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1
Falk, Anna. "Stem cells : proliferation, differentiation, migration /." Stockholm, 2005. http://diss.kib.ki.se/2006/91-7140-497-X/.
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Brigham, Lindy Andersen 1951. "Root border cell differentiation." Diss., The University of Arizona, 1996. http://hdl.handle.net/10150/290689.
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The inability of a plant to run from danger or seek nutrients necessitates its capacity to change the environment of the surrounding soil for protection and sustenance. A unique plant process, the release of thousands of autonomous cells from the root cap, called root border cells, may play a role in the ability of the plant to regulate microbial populations and nutrient availability in the rhizosphere. In this study, evidence is presented showing that root border cells are a differentiated tissue, that the production of border cells is highly regulated and tied to cell turnover in the root cap and that products of border cells regulate cell division in the root cap meristem. In vivo labeling experiments demonstrate that 13% of the proteins that are abundant in preparations from border cells are undetectable in root tip cells. Differences between the two cell populations are apparent as soon as border cells separate from each other, even when they are still adhered to the root tip. Twenty-five percent of the proteins synthesized by border cells in a 1-hour period are rapidly excreted into the incubation medium. Border cells arise within the root cap meristem by cell division and their production is tightly regulated both developmentally and in response to border cell removal. Removal of border cells results in the induction of cell division in the transverse root cap meristem to 400% of the basal rate within 30 minutes. This elevated rate of mitosis is maintained for 1.5 h and falls to basal levels within 6 hours. During this time, mitosis in the root apical meristem remains constant. mRNA differential display analysis showed changes in gene expression in the root cap within 5 to 15 minutes of removal of border cells. Genes putatively involved in cell functions in three regions of the cap showed expected distribution patterns by in situ hybridization and RNA blot analysis revealed changes in their expression patterns were seen in response to border cell removal. The presence of border cells acts as an inhibitor to continued mitosis and border cell production in the root cap. Evidence from fractionation studies shows that a heat stable, protease insensitive molecule in the range of 25 to 80 kDa, produced by the border cells themselves, is responsible for this inhibition.
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Loop, Franciscus Theodorus Lambertus van der. "Cell biological aspects of muscle cell differentiation." Maastricht : Maastricht : Universitaire Pers Maastricht ; University Library, Maastricht University [Host], 1996. http://arno.unimaas.nl/show.cgi?fid=7288.
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Li, Victor Chun. "The Cell Cycle and Differentiation in Stem Cells." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10536.
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The relationship between cellular proliferation and differentiation is a major topic in cell biology. What we know comes from models of somatic cell differentiation, where it is widely viewed that cycling and differentiation are coupled, antagonistic phenomena linked at the G1 phase. The extension of this view to stem cells, however, is unclear. One potential possibility is that stem cells also tightly link their G1 phase with their differentiation, indicating a similarity between the differentiation of stem cells and the differentiation of more mature somatic cells. On the other hand, stem cells may utilize different mechanisms or adaptations that confer on them some aspect of uniqueness or "stemness." In this case, stem cells will not exhibit the same coupling with the cell cycle as in many somatic cell models. In this thesis, we examined mouse embryonic stem cells (mESCs), a stem cell that is pluripotent and rapidly cycling with a highly condensed G1 phase. Direct extension of the somatic view posits that elongation of their G1 phase to somatic lengths by cyclin-dependent kinase (CDK) activity inhibition should induce or increase differentiation of these stem cells. Evidence supporting this claim has been contradictory. We show that elongation of the cell cycle and elongation of G1 to somatic lengths is fully compatible with the pluripotent state of mESCs. Multiple methods that lengthen the cell cycle and that target CDK activity or that trigger putative downstream mechanisms (i.e. Rb and E2F activity) all fail to induce differentiation on their own or even to facilitate differentiation. These results indicates that the model of linkage between the G1 phase and differentiation in mESCs is incorrect and leads us to propose that "stemness" may have a physiological basis in the decoupling of cell cycling and differentiation. In summary, we provide evidence that there is a resistance of mESCs to differentiation induced by lengthening G1 and/or the cell cycle. This could allow for separate control of these events and provide new opportunities for investigation and application.
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Ellison, David William. "Cell proliferation, cell death, and differentiation in gliomas." Thesis, University of Southampton, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295912.
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Xue, Yintong. "Glucocorticoid in T cell differentiation /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3950-0/.
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Locklin, Rachel M. S. "Biochemistry of bone cell differentiation." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363755.
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Giddings, Ian. "Analysis of myeloid cell differentiation." Thesis, King's College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285145.
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Jones, Philip Anthony. "B cell differentiation in sheep." Thesis, University of Edinburgh, 1988. http://hdl.handle.net/1842/30327.
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The ileal Peyer's patch (IPP) of lambs is a region of intense lymphopoiesis and B cell development. Monoclonal antibodies against ovine lymphoctye antigens have been used to characterise the IPP lymphocyte. Three murine monoclonal antibodies against ovine IgM, IgG1 and Ig light chain were produced and are described fully. IgM and MHC class II antigens are expressed on the vast majority of IPP cells whilst cells bearing other serum Ig isotypes and T cell antigens are rare. A novel Ig molecule appears to be coexpressed with IgM, it is proposed that this is the ovine equivalent of IgD. IPP cells can be induced to proliferate and differentiate when cultured with lipo-polysaccharide (LPS) and interleukin 2 (IL2). Proliferation is inhibited by rabbit anti-sheep Ig antibodies. Using an ELISA for Ig, it has been possible to quantitate Ig synthesis and secretion. Mean cellular Ig increases greater than 25-fold during differentiation. High-rate secretion begins 4 days after initiation of culture and is virtually complete by day 7. As IPP B cells differentiate to IgM secretion, membrane Ig is rapidly lost so that by day 6, only 15% of cells express Ig on their surfaces. Changes in MHC class II antigens were also studied. Surface expression of MHC class II molecules doubled by 24 hours and slowly declined to resting levels as differentiation proceeded. A large increase in cytoplasmic MHC class II content was noted on day 3. The reasons for this increase are discussed. Kinetic studies suggest that IL2 responsiveness is acquired approximately 20 hours after activation by LPS. The concentration required to give half maximal Ig secretion is 125 pM indicating that the interaction between IL2 and its receptor is one of high affinity. During differentiation, the cells enlarge and show an increase in the cytoplasmic:nuclear ratio. The formation of extensive rough endoplasmic reticulum and additional mitochondria is indicative of the functional changes occurring. This is the first description of a sheep B cell differentiation assay. It is proposed that this system is a suitable model on which to base further studies into the molecular biology of sheep Ig genes, Ig isotype switching and lymphokines and their receptors.
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Park, Jaesang. "Automatic white blood cell differentiation /." free to MU campus, to others for purchase, 2002. http://wwwlib.umi.com/cr/mo/fullcit?p3074435.
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Holm, Pontus. "Survival and differentiation of central noradrenergic neurons /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-277-9.
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Sankar, Uma. "Coordination of cell cycle and cell differentiation by receptor activator of NF-KAPA-B ligand during osteoclast differentiation." Columbus, Ohio : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1056980709.
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Thesis (Ph. D.)--Ohio State University, 2003.<br>Title from first page of PDF file. Document formatted into pages; contains xvii, 292 p.; also includes graphics (some col.). Includes abstract and vita. Advisor: Mihael C. Ostrowski, Dept. of Molecular, Cellular and Developmental Biology. Includes bibliographical references (p. 259-292).
