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Molecular and immunological characterization of glycolytic enzymes : FBA and GAPDH-1 of Neisseria meningitidisTunio, Sarfraz Ali January 2010 (has links)
There is growing evidence that several glycolytic enzymes, so-called housekeeping enzymes, including fructose bisphosphate aldolase (FBA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), despite being devoid of any apparent secretion signal, may be localised to the cell surface of several bacterial and fungal species, where they exhibit diverse non-glycolytic biological functions. However, the mechanism(s) of secretion of such signal-less proteins to the cell surface or to external environment is not well understood. Whilst their intracellular functions are well known, it is unclear whether they perform any additional functions, unconnected to their central role in glycolysis, on the bacterial surface. It is becoming apparent that such proteins may be immunogenic and they may be capable of eliciting protective immunity in animal models. As such, they represent potential vaccine candidates. In a search for novel surface exposed proteins as potential vaccine candidates against N. meningitides serogroup B, and in accordance with the fact that glycolytic enzymes are putative virulence factors in some bacterial species, it is hypothesised that meningococcal FBA and GAPDH-l, may be present on the cell surface and thus may contribute to the pathogenesis of disease. In N. meningitidis serogroup B, there is a single gene cbbA (NMB 1869) and two genes gapA-l and gapA-2, predicted to encode fructose bisphosphate aldolase and glyceradehyde 3-phosphate dehydrogenase (GAPDH) enzymes, respectively. Sequence analysis shows that FBA and GAPDH-l are highly conserved at the amino acid level. The amino acid sequences of FBA from N. meningitidis and those from Xanthobacter flavus and Synechocystis sp. displayed high identities (67 and 65%, respectively), which suggests that the meningococcal FBA (like those of X flavus and Synechocystis sp.) belongs to bacterial Class-II FBP aldolases. The cbbA and gapA-l genes were cloned and over-expressed in host E. coli. FBA was purified under non-denaturing and denaturing conditions, whilst GAPDH-l was purified under denaturing conditions. Recombinant native FBA was used in a coupled enzymic assay confirming that it has fructose bisphosphate aldolase activity. The purified FBA and GAPDH-l proteins were then used to raise polyclonal monospecific rabbit antiserum (RaFBA and RaGAPDH-l) for subsequent characterisation of enzymes with the aim to determine their subcellular localization as well as potential roles in pathogenesis of meningococcal disease. RaFBA and RaGAPDH-l reacted with ca. 38-kDa and 37-kDa proteins, respectively, in immunoblot analysis against whole cell lysates from meningococcal strain MCS8 but not the cbbA and gapA-l isogenic mutants, respectively, confirming that cbbA and gapA-l are naturally-expressed proteins in N. meningitidis. Furthermore, expression of cbbA was detected in 26/26 and GAPDH-l in 17/17 diverse meningococcal strains. Cell fractionation experiments showed that meningococcal FBA and GAPDH-l are localized both to the cytoplasm and to the outer membrane. These results were validated by flow cytometry. The data demonstrated that outer membranelocalized FBA was surface-accessible to FBA-specific antibodies in encapsulated N. meningitidis, whereas flow cytometry analysis confirmed that GAPDH-I could be detected on the cell surface, but only in a siaD-deficient background, suggesting that GAPDH-I is inaccessible to antibody in encapsulated meningococci. Mutational analysis and functional complementation was used to identify additional functions of FBA and GAPDH-l. The cbbA and gapA-l knock-out mutant strains were unaffected in their ability to grow in vitro, but showed a significant reduction in adhesion to HBME and HEp-2 cells compared to their isogenic parent and complemented derivatives. In a transgenic mouse model, cbbA mutant strains were shown to be less able to establish bacteraemia compared to their wild-type parent strains. In summary, in this study, expression of FBA and GAPDH-l was shown to be well conserved across diverse isolates of Neisseria species. This study also demonstrates for the first time that meningococcal glycolytic enzymes, FBA and GAPDH-l. are surface localised proteins and required for optimal adhesion of meningococci to host cells. Taken together, these results suggest that FBA and GAPDH-l may be involved in the pathogenesis of meningococcal disease.
