Dissertations / Theses on the topic 'Mammalian cells'
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Iqbal, Syed Amir. "Asymmetric Cell Division in Mammalian Cells." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503635.
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Lau, Stephen S. K. "Gene silencing in mammalian cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ28435.pdf.
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Renglin, Lindh Anna. "Mitotic aberrations in mammalian cells /." Stockholm : Dept. of genetics, microbiology and toxicology, Stockholm university, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-522.
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Xue, Yue 1978. "Iron metabolism in mammalian cells." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79216.
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Iron, known for its versatility, is an essential element in the metabolism of mammalian cells. One of the most common iron disorders is autosomal recessive disease---hereditary hemochromatosis, which leads to the iron overload in population of northern European descent. During years of my graduate research, I focused on the study of Hemochromatosis gene Hfe and a point mutation C282Y that leads to more than 80% of all hemochromatosis cases.Iron Regulatory Proteins (IRPs), which serve as main posttranscriptional regulators of cellular iron homeostasis, are the other interest of research. Iron regulatory proteins reversibly interact with iron regulatory elements (IREs) within ferritin and transferrin receptor (TfR) mRNAs. The binding ability of IRPs is under tight control so that they respond to the changes in the intracellular iron requirements in a coordinate manner by differentially regulating ferritin mRNA translational efficiency and TfR mRNA stability. Besides intracellular iron levels, some other stimuli, such as oxidative stress, are capable of regulating this RNA-protein interactions.
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Hawley, Patricia. "Oligodeoxynucleotide interaction with mammalian cells." Thesis, University of East Anglia, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338058.
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Gohari, Nasrollah Saleh. "Homologous recombination in mammalian cells." Thesis, University of Sheffield, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414660.
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Jiang, Lei. "Mitochondrial Distribution in Mammalian Cells." University of Dayton / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1259968456.
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Heikinheimo, Liisa. "Phosphatidylserine translocation in mammalian cells." Helsinki : University of Helsinki, 2002. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/heikinheimo/.
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Bekei, Beata [Verfasser]. "In-cell NMR Spectroscopy in Mammalian Cells / Beata Bekei." Berlin : Freie Universität Berlin, 2013. http://d-nb.info/1043198059/34.
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Fengler, John Josef Paul. "Respiration induced oxygen gradients in cultured mammalian cells." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/28381.
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Oxygen is known to sensitize X-irradiated cells to lethal radiation damage. At low ambient oxygen tensions, however, the molecular mechanisms of the sensitization process and the metabolic requirements of the cell may be forced to compete for the cellular oxygen supply. The effect of cell respiration on the availability of intracellular oxygen during irradiation was consequently investigated by comparing the radiosensitivities of respiring and non-respiring cells. Cultured mammalian cells were irradiated in single cell suspensions and thin film monolayers at respiration inhibiting (4°C) and at normal cell culturing (37°C) temperatures. Due to oxygen equilibration and radiolytic depletion problems, the results of the suspension culture experiments were inconclusive. By subsequently analyzing the diffusive mass transfer of oxygen in the suspension medium, the stirrer flask was determined to be an inappropriate culture vessel in which to irradiate cells at constant low oxygen concentrations. A thin film cell culture system in which the oxygen concentrations to which the cells were exposed during irradiation could be more accurately controlled was then developed. A comparison of the oxygen enhanced radiosensitivities of the respiring and non-respiring cells in thin film monolayers suggested that the metabolic depletion of oxygen at low oxygen tensions has a significant effect on the local and intracellular oxygen distribution. These effects are representative of those that would be produced if respiration induced oxygen gradients existed inside and immediately around respiring cells. The magnitude of the differential radiosensitivities was found to be dependent on cell shape and to have values that agreed very well with theoretical predictions based on the existence of such gradients.Science, Faculty of
Physics and Astronomy, Department of
Graduate
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Godart, Helene. "KCI cotransport regulation in mammalian erythrocyctes." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318547.
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Savolainen, Linda. "Transcription Associated Recombination in Mammalian Cells." Doctoral thesis, Stockholm : Department of Genetics, Microbiology and Toxicology, Stockholm University, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-38931.
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Diss. (sammanfattning) Stockholm : Stockholms universitet, 2010.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.
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Oswald, Corina. "Mitochondrial copper homeostasis in mammalian cells." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-61580.
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Assembly of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the correct insertion of subunits, accessory proteins, cofactors and prosthetic groups. Most of the fundamental biological knowledge concerning mitochondrial copper homeostasis and insertion of copper into COX derives from investigations in the yeast Saccharomyces cerevisiae. In this organism, Cox17 was the first identified factor involved in this pathway. It is a low molecular weight protein containing highly conserved twin Cx9C motifs and is localized in the cytoplasm as well as in the mitochondrial intermembrane space. It was shown that copper-binding is essential for its function.
So far, the role of Cox17 in the mammalian mitochondrial copper metabolism has not been well elucidated. Homozygous disruption of the mouse COX17 gene leads to COX deficiency followed by embryonic death, which implies an indispensable role for Cox17 in cell survival.
