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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

Metabolism and effects of dietary phenolic acids

Poquet, Laure January 2008 (has links)
Ferulic acid is a secondary metabolite usually found as esters in plants and dihydrocaffeic acid results from the microbial metabolism of flavonoids and of caffeic acid, both widely distributed in food. Even though ferulic acid and flavonoids have been proposed to exert several beneficial effects on health, their in vivo activities could partly result from their microbial metabolites and strongly depend on their bioavailability. The absorption and metabolism of phenolic acids were studied in vitro with a model for the colonic epithelium composed of absorptive and mucus secreting cells, ex vivo with everted colonic sacs and liver slices, and in vivo with rats. The photoprotective effect of phenolic acids was tested in vitro on the keratinocytes HaCaT.The ferulic acid permeation was mainly by transcellular diffusion and also by a facilitated transport (S-MCT and MCTI). Intestinal cells conjugated ferulic acid with sulphate or glucuronide and reduced its unsaturated side chain. In rats, intestinal cells were more potent for glucuronidation of dihydrocaffeic acid, whereas the liver favoured sulphation, the methylation being regio-selective.

Catalytic Properties of a Periplasmic Nitrate Reductase : A Voltammetric Perspective

Gates, Andrew James January 2008 (has links)
In Paracoccus pantotrophus, periplasmic nitrate reduction is performed by NapAB. This dissimilatory system is implicated in maintaining membrane redox homeostasis by linking ubiquinol oxidation with the reduction of nitmte, during aerobic metabolism of highly reduced carbon sources. NapA contains a bis-molybdopterin guanine dinucleotide cofactor and an iron-sulphur cluster. The associated di-haem subunit, NapB mediates catalytic electron flow from the quinol-oxidase electron donor, Nape. Protein film voltammetry (PFV) revealed steady-state electrocatalysis from NapAB in the presence of nitrate. At pH 7, nitrate reduction is onset <0.15 V and increases across the potential mnge where ubiquinollevels predominate in vivo. Though NapAB cannot opemte upstream of cytochrome be], its opemting potential is entirely consistent with the proposed physiological role of this enzyme in productive redox balancing. The magnitude of the electrocatalytic current-potential profile was greatest at acidic pH (~7) and responded readily to micromolar concentmtions of nitrate. At pH 6 and 7, KM values of ca. 23 and 45 J.lM were defined at the activity peak potential and 40 V, respectively. These values are in good agreement with the KM value of <15 J.lM defined for NapAB in intact cells of P. pantotrophus M-6 and thus bring the KM value for this enzyme into line with those of related proteins. Electrocatalytic activity was further modulated below ca. -0.15 V, where attenuation was observed. Such 'tunnel-diode' behaviour was maintained at [S]»KM and the high potential wave flank showed greatest pH dependence. At pH 6, potentiometric titration confirmed that activity attenuation occurred in the vicinity of the [4Fe-4Sf+/I + redox couple. A competitive inhibitor, thiocyanate had little impact on wave shape. However, NapAB purified from bacteria grown in tungsten-supplemented media showed a similar waveform to the molybdoenzyme, but where catalytic onset was displaced to lower potentials, while the position of the low potential wave flank was unchanged. With activity attenuation attributed to a redox couple distinct from the active site, modelling indicates the M06 + 15 + and M05 +/ 4+ potentials likely lie in close proximity in the high potential wave flank, thus a semi-cooperative redox process may describe the redox chemistry of the molybdenum site, consistent with the presence of sub-stoichiometric M05+ populations in EPR experiments.

Fluorescence approaches to understanding the oligomeric state and dynamics of the SecYEG translocon

Deville, Karine January 2010 (has links)
In Gram-negative bacteria such as Escherichia coli, signal sequence-bearing secretory preproteins are targeted post-translationally from the cytosol to their final destinations. This mechanism is mainly performed by the ubiquitous Sec machinery, a multiprotein complex containing the molecular motor ATPase SecA, the secretion-dedicated chaperone SecB and a heterotrimeric protein-conducting channel consisting of the SecY, SecE and SecG subunits. Crystal structures have been obtained for rnonomeric, detergent-solubilised SecYEG in its 'closed' and SecA-bound states, revealing that the channel lies at the centre of a single protomer. However, many aspects of preprotein translocation remain uncertain, including the functional significance of the observation that in membranes SecYEG is predominantly dimeric. To address these uncertainties, total internal reflection fluorescence microscopy (TIRFM) was exploited to investigate the functional oligomeric state and monitor dynamics of the translocon at a single molecule level. This approach revealed that while monomers are sufficient for the SecA- and ATPdependent association of SecYEG with preproteins, active transport requires SecYEG dimers associated through the SecE subunit. In collaboration with the Collinson group (University of Bristol, UK), a molecular model of the functional translocon was proposed, rationalising the need for both SecYEG copies. The SecY channel is closed at the periplasmic side of the membrane by a small helical region termed the 'plug'. Relocation of the latter towards SecE during polypeptide translocation was investigated kinetically and spatially by ensemble and single molecule Forster resonance energy transfer (FRET), respectively. Intra-molecular conformational changes within SecA were also probed using FRET, the results suggesting that monomerisation of the SecA dimer occurs as a pre-activation step upon binding to SecYEG. Overall, the results presented in this Thesis describe the first use of single molecule imaging to study the bacterial Sec-translocon, and represent an integral part of the emerging applications of single molecule techniques in the membrane protein field.

