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1
Einarsson, Elin. "Comparative Cell Biology in Diplomonads." Doctoral thesis, Uppsala universitet, Institutionen för cell- och molekylärbiologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-264541.
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The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections with high mortality. We created a transfection system for S. salmonicida to study cellular functions and virulence in detail (Paper I). The system was applied to explore the mitochondrion-related organelle (MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome. Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular structures, including a putative adhesion structure anterior in the cell. The disease-causing Giardia trophozoites differentiate into infectious cysts, a process essential for transmission and virulence of the parasite. Cysts are often spread via contaminated water and exposed to environmental stressors, such as UV irradiation. We studied the survival and transcriptional response to this stress factor (Paper IV) and results showed the importance of active DNA replication machinery for parasite survival after DNA damage. In addition, we studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as putative regulators. Large transcriptional changes appeared late in the process with the majority of differentially regulated genes encoding hypothetical proteins. We studied the localizations of several of these to gain information of their possible function. To conclude, the diplomonads are complex eukaryotic microbes with cellular processes adjusted to match their life styles. The work in this thesis has provided insight of their adaptations, differences and similarities, but also new interesting leads for future studies of diplomonad biology and virulence.
2
Lewis, Beverley Anne. "Cell biology of rat spermatozoa." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/23087.
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The purpose of the research presented in this thesis was to investigate the cell biology of rat spermatozoa. An additional aim was to utilise the knowledge obtained to aid the development of in vitro functional tests for the assessment of rat sperm fertility and identify potential markers of normal epididymal maturation. As mammalian spermatozoa migrate through the epididymis, they acquire the potential for fertilisation, characterised by the acquisition of the ability to express co-ordinated movement and the competence to undergo capacitance. The mechanisms by which epididymal maturation confers upon mammalian spermatozoa the potential to capacitate is poorly understood. These studies investigated the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation using the laboratory rat as an animal model, since this signal transduction pathway is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility, both of which are prerequisites for fertilisation. Western Blot and immunocytochemical analysis demonstrated that epididymal maturation is associated with a progressive loss in phosphotyrosine expression located to the acrosomal domain. These differences in phosphotyrosine expression between caput and caudal epididymal spermatozoa appeared to reflect the normal in vivo situation. In addition, epididymal maturation of rat spermatozoa is also associated with an acquired competence to respond to high levels of intracellular cAMP by phoshorylating tyrosine residues on the sperm tail. Epididymal maturation also led to unique differences in the generation of reactive oxygen species (ROS) by spermatozoa obtained from the caput and caudal regions of the epididymis. Spermatozoa from both regions of the epididymis spontaneously generated equal levels of O2 whereas only mature caudal spermatozoa generated significant levels of H2O2. In contrast, although both caput and caudal spermatozoa generated increased O2-. in response to NADPH, induced levels were significantly greater in the immature caput cells.
3
Yu, Yang. "Lipidomics Investigations in Cell Biology." Doctoral thesis, Università degli studi di Trento, 2014. https://hdl.handle.net/11572/368594.
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Cell membrane is the biological barrier serving as both territorial defense and the communication hinge for the interior of cell from its surroundings. As building blocks of cellular membranes and also precursor for second messengers, a variety of lipids play essential roles in cellular membrane dynamics as well as important functions such as cell proliferation, apoptosis, signal transduction and membrane trafficking modulation. Lipidomics, representing the systematic and integrative studies of diversified lipids (lipidome) in a biological system, is an emerging yet rapid developing field and hence requires advanced and complementary analytical techniques as well as multiple statistical tools. Our development of reliable analytical methodology (the advanced Mass Spectrometric and high-resolution NMR techniques) and application of multiple statitistical approaches (multivariate data analysis and univariate t-test) enable us to achieve these comprehensive understandings.
We have investigated, first of all, the effects induced by hypoxia on cervical cancer derived cells (HeLa cells) to see how and how much the changes in phospholipids profile are able to get light into the targeted biological problem (hypoxia) and provide a preliminary insight into the underlying mechanisms. We found that hypoxia stimulation dramatically reduced the total amount of cellular phosphoinositols (PI) but prominently increased the amount of lyso phosphocholines (lyso-PC) and lyso phosphoethanolamines (lyso-PE). Moreover, our studies suggested the polyunsaturated phospholipids species as stronger biomarkers upon hypoxia treatment. The evaluation of changes in the average unsaturation index (UI) of the membrane lipids acyl chains revealed that UI slightly increased in several lipid classes, thus affecting membrane fluidity and further membrane-dependent functions. The plausible mechanisms by HeLa cells to adapt to hypoxia conditions are briefly reported as well.
We have also conducted the comparative lipidomic studies of urothelial cancer cell line RT4 (a model system of a benign tumor) and T24 (a model system of a metastatic tumor) aiming to reveal probable roles and relevant differential changes of membrane lipids with respect to urinary bladder metastasis progress. Significant changes of lipids metabolism were found to correlate with urothelial nonmetastatic and metastatic cell models. The most remarkable finding was that the malignant cell type (T24) showed a strong decrease of ether PC species complemented by a sharp increase of the length and the average unsaturation number of lipids acyl chains. Ceramide-based sphinglipids also showed altered profiles in these two cell types. Such analyses suggest a certain significant re-organization of cellular membrane in malignant cell transformation, involving variations in compositional lipid structures and possible signaling transduction pathways. Observations of such reduction of the 1-alkyl PC species and the chain shortening of lipid species might serve as a tool in urinary bladder cancer intervention.
4
Yu, Yang. "Lipidomics Investigations in Cell Biology." Doctoral thesis, University of Trento, 2014. http://eprints-phd.biblio.unitn.it/1294/1/Yang_Yu__Lipidomics_Investigations_in_Cell_Biology.pdf.
