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14-3-3 mediated forward transport and heterodimerisation of acid-sensitive K2p channelsLowry, Eleanor J. January 2010 (has links)
No description available.
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An investigation into the performance of bioluminescence-based measurementsDriscoll, Mark B. January 2002 (has links)
This work describes an amplification system, which can greatly increase the amount of ATP available for detection by bioluminescent methods, increasing the sensitivity. The system makes use of an enzyme, adenylate kinase (AK), present in virtually all living cells. The sensitivity of the assay was increased by l0 to 100-fold. A suitable extractant needed to be developed, which had the role of allowing intracellular AK to be detected and assayed, but which was not harmful to the enzyme or ATP, and did not interfere with the assay in any way. The assay required ADP as a substrate, and contaminant ATP from commercial ADP preparations needed to be removed beforehand. Initially, HPLC was used to do this on an analytical scale but was developed into a large-scale preparative procedure employing ion-exchange chromatography. The ATP contamination was reduced to a concentration of less than 1 x 10-6 %. Similarly, commercial luciferase enzymes for the detection of ATP contain contaminating AK, and several approaches were developed to remove this, including Iso-electric Focusing and low-ionic strength precipitation. Over 98% of contaminant AK was successfully removed. Industrial applications of the system were successfully proven, such as incorporation of the assay into a continuous flow luminometer, equipped with a timed 'delay loop' to allow ATP levels to be amplified prior to detection with luciferase in a 'flowcell' detection system. The assay has also been shown to be of significant use in the brewing industry, for the assessment of yeast quality. Current methods involve the assay of protease activity, which is time consuming, expensive, and of poor sensitivity. The AK assay was found to provide an extremely sensitive method for the determination of yeast leakage and autolysis, allowing selection of yeast for re-pitching as part of yeast management in the brewing sector.
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The effect of casein hydrolysate formed by trypsin or bifidobacterium animalis subsp. lactis (Bb-12) culture on cholesterol and angiotensin converting enzyme (ACE) inhibitionAl-Haj, Omar Amin January 2008 (has links)
The hypocholesterolemic effect of peptides present in crude casein hydrolysate using trypsin from Bifidobacterium animalis subsp. lactis (Bb-12) culture grown in casein solution or in MRS broth were compared with the control (unhydrolysed crude casein). These samples have shown to reduce cholesterol level in vitro to varying degree between 24-87%. The unhydrolysed crude casein (control) has shown not to have a reducing effect on cholesterol level. The reduction level of cholesterol was found to be dependant on the degree of hydrolysis. Fractions obtained after size exclusion from crude casein hydrolysate formed by trypsin after 48h hydrolysis have shown to reduce cholesterol level to degree vary between 2.7% to 50%. Bb-12 or trypsin crude casein hydrolysates were also found to possess angiotensin-converting enzyme (ACE) inhibitory activity in vitro to a varying degree between 29% and 38%, respectively. Unhydrolysed crude casein showed no real effect on ACE inhibitory activity. Trypsin hydrolysate has slightly more effect on ACE activity than Bb-l2. A relationship was also found between ACE-inhibitory activity and degree of hydrolysis. Fractions of crude casein hydrolysate after 48h hydrolysis with trypsin by size exclusion have shown to have ACE-inhibitory activity varying between 37% and 50%. Several identified ACE-inhibitory peptides from active fractions were found to have Phe, Trp, Tyr, Arg, Lys or Pro at their ultimate C-terminal position, making them a possible candidate for ACE-inhibitory activity. Further analysis on the profiles of casein and whey protein fractions, obtained by fast protein liquid chromatography (FPLC), of skimmed milk and four different commercial types of probiotic health drinks, and casein profile of added Bb-12 culture were investigated to study the possible effect of probiotics on milk proteins. Differences in protein-profile were found between the control and the four commercial probiotic health drinks as well as with added Bb-12 culture. Whey proteins were more resistance to hydrolysis than caseins.