In this thesis, the role of COX17 in the biogenesis of the respiratory chain in HeLa cells was explored by use of siRNA. The knockdown of COX17 results in a reduced steady-state concentration of the copper-bearing subunits of COX and affects growth of HeLa cells accompagnied by an accumulation of ROS and apoptotic cells. Furthermore, in accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper center of COX, COX17 siRNA knockdown affects COX-activity and -assembly. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so called supercomplexes. While the abundance of COX dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. Accumulation of a novel ~150 kDa complex containing Cox1, but not Cox2 could be observed. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. Data presented here suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. Instead an interdependent assembly scenario for the formation of supercomplexes is proposed that requires the coordinated synthesis and association of individual complexes.
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Elvers, Ingegerd. "Replication Fork Stability in Mammalian Cells." Doctoral thesis, Stockholms universitet, Institutionen för genetik, mikrobiologi och toxikologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-56697.
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Maintaining replication fork integrity is vital to preserve genomic stability and avoid cancer. Physical DNA damage and altered nucleotide or protein pools represent replication obstacles, generating replicative stress. Numerous cellular responses have evolved to ensure faithful DNA replication despite such challenges. Understanding those responses is essential to understand and prevent or treat replication-associated diseases, such as cancer. Re-priming is a mechanism to allow resumption of DNA synthesis past a fork-stalling lesion. This was recently suggested in yeast and explains the formation of gaps during DNA replication on damaged DNA. Using a combination of assays, we indicate the existence of re-priming also in human cells following UV irradiation. The gap left behind a re-primed fork must be stabilised to avoid replication-associated collapse. Our results show that the checkpoint signalling protein CHK1 is dispensable for stabilisation of replication forks after UV irradiation, despite its role in replication fork progression on UV-damaged DNA. It is not known what proteins are necessary for collapse of an unsealed gap or a stalled fork. We exclude one, previously suggested, endonuclease from this mechanism in UV-irradiated human fibroblasts. We also show that focus formation of repair protein RAD51 is not necessarily associated with cellular sensitivity to agents inducing replicative stress, in rad51d CHO mutant cells. Multiple factors are required for replication fork stability, also under unperturbed conditions. We identify the histone methyltransferase SET8 as an important player in the maintenance of replication fork stability. SET8 is required for replication fork progression, and depletion of SET8 led to the formation of replication-associated DNA damage. In summary, our results increase the knowledge about mechanisms and signalling at replication forks in unperturbed cells and after induction of replicative stress.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 2: Submitted. Paper 3: Manuscript. Paper 5: Submitted.
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Schuliga, Michael, and michael schuliga@deakin edu au. "Steroidogenesis in cultured mammalian glial cells." Deakin University. School of Biological and Chemical Sciences, 1998. http://tux.lib.deakin.edu.au./adt-VDU/public/adt-VDU20061207.154152.
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A protocol for culturing mammalian type 1 astrocytic cells, using female post-natal rat cerebral cortical tissue, was established and refined for use in steroidogenic metabolic studies incorporating progestin radioisotopes. Cultures were characterised for homogeneity using standard morphological and immunostaining techniques. Qualitative and quantitative studies were conducted to characterise the progesterone (P) metabolic pathways present in astrocytes in vitro. Of particular interest was the formation of the P metabolite, 5á-pregnan-3á-ol-20-one (THP). THP is a GABA(A) receptor agonist, believed to play a vital role in neural functioning and CNS homeostasis. One aim of this study was to observe any modulatory effects selected neuroactive ligands have on the conversion of P into THP, in an attempt to link astrocytic steroidogenesis with neuronal control.
In qualitative studies, chromatographic procedures were used to establish the progestin profile of cerebral cortical astrocytes. Tritiated P, DHP (5á-pregnan-3,20-dione) and THP incurbates were preliminary fractionated by either normal phase (NP) or reverse phase (RP) high performance liquid chromatography (HPLC). The radiometabolites associated with each fraction were further chromatographed, before and/or after chemical derivatistation, by the aforemention HPLC procedures and thin layer chromatography (TLC). Steroid radiometabolites were tentatively identified by comparing their chromatographic mobility with authentic steroids. The identity of the main putative 5á-reduced P metabolities, DHP, THP and 5á-pregnan-3á,20á-diol (20áOH-THP) were further confirmed by isotopic dilution analysis. Their conclusive identification, along with the tentative identification of 20á-hydroxypreg-4-en-3-one (20áOH-P) and 20á-hydroxy-5á-pregnan-3-one (20áOH-DHP), verify the localisation of 5á-reductase, 3á-hydroxy steroif oxidoreductase (HSOR), and 20á-HSOR activity in the cultured astrocytes utilised in this study programme. Other minor metabolites detected were tentatively identified, including 5á-pregnan-3á,21-diol-20-one (THDoc), indicating the presence of 21-hydroxylase enzymatic activity. THDoc, like THP, is a GABA(A) receptor agonist. The chemical and physical characterisation of several yet unidentified progestin metabolites, associated with a highly polar RP HPLC fraction (designated RP peak 1*), indicate the presence of one or more extra hydroxylase enzymes.
Quantitative analysis included a preliminary study. In this study, the percentage yields of radiometabolites formed in cultures incubated with increasing substrate concentrations of (3)H-P for 24 hours were determined. At the lower concentrations examined (ie 0.5 to 50nM), the metabolites associated with the polar RP HPLC fraction (RP peak 1*) collectively have the highest percentage yield. They are subsequently considered metabolic end products of degradative catabolic P pathways. The percentage yield of THP peaks in the medium concentration ranges (ie 5 to 500nM), whereas DHP remains fairly static at a low level with increasing concentration. Both DHP and THP are considered metabolic pathway intermediates. The percentage yield of 20áOH-THP continues to increase with increasing concentration over 5nM, superseding THP approaching the highest concentration examined (5000nM). This indicated the formation of 20áOH-THP does not occur entirely via THP. 20áOH-THP also possibly serves as the direct intermediate in the formation of the main radiometabolites associated with RP peak 1*.
A time/yield study incorporating incubation times from one to 24 hours was also conducted. The full array of radiometabolites (individually or in groups) formed in astrocyte cultures incubated with 50nM tritiated P, DHP of THP, were assayed. Cultures were observed to rapidly convert any DHP into THP, showing astrocytic 3á-HSOR activity is very high. The study also showed 5á-reduction (ie the conversation of P into DHP) is the rate limiting reaction in the two step conversion of P into THP. 5á-Reduction also appears to be a rate limiting step in the formation of 20á-hydroxylated metabolites in astrocytes. Cultures incubated with the tritiated 5á-reduced pregnanes from one to four hours form greater quantities to 20á-hydroxylated radiometabolites compared to cultures incubated with (3)H-P.
The time yield/studies also provided further evidence the unidentified polar radiometabolites associated with RP peak 1* are metabolic end products. For the P and DHP incubates, the collective formation of the aforementioned polar radiometabolites initially lags behind the formation of THP. As the formation of the latter begins to plateau with increasing time between four to 24 hours, the net yield of radiometabolites associated with RP peak 1* continues to rise. The time/yield studies also indicate 5á-reduction and perhaps 3á-hydroxylation are pre-requisite steps in the formation of the polar metabolites. Cultures incubated with the 5á-reduced progestins from one to four hours form higher yields of the radiometabolites associated with RP peak 1* compared to cultures incubated with P as substrate. The net yields of the radiometabolites associated with RP peak 1* for cultures incubated with THP were substantially higher compared to cultures incubated with DHP after equivalent times.
The effect selected neuroligands have on the yield of radiometabolites formed by cultured astrocytes incubated with 50nM (3)H-P was also examined. Dibutyryl cyclic adenosine monophosphate (DBcAMP), not actually a neuroligand per se, but an analog of the intracellular secondary messenger cAMP, was also utilised in these studies. The inhibitory neurotransmitter ã-amino-nbutyric acid (GABA), DBcAMP and isoproterenol (a â-adrenergic receptor agonist) all quickly induce a transient but substantial increase in 20á-HSOR activity in cultured astrocytes. Cultures pretreated with these three compounds (10, 20 and 1µM respectively) form substantially higher yields of 20á-hydroxylated metabolites, including 20áOH-THP (between 200 to 580% greater), when incubated with 50nM (3)H-P for one to four hours. These increases also coincide with increases in the net yield of metabolites formed (by 16 to 48%). The same pre-treated cultures form significantly lower yields of THP, by 25 to 41%, after one hour. This is most likely due to the increased metabolism of any formed THP into 20áOH-THP.
Octopamine (an á-adrenergic agonist) only induces a slight increase in 20á-HSOR activity, having relatively little effect on the yield of 20áOH-THP formed. Pretreatment with octopamine induces a significant increase in the yield of THP for cultures incubated with (3)H-P for four hours (by 24%). The increase in THP formation appears to be due to an increase in 3á-HSOR activity, as judged by the concomitant drop in the yield of the 5á-reduced, 3-keto substrates. An increase in 5á-reductase activity cannot be excluded however. Isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol one and four hour incubates have higher yields of DHP. This is in contrast to the other three incubates. After 12 hours, all incubates have higher yields of THP (15-30%).
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Korsieporn, Pira. "Interaction of plasticizers with mammalian cells." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98982.
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This investigation was focused on the in vitro metabolism of DEHA, DEHP and other common plasticizers by mammalian cell lines. The metabolic products and cell viability of hepatocytes (mouse and human) and human umbilical endothelial cells exposed to plasticizers was investigated in static culture.Gas chromatography and mass spectrometry showed that all of the plasticizers investigated were partially degraded, but at differing rates, depending on the plasticizer and cell line. Solubility and stearic effects were found to play important roles in determining the rate of hydrolysis. The only metabolic product observed was 2-ethyl hexanol, which accumulated in culture. This was due to the lack of alcohol dehydrogenase production in the human hepatocyte cell line used.
