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Cytology and ultrastructure of new, rare and interesting algae from TasmaniaPipes, L. D. January 1988 (has links)
No description available.
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Changes in functional chromatin organisation of the delta crystallin gene region during retinal transdifferentiationMcLaughlin, M. H. January 1988 (has links)
No description available.
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Novel approaches to the isolation of farm animal embryonic stem cellsThansa, Kwanta January 2009 (has links)
The establishment of stable immortal ES cell lines using embryos as a source of isolation in domesticated farm animals, in particular for pigs, which are closer to humans than other ungulates, has not been reported; hence this information could contribute to the improvement of regenerative medicine in humans, biotechnology and agriculture. Therefore, the discovery of effective protocols to derive and maintain ES cells and the induction of purified somatic cells from ES cells in pigs is of importance. The objectives of this study were to produce pES-like cells and direct differentiation of the ES-like cells obtained by improving the culture conditions. In vivo-derived porcine blastocysts at day 6-8 were classified into two groups distinguished by the exhibition of ICMs and epiblasts of the embryos. In each group, intact blastocysts and isolated ICMs or epiblasts were designed to culture in either KO4bh or DM40bh medium on mitotically inactivated MEFs under the humidified air of 5%CO2 at 39°C until the primary outgrowth of ES-like cells was observed. Two morphologically distinct pES-like cells, pESA-like and pESB-like cells were isolated from the epiblasts, whereas no cell lines were generated from ICMs. pESA-like cells were observed as individual small round cells containing one or multiple nucleoli along with a high ratio of nucleus to cytoplasm, while pESB-like cells formed dome-like colonies. The pESA-like cells were stained both negative and positive with the alkaline phosphatase enzyme, while pESB-like cells were all stained positive. With immunofluorescence staining of OCT-4 and nanog, the nuclei of pESB-like cells appeared not to be stained positive with these two antibodies, while the designed self-renewing genes such as OCT-4, nanog, SOX-2, REX-1 and DPPA-3 were detectable as similar to mES cells. Regarding the pluripotent abilities of pESB-like cells, they could be induced to form neuronal-like, neuronal supporting-like, smooth muscle-like and hepatic-like cells in a variety of desirable differentiation media under the feeder-free culture system. The cytoplasmic contents of certain induced mature cells were stained positive with nestin, α-smooth muscle actin and α-fetoprotein in association with the expression of differentiated genes specific to each germ layer such as nestin, α-smooth muscle actin, smooth muscle myosin heavy chain, α-cardiac actin, transthyretin, α-fetoprotein, albumin and HGF1β. In conclusion, pESB-like cells obtained in this study may possibly have the potential to be authentic ES cells isolated from early epiblast origin as mES cells.
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The role of extracellular signal-regulated kinase in beta-adrenoceptor-mediated vasodilatationUhiara, Chukwuemeka Obinna January 2012 (has links)
Beta-Adrenoceptors (B-ARs) mediate vasodilatation by activating various mechanisms that collectively contribute to vascular smooth muscle (VSM) relaxation. It has been shown that B2-AR stimulation in cultured cells results in activation of extracellular signal-regulated kinase (ERK). As the functional relevance of this was not known, the aim of the current investigation was determine the role of ERK in beta-AR-mediated vasodilatation. Isoprenaline-induced relaxation of porcine coronary artery (PCA) segments pre-contracted with the thromboxane mimetic U46619 was significantly enhanced by inhibition of ERK activation. Relaxations to the beta2-AR agonist salbutamol, but not those to the beta1-AR agonist xamoterol or the adenylyl cyclase activator forskolin, were also enhanced. The intermediate-conductance Ca2+-activated K+ (IKCa) channel blocker TRAM-34 prevented the enhancement of beta2-AR-mediated responses. Taken together, the data indicate that ERK inhibits beta2-AR-mediated vasodilatation by interacting with a cyclic 3’, 5’-adenosine monophosphate-independent relaxation pathway involving K+ channels. This may occur through a direct regulatory action on the IKCa channel via phosphorylation. Furthermore, the finding that increased ERK activation in a rat model of Type II diabetes was associated with significantly impaired beta-AR-mediated vasodilatation raises the possibility that ERK may represent a promising therapeutic target in the treatment of disease states characterised by abnormal vascular function.
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Sourcing cells for gut tissue engineering : understanding and inducing embryonic stem cell differentiation to the intestinal cell lineageCarter, David Andrew January 2012 (has links)
Tissue engineering of any tissue type requires the combination of a supporting scaffold, a range of biological factors and a suitable source of cells. This source of cells must satisfy a number of criteria:- • The ability to form all of the mature/specialised cell types found in the target tissue. • Readily obtainable. • Readily maintainable in the laboratory without requiring excessive resources or time. For intestinal tissue engineering there are a number of issues associated with the use of tissue derived stem cells particularly quantity of normal tissue available (from the patient) and maintenance and expansion of the cells when cultured in vitro. Using embryonic stem cells offers a potential alternative strategy but methods must be established to efficiently differentiate the cells towards the desired fate. Many strategies for differentiating embryonic stem cells are based upon treatment with growth factors in vitro. There is a (logistical) limit to the degree of complexity that these systems can achieve and therefore a limit to the number of differentiation signals that occur during in vivo development that can be mimicked. In recent years using embryonic tissue to provide signals to undifferentiated cells has proved a successful method of directing the differentiation of naïve cells towards a particular fate (with the choice of tissue determined by the desired target cell type). The aims of this thesis were to explore the potential of differentiating embryonic stem cells towards the intestinal progenitor fate using a combination of in vitro cell culture treatment with the growth factor Activin-A and ex vivo co-culture with embryonic chick gut tissue. Previous studies [Kubo et al 2004, Tada et al 2005, Yasunaga et al 2005, D’Amour et al 2005, MacClean et al 2007] have shown that Activin-A treatment will induce embryonic cells to more efficiently differentiate to definitive endoderm, the germ layer from which the intestines (and other visceral organs) arise. These techniques were applied to the Columnar Epithelial Epiblast murine embryonic stem cell line and cell differentiation was then evaluated at the molecular level using Reverse Transcription-Polymerase Chain Reaction, immunocytochemistry and Western blotting. Activin-A treatment produced an upregulation of definitive endoderm markers at both the mRNA and proteomic levels compared to the control conditions. However the cell population produced retained expression of pluripotent markers and showed some expression of markers of other cell lineages. Further studies [Sugie et al 2005, Fair et al 2003, Van Vranken et al 2005, Coleman et al 2007, Krassowska et al 2006] have shown that co-culture of embryonic stem cells with early stage embryonic tissue can induce the formation of particular tissue types; the tissue must be selected based on proximity to the target cell type during development. This exposes the embryonic stem cells to the signals that prompt differentiation towards the target tissue during normal development. With gut tissue much signalling occurs between the different tissue layers that make up the whole organ both during development and in adult tissue. Ex vivo co-culture of murine embryonic stem cells with embryonic chick gut tissue was used to direct their differentiation to the intestinal epithelial stem cell fate. Before the co-culture was carried out various experiments were carried out to establish if the proposed protocol was viable e.g. defining how long chick gut tissue explants could survive in culture. Once this had been established co-culture experiments were undertaken and cell differentiation was then evaluated at the molecular level using Reverse Transcription-Polymerase Chain Reaction, immunocytochemistry and Western blotting. The cells showed some expression of intestinal epithelial stem cell markers at both the mRNA and proteomic levels following co-culture. The cells were also assessed at a physiological/functional level by evaluating their ability to form a functional intestinal epithelial barrier. This was achieved using an in vitro co-culture model with intestinal subepithelial myofibroblasts by measuring transepithelial resistance, permeability to protein and morphology in a simple tissue model co-culture. The cells did not display the morphological or physiological characteristics associated with intestinal epithelial cells in the model system. Overall this work has shown that co-culturing pluripotent mES cells with embryonic chick gut tissue can induce differentiation towards the ISC fate. Pre-treating the cells with growth factors in vitro did not seem to enhance this differentiation but there was scope to refine these techniques. Following the differentiation protocols the cells did not display the desired physiological characteristics but again there was scope to refine the techniques particularly with regard to selecting cells positive for the expression of the chosen molecular markers. These techniques show promise but do require some further development.
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Using biophysical techniques to study the mechanism of ligand-gated potassium efflux systems (KEF) from bacteriaPliotas, Christos January 2011 (has links)
The ligand-gated potassium channels KefC and KefB of <i>Escherichia coli </i>are critical components in protecting cells from toxic electrophiles. Potassium efflux through these channels is coupled to a decrease in cytoplasmic pH which in turn reduces the damage to DNA by electrophiles. KefC and KefB are both inhibited by cytoplasmic glutathione and activated by glutathione adducts, such as ESG, formed by conjugation of glutathione with electrophilic compounds. Robust membrane purification protocols were developed to isolate both the wild type full-length KefC and KefB and the mutants required for biophysical analysis. <i>In vivo </i>K<sup>+</sup> measurements were performed to ensure that all of the constructs used were fully functional. Structural and functional analysis used electron paramagnetic resonance (EPR) and stead state emission fluorescence measurements <i>in vivo</i>, on wild type and mutated full-length proteins to elucidate the gating mechanism and test the model generated from crystallographic data. In particular, EPR spectroscopy combined with site-directed spin labelling revealed a substantial conformational change and thus provided the first insight into coupling between sensing and gating. Steady state fluorescence spectroscopy was used to precisely measure binding affinities for both activating and inhibitory ligands and characterise nucleotide binding to KefC. Finally, a variety of chemically diverse glutathione adducts was tested on KefC <i>in vitro </i>to elucidate the mechanism by which these ligands initiate K<sup>+</sup> flux through the associated transmembrane domain.
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Phospholipids and the beta-adrenergic responseMcKenzie, Roderick Colin January 1983 (has links)
The phospholipid polar headgroup composition of membranes of C6 cells was modified in vivo by growth for 24h in media supplemented with the polar headgroup precursors, N, N'-dimethylethanolamine, N-monomethylethanolamine or ethanolamine. These modifications were achieved without alteration of the cholesterol and phospholipid content of the membranes, and without changes in the fatty acyl or protein composition of these membranes. No changes were found in the physical properties of membranes isolated from these cells . Enriching the cell membranes with PDME, PNME or PE elevated the basal intracellular cAMP content, but decreased the degree of stimulation of intracellular cAMP content in response to β-adrenergic stimulation. This reduction of the β-adrenergic response appeared to be due to enhanced phosphodiesterase activity. β-adrenergic stimulation did not affect bulk physical properties of the membrane. Exposure to isoproterenol increased the amount of [3H] methyl label recovered from TLC plates but the different distribution of label indicated that such analysis is insufficient to demonstrate specific phospholipid methylation. This system is a useful means of studying the relationship between phospholipid composition, phospholipid metabolism and the β-adrenergic response in the C6 cell line.
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PPARalpha in peroxisome proliferation : molecular characterisation and species differencesChoudhury, Munim January 2000 (has links)
Peroxisome proliferators (PPs) cause proliferation of peroxisomes and hepatocarcinogenesis in rodent liver, mediated by Peroxisome Proliferator-Activated Receptor-alpha (PPARalpha). There are marked species differences in peroxisome proliferator-induced responses, and the functionality of PPARalpha may be an important determinant factor in species sensitivity to PPs. Primary hepatocytes were investigated for a highly responsive marker induced by PPs to study the effects of transfected PPARalphaCYP4A1 was highly induced in rat hepatocytes that require hydrocortisone for maximal induction. Hepatocytes were cultured in hydrocortisone-deficient media to determine if reduced endogenous PPARalpha was associated with lowered induction of CYP4A1. However, there was residual induction of CYP4A1 by peroxisome proliferators. Primary hepatocytes from PPARalpha knock-out (-/-) mice were investigated as they lack endogenous PPARalphaIn vitro and in vivo studies demonstrated that Cyp4a10 and 14 were highly inducible by PPs in the hepatocytes of wild-type but not in -/- mice. However, addition of either mouse or guinea pig PPARalpha in -/- hepatocytes did not induce the expression of these marker genes, although both receptors showed trans-activation ability in a reporter assay. The failure of added PPARalpha to activate endogenous genes responsive to PPs, whilst at the same time activating episomal DNA containing response elements of PP-inducible gene, suggests that the endogenous genes require PPARalpha to remain in an accessible conformation. Although hamster is considered to be a partially-responsive species to PPs, their response to PPs is poorly characterized. Three CYP4A genes (CYP4A17, 18 and 19) were cloned from hamster liver cDNA, and hepatic CYP4A17 was found to be highly inducible by PPs. In addition, PPARalphawas cloned from hamster liver and shows higher identity to rat and mouse PPARalpha than to human and guinea pig. Hepatic expression of PPARalpha mRNA was compared between mouse, hamster and guinea pig. The level of PPARalpha transcript was found to correlate well with species response to PPs, i.e. mouse (highly responsive species) has the highest level and guinea pig (non-responsive) the lowest, while hamster (partially-responsive) has an intermediate level. This is consistent with a model where the level of expression of hepatic PPARalpha determines species response to PPs. Expression of PPARalpha and transcriptional coactivators, such as PBP, SRC-1 and CBP/p300, were confined to mouse liver at the RNA level, but in each case expression showed homogenous distribution within the liver acinus and was non-inducible by PPs. Mouse PPARalpha ligand binding domain (LBD) was bacterially expressed as a histidine-tagged protein and soluble proteins were purified using affinity and column chromatography. Functional LBD may serve as a useful bait in protein-protein interaction studies for the identification of any novel PPARalpha interacting coactivator protein.
