Protein ubiquitinatlon is a dynamic and reversible post-translational modification implicated In the regulation of a number of cell signalling pathways. Nevertheless, its involvement in the Hepatocyte Growth Factor (HGF)-mediated cell scattering has not been studied before. Here, I tested the hypothesis that ubiquitin signalling regulates HGF-mediated scattering. In the first part of this project, I carried out an HGF-dependent scattering screen in A549 cells using an sIRNA library targeting the deubiquitinating enzymes (DUBs). My screen of 83 DUB genes identified twelve potential regulators that inhibited scattering upon knockdown, Subsequently, I performed a series of experiments to characterise the candidate DUBs identified in the screen. I associated distinct cell morphologies with the depletion of different DUBs and also, where possible I correlated the level of DUBs with the HGF-mediated motility response.

Cells in the human body show drastic differences in shape and morphology depending on the tissue that they are embedded in, and pathological tissue is often accompanied by changes in cell architecture. However, the question of whether changes in cell architecture play a regulatory role in physiology and disease is still mostly unexplored. As the cell architecture is influenced by the cell micro environment, such as the surrounding ECM, it might play a central role as a regulator and mediator of physical, or even chemical, environmental cues in vivo. As it is not possible to study the effect of cell architecture in vivo, various in vitro bioengineering systems based on microfabrication were developed in this thesis. The use of cell micropatterns allowed precise engineering of cell geometry, which was found to result in specific cell cytoskeletal arrangements and changes in cell contractility. It was found that those changes in contractility mediate cell geometry-dependent lineage commitment of mesenchymal stem cells. Development of several new analytical techniques and subsequent application to cell micropattern systems resulted in many new and unexpected cell geometry-dependent effects. For the first time, a link between lipid raft formation and cellcell geometry was observed. Additionally, plasma membrane curvature as a new cell characteristic is described and reported to be cell geometry-dependent in this thesis. Furthermore, a new mechanism involving lipid raft dependent Akt signalling was found to mediate cell geometry-dependent stem cell lineage commitment independent of PI3K activity in mesenchymal stem cells. Considering the involvement of Akt signalling in a wide variety of cell physiological events, and in many challenging diseases such as type-2 diabetes and cancer, the evidence of a link between cell geometry and Akt signalling could have broader implications and warrants further investigation in other cell systems. Moreover, depending on the soluble factor environment, cell geometry was observed to have different, seemingly antagonistic effects, on cell differentiation. In the absence of soluble differentiation cues, β-catenin was found to mediate cell differentiation in a most likely Wnt independent mechanism. Furthermore, by developing a novel system based on decellularized ECM micropatterns, it was reported that cell architecture also changes ECM structure by cell shape-dependent secretion of ECM proteins, suggesting a bi-directional regulatory mechanism between cell shape and ECM. It was also found that downstream effects caused by other mechanical cues in the cell microenvironment, such as matrix elasticity and topography, are indirect effects mediated by changes in cell geometry. Therefore, cell architecture seems to have an important role in mediating cell physiological effects of ECM and might be a main component that transduces effects of mechanobiological cues into specific downstream behaviour. The work in this thesis suggests a possible key role of cell geometry within the cell microenvironment, and is highly relevant for improved targeted therapies, especially in cancer, the design of tissue engineering scaffolds and in vitro drug screening systems.