Hepatocyte cell viability was not significantly affected at 4 days of exposure to DEHA. By 12 days, only 50% of the cells remained viable when compared to control experiments. These results suggest that the accumulation of plasticizers metabolites, specifically 2-ethyl hexanol, may have potentially toxic effects.
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McGlynn, Andrea. "Interaction of DEHA with mammalian cells." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111939.
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This project studied the biodegradation of a plasticizer, di-(2-ethylhexyl) adipate (DEHA), by two mammalian cell lines, HepG2 and WIF-B, in vitro . An MTT assay showed that DEHA had a toxic effect on both cell lines. Despite this, both hepatocyte cell lines successfully degraded the plasticizer. Metabolites were identified and quantified by gas chromatography. HepG2 cells showed minimal alcohol dehydrogense activity and this resulted in the accumulation of 2-ethylhexanol. WIF-B cells were able to breakdown the alcohol and produced 2-ethylhexanoic acid. It is important to note that an enzyme was essential for this step in the degradation of the plasticizer, as this proves that it was biodegradation and not physical degradation. By comparing the metabolites formed and the order of their appearance, the degradation pathway in these mammalian cells was found to be similar to the established degradation pathways for bacteria, fungi and yeast.
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Terrone, Donato Gerardo. "Ras protein targeting in mammalian cells." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98506.
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The Ras proteins (H-Ras, N-Ras and K-Ras4B) are monomeric GTP-binding proteins that play key roles in cell regulation. It has been proposed that correct plasma membrane localization of the Ras proteins requires 'two signals', the processed farnesylation sequence and an adjacent palmitoylation site(s) or polybasic sequence. First, to investigate potential proteinaceous binding partners for the K-Ras4B targeting sequence at the plasma membrane, an in vivo crosslinking analysis was carried out. Consistent with current suggestions that K-Ras4B interacts via electrostatic interactions with plasma membrane lipids, no indication of a K-Ras4B plasma membrane binding partner was found. In the second portion of this thesis, I describe the development of a new approach, using fluorescence microscopy and the technique of rapamycin-induced protein heterodimerization, to demonstrate that prenylated proteins lacking a 'second signal' are not specifically sequestered in the endoplasmic reticulum and Golgi compartments as suggested previously but can distribute freely and rapidly between different cellular membranes.
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Lao, Yuanzhi. "Calcium signaling in apoptotic mammalian cells /." View abstract or full-text, 2008. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202008%20LAO.
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Zheng, Yi. "Nucleotide excision repair in mammalian cells /." Digital version accessible at:, 1999. http://wwwlib.umi.com/cr/utexas/main.
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Wiese, Meike. "Characterisation of HP1γ in mammalian cells." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/273362.
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The degree of chromatin compaction plays a fundamental role in controlling the accessibility of DNA to the transcription machinery as well as other DNA-dependent biological pathways. The mammalian HP1 (Heterochromatin protein 1) protein family consists of three members: HP1α, β and γ. Each paralogue regulates formation and maintenance of heterochromatin by binding to the repressive chromatin marks H3K9me2/3 with their chromodomains (CDs). Despite high sequence conservation, each HP1 paralogue possesses specific functions, which are likely to be cell type specific. The aim of my thesis was to find novel functions for HP1γ in mouse embryonic stem cells (mESCs) and breast cancer cells. Mass spectrometry analysis identified citrullination of residues R38 and R39 within the CD of HP1γ. I show that these residues are citrullinated by peptidyl arginine deiminase 4 (PADI4) in vitro and in vivo. Mutations in HP1γ (R38/9A), designed to mimic the loss of charge accompanied with citrullination, affect HP1γ’s binding to H3K9me3 peptides and reduce its residence time on chromatin in differentiated mESCs, indicating a role for citrullination in regulating HP1γ binding to chromatin during differentiation. Furthermore, I studied the phenotype of HP1γ depletion in two human breast cancer models and found that HP1γ is essential for cell proliferation and viability of cancer, but not of normal epithelial cells. I performed whole transcriptome analysis in breast cancer cells depleted of HP1γ and cross-referenced it with its genomic localisation, which identified increased expression of interferon/antiviral defense genes and activation of pro-apoptotic pathways. Whilst genes involved in these pathways were not directly bound by HP1γ, this analysis also identified HP1γ as a novel regulator of zinc finger (ZNF) genes. In summary, I identified novel post-translational modifications in HP1γ and characterised them in mESCs. I further demonstrated a role for HP1γ regulating breast cancer cell viability and identified HP1γ as a novel regulator of ZNF genes. My findings highlight HP1γ as a potential target for breast cancer therapy.
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Wong, Ching-hang. "Cell-cell interactions and cell junction dynamics in the mammalian testis." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B31993084.
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Beckmann, Roland. "Expression of the red cell anion exchanger in mammalian cells." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313008.
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Zoro, Barnaby James Henry. "Bioprocessing of mammalian cells for tissue engineering and cell therapies." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1446824/.