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Bioactivity of casein hydrolysates fractions with reference to their in-vitro radical scavenging, hypocholesterolemic and hypotensive propertiesIrshad, Imran January 2011 (has links)
The bioactivities of casein hydrolysates for hypocholesterolemic (HC) and hypotensive (HT) properties, using Angiotensin Converting Enzyme (ACE) inhibition as an indicator, as well as antioxidant properties have been investigated. Sodium caseinate was used as the source for the production of bioactive peptides by enzymatic hydrolysis using trypsin at pH 7.5 and a combination of pepsin at pH 2.0 followed by trypsin (P+T) at different time intervals and up to 4 hours. Crude casein hydrolysate (CH) was further fractionated by membrane separation technique, ultrafiltration, using two membranes with two different molecular weight cut-off at 10 kDa and 1 kDa. The crude hydrolysate was compared with the two collected permeates; one containing a mixture of peptides with molecular weight below 10 kDa and the second with peptides below 1 kDa. In general, the results indicated that the higher level of hydrolysis exhibited higher bioactivities for all the tested samples. Hypocholesterolemic effect of the hydrolysates and the permeates, CH, 10 kDa and 1 kDa, using trypsin only for 4 h shown to reduce cholesterol level by 39.5, 50.1 and 69.6%, respectively. On the other hand, the hydrolysates and the permeates from the combined action of pepsin and trypsin showed higher cholesterol reduction of 44.9, 52.2 and 87 .0%, respectively. ACE inhibition activities of the hydrolystate and its permeates, CH, 10 kDa and 1 kDa from trypsin hydrolysis after 4 hours were 18.9, 29.7 and 5l.4%, respectively. ACE inhibition activities were greatly increased when the combined P+T were used and exhibited 31.1, 52.7 and 82.4% inhibitions, respectively. The antioxidant activity of the three samples (crude casein hydrolysate, 10 kDa & 1 kDa permeates) after 4h hydrolysis using trypsin showed radical scavenging activities of 13.7, 17.3 and 24.4% respectively. However, when using the combined enzymes P+T, the activities were reduced to 13.1, 15.5 and 19.6% respectively.
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Using Drosophila to study the contribution of human kinases to tau toxicityPovellato, Giulia January 2012 (has links)
The major neuropathological feature associated with tauopathies is the aggregation of the microtubule associated protein tau within neurons. Aberrant phosphorylation events on tau contribute to the aggregation during disease. The kinases and the phosphorylation sites on tau responsible for the generation of pathogenic tau forms are still unclear. My thesis proposes to establish a Drosophila model to investigate the human kinases and phosphorylation sites responsible for the generation of human tau toxicity. Expression of human tau in the photoreceptors of transgenic Drosophila leads to a degenerative phenotype proportional to the transcriptional level of the transgene. Since the genomic position of the transgene influences its expression, I used a novel method of transgenesis to insert the human tau transgene in a reproducible position within the fly genome. Using this system, tau isoforms differing for the presence of two N-terminal domains were shown to generate the same level of toxicity in the fly eye. Moreover, in contrast with other studies, the tauopathy-associated mutation R406W on human tau did not cause an enhancement of tau-mediated degeneration of the fly eye. Selected human kinases relevant to tau pathology were tested for their ability to enhance tau toxicity in flies. Only human GSK3β showed a robust enhancement of tau-mediated eye degeneration. For the first time I showed that a human kinase, GSK3β, could specifically phosphorylate human tau in Drosophila on sites not targeted by endogenous fly kinases. In particular, tau phosphorylation of T181, S396 and S404 was found to significantly increase upon GSK3β expression. Site-directed mutagenesis on tau suggested that S404 might play a central role in mediating human tau toxicity in the fly eye. Finally this thesis demonstrates that the Drosophila model of tauopathy here created can be used to assess the importance of human kinases and of single phosphorylated sites in the generation of tau toxicity.
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Investigating the role of the post-transcriptional regulator protein ZFP36L1 in B-cell functionsNasir, Asghar January 2012 (has links)
ZFP36L1 belongs to a class of RNA-binding proteins known as the ZFP36 protein family which consist of three other members namely ZFP36, ZFP36L2 and ZFP36L3. All the members of the ZFP36 protein family have been reported to bind to the ARE in the 3’ UTR regions of the target mRNA (via the zinc finger domains) and subsequently result in the destabilization and degradation of the mRNA. Established targets of ZFP36L1 include TNF-a, GM-CSF, VEGF, IL-3, C-IAP2, NOTCH 1, STAR, STATSb and DIM. The knowledge about the function of ZFP36L1 and its role in post-transcriptional regulation in B-cell development is extremely limited. The primary aim of this project was to investigate role of ZFP36L1 in regulating B-cell development, in particular late B-cell development (plasma-cell differentiation). BCL-1 cells (provide a model system to study plasma-cell differentiation) were found to express relatively high levels of ZFP36L1 and the cytokine-induced plasmacytic differentiation was associated with downregulation of ZFP36L1 expression. In order to determine a direct involvement of a downregulation of ZFP36L1 expression in plasmacytic differentiation process, lentiviruses expressing shRNAs targeting the zfp3611 mRNA were employed to downregulate ZFP36L1 expression in BCL-1 cells. Efficient downregulation of zfp3611 mRNA expression and ZFP36L1 protein expression was established in unstimulated BCL1 .zfp3611 .RNAi cells. It was observed that BCL1 .zfp3611 .RNAi cells produced higher amounts of IgM compared with control cells. This result suggested that a downregulation of ZFP36L1 expression in BCL-1 cells results in an increase in IgM production (a phenotype associated with BCL-1 cells undergoing plasmacytic differentiation). The results seem to be consistent with other studies suggesting a role of ZFP36L1 in negatively regulating differentiation, although this would need to be investigated further.
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Community structure detection in complex biological networksBennett, Laura January 2013 (has links)
With the advent of high-throughput technology there has been a large increase in the availability of biological data, such as interaction data of genes, proteins and metabolites. It is therefore necessary to develop ways in which these data can be efficiently modelled and analysed. Networks over a natural modelling framework for complex biological systems and as such, network theory and related computational approaches have proven important in bioinformatics. A particular facet of network theory that has been employed to analyse biological networks is community structure detection. Community structure is a modular network topology where communities are defined as groups nodes with dense intra-community connections and less dense inter-community connections. Methods to uncover such communities in complex biological networks have the potential to contribute towards a better understanding of the underlying organisation of a system. Consequently, this thesis focuses on the development of a series of mathematical programming models to address various manifestations of the community structure detection problem. The aim is to produce more information-rich models that can accurately represent the features of biological systems; with weighted and unweighted interactions, disjoint and overlapping communities and network dynamics all being considered. First, the detection of disjoint communities in unweighted networks is approached through a two-stage procedure, known as iMod. A mixed integer nonlinear programming (MINLP) model optimises modularity to find an initial partition which is then improved by iteratively solving a mixed integer quadratic programming (MIQP) model. A comparative analysis shows that iMod finds globally optimal solutions for networks of up to 512 nodes and outperforms all other methods tested when applied to larger networks. Subsequently, the MINLP model is generalised to weighted networks, known accordingly as WeiMod. Competitive results are found when WeiMod is compared with several other well known methods from the literature. Next, the work on disjoint community structure is extended to _nd overlapping modules. An MINLP model, known as OverMod, transforms disjoint to overlapping communities through the optimisation of the community strength metric. OverMod is compared with similar methods from the literature and is further assessed on protein-protein interaction (PPI) networks to test the method's ability to extract meaningful biological results. It is shown that proteins assigned to more than one module exhibit topological and functional properties indicative of their strategically important role in the organisation of the PPI networks. The work on disjoint and overlapping community structure concludes with the investigation of a network generated from sequence, protein interaction and co-expression data, for the fungal pathogen, Fusarium graminearum. The functional coherence of communities, properties of multiclustered genes and aspects of virulence-associated genes are all explored in an attempt to link topological and functional features. Finally, the concept of community structure in dynamic networks is explored. Consensus clustering is tackled; de_ned as detecting a single partition of a dynamic network that is relevant across multiple snapshots. This is addressed by extending the previously proposed MIQP and MINLP models such that average modularity across network snapshots is now optimised. A comparison is made with a similar method from the literature showing that the proposed approaches achieve competitive results for small to medium sized networks. Overall, this thesis demonstrates that the exible nature of mathematical programming lends itself well to developing versatile solution procedures for community structure detection. The method evaluations show the proposed algorithms to be comparable to other approaches from the literature and able to detect meaningful results in biological applications. Finally, the methods described in this thesis have the potential to infer important topological-functional relationships and help to provide insight into the organisation and evolution of biological systems.