Motor cooperation in bi-directional early endosome motility

Schuster, Martin January 2011 (has links)
In mammalian cells and fungi, early endosomes form a dynamic compartment that undergoes bi-directional motility along microtubules. Previous work has shown that in the model system Ustilago maydis early endosome motility involves the opposing motor proteins dynein and kinesin-3. Here I performed a detailed analysis of the role of the motors in early endosome motility, using quantitative live cell imaging of kinesin-3, dynein and the endosomal GTPase Rab5a. In the first part of my work, I analysed the role of dynein at MT plus-ends, where the motor forms a strong accumulation that was thought to be involved in capturing early endosomes. I could demonstrate that ~55 dynein motors build up the dynein accumulation. In collaboration with Ms. Congping Lin and Prof. Peter Ashwin (Institute for Mathematics, Exeter), I found theoretical evidence that ~25 dynein motors concentrate and leave the plus-ends stochastically. In addition, dynein motors are captured by an interaction of dynactin and the plus-end binding protein EB1. Together both mechanisms increase the number of motors, which ensures that EEs will be loaded onto dynein before they reach the end of their track. In a second project, I provide evidence that loading of dynein is not restricted to the plus-ends. Instead, dynein leaves the plus-ends and is able to bind to kinesin-3 delivered early endosomes, which changes their transport direction from anterograde to retrograde. Kinesin-3 remains bound to these retrograde EEs. When dynein leaves the organelle, it switches back to anterograde motility. Interestingly, a single dynein wins over three to five kinesin-3 motors. I discuss these findings in the light of current motor cooperation concepts. In a third part, I demonstrated that kinesin-3 has an unexpected role in long-range retrograde endosome motility. In contrast, dynein is only responsible for the distal 10-20 µm. This is possible because most of the hyphal cells contain a symmetric and bi-polar MT array. This MT organization is reminiscent of that in dendrites. Kinesin-3-based retrograde motility is required to mix the organelles and might support long-range communication between both cell poles.

Single-ended resonant pre-converters for high-frequency induction heaters

Pforr, Johannes January 1992 (has links)
No description available.

Characterisation of proteinase-activated receptor-4 (PAR-4) signalling in NCTC-2544 cells stably expressing PAR-4 (Clone 10H9) and PAR-4 expressing cells, EAhy-926

Nilsson, Mary Francis January 2010 (has links)
No description available.

Bifurcation and stability of elastic membranes : theory and biological applications

Pearce, S. P. January 2010 (has links)
No description available.

Expression and characteristics of ion channels in osteoblasts : putative roles for TRP and K+ channels

Henney, Neil C. January 2008 (has links)
Bone turnover is regulated by a cocktail of hormones and signalling factors controlling key cell processes such as proliferation, differentiation, mineralisation and apoptosis. Disruption to the overall mineralisation-resorption balance leads to bone disorders, such as osteoporosis - a 'silent' disease affecting around 7 million people in England and Wales. Ion channels that are presumed targets for bone signalling factors include voltage-gated K channels, ATP-dependent K channels and transient receptor potential TRP channels, and several of these channel-types reportedly have roles in cell proliferation, apoptosis, and differentiation in various tissues. This Thesis shows that human osteoblasts express a number of channels in these families, including maxi-K, ATP-dependent K channels, TRPV1 and TRPM7. The maxi-K channel, displaying characteristic electrophysiological hallmarks, is abundant in patch-clamp recordings of primary human osteoblasts implying a functional role, and the Katp agonist pinacidil is shown to promote osteoblast proliferation. Electrophysiological evidence for the TRPVI channel is not found, although the mRNA signal for a TRPVI splice variant TRPVlb may provide an answer, as it renders the channel less sensitive to capsaicin and protons. However, Ca imaging indicates that osteoblastic TRPV1 channels allow Ca2 influx, and are sensitive to 1 &micro;M capsaicin and protons. In functional studies the TRPVI ligands capsaicin and capsazepine do not influence mineralisation, but interestingly the TRPVI agonists capsaicin, resiniferatoxin and anandamide appear to prevent differentiation of osteoblastic pre-cursor cells to adipocytes, and instead encourage maturation along the osteoblast pathway, whilst TRPV1 antagonists do not affect adipocyte differentiation. In conclusion, a number of K channels and the TRPV1 channel are expressed in osteoblasts and may have important putative roles in osteoblast cell function. Further steps are required to confirm this before the channels can be considered targets for drug development to treat bone disorders.

Determining a role for the calcium-sensing receptor (CaR) in pulmonary development

Finney, Brenda A. January 2008 (has links)
In the adult, changes in free ionized plasma calcium concentration (Ca2+ G) are monitored by the G-protein-coupled, extracellular calcium-sensing receptor, CaR, but whether CaR plays a role in lung development is unknown. CaR has the potential to be a key regulator of Ca2+ dependent cell fate during development. It is hypothesized that extracellular calcium is an important extrinsic factor that modulates the intrinsic lung developmental programme, through activation of the CaR. CaR is expressed in the developing mouse lung in the pseudoglandular phase, from embryonic day 10.5 (El0.5), with a peak of expression at El2.5 and a subsequent decrease by El8, after which the receptor is absent. Lung branching morphogenesis in vitro is sensitive to Ca2+ G, being negatively modulated by the higher, fetal (i.e., 1.7 mM) Ca2+ 0 yet optimal at physiological adult Ca2+ Q (i.e., 1.05-1.2 mM). Administration of the specific CaR positive allosteric modulator, the calcimimetic R-568, mimics the suppressive effects of high Ca2+G on branching morphogenesis while both phospholipase C and PI3 kinase inhibition reverse these effects. CaR activation suppresses cell proliferation while it enhances lung distension, fluid secretion and intracellular calcium signalling. Conditions which are restrictive to branching and fluid secretion can be rescued by manipulating Ca2+ 0 in the culture medium. Lung explant cultures from the current mouse model of CaR inactivation respond in a similar manner to Ca2+ Q and the calcimimetic R-568. These results indicate the presence of expression of a functional CaR splice variant, which is detected at El 1.5, 12.5 and 15.5 in CaR knockout lungs. The observations presented here support a novel role for the CaR in preventing hyperplastic lung disease in utero and present two potential models for its mode of action within this system.

Engineering aequorin as an indicator of calcium signals near the BK channel

Reviriego Santos, Pablo January 2009 (has links)
The BK channel is a large conductance calcium-activated voltage- dependent potassium channel. This channel plays a key role as a negative feedback mechanism of membrane excitability and cellular Ca2+. There is substantial evidence suggesting that the Ca2+ activation of the BK channel is regulated by localised Ca2+ release from intracellular stores. The aim of the work presented in this thesis was to develop a novel method of measuring the local Ca2+ concentration controlling the BK channel activation. The p2 subunit, an auxiliary protein of the BK channel, was extracted from MG63 cells and cloned. Subsequently, the aequorin sequence was attached to its C-terminus using splicing by overlapping extension. The recombinant protein retained the features of the native proteins emitting light in response to Ca2+ and showed correct targeting to the ceil membrane. The resultant light emission of the new protein was diminished in comparison to the native aequorin. The p2-Aequorin and a cytosolic Luciferase-aequorin were successfully transfected in a HEK293 cell line which stably express the BK channel a subunit. The expression of the aequorin constructs in HEK293 cells in suspension revealed the presence of intracellular mechanosensitive Ca2+ channels. The main finding of this thesis was that the Ca2+ affecting the BK channel is regulated independently of cytosolic Ca2+ in HEK293 cells. Stimulation with agonists such as carbachol, ATP and cyclopiazonic acid demonstrated clear differences in the magnitude of BK channel microdomain and cytosolic Ca2+ signals. Short term exposure to caffeine induced a significant decrease in the Ca2+ signals near the channel. The addition of extracellular Ca2+ led to large Ca2+ transients close to the BK channel suggesting a store-operated Ca2+ mechanism. The Ca2+ effects produced by carbachol, ATP, caffeine and cyclopizaonic acid indicate a coupling between IP3-induced Ca2+ release from the ER and Ca2+- activation of the BK channel.

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