Dissertations / Theses on the topic 'Animal cell culture'
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Berdugo, Claudia. "Cell Damage Mechanisms and Stress Response in Animal Cell Culture." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1269467441.
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Sage, Elizabeth Ann. "The functional competence of animal cells in culture : the NSO cell proteome." Thesis, University of Kent, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399618.
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Amos, B. "Dialysis culture in animal cell growth and protein production." Thesis, University of Birmingham, 1995. http://etheses.bham.ac.uk//id/eprint/5350/.
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Hybridoma cells were grown in dialysis perfusion culture using a stirred reactor within which a tubular membrane was suspended. Nutrient and product flows occurred by diffusion processes alone, and were both to and from the culture environment A mathematical model of the transfer and reaction allowed prediction of steady state cell and metabolite concentrations. Steady states in cell concentration were observed for a range of perfusion rates and membrane areas. However the model could not be applied to predict steady state cell concentrations between changes in the medium. The perfusate consisted of basal medium only. Serum addition to the reactor itself resulted in decreased steady state cell densities except when it relieved a glucose limitation. Antibody was accumulated to high concentrations and yields on both basal medium and serum were many times those achieved in standard batch cultures. Cell viability fell to 30-50% but product quality did not appear to be adversely affected by the low viability. Recombinant CHO-320 cells also grew successfully under dialysis conditions and produced 7-interferon. Cell concentrations and viabilities were higher than those seen with the hybridoma. The insect cell line SF9 did not grow during dialysis perfusion, but post infection with a recombinant Baculovirus permitted the yield of \(\beta\)-galactosidase to double in dialysis culture.
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Perry, Steven D. (Steven David). "Packed fiber bed reactor design for animal cell culture." Thesis, Massachusetts Institute of Technology, 1987. http://hdl.handle.net/1721.1/17220.
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Kellokoski, E. (Eija). "Ghrelin and atherosclerosis:human, experimental animal and cell culture studies." Doctoral thesis, University of Oulu, 2009. http://urn.fi/urn:isbn:9789514292590.
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Abstract Atherosclerosis is the major cause of cardiovascular diseases and the leading cause of death globally. Atherosclerosis is a complex, chronic disease characterized by lipid accumulation and inflammation within the intima layer of vessel wall. Novel biomarkers and therapeutics are still being sought to provide both better diagnosis and treatment. Ghrelin represents an attractive target for studies into atherosclerosis. Ghrelin is a gastric peptide hormone, which has multiple functions, including regulation of appetite and energy metabolism. Emerging evidence suggests that it may also have a role in the cardiovascular and immune systems. The aim of the present study was to explore the role of ghrelin in atherosclerosis. The specific aims were 1) to investigate the association between the plasma ghrelin level and early atherosclerosis as determined by carotid artery intima media thickness (IMT) in a large (n = 1024) cross-sectional population-based study of middle-aged subjects, 2) to measure the associations between plasma ghrelin levels and already established risk factors of atherosclerosis in human subjects, 3) to assess the effects of ghrelin on atherogenesis in vitro by analyzing monocyte adhesion to endothelial cells, oxidized low density lipoprotein (LDL) binding and acetylated LDL uptake using macrophages, and 4) to study the influence of ghrelin on atherosclerosis using ghrelin vaccination in a mouse model of atherosclerosis. Plasma total ghrelin levels were positively associated with carotid IMT in male subjects. Association studies demonstrated plasma ghrelin levels to be negatively associated with total and LDL cholesterol, and triglyceride concentrations as well as with body mass index (BMI), and positively assocated with high density lipoprotein (HDL) cholesterol concentration in postmenopausal women and in a population-based study. In addition, estrogen increased plasma acylated ghrelin levels in postmenopausal women. Cell culture studies demonstrated that ghrelin could increase the binding of oxidized LDL and monocytes to endothelial cells. Interestingly, when endothelial cells were stimulated with tumor necrosis factor α (TNFα), then ghrelin prevented monocyte adhesion. The study with LDL receptor knockout mice, revealed that ghrelin vaccination could increase plasma ghrelin levels but had no effects on the development of atherosclerosis. However, the plasma MCP-1 level decreased in mice immunized with ghrelin vaccine. In conclusion, these studies suggest that ghrelin has modulatory functions in the vascular system and atherogenesis though the effect may not be as dominant as that of the known traditional risk factors. Whether this effect of ghrelin is positive or negative in atherogenesis will be clarified in further studiesTiivistelmä Sydän- ja verisuonitaudit ovat suurin kuolinsyy niin Suomessa kuin useimmissa länsimaissakin. Näiden sairauksien taustalla on yleensä valtimonkovettumatauti eli ateroskleroosi, joka voi kliinisesti ilmentyä mm. sepelvaltimotautina, aivoveritulppana ja laskimotautina. Ateroskleroosissa tulehdussoluja ja kolesterolia kertyy verisuonen seinämään muodostaen ahtauman eli ateroomaplakin valtimoon. Valtimonkovettumataudin riskitekijäitä tunnetaan jo hyvin, mutta uusia tautia ennustavia merkkiaineita sekä hoitomuotoja tarvitaan yhä. Greliini on mahalaukusta eritettävä peptidihormoni, joka osallistuu elimistössä mm. ruokahalun, energiametabolian, tulehdustekijöiden sekä sydän- ja verenkiertoelimistön toiminnan säätelyyn. Tämän työn tavoitteena oli tutkia greliinin yhteyttä ihmisen valtimonkovettumatautiin. Tutkimus toteutettiin käyttämällä kahta eri potilasaineistoa, soluviljelykokeita sekä valtimonkovettumataudin hiirimallia. Laajassa väestöpohjaisessa potilasaineistossa tutkittiin veren greliinipitoisuuden yhteyttä kaulavaltimon seinämän paksuuteen, jota pidetään valtimonkovettumista kuvaavana tekijänä. Veren greliinipitoisuuden yhteyttä valtimonkovettumataudin tunnettuihin riskitekijöihin tutkittiin myös laajassa potilasaineistossa sekä vaihdevuosi-ikäisillä naisilla, joille annettiin estrogeenikorvaushoitoa. Solukokeilla selvitettiin greliinin vaikutusta tärkeisiin valtimonkovettumataudin syntyvaiheisiin käyttäen monosyytti-, endoteelisolu- sekä makrofaagi-soluviljelmiä. Greliinin vaikutusta ateroskleroosiin in vivo selvitettiin rokottamalla LDL-reseptoripuutteiset hiiret greliini-rokotteella. Tutkimuksessa havaittiin yhteys veren korkean greliinipitoisuuden ja kaulavaltimon seinämän paksuuden välillä miehillä laajassa potilasaineistossa (n = 1024). Tulosta tukivat soluilla tehdyt kokeet, joissa greliini lisäsi hapettuneen LDL:n sitoutumista makrofaageihin sekä monosyyttien tarttumista endoteelisolujen pinnalle. Greliinin vaikutukset monosyyttien tarttumiseen endoteelisolujen pinnalle olivat päinvastaiset silloin, kun endoteelisolut käsiteltiin tulehdusta stimuloivalla tekijällä. Matalat veren greliinipitoisuudet olivat myös yhteydessä korkeisiin LDL-kolesteroli- ja triglyseriditasoihin sekä painoindeksiin ja matalaan HDL-kolesterolitasoon potilasaineistoissa. Estrogeeni nosti veren greliinipitoisuutta vaihdevuosi-ikäisillä naisilla. Greliinirokote ei vaikuttanut ateroskleroosin kehittymiseen hiirimallissa. Tutkimustulosten perusteella greliinillä näyttäisi osallistuvan valtimonkovettumataudin kehitykseen, vaikkakin sen vaikutus on pienempi kuin aiemmin tunnetuilla taudin riskitekijöillä
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Chiou, Tzyy-Wen. "A modified airlift fiber-bed bioreactor for animal cell culture." Thesis, Massachusetts Institute of Technology, 1992. http://hdl.handle.net/1721.1/16508.
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Chambers, M. E. W. "Growth of animal cells on a novel substratum." Thesis, University of Strathclyde, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382324.
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Barry, Megan M. Crockett Robert S. "Three-dimensional scaffolds for mammary epithelial cell growth : a thesis /." [San Luis Obispo, Calif. : California Polytechnic State University], 2008. http://digitalcommons.calpoly.edu/theses/12/.
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Thesis (M.S.)--California Polytechnic State University, 2008.Major professor: Robert S. Crockett, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Engineering." "May 2008." Includes bibliographical references (leaves 38-45). Also available on microfiche (1 sheet).
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Cheung, Caleb Kin Lok Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Effects of imperfect mixing in suspended plant and animal cell cultures." Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences, 2006. http://handle.unsw.edu.au/1959.4/25200.
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A common problem observed in large-scale cell cultivation is reduced culture performance compared with small-scale processes due to the existence of concentration gradients caused by poor mixing. Small-scale simulations using microbial cell suspensions have shown that circulation of cells through concentration gradients of oxygen, pH and glucose can result in reduction of cell growth and product formation similar to the effects observed in large-scale bioreactors. This study was aimed at using scale-down studies to investigate poor mixing in large-scale bioreactors used for suspended plant and animal cell culture. Two plant cell suspensions and a hybridoma cell line were used in this work. The range of oxygen transfer coefficients achieved in the hybridoma and plant suspensions were about 50???20 h-1 and 12???6 h-1, respectively. One-vessel simulation was developed to induce fluctuations of dissolved oxygen tension in a 2-L bioreactor using intermittent sparging of air and nitrogen. The effect of dissolved oxygen fluctuations on the cells was examined by comparing the performance of the cultures with those operated at constant dissolved oxygen tension. In the hybridoma suspension culture, only slight effects on cell growth were observed at circulation times above 300 s. No effect on the specific glucose uptake rate or antibody production was observed at the circulation times tested. Analysis of gene expression for selected hypoxia-related genes also suggested that the overall effect was limited. In plant cell suspensions, the specific growth rates and biomass yields on total sugar in the cultures under fluctuating dissolved oxygen tension were essentially the same as those at constant dissolved oxygen tension for both transgenic Nicotiana tabacum and Thalictrum minus. Under fluctuating dissolved oxygen tension, no effect on antibody accumulation was observed in transgenic N. tabacum suspensions, but a decrease in berberine accumulation was observed in T. minus. From the results, it can be concluded that only minimal effects due to the development of concentration gradients would be expected in large-scale bioreactors used for the cultivation of the hybridoma and plant cell suspensions tested in this work.