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Translational control : mTOR signalling and the use of next-generation sequencing methodsJackson, Thomas John January 2013 (has links)
Translation is a multi-stage process comprising initiation, elongation and termination. It has been suggested that the initiation phase is the rate limiting step of this process. In this thesis the contributions of how changes in initiation and elongation rates lead to overall alterations in gene expression pathways were investigated in a number of different systems. It has been shown previously that increased expression of tRNAiMet is associated with tumourigenesis however the precise role of tRNAiMet in this process was unclear. Data obtained from cells with increased tRNAiMet copy number show that the associated increase in proliferation is transient and unlikely to play a major role in cancer. In collaboration with Dr Owen Samson’s group, it was shown that early dysplastic changes in intestinal tumourigenesis are driven by increased translation elongation via mammalian target of rapamycin 1 (mTORC1). It was found that constitutively active mutant K-Ras confers resistance to mTORC1 inhibition, and combined mTORC1/2 inhibition but that these tumours are acutely sensitivity to loss of the mTORC2 component, Rictor. Interestingly, changes in translation elongation rate were also identified in cells cooled to 32oC and this was associated with the reprogramming of gene expression under these conditions. Finally, the use of the next-generation sequencing technique ribosome profiling illustrated some potential challenges of using this approach to infer biologically relevant conclusions. These include: biases in fragment and library generation, limited read depth and statistical inference from low-count data. An alternate library generation method reduced bias, but reads predicted to be highly structured were still over-represented. The use of a newly developed thermostable ligase did not remedy this problem, but this may be due to additional biases associated with the particular ligase used.
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A click-chemistry based approach for the synthesis of new BODIPY-labelled fluorescent ligandsSpeed, Daniel January 2013 (has links)
Fluorescent ligands have found numerous applications for studying interactions of drug molecules with their target and as a probe of biological systems. A common approach when designing and synthesising a fuorescent ligand is to separate the fluorophore and pharmacophore via a linker. One novel approach is to utilise click chemistry to allow the coupling of fluorophore to a pharmacophore. This thesis reports the results of an investigation into utilising click chemistry, specifically the alkyne-azide copper (I) cycloaddition to synthesis novel fluorescent GPCR ligands. Targets included the β1, β2 adrenoceptor and the muscarinic M3 receptor. Investigations into the introduction of a 1,2,3-triazole within the linker to the fluorophore resulted in 14 novel fluorescent antagonists active at the β1 and β2 adrenoceptor. The most promising ligand had log Ki values of -6.77 ± 0.20 (β1) and -7.32 ± 0.05 (β2). These ligands were used in a confocal microscopy studies to visualise the β1 and β2 adrenoceptors on the surface of CHO cells. However the ligands internalistion, and receptor visualisation was not possible. A range of structural modifications were made to reduce this with the introduction of a polar linker but this did not reduce the intracellular accumulation. The change to a longer wavelength fluorophore stopped intracellular accumulation but reduced the binding log Ki to - 5.16 ± 0.06 (β1) -5.96 ± 0.20 (β2). Twenty two novel fluorescent M3 ligands were synthesised and their inhibitory properties were investigated. An initial screen showed four promising ligands and further study into the binding affinities showed the ligands to have high potency (log Kb -7.97 ± 0.07 to -8.89 ± 0.11). These ligands were studied with confocal microscopy and intracellular accumulation did not occur. Structural changes to include a polar side chain or a sulfonic acid onto the fluorophore were investigated and led to three novel fluorescent ligands that had reduced lipophilicity. With this reduced lipophilicity, binding affinities were also reduced by ten fold compared to the original fluorescent ligand. The seven ligands were fully profiled physiochemically and kinetically. The physioschemical properties of these seven ligands gave a wide variety of lipophilic values. The kinetic profiles of the ligands exhibited very similar dissociation properties to those of the parent ligand with varying association rates. The Muscarinic M3 ligands synthesised show great binding affinities for fluorescent ligands and kinetic profiles that are extremely similar to the parent ligand. These fluorescent ligands hold characteristics that can be used to further examine the pharmacology of muscarinic receptors and be used to replace radioligands for binding studies.
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The role of very long chain fatty acids in Arabidopsis growth and developmentSeamons, Laura Elizabeth January 2015 (has links)
Very long chain fatty acids (VLCFAs) are essential to Arabidopsis growth and development. VLCFAs are found in sphingolipids, glycerophospholipids, triacylglycerols, suberin and cuticular waxes. VLCFAs are synthesized by the addition of 2 carbons from malonyl-CoA to pre-existing acyl-CoAs to produce chain lengths of greater than 18 carbon atoms. VLCFA synthesis involves four consecutive reactions that are catalysed by the microsomal Fatty Acid Elongase. In Arabidopsis the first reaction is catalysed by one of 21 different Keto-CoA Synthases (KCS) with diverse levels of expression and overlapping tissue specificities. The other three enzymes are ubiquitously expressed throughout the plant, and form the core components of the elongase. Lipidomic profiling has been performed on roots and shoots of plants with reduced levels of VLCFAs. Mutants of the core components of the elongase were analysed along with herbicides that inhibit a number of KCS enzymes, this allowed the whole elongase complex to be analysed. Differences were seen in the lipidomic profiles of the different elongase mutants and between the roots and shoots of the same mutants. This has revealed correlations between phenotypic differences and lipidomic changes giving insight into which lipid classes might be responsible for the various phenotypes. A forward genetic screen has been conducted in the Arabidopsis cer10-1 mutant to identify novel genes involved in VLCFA metabolism. CER10 encodes for the fourth component of the elongase complex. One suppressor mutant that has been identified has flower buds and fertility comparable to wild type plants but still displays the reduced size of the cer10-1 mutant. The second suppressor mutant identified showed restored size of aerial organs but the flower buds remained fused. Whole genome sequencing allowed localisation of these suppressor mutations on Chromosome 3. Partial biochemical characterisation of these mutants revealed interesting changes in their acyl-CoA and cuticular lipid profiles.
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The role of VGF and its derived peptides in the regulation of energy homeostasisLewis, Jo Edward January 2015 (has links)
The VGF gene (non-acronymic) was first implicated in energy homeostasis by VGF-/- null mice, which were lean, hypermetabolic and hyperactive, suggesting an anabolic role for VGF (Hahm et al., 1999). Furthermore, VGF-/- mice were resistant to obesity induced by diet and genetic manipulation (Hahm et al., 2002). While VGF mRNA was reduced in response to short photoperiod, which is associated with reduced food intake and the utilisation of intra-abdominal fat stores in the Siberian hamster (Barrett et al., 2005; Ebling, 2014). However subsequent studies in Siberian hamsters and mice have suggested a catabolic role for the VGF derived peptide TLQP-21 (Bartolomucci et al., 2006; Jethwa et al., 2007). Thus the aim of this thesis was to further investigate the role of VGF in the regulation of energy homeostasis in the mouse and Siberian hamster. The studies presented in this thesis have shown that VGF derived peptide HHPD-41 can affect short term food intake in the Siberian hamsters, while overexpressing VGF mRNA in the hypothalamus of both Siberian hamsters and mice reduced bodyweight. However, this reduction in body weight was associated with an increase in both food intake and energy expenditure. These effects of VGF overexpression were attenuated in disrupted models of energy regulation. Finally these studies identified novel regulators of the VGF gene in vitro to postulate a possible mechanism for the seasonal regulation of appetite in the Siberian hamster. Collectively, the studies described in this thesis demonstrate a role for VGF in the regulation of energy homeostasis and contribute to increasing our understanding of how the brain regulates food intake.
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Investigation of polyamidoamine dendrimers induced DNA condensation and enzymatic degradation of these complexes : an atomic force microscopy studyGharib Abdelhady, Hosam January 2004 (has links)
Extensive investigations have been made to try and understand the physical properties and structures of condensed DNA phases in vitro during the past decades (Bloomfield, 1991, Marquet and Houssier, 1991, Bloomfield, 1996). The packing pathways of DNA molecules are three dimensional processes, and are not yet fully understood (Yoshikawa et al., 1997). Distinguishing different single DNA molecules at different locales in time in the presence of condensating or dissociating agents is crucial for understanding the mechanisms of packing and unpacking of DNA molecules. The aim of this study is to provide an increased understanding of the some of the pathways of packing and unpacking of DNA. This aim was achieved by monitoring in time and at molecular scale the interaction between the DNA and polyamidoamine dendrimers, as condensating poly cations, and by observing the dissociation of some of these condensates in time when they were exposed to DNase I enzyme, as a dissociating in vivo agent. The main techniques used were atomic force microscopy (AFM) and gel electrophoresis. We believe that the results could be beneficial to the understanding of the in vivo condensation and dissociation process of certain DNA morphologies. Chapter 1 will focus on providing an overview of the single molecule techniques used, atomic force microscopy as a means of detecting individual biomolecules in near physiological conditions with time and its application in monitoring non-viral gene delivery systems on surfaces. Methods used for gene delivery, and the PAMAM dendrimers as one of the recently applied polymer in non-viral gene delivery are also reviewed. The materials and methods used in this thesis were considered in chapter 2. Chapter 3 will concentrate on the