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It is envisaged that in order to achieve the stringent quality requirements of future human cell and tissue manufacturing processes, implementation of automated processing systems will be required. As automated systems are capable of operating at high speeds, throughput is likely to be constrained by sensitivity of cellular materials to mechanical stress. The effect of laminar capillary shear stress (pipe flow) on rat aortic smooth muscle cells was investigated. Initial studies showed that repeated exposure to high shear stress (>100 Pa) causes substantial cell damage, with surviving cells able to grow normally. A flow cytometry assay for cell membrane integrity was developed and exhibited high assay resolution, precision and speed. Further shear studies indicated minimal cell damage or loss in the shear stress range 5-25 Pa, with some evidence of cell damage at high shear stresses (35-75 Pa). Cell loss at very low shear stress ( 2 Pa) was attributed to surface adhesion. Manufacture of cell based therapeutics requires the generation and processing of concentrated cell suspensions. Volume mean cell diameter for rat aortic smooth muscle cells was measured to be 22.4 mum and enabled conversion between cell concentration and cell volume fraction in suspensions. Concentrated cell suspensions behaved as 'power law' (shear thinning) fluids and showed similarity with reported rheology of concentrated yeast and plant cell suspensions. Predictions based on literature values for oxygen uptake rate of mammalian cells indicated that oxygen depletion is likely to occur during processing of cell pellets and concentrates. Two semi-automated cell concentration protocols (centrifuge-resuspend) were developed and compared. Both protocols achieved cell volume fraction of around 0.3-0.4 (wet pellet) and the superior protocol exhibited intact cell yield of ~75%. Apparent loss of cells was mainly attributed to formation of large aggregates during concentration. A 'windows of operation' analysis was constructed to examine process feasibility in the context of experimental results. Three different processing scenarios were predicted to be feasible and it was shown that processing yield is a critical factor in determining overall process feasibility and manufacturing costs.
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Norman, M. J. "Loss of scribble causes cell competition in mammalian epithelial cells." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302397/.
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Cancer is a disease caused by transformation of cells by the activation or over-expression of oncogenes such as Ras and c-myc, and the loss of tumour suppressor genes such as E-cadherin and scribble. The initial stage of tumourigenesis is the transformation of a single cell in an otherwise normal epithelium. What occurs at this stage is largely unknown - do the transformed cells and normal cells co-exist or is there an antagonism between them? This thesis examines the fate of epithelial cells that lose the tumour suppressor scribble when in an otherwise normal epithelium. The fate of scribble knockout clones has been studied in Drosophila melanogaster larval imaginal discs. It has been observed that scribble knockout clones are removed from the larval tissues by c-Jun N-terminal kinase (JNK) dependent apoptosis. It is though that this is an innate tumour suppressive mechanism. It is therefore of great interest and importance to understand if a similar phenomenon can be seen in mammalian cells. Scribble knockdown Madin-Darby canine kidney (MDCK) epithelial cells die only when surrounded by normal MDCK cells. Dead scribble short-hairpin RNA (shRNA) cells are apically extruded from the epithelium after cell death and exhibit classical apoptotic markers such as cytoplasmic condensation, caspase 3 activation and DNA fragmentation. Extrusion of dead scribble knockout cells occurs after initiation of apoptosis as blocking myosin activation results in many dead scribble knockout cells staying in the epithelial monolayer. Prior to cell death they maintain normal cell-cell adhesion with their normal MDCK neighbours and activate the stress induced protein kinase p38, but not c-‐Jun N‐- terminal kinase (JNK).
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Hall, James Anthony. "Swelling-activated transport of diverse solutes in mammalian cells." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320647.
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Wong, Ching-hang, and 黃政珩. "Cell-cell interactions and cell junction dynamics in the mammalian testis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B31993084.
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Hattori, Kimihiko, Shinji Hayano, and Hisao Seo. "Expression of ZAKI-4 in Mammalian Cells." Research Institute of Environmental Medicine, Nagoya University, 2003. http://hdl.handle.net/2237/7558.
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Rahman, Ruman. "Regulation of telomere length in mammalian cells." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/25107.
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Mammalian telomere length is regulated in a dynamic manner through multi-factorial elements. This project focuses on specific aspects of both extrinsic and intrinsic factors that govern telomere length. Expression of the human telomerase and reverse transcriptase (hTERT) gene in sheep fibroblasts reconstitutes telomerase activity and extends their lifespan. Although stabilization of telomere length is directly correlated to hTERT expression level, this was not reflected in telomerase activity. Data presented here shows that overexpression of non-catalytic domains decreased telomerase activity, compromised senescence bypass status and did not enhance telomere maintenance, which may result from competitive binding to sheep TR molecules and/or disruption of hTERT homodimeric structure. These results are consistent with the notion that telomere maintenance requires a functional telomerase complex which contains an inter-dependent hTERT homodimeric structure. The single-stranded 3’ G-rich overhang has been implicated as a key feature with a telomeric end-protection configuration. Data presented here show that the 3’ overhand is eroded during culture of sheep fibroblasts, indicating that the 3’ overhang length shortens prior to replicative senescence in cultured sheep fibroblasts. Furthermore, a correlation between hTERT expression level, karyotypic stability and 3’ overhang length is observed. This project also demonstrated that first application of the Quantitative-FISH (Q-FISH) and Telomere-Oligonucleotide Ligation Assay (T-OLA) techniques in the sheep model system.