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Erlandsson, Anna. "Neural Stem Cell Differentiation and Migration." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl.[distributör], 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3546.
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Ansari, Naser A. (Naser Awni). "Purification and Characterization of a Differentiation Factor From Rat Lung Conditioned Medium." Thesis, North Texas State University, 1988. https://digital.library.unt.edu/ark:/67531/metadc798062/.
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A Differentiation Factor (DF) was purified from rat lung conditioned medium by a four-steps procedure. The DF has a molecular weight of 27000, and an isoelectric point of 4.70. Although DF is stable up to 60°C, it is sensitive to digestion by trypsin, chymotrypsin and subtilisin. DF forms granulocyte colonies in soft agar. Studies using anti-NRK CSF antibody demonstrated that DF is distinct from GM-CSF.
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Muguruma, Yukari. "The origin and differentiation of the osteoclast /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/5681.
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Liu, Haoyu. "Goblet cell differentiation in human colorectal cancer cell lines." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:d907bbf2-76fc-409a-877c-529fc04ca042.
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Goblet cells are one of the three fully differentiated lineages in human colonic crypts. They play an important role in protecting epithelial cells from direct contact with luminal contents by secreting mucus, which consists of MUC2, TFF3 and other constituents. The goblet cell differentiation, however, is largely dysregulated in human colorectal cancers. Abundant expression of MUC2 is often observed in signet ring carcinomas and mucinous carcinomas that are associated with unsatisfactory prognosis. Despite the significant roles of goblet cells, its genetic nature and exact regulatory mechanisms remain incompletely understood. In this thesis, the goblet cell differentiation at mRNA and protein levels was characterised in a panel of 64 human colorectal cancer cell lines. Microarray analysis on the bulk population of these cell lines reveals the genes differentially expressed in goblet cell-positive cell lines, including AKR1B10, AGR3 and the cellular surface protein CA12 (Chapter 3). In addition, a novel protocol is developed for isolation and sequencing of RNA from the fixed and FACS purified cells. Using this protocol, the first goblet cell transcriptome is profiled in LS180 cancer cell line, and the goblet cell-specific genes are identified, including TFF3 and SPDEF (Chapter 4). The co-expression patterns of TFF3 and MUC2 is further investigated. In a subset of cancer cell lines, TFF3 recognises the goblet cells that cannot produce MUC2, suggesting additional regulatory mechanisms are required for its expression. The dysregulated regulation of TFF3 may provide additional evidence in colorectal cancer classification. The transcriptional regulation of ATOH1 and SPDEF on goblet cell differentiation is also demonstrated. Knock-down of either transcriptional factor decreases the goblet cell numbers, while double knock-down completely depletes goblet cell formation, suggesting the co-operative regulation of SPDEF and ATOH1 on goblet cell differentiation. In addition, CA12-positive but not -negative cells can give rise to goblet cells with the expression of MUC2, TFF3 and SPDEF. This indicates CA12 may act as a potential cellular surface marker to identify goblet cell progenitors (Chapter 5). In summary, this thesis screens goblet cells in colorectal cancer cell lines and characterise the first goblet cell transcriptome, which provides the foundation to understand regulatory control of goblet cell differentiation.
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Lu, Xibin, and 盧希彬. "Quantitative characterization of mouse embryonic stem cell state transition." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208049.
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Lyons, Alan Bruce. "Human myeloid differentiation antigens /." Title page, table of contents and abstract only, 1987. http://web4.library.adelaide.edu.au/theses/09PH/09phl991.pdf.
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Rennel, Emma. "Molecular Mechanisms in Endothelial Cell Differentiation." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4059.
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Graaff, Wiebo Leendert van der. "T cell differentiation in autoimmune diseases." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2003. http://dare.uva.nl/document/70775.
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Masciarelli, Silvia. "Molecular physiology of plasma cell differentiation." Thesis, Open University, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491475.
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Plasma cells are central to an effective immune response, being the sole producers of the antibodies, yet they can caqse severe disease in autoimmunity and multiple myeloma. Therefore their differentiation and survival must be tightly regulated. In this work I investigated some of the mechanisms regulating plasma cell differentiation and life span. The differentiation of a long lived B cell to a short lived plasma cell entails a profound structural and functional metamorphosis finalized to the massive production of immunoglobulins (Ig). Exuberant Ig synthesis causes several types of stress in differentiating plasma cells. My work deals with the characterization of. the C/EBP transcription factor CHOP in plasma cell differentiation. Comparing differentiation of B cells harvested from chop'!' mice to wt cells I found a mild phenotype, consisting in an increased accumulation of intracellular IgM aggregates and a decreased secretion of this antibody class, in vitro and in vivo. These findings' reveal a novel role for CHOP in ensuring optimal functionality of the secretory pathway in the course of plasma cell differentiation. CHOP is involved in the differentiation of various cell types, where it interacts with other members of the C/EBP family favoring or impeding differentiation and it is an important factor in the ER stress response named unfolded protein response (UPR), in which it plays a pro-apoptotic role in most of the systems tested. I extended my investigation on the functions of CHOP in B cells by examining the resistance to ER stress-induced apoptosis in wt and in chop-I' cells. Surprisingly, I observed that in B cells CHOP expression in the UPR plays an anti-apoptotic function. Altogether my data suggest a cell-type specific role for CHOP in B cells and add information on the multi-faceted role of this transcription factor. Most plasma cells exhibit a short life span. The mechanisms at the basis of plasma cell apoptosis are still obscure. I propose that multiple forms of stress, linked to the massive antibody production, contribute to plasma cell death.
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Jermyn, Keith A. "Prestalk cell differentiation in Dictyostelium discoideum." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314869.
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Marek, Carylyn Jane. "Trans-differentiation and liver cell biology." Thesis, University of Aberdeen, 2004. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU186421.
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Trans-differentiation is the term applied where one fully differentiated cell type changes into another fully differentiated cell type. The AR432J-B13 cell line has been shown to demonstrate this phenomenon in vitro. These cells of exocrine pancreatic lineage, can trans-differentiate into hepatocytes upon treatment with the synthetic glucocorticoid dexamethasone. This thesis demonstrates that the generation of these cells (B13-H) from AR42J-B13 cells could prove to be a novel source of hepatocytes in culture. B13-H cells express functionally active and inducible CYP enzymes for at least 30 days in culture, an attribute that primary hepatocytes do not hold. In addition, B13-H cells have shown to be a useful alternative to primary hepatocytes in the investigation of protective mechanisms against paracetamol toxicity. The pancreas and liver have a close association developmentally which helps to explain their relationship in adulthood. As primary hepatocytes rapidly dedifferentiate in culture, the use of these or other pancreas-derived hepatocytes would be beneficial, both in a clinical (e.g. bioartificial liver device as a 'bridge' until transplant) and pharmacological (e.g. drug metabolism studies) settings. Another liver cell affected by disease is the hepatic stellate cell. These cells trans-differentiate into a myofibroblast-like cell and are pivotal in the formation of liver fibrosis. By inhibiting this trans-differentiation event, liver fibrosis can resolve, thereby preventing the terminal state of cirrhosis. PCN is such a compound capable of achieving this outcome both in vitro using isolated hepatic stellate cells and in vivo carbon tetrachloride-induced rodent liver-injury models. Transgenic mice enabled the determination that this effect is both dependent and independent upon the nuclear receptor PXR of which PCN is a ligand.