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Chen, Chunnuan. "Animal cell culture in a fibrous-bed bioreactor : protein production, cell immobilization, and cell cycle and apoptosis /." The Ohio State University, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=osu148819366523437.
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Dey, Sabrina. "Thermo-responsive surfaces for enzyme free mammalian cell culture." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/13416/.
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Embryonic stem cells are of great interest to scientists as they can differentiate into any somatic cell lineage making them excellent candidates for tissue regeneration and cell based treatment therapies. Currently, human embryonic stem cells (hESCs) are cultured using feeder fibroblasts or protein substrates such as matrigel, fibronectin or laminin in conditioned media. hESCs are then subcultured using enzymes to detach them from the culture substrate. However, the use of the xenosupport systems makes the hESCs therapeutic applications difficult due to cross-contamination with animal pathogens from the animal derived feeders, matrix or conditioned media to the hESCs. Moreover, the use of enzymes to recover hESCs can damage these cells. For the mentioned reasons, development of completely synthetic surfaces is desirable for hESCs culture. Thermo-responsive surfaces have been extensively studied for cell culture using the well know thermo-responsive polymer poly (N-isopropylacrylamide) (PNIPAAM) which has a switchable properties across its lower critical solution temperature (LCST) at 32°C. However, more biocompatible polymers that have similar thermo-responsive properties to PNIPAAM and biocompatible properties, such as PEG based materials, have been proposed for use as switchable surfaces. Within this thesis, thermo-responsive copolymer brushes composed of 2-(2- methoxyethoxy) ethylmethacrylate (ME02MA) and oligo (ethylene glycol) methacrylate (OEGMA) that have LCST close to body temperature (37°C) were investigated for use as a cell culture surface for temperature sensitive passaging. Poly (ME02MA-co-OEGMA) brushes were grafted from the surface using atom transfer radical polymerisation (ATRP). Hydroxyl plasma-polymer functionalised glass slides were prepared using plasma polymerisation and then an ATRP initiator was reacted to these surfaces. ATRP of the copolymers was then performed from these initiation sites. These surfaces were characterised using X-ray photoelectron spectroscopy (XPS), Time of Flight Secondary Ion Mass Spectroscopy (ToF-SIMS), atomic force microscopy (AFM) and water contact angle (WCA). Using 3T3 fibroblasts as a model cell type for initial studies, it was demonstrated that these cells adhered and proliferated on the poly (ME02MA-co-OEGMA) thermo-responsive surfaces at 37°C when the polymer brushes are in their hydrophobic state. Subsequent detachment assays were conducted when the temperature was lowered to 20°C i.e. the hydrated conformation of the copolymer brushes. Mouse embryonic stem cells (mESCs) were then cultured on these surfaces following adsorption of fibronectin (to encourage cell attachment) and cultured for 3 days. Passaging experiments were performed for 10 passage cycles and the cells analysed for retention of the undifferentiated stem cell status. These thermoresponsive polymer/fibronectin surfaces are found to be suitable for mESCs culture but evidence of differentiation was observed. Attachment of a novel gelatine based peptide to the poly (ME02MA-co-OEGMA) was also investigated for mESCs culture to avoid the use of fibronectin (which was thought to be a contributing factor to the stem cell differentiation seen). mESCs adhesion was observed both to the peptide adsorbed on TCPS and on the peptide coupled to the thermo-responsive poly (ME02MA-co-OEGMA) surface. This research indicates that these smart stimuli biomaterials have promise as a new generation of culture surfaces for enzyme free cell culture and passage suitable for generating cell populations for clinical applications.
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Basu, Shubhayu. "Effects of three dimensional structure of tissue scaffolds on animal cell culture." Connect to this title online, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1092689986.
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Thesis (Ph. D.)--Ohio State University, 2004.Title from first page of PDF file. Document formatted into pages; contains xviii, 236 p.; also includes graphics (some col.). Includes bibliographical references (p. 194-211). Available online via OhioLINK's ETD Center
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Godoy, Ruben D. "Lethal and sub-lethal effects of hydrodynamic forces on animal cell culture." Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1206393724.
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Mollet, Michael A. "Physiological effects of hydrodynamic forces on animal cells." Connect to this title online, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1101175313.
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Thesis (Ph. D.)--Ohio State University, 2004.Title from first page of PDF file. Document formatted into pages; contains xv, 145 p.; also includes graphics (some col.) Includes bibliographical references (p. 127-135).
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Bickerton, Erica Jane. "Cellular tropism and cell-to-cell fusion properties of the infectious bronchitis virus spike glycoprotein." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/35165/.
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There are numerous vaccines available for the control of infectious bronchitis virus (IBV) in poultry, however protection is short-lived and poorly cross-protective between strains. The vaccines must currently be grown in embryonated eggs, a cumbersome and expensive process. The ability to grow vaccines on a cell-line such as Vero cells would be highly advantageous. The spike (S) glycoprotein of IBV is comprised of two subunits, S1 and S2, has a vital role in virulence in vivo and is responsible for cellular tropism in vitro. This project aims to identify the amino acids present in the S glycoprotein involved in determination of cellular tropism and cell-to-cell fusion. The IBV Beaudette strain is able to replicate in both primary chick kidney (CK) cells and Vero cells, whereas the IBV M41 strain replicates in primary cells only. Recombinant IBVs with chimaeric S genes were generated using a reverse genetics system with the genomic background of Beaudette and part of the S gene from M41. Their growth characteristics and cellular tropism were investigated. The S2 subunit of Beaudette was found to be sufficient to confer the ability to grow on Vero cells and swapping just three amino acids with corresponding ones from M41 was sufficient to remove the ability of the Beaudette S glycoprotein for growth on Vero cells. Beaudette was further adapted to syncytia formation on Vero cells by serial passage and isolates were sequenced to identify amino acid changes between parent and Vero-adapted viruses that are potentially involved in cell-to-cell fusion. Understanding the way in which IBV infects host cells is vital in order to rationally design better vaccination and treatment strategies and help to reduce the prevalence of IBV infection in poultry worldwide. Using the IBV reverse genetics system, we now have the potential to grow IBV vaccines on Vero cells.
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McDermott, Ruth Helen. "The adaptation of anchorage-dependent cells to glutamine-free medium." Thesis, Manchester Metropolitan University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294056.
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Mardikar, Sudhanshu H. "Shear damage to animal cells due to disengagement of spherical cap bubbles." Thesis, University of Reading, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242331.
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Pan, Yuanlong. "Peptides can be utilized as amino acid sources for protein accretion and cell proliferation by cultured animal cells." Diss., Virginia Tech, 1993. http://hdl.handle.net/10919/38647.
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Hutter, Victoria. "Characterisation of ATP-binding cassette (ABC) transporters in bronchial epithelial cell culture models." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12442/.
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In vitro epithelial cell cultures are increasingly used to model drug permeability, as predictive tools for absorption in humans. Medical regulatory agencies recommend in vitro permeability screening for biopharmaceutical classification of novel therapeutic compounds, and recently published guidelines on investigating interactions of novel therapeutic compounds with clinically relevant transporters. The expression and functionality of drug transporters in the lung is poorly characterised, and insufficient to allow detailed understanding of drug-transporter interactions in the airways. Additionally, as human in vitro permeability is used to predict absorption from rat in vivo, a rat bronchial epithelium in vitro cell line would aid the understanding of interspecies differences in transporter-mediated drug trafficking. This thesis investigates the morphological and physiological barrier properties of Calu-3, normal human bronchial epithelial (NHBE) cell layers and rat airway epithelial cell (RL-65) cultures. The morphology and barrier integrity of RL-65 layers were shown to be in agreement with existing human bronchial epithelial cell models after culture for 8 days at air-liquid interface. The expression of >30 ABC, SLC and SLCO transporters in human models was in general agreement with published expression levels in human lungs. MDR1 functionality was investigated, and whilst no asymmetric transport of 3H-digoxin was observed in RL-65 cell layers, net secretory transport was observed for Calu-3 cell layers at both low (25-30) and high (45-45) passage number and for some batches of NHBE cell layers. Chemical, metabolic and biological inhibitors were employed to evaluate MDR1 contribution to 3H-digoxin trafficking, however the exact transporter(s) involved could not be determined. Whilst MDR1 functionality could not be ruled out, results suggest that it is unlikely to be the main transporter involved in 3H-digoxin trafficking in the bronchial epithelium. These studies have highlighted the need for more specific approaches to investigating transporter functionality in in vitro systems.
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Stevenson, David M. "Use of the regulated secretory pathway to ease product recovery in animal cell culture." Thesis, Massachusetts Institute of Technology, 1994. http://hdl.handle.net/1721.1/33497.
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Barata, David Miguel Baião. "Production of recombinant protein hLIF in static and dynamic systems of animal cell culture." Master's thesis, FCT - UNL, 2009. http://hdl.handle.net/10362/3324.
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Thesis for the Degree of Master of Science in Biotechnology Universidade Nova de Lisboa,
Faculdade de Ciências e TecnologiaThe present work focuses the optimization of the production of human Leukemia Inhibitory Factor (hLIF) by human embryonic kidney 293 (HEK293) - EBNA1 cell line cultured as suspension aggregates in spinner flask. The effects of initial cell density and feeding-regime in cellular growth and productivity were evaluated. A first phase occurs until the end of exponential growth in medium containing serum, being followed by puromycin selection of cells containing hLIF plasmid. A second phase, the production phase, is developed under serum-free conditions. Three initial cell densities (2´104 cells.mL-1, 2´105 cells.mL-1 and 2´106 cells.mL-1) were tested and the effect on maximum cell density and cell aggregate size distribution was evaluated. The inoculum of 2´105 cells.mL-1 led to final higher cellular densities around 7´106 cells.mL-1 and homogeneous aggregates around 278 μm were observed. The effect of the feeding-regime was then studied for the production of recombinant hLIF with an initial cell density of 2´105 cells.mL-1 by performing metabolite analysis Metabolite analysis revealed the occurrence of glucose and glutamine starvation upon 9 and 7 days, respectively, in 25% FR. Therefore, glutamine acted as an alternative carbon, energy and aminoacid source in HEK293-EBNA1 growth. Observed lactate levels were bellow the inhibition limit concentrations (30 mM 1).HEK293-EBNA1 did not seem to be affected by the produced levels of ammonia. In conclusion, the 25% feeding regime at 2´105 cells.mL-1 initial cell density lead to the higher viable cell densities, either along culture time at growth and at production phase, with aggregates in suspension below necrotic diameters. This experiment conditions also allowed achieving the higher LIF volumetric productivity, 125 ± 2.2 ng.mL-1.
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Archer, Simon C. "Influence of somatic cell count in heifers on lifetime milk yield and disease management." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/13747/.
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The aim was to assess the impact of milk somatic cell count (SCC) during the first lactation on the lifetime milk production of cows, and therefore estimate potential savings through heifer mastitis control. Cow level SCC over the first lactation was summarised as SCC between 5 and 30 days in milk (SCC1), and the geometric mean and variance of first lactation SCC. The impact of SCC1 on cumulative milk yield over different time periods was assessed for cows in Irish, English, and Welsh dairy herds. The impact of SCC1 and the geometric mean and variance of first lactation SCC on lifetime milk yield, and the association between SCC1 and disposal risk were assessed for cows in Irish dairy herds. Increase in SCC throughout the first lactation was associated with large reductions in the milk yield of cows, and increased disposal risk. Bayesian micro-simulation was used to demonstrate the impact in different herd scenarios. This was extended to synthesise evidence on potential savings using previous research, to estimate the economic impact of specific interventions to reduce the prevalence of cows with high SCC1. There was considerable variation between herds in the apparent impact of SCC1 on SCC throughout the first lactation, indicating the importance of a herd specific approach to control. ‘Cost effectiveness’ of interventions to reduce the prevalence of cows with high SCC1, was found to be highly dependent on the willingness of decision makers to pay for control measures. Increase in herd size was associated with increase in cow SCC, highlighting a need for improved management of mastitis when expansion is planned. An important component of this should be through monitoring and control of mastitis in heifers, especially those in spring-calving Irish dairy herds.
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Dubé, Gilles. "Post-translational processing of atrial natriuretic factor. A study using a novel cell culture system." Thesis, University of Ottawa (Canada), 1992. http://hdl.handle.net/10393/7779.
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In the present work, a reproducible cell culture system using adult rat atrial cardiocytes was developed to study ANF processing. Freshly isolated atrial cardiocytes stored high molecular weight ANF (38.0 $\pm$ 4.5 pg/$\mu$g of DNA) and released almost exclusively (83.3% $\pm$ 6.7%) low molecular weight ANF, at an average rate of 12 pg/hour/$\mu$g of DNA. The cell content and the rate of release of ANF decreased over 15 days in culture to 3.9 $\pm$ 1.2 pg/$\mu$g of DNA and 0.32 pg/h/$\mu$g of DNA $\pm$ 0.08 respectively and 62.7% $\pm$ 6.3% of the released peptide was of a low molecular weight. Cultures of non-cardiocytes, superfused with exogenous proANF, did not process the peptide. There was no correlation between the changes in cell population and the reduction in processing. Therefore, atrial non-cardiocytes are not involved in ANF processing. The results presented in this work vary from other reports which found that ANF processing in cultures is absent. The discrepancies may be due to differences related to serum-free culture conditions versus serum supplemented cultures. This suggests that factors present in the serum may be responsible for maintaining ANF processing activity in culture. (Abstract shortened by UMI.)
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Lu, Zhongyan. "Genetic Mechanisms of Porcine Sapovirus Adaptation to Cell Culture." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449149533.
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Sebastian, Sujith. "Development of an extended 2D porcine muscle cell culture system and impact of growth promoters on muscle's innate immune resistance." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33778/.
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Skeletal muscle comprises about half of the body weight in mammals and its diverse roles in metabolism and innate immune functions makes cultured muscle as a useful tool in biomedical research. In this thesis, some of the major technical obstacles in porcine primary muscle cell cultures such as inadequate differentiation of myoblasts into myotubes and the premature detachment of the formed myotubes have been overcome with the achievement of high differentiation and myotube fusion rate of over 85% along with prolonged maintenance of myotubes in excess of 70 days. Myosin heavy chain (MyHC) expression profile of differentiated myotubes recapitulated adult muscle fibres and displayed phenotypic plasticity of gene expression in response to different media. Growth factor ractopamine (Ract) treatment (1h and 6h) of myotubes followed with subsequent bioinformatics analysis of a stable isotope labelling by amino acids in cell culture (SILAC) based proteomics study suggested differential expression of proteins associated with anti-viral innate immune response and increased protein accretion. Porcine muscle cells were infected with influenza A viruses to evaluate their immune function. Porcine muscle cells expressed influenza virus sialic acid α-2,3 and α-2,6 receptors and were fully permissive to influenza virus infection. Myoblasts produced more virus particles than myotubes. Muscle cells expressed the pro-inflammatory genes tumor necrosis factor alpha (TNF-α) and antiviral gene Mx-1 and the infected cells had elevated caspase 3/7 activity to indicate apoptosis. However, myotubes pre-treated with Ract appeared to confer no reduction in influenza virus output. Evidence presented herein suggests that the functional myotubes developed by this work can be used as a tool to study the molecular mechanisms of growth and innate immune pathways in muscle.
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Parker, Steven W. "FBS free culture of porcine umbilical cord matrix cells." Thesis, Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/2319.
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Nikolay, Alexander Verfasser], and Udo [Gutachter] [Reichl. "Intensified yellow fever and Zika virus production in animal cell culture / Alexander Nikolay ; Gutachter: Udo Reichl." Magdeburg : Universitätsbibliothek Otto-von-Guericke-Universität, 2020. http://d-nb.info/1220035750/34.
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Nikolay, Alexander [Verfasser], and Udo [Gutachter] Reichl. "Intensified yellow fever and Zika virus production in animal cell culture / Alexander Nikolay ; Gutachter: Udo Reichl." Magdeburg : Universitätsbibliothek Otto-von-Guericke-Universität, 2020. http://d-nb.info/1220035750/34.
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Middlebrook, Aaron J. "Nicotine and TNF alpha, modulators of T cell signaling-effects on T cell development in fetal thymus organ culture." Diss., The University of Arizona, 2004. http://hdl.handle.net/10150/280628.
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T cell development is regulated by signals generated in the interactions between developing thymocytes and the thymic stroma. Using fetal thymus organ culture (FTOC) as a model of T cell development, we investigated the ability of two potent signal modulators to influence this process. These studies show that both nicotine and tumor necrosis factor-alpha have the ability to influence T cell receptor (TCR) signaling and the maturational capacity of treated cultures. FTOC treated with low concentrations of nicotine produced more immature T cells and fewer mature T cells. These expanded populations of cells also expressed CD69, CD95 (FAS) and elevated levels of recombinase activating genes (RAG). This phenotype reflects the fact that these cells have received a positive selection signal, are for apoptosis and are likely attempting secondary TCR rearrangements. Nicotine effects were partially blocked by the nicotinic antagonist, d-tubocurarine. Furthermore, d-tubocurarine alone blocked the development of T cells entirely, suggesting the presence of an endogenous ligand that may engage nicotinic acetylcholine receptors and regulate normal thymopoeisis. These observations underscore the linkage between the nervous and the immune systems, not only in terms of shared resources, but also in terms of direct interactions between these two systems. In another study we used FTOC and an associated in vitro Type 1 diabetes mellitus model to reconcile the role of TNF-alpha in thymopoiesis with its role in diabetes. Our data indicate that thymocytes from NOD FTOC express lower levels of TNF receptors and produce more TNF-alpha compared to non-diabetic C57BL/6 (B6) FTOC. Neutralization of endogenous TNF-alpha in NOD FTOC with a soluble TNF receptor (sTNF R1) rescued insulin production in our in vitro diabetes model. NOD FTOC treated with TNF-alpha produced greater numbers of mature T cells and a higher percentage of cells expressing CD95L (Fas ligand). Treatment with sTNF R1 had the opposite effect. TNF-alpha's known ability to attenuate TCR signaling coupled with these observations suggest that its overproduction in these animals may be driving T cells to maturity, altering the process of negative selection and ultimately enhancing the survival of potentially diabetogenic T cells resulting in disease susceptibility in these animals.
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Janes, George. "Factors regulating cortex cell file proliferation under low phosphorus stress in Arabidopsis thaliana roots." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/52299/.
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Radial patterning of root hair bearing cells (trichoblasts) in Arabidopsis thaliana (arabidopsis) follows the type 3 root hair patterning mechanism whereby the radial positioning of trichoblasts is coordinated according to the position of underlying cortex cells (Pemberton et al. 2001). Epidermal cells which are positioned over the cleft between two underlying cortex cell files adopt trichoblast identity. Low phosphate stress in arabidopsis roots has been show to result in an increase in the number of cortex cell files from 8 to 12-16, which in turn results in an increase in the number of trichoblast position cells (Zhang et al. 2003, Cederholm and Benfey, 2015). However, little is known about what mechanisms control this proliferation in cortex tissue. Zhang et al. (2003) demonstrate that eir1 (an allele of pinformed2, pin2) mutants are deficient in this response to low P. This finding suggests that PIN2 (an efflux facilitator of directional auxin transport) is required to orchestrate proliferation of cortex cell files under low P. This implies that directional auxin transport and, ultimately, auxin response are required to enable proliferative divisions in cortex tissues under low P. First of all, a deeper anatomical understanding of the cortex cell file number response to low P was developed. That showed that the divisions which lead to the increase in cortex cell file number occur in the first cortex cell next to the quiescent centre. It was also found that the anatomical changes only significantly affect the number of cortex cell and trichoblast cell files, suggesting that it is a cortex specific response to increase radial root hair density. It was further discovered that the root is sensitive to up to 400μM P and responds with increased cortex cell file number within 24 hours. I also showed that this response is independent of iron concentration. It was next hypothesised that the role of directional auxin transport implied that other PIN and AUX/LAX genes may be required as well as activating AUXIN RESPONSE FACTORs (ARFs). These experiments revealed that no other PINs or ARFs play a crucial role in this response. However it was found that PIN2:GFP protein changes its subcellular localisation in response to low P, suggesting that changes in auxin flux direction is required. Based on this, it was hypothesised that PIN2 complementation in the cortex in pin2 would rescue the phenotype. This was indeed the case, but also for PIN2 under the AUX1 promoter, suggesting an additional role for PIN2 in the epidermis and root cap during the low P response. Mathematical modelling suggested that auxin flux out of the cortex, mediated by PIN2, would be required for the response. However, results testing this in vivo with the DII:VENUS auxin reporter were inconclusive. It was suspected that ground tissues patterning regulators, BIRD genes, may also play a role in tissue patterning under low P. jkd (jackdaw) and mgp (magpie) mutants did not show any strong phenotype on low P, but expression analysis using reporter lines and reverse transcription qPCR did suggest changes in regulation. It was then hypothesised that BIRD genes and PIN2 may interact, by crossing pin2 and jkd I found that the two interact to a small degree but it is not evident that they interact directly within the same pathway. It was concluded that this response to low P was controlled by a number of regulatory networks which definitely involves PIN2 mediated auxin transport with a contribution from BIRD genes.
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Madouasse, Aurélien. "An evaluation of milk recording, somatic cell counts and reproductive performance in a large cohort of dairy herds in England and Wales." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/11203/.
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Milk recording consists in the regular, usually monthly, collection of a milk sample from all the lactating cows of a dairy herd. A large sample of milk recording data collected in England and Wales between 2004 and 2006 was used in this thesis. A sample of 8,211,988 recordings in 2,128 herds, representing 16 % of the dairy herds in activity in December 2006, were described and analysed. Calvings followed a seasonal pattern with 80 % more calving in September than in May. Milk production was highest in May (26.5 kg) and lowest in October (24.1 kg). Butterfat was stable, close to 4 % from October to March and reached a minimum at 3.7 % in June and July. Protein stayed between 3.2 and 3.3 % all the year. Geometric mean somatic cell count was between 177 and 180 between October and March and reached 205,000 cells/mL in July and August. At the individual cow level, the mean milk yield, percentage of butterfat, percentage of protein, fat to protein ratio and somatic cell count (geometric mean) were 26.4 kg, 3.96 %, 3.29 %, 1.21 and 90,000 cells/mL, between 5 and 305 days in milk. Changes in individual cow somatic cell counts around a threshold of 200,000 cells/mL between consecutive recording dates were used to predict bulk milk somatic cell count at both the herd-year and test-day levels. The main contributors to bulk milk somatic cell counts were cows staying above the threshold for 2 consecutive test-days. Milk yields and composition at the start of lactation were used to predict the calving to conception interval. Higher milk yield, lower percentage of protein, lower percentage of lactose, higher somatic cell count and higher percentage of butterfat were associated with lower probabilities of conception before 145 days in milk.
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Daniel, Pablo Gabriel. "The Influence of Embryo Cell Culture Systems on Pretransfer Development of Early Ovine Embryos." DigitalCommons@USU, 1989. https://digitalcommons.usu.edu/etd/4061.
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The complete requirements for early embryo development in vitro of the ovine and other domestic species remain unknown. Many studies have concentrated on new media, supplementation, and gas atmosphere formulations.
A newer approach is to coculture early embryos with different cell types to provide the physico-chemical requirements for their development.
In this study, oviduct epithelial (OEC) and dissociated embryo cell (DEC) growth were tested in minimum essential media (MEM) and RPMI. Media were supplemented with fetal calf serum (FCS) and equine derived serum (EDS). Fetal calf serum supported maximum cell confluence in OEC collected on day 3 and day 13 post-estrus. Although at a slower growth rate, DEC developed faster in FCS-supplemented media. Cell growth was slower for EDSsupplemented media in all treatments.
As a result, FCS-supplemented media were used to evaluate early embryo growth in various coculture systems. In MEM + OEC, 67% of 1- to 1 0- cell embryos developed to the hatched blastocyst stage (following 8 days of culture). In MEM+DEC, 66% hatched after the same time period. In control treatments (no exogenous cell layers), all embryos degenerated. When early embryo development was compared between St.Croix and Targhee-type breeds in MEM+OEC and RPMI+OEC, no significant differences were observed. The improved results obtained with coculture systems may provide an important method for assessing the viability of embryos following micromanipulation techniques (such as splitting, gene transfer, or following long periods of freezing). The nature of the beneficial action of these coculture systems remains unknown.
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Cho, Hyung Joon. "Pro-oxidative and Pro-inflammatory Mechanisms of Brain Injury in Experimental Animal and 3D Cell Culture Model Systems." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/73476.
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The pro-oxidative and pro-inflammatory mechanisms have been implicated in various human diseases including neurological and psychiatric disorders. However, there is only limited information available on the etiology in the progression of neurological damage to brain. The emergence of tissue engineering with the growing interest in mechanistic studies of brain injury now raises great opportunities to study complex physiological and pathophysiological process in vitro. Therefore, the prime goals of this study include: (1) Determination of the molecular and cellular mechanisms responsible for blast- and radiation-induced brain injuries and (2) Development of a three-dimensional (3D) model system in order to mimic in vivo-like microenvironments to further broaden our knowledge in pro-oxidative and pro-inflammatory mechanisms and their cellular responses within 3D constructs.
In the first study, we demonstrated that blast exposure induced specific molecular and cellular alterations in pro-oxidative and pro-inflammatory environments in the brain and neuronal loss with adverse behavioral outcome. The results provide evidence that pro-oxidative and pro-inflammatory environments in the brain could play a potential role in blast-induced neuronal loss and behavioral deficits.
In the second study, we investigated that fractionated whole-brain irradiation induced specific molecular and cellular alterations in pro-oxidative and pro-inflammatory environments in the brain along with elevation of reactive oxygen species (ROS)-generating protein (NOX-2) and microglial activation. Additionally, the contribution of NOX-2 in fractionated whole-brain radiation-induced oxidative stress was observed by dramatic amelioration of ROS generation after pharmacological inhibition of NOX-2. These results support that NOX-2 may play a pivotal role in fractionated whole-brain radiation-induced pro-oxidative and pro-inflammatory pathways in mouse brain.
In the third study, we developed an in vitro 3D experimental model of brain inflammation by encapsulating microglia in collagen hydrogel with computational analysis of 3D constructs. The results indicated that our newly developed in vitro 3D model system provides a more physiologically relevant environment to mimic in vivo responses.
In conclusion, these data may be beneficial in defining a cellular and molecular basis of pathophysiological mechanisms of brain injuries. Furthermore, it may provide new opportunities for preventive and therapeutic interventions for patients with brain injuries and associated neurological disorders.Ph. D.
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Grant, Jennifer Sarah. "The role of microRNA in the development of pulmonary arterial hypertension : studies in cell culture and animal models." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5261/.
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Pulmonary arterial hypertension (PAH) is a complex disease characterised by narrowing and remodelling of the small pulmonary arteries. This process involves all cell types within the vessel wall and results in an increase in pulmonary artery pressure, right heart failure and can eventually lead to premature death. Diagnosis of PAH occurs late in disease progression with patients already displaying severe hemodynamic compromise and mortality rates remain unacceptably high despite current treatment. Therefore the development of new therapies is required to manage the symptoms and treat the underlying causes of this multifaceted disease. Recent studies have highlighted a role for microRNAs (miRNAs) in the initiation, development and progression of PAH. MiRNAs are small non-coding RNA molecules ~22 nucleotides long that negatively regulate gene expression. Previous work from our laboratory has shown that miRNAs are dysregulated within the lung during the development of experimental pulmonary hypertension (PH). Consequently, the aim of this study was to assess the involvement of specific miRNAs in the development of PAH using cell culture and experimental models of PH. The first miRNA focused on was miR-451 which is up-regulated in the lungs from animal models of PH. In human pulmonary artery smooth muscle cells (hPASMCs), miR-451 over-expression promoted migration in the absence of serum but had no effect on cellular proliferation. Silencing of miR-451 was performed in vivo using antimiR-451 and miR-451 knockout mice. Indices of PAH were assessed after exposure to hypoxia via measurement of right ventricular pressure (RVP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. There was a reduction in systolic RVP in hypoxic rats pre-treated with antimiR-451 compared to control antimiR (47.7 ± 1.36 mmHg and 56.0 ± 2.03 mmHg respectively, p<0.01). MiR-451 knockout mice exposed to chronic hypoxia displayed no significant differences for PAH indices compared to wild type hypoxic mice. Thus illustrating that transient inhibition of miR-451 attenuates the development of PH in hypoxic rats however, genetic deletion of miR-451 has no beneficial effect on the development of PH. This may be due to compensatory mechanisms present in the miR-451 knockout mice. Previous work has also shown that miR-145 is up-regulated in the lungs and pulmonary arteries from animal models of PH as well as PAH patients. Therefore miR-145 expression was modulated in rats using antimiR-145 both prior to and post exposure to hypoxia and SU5416 administration. Prophylactic silencing of miR-145 in the hypoxia/SU5416 model of PH showed no beneficial effect on the development of PH compared to control antimiR treated rats exposed to hypoxia. Therapeutic modulation of miR-145 also demonstrated no protective effect on RVP, RVH or muscularisation of pulmonary arteries in the rat hypoxia/SU5416 model. There was however a significant reduction in the number of occluded vessels in rats with established PH treated with antimiR-145. This reduction in occluded vessel count is interesting as it was not observed in the prevention study. Further work is required to pinpoint the exact mechanisms through which antimiR-145 is producing this positive effect on pulmonary vessels with therapeutic silencing of miR-145. The role of miR-145 on PAH development was further investigated with the use of miR-145 knockout mice. Recent studies show that genetic ablation of miR-145 protects female mice from developing hypoxia-induced PH. We therefore sought to establish whether this beneficial response was also observed in male miR-145 knockout mice. Hypoxic male miR-145 knockout mice showed similar indices of PAH as hypoxic miR-145 wild type mice, with increased RVP and RVH compared with normoxic mice. Pulmonary vascular remodelling analysis indicates that miR-145 knockout mice exposed to hypoxia may have a reduction in remodelling compared to wild type hypoxic mice however this does not reach significance. Thus it appears from this study that male miR-145 knockout mice are not protected against developing PAH as the female knockout mice are. The results from this study on male miR-145 knockout mice demonstrate that the effects of silencing miR-145 in vivo are indeed gender specific. As well as affecting the pulmonary arteries, PAH also induces changes within the right ventricle culminating in right ventricular dysfunction and failure. Therefore a miRNA profile was established for the PAH diseased right ventricle. MiR-27a and miR-27b were up-regulated within the right ventricle of hypoxia/SU5416 mice and rats, respectively. This response appears to be cardiac specific and may help to establish therapies to maintain and stabilise RV function. In summary of these findings, we have confirmed that miRNAs are dysregulated within the lung and right ventricle during PH development. Results suggest that there are complex mechanisms regulating miRNA processing within the lung during the development of PAH and that these pathways may be gender specific. Further work is required to understand the genes targeted, and therefore the pathways modulated, by miRNAs during PAH development to enhance our understanding of the intricate systems involved in disease progression. MiRNAs represent a potential therapeutic target for the treatment of PAH with further work required to pinpoint the exact mechanistic pathways through which they exert their effects.
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Stålhös, Lars. "Marknadsundersökning av grisplättlysat för att ersätta serum i cellodling." Thesis, KTH, Skolan för bioteknologi (BIO), 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-168990.
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Bryan, Kelley Elizabeth. "Effects of extracellular matrices on porcine umbilical cord matrix stem cells." Thesis, Manhattan, Kan. : Kansas State University, 2008. http://hdl.handle.net/2097/1081.
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Misztal, David Richard Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Identification of cellular changes associated with increased production of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell line." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2008. http://handle.unsw.edu.au/1959.4/41326.
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A proteomics approach was used to identify proteins potentially implicated in the cellular response concurrent with elevated production levels of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell line (Darren cells), using zinc and sodium butyrate in the production media to increase expression. To this end, 2-dimensional gel electrophoresis (2-DGE) was utilized. Firstly, several aspects of 2-DGE were developed for this investigation. Gel drying conditions were optimized, and a glycine-free blotting method is described which achieved greater efficiency in rapid transfer of proteins than those previously described. Next, hFSH expression was characterized in Darren cells. An ELISA developed for this investigation examined intracellular (expression) and extracellular (secretion) of hFSH during increased expression. These results show a disproportionate increase in intracellular hFSH (188%) expression above extracellular hFSH (41%).
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Korecki, Casey. "Effects of Compression Loading, Injury, and Age on Intervertebral Disc Mechanics, Biology and Metabolism Using Large Animal Organ and Cell Culture Systems." ScholarWorks @ UVM, 2008. http://scholarworks.uvm.edu/graddis/126.
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The intervertebral disc (IVD) is a complex orthopaedic tissue that is located between the vertebrae in the spine. Degeneration of the IVD is thought to be a contributor to low back pain (LBP), which affects up to 80% of the population at enormous economic cost. The role of the intervertebral disc in supporting and resisting applied loading to the spine, along with the observation of disorders associated with abnormal spinal loading, provide support to the theory that applied mechanical loading is crucial in maintaining the health of the intervertebral disc. The encompassing goal of this work was to examine the biological response of the intervertebral disc to changes in the surrounding mechanical environment in a large animal model. Aim 1 utilized an organ culture model to explore the relationship between disc mechanics and biology in needle puncture injury, a commonly used model of experimentally induced disc degeneration, thus providing a possible mechanism for in vivo injury induced disc degeneration models. Aim 2 was to explore the interaction between the amplitude of applied mechanical loading and intervertebral disc cell signaling, also performed in an organ culture model to include cell-matrix signal transduction. Aim 3 addressed frequency and age effects on the IVD response to mechanical stimulation, performed in vitro to control for the effects of varying matrix compositions between old and young animals. Finally, Aim 4 utilized kmeans and fuzzy c-means clustering techniques to reveal patterns in experimental phenotype (determined by gene expression data) and gene response to experimental conditions. The application of biclustering, where the gene responses within experimental phenotypes are clustered to elucidate possible mechanisms for different gene level-responses to experimental conditions, was also accomplished. Finally, the ability for the model to predict the behavior of other genes critical to IVD mechanobiology, or in determining the membership of an unexamined experimental phenotype was explored. Overall, applied dynamic compression was not found to significantly alter disc mechanics, while a disruption in the annulus through needle puncture rapidly decreased the compressive modulus. Changes in disc mechanics may precede biological remodeling, with little evidence of remodeling present without mechanical alteration. Aging, however, crucially impacts disc cell biology, particularly in the nucleus pulposus, and will interact with applied loading to further impact the ability for the intervertebral disc cells to maintain a healthy extracellular matrix.
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Rutt, Julianne Eileen. "Molecular Analysis Of The Epiphyseal Growth Plate In Rachitic Broilers: Evidence For The Etilogy Of The Condition." Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1223319403.
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Thomas, Mark Peter. "Differential tolerance of a cancer and a non-cancer cell line to amino acid deprivation : mechanistic insight and clinical potential." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/19912.
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Thesis (PhD)--Stellenbosch University, 2012.ENGLISH ABSTRACT: Introduction – Due to spatial separation from the native vascular bed, solid tumours develop regions with limited access to nutrients essential for growth and survival. The promotion of a process known as macroautophagy may facilitate in the maintenance of intracellular amino acid levels, through breakdown of cytoplasmic proteins, so that they remain available for macromolecular biosynthesis and ATP production. Several studies point to the potential ability of some cancers to temporarily increase autophagy and thereby prolong cell survival during metabolic stress. The validity of these claims is assessed when a commonly used breast cancer cell line and an epithelial breast cell line are starved of amino acids in this study. Furthermore, we go on to hypothesize that acute amino acid deprivation during treatment will result in an elevated sensitivity of MDAMB231 cells to doxorubicin toxicity but limit its cytotoxic side-effects in MCF12A cells. Methods and study design- Human breast cancer cells (MDAMB231) and breast epithelial cells (MCF12A) cultured in complete growth medium were compared to those incubated in medium containing no amino acids. Steady state autophagy levels were monitored using classical protein markers of autophagy (LC3-II and beclin-1) and the acidic compartmentalization in cells (Lysotracker™ red dye) in conjunction with autophagy inhibition (bafilomycin A1 and ATG5 siRNA). Cell viability was monitored using several techniques, including caspase 3/7 activity. ATP levels were assessed using a bioluminescent assay, while mass spectrometry based proteomics was used to quantify cellular amino acid levels. Similar techniques were used to monitor autophagy during doxorubicin treatment, while cellular doxorubicin localization was monitored using immunofluorescence microscopy. Finally, a completely novel GFP-LC3 mouse tumour model was designed to assess autophagy and caspase activity within tumours in vivo, during protein limitation and doxorubicin treatment. Results - Amino acid deprivation resulted in a transient increase in autophagy at approximately 6 hours of amino acid starvation in MDAMB231 cells. The amino acid content was preserved within these cells in an autophagy-dependent manner, a phenomenon that correlated with the maintenance of ATP levels. Inhibition of autophagy during these conditions resulted in decreased amino acid and ATP levels and increased signs of cell death. MCF12A cells displayed a greater tolerance to amino acid starvation during 24 hours of amino acid starvation. Evidence indicated that autophagy was important for the maintenance of amino acid and ATP levels in these cells and helped prevent starvation-induced cell death. Furthermore, data showed that concomitant amino acid withdrawal resulted in decreased cellular acidity in MDAMB231 cells, and increased acidity in MCF12A cells, during doxorubicin treatment. These changes correlated with evidence of increased cell death in MDAMB231 cells, but a relative protection in MCF12A cells. A novel model was used to apply these techniques in vivo, and although mice fed on a low protein diet during high dose doxorubicin treatment had increased mean survival and smaller tumour sizes, evidence suggested that autophagy is protecting a population of cells within these tumours. Conclusions - This novel approach to tumour sensitization could have several implications in the context of cancer therapy, and given the delicate relationship that autophagy has with the cancer microenvironment, efforts to determine the mechanisms involved in autophagy and sensitization could lead to new and innovative treatment opportunities for cancer management.
AFRIKAANSE OPSOMMING: Inleiding – As gevolg van hul skeiding van die oorpronklike vaskulêre netwerk, ontwikkel soliede gewasse areas met beperkte toegang tot noodsaaklike voedingstowwe. Die bevordering van 'n proses wat as makro-autofagie bekend staan, kan die handhawing van intrasellulêre aminosuur vlakke fasiliteer. Voorafgenoemde proses word waarskynlik deur die afbreek van sitoplasmiese proteïene teweegebring om sodoende vir makro-molekulêre biosintese en ATP produksie beskikbaar te kan wees. Verskeie studies dui daarop dat sommige kankersoorte die vermoë het om autofagie tydelik te verhoog, en daarby sel oorlewing gedurende metaboliese stress te verleng. Die geldigheid van hierdie eise word evalueer wanneer 'n algemeen beskikbare borskanker sellyn, en 'n borsepiteelsellyn in hierdie studie van aminosure verhonger word. Verder, veronderstel ons dat akute aminosuur ontneming gedurende behandeling 'n verhoogde sensitiwiteit van MDAMB231 selle tot doxorubicin toksisiteit tot gevolg sal hê, maar terselfdetyd die middel se sitotoksiese newe-effekte in MCF12A selle sal beperk. Metodes en studie ontwerp – Menslike borskanker- (MDAMB231) en bors epiteel selle (MCF12A) wat in volledige groeimedium gekweek is, is vergelyk met selle wat in aminosuur vrye medium gekweek is. Basislyn autofagie-vlakke is gemonitor deur die gebruik van klassieke autofagie proteïen merkers (LC3-II en beclin-1) en die asidiese kompartementalisering in selle (Lysotracker™ rooi kleurstof) saam met autofagie inhibisie (bafilomycin A1 and ATG5 siRNA). Sellewensvatbaarheid is deur die gebruik van verskeie tegnieke, insluitend caspase 3/7 aktiwiteit, gemonitor. ATP-vlakke is deur die gebruik van 'n bioluminiserende tegniek gemeet, terwyl massa-spektrometrie-gebaseerde “proteomics” gebruik is om sel aminosuur vlakke te kwantifiseer. Soortgelyke tegnieke is gebruik om autofagie gedurende doxorubicin behandeling waar te neem, terwyl sellulêre doxorubicin lokalisasie deur die gebruik van immunofluoresensie mikroskopie gemonitor is. Ten slotte, is 'n unieke GFP-LC3 muismodel in hierdie studie ontwikkel. Hierdie model is gebruik om autofagie en caspase aktiwiteit in gewasse in vivo te bestudeer tydens proteïen beperking en doxorubicin behandeling. Resultate – Aminosuur ontneming het tot 'n tydelike verhoging in autofagie na ongeveer 6 ure van aminosuur verhongering in MDAMB231 selle gelei. Die aminosuur inhoud van hierdie selle het op 'n autofagie-afhanklike manier behoue gebly. Hierdie verskynsel het met die handhawing van ATP-vlakke gekorreleer. Autofagie inhibisie gedurende hierdie kondisies het 'n verlaging in aminosuur en ATP-vlakke teweeggebring, sowel as vermeerderde tekens van seldood tot gevolg gehad. MCF12A selle het 'n groter toleransie tot aminosuur verhongering tydens die 24 uur aminosuur verhongeringsperiode getoon. Getuienis het aangedui dat autofagie belangrik vir die handhawing van aminosuur en ATP-vlakke in hierdie selle was, en gehelp het om verhongerings-geïnduseerde seldood te voorkom. Verder het data gewys dat aminosuur ontrekking tot verminderde sellulêre asiditeit in MDAMB231 selle, en verhoogde asiditeit in MCF12A selle gedurende doxorubicin behandeling gelei het. Hierdie veranderinge stem ooreen met getuienis van toenemende seldood in MDAMB231 selle, maar 'n relatiewe beskerming in MCF12A selle. 'n Unieke model was gebruik om hierdie tegnieke in vivo toe te pas. Alhoewel verhoogde oorlewing en kleiner gewasse in muise op 'n lae proteïen dieet gedurende hoë dosis doxorubicin behandeling opgemerk is, het bewyse voorgestel dat autofagie 'n populasie selle binne die gewasse beskerm. Gevolgtrekkings – Hierdie unieke benadering tot tumor sensitisering kan verskeie implikasies in die konteks van kanker behandeling hê. Gegewe die delikate verhouding van autofagie met die kanker mikro-omgewing, kan pogings om die meganismes betrokke in autofagie en sensitisering te bepaal, tot nuwe en innoverende behandelings vir kanker lei.
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Berglund, Joseph Delore. "Elastin and viscoelasticity in cell-seeded collagen constructs cultured in virto : implications for tissue-engineered blood vessels." Diss., Georgia Institute of Technology, 2002. http://hdl.handle.net/1853/11722.
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Barbouche, Naziha. "Réponse biologique de cellules animales à des contraintes hydrodynamiques : simulation numérique, expérimentation et modélisation en bioréacteurs de laboratoire." Thesis, Vandoeuvre-les-Nancy, INPL, 2008. http://www.theses.fr/2008INPL075N/document.
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La réponse globale de cellules animales à des contraintes hydrodynamiques lors de leur culture en suspension dans des réacteurs agités a été étudiée grâce à une approche intégrative couplant les outils du génie biochimique à ceux de la mécanique des fluides numérique. En premier lieu, la description de l’hydrodynamique moyenne et locale de deux systèmes de culture agités de laboratoire, spinner et bioréacteur, a été réalisée. Puis, l'étude des cinétiques macroscopiques de cellules CHO cultivées en suspension, en milieu sans sérum et sans protéine, a été réalisée avec différentes vitesses d’agitation, pour évaluer l'impact de l'agitation sur les vitesses de croissance et de mort cellulaires, ainsi que de consommation des substrats et de production des métabolites et de l'interféron-gamma recombinant. Des caractérisations supplémentaires des cellules (apoptose, protéines intracellulaires) et de l'interféron ont également été réalisées. Les effets de l'intensification de l'agitation ont été représentés avec plusieurs corrélations globales reliant : (i) en milieu contenant du pluronic, l'intégrale des cellules viables au nombre de Reynolds, et la proportion de cellules lysées à la valeur moyenne de l'énergie de dissipation, <[epsilon]? (ii) en milieu sans pluronic, les vitesses spécifiques de croissance et de mort cellulaires à <[epsilon]. De plus, l'analyse par CFD de la distribution spatio-temporelle des contraintes indique que la lyse cellulaire, observée dans le réacteur aux conditions extrêmes d'agitation, serait plutôt liée à des valeurs locales très élevées de [epsilon], ainsi qu’à la fréquence d'exposition des cellules dans ces zones énergétiques. Un modèle hydro-cinétique original, couplant l’hydrodynamique locale aux cinétiques cellulaires de croissance et de mort, et basé sur l’intermittence de la turbulence permet la prédiction de la lyse massive observée en réacteur sous certaines conditions. Pour confirmer le fait que les effets liés à l'intensification de l'agitation sont bien le résultat d'une augmentation des contraintes hydrodynamiques, et non d'une amélioration du transfert d'oxygène, ce dernier a été mesuré et modélisé par couplage avec une simulation numérique de type Volume Of Fluid , concluant en une absence de limitation d'oxygène. Enfin, la conception, le dimensionnement et la caractérisation hydrodynamique d'un réacteur innovant de type Couette-Taylor, sont proposées pour la mise en œuvre de cultures perfusées dans un environnement hydrodynamique mieux contrôléThe global response of animal cells to hydrodynamic stress when cultivated in suspension in stirred tank reactors was studied. To do this, an integrative approach coupling biochemical engineering and fluid mechanics tools were used. First, the description of the global and local hydrodynamics of two bench-scale agitated reactors, a spinner flask and a bioreactor, was carried out. Then, macroscopic kinetics of CHO cells cultivated in a serum and protein-free medium were obtained at various agitation rates, in order to evaluate the impact of agitation on cellular growth and death, as well as substrates consumption and metabolites and recombining IFN-[gamma] production. IFN-[gamma] and cells physiological state were more precisely characterised by glycosylation, apoptosis state and intracellular proteins measurements. The effects of the agitation increase were represented by several global correlations that related: (i) in a medium containing Pluronic F68, the Integral of the Viable Cells Density to the Reynolds number, and the proportion of lysed cells with the average value of energy dissipation rate <[epsilon]? (ii) in a medium without pluronic, specific cell growth and death rates to <[epsilon]. Moreover, CFD analysis of the stress distribution indicated that the cellular lysis observed in the bioreactor at the highest agitation rate, would be related to very high local values of [epsilon], and to the exposure frequency of the cells in these energetic zones. An original hydro-kinetic model based on the intermittency of turbulence and coupling the local hydrodynamics with cell growth and death kinetics, allowed the prediction of the massive cell lysis observed in the bioreactor under some mixing conditions. To decouple shear stress effects from oxygen transfer improvement, the oxygen transfer coefficient was experimentally measured and modelled using a Volume Of Fluid numerical simulation. Our results indicated the absence of an oxygen limitation, which confirmed that this cell response resulted from the hydrodynamic stress increase alone. Lastly, an innovative continuous and perfused Couette-Taylor reactor, allowing a better-controlled hydrodynamic environment was designed and sized. Its hydrodynamic description was carried out using CFD calculations
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Haldankar, Raj. "A kinetic study of the growth of anchorage-dependent mouse L cells." Ohio : Ohio University, 1994. http://www.ohiolink.edu/etd/view.cgi?ohiou1177097944.
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Mitchell, Sheila. "Development of a Canine Coccidiosis Model and the Anticoccidial Effects of a New Chemotherapeutic Agent." Diss., Virginia Tech, 2008. http://hdl.handle.net/10919/27600.
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Coccidia are obligate intracellular parasites belonging to the phylum Apicomplexa. Many coccidia are of medical and veterinary importance such as Cystoisospora species and Toxoplasma gondii. The need to discover new anticoccidial therapies has increased due to development of resistance by the parasite or toxicity issues in the patient. The goals of this work were to develop a model for canine coccidiosis while proving that Cystoisospora canis is a true primary pathogen in dogs and to determine the efficacy of a new anticoccidial agent. A canine coccidiosis model would be useful in evaluating new anticoccidial treatments. Oral infections with 5 X 104 (n=2) and 1 X 105 (n=20) sporulated C. canis oocysts were attempted in 22 purpose bred beagle puppies. Clinical signs associated with disease were observed in all dogs. Bacterial and viral pathogens were ruled out by transmission electron microscopy (TEM) and bacterial growth assays. Development of C. canis in cell culture was also evaluated.
The efficacy of ponazuril, a new anticoccidial drug, was examined in T. gondii. In vitro studies were conducted to determine the activity of ponazuril on tachyzoites and how this agent affects development of apicomplexcan parasites. The tachyzoite production assay was conducted. Ponazuril at a dose of 1.0 µg/ml had a significant affect on tachyzoite reproduction. Comparisons were made on how ponazuril affects T. gondii and Neospora caninum. Inhibition of T. gondii tachyzoites occurred after the second round of replication and with N. caninum tachyzoites after 4 rounds of replication. Results of TEM revealed ponazuril affects replication of T. gondii and N. caninum differently.
The efficacy of ponazuril to prevent and treat acute and chronic toxoplasmosis was investigated. Mice treated prophylactically with ponazuril were completely protected from developing an acute T. gondii infection. Fatal toxoplasmosis was prevented in mice starting treatment 3 and 6 days post infection at a dose of 20 mg/kg. Immunohistochemistry was used to evaluate ponazurilâ s effect on chronic toxoplasmosis. Sections of brain were scored according to the number of tissue cysts present. Ponazuril also proved to be highly active against toxoplasmic encephalitis in an interferon-gamma knockout mouse model.Ph. D.
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Pelletier, François. "Mise au point d'observateurs d'état pour le suivi de cultures de cellules animales." Vandoeuvre-les-Nancy, INPL, 1995. http://www.theses.fr/1995INPL135N.
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L’objectif de ce travail est l'élaboration d'outils d'estimation d'état simples d'emploi pour l'évaluation de la composition des milieux de culture cellulaires. La première partie concerne le développement d'un nouvel estimateur appelé observateur à base de bilans de matière construit sur le principe de l'observateur asymptotique. Il peut être appliqué à n'importe quel type de culture. Il ne nécessite aucun réglage. La possibilité de prendre en compte certaines lois cinétiques connues permet de le faire fonctionner avec un nombre restreint de mesures. La seconde partie traite de la mise au point d'une nouvelle variante du filtre de Kalman, le filtre de Kalman auto-ajustable. Le réglage du filtre de Kalman est délicat et peut conduire à des problèmes de stabilité de l'erreur d'estimation et de convergence des valeurs estimées vers les valeurs réelles. Le filtre de Kalman auto-ajustable assura la stabilité des erreurs d'estimations avec un nombre réduit de réglages. Les deux nouveaux observateurs ont été appliqués à trois cultures d'hybridomes productrices d'anticorps monoclonaux. À partir de deux mesures expérimentales (glucose et lactate déshydrogénase ou ammoniaque et lactate déshydrogénase), la composition du milieu de culture est évaluée par les deux techniques. L’observateur à base de bilans de matière a donné de bons résultats et ceux obtenus avec le filtre de Kalman auto-ajustable sont acceptables tant que l'on ne sort pas du domaine de validité du modèle
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Petiot, Emma. "Procédés de cultures de cellules VERO en milieu sans sérum : contributions au développement d'une stratégie PAT." Thesis, Vandoeuvre-les-Nancy, INPL, 2009. http://www.theses.fr/2009INPL071N/document.
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Ce travail apporte une contribution au développement de la stratégie PAT pour les procédés de culture de cellules animales. Il propose l'amélioration de la compréhension et de la maîtrise de la culture de cellules Vero, dédiées à la production de vaccins viraux, et cultivées sur microporteurs dans un milieu sans sérum. Une première partie a permis de cribler les effets de certains groupes de composés du milieu de culture par le suivi de la croissance en microplaques. Puis, des études cinétiques et métaboliques plus approfondies, réalisées en spinners, ont montré que le métabolisme carboné des cellules Vero est saturé par l'accumulation intracellulaire de pyruvate et qu’il est peu orienté vers la croissance. Alors que le renouvellement du milieu ou l'ajout ponctuel de glutamine amélioraient la croissance cellulaire sans ré-équilibrer le métabolisme, la substitution du glucose et de la glutamine a permis de réduire l’apoptose et d'améliorer les performances métaboliques et de croissance. Par ailleurs, les spectroscopies diélectrique et proche-infrarouge ont été évaluées pour le contrôle en-ligne du procédé, en prenant en compte les particularités des cellules adhérentes. Nous avons montré leur capacité à évaluer les concentrations de cellules, de composés du milieu, et à détecter l'apoptose. Enfin, les principales améliorations par substitution de la glutamine ont été appliquées en bioréacteurs, à la production d'un vaccin prototype contre la dengue, dans des conditions proches de celles d'un procédé industriel. Ceci a permis de limiter les renouvellements de milieu pendant l’expansion cellulaire, sans compromettre la production de particules virales infectieusesThis work contributes to the development of the PAT strategy for animal cell culture processes. The aim of this study was to improve the understanding and the control of Vero cell culture, dedicated to the production of viral vaccines, and grown on microcarriers in serum-free medium. An initial study was performed to screen the effects of certain groups of compounds of the culture medium, by the cell growth monitoring in microplates. Then, kinetic and metabolic studies conducted in spinners flasks allowed to go further and to show that the Vero cell metabolism is saturated through the pyruvate intracellular accumulation and that it is not oriented toward growth. While media renewal or punctual addition of glutamine improve the cell growth without improving the metabolism balance, the substitution of glucose and glutamine allowed to reduce apoptosis and to improve growth and metabolic performances.Furthermore, dielectric and near-infrared spectroscopies have been evaluated for the in-line process monitoring, taking into account the particularities of adherent cells. We have demonstrated their ability to quantify cell concentrations, medium component concentrations, and to detect apoptosis. Finally, major improvements by substitution of glutamine have been applied to bioreactor culture to produce a dengue vaccine prototype, with culture conditions close to industrial process. In these cases, medium renewal during the cell expansion was removed without compromising the production of infectious viral particles
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Ortiz, Manoel. "Dry Powders Inhalers (DPI) obtidos a partir de nanocápsulas de núcleo lipídico contendo budesonida : caracterização, avaliação in vivo em modelo animal de asma e da toxicidade in vitro em cultura celular." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2016. http://hdl.handle.net/10183/159497.
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A asma é definida como uma doença inflamatória crônica de caráter multifatorial, caracterizada pela obstrução reversível das vias aéreas, denso infiltrado inflamatório e hiper-reatividade brônquica a estímulos externos. Clinicamente, a doença é marcada por sintomas episódicos de dispneia, sibilo, tosse seca e sensação de aperto no peito. A terapia convencional da asma compreende o uso de anti-inflamatórios e broncodilatadores. A budesonida é um glicocorticoide esteroide e é dos fármacos mais utilizados na terapêutica da asma. No entanto, a budesonida apresenta baixa biodisponibilidade oral e o uso prolongado pode levar a efeitos adversos graves como afinamento da pele e supressão adrenocortical. No desenvolvimento de novas formulações, a avaliação da toxicidade é de extrema importância. Por conseguinte, o uso de cultura celular é de grande valia no desenvolvimento de protocolos para avaliação da toxicidade de novas formulações. Adicionalmente, a nanotecnologia é uma ferramenta importante para resolver problemas de biodisponibilidade e para contornar efeitos adversos da terapêutica convencional. Desta forma, o objetivo desta tese foi desenvolver um novo sistema nanoestruturado na forma de pó seco para inalação (Dry powders inhalers – DPI), obtido por aspersão contendo budesonida encapsulada, visando o tratamento da asma aguda e crônica. Essa proposta foi baseada na obtenção de um sistema pulverulento nanoestruturado com tamanho reduzido e controlado, visando a entrega pulmonar da budesonida. Na etapa de pré-formulação foi realizado um estudo fatorial avaliando diferentes métodos de preparação das nanocápsulas e os adjuvantes de secagem utilizados. As análises de tamanho de partícula, da formulação selecionada (nanocápsulas contendo budesonida e secas por aspersão com leucina) mostraram um tamanho reduzido e adequado para a administração pulmonar (2,7 μm). A morfologia demonstrou que estas partículas possuem um tamanho reduzido, forma esférica e superfície irregular, características importantes para a administração pulmonar. Quando analisada a distribuição pulmonar in vitro, em Impactador de Andersen, a formulação apresentou uma fração de partículas finas (Fine Particle Fraction – FPF) de 28%. Analisando os resultados dos experimentos em modelos de asma aguda e crônica induzidos por ovalbumina, os resultados da mecânica respiratória e função pulmonar mostraram uma diminuição na resistência e na elastância pulmonar, quando a budesonida nanoencapsulada foi utilizada, quando comparada com uma formulação comercial de budesonida, nas duas doses utilizadas (0,5 e 1,0 mg/Kg). Esse tratamento com nanocápsulas também mostrou eficiência na redução da inflamação, pela redução do número de leucócitos totais no fluido de lavagem bronco alveolar (Broncho Alveolar Lavage Fluid – BALF) e, principalmente, redução significativa no número dos eosinófilos no infiltrado pulmonar. Corroborando esses resultados, a quantificação da eotaxina – 1 e das citocinas pró-inflamatórias foram reduzidas, quando comparadas ao tratamento comercial. A análise histopatológica mostrou que quando o tratamento com as nanocápsulas foi utilizado, a produção de muco foi reduzida, bem como a produção de fibrose sub-epitelial, sugerindo um possível efeito sobre o remodelamento tecidual. Os resultados de toxicidade utilizando linhagem celular epitelial pulmonar (H441) mostrou uma redução na toxicidade da budesonida, quando encapsulada nas nanopartículas, tanto na forma de suspensão como na forma pulverulenta. Essa redução da toxicidade foi de 75% e de 50%, na dose de 100 μg/mL, para a suspensão e para o DPI, respectivamente. O conjunto dos resultados obtidos mostrou a potencial aplicabilidade da budesonida nanoencapsulada para o tratamento da asma, utilizando esse novo sistema DPI.Asthma is characterized as a chronic inflammatory disease developed by multifactorial aspects such as genetic predisposition and exposure to environmental factors such as pollution, smoke and microorganisms. The conventional asthma therapy comprises the use of bronchodilators and anti-inflammatory. Budesonide is a glucocorticoid and is the most frequently used therapy in the treatment of asthma. However, this drug has low oral bioavailability and long term use may lead to adverse effects such as skin thinning and adrenal suppression. The evaluation of the toxicity of new formulation has critical role in the pharmaceutical development. The use of cell culture experiments can help this aspect. Additionally, nanotechnology is an important tool to solve problems regarding bioavailability and to circumvent adverse effects of conventional therapy. The aim of this work was to develop a nanostructured system as dry powder inhaler (DPI) containing budesonide loaded, obtained by spray-drying, targeting the treatment of acute and chronic asthma. This proposal was based on obtaining a nanostructured powder system with reduced and controlled size, aiming an alternative to treatment of asthma. A factorial study comparing different methods to produce the nanocapsules as well as the type of drying adjuvants was performed. The particle size of the selected formulation was 2.7 μm, an adequate reduced size suitable for pulmonary administration. The morphology of these particles showed a small size, spherical shape and irregular surface. All these characteristics are important for pulmonary administration. When analyzed the in vitro pulmonary distribution of the DPI, using an Andersen Cascade Impactor, showed a fine particle fraction (FPF) of 28%. Analyzing the results of the biological experiments, the mechanical respiratory and pulmonary function showed a decrease in lung elastance and resistance when budesonide was used nanoencapsulated compared with a commercial formulation of budesonide in two doses (0.5 and 1.0 mg / kg). Both treatments also showed nanocapsules efficiency in reduction of inflammation by reducing the total of leukocytes in the bronchial alveolar lavage fluid (BALF) and especially significant reduction in eosinophil infiltration in the lung tissue. Corroborating with these results, the quantification of eotaxin - 1 and proinflammatory cytokines was reduced when compared to commercial budesonide treatment. Histopathological analysis showed that when treatment with the nanocapsules was used, mucus production was reduced and reversed the phenomena of airway remodeling. The cytotoxicity assay by Alamar blue using the bronchial epithelium cell line (H441) showed a reduction on the toxicity of budesonide when the nanocapsules were used even in suspension or in the DPI. The cytotoxicity reduction were 75 and 50%, at 100 μg/mL, for the suspension and the DPI, respectively. All these results show that budesonide-loaded nanocapsules in dry powder inhaler is a promising approach for the treatment of asthma.
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Alves, Chrystian Junqueira. "Caracterização do papel da célula de Schwann no processo de neurodegeneração do neurônio motor na esclerose lateral amiotrófica no modelo animal transgênico e no nervo periférico de pacientes: estudo in vitro." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/5/5138/tde-24112015-082439/.
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A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa progressiva de evolução rápida, caracterizada pela perda seletiva dos neurônios motores (NM) superiores e inferiores. Recentemente, as células gliais centrais (astrócito, microglia e oligodendrócito) mostraram-se tóxicas aos NM, porém os detalhes moleculares não estão completamente elucidados. Em relação às células gliais periféricas, alterações eletrofisiológicas no nervo ciático do modelo animal da ELA na idade pré-sintomática foram reportadas pelo nosso grupo e os achados de denervação precoce tanto no modelo animal quanto em pacientes sugerem a participação das células de Schwann (CS) na morte neuronal retrógrada na ELA, teoria conhecida como dying back. Nesse contexto, as CS mostraram-se capazes de induzir a retração axonal e a denervação das junções neuromusculares, eventos precoces na doença, ocorrendo possivelmente na fase présintomática. O objetivo deste trabalho foi verificar a influência das CS do modelo experimental na fase pré-sintomática e do paciente com evolução recente da forma esporádica da ELA, na sobrevida e no tamanho dos prolongamentos dos NM in vitro e entender a natureza molecular do fenômeno. Culturas de CS altamente purificadas foram obtidas a partir do nervo ciático do camundongo modelo animal e do nervo periférico de pacientes com ELA. Os NM da medula espinal de camundongos neonatos foram co-cultivados com as CS. A neurodegeneração foi avaliada pela presença do marcador Fluoro-Jade C (FJC). Os NM também foram tratados com o meio condicionado das culturas de CS do modelo animal ou dos pacientes com ELA. Os motoneurônios tiveram os seus prolongamentos contados e a morte neuronal foi identificada pela presença do FJC. Diversos fatores neurotróficos foram quantificados no meio condicionado das culturas de CS pela técnica de ELISA. A reação em cadeia da polimerase quantitativa (do inglês, quantitative polymerase chain reaction - qPCR) foi realizada para detectar alterações nas CS e no nervo periférico que pudessem estar relacionadas com disfunção na unidade CS/NM. Os resultados mostraram que os NM cultivados na ausência das CS mostraram-se mais susceptíveis à morte. Os NM cocultivados com as CS ELA mostraram maior número de perfis neurodegenerativos em comparação com os NM co-cultivados com as CS controle. Após o tratamento com o meio condicionado das CS ELA, os NM mostraram redução no tamanho dos prolongamentos e aumento do número de células em neurodegeneração em comparação com o grupo controle. Quantidades reduzidas dos fatores neurotróficos foram encontradas no meio condicionado das culturas de CS ELA. Alterações na expressão gênica das CS e no nervo periférico evidenciaram disfunções na unidade CS/NM que podem estar contribuindo para o processo neurodegenerativo visto na ELA. Conclui-se que a falência nos mecanismos de neuroproteção pelas CS ELA é um importante mecanismo implicado na morte neuronal, com grande potencial terapêuticoAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of upper and lower motor neurons (MN). Recently, central glia (astrocytes, microglias and olygodendrocytes) were toxic to the MN, but the molecular aspects have not fully described. In relation to the peripheral glia, electrophysiological changes in the sciatic nerve of ALS animal model in the presymptomatic stage have been reported by our group and early denervation findings in both animal models and patients suggests the participation of Schwann cells (SC) in the retrograde neuronal death of ALS , theory known as dying back. In this context, the SC proved to be able to induce axonal retraction and denervation of the neuromuscular junctions, early events in the disease, possibly occurring in the pre-symptomatic phase. The aim of this thesis was to investigate the influence of SC of pre-symptomatic experimental model and from patient with recent evolution of ALS sporadic form, in the survival and axonal length of MN in vitro and understand the molecular nature of the phenomenon. Highly purified SC cultures were obtained from the sciatic nerve of the animal model and from ALS patient\'s peripheral nerve. MN from the newborn mouse spinal cord were co-cultured with SC and the neurodegeneration was assessed by the presence of the marker Fluoro-Jade C (FJC). MN were also treated with conditioned medium from cultures of SC of the animal model or ALS patients. MN had their neuronal length measured and neuronal degeneration was identified by the presence of the FJC. Several neurotrophic factors were measured in conditioned medium of mice and ALS patient\'s SC cultures by ELISA. The chain reaction quantitative polymerase (qPCR) was performed to detect changes in the SC and peripheral nerve that could be related with dysfunction in the functional unit SC/MN. The MN co-cultured with ALS SC showed a greater number of neurodegenerative profiles compared with MN cocultured with control SC. After treatment with ALS SC conditioned medium, MN showed a reduction in the neuronal length and increased number of cells in neurodegeneration compared with the control group. Lower levels of neurotrophic factors were found in the conditioned medium of ALS SC cultures. Changes in the gene expression of SC and peripheral nerve showed dysfunctions in SC/MN unit, which may be contributing to the neurodegenerative process seen in ALS. In conclusion, the failure of neuroprotection by ALS SC is an important mechanism implicated in the MN cell death, with great therapeutic potential
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El, Kouchni Samira. "Towards an Animal-Derived Component Free Medium for Sp2/0 Fed-batch Culture : requirements and Challenges for an Effective Lipid Supplementation." Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21891.
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Les anticorps monoclonaux (mAb) sont d’importants agents thérapeutiques largement utilisés dans le traitement de cancers. Ces protéines recombinantes complexes sont généralement produites dans des lignées cellulaires de mammifères et l’industrie pharmaceutique a développé des procédés robustes permettant d’obtenir de grandes quantités d’anticorps monoclonaux de qualité constante. Une tendance générale observée aujourd’hui est d’éviter l’utilisation de produits d’origine animale dans ces procédés. En effet, ces composés représentent un risque de contamination du médicament par des agents infectieux et les autorités règlementaires renforcent leurs exigences pour leur retrait des procédés de fabrication. Ces composés sont par ailleurs mal définis et posent des problèmes de variabilité des procédés de production. L’objectif de ce projet était de développer un milieu sans dérivés animaux pour la culture d’une lignée cellulaire Sp2/0 utilisée par la société Merck Serono pour exprimer un anticorps monoclonal thérapeutique. Le procédé de fabrication actuel contient de la sérum albumine bovine (BSA) et de l’EX-CYTE (concentré commercial de lipoprotéines et acides gras) extraits de sérum bovin. Le retrait des deux composés du milieu de culture a entrainé une diminution de la productivité du procédé de 87% et il a été observé que l’EX-CYTE et la BSA étaient essentiels pour la survie de notre lignée cellulaire Sp2/0. La BSA a permis à elle seule de remplacer l’EX-CYTE dans le procédé et a été utilisée comme modèle pour le développement d’un remplacement sans dérivés animaux. Une étude de caractérisation de la préparation de BSA a été effectuée afin d’identifier les facteurs responsables de son activité promotrice pour la croissance cellulaire. Les lipides représentaient une partie importante de cette activité mais un rôle significatif d’autres protéines contaminantes a été révélé. Enfin, un supplément lipidique sans dérivés animaux a été développé. Ce supplément était constitué d’un mélange de quatre acides gras (les acides oléique, linoléique, palmitique et stéarique) couplés à de la sérum albumine humaine recombinante (rHSA). Le supplément acides gras-rHSA a permis de remplacer l’EX-CYTE et la BSA et un milieu sans composés d’origine animale a finalement été obtenuMonoclonal antibodies (mAbs) are important therapeutics widely used for cancer therapy. Mammalian cell lines are usually employed to produce these complex recombinant proteins and the pharmaceutical industry has developed robust processes that deliver large quantities of mAbs with a sustained quality. A general trend observed today is to avoid the use of animal-derived components in such processes. Indeed, these compounds represent a potential risk of contamination of the final drug product with infectious agents and regulatory authorities are putting pressure for their removal from manufacturing processes. Such compounds are also ill defined and source of variability for the production processes. The goal of this project was to develop an animal-derived component free (ADCF) medium for the culture of an Sp2/0 cell line used by the company Merck Serono to express a therapeutic mAb. The manufacturing process currently used contains bovine serum albumin (BSA) and EX-CYTE (a commercial concentrate of lipoproteins and fatty acids) sourced from bovine serum. The removal of both components from the cell culture medium decreased the productivity of the process by 87%. EX-CYTE and BSA were found to be essential for the survival of our Sp2/0 cell line. BSA, which was found to replace EX-CYTE in the process, was used as a model for the development of an animal-derived component free replacement. A characterization of the BSA preparation was carried out to identify the factors responsible for its growth-promoting activity. Lipids accounted for a major part of the activity of the BSA preparation but a significant role of other protein contaminants was revealed. Finally, an animal-derived component free lipid supplement was developed. This supplement consisted in a mixture of four fatty acids (FA) (oleic, linoleic, palmitic and stearic acids) complexed with recombinant human serum albumin (rHSA). The FA/rHSA supplement could substitute for EX-CYTE and BSA and an ADCF medium was finally obtained
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Zemaryalai, Khatera. "Investigations into the roles of potassium channels in hair growth : studies confirming the presence of several ATP-sensitive potassium (K+ATP) channels in hair follicles and exploring their mechanism of action using molecular biological, cell culture, organ culture and proteomic approaches." Thesis, University of Bradford, 2010. http://hdl.handle.net/10454/4461.
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Hair disorders cause significant distress. The main, but limited, treatment for hair loss is minoxidil, an ATP-sensitive potassium (KATP) channel opener whose mechanism of stimulation is unclear. The regulatory component of KATP channels has three forms: SUR1, SUR2A and SUR2B which all respond to different molecules. Minoxidil only opens SUR2B channels, though SUR1 and SUR2B are present in human hair follicles. To expand our understanding, the red deer hair follicle model was used initially. Deer follicles expressed the same KATP channel genes as human follicles when growing (anagen), but no channels were detected in resting follicles. This reinforces the importance of KATP channels in active hair growth and the usefulness of the deer model. To assess whether SUR1 KATP channels are actually involved in human hair growth, the effects of a selective SUR1 channel opener, NNC55-9216, on scalp follicle growth in organ culture was examined. NNC55-9216 stimulated anagen; its effect was augmented by minoxidil. This creates the potential for more effective pharmaceuticals to treat hair loss via SUR1 channels, either alone or in combination with minoxidil. The dermal papilla plays a crucial regulatory role in hair follicle activity determining the type of hair produced. Minoxidil had no effect on dermal papilla cell proliferation, but altered the profile of proteins produced when assessed by proteomics. Further research into the roles of KATP channels and greater understanding of the significance of these protein changes should enhance our knowledge of hair biology and help the development of new, improved therapies for hair pathologies.