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Brown, Stephanie Marie. "Replication of damaged DNA in mammalian cells." Thesis, University of Sussex, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445620.
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Bell, Patricia Louise. "Ectopic gene conversion in mammalian somatic cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ61872.pdf.
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Braddock, Peta S. H. "NMR studies of genetically modified mammalian cells." Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.279910.
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Shamsher, M. K. "Molecular analysis of mutation in mammalian cells." Thesis, Swansea University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.638809.
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Most of the currently available mammalian mutation systems depend on the selection of a mutant phenotype. These selective systems include resistance to the effects of toxic drugs, as is widely used in various mammalian cultured cell systems, and the outgrowth of mutant cells. These systems have served us well for many years but they have their limitations, especially as they can only be used to detect selected target genes in the genome. Here, the restriction site mutation (RSM) assay is proposed as a methodology for the detection of DNA base changes in restriction enzyme recognition sites, without the need for prior selection methods. Changes within a restriction site, created by base mutation renders an inherent restriction site on target genomic DNA resistant to digestion with the particular endonucleases. These unrestricted sequences are then amplified with the polymerase chain reaction where enrichment of the mutated sequence occurs. The developmental stages of the RSM, its applications and limitations are described here when applied to the study of point mutations in the ras oncogene from various sources, namely tissue culture cells, vectors and various tissues from intact mammals. The relative ease and high sensitivity of this technique makes it a suitable method for identifying mutations at specific sites in DNA, especially when the mutated target sequences are present at low frequencies. Here. the methodology has been shown to be applicable to any species for which the DNA sequence information is available.
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Kukushkin, Nikolay V. "Pathways for glycoprotein degradation in mammalian cells." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.556211.
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/ In mammalian cells, N-glycoproteins comprise the bulk of secretory cargo. N-glycosylation is tightly linked to the mechanisms of eo- and post-translational folding and quality control in the endoplasmic reticulum (ER). Aberrant glycoproteins must be cleared from the secretory pathway. Several mechanisms for glycoprotein degradation exist, of which ER-associated degradation (ERAD) is most well-studied. The latter involves retrotranslocation of misfolded glycoproteins into the cytosol, followed by proteasomal destruction of the polypeptide. N-linked oligosaccharides are removed from glycoproteins during degradation to form free oligosaccharides (FOS). FOS analysis has been employed to gain a global, rather than single protein-centred view of the pathways of ERAD and the mechanisms of its regulation. It was established that convergent ERAD pathways, distinct in the site of deglycosylation, as well as sensitivity to a number of modulators, exist in the cell. It was further demonstrated that the pathway leading to the production of ER-localised FOS is linked to retrograde Golgi-to-ER transport and is masked by the activity of Golgi endomannosidase. Investigating the involvement on the latter in ERAD has uncovered an endomannosidase-mediated e pre-degradative glycoprotein processing route and showed that endomannosidase can rescue a fraction of glycoproteins from degradation following inhibition of ER glucosidases. Further investigation of endomannosidase activity and specificity in different cell lines showed that the ability of the enzyme to process truncated monoglucosylated oligosaccharides appears' to be necessary and sufficient for evolutionary conservation in mammals. Overall, the data presented have demonstrated on the global level the connections between the pathways for glycoprotein degradation and processing in the Golgi apparatus, underlining the complexity of biosynthetic regulation in the secretory pathway.
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朱瑞中 and Sui-chung Chu. "Regulation of lipoprotein uptake in mammalian cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31969707.
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Rios, Villanueva Xavier. "Toward Multiplex Genome Engineering in Mammalian Cells." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11179.
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Given the explosion in human genetic data, new high-throughput genetic methods are necessary for studying variants and elucidating their role in human disease. In Chapter I, I will expand on this concept and describe current methods for genetically modifying human cells. In E. coli, Multiplex Automatable Genome Engineering (MAGE) is a powerful tool that enables the targeting of multiple genomic loci simultaneously with synthetic oligos that are recombined at high frequencies in an optimized strain. MAGE as a method has two components: organism-specific optimization of oligo recombination parameters and a protein capable of increasing recombination frequencies.
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Al-Rekabi, Zeinab. "Investigating Mechanotransduction and Mechanosensitivity in Mammalian Cells." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/30256.
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Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.
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Gedge, Lucinda J. E. "Actin in the nuclei of mammalian cells." Thesis, University of Leeds, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400260.
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Keen, Michael John. "Low-protein media for specialised mammalian cells." Thesis, London South Bank University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336367.
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Heinrichs, Arianne Augustina Johanna. "Antibody display and selection on mammalian cells." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627100.
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Hannibal, Luciana. "Intracellular Processing of Cobalamins in Mammalian Cells." Kent State University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=kent1247938485.