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Shah, Nadia Nisa. "Human embryonic stem cells : prospects for pancreatic β-cell differentiation". Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.495052.
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The focus of this thesis was to explore different strategies in trying to generate putative pancreatic β-cells using one of the initial Wisconsin H7 hES cell lines. Prior to this, human pancreas development was assessed during the first trimester of pregnancy in an attempt to determine the spatial and temporal expression of development and mature pancreatic β-cell markers during this period. Spontaneous differentiation of hES cells can be induced by the formation of embryoid bodies (EBs) in suspension culture. EBs began to express markers of pancreatic β-cell development and function at a molecular, protein and functional level upon differentiation over a 3-week period. The constitutive over-expression of the terminal β-cell marker PAX4 enhances this process, whereas karyotypic abnormalities induced in hES cells over prolonged culture can hinder differentiation potential towards pancreatic β-cells. Directed differentiation strategies which mimic mouse pancreas development have led to the elucidation of an in vitro protocol to generate putative definitive endoderm from hES cells through the application of Wnt3a and Activin A in the presence of low serum. Indirect co-culture of this H7 hES cell-derived putative definitive endoderm with mouse islets did not lead to the differentiation of fully functional pancreatic β-cells. The hES cell-derived putative definitive endoderm did however influence the aging mouse islets in a positive manner by allowing the maintenance of insulin secretagogue-induced functional responses which are usually lost in culture. This may prove useful in maintaining viability of human islets during culture to be used for transportation therapies.
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VanOudenhove, Jennifer J. "Mechanisms Regulating Early Mesendodermal Differentiation of Human Embryonic Stem Cells: A Dissertation." eScholarship@UMMS, 2016. http://escholarship.umassmed.edu/gsbs_diss/849.
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Key regulatory events take place at very early stages of human embryonic stem cell (hESC) differentiation to accommodate their ability to differentiate into different lineages; this work examines two separate regulatory events.
To investigate precise mechanisms that link alterations in the cell cycle and early differentiation, we examined the initial stages of mesendodermal lineage commitment and observed a cell cycle pause that occurred concurrently with an increase in genes that regulate the G2/M transition, including WEE1. Inhibition of WEE1 prevented the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. These findings reveal a novel G2 cell cycle pause required for endodermal differentiation.
A role for phenotypic transcription factors in very early differentiation is unknown. From a screen of candidate factors during early mesendodermal differentiation, we found that RUNX1 is selectively and transiently up-regulated. Transcriptome and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that RUNX1 knockdown impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion compromised TGFβ2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFβ2, but not TGFβ1. These findings identify novel roles for RUNX1-TGFβ2 signaling in mesendodermal lineage commitment.
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Bennett, Jonathan Hilary. "The differentiation of osteogenic cells from bone marrow." Thesis, University of Oxford, 1991. http://ora.ox.ac.uk/objects/uuid:3460f26e-a124-4605-8601-2e300241de14.
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Beckstead, Benjamin L. "Control of epithelial differentiation by cell-instructive scaffolds /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/8096.
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Ortmann, Daniel. "Reporter cell lines to study cell populations and fate decisions during human pluripotent stem cell differentiation in vitro." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648356.
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Watson, Andrea. "Heat shock proteins in leukaemia cell differentiation and cell death." Thesis, Aston University, 1990. http://publications.aston.ac.uk/12533/.
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When HL60 cells were induced to differentiate to granulocyte-like cells with the agents N-methylformamide and tunicamycin an concentrations marginally below those which were cytotoxic, there was a decrease in the synthesis of the glucose- regulated proteins which preceded the expression of markers of a differentiated phenotype. There was a transient increase in the amount of hsp70 after 36 hours in NMF treated cells but in differentiated cells negligible amounts were detected. Inducers which were known to modulate hsp70 such as azetadine carboxylic acid did not induce differentiation suggesting early changes in the endoplasmic reticulum may be involved in the commitment to terminal differentiation of HL60 cells. These changes in group synthesis were not observed when K562 human chronic myelogenous leukemia cells were induced to differentiate to erythroid-like cells but there was a comparable increase in amounts of hsp70. When cells were treated with concentrations of drugs which brought about a loss in cell viability there was an early increase in the amount of hsp70 protein in the absence of any increase in synthesis. HL60 cells were treated with NMF (225mM), Adriamycin (1 jiM), or CB3717 (5iM) and there was an increase in the amounts of hsp70, in the absence of any new synthesis, which preceded any loss of membrane integrity and any significant changes in cell cycle but was concomitant with a later loss in viability of > 50% and a loss in proliferative potential. The amounts of hsp70 in the cell after treatment with any of the drugs was comprable to that obtained after a heat shock. Following a heat shock hsp70 was translocated from the cytoplasm to the nucleus, but treatment with toxic concentrations of drug caused hsp70 to remain localised in the cytoplasm. Changes in hsp70 turn-over was observed after a heat shock compared to NMF-treated cells. Morphological studies suggested that cells that had been treated with NMF and CB3717 were undergoing necrosis whereas the Adriamycin cells showed characteristics that were indicative of apoptosis. The data supports the hypothesis that an increase in amounts of hsp70 is an early marker of cell death.
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Sancho, Maria Margarida Gouveia. "Function of Bmpr1a in ES cell differentiation and cell competition." Thesis, Imperial College London, 2009. http://hdl.handle.net/10044/1/5885.
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Bone morphogenetic protein (BMP) 4 signalling via BMPR1A is required for the maintenance of the epiblast in the early embryo, and for self-renewal of pluripotent mouse embryonic stem (ES) cells by inhibiting neural differentiation. In this study, the self-renewal and differentiation abilities of ES cells lacking BMPR1A were investigated. Bmpr1a-null ES cells did not respond to BMP4 but retained a degree of SMAD1/5/8 activation and Id1 expression. This activation was likely due to BMP7 signalling via ACVR1. The observation that Bmpr1a-/- ES cells showed no selfrenewal or pluripotency defects suggested that signalling by BMPs of the 60a subgroup (such as BMP7) can also maintain pluripotency. When Bmpr1a-/- ES cells were differentiated, although they did form derivatives of the three germ layers, they displayed a higher propensity to undergo neurectodermal specification than control cells, likely due to their lower levels of BMP signalling. Cell Competition is the process by which viable cells are eliminated in the presence of metabolically more active or fitter cells. In Drosophila this process depends on dMyc levels and on limiting amounts of the survival factor Decapentaplegic (homologous to the mammalian BMPs). When Bmpr1a-/- ES cells were co-cultured with wild-type cells, they gradually disappeared from the culture and were therefore out-competed. This cell competition was enhanced by limiting the amounts of survival and growth factors and could be rescued by restoring BMP4 signalling in Bmpr1a-/- cells. In co-culture, Bmpr1a-/- ES cells showed no significant changes in apoptosis but had a decreased cell cycle rate and increased levels of differentiation. Concomitantly, higher c-MYC levels were observed in wild-type cells due to increased protein stability. The out-competition of Bmpr1a-/- cells was dependent on differentiation as it could be prevented by inhibiting this process. These results suggest that during development cell competition may be an important mechanism controlling cell fate and survival.
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Leung, Y. L., and 梁宇亮. "Transcriptional regulators of col10al in chondrocyte differentiation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B31244440.
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Li, Jing. "Effects of intrinsic & extrinsic factors on the growth and differentiation of human mesenchymal stem cells." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36434450.
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Fan, Ngo-yin, and 樊傲賢. "The role of protein kinase D in osteoblast differentiation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41508488.
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Apperly, James A. "The relationship between proliferation and differentiation during oligodendrocyte development." Thesis, University College London (University of London), 2001. http://discovery.ucl.ac.uk/1349376/.
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How do precursor cells know when it is time to stop dividing and differentiate? The phenomenon of lineage-specific progenitor cells undergoing a limited period of proliferation prior to terminal differentiation is a common theme in multicellular development. Despite this, little is understood about how these two events are co-ordinated during the normal schedule of development. I have studied the question of how proliferation and differentiation are co-regulated in the oligodendrocyte lineage in the rodent optic nerve. Oligodendrocytes are post-mitotic cells that myelinate axons in the vertebrate central nervous system. They develop from precursor cells whose maturation is controlled by a timer, which is an intrinsic property of the cells, that limits proliferation. The timer seems not to control the number of divisions the cell can undergo but rather the length of time during which divisions can occur. Significant effort has been devoted to understanding how the intracellular timer regulates oligodendrocyte development. The timer consists of two components that are modulated by distinct kinds of extracellular signals. Mitogens drive a timing component whose value increases as precursor cells continue to divide. Once this value exceeds a critical threshold, it signals that the proliferative period has elapsed, and hydrophobic signalling molecules trigger an effector component that elicits cell-cycle arrest and differentiation. The value of the timing component is determined by several intracellular molecules whose activities change as the timer runs. One of these molecules is the cell-cycle inhibitor p27: it accumulates in oligodendrocyte precursor cells as they proliferate in culture. When p27 expression is high the precursor cells are more likely to stop dividing and differentiate than when it is low. In oligodendrocyte precursor cells derived from mice that lack p27, the timer runs aberrantly and cell-cycle arrest and terminal differentiation are delayed. It is not understood how the molecular mechanics of the timer control oligodendrocyte development. Does the timer serve to arrest the cell-cycle, with differentiation following by default, or is cell-cycle arrest subordinate to the programme of terminal differentiation? These questions remain unanswered, largely because of a persistent inability to experimentally manipulate the genome of oligodendrocyte precursor cells. The present study was an attempt to overcome these problems and had two aims - first, to devise a reliable system for transfecting oligodendrocyte precursor cells and second, to determine whether the timer primarily controls the timing of cell-cycle arrest. I developed a new retroviral vector that co-expresses p27 and green fluorescent protein (GFP) in precursor cells. The use of GFP allows the identification of living precursor cells that over-express p27, which can then be followed over many days in culture. My findings support previous work showing that p27 plays a role in governing the timing of oligodendrocyte differentiation. They show that over-expression of p27 promotes oligodendrocyte differentiation by advancing the value of the timing component, although it does not promote differentiation if the effector component is inoperative. The cell-cycle time of precursor cells that over-express p27 is dramatically extended, but not stopped. It appears that a firm cell-cycle arrest and entry into a quiescent state may be required to elicit terminal differentiation.
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Fan, Ngo-yin. "The role of protein kinase D in osteoblast differentiation." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41508488.
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Constant, Vanessa Auguste. "Macrophage-conditioned medium inhibits adipocyte differentiation." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27967.
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Obesity is accompanied by a reduced adipogenic capacity that promotes a dipocyte hypertrophy, low-grade inflammation, and insulin resistance. Macrophages infiltrate adipose tissue and may contribute to the low-grade inflammation associated with obesity and insulin resistance, suggesting that they could also play an anti-adipogenic role. I hypothesized that macrophage-secreted factors inhibit adipogenesis. My objectives were to assess if macrophage-conditioned medium (MacCM) inhibits adipocyte differentiation and to determine the mechanism by which the inhibition occurs. Murine J774 or human THP-1 MacCM was added to murine 3T3-L1 or human abdominal subcutaneous or omental preadipocytes. Either type of MacCM impaired murine and human adipogenesis as measured by triglyceride accumulation and protein expression of adipogenic markers. Time course studies revealed that THP-1-MacCM was required during the early phase of 3T3-L1 adipogenesis for its inhibitory effect. THP-1-MacCM stimulated the phosphorylation of ERK1/2 and IKKbeta in 3T3-L1 preadipocytes. Pharmacological inhibition of ERK1/2, with the specific MEK1 inhibitor PD98059, alleviated the inhibitory effect of THP-1-MacCM on TG accumulation. In conclusion, MacCM inhibits adipocyte differentiation in culture. The antiadipogenic effect depends on early exposure of THP-1-MacM to differentiating 3T3-L1 preadipocytes, and ERK1/2 is required for the inhibitory effect of THP-1-MacCM on TG accumulation.
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Capra, J. (Janne). "Differentiation and malignant transformation of epithelial cells:3D cell culture models." Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526218236.
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Abstract The epithelial cells form barriers that compartmentalize the organs. Carcinomas are cancers stemming from epithelial cells and are the most common cancer type. The aim of this study was to understand the differentiation and malignant transformation of epithelial Madin-Darby canine kidney (MDCK) cells and to analyse the electrophysiological parameters which regulate their transport capacity. Emphasis was placed on comparing different culture environments, both in 2D and 3D. First, the effects of drugs or basal extracellular fluid composition on MDCK cell, cyst and lumen volumes were analysed using time-lapse microscopy. The results showed that MDCK cells were capable of both water secretion and reabsorption. The cells were able to perform these functions in a hyperpolarizing or depolarizing environment; change in osmolality of basal fluid was not required. Taken together, these results validate MDCK cells as a good basic model for studying kidney function. Next, the aim was to analyse the effect of 2D and 3D culture environments on the gene expression of untransformed MDCK and temperature sensitive ts-Src -transformed MDCK cells and the changes a single oncogene can induce. Microarray analysis revealed a decrease in the expression of survivin, an inhibitor of apoptosis protein, when switching the untransformed cells from 2D environment to 3D. This downregulation of survivin occurs in adult tissues as well, indicating that the cells grown in 3D are closer to the in vivo state than 2D cells. Src oncogene induced disintegration of cell junctions, but did not downregulate E-cadherin expression. The last part was to study further the factors controlling survivin expression and its significance to cell survival. MDCK cells grown in 3D did not suffer apoptosis if the cells remained in contact with the extracellular matrix. If MDCK cells were denied of ECM contacts they were more susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, if cells were denied of cell-cell junctions, cells lacking survivin suffered apoptosis even though they had proper cell-matrix contacts. Taken together, these results highlighted the importance of cellular contacts to the cells: MDCK cells needed ECM contacts to differentiate and cell-cell contacts to avoid apoptosis<br>Tiivistelmä Epiteelisolut ovat erikoistuneet toimimaan rajapintana elimen ja ympäristön välillä. Ihmisten yleisin syöpä on epiteelisoluista alkunsa saanut karsinooma. Tämän tutkimuksen tarkoituksena oli ymmärtää Madin-Darby-koiran munuaisen solujen (MDCK) erilaistumista ja pahanlaatuistumista sekä analysoida sähköfysiologisia tekijöitä, jotka säätelevät näiden solujen kuljetustoimintaa. Erityisenä kiinnostuksen kohteena oli erilaisten kasvuympäristöjen vertailu. Farmakologisten aineiden tai basaalisen, solunulkopuolisen nesteen koostumuksen vaikutusta MDCK-solujen, -kystan sekä luumenin kokoon tutkittiin valomikroskooppisten aikasarjojen avulla. Tulokset osoittivat MDCK-solujen olevan kykeneviä sekä veden eritykseen että absorptioon, niin hyperpolarisoivassa kuin depolarisoivassakin ympäristössä. Basaalisen nesteen osmolaliteetin muutosta ei tarvittu. Nämä tulokset osoittavat MDCK-solujen olevan hyvä munuaisen tutkimuksen perusmalli. Seuraavaksi analysoitiin kaksi- ja kolmiulotteisten (2D ja 3D) viljely-ympäristöjen vaikutusta ei-transformoitujen MDCK-solujen ja lämpötilaherkkien ts-Src-transformoitujen MDCK-solujen geenien ilmentymiseen sekä yhden onkogeenin aktivoimisen aikaansaamia muutoksia. Microarray-analyysi osoitti apoptoosin estäjän, surviviinin, ilmentymisen vähenemisen, kun kasvuympäristö vaihdettiin 2D-ympäristöstä 3D-ympäristöön. Koska surviviinin väheneminen on normaali tapahtuma aikuisissa kudoksissa, voitiin todeta, että 3D-ympäristössä kasvatetut solut ovat lähempänä luonnonmukaista olotilaa kuin 2D-ympäristössä kasvaneet. Src-onkogeeni sai aikaan soluliitosten hajoamisen, mutta ei vähentänyt E-kadheriinin ilmentymistä. Tutkimuksen viimeinen osa keskittyi surviviinin ilmentymistä säätelevien tekijöiden analysoimiseen ja surviviinin merkitykseen solujen eloonjäämiselle. 3D-ympäristössä kasvaneet MDCK-solut eivät kärsineet apoptoosista edellyttäen, että solut pysyivät kosketuksissa soluväliaineeseen. Jos solut irtautuivat soluväliaineesta, ne päätyivät herkemmin apoptoosiin kuin surviviinia ilmentävät ts-Src MDCK-solut. Mikäli solujen väliset liitokset pakotettiin avautumaan, solut joutuivat apoptoosiin, vaikka ne olivat kosketuksissa soluväliaineeseen. Yhteenvetona nämä tulokset korostavat solujen kontaktien merkitystä: MDCK-solut tarvitsevat soluväliainekontakteja erilaistumiseen ja solujen välisiä kontakteja välttyäkseen apoptoosilta
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Fong, Jenna. "Breast cancer cells affect bone cell differentiation and the bone microenvironment." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=104758.
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Breast carcinoma is the most commonly diagnosed cancer among women worldwide, with approximately 1 in 7 expected to be affected during her lifetime. The spread of breast cancer to secondary sites is generally incurable. Bone is the preferred site of metastasis, where the development of a secondary tumour causes severe osteolysis, hypercalcemia and a considerable pain burden. However, how breast cancer cells establish supportive interactions with bone cells is not well understood. We have examined the effects of factors released from MDA-MB-231 and 4T1 breast cancer cells on the differentiation of C57BL6 mouse bone marrow cells. Treatment with cancer-derived factors resulted in a sustained 40–60% decrease in osteoblast differentiation markers, and induced an osteoclastogenic change in the ratio of receptor activator of NF-κB ligand (RANKL) to osteoprotegerin (OPG). Importantly, exposure of bone cells to breast cancer-derived factors stimulated the subsequent attachment of cancer cells to immature osteoblasts. Inhibition of γ-secretase using pharmacological inhibitors DAPT and Compound E completely reversed cancer-induced osteoclastogenesis as well as cancer-induced enhancement of cancer cell attachment, identifying γ-secretase activity as a key mediator of these effects. We next evaluated the effects of breast cancer cells on the energy metabolism of bone cells. Treatment of bone marrow cells with conditioned medium from 4T1 breast cancer cells resulted in an increase in glucose consumption by bone cells, higher mitochondrial transmembrane potential, and a 2.3-fold rise in cellular ATP content. In addition, breast cancer derived factors stimulated the expression of mRNA and protein levels of metabolic sensor, AMP-regulated protein kinase (AMPK). To assess if such change in cell bioenergetics may have consequences for cell differentiation and activity, we used defined models of osteoclastogenesis, and increased precursor metabolic activity by providing excess energy substrates. We have found that an increase in mitochondrial transmembrane potential and cellular ATP levels during osteoclastogenesis resulted in the formation of larger osteoclasts that demonstrate higher resorptive activity. Thus, we have uncovered that osteoblasts act as a critical intermediate of premetastatic signalling by breast cancer cells, and pinpointed γ-secretase as a robust target for developing therapeutics potentially capable of reducing both the homing and progression of cancer metastases to bone. In addition, we have discovered heightened energetics in bone cells exposed to breast cancer cell-released factors, which may contribute to the formation of larger, more active osteoclasts. Modification of the AMPK pathway may prove an important therapeutic target for breast cancer metastasis to bone.<br>Le cancer du sein est le cancer plus diagnostiqué chez les femmes. On estime qu'environ une femme sur sept en sera affectée. La diffusion du cancer du sein aux emplacements secondaires est généralement incurable. L'os est l'emplacement préféré de la métastase, où le développement d'une tumeur secondaire cause de l'osteolyse, de l'hypercalcemie, et une douleur considérable. Cependant, comment les cellules de cancer du sein établissent des interactions supportifs avec des cellules d'os n'est pas bien compris. Nous avons examiné les effets des facteurs libérés des cellules du cancer du sein MDA-MB-231 et 4T1 sur la différentiation des cellules de moelle de la souris C57BL6. Le traitement avec des facteurs cancer-dérivés a produit une diminution de 40-60% des marqueurs de différentiation d'osteoblast, comparé au traitement par l'acide ascorbique, et a induit un changement osteoclastogenique dans le rapport du RANKL/osteoprotegerin. L'exposition des cellules d'os à des facteurs dérivés du cancer du sein a ensuite stimulé l'attachement des cellules cancéreuses aux osteoblasts non mûrs. L'inhibition du γ-secretase utilisant les inhibiteurs pharmacologiques DAPT et le Compound E a complètement inversé l'osteoclastogenise cancer-induit aussi bien que le perfectionnement cancer-induit de l'attachement de cellules cancéreuses, identifiant l'activité de le γ-secretase comme étant le médiateur principal de ces effets. Nous avons ensuite évalué les effets des cellules cancereuse sur le métabolisme énergétique des cellules d'os. Le traitement des cellules de moelle avec le medium conditionné des cellules du cancer du sein 4T1 a eu comme conséquence une augmentation des mitochondries à haut-potentiel de membrane, une augmentation de 2.3 fois le contenu cellulaire de triphosphate d'adénosine, et une consommation plus rapide du glucose. Ce changement de l'énergétique a été accompagné d'une stimulation d'AMPK dans la protéine et l'ADN messagère. Pour évaluer les effets du statut de haute énergie dans les osteoclasts, nous avons élevé l'énergique des osteoclasts avec du pyruvate de sodium. Cette addition a causée une croissance des osteoclasts, avec des plus grands nucleus, et la résorption de plus de substrat. Ainsi, nous avons découvert l'osteoblast comme étant un intermédiaire clé à la signalisation prémetastatique par des cellules du cancer du sein. Nous avons aussi indiqué le γ-secretase comme cible robuste pour le developpement de thérapeutique potentiellement capable de réduire l'autoguidage et la progression des métastases de cancer à l'os. Additonellement, nous avons découvert l'énergétique intensifiée chez les cellules d'os exposées aux facteurs cellule-libérés par le cancer du sein, qui mène à une osteoclastogenesise plus active et plus importante. La modification de la voie d'AMPK peut s'avérer être une cible thérapeutique importante pour que la métastase de cancer du sein aux os.
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Cowan, Gillian. "Fetal germ cell differentiation and the impact of the somatic cells." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4164.
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Specification of a germ cell lineage and appropriate maturation are essential for the transfer of genetic information from one generation to the next. Germ cells form from pluripotent precursor cells that migrate into the gonadal ridge and undergo commitment to either the female or male lineage. In the fetal ovary, germ cells enter meiotic prophase I, then arrest at the diplotene stage; in the testis germ cells do not begin meiosis until puberty. Abnormal differentiation of germ cells can result in malignant transformation. Somatic cells play a key role in modulating the developmental fate of the germ cells. Research into germ cell development during fetal life has almost exclusively focused on studies in rodents, but we, and others, have reported several fundamental differences in the expression of germ cell specific markers in the human compared with the mouse. The studies described in this thesis have investigated germ cell-specific gene expression and the possible impact of the somatic cells during development. This was achieved by studying human fetal gonads obtained during the 1st and 2nd trimesters of pregnancy and through the use of both wild-type and mutant mouse ES cell lines. Studies on germ cells in the human fetal testis have extended the findings of others, and confirmed that germ cell populations at different stages of maturation co-exist in the human fetal testis, a situation that is in contrast to that in rodents. For example expression of M2A and AP2γ was restricted to the OCT4-positive gonocyte population, while VASA and NANOS1 were localised exclusively to the to the OCT4-negative prespermatogonia. DAZL was expressed in both populations. Analysis also revealed that both the gonocyte and prespermatogonial populations proliferate throughout the 2nd trimester. Recent studies have implicated retinoic acid (RA) in the control of meiotic entry in germ cells of the fetal mouse ovary. In this study we demonstrated for the first time that two genes implicated in the action of RA in mouse gonad, STRA8 and NANOS2, are also expressed in a similar sexspecific- manner in the human fetal gonads, and that the RA receptors are present in both somatic and germ cells suggesting that RA may regulate germ cell function in the human as well as the mouse. However, whilst the mesonephros appears to be the primary site of RA synthesis in the mouse our initial studies indicate that in the human the gonad itself may be a more likely site of RA biosynthesis. In the fetal mouse testis, RA is degraded by the enzyme Cyp26b1 present in the somatic cells and germ cells do not enter meiosis, our novel findings suggest that CYP26B1 is more abundant in the human fetal ovary than the testis, suggesting that meiotic entry may be controlled by an alternative signalling pathway in the human. One of the methods that can aid our understanding of somatic cell gene expression in the gonad is in vitro culture. To date, there have been no published reports of the successful in vitro culture of somatic cells from the human fetal testis. In the current study, populations of human somatic cells were dissociated and maintained in vitro and characterised. Analysis demonstrated that cells expressing mRNAs characteristic of Sertoli cells, Leydig cells and peritubular myoid (PTM) cells were present initially, but long-term culture resulted in downregulation in expression of mRNAs specific for Sertoli cells and Leydig cells, suggesting that these cells either failed to survive or underwent alterations to their phenotype. In contrast PTM/fibroblast cells proliferated in vitro and initially maintained androgen receptor expression. These cultures therefore hold promise for studies into the signalling or cell-cell interactions in testicular somatic cells especially those relevant to the PTM population. Several studies have claimed differentiation of putative germ cells from ES cells. In the current study, analysis of mouse ES cell lines has expanded on results showing that ES cells and early germ cells express a number of genes in common. Kit signalling was shown to be important for ES cell survival as they differentiate although expression of Kit was heterogeneous. We also demonstrated that ES cells that did not express Kit displayed a decreased expression of the early germ cell genes Blimp1, Fragilis and Stella, implicating Kit signalling in the control of germ cell-associated gene expression in ES cells. This may be important to future studies optimising germ cell derivation from ES cells. In conclusion, this study has demonstrated important differences in protein expression patterns in germ cells of the human fetal testis compared to the mouse, and has raised questions about whether the proposed mechanism controlling meiotic entry of germ cells in the mouse can be applied to the human. The establishment of a system for culturing human fetal gonadal somatic cells may lead to further understanding of gene expression and development in the human fetal testis, and data suggest that the Kit/Kitl signalling system may influence germ cell gene expression in mouse ES cells.
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Yang, Liu, and 楊柳. "Genetic analyses of terminal differentiation of hypertrophic chondrocytes." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hdl.handle.net/10722/210320.
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Yang, Liu. "Genetic analyses of terminal differentiation of hypertrophic chondrocytes." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43223758.
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Nsiah, Barbara Akua. "Fluid shear stress modulation of embryonic stem cell differentiation." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/47552.
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Vascularization of tissue-engineered substitutes is imperative for successful
implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.
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Lemos, Sara Sofia de Campos Pereira. "CD8 T cell differentiation during immune responses." Thesis, Paris 5, 2014. http://www.theses.fr/2014PA05T009/document.
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Les lymphocytes T CD8 ont un rôle essentiel dans la protection contre les agents pathogènes intracellulaires et la progression tumorale. Ainsi, la compréhension de la diversité des mécanismes de différenciation des lymphocytes T CD8 naïfs en cellules effectrices, ainsi qu’en cellules mémoires compétentes, est fondamentale pour le développement efficace de vaccins à cellules T. Dans ce travail de thèse, nous avons abordé deux questions centrales : (1)Très tôt après l’activation des cellules T CD8, quels sont les mécanismes par lesquels les cellules T effectrices agissent comme effecteurs pro-inflammatoires en recrutant d’autres cellules? Et quel est leur rôle dans la réponse immunitaire? (2) Quel est le rôle du contexte infectieux dans le programme de différenciation des lymphocytes T CD8 ? Est-il responsable de l’hétérogénéité des cellules répondeuses et a-t-il un rôle dans les différents effets protecteurs des cellules mémoires? Afin de répondre à ces questions, nous avons choisit d’utiliser des cellules T CD8 exprimant un récepteur pour l’antigène transgéniques (TCR-Tg) pour suivre la différentiation in vivo des lymphocytes T CD8. De plus, la méthode de RT-PCR sur des séries de cellules uniques, nous a permis d’analyser la co-expression des ARNm dans ces cellules. Comme l’utilisation à haute fréquence de cellules TCR-Tg a été fortement critiquée, nous avons comparé la différenciation de ces cellules avec celle des cellules endogènes (non transgéniques et rares). Dans ce premier manuscrit nous avons observé un comportement similaire, ce qui a renforcé l'avantage d'utiliser des cellules TCR Tg pour étudier les réponses immunitaires des lymphocytes T CD8. De plus, nous avons conclu que la diversité des réponses immunitaires des lymphocytes T CD8 n’est pas conditionnée par la fréquence de cellules naïves. Dans un deuxième manuscrit, nous avons comparé la réponse des cellules OT1 TCR-Tg (spécifiques de l’antigène OVA) à l'infection bactérienne LM-OVA (Listeria Monocytogènes exprimant OVA) avec la réponse des cellules P14 TCR-Tg (spécifiques de l’épitope GP33) à l’infection par le virus LCMV. Nous avons montré que les cellules OT1, stimulées par l’OVA dans un contexte bactérien (LM-OVA), présentent un profil d’expression génique distinct de celui des cellules P14 stimulées par le GP33 dans un contexte viral (LCMV). Nous avons également co-stimulé les cellules P14 et OT1 dans une même souris suivant le même contexte bactérien avec LM-GP33 et LM-OVA. Dans ce cas, nous n’avons pas observé de différence dans le profil d’expression génique. L’ensemble des résultats démontrent que les stimulations spécifiques des cellules T CD8 par différents agents pathogènes génèrent des cellules T CD8 présentant des caractéristiques différentes qui ne sont pas déterminées par la spécificité du TCR mais plutôt par le contexte infectieux. De plus, nous avons montré que les cellules mémoires endogènes résultant de la stimulation des CD8 en présence de LCMV ont été plus efficaces après une deuxième réponse immunitaire que des cellules mémoires générées après stimulation avec LM-GP33 (bactérie). Nous avons également observé que la protection plus efficace dans le contexte viral est associée à des cellules T CD8 qui présentent un phénotype de cellules T mémoires effectrices (TEM) tandis que les cellules T CD8 générées dans un contexte bactérien ont plutôt un phénotype associé aux cellules T mémoires centrales (TCM). Ces résultats démontrent que différents pathogènes induisent différents profils de différentiation des cellules T CD8 et que malgré l’élimination efficace des différents pathogènes dans une réponse primaire, la qualité des cellules mémoires générées au cours de cette réponse peut être différente. Dans un troisième manuscrit, nous avons étudié les mécanismes de recrutement d’autres cellules par les lymphocytes T CD8 activés à un temps précoce de la réponse immunitaire. (...)<br>CD8 T cells are essential for the elimination of intracellular pathogens and tumor cells. Understanding how naïve CD8 T cells differentiate into effector cells capable of eliminating pathogens and to generate adequate memory cells during immune responses is fundamental for optimal T cell vaccine design. In this PhD thesis work we addressed two central questions: 1) What are the mechanisms by which early effector T cells could act as pro-inflammatory effectors? And what is their role in the immune response? 2) How heterogeneous are CD8 responses? Could different pathogens modulate CD8 T cell differentiation programs and be responsible for CD8 cell-to-cell heterogeneity? Could they also generate memory cells with different protection capacities? To address these questions related to the diversity of CD8 T cell differentiation during immune responses, we used the single cell RT-PCR technique to detect ex vivo expression of mRNA in each individual cell, and Brefeldin A injected mice to detect ex vivo intracellular proteins. As experimental system to evaluate in vivo cell activation we used T cell receptor transgenic (TCR-Tg) CD8 T cells. Since the use of TCR-Tg cells to study immune responses has been subjected to criticism (due to high frequency of naïve-precursor transfers), in a first Ms. we compared the behavior of TCR-Tg and endogenous (non-transgenic and present at low frequency) cells in the same mouse. We found fully overlapping behavior between these two cell populations, which reinforced the advantage of using TCR-Tg cells to study CD8 immune responses. In addition, we concluded that the frequency of naïve-precursors do not induce diversity on CD8 T cell differentiation patterns. In a second Ms. we evaluated the impact of different pathogens in the diversity of CD8 T cell properties during two different immune responses: OT1 TCR-Tg cells (specific for OVA antigen) in the response to LM-OVA (Listeria Monocytogenes expressing OVA) infection; and P14 TCR-Tg cells (specific for GP33 epitope) in the response to Lymphocytic choriomeningitis vírus (LCMV) infection. We found that OT1 and P14 cells had different properties. As this difference could also be attributed to the different TCR avidity between OT1 and P14 cells, we then compared the behavior of P14 and OT-1 cells in the same mouse, co-injected with LM-OVA and LM-GP33. Since no differences were then detected, these results demonstrated that priming with different pathogens generates CD8 T cells with different characteristics that are not determined by TCR usage, but rather by the infection context. In addition, when looking for the protection capacity of endogenous CD8 memory cells generated in bacterial or viral context, we found that memory cells generated after LCMV priming were more efficient in responding to a second challenge, than memory cells generated after LM-GP33 priming. We also found that this better protection is associated with a T cell effector memory (TEM) phenotype associated with the LCMV infection, in contrast with a T cell central memory (TCM) phenotype generated after LM-OVA infection. These results demonstrate that different pathogens are responsible for diversity of CD8 T cell differentiation patterns and that even when distinct pathogens are efficiently eliminated during the primary immune response the quality of the memory generated may differ. In a third Ms. we studied the mechanisms by which effector CD8 T cells attracted other cell types in the early days of an immune response. We used two experimental systems: the response of OT1 TCR-Tg cells to LM-OVA infection; and the response of anti-HY TCR-Tg cells to male cells (“sterile”-non infectious context). In both cases we found that immediately after activation, CD8 T cells expressed high levels of pro-inflammatory cytokines and chemokines (such as TNFα, XCL1, CCL3 and CCL4). (...)
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Syvertsson, Simon. "Bistable differentiation in an isogenic cell population." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/3002.
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Single-cell organisms such as bacteria have traditionally been regarded as discrete units, which in turn has been reflected by the bulk-level methods used to study them. A growing culture of the bacterium Bacillus subtilis will exhibit a range of heterogeneous genetic developmental programmes such as motility, competence, and finally sporulation. As a popular choice for production of compounds in bioreactors, the bistable behaviours of B. subtilis may be undesirable traits, as they divert resources from their intended activity of synthesising a product. This thesis investigates a novel observation that expression of a ribosomal subunit gene (rpsD) is elevated in the non-motile state of B. subtilis, using unstable GFP reporter constructs. The implications of using a proteolytically unstable protein as a reporter are also investigated with regard to the effect of protein degradation rates on the reporter construct, as well as presenting evidence for modulation of ClpXP activity in a pnpA background. Investigation of the motile/non-motile heterogeneous phenotype of B. subtilis posed a challenge for automated analysis pipelines. This thesis addresses this problem by developing and testing microscopy analysis pipelines designed to circumvent the traditional requirement for physically separated objects in a phase contrast channel, and instead using nucleoid or membrane stains to identify cells in a microscopy image. Other factors impacting the activity of a proteolytically unstable PrpsD reporter construct were investigated, including the rate of degradation of the reporter, and integration locus of the reporter construct. To assess the impact of locus positioning, a genetic tool was also created to survey changes in noise and overall expression levels from two homogeneously expressed promoters across different positions on the chromosome.
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Rijal, Dikchha. "Cell Death Signaling Complexes During Macrophage Differentiation." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36455.
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Monocytes migrate to various tissues and differentiate to macrophages and mediate early control of pathogens. Various alternative pathways of cell death have been discovered which have been shown to play a key role in host survival. Herein, we investigated the impact of differentiation of monocytes to macrophages on their susceptibility to two distinct cell death inducing complexes, ripoptosome and necrosome. Our results indicate that differentiation of macrophages results in resistance to ripoptosome- but not necrosome- induced cell death. Additional experiments indicated that the resistance to ripoptosome signaling correlated with reduced caspase activation and enhanced expression of anti-apoptotic mediators XIAP and cFLIPL. Our results also reveal the contradictory roles of p38 MAPK/MK2 in stimulating (phosphorylating RipK1) or inhibiting (reducing TNF-α expression and caspase 8 activation) ripoptosome signaling. These findings reveal novel mechanistic insights that can be exploited for development of therapeutics.
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Gore, S. "Neuronal differentiation markers in basal cell carcinoma." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1445574/.
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Basal cell carcinoma (BCC) is the most common skin cancer in humans. The demonstration of genetic and protein alterations has, so far, had limited correlation with either biological behaviour or histological classification of these tumours. It was observed that Glil-overexpressing keratinocytes express elevated levels of genes known to be associated with neuronal development, including p-tubulin III, GAP-43, Arc and neurofilament. It was proposed that these genes may similarly be overexpressed in BCCs and that different levels of expression may be present in different BCC subtypes Immunohistochemistry of BCCs demonstrated that neuronal differentiation marker proteins are expressed in BCCs, but that this expression is significantly reduced in tumours that behave aggressively. Elevated neuronal differentiation marker gene expression was shown in BCCs. Again, expression was more prominent in tumour types that behave indolently. Results were obtained for tumour samples processed by laser capture microdissection, needle microdissection and homogenised tissue. Expression of neuronal differentiation marker genes in Gli-overexpressing keratinocytes was examined by semi-quantitiative PCR. Neuronal differentiation marker expression was associated with GUI and GH2 over-expression in some cases {P-tubulin III and Arc). GUI and GH2 also promoted the expression of each other in a positive-feedback loop. Expression of these markers was examined in archival tumours for which the clinical outcome was known in terms of recurrence. In completely excised tumours P-tubulin III was significantly reduced in tumours that went on to subsequently recur. Other markers were not expressed in significantly different amounts. In summary, I have shown that expression of markers associated with neuronal development is a feature of Basal Cell carcinoma, and that the expression of these markers correlates strongly with the tumour histological subtype but only weakly with tumour recurrence. More work will be required to discover further alterations in BCC molecular biology that impact significantly on tumour behaviour.
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Wurff, A. A. M. van der. "Cell differentiation and adhesion in colorectal cancer." [Maastricht : Maastricht : Universiteit Maastricht] ; University Library, Maastricht University [Host], 1998. http://arno.unimaas.nl/show.cgi?fid=8253.
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Henderson, Andrew Paul. "Small molecules for controlling stem cell differentiation." Thesis, Durham University, 2011. http://etheses.dur.ac.uk/3559/.
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Stem cell homeostasis and differentiation are controlled by the complex interplay of a wide range of signalling pathways and small molecules, including all-trans-retinoic acid (ATRA). The endogenous effects elicited by ATRA, have led to its use in numerous in vitro protocols as a tool for cell differentiation. However, ATRA isomerises and degrades under standard laboratory conditions and furthermore, is rapidly metabolised in vivo, which leads to pleotropic effects and a high efficacious dose response. Consequently, synthetic analogues that are structurally and/or functionally equivalent to ATRA have been developed, as alternative pharmacological tools to further the understanding of this molecular pathway and control cell differentiation.In this study a small library of synthetic retinoids were prepared, which were designed to probe structural size, conformation and biological function, while being more resistant to cellular metabolism and isomerisation. Their stability towards fluorescent light was examined along with their activity in four different stem cell models. Two compounds, AH60 and AH61 were found to inhibit cellular proliferation and induce neural differentiation, through acting on the retinoic acid receptor pathway. Compared to ATRA, AH60 was approximately 10-fold more active, while AH61 was 100-fold more active in two of the cell models tested. These compounds are described comprehensively herein, and should be suitable and convenient alternatives to ATRA and 13cRA for use in in vitro studies carried out by cell and molecular biologists. In addition, an unrelated small molecule, neuropathiazol, has been synthesised to further characterise both the chemistry involved in its production and its biological activity in controlling cell differentiation. This compound was highlighted in the literature as an alternative to ATRA, for inducing neural differentiation in neural progenitor cells. We have further investigated its potential to differentiate other neural stem cell types and pluripotent stem cells. In addition potential analogues of neuropathiazol are discussed, as compounds of this nature are potentially highly useful for selectively controlling neural differentiation.
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De, Campos Pereira Lemos Sara Sofia. "CD8 T cell differentiation during immune responses." Phd thesis, Université René Descartes - Paris V, 2014. http://tel.archives-ouvertes.fr/tel-01059806.
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CD8 T cells are essential for the elimination of intracellular pathogens and tumor cells. Understanding how naïve CD8 T cells differentiate into effector cells capable of eliminating pathogens and to generate adequate memory cells during immune responses is fundamental for optimal T cell vaccine design. In this PhD thesis work we addressed two central questions: 1) What are the mechanisms by which early effector T cells could act as pro-inflammatory effectors? And what is their role in the immune response? 2) How heterogeneous are CD8 responses? Could different pathogens modulate CD8 T cell differentiation programs and be responsible for CD8 cell-to-cell heterogeneity? Could they also generate memory cells with different protection capacities? To address these questions related to the diversity of CD8 T cell differentiation during immune responses, we used the single cell RT-PCR technique to detect ex vivo expression of mRNA in each individual cell, and Brefeldin A injected mice to detect ex vivo intracellular proteins. As experimental system to evaluate in vivo cell activation we used T cell receptor transgenic (TCR-Tg) CD8 T cells. Since the use of TCR-Tg cells to study immune responses has been subjected to criticism (due to high frequency of naïve-precursor transfers), in a first Ms. we compared the behavior of TCR-Tg and endogenous (non-transgenic and present at low frequency) cells in the same mouse. We found fully overlapping behavior between these two cell populations, which reinforced the advantage of using TCR-Tg cells to study CD8 immune responses. In addition, we concluded that the frequency of naïve-precursors do not induce diversity on CD8 T cell differentiation patterns. In a second Ms. we evaluated the impact of different pathogens in the diversity of CD8 T cell properties during two different immune responses: OT1 TCR-Tg cells (specific for OVA antigen) in the response to LM-OVA (Listeria Monocytogenes expressing OVA) infection; and P14 TCR-Tg cells (specific for GP33 epitope) in the response to Lymphocytic choriomeningitis vírus (LCMV) infection. We found that OT1 and P14 cells had different properties. As this difference could also be attributed to the different TCR avidity between OT1 and P14 cells, we then compared the behavior of P14 and OT-1 cells in the same mouse, co-injected with LM-OVA and LM-GP33. Since no differences were then detected, these results demonstrated that priming with different pathogens generates CD8 T cells with different characteristics that are not determined by TCR usage, but rather by the infection context. In addition, when looking for the protection capacity of endogenous CD8 memory cells generated in bacterial or viral context, we found that memory cells generated after LCMV priming were more efficient in responding to a second challenge, than memory cells generated after LM-GP33 priming. We also found that this better protection is associated with a T cell effector memory (TEM) phenotype associated with the LCMV infection, in contrast with a T cell central memory (TCM) phenotype generated after LM-OVA infection. These results demonstrate that different pathogens are responsible for diversity of CD8 T cell differentiation patterns and that even when distinct pathogens are efficiently eliminated during the primary immune response the quality of the memory generated may differ. In a third Ms. we studied the mechanisms by which effector CD8 T cells attracted other cell types in the early days of an immune response. We used two experimental systems: the response of OT1 TCR-Tg cells to LM-OVA infection; and the response of anti-HY TCR-Tg cells to male cells ("sterile"-non infectious context). In both cases we found that immediately after activation, CD8 T cells expressed high levels of pro-inflammatory cytokines and chemokines (such as TNFα, XCL1, CCL3 and CCL4). (...)
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Cardoso, Sandra Pinto. "Control of cell differentiation during secondary myogenesis." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250587.
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