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Inhibition of mammalian nicotinic acetylcholine receptors by philanthotoxin analogues is strongly influenced by subunit compositionKachel, Hamid Saeid January 2015 (has links)
Philanthotoxin-433 (PhTX-433) is an active component of the Egyptian solitary digger wasp, Philanthus triangulum, venom which non-selectively inhibits several excitatory ion channels. With the aim of improving potency and selectivity, several synthetic analogues were developed based on the single or multiple modifications in the hydrophilic polyamine tail and hydrophobic aromatic head group of the PhTX. In the first part of this study, we investigated the pharmacological actions of two synthetic analogues, Philanthotoxin-343 (PhTX-343) and Philanthotoxin-12 (PhTX-12), on mammalian hetero- and homooligomeric nicotinic acetylcholine receptor (nAChR) subunit combinations expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX-analogue were studied electrophysiologically using two-electrode voltage-clamp at three different membrane holding potentials (VH = -60 mV, -80 mV and -100 mV). Concentration-inhibition curves were constructed and IC50 values estimated for each holding potential. The IC50 (95% CI, n=oocytes) values for PhTX-343 inhibition of α3β4, α3β2, α4β2, α4β4, α7, α3β4(F255V), α4β2(V253F) and α1β1δγ peak currents at -100 mV were 0.07 µМ (0.05-0.10 µМ, n=9), 3.20 µМ (1.69-6.26 µМ, n=8), 0.14 µМ (0.09-0.21 µМ, n=7), 0.28 µМ (0.17-0.46 µМ, n=6), 8.7 µМ (6.9-11.0 µМ, n =9), 0.01 µМ (0.01-0.02 µМ, n=8), 0.10 µМ (0.06-0.16 µМ, n=8) and 3.2 µМ (2.5-4.1 µМ, n=9) respectively; for PhTX-12 they were 2.03 µМ (1.22-3.26 µМ, n=8), 74.0 µМ (21-259 µМ, n=10), 1.6 µМ (0.7-3.6 µМ, n=11), 1.82 µМ (0.81-4.11 µМ, n=9), 12.1 µМ (9.0-16.0 µМ, n =10), 1.0 µМ (0.5-2.0 µМ, n=5), 3.4 µМ (1.3-8.7 µМ, n=9) and 5.0 µМ (2.8-8.8 µМ, n=8) respectively; i.e. in contrast to M-nAChR, PhTX-343 was more potent than PhTX-12 in all cases. All IC50s were lower when inhibition was measured after 1 minute of application indicating use dependence. For inhibition of heteromeric nAChRs, the peak potency of PhTX-343 was strongly augmented by holding the cell at more negative VH while this was not the case for PhTX-12 where only weak voltage-dependence was observed. The inhibition of homomeric α7 receptors by PhTX-343 was voltage-independent, whereas the block by PhTX-12 was voltage-dependent. In addition, these two synthetic analogues were different in their recovery rates (PhTX-12 was generally faster than PhTX-343) but they were similar in their mechanism of action, non-competitive inhibition. In the second part of the project, I explored the structure-activity relations of twenty one synthetic analogues, with a view to improving their activity and selectivity on rat neuronal α4β2 and α3β4 nAChRs at VH = -100 mV. We showed that the presence of positive charge in the polyamine tail of PhTX compounds is essential for nAChR subtype selectivity and its removal made the molecule lose its selectivity. Also, it appears that adding a bulky group to the terminal ammonium drastically reduced activity whereas a similar addition to the head region it increased their potency. In addition, we identified the key regions and substitutions responsible for increasing PhTX activity, cyclohexylalanine in place of tyrosine, and selectivity, phenyl group. Analogues having cyclohexylalanine and a phenolic group in the head region showed IC50 values in the low nano-molar and pico-molar (160-400 pM) range. These data suggest that PhTXs could serve as lead compounds for highly potent and selective inhibitors of N-nAChRs.
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Studies on the morphology and mapping of human chromosomes.Bobrow, M. January 1978 (has links)
Submitted for the degree of MD, University of Witwatersrand. / The published works submitted in this thesis fall into three groups, covering different aspects of the study of the morphology and structure of human chromosomes.
Session 1: Studies on chromosome banding and structural polymorphisms of human chromosomes.
Session 2: Human gene mapping
Session 3: Miscellaneous / WHSLYP2017
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