factors effecting the interactions of generation 4, 6 and 8 PAMAM dendrimers on surfaces. An understanding of these interfacial interactions is important to understand their effects on the individual DNA molecules. This aim was achieved by using AFM as an imaging and force measuring tool to visualize and characterize the adsorption of these dendrimers on mica, gold and on alkanethiol self assembled monolayers (SAMs). Developing a deep understanding of the adsorption of DNA onto oppositely charged substrates would be of fundamental importance in understand the packing and unpacking pathways of these molecules. This philosophy is demonstrated in Chapter 4 in which the ability of the monovalent cations to facilitate imaging of DNA, and the effect of these monovalent cations in the partial condensation of DNA is explored. In addition, Chapter 4 introduces DNA imaging in the presence of divalent cations in liquid and in air. The folding pathways of dendrimer-induced DNA condensation with time on the surface of mica in aqueous environment were the targets of Chapter 5. In addition, the surface-influenced DNA condensation in the presence and absence of sufficient soluble cations and the ionic strength dependence were also studied. Structural volume and hence information regarding the number of plasmid molecules in each condensate was explored. Furthermore, the effect of loading ratio and generation type on the complex retardation in gel electrophoresis was investigated. Chapter 6 investigates the different mechanisms of the DNA-PAMAM dendrimer condensate relaxation and fragmentation by DNase I with time and explores the mechanisms of wrapping and unwrapping of the DNA on the larger generations of dendrimers. The final chapter, Chapter 7, discuses the progress made towards the aims of this thesis. Interestingly, this investigation is one of the first to apply atomic force microscopy operating in liquid to visualize at the molecular scale and in real time DNA molecules in the absence of multivalent cations, to explore the formation of DNA complexes with ethylene di amine PAMAM dendrimers that have real potential as gene delivery vectors and to investigate the different condensation and dissociation pathways of individual DNA molecules. Overall it is hoped that the work described in this thesis provides a step forward in the methods applied for AFM based nano-force biomolecular imaging with time and provides a valuable information that aid in developing a successful non-viral gene delivery system.
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Protein polysaccharide complexes : permanent/nonpermanent interactions between polysaccharides and polypeptidesGillis, Richard Benjamin January 2015 (has links)
This investigation looks at the hydrodynamic characterisation of both covalent and non-covalent protein polysaccharide complexes in the context of novel treatments and healthcare. New techniques were employed and evaluated, such as the MUTLTISIG and SEDFIT-MSTAR algorithms for sedimentation equilibrium analysis, as well as the Extended Fujita Approach for sedimentation velocity. Other characterisation techniques were used such as viscometry, density measurement, Dynamic Light Scattering and Size Exclusion Chromatography coupled to Multi Angle Light Scattering. Therapeutics for the treatment of Diabetes Mellitus and Coeliac Disease were considered. There is evidence to suggest that a protein polysaccharide complex extracted from the pulp of pumpkins has a hypoglycaemic effect in human physiology. This extract was assessed in terms of molecular integrity as a precursor to human trial studies. Equally, a novel treatment for Coeliac Disease, gliadin intolerance found in approximately 1% of the population, was assessed in terms of protecting the immune system from gliadin. Well-established methods, along with newly developed methods, were also used to characterise two glycoproteins relevant to the healthcare and food industries: Human gastric mucin, a natural lubricant found in the human stomach, and gum arabic, a plant extract from the Acacia tree. Findings from these investigations were able to add to our current understanding of these two macromolecules.
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Swap70b acts downstream of Wnt11 signalling to regulate zebrafish convergence and extension cell movementsXu, Xiaoou January 2015 (has links)
During vertebrate gastrulation, convergence and extension (CE) cell movements narrow the body axis medial-laterally (convergence) and extend it anterior-posteriorly (extension). In zebrafish, Wnt11 (silberblick) and Wnt5b (pipetail) mutants exhibited widening and shortening of the body axes characteristic for CE cell movements defects during gastrulation. It was subsequently reported that Wnt signalling resulted in activation of Rho GTPases and rearrangements of the F-actin cytoskeleton, establishing that CE cell movements are regulated through the non canonical Wnt/PCP pathway. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and it was recently found that Def6a, a GEF for Rac1, Cdc42, and RhoA, was the downstream target of Wnt5b signalling. In this thesis, morpholino-mediated knockdown of gene expression in conjunction with phenotypic rescue experiments revealed that Swap70b, a GEF protein closely related to Def6a, functions downstream of Wnt11. Swap70b morphants exhibited broader and shorter body axis with no apparent defect in cell fate specification, and ectopic Swap70b expression robustly rescued wnt11 morphants, establishing Swap70b as a novel member of the non-canonical Wnt/PCP pathway downstream of Wnt11. The following phenotypic rescue experiments demonstrated that Swap70b and Def6a execute both distinct and overlapping functions in the modulation of CE cell movements. In addition, as previously shown for Wnt11 (silberblick) and Wnt5b (pipetail) double mutants, Swap70b and Def6a double mutant morphants exhibited a more severe CE cell movement defect, suggesting that Swap70b and Def6a delineate Wnt11 and Wnt5b signalling pathways.
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RsmN : a new atypical RsmA homologue in Pseudomonas aeruginosaLovelock, Laura Charlotte January 2012 (has links)
The RsmA/CsrA family of global post-transcriptional regulators are small RNA-binding proteins involved in the regulation of a large number of genes such as those involved in quorum sensing, virulence factor production, secondary metabolism, motility and biofilm formation. They bind to target mRNAs and hence modulate their stability and translation rates. Their effects are antagonised by small non-coding regulatory RNAs. The control of expression of target genes via this post-transcriptional regulatory network is mostly operated in Pseudomonas spp. via the GacS/GacA two component system. This study aimed to perform a biophysical analysis of RsmA and to obtain a preliminary understanding of the structure, function and regulation of RsmN, a new atypical RsmA homologue from Pseudomonas aeruginosa. RsmA was purified and biophysical analysis confirmed that RsmA exists as a dimer and is highly stable at high temperatures (75 °C) and low pH (5.2). Although RsmN was found to be structurally similar to RsmA, no functional phenotypes have been identified. Consequently, rsmN was mutated and transcriptional fusions to rsmN and its anti-sense transcript were constructed for expression studies. Phenotypic analysis indicated that RsmN was not involved in the control of swarming, pyocyanin, elastase and protease production or glycogen accumulation. Unlike RsmA, RsmN does not have a control on the restriction modification system of P. aeruginosa. Transcriptional fusions revealed RetS, LadS and GacA all appear to have a 21 significant effect as activators of both the rsmN and nmsR promoters. 2-Alkyl-4(1H)-quinolone (AQ) signalling also modulate rsmN expression possibly via the iron chelating properties of 2-alkyl-3-hydroxy-4(1H)-heptyl-quinolone (PQS). RsmN targets identified from Deep Sequencing include those required for structural outer membrane proteins, transcriptional regulators as well as genes involved in motility, secretion, flagellar structure and biofilms. RsmA, RsmZ and RsmY were all identified as targets together with the small RNAs RgsA (indirectly gac-controlled) and the antagonistic RNA CrcZ (represses catabolite repression control protein Crc). Targets common to both RsmN and RsmA include the transcriptional regulators Vfr, PqsR, MvaT and Anr, regulatory RNAs RsmZ and RsmY together with transcripts corresponding to the pqsABCDE operon, LasB, LecA/B, RhlI, LasR/I, Crc and CrcZ. The identification of many mRNA targets for RsmN which are shared with targets of RsmA provides further evidence that RsmN is involved in global-post-transcriptional regulation of gene expression.
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Playing Tetris with porphyrins : the synthesis of porphyrinic materials for self-sssembly studiesHames, Tim January 2016 (has links)
This thesis focuses on the synthesis of porphyrinic materials for self-assembly studies. It begins with the synthesis of a large number of ‘building block’ molecules, terminated with bromo-, alkyne- and TMS- groups. These molecular building blocks are combined to form porphyrin compounds also terminated with bromo- alkyne- and TMS- groups at carefully selected positions. A series of porphyrin arrays inspired by the computer game Tetris was designed to be synthesised from these porphyrin units. The Tetris compounds are comprised of four square-like tetraphenylporphyrin (TPP) units, linked together with alkyne bridges to create arrays shaped like the T, I, L/J, S/Z and O Tetriminos. The NMR spectroscopy of the Tetris compounds is thoroughly explored to highlight the slight structural differences between the molecules. The synthesis of two bromoaryl-terminated porphyrin dimers for ultimate incorporation into covalent organic frameworks (COFs) is also described. The X-ray diffraction data collected showed that crystals grown were of trifluoroacetic acid (TFA) salts of the target compounds. The presence of the TFA anions resulted in significant distortion of the porphyrin ring which is described in full. Finally, the synthesis of a series of poly(acetylene) anthracene dimer compounds that have potential use in the field of organic electronics has also been explored. The electrochemical and fluorescence properties of the molecules across the series are investigated and the crystal structures are described.
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