The yeast Saccharomyces cerevisiae is an important model organism for the study of cell biology. The similarity between yeast and human genes and the conservation of fundamental pathways means it can be used to investigate characteristics of healthy and diseased cells throughout the lifespan. Yeast is an equally important biotechnological tool that has long been the organism of choice for the production of alcoholic beverages, bread and a large variety of industrial products. For example, yeast is used to manufacture biofuels, lubricants, detergents, industrial enzymes, food additives and pharmaceuticals such as anti-parasitics, anti-cancer compounds, hormones (including insulin), vaccines and nutraceuticals. Its function as a cell factory is possible because of the speed with which it can be grown to high cell yields, the knowledge that it is generally recognized as safe (GRAS) and the ease with which metabolism and cellular pathways, such as translation can be manipulated. In this thesis, these two pathways are explored in the context of their biotechnological application to ageing research: (i) understanding translational processes during the high-yielding production of membrane protein drug targets and (ii) the manipulation of yeast metabolism to study the molecule, L-carnosine, which has been proposed to have anti-ageing properties. In the first of these themes, the yeast strains, spt3?, srb5?, gcn5? and yTHCBMS1, were examined since they have been previously demonstrated to dramatically increase the yields of a target membrane protein (the aquaporin, Fps1) compared to wild-type cells. The mechanisms underlying this discovery were therefore investigated. All high yielding strains were shown to have an altered translational state (mostly characterised by an initiation block) and constitutive phosphorylation of the translational initiation factor, eIF2a. The relevance of the initiation block was further supported by the finding that other strains, with known initiation blocks, are also high yielding for Fps1. A correlation in all strains between increased Fps1 yields and increased production of the transcriptional activator protein, Gcn4, suggested that yields are subject to translational control. Analysis of the 5´ untranslated region (UTR) of FPS1 revealed two upstream open reading frames (uORFs). Mutagenesis data suggest that high yielding strains may circumvent these control elements through either a leaky scanning or a re-initiation mechanism. In the second theme, the dipeptide L-carnosine (ß-alanyl-L-histidine) was investigated: it has previously been shown to inhibit the growth of cancer cells but delay senescence in cultured human fibroblasts and extend the lifespan of male fruit flies. To understand these apparently contradictory properties, the effects of L-carnosine on yeast were studied. S. cerevisiae can respire aerobically when grown on a non-fermentable carbon source as a substrate but has a respiro-fermentative metabolism when grown on a fermentable carbon source; these metabolisms mimic normal cell and cancerous cell metabolisms, respectively. When yeast were grown on fermentable carbon sources, in the presence of L-carnosine, a reduction in cell growth and viability was observed, which was not apparent for cells grown on a non-fermentable carbon source. The metabolism-dependent mechanism was confirmed in the respiratory yeast species Pichia pastoris. Further analysis of S. cerevisiae yeast strains with deletions in their nutrient-sensing pathway, which result in an increase in respiratory metabolism, confirmed the metabolism-dependent effects of L-carnosine.

The Cys-loop or nicotinic superfamily is an important group of ligand-gated ion channels. The pentameric channels in this family commonly mediate fast synaptic transmission in eukaryotes (cf. nicotinic acetylcholine, γ-aminobutyric acid receptor and glycine receptor). Related channels are also found in prokaryotes and these channels include Gloeobacter violaceous ligand-gated ion channel (GLIC) and Erwinia chrysanthemi ligand gated ion channel (ELIC). Cys-loop channels share a common structural fold and are thought to activate in a similar way: the ligand binds at the interface between the extracellular domains of adjacent subunits and causes the receptor to undergo a conformational change. This is conveyed to the transmembrane region of the receptor where it results in the opening of the channel pore. The energy landscape of the channel protein can be characterised by determining a kinetic mechanism, which explicitly details the functional states that the channel can visit and quantifies the transition rates between them. This is possible in ion channels because the current generated by a single channel molecule can be detected with high temporal resolution. Establishing a detailed kinetic mechanism allows a better understanding of the structure function relation for the protein, because it allows us to determine which step of the activation is affected when we change agonist or when we mutate the channel protein itself. The aim of my work was to establish the kinetic mechanism that best describes two homomeric ligand-gated ion channels, the α3 glycine receptor and ELIC receptor. This work involved recording single channel currents and the currents elicited by fast agonist applications and analysis by direct model fitting to the data. The physiological role of α3 glycine receptor is not currently known. The α3 expression is concentrated in areas involved in pain processing, such as the superficial dorsal horn (Harvey et al., 2004), suggesting that this isoform could be involved in the nociceptive pathway. Characterising the kinetics of a synaptic receptor is important also in understanding synaptic transmission, because it is the lifetime of the activated channel that is largely responsible for setting the time course of synaptic currents. We investigated the activation mechanism of this channel in HEK293 cells by maximum likelihood fitting of single-channel data, at a wide range of glycine concentrations. The mechanism we propose suggests that α3 channels can open only when more than 3 binding sites are occupied by glycine, and only after the channel undergoes a conformational change (‘flip’) that links binding to gating. The scheme can describe adequately macroscopic currents from fast concentration jumps experiments. The function of the prokaryotic channel ELIC is unknown, but ELIC is important as it has been recently crystallized (Hilf and Dutzler, 2008) in non-conductive state (the highest resolution structure currently available for a non-conductive state). Establishing a kinetic mechanism for this channel is particularly important, because combining structure and function offers the best possibility to investigate how the perturbation induced by the binding of the transmitter opens the channel. We investigated the activation mechanism of this channel in HEK293 cells by maximum likelihood fitting of single-channel data, at a wide range of propylamine concentrations. The mechanism we propose suggests that ELIC can open from partially primed conformational states, and only after the channel undergoes a conformational change (‘priming’), which links binding to gating. The scheme describes adequately both macroscopic currents from fast concentration jumps experiments and single channel activity.

There are considerable differences in the timing, type and extent of age-related decline between individuals who share the same chronological age, which may be driven by a combination of genetic, stochastic and environmental factors. Biomarkers of ageing (BoA) that can discriminate between individuals who differ in their biological age will therefore be useful to understand biological mechanisms, develop and test interventions and allow the prediction of age-related events so interventions can be implemented. In recent years, a variety of mechanistic candidate BoA have been discovered on the basis of a greatly improved understanding of the cellular and molecular biology of ageing. These include various measures of oxidative stress, which is thought to contribute causally to the ageing of organisms via its acceleration of cellular senescence. However, their reliability and validity as BoA, especially within population based cohorts are scarce. This study therefore focused on various oxidative stress-related measures as candidate BoA including: reactive oxygen species (ROS) production from dysfunctional mitochondria, by measuring superoxide levels, mitochondrial mass and mitochondrial membrane potential in blood mononuclear cells by flow cytometry; and also markers of lipid peroxidation, F2-isoprostanes, by measuring 8-iso Prostaglandin F2 by Automated Dissociation Enhanced Lanthanide Fluorescence Immunoassay (AutoDELFIA). Despite providing evidence of experimental reliability for all measures and also some evidence of construct validity for ROS production from dysfunctional mitochondria in terms of: associations with chronological age, associations with some markers of oxidative stressinduced cellular senescence, validation in a dietary restricted animal model of ageing and a role in an immunosenescent phenotype; there was no evidence of predictive validity in terms of longevity or age-related health outcomes in a population based cohort of the very old, the Newcastle 85+ study. This questions the predictive validity of these parameters as candidate BoA in the very old population.

No description available.

Platelet ion channels are essential for Ca2+-influx, maintaining the resting membrane potential and cell volume regulation. Cation channels have been widely studied but few reports of platelet anion channels exist. A recent ‘channelome’ screen has suggested that human platelets express several anion-permeable channels of unknown function. This thesis explores the function of two such channels, Pannexin-1 and TMEM16F, in platelets, primary megakaryocytes (MKs) and related cell lines. Using pannexin-1 inhibitors, these channels were shown to open in response to stimulation by thrombin, contribute to Ca2+-influx and release cytosolic ATP following stimulation by threshold concentrations of platelet agonists. Experiments also suggested that ATP release by pannexin-1 channels contributes to Ca2+-influx via stimulation of ATP-gated P2X1 receptors. Anion-selective TMEM16F channels have been recorded in a variety of cell types and activate in response to sustained elevation of [Ca2+]i to 100 μM. Controversially, these channels were reported to be cation-selective in mouse MKs. Thus, whole cell patch clamp recordings were performed to assess the biophysical properties of TMEM16F channels in HEL cells and primary mouse and rat MKs. Elevating [Ca2+]i to 100 μM in HEL cells and rat MKs induced a Ca2+-dependent, outwardly rectifying anion-permeable conductance, which was blocked by the TMEM16F inhibitor A01. Recordings of mouse MKs identified an equally Ca2+-dependent, outwardly rectifying and A01-sensitive conductance, however this was predominantly permeable to cations. Thus, a major interspecies difference exists in the ionic selectivity of MK TMEM16F channels; possible explanations for this difference, such as mutations within the pore region, are discussed. In summary, this thesis has explored the biophysical properties and function of platelet and MK anion channels using in vitro assays. These studies have relied heavily upon pharmacological tools and future studies of platelet function would benefit from the use of transgenic models.

This investigation explores the basis of the susceptibility to hearing loss from a neurobiological stand point, in different genetically modified and inbred strains of mice: CBA/Ca (CBA), C57BL/6J (C57), Kv2.2 knock-out (Kv2.2 KO) and USP42 KO. Kv2.2, a delayed rectifier potassium channel, is expressed in the ventral nucleus of the trapezoid body (VNTB) in the auditory brainstem, in which originates the medial olivocochlear system (MOC), an efferent system which protects and regulates the cochlea. Preventing expression of Kv2.2 predisposes mice to noise induced hearing loss. Labelling experiments presented here show Kv2.2 expressing neurons in the VNTB contribute to the population of MOC neurons, implicating regulation of excitability of MOC neurons in protection from hearing loss. The MOC system of the C57 is ineffective, thought to lend this strain predisposition to noise-induced and age-related hearing loss. Whole cell patch clamp was used to compare VNTB neurons of the C57 with those of the CBA, a different inbred strain with an effective MOC, in order to determine if differences in excitability exist between strains that may explain the differing strength of their respective MOC systems. Pre-hearing onset, the neurons of the VNTB were identical. Post hearing onset, electrophysiological characteristics diverged: the VNTB neurons of the CBA were able to support higher firing rates due to slower inactivation of its voltage-gated potassium currents. The role of inflammation in hair cell loss in the C57 was probed by knocking out USP42, a gene involved in cell-cycle arrest in response to metabolic damage, highlighting changes in the cochlear nuclei following peripheral loss of sensitivity. The changes observed in the VNTB and cochlear nuclei highlight the importance of activity dependent changes and homeostatic regulation of excitability in the central auditory system.

Dame Honor Bridget Fell, DBE, Ph.D, D.Sc, FRS (22 May 1900 - 22 April 1986) was a British scientist and zoologist. Her contributions to science included the development of experimental methods in organ culture, tissue culture, and cell biology. Her PhD thesis presented as a collection of papers was awarded in 1924 and is archived online here. More information about her life can be found on Wikipedia at https://en.wikipedia.org/wiki/Honor_Fell

This thesis seeks to demonstrate technologies capable of improving the purity of pluripotent-derived cells. Such cells offer an enormous opportunity for medical science. However, it is anticipated that a higher proportion of undifferentiated pluripotent cells will remain when manufacturing at scale. As indicated in the literature, the in vivo transplantation of undifferentiated cells is a threat to patient safety and is considered a limiting factor for large-scale manufacture. Pluripotent undifferentiated cells can be identified and removed based on an affinity interaction with the SSEA-4 antigen, which is down-regulated on differentiated cells. Using CD20+ and CD20- (HLA-A2+) lymphocyte cells as a cost-effective alternative, where the CD20+ cells are target impurities and the HLA-A2+ cells are product-designated cells, this thesis presents two technologies to minimise the proportion of undifferentiated cells during manufacture. These technologies rely upon shear stress-induced affinity separation to differentiate between cells with and without target antigens. A small-scale model is used to identify a range of shear stresses (0 25 dynes/cm2) with which these differences can be elucidated. These technologies are: (1) a quartz crystal microbalance (QCM) biosensor to detect pluripotent cell differentiation over a multi-day period, particularly during process development. The limit of quantitation (LoQ) was estimated to be 5,000 cells, which would enable the measurement of target cell purities in excess of 4 %. Findings provide the basis for such a system, but also highlight the technical challenges of development, in particular variability. (2) two affinity membranes (hollow fibre and flat sheet) were used to deplete cells positive for the target antigen, demonstrating a possible downstream-purification tool in instances where clinical purity does not suffice. The shear stress-induced detachment of adsorbed cells incubated for 30 minutes was investigated over 1 25 dynes/cm2 and 1 10 dynes/cm2 for the hollow fibre and flat sheet membranes respectively. Measured output included cell purity, which showed an increase in the relative change in purity (RCP) of 0.2 0.5 for the hollow fibre modules at 5 dynes/cm2 (n = 5) and was as high as 11.8 at 10 dynes/cm2 for the flat sheet membrane cassette (n = 1). A loss of cell membrane integrity, where up to 5 % in hollow fibres and up to 55 % in the flat sheet membrane were observed.