall folate profile in rice which can be exploited in breeding programmes designed to enhance folate content in staple crops like rice.
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Characterization and utilization of self-assembled diphenylalanine nanotubesXu, Kairuo January 2011 (has links)
Diphenylalanine (FF) peptide is the core-recognition motif of β-amyloid polypeptide, a peptide associated with diseases such as Alzheimer’s and which is known to be capable of self-assembly. FF has attracted interest in nanotechnology due to the physical and chemical stability and mechanical rigidity of the self-assembled nanotube form of the peptide. A number of promising applications of FF nanotubes have previously been explored. To extend this work to biomedical and pharmaceutical areas, an improved understanding of the physicochemical properties of FF tubes, together with the influence of assembly conditions, cytotoxicity properties and potential in drug delivery field are presented in this thesis. The studies presented in Chapter 2 address the self-assembly of FF peptide prepared by two known methods of preparation, one aqueous based, the other utilizing an organic solvent. A range of complementary characterization methods is applied including atomic force microscopy, scanning electron microscopy, focused ion beam-scanning electron microscopy, X-ray powder diffraction, and Raman Spectroscopy. The investigations reveal differences in morphology of the tubes formed by the different preparation methods. The aqueous based method produces tubes that are long, straight and unbranched and are consistent with previous work. The alternative organic solvent method produced tubes that are shorter and narrower. In addition, these tubes displayed flexibility and nucleation points. Following on from these findings, a proposed mechanism of tube growth is discussed. Chapter 3 further extends the investigation to the biological field. Possible cytotoxicity issues are studied using a MTT assay on a HeLa cell line. Moreover, total internal reflection microscopy was applied to investigate HeLa cell behaviour in the presence of FF nanotubes. The results from these studies reveal that the nanotubes and FF peptide do not cause any mitochondrial related damage to HeLa cells. Furthermore, short tubes were observed to be taken up by cells through a suggested macropinocytosis pathway. Finally, in Chapter 4 the focus turns to the investigation of the potential of FF tubes as drug carriers in drug delivery. Here, successful synthesis of drug-loaded FF tubes is presented with two model drugs. The physical characterization of the complex formed under different conditions using scanning electron microscopy reveals FF nanotube self-assembly is a drug concentration and solvent type dependent process. Finally, in vitro drug release from FF nanotubes is performed and compared to that of the drug alone. Extended drug release is observed for both drug candidates and release mechanisms are proposed. The results presented throughout this thesis demonstrate the versatility of self-assembling FF peptides for the formation of tubular nanostructures with different morphologies and physical properties under different conditions. The assembled nanostructures appear non-toxic to cells and offer promise in drug delivery as novel drug carriers.
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The nematicidal effect of cysteine proteinases on the root knot nematode Meloidogne incognitaGorny, Samuel Victor January 2013 (has links)
Despite current control measures, plant parasitic nematodes are estimated to be responsible for > $100 billion of damage to worldwide crop production per annum. Current nematicides are highly toxic, and due to health and environmental safety concerns, many are being withdrawn from the market under directive 914/414/EEC. Alternative control strategies are urgently required. The cysteine proteinases papain, actinidain and recombinant endoproteinase B isoform 2 (R.EP-B2) have been demonstrated to affect the mobility of M. incognita J2s; 50.7 μM R.EP-B2, 101.7 μM papain and 200.3 μM actinidain immobilised 50% of the M. incognita population. Papain has also been demonstrated to affect the infection of plants by M. incognita, 5 μM papain reduced the attraction to and invasion of A. thaliana by 41.2 + 25.6% and 80.4 + 10.5% respectively. M. incognita J2s showed extensive damage to and removal of the cuticle when treated with 100 μM papain. MALDI-TOF analysis identified a number of M. incognita proteins affected by the papain treatment; of particular interest were a cuticle preprocollagen and a rhodopsin-like GPCR chemoreceptor. Proteins of these types are essential for movement and host location, disrupting their function helps to explain the loss of mobility and reduction in A. thaliana infection observed in the bioassays. Finally transgenic A. thaliana was generated with the barley cysteine proteinase endoproteinase B isoform two under the control of the root cap specific MDK4-20. The preliminary testing of these plants showed a reduction in root invasion similar to that obtained with papain.
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Nutritional modulation of hepatic lipid metabolism in health and diseaseJohnston, Richard David January 2012 (has links)
The objective of this thesis was to assess the impact of altering macronutrient intakes on hepatic lipid metabolism. Two separate studies were performed, with liver triglyceride content being the principal outcome of both. In the first study 32 healthy and centrally overweight males were randomised to 2 periods, each of 2 weeks, of either a high fructose or glucose intake in a non-crossover fashion. Isoenergetic status was maintained by providing foodstuffs during the first period, followed by a 6 week washout and then a second period of ad libitum overfeeding. In the second study 55 patients with biopsy proven non-alcoholic fatty liver disease were randomised to 3 months 5g a day of capsules containing either n-3 polyunsaturated fatty acid or oleic enriched sunflower oil. The main findings are summarised. High intakes of fructose and glucose in the isoenergetic period resulted in a stable weight, and no change in hepatic, serum and ectopic triglyceride content. There was a raised serum uric acid with fructose. During the hyperenergetic period there was a tendency for greater uric acid with fructose, whilst both groups had a matched weight gain, elevation of liver biochemistry and an increase in hepatic, serum and muscle triglycerides. Changes in liver biochemistry and triglycerides were associated with changes in weight. During both periods there was calorimetric evidence for a shift in whole body metabolism towards that reflective of a high carbohydrate intake. There was no alteration in renal function or cardiovascular haemodynamic parameters or consistent change in insulin resistance. The n-3 polyunsaturated versus oleic acid study resulted in significant alterations of serum fatty acid profiles between the groups, which were in line with the capsules’ contents. These changes however failed to translate, in the whole group, to any detected metabolic or hepatic changes beyond a reduction in serum triglyceride with n-3 polyunsaturated fatty acids. Only 43 of the 55 patients had elevated liver triglycerides on baseline MRI. Amongst this 43 there was a reduction in liver triglyceride with n-3 polyunsaturated fatty acids, but no other associated metabolic changes. The uric acid findings support the notion of fructose and glucose differing in their pre triose metabolism. There was however no differing outcomes in terms of lipid synthesis or storage. There was a suggestion of reduced liver triglycerides with n-3 polyunsaturated fatty acids though this was an isolated result only found amongst those with a steatotic liver at baseline. Ultimately the exquisite sensitivity of the liver to nutrient intakes was highlighted by the 0.8% gain in weight in the fructose / glucose study resulting in a 24% increase in liver lipid. This affirms the notion that dietary energy intakes have a profound influence on hepatic metabolism, but there is no evidence from this thesis that this influence is macronutrient specific. In the future macronutrient comparisons need to be made.
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The role of aquaporins in the developing ovarian follicleWilliams, Leanne January 2012 (has links)
The growth of ovarian follicles is well documented in terms of hormonal control, however the fluid dynamics of antral follicle growth is less well understood. Aquaporins (AQP) are transmembrane water channels which facilitate the passive movement of water. In mammals 13 AQPs have been identified in a vast range of tissue types. In terms of ovarian AQPs there is currently a paucity of information. Recent studies in rat, pig and human have revealed the presence of ovarian AQPs, but in doing so have also highlighted a lack of consensus on AQP-type and location. The main aim of this study was to investigate the potential role of AQP in antral follicle growth. The first objective was to identify tissue expression and localisation of AQP proteins in the bovine ovary. This required the characterisation of a panel of polyclonal serum antibodies. Immunohistochemistry (IHC) was then used to identify AQPs and to detect changes in protein expression during follicular growth. Aquaporin 1 was found in most vascular endothelium; it was plentiful in capillaries surrounding antral follicles and increased in abundance as vasculature increased with follicle development. Aquaporin 2 was not found in bovine ovarian tissue and the remaining antibodies were deemed too nonspecific to permit reliable conclusions. The second objective was to investigate, via RT-qPCR, mRNA levels of AQPs in granulosa and theca cells isolated from preantral, through to large preovulatory follicles. Transcripts of AQP 1, -3, -4, -5, -7 and -9 were detected in both the granulosa and theca of antral follicles with expression levels generally higher in theca. The expression of AQP 1, -5, -7 and -9 was initiated in the theca cells of early antral follicles. Finally, swelling assays using bovine and porcine granulosa cells demonstrated the ability of granulosa to swell. This was inhibited by HgCb which is characteristic of AQP function. Porcine granulosa cells incubated with androgen swelled by 27%, this effect was inhibited by hydroxyflutamide. Protein analysis of AQP5 via IHC and Western blotting showed possible up-regulation in porcine follicles. RTqPCR did not reveal AQP5 transcript, the reasons for this currently remain unclear. In conclusion, this study has revealed for the first time the involvement of AQPs in bovine ovarian follicle development, with AQPI, -5, -7 and -9 potentially playing a pivotal role in antrum formation. The AQP system in porcine granulosa cells is androgen sensitive however identification of the AQP/s responsible needs further investigation. The evidence from this investigation suggests a role for AQPs in facilitating follicle growth. The stage-dependent expression of certain AQPs and the androgen sensitive porcine granulosa cells reveals the possibility that AQPs may be modulated by follicle-regulating hormones.
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Characterising the binding interactions and thermodynamics of odour binding protein 3Portman, Katherine Louise January 2012 (has links)
Odour Binding Proteins (OBPs) are found in the olfactory system of a range of species. Whilst invertebrate OBP function is well understood, the exact function of these proteins in the vertebrate nasal mucus is not fully understood. Multiple subtypes of rat OBPs have been identified and found to share less than 30% sequence identity. Studies have suggested each rat OBP binds to particular sets of odours, which may afford them a particularly important role within the olfactory system, pre-sorting odours. This study focuses on OBP3, closely examining the binding interaction of this protein with a range of odours. This has been done using Isothermal Titration Calorimetry which revealed that the binding of the highest affinity ligands, the heterocyclic compounds, is enthalpically driven. A defined odour series, the gamma-lactones showed that despite increasing ligand size and hydrophobicity, the free energy of binding of these ligands is maintained. Interactions with both 2-isobutylthiazole and the gamma-lactones were examinedusing NMR spectroscopy, which required the NMR assignment of OBP3 to be determined. In addition a homology model of OBP3 was created in order to structurally map the per-residue changes of OBP3 upon binding. It has been found that OBP3 is able to subtly adjust in order to accommodate each of these ligands. Protein engineering of the OBP3 binding pocket has been used to highlight the importance of its size and hydrophobicity. The importance of a tyrosine residue that appears to cover the opening to the binding pocket and is conserved across both the aBPs and the lipocalins family they are part of, has been demonstrated. Mutagenesis has also revealed the importance of a number of key residues for the binding of 2-isobutylthiazole. The ability to rationally improve the affinity of OBP3 for a particular odour has also been demonstrated.
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