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Sensory transduction in enteroendocrine cellsBircher, Jack January 2013 (has links)
The sensing of luminal nutrients is a central function of the gut epithelium where enteroendocrine cells act as sensor cells to secrete hormones in response to the luminal contents. This thesis focuses on voltage gated ion channels and the transient receptor potential (TRP) superfamily in I cells and enterochromaffin cells using the model tumour cell lines STC-1 and KRJ-I respectively. Electrophysiology and PCR was performed on STC-1and KRJ-I model cell lines to elucidate the channels that may regulate hormone release. In KRJ-I cells two outward currents were observed. In STC-1 cells three types of ionic currents were identified; a large outward voltage gated potassium current, a tetrodotoxin-sensitive voltage gated sodium current and a nifedipine-sensitive persistent calcium current. In intracellular calcium studies of STC-1 cells, the TRPA1 agonists cinnamaldehyde and AITC evoked increases in intracellular calcium concentration that could be inhibited by the TRPA1 antagonist AP18. Calcium responses evoked by fatty acids, the bitter tastant denatonium and cold temperatures below 25°C were also attenuated by the TRPA1 antagonist AP18. Studies to elucidate the mechanism of action of these compounds were carried out using tools to block signalling pathways. As enterochromaffin cells are thought to be mechanically active, KRJ-I cells were used as a model to study the potential molecular mechanism. PCR experiments showed the presence of the novel mechanosensitive channels. Three methodologies were developed to stimulate KRJ-I cells directly: intracellular calcium measurements with stimulation by either jets of saline or shear flow, and whole cell patch clamp with direct mechanical stimulation. No evidence for mechanical responsiveness was found. These studies provide valuable insight into these two model cell lines on which a large amount of work is being carried out. Although cell models provide valuable insight into transduction mechanisms, studies on primary cell cultures remain paramount.
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The microbial synthesis of biotinCamp, Dominic J. January 1993 (has links)
Biotin synthetase was expressed in <I>E.coli</I> using the <I>bioB </I> gene cloned in a plasmid vector. Biotin production in these cells was determined using a <I>Lactobacillus plantarum </I>bioassay. It was found that overexpression of biotin synthetase led to an increase in both the rate and quantity of biotin produced compared to wild type bacteria. However the rate of biotin produced decreases sharply as the cells grow from logarithmic to stationary phase. An explanation for this phenomenon was sought by supplementing the growth medium with various additives and analysing their effect on biotin production. Biotin production in cell-free extracts was assayed using a radiometric, chromatographic assay technique. Various additives were assayed for their effect on dethiobiotin to biotin conversion in an attempt to define the exact nature of substrates and cofactors required for biotin production <I>in vitro.</I> Various chromatographic methods were performed on cell-free extracts of biotin synthetase overproducing cells in an attempt to purify the enzyme. Results from these experiments point toward the requirement of other hitherto unidentified proteins to produce biotin from dethiobiotin <I>in vitro.</I> The amino acids sequences of three known biotin synthetase enzymes were compared in order to define the contribution of specific amino acid residues to biotin synthetase structure and function. This led to the synthesis of <I>bioB </I>deletions mutants using recombinant DNA technology. The biotin synthetase amino acid sequence was used to probe available protein databases to search for proteins with similar amino acid sequence motifs. Biotin synthetase shows strong homology to a number of proteins but in particular to enzymes involved in lipoic acid and thiamine biosynthesis.
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Synthesis and structure of ubiquitinWilken, Jill January 1995 (has links)
The protein ubiquitin has been used as a model for folding studies with the synthesis of ubiquitin analogues including both single and double substitutions. Chemical synthesis has allowed for the facile incorporation of both unnatural amino acids and a novel synthetic fluorinated amino acid. Purification of synthetic ubiquitin by a variety of protocols has identified important steps in the folding pathway. We have investigated the placement of fluorine within the hydrophobic core of ubiquitin through the synthesis of three double substitutions at positions 43&67, 50&67, 56&67 and one single substitution at position 67 using (2S,4S)-5-fluoroleucine to replace leucine. Methyl transfer across the hydrophobic core has been considered using the double substitution analogue [3-Norleucine,43-norvaline]ubiquitin involving a modification in the distribution of alkyl groups. The importance of the chirality of the single histidine on the final strand of β-sheet has been examined in the synthesis of the analogue [68-DHistidine]ubiquitin. The contribution of secondary structural features in protein folding has been investigated by studying peptide fragments corresponding to the N and C-terminal halves of ubiquitin and three overlapping peptides on the final strand of β-sheet. The complementation of the N and C-terminal halves to regenerate the native fold has also been studied.
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