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Brunton, Valerie G. "Polyamine toxicity in mammalian cells in culture." Thesis, University of Aberdeen, 1990. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU546698.
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The polyamines, spermidine and spermine, and their diamine precursor putrescine, act as both positive and negative regulators of cell growth. Their oxidation to growth inhibitory aminoaldehydes by a copper-dependent amine oxidase present in ruminant serum was initially thought to be responsible for their toxicity. However, more recently, it has been shown that polyamines are toxic in the absence of ruminant serum and that oxidation by an intracellular amine oxidase may be involved. It is this area of polyamine toxicity that this work has focused on. The model used for a normal fibroblast cell line derived from baby hamster kidney cells (BHK-21/C13). They are routinely grown in Dulbecco's medium supplemented with 10&'37 (v/v) horse serum. There was no extracellular metabolism of spermine in this system. The ID50 value for spermine in BHK cells was 1.03 0.07mM following a 24h exposure to the amine. 2mM-Spermine was cytotoxic, resulting in a significant decrease in cell number, protein content and DNA synthesis after an 8h exposure. Treatment of cells with this concentration of spermine resulted in the accumulation of spermine intracellularly, with levels reaching 11 fold higher than in control cells. Pretreatment of cells with aminoguanidine, an inhibitor of copper-containing amine oxidases, attenuated spermine toxicity, suggesting that oxidation by an intracellular copper-containing amine oxidase plays an important role in spermine toxicity. As well as the copper-dependent amine oxidases, polyamines can also be oxidized by a FAD-dependent peroxisomal amine oxidase. This enzyme is involved in the polyamine retroconversion pathway which converts spermine back to spermidine and putrescine by a series of acetylation and oxidation reactions. Inhibition of this enzyme enhanced the toxicity of spermine, which indicates a protective role for the retroconversion pathway in the removal of the high intracellular spermine pool. This may be linked to enhanced excretion of spermidine and N. 1-acetylspermidine which have been shown to be the main excretory products of BHK cells. Intracellular reduced glutathione levels were depleted in spermine-treated cells. The depletion of glutathione preceded any effect on cell growth and prior depletion of the thiol, by inhibiting its synthesis, enhanced the toxicity of spermine, both suggestive of an important cytoprotective role for glutathione within the cells against spermine toxicity. This may be in the removal of H_2O_2 formed during spermine oxidation, however inactivation of the glutathione redox cycle which is responsible for this did not alter the toxicity of spermine. Glutathione may conjugate with spermine or an oxidative metabolite of spermine, which would act to detoxify the amine and also result in a depletion of intracellular glutathione. When cells that were preloaded with [. 35S]cysteine, were treated with [. 14C]spermine, there was no indication of a double labelled compound in the acid soluble material, indicative of a glutathione spermine conjugate. More studies are required before the formation of such an adduct can be dismissed, as the conjugate may be excreted or broken down, or may be associated with protein thiol groups.
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Termanis, Ausma. "Regulators of DNA methylation in mammalian cells." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/11749.
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Although the many cells within a mammal share the same DNA sequence, their gene expression programmes are highly heterogeneous, and their functions correspondingly diverse. This heterogeneity within an isogenic population of cells arises in part from the ability of each cell to respond to its immediate surroundings via a network of signalling pathways. However, this is not sufficient to explain many of the transcriptional and functional differences between cells, particularly those that are more stable, or, indeed, differences in expression between parental alleles within the same cell. This conundrum lead to the emergence of the field of epigenetics - the study of heritable changes in gene expression independent of DNA sequence. Such changes are dependent on “epigenetic modifications”, of which DNA methylation is one of the best characterised, and is associated with gene silencing. The establishment of correct DNA methylation patterns is particularly important during early development, leading to cell type specific and parental allele specific gene regulation. Besides DNA methyltransferases, various other proteins have recently been implicated in DNA methylation. The absence of these proteins leads to defects in DNA methylation and development that can be even more severe than those in DNA methyltransferase knockouts themselves. In this study I focus on three such accessory proteins: LSH (a putative chromatin remodelling ATPase), G9a (a histone lysine methyltransferase) and SmcHD1 (a structural maintenance of chromosomes protein). To compare DNA methylation between WT cells and cells knocked out for each of these proteins, I used whole genome methylated DNA affinity purification and subsequent hybridization to promoter microarrays. This enabled me to compare the requirement for each protein in DNA methylation at specific genomic regions. The absence of LSH in mouse embryonic fibroblasts (MEFs) resulted in the loss of DNA methylation at 20% of usually methylated promoters, and the misregulation of associated protein coding genes. This revealed a requirement for LSH in the establishment of DNA methylation at promoters normally methylated during pre-implantation as well as post-implantation development. Secondly, I identified hypomethylation at 26% of normally methylated promoters in G9a-/- compared to WT ES cells. Strikingly, this revealed that G9a is required for maintenance of DNA methylation at maternal as well as paternal imprinting control regions (ICRs). This is accompanied by expression defects of imprinted genes regulated by these ICRs. Finally, in collaboration with the Brockdorff lab at the University of Oxford I identified a role for SmcHD1 in establishing DNA methylation at promoters on the X chromosome normally methylated slowly during X chromosome inactivation. Interestingly, SmcHD1 was also required for DNA methylation at autosomal gene promoters, contrary to previous reports that it is mainly involved in X chromosome methylation. I conclude that different accessory proteins are required to facilitate correct DNA methylation and gene repression at distinct regions of the genome, as well as at different times during development. This function of accessory proteins may be in part dependent on the prior establishment of specific chromatin signatures and developmental signals, together comprising a precisely regulated system to establish and maintain appropriate DNA methylation throughout development.
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Cook, April D. "Characterization of nucleosome occupancy in mammalian cells." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13070019.
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Chromatin is a complex of genomic DNA, RNA, and associated proteins. Many of the processes that occur on chromatin regulate the accessibility of the genetic material of a cell. The nucleosome is the basic subunit of chromatin, composed of a histone octamer wrapped with approximately 150bp of DNA. Alterations to chromatin structure, including to nucleosomes and their location, underlie global transcriptional diversity. A striking example of this is the so-called "open" chromatin state in pluripotent cells, characterized by loosely bound chromatin proteins and rapid nucleosome turnover, that allows transcriptional flexibility for subsequent differentiation. In contrast, differentiated cells contain compacted chromatin that can selectively block access to DNA and subsequent transcription. Thus, characterizing the physical state of chromatin is important to understanding its regulatory state.
Digestion of chromatin with micrococcal nuclease (MNase) and subsequent sequencing of the protected DNA fragments produces a map of nucleosome occupancy. Traditional MNase mapping experiments capture a snapshot of nucleosome occupancy, providing information about nucleosomes that are accessible at the level of digestion used. We analyzed regions of difference in nucleosome occupancy between embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and differentiated cell types using traditional MNase-seq and found that differences in pluripotent and differentiated cells are punctate and correlate with regulatory regions important for pluripotency and development. Further, our analysis shows ESCs and iPSCs to be vastly more similar to each other in their chromatin structure than to the differentiated cells.
We then developed a new way of collecting and analyzing MNase-seq data that allows us to determine both nucleosome occupancy as well as the accessibility of DNA to regulatory factors. Our methodology discerns distinct physical states of chromatin and provides novel insights into the accessibility of regulatory regions. Additionally, we present a quantitative metric useful for characterizing local and global regions of the genome that should be useful in future cell type comparisons.
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Devary, Yoram. "Response of mammalian cells to ultraviolet irradiation /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1997. http://wwwlib.umi.com/cr/ucsd/fullcit?p9724890.
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Chu, Sui-chung. "Regulation of lipoprotein uptake in mammalian cells." Hong Kong : University of Hong Kong, 2000. http://sunzi.lib.hku.hk/hkuto/record.jsp?B22088982.
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Ives, Katherine Jane. "The effects of ultrasound on mammalian cells." Thesis, University of Bath, 1999. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301523.
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Calegari, Federico, and Julieta Aprea. "Bioelectric State and Cell Cycle Control of Mammalian Neural Stem Cells." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-185623.
Full textAbstract:
The concerted action of ion channels and pumps establishing a resting membrane potential has been most thoroughly studied in the context of excitable cells, most notably neurons, but emerging evidences indicate that they are also involved in controlling proliferation and differentiation of nonexcitable somatic stem cells. The importance of understanding stem cell contribution to tissue formation during embryonic development, adult homeostasis, and regeneration in disease has prompted many groups to study and manipulate the membrane potential of stem cells in a variety of systems. In this paper we aimed at summarizing the current knowledge on the role of ion channels and pumps in the context of mammalian corticogenesis with particular emphasis on their contribution to the switch of neural stem cells from proliferation to differentiation and generation of more committed progenitors and neurons, whose lineage during brain development has been recently elucidated.
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Bodey, Elijah D. "Evaluation of Cell Permeability of Intact Histone Complexes in Mammalian Cells." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1525705861000928.
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Calegari, Federico, and Julieta Aprea. "Bioelectric State and Cell Cycle Control of Mammalian Neural Stem Cells." Sage-Hindawi, 2012. https://tud.qucosa.de/id/qucosa%3A27972.
Full textAbstract:
The concerted action of ion channels and pumps establishing a resting membrane potential has been most thoroughly studied in the context of excitable cells, most notably neurons, but emerging evidences indicate that they are also involved in controlling proliferation and differentiation of nonexcitable somatic stem cells. The importance of understanding stem cell contribution to tissue formation during embryonic development, adult homeostasis, and regeneration in disease has prompted many groups to study and manipulate the membrane potential of stem cells in a variety of systems. In this paper we aimed at summarizing the current knowledge on the role of ion channels and pumps in the context of mammalian corticogenesis with particular emphasis on their contribution to the switch of neural stem cells from proliferation to differentiation and generation of more committed progenitors and neurons, whose lineage during brain development has been recently elucidated.