Full textAbstract:
Cell membrane is the biological barrier serving as both territorial defense and the communication hinge for the interior of cell from its surroundings. As building blocks of cellular membranes and also precursor for second messengers, a variety of lipids play essential roles in cellular membrane dynamics as well as important functions such as cell proliferation, apoptosis, signal transduction and membrane trafficking modulation. Lipidomics, representing the systematic and integrative studies of diversified lipids (lipidome) in a biological system, is an emerging yet rapid developing field and hence requires advanced and complementary analytical techniques as well as multiple statistical tools. Our development of reliable analytical methodology (the advanced Mass Spectrometric and high-resolution NMR techniques) and application of multiple statitistical approaches (multivariate data analysis and univariate t-test) enable us to achieve these comprehensive understandings.
We have investigated, first of all, the effects induced by hypoxia on cervical cancer derived cells (HeLa cells) to see how and how much the changes in phospholipids profile are able to get light into the targeted biological problem (hypoxia) and provide a preliminary insight into the underlying mechanisms. We found that hypoxia stimulation dramatically reduced the total amount of cellular phosphoinositols (PI) but prominently increased the amount of lyso phosphocholines (lyso-PC) and lyso phosphoethanolamines (lyso-PE). Moreover, our studies suggested the polyunsaturated phospholipids species as stronger biomarkers upon hypoxia treatment. The evaluation of changes in the average unsaturation index (UI) of the membrane lipids acyl chains revealed that UI slightly increased in several lipid classes, thus affecting membrane fluidity and further membrane-dependent functions. The plausible mechanisms by HeLa cells to adapt to hypoxia conditions are briefly reported as well.
We have also conducted the comparative lipidomic studies of urothelial cancer cell line RT4 (a model system of a benign tumor) and T24 (a model system of a metastatic tumor) aiming to reveal probable roles and relevant differential changes of membrane lipids with respect to urinary bladder metastasis progress. Significant changes of lipids metabolism were found to correlate with urothelial nonmetastatic and metastatic cell models. The most remarkable finding was that the malignant cell type (T24) showed a strong decrease of ether PC species complemented by a sharp increase of the length and the average unsaturation number of lipids acyl chains. Ceramide-based sphinglipids also showed altered profiles in these two cell types. Such analyses suggest a certain significant re-organization of cellular membrane in malignant cell transformation, involving variations in compositional lipid structures and possible signaling transduction pathways. Observations of such reduction of the 1-alkyl PC species and the chain shortening of lipid species might serve as a tool in urinary bladder cancer intervention.
5
Bair, Elisabeth Laurine. "Cell-cell and cell-matrix interactions involved in cancer invasion." Diss., The University of Arizona, 2004. http://hdl.handle.net/10150/280673.
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In order for a cancer to metastasize, it must first invade through the basement membrane that surrounds it, invade blood vessels and travel through the bloodstream to a new location where it extravasates the vessel and begins growing at the new site. The mechanisms by which a cancer becomes able to invade and metastasize are currently under intense study. Interactions of the cell with its environment via cell-cell contacts, extracellular matrix (ECM) interactions, and circulating proteins are thought to play a major role in signaling for these invasive processes to occur. Upregulation of proteolytic enzymes, such as the matrix metalloproteases, is suspected of being involved in the metastatic process. Cell-cell and cell-matrix contacts via integrins and cadherins are necessary for upregulation of the matrix metalloprotease matrilysin in oral squamous cell carcinoma. In an effort to identify the factors involved in upregulation of matrilysin expression detected in a co-culture of oral squamous cell carcinoma (SCC) cells and fibroblast cells, a coculture model designed to represent the actual tumor environment, we show that inhibition of beta1 integrin, E-cadherin, and N-cadherin with blocking antibodies thoroughly decreases the induction of matrilysin in the co-culture model. This demonstrates that interactions between cancer cells and normal cells surrounding them may allow for invasion and metastasis. The protein 90K may also play a role in the invasive process of prostate cancer. It functions as an immune modulator upregulating cytokines that induce MMPs and we show that it can induce matrilysin expression in prostate cancer cells. It also functions in cell aggregation, which can help cells survive during metastasis. For this reason, expression of 90K in prostate cancer, which we examined, may be indicative of aggressive disease, making 90K a potentially useful tumor marker. Cell-matrix contacts are also important for the transmembrane matrix metalloprotease MT1-MMP cleavage of laminin-10. We demonstrate that recombinant MT1-MMP is able to cleave human laminin-10 into four distinct products. This allows for prostate cancer cell migration on laminin-10 coated substrates, which can be inhibited with the addition of MT1-MMP antisense oligonucleotides. Ln-10 cleavage also occurs in vivo in human prostate tissue, indicating that this cell-matrix interaction has in vivo relevance in human prostate cancer.
6
Zhang, Yuan. "Cell-cell interactions and Gossypol's effects on cell functions of primary cultured Human Breast Epthelial, Stromal and Adipose Stromal cells /." The Ohio State University, 1999. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488187763845791.
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Agüera-González, Sonia. "Cell biology on NKG2D ligands and NK cell recognition." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609348.
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Camacho, Diogo Mayo. "In silico cell biology and biochemistry: a systems biology approach." Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/27960.
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In the post-"omic" era the analysis of high-throughput data is regarded as one of the major challenges faced by researchers. One focus of this data analysis is uncovering biological network topologies and dynamics. It is believed that this kind of research will allow the development of new mathematical models of biological systems as well as aid in the improvement of already existing ones. The work that is presented in this dissertation addresses the problem of the analysis of highly complex data sets with the aim of developing a methodology that will enable the reconstruction of a biological network from time series data through an iterative process.
The first part of this dissertation relates to the analysis of existing methodologies that aim at inferring network structures from experimental data. This spans the use of statistical tools such as correlations analysis (presented in Chapter 2) to more complex mathematical frameworks (presented in Chapter 3). A novel methodology that focuses on the inference of biological networks from time series data by least squares fitting will then be introduced. Using a set of carefully designed inference rules one can gain important information about the system which can aid in the inference process. The application of the method to a data set from the response of the yeast Saccharomyces cerevisiae to cumene hydroperoxide is explored in Chapter 5. The results show that this method can be used to generate a coarse-level mathematical model of the biological system at hand. Possible developments of this method are discussed in Chapter 6.Ph. D.
9
Buffa, Laura. "Cell Biology of the ICA69 protein family in Neurosecretory cells." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2007. http://nbn-resolving.de/urn:nbn:de:swb:14-1174057636463-96361.
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In type 1 diabetes (T1D), an autoimmune disease, autoantibodies are preferentially directed against proteins associated with Golgi and post-Golgi secretory vesicles, including insulin secretory granules and synaptic-like microvesicles. Thus, the study of beta-cell autoantigens with yet unknown function may provide novel insight into the secretory machinery of beta-cells and led to the discovery of novel pathways. Islet cell autoantigen of 69 kDa (ICA69) is a T1D autoantigen. It is a cytosolic protein of still unknown function. An impairment in neurotransmitter release upon mutation of its homologue in C. elegans suggests, however, an involvement of ICA69 in neurosecretion. Interestingly, ICA69 contains a BAR domain, present in several proteins involved in intracellular transport. The BAR domain functions as a dimerization motif, provides a general binding interface for different types of GTPases, and is a membrane binding/bending module. Its presence in ICA69 is a further hint supporting the putative involvement of ICA69 in intracellular membrane trafficking. The first part of this thesis was concerned with the characterization of ICA69, and the elucidation of its role in membrane traffic in pancreatic beta-cells. ICA69 was shown to be enriched in the perinuclear region, where also markers of the Golgi region are found. ICA69 was shown to interact with several membrane lipids, preferentially with PI(4)P, enriched on the Golgi complex. During the course of this thesis a combination of biochemical and imaging techniques were applied to investigate the interaction between ICA69 and Rab2, a small GTPase associated with the intermediate compartment and involved in the trafficking between the ER and the Golgi complex. ICA69 was shown to co-immunoprecipitate with Rab2 from INS-1 cells extracts. GST-pull down assays demonstrated that this interaction is GTP-dependent. Furthermore, confocal microscopy indicated that ICA69 and Rab2 extensively colocalize in particulate structures throughout the cytoplasm. Immunocytochemistry and subcellular fractionation experiments suggested that Rab2 recruits ICA69 to membranes. Functional studies indicated that ICA69 over-expression in INS-1 cells has effects that resemble, and in some cases amplify those observed upon Rab2 over-expression. Specifically, it impairs the trafficking between ER and Golgi, measured through the appearance and the conversion of the pro-form of ICA512 in the mature form of the protein. Moreover, it correlates with a redistribution of the beta-COP subunit of the coatomer, participating in the early secretory pathway, between membrane-bound compartments and the cytosol and it reduces stimulated insulin secretion. The data reported in this thesis conclusively point to ICA69 as a novel Rab2 effector, and may therefore contribute to the elucidation the yet poorly understood mechanism of action of Rab2 in the secretory pathway. The second part of the thesis was devoted to the study of an ICA69 paralogue gene, called ICA69-RP. Similarly to ICA69, ICA69-RP mRNA was shown to be primarily present in tissues such as brain and pancreatic islets, showing the expression pattern of a gene preferentially expressed in neuroendocrine cells. Unlike ICA69, however, and similar to other genes associated with the secretory machinery of beta-cells, ICA69-RP appeared to be glucose regulated, as shown by a 1.55 fold increase in mRNA levels upon stimulation of the cells with 25 mM glucose for two hours.Glucose stimulation of beta-cells prompts the activation of post-transcripional mechanisms which quickly up-regulate the expression of secretory granule genes and consequently renew granule stores. The increased expression of ICA69-RP upon glucose stimulation of cells may be part of this process. Unfortunately, all attempts to elucidate the intracellular localization of endogenous ICA69-RP failed, and it was not possible to obtain significant insights about its localization by over-expressing a fusion protein between ICA69-RP and GFP. Unlike other paralogues containing the BAR domain, such as amphiphysin 1 and 2 or Rvs167p and Rvs161p, ICA69 and ICA69-RP were shown not to form heterodimers. Furthermore, ICA69-RP did not show any interaction with Rab2 or Rab1, involved in the anterograde transport between ER and Golgi. Thus, its physiological role remains to be investigated.
10
Edwards, T. L. "The molecular cell biology of spartin." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598783.
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This thesis has focused on the protein spartin, which is mutated in a form of autosomal recessive hereditary spastic paraplegia (HSP) called Troyer syndrome. Upon commencement of this project little was known about the likely mechanism of axonopathy in spartin HSP, indeed there was considerable disagreement in the literature as to its precise subcellular localisation. However, a putative role for spartin in endocytosis was proposed due to sequence homology identified with several proteins known to function in this area. This suggested that spartin belonged to an enlarging group of HSP proteins with membrane traffic and transport-related roles. This thesis had three primary aims: to clarify the subcellular localisation of endogenous spartin, to identify proteins that interacted with spartin, and to investigate a possible functional role(s) for spartin. Spartin was found to localise predominantly in the cytoplasm, with a cystosolic pool that was recruited to endosomes. Additionally, a proportion of spartin was found in mitochondria. Furthermore, spartin was shown to undergo multiple monoubiquitination and to interact with ubiquitin, two ankyrin repeat domain-13 family members, and also with the E3 ubiquitin ligase AIP4. Functional assays suggest that spartin is an inhibitor of epidermal growth factor receptor degradation, and negatively regulates bone morphogenetic protein (BMP) signalling. A putative role in BMP signalling is interesting because this pathway is emerging as an important regulator of distal axonal morphology and function, and has been implicated in the pathogenesis of another endosomal HSP protein, NIPA1. This suggests that altered BMP signalling may be a key pathological component of spartin HSP.
11
Yaacob, Nik Soriani. "Molecular cell biology of peroxisome proliferators." Thesis, University of Surrey, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244831.
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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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Scutt, Nanette. "Investigations into the cell biology of tendon & ligament derived cells." Thesis, University of Sheffield, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505569.
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Maghzal, Nadim. "The epithelial cell adhesion molecule (EpCAM) regulates cell motility and cell-cell adhesion by inhibiting PKC signaling." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114215.
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Tissue cohesion is achieved in part by a large family of plasma membrane-bound cell adhesion molecules (CAMs). During morphogenesis, CAM-mediated interactions provide adhesive forces required for cells to aggregate and form tissues. CAM-mediated adhesions in developing cells are highly dynamic, which provides the fluidity required for cellular movements that drive morphogenesis. Xenopus laevis gastrulation is an established model to study morphogenetic movements. During this phase of development, the mesoderm moves inside the embryo through involution, and migrates along the inner surface of the ectoderm while remaining separated from this tissue. Members of the Fagotto lab have identified the Xenopus orthologue of the Epithelial Cell Adhesion Molecule (EpCAM) in a gain-of-function screen to find gene products that cause aberrant ectoderm/mesoderm tissue mixing in the gastrula. EpCAM is a well known tumor-associated antigen that is specifically expressed in epithelial tissues, where its overexpression often correlates with malignancy. The initial aim of this thesis was to understand the molecular mechanism through which EpCAM promotes ectoderm/mesoderm tissue mixing. Overexpression of EpCAM in cells at the boundary increases their "invasive" behavior via a signaling property of its cytoplasmic domain (EpTAIL) that inhibits PKC signaling to promote cell motility. The most important findings of this thesis are that 1) EpTAIL inhibits PKC activity to promote cell motility and cell-cell adhesion by acting as a PKC pseudosubstrate domain that binds the enzyme on its catalytic site, and 2) this previously unknown mode of PKC inhibition is not specific to EpCAM as other PKC pseudosubstrate-mimicking plasma membrane proteins were identified and could potentially play important roles in the regulation of PKC activity. The data presented in this thesis further our understanding of EpCAM biology and unravel a new mode of PKC regulation that is valuable as PKCs are one of the major families of cytoplasmic kinases in cells.Les mécanismes de liaison cellulaire sont établis en partie par une vaste famille de protéines d'adhésion cellulaire ou CAMs. Lors de la morphogenèse, les interactions induites par les CAMs créent des forces d'adhésion nécessaires afin que les cellules puissent s'agréger et former des tissues. Les adhésions induites par les CAMs dans les cellules en développement sont très dynamiques et offrent ainsi la fluidité nécessaire aux mouvements cellulaires qui régissent la morphogenèse. La gastrulation chez la grenouille Xenopus laevis sert de modèle d'étude des mouvements morphogéniques. Durant ce stade de développement, le mésoderme se déplace vers l'intérieur de l'embryon via un mouvement d'involution et migre le long de la paroi interne de l'ectoderme tout en maintenant une séparation des deux tissues. Des membres du laboratoire de Dr. Fagotto ont réussi à identifier un orthologue de la protéine «Epithelial Cell Adhesion Molecule (EpCAM) » chez Xenopus dans un tri de gain de fonction permettent d'identifier des protéines pouvant être à l'origine d'aberrations au niveau du maintien de la séparation de l'ectoderme et du mésoderme durant la gastrulation. EpCAM est un antigène associé aux tumeurs exprimé dans les cellules épithéliales et dont la surexpression corrèle avec des tumeurs malignes. L'objectif initial de cette thèse était de découvrir les mécanismes moléculaires pouvant expliquer l'effet de EpCAM sur les aberrations entre la séparation des tissues de l'ectoderme et du mésoderme. Une surexpression de EpCAM dans les cellules à la bordure de l'ectoderme et du mésoderme cause une augmentation du comportement « invasif » entre les deux tissues, via la fonction de transduction du signal de son domaine cytoplasmique (EpTAIL), qui inhibe le signal de la protéine PKC afin de promouvoir le mouvement cellulaire. Les principales contributions de cette thèse ont été 1) EpTAIL inhibe l'activité de PKC en jouant le rôle d'un pseudosubstrat de PKC en interagissant avec le site catalytique de l'enzyme, et 2) ce mécanisme d'inhibition jusqu'à présent inconnu pour PKC n'est pas seulement spécifique à EpCAM, car d'autres protéines membranaires possède également cette capacité à imiter le pseudosubstrat de PKC et pourraient potentiellement avoir un rôle important à jouer au niveau de la régulation de l'activité de PKC. Les donnée présentées dans cette thèse contribuent à approfondir davantage notre connaissance d'EpCAM et dévoilent un nouveau mécanisme de régulation de PKC qui pourrait être important puisque les molécules PKC forment l'une des plus importantes familles de kinases cytoplasmiques dans les cellules.
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Lodato, Michael A. (Michael Anthony). "Sox2 co-occupies distal enhancer elements with cell-type-specific POU factors to specify cell identity in embryonic stem cells and neural precursor cells." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/72631.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, June 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
"June 2012." Cataloged from student submitted PDF version of thesis.
Includes bibliographical references.
Sox2 is a master regulator of two distinct cellular states, that of pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs), but what common or distinct roles Sox2 may play in these cell types not fully understood. Further, the molecular mechanisms by which Sox2 can specify two distinct cell identities are as of yet unclear. This thesis is aimed at answering these fundamental questions. In ESCs, Sox2 was associated with a subset of poised regulators of nervous system development, and upon differentiation into NPCs Sox2 selectively activates those which are important for progenitor cell state, while keeping others poised to become activated in later neural development. These data suggested that Sox2 might act as a pioneer factor for neural development throughout embryogenesis. While Sox2 is known to co-occupy target loci in ESCs with the POU factor Oct4, in NPCs Sox2 interacts with the central-nervous-system-expressed POU factors Brn1 and Brn2. By utilizing distinct composite Sox:Octamer motifs in each cell type, Sox2:POU modules control the expression of thousands of genes involved in the development of the neural lineage in a cell-type-specific manner. These data advance our understanding of the mechanism by which transcription factors control cell fate transitions, and indicate that combinatorial interactions between transcription factors may be a pervasive mechanism of transcriptional control in development
by Michael A. Lodato.
Ph.D.
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Bergström, Tobias. "Modeling Neural Stem Cell and Glioma Biology." Doctoral thesis, Uppsala universitet, Cancer och vaskulärbiologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-204949.
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This thesis is focused on neural stem cell (NSC) and glioma biology. I discuss how NSCs interact with extracellular matrix (ECM) proteins in the stem cell niche, and investigate the consequences of deregulated Platelet-derived growth factor (PDGF) signaling for embryonic NSCs in transgenic mice. Furthermore I present cell cultures of human glioblastoma multiforme (GBM) that models human disease, taking into account the heterogeneity of GBM. Finally, interactions between brain tumors and mast cells are studied using the glioma cultures. In paper I, the importance of NSC interactions with the ECM in the stem cell niche during development is discussed. Contacts between NSCs and the ECM in the subventricular zone (SVZ) are emerging as important regulatory mechanisms. We show that early postnatal neural stem and progenitor cells (NSPC) attach to collagen I, and that the adhesion is explained by higher expression of collagen receptor integrins compared to adult NSPC. Further, blood vessels in the SVZ express collagen I, indicating a possible functional relationship. Growth factors, e.g. PDGF, regulate NSC proliferation and differentiation. Aberrant activation of growth factor signaling pathways also plays a role in brain tumor formation. Paper II demonstrates that transgenic mice expressing PDGF-B at high levels in embryonic NSCs displayed mild neurological defects but no hyperplasia or brain tumors. This suggests that a high level of PDGF is not sufficient to induce brain tumors from NSCs without further mutations. Paper III presents a novel panel of human glioma stem cell (GSC) lines from GBM that display NSC markers in vitro and form secondary orthotopic tumors in vivo. GBM has recently been categorized in molecular subclasses and we demonstrate, for the first time, that these subclasses can be retained in vitro by stem cell culture conditions. We have thus generated models for research and drug development aiming at a focused treatment depending on GBM subtype. Interactions with the immune system are integral parts of tumorigenesis. Mast cells are found in glioma and in paper IV we demonstrate that the grade-dependent infiltration of mast cells is in part mediated by macrophage migration inhibitory factor and phosphorylation of STAT5.
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鍾穗華 and Shui-wah Chung. "Cell-cell interactions in the rat testis: biology and future perspectives." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1999. http://hub.hku.hk/bib/B31238385.
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Chung, Shui-wah. "Cell-cell interactions in the rat testis : biology and future perspectives /." Hong Kong : University of Hong Kong, 1999. http://sunzi.lib.hku.hk/hkuto/record.jsp?B2056692X.
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San, Roman Adrianna Katrina. "Mechanisms of Stem Cell Maintenance and Cell Differentiation in the Intestinal Epithelium." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226057.
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Constant regeneration of the intestinal epithelium, a dynamic tissue with vital digestive and barrier functions, depends on proliferation of resident stem cells and their differentiation into mature cell types. This epithelium thus provides an ideal model to study stem cells and mechanisms of cell differentiation in an adult tissue.
The identification of a proliferative population of intestinal stem cells (ISCs) at the base of intestinal crypts presents the prospect of understanding their regulation by extrinsic and intrinsic factors. Although activation of Wnt signaling in ISCs is thought to be one crucial function of the ISC niche, the cellular source of Wnt ligands is uncertain. Chapter 2 addresses this question through genetic elimination of Wnt ligand secretion in candidate niche cell populations. The data reveal that Wnts originating in any of the sources considered in the literature – the epithelium (including Paneth cells) and sub-epithelial myofibroblasts – are not required for ISC function. These data support models of highly complex cell redundancy or alternative, non-Wnt ligands. Chapter 3 investigates the cell-intrinsic contributions of an intestine-restricted transcription factor (TF), CDX2, to important ISC behaviors. Cdx2 loss in vivo perturbs ISC proliferation and differentiation, distinct from its functions in mature enterocytes. Analysis of candidate direct CDX2 target genes in ISCs suggests that CDX2 modulates Fibroblast Growth Factor signaling, thus opening new avenues of investigation.
Although cells that differentiate from ISCs rely on gene expression changes mediated by TFs, loss of several individual TFs in vivo has modest effects on intestinal function. Chapter 4 characterizes the functional interactions of CDX2, which has many properties of a master regulator, with two other intestinal TFs: GATA4 and Hepatocyte Nuclear Factor 4 alpha (HNF4A). Analysis of compound mutant mouse intestines elucidated combinatorial roles for CDX2 with GATA4 in crypt cell proliferation and with HNF4A in enterocyte differentiation. Building on this foundation, Chapter 5 describes preliminary investigations into another TF, HNF1A, in intestinal gene regulation and its relationship with CDX2 in controlling cell differentiation and tissue architecture. These studies highlight the complexities of TF interactions and the functions of diverse TF complexes in controlling tissue-specific genes during cell differentiation.
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Faddah, Dina Adel. "Single-cell analyses of cellular reprogramming and embryonic stem cells." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/89941.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.Vita. Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Three years before the start of this thesis, Yamanaka and Takahashi published a groundbreaking paper entitled "Induced of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors." A mere two scientists reprogrammed somatic cells to an embryonic stem-cell like state (termed induced pluripotent stem cells, iPSCs) by simply overexpressing four transcription factors: Oct4, Sox2, c-Myc, and Klf4. During cellular reprogramming, only a small fraction of cells become iPSCs. Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. Using single-cell analysis, we found Esrrb, Utf1, Lin28 and Dppa2 to be predictive markers of reprogramming. We found that single cells exhibit high variation in gene expression early in reprogramming and this heterogeneity decreases are the cell reaches pluripotency. Our results show that a stochastic phase of gene activation is followed by a late hierarchical phase, initiated by activation of the Sox2 locus, leading to the activation of the pluripotency circuitry. Finally, we reprogram cells without Oct4, Klf4, Sox2, c-Myc, and Nanog. Embryonic stem cells (ESCs) are the gold standard comparison for iPSCs. Our investigation of ESCs must continue in parallel to that of iPSCs since we cannot truly understand iPSCs if we do not understand the molecular mechanisms that regulate ESC pluripotency. The homeodomain transcription factor Nanog is a central part of the core pluripotency transcriptional network and plays a critical role in ESC self-renewal. Several reports have suggested that Nanog expression is allelically regulated and that transient downregulation of Nanog in a subset of pluripotent cells predisposes them toward differentiation. Using single-cell gene expression analyses combined with different reporters for the two alleles of Nanog, we show that Nanog is biallelically expressed in ESCs independently of culture condition. We also show that the overall variation in endogenous Nanog expression in ESCs is very similar to that of several other pluripotency markers. Our analysis suggests that reporter-based studies of gene expression in pluripotent cells can be significantly influenced by the gene-targeting strategy and genetic background employed. Our results show that single-cell analysis is essential for deciphering the mechanisms of reprogramming and understanding gene regulation of ESCs, exposing important rarities typically masked by population-based assays.
by Dina Adel Faddah.
Ph. D.
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Durán, Ibáñez Sara. "Suspended micro- and nanotools for cell biology." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/285575.
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Esta tesis presenta el diseño, desarrollo tecnológico, caracterización y aplicaciones tanto químicas como biológicas de micro- y nanodispositivos destinados a ser herramientas funcionales en biología celular.
Esta línea de investigación es posible gracias a los avances obtenidos en el campo de las Micro- y Nanotecnologías, donde la aplicación de técnicas de miniaturización en la fabricación de sus dispositivos a escala celular es ya una realidad. Estas herramientas son lo suficientemente pequeñas como para etiquetar y permitir el seguimiento de embriones vivos o incluso actuar como sensores permitiendo el estudio de células vivas únicas de forma intra- y extracelular. Además, una de las características más relevantes de las micro- y nanoherramientas presentadas en esta tesis es su capacidad de suspensión, es decir, que estos dispositivos pueden ser liberados de la oblea en la que están fabricados y directamente actuar con las células en su mismo medio y en su misma escala.
Estas herramientas presentan diferentes formas, tamaños, materiales y funcionalidades específicas, ya que la combinación de todas estas características o incluso la incorporación de partes nanoestructuradas en un solo dispositivo nos permite la obtención de herramientas multi-funcionales.
Por todo ello, las micro- y nanoherramientas presentadas en esta tesis implican un gran número de aplicaciones como sensores y actuadores en biología celular. O incluso en el futuro estos dispositivos podrán ser aplicados en el campo de la nanomedicina como sistemas de diagnosis y drug delivery.This thesis presents the design, technological development, characterization and chemical and biological applications of micro- and nanodevices which are focused on being functional tools for cell biology. This research is possible thanks to the recent advances in Micro- and Nanotechnologies, where the application of miniaturization techniques at cell scale is already a reality. These tools are small enough to label and track living embryos or even sense and operate in single living cells in an extra- and intracellular way. Furthermore, one of the most relevant new features of the micro- and nanotools presented in this thesis is the capability of being suspended, meaning that these devices can be released from the wafer and directly interact with cells in their same medium and at cell scale. These micro- and nanotools present different shapes, sizes, materials and specific functionalities, as the combination of these features or even the incorporation of nanostructured parts in a single device can let us obtain multi-tasking devices. Summarizing, the extensive capabilities of the presented micro- and nanotools imply a broad number of applications as sensors and actuators in cell biology. Or even in the near future, these devices can be applied in the nanomedicine field as diagnosis and drug delivery systems.
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Diks, Sander Henricus. "Analysis of protein superhighways in cell biology." [S.l. : [Groningen : s.n.] ; University Library Groningen] [Host], 2006. http://irs.ub.rug.nl/ppn/298197421.
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Campbell, James Kyle. "Microfluidics for optics and quantitative cell biology." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3290680.
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Thesis (Ph. D.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed February 4, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 145-150).
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Hinton, I. E. "The developmental biology of Drosophila cell surfaces." Thesis, University of Oxford, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233464.
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McNae, Fiona. "The cell biology of non-genotoxic hepatocarcinogens." Thesis, University of Surrey, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260350.
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Patterson, James Oliver. "Quantitative biology of cell cycle decision making." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10056386/.
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In the fission yeast Schizosaccharomyces pombe as well as metazoans, the G1/S and G2/M transitions are a focal point of cell cycle regulation. Cells perform these transitions at defined cell sizes, and if born large or small adjust their growth to compensate. The cell cycle is driven by cyclin-CDK activity, and multiple signals are integrated by a single inhibitory phosphorylation site on the CDK1 enzyme. This phosphorylation is regulated by Wee1 kinase and Cdc25 phosphatase. I sought to quantify how and if cell size directly regulates CDK activity in single cells, and to what extent cyclin levels may inform cell size at division. I show that by altering the activity of Wee1 and Cdc25, the cell establishes a certain threshold cyclin-CDK for G2/M entry. Cyclin levels scale with cell size, and the probability of a cell having suprathreshold levels of cyclin dictates its probability of entering mitosis. Cell-cell variability in cyclin levels are rate limiting for cell size fidelity. The G1/S transition is similarly probabilistically controlled by cell size. Using a new single cell fluorescent biosensor of CDK activity, I show that the dose response of cyclin-CDK1 levels on CDK activity is ultrasensitive. The larger a cell, the more switch-like the dose response. This size dependent ultrasensitivity is dependent on CDK tyrosine phosphorylation. Intriguingly, in the absence of all canonical CDK control pathways, small cell size decreases intrinsic cyclin-CDK1 enzyme activity. Upon entry to mitosis, progressive substrate phosphorylation appears to be coordinated by substrate specific integration of CDK activity, rather than defined activity thresholds. This integrated-activity model of mitotic progression generates robustness to CDK inhibition. Finally, I applied the new computational and molecular tools developed throughout this thesis to demonstrate the functional ramifications of cancer associated APC/C mutations. APC/C mutations likely decrease the lethality of chromosomal instability in cancer.
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Larocque, Gabrielle. "Cell biology of tumor protein D54 (TPD54)." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/107000/.
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The expression of Tumor protein D52 (TPD52) family members is deregulated in many types of cancer. When overexpressed, it is suggested that they increase cell proliferation and migration/invasion as well as avoid apoptosis. Deregulation in the expression of the TPDs is therefore linked to poor prognosis. Little characterisation has been carried out to date, but it is known that the TPDs are found in association with components of the membrane trafficking pathway. The aim of this work is to uncover how the least studied member of the family, TPD54, affects cellular processes involved in carcinogenesis, such as cell migration and invasion. By using the knocksideways method, we have been able to map the cellular localisation of TPD54 and have identified association partners. These associations have been confirmed by immunoprecipitation and mass spectrometry analysis. Amongst these was the small GTPase Rab14. We have also found that TPD54 is involved in the trafficking of receptors containing a dileucine motif in their cytosolic tail, but not a tyrosine-based or NPXY motif. With the mapping of the localisation of TPD54, we hypothesise that TPD54 is on the recycling route following the Golgi apparatus, and in association with Rab14, regulates the trafficking of receptors containing a dileucine motif. Integrins are receptors controlling cell migration. They can be trafficked through the Golgi apparatus before being recycled back to the plasma membrane. This recycling route is not well characterised. We therefore hypothesise that TPD54 regulates this route with Rab14, and that this is the reason why TPD54 is important for cell migration, and that a defect in its function can cause cancer.
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Trundley, Anita Elizabeth. "Aspects of human uterine NK cell biology." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620028.
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Shannon, Matthew Frederick. "The molecular biology of sickle cell anaemia." Thesis, King's College London (University of London), 2018. https://kclpure.kcl.ac.uk/portal/en/theses/the-molecular-biology-of-sickle-cell-anaemia(d4d29fde-2799-4f3f-b499-b5ae0f5b6782).html.
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Sickle cell anaemia (SCA) is a haemolytic anaemia that reduces life expectancy and places a great burden on healthcare systems worldwide. Despite being a monogenic disorder, the phenotypic severity varies greatly between patients, ranging from patients that experience multiple strokes and organ failure during childhood, to those that live largely unaffected lives. Some genetic variants that affect globin gene expression are known to influence phenotype severity, but most of this variation remains unaccounted for. We conducted whole exome sequencing analyses, comparing SCA patients with mild and severe clinical phenotypes, with the aim of identifying novel genetic modifiers of the disease. SCA patient exomes were sequenced from a cohort at King’s College Hospital, and combined with publicly available SCA exomes recruited in the United States. Nine candidate variants were identified in genes with plausible mechanisms to influence the pathophysiology of the disease. The genes identified in this study affected nitric oxide signalling, haematopoietic regulation, globin gene expression and recovery from ischaemic injury. In order to evaluate these variants, a CRISPR genomic editing pipeline was established and tested on two previously identified candidate modifiers of SCA, in the genes ASH1L and KLF1. These variants were successfully introduced into erythroleukaemic cells and provide a pathway for testing the novel modifier genes identified in the exome sequencing analysis. Preliminary studies indicate that both ASH1L and KLF1 variants alter globin gene expression. In addition to genetic factors, we also hypothesised that epigenetic factors affect the SCA phenotype, and play a role in the therapeutic mechanism of hydroxyurea treatment. We optimised a method for isolating CD45+CD71+GPA- nucleated erythroid progenitors from small volumes of SCA peripheral blood. This was undertaken to evaluate the role of the epigenome in SCA phenotype severity and drug action, but for which patient sample collection proved too challenging within our clinical cohort.
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Szymanska, Katarzyna. "Molecular genetics and cell biology of ciliopathies." Thesis, University of Leeds, 2015. http://etheses.whiterose.ac.uk/8891/.
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Defects in cilia structure and/or function are now known to be the cause of an important group of Mendelian developmental conditions called ciliopathies. Meckel-Gruber syndrome (MKS) and Joubert syndrome-related disorders (JBTS) are the focus of this work. The research comprised genetic screening of an established MKS/JSRD patient cohort for mutations in seven known genes, and different approaches to identify new causes for these disorders. The latter included whole exome sequencing (WES) of mutation-negative patients, and a high-throughput whole genome siRNA-based reverse genetics screen to identify novel ciliopathy genes and genes implicated in the process of ciliogenesis. Mutation screening in the University of Leeds MKS/JSRD patient cohort showed that about 50% patients (n=29/65) were mutation-negative for known genes and confirmed mutations in TMEM67 as a major cause of MKS/JSRD. WES gave a conclusive molecular diagnosis for n=4/7 families. WES allowed the identification of mutations in TMEM237 as a new cause of JSRD. In vitro assays showed that the TMEM237 protein is required for correct cilia formation and function. Loss of the protein in patient fibroblasts and after transcript knockdown caused defects in ciliogenesis and the Wnt signaling pathway. The whole genome reverse genetics screen identified new functional modules that were not previously linked to cilia (components of the spliceosome and proteasome) or had a poorly characterized ciliary function (several neuroactive GPCRs). Cross-comparison of screen hits with available WES data allowed the prioritisation and confirmation of mutations in PIBF1 and C21orf2 as new causes of JBTS and the skeletal ciliopathy Jeune syndrome, respectively. In summary, the multiple approaches presented in this work have allowed further insights into the structure and function of the primary cilia, as well as the disease mechanisms of human ciliopathies.
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Chen, Luxi. "Human Innate Lymphoid Cell Biology and Development." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1551901769401192.
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Szekely, Tamas. "Stochastic modelling and simulation in cell biology." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:f9b8dbe6-d96d-414c-ac06-909cff639f8c.
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Modelling and simulation are essential to modern research in cell biology. This thesis follows a journey starting from the construction of new stochastic methods for discrete biochemical systems to using them to simulate a population of interacting haematopoietic stem cell lineages. The first part of this thesis is on discrete stochastic methods. We develop two new methods, the stochastic extrapolation framework and the Stochastic Bulirsch-Stoer methods. These are based on the Richardson extrapolation technique, which is widely used in ordinary differential equation solvers. We believed that it would also be useful in the stochastic regime, and this turned out to be true. The stochastic extrapolation framework is a scheme that admits any stochastic method with a fixed stepsize and known global error expansion. It can improve the weak order of the moments of these methods by cancelling the leading terms in the global error. Using numerical simulations, we demonstrate that this is the case up to second order, and postulate that this also follows for higher order. Our simulations show that extrapolation can greatly improve the accuracy of a numerical method. The Stochastic Bulirsch-Stoer method is another highly accurate stochastic solver. Furthermore, using numerical simulations we find that it is able to better retain its high accuracy for larger timesteps than competing methods, meaning it remains accurate even when simulation time is speeded up. This is a useful property for simulating the complex systems that researchers are often interested in today. The second part of the thesis is concerned with modelling a haematopoietic stem cell system, which consists of many interacting niche lineages. We use a vectorised tau-leap method to examine the differences between a deterministic and a stochastic model of the system, and investigate how coupling niche lineages affects the dynamics of the system at the homeostatic state as well as after a perturbation. We find that larger coupling allows the system to find the optimal steady state blood cell levels. In addition, when the perturbation is applied randomly to the entire system, larger coupling also results in smaller post-perturbation cell fluctuations compared to non-coupled cells. In brief, this thesis contains four main sets of contributions: two new high-accuracy discrete stochastic methods that have been numerically tested, an improvement that can be used with any leaping method that introduces vectorisation as well as how to use a common stepsize adapting scheme, and an investigation of the effects of coupling lineages in a heterogeneous population of haematopoietic stem cell niche lineages.
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Kobert, Antonia. "CNS-resident cells support MS-relevant B-cell responses." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114274.
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The therapeutic success of peripheral B-cell depletion strategies in MS patients has identified B cells as important contributors to new relapsing disease activity in the periphery. However, they may also drive the CNS-compartmentalized inflammatory processes thought to underlie the chronic-progressive stages of disease. The long-term persistence of plasma cells in the CNS of MS patients, and cellular aggregates rich in B cells, T cells and FDC-like cells that have recently been identified in the meningeal compartment, suggest the inflamed MS CNS may be a B-cell fostering environment. The factors that contribute to such a permissive environment, however, have remained poorly understood.Here, we demonstrate that glial cells and their soluble products can support B-cell survival and MS-relevant B-cell responses, including co-stimulatory molecule expression and T-cell activation, effector-cytokine secretion and immunoglobulin production. Glial-cell derived factors have been shown to be abnormally elevated in CSF of MS patients, and we hypothesized that MS CSF may be able to support B-cell survival in-vitro. We demonstrate that CSF alone, in isolation of the complex cellular environment of the inflamed MS CNS, is not sufficient to support B-cell survival in culture. We then considered CNS-resident ECs as another source of B-cell support, and we demonstrate that soluble factors secreted by BBB and meningeal ECs can either enhance or regulate B-cell survival, and increase expression of the T-cell co-stimulatory molecule CD86.Our observations suggest that CNS-resident glial and endothelial cells and their soluble products may significantly contribute to a B-cell permissive environment and support MS-relevant B-cell effector functions within the inflamed MS CNS.L'appauvrissement des cellules B en périphérie est un traitement effectif chez les patients atteints de la SP et ce type cellulaire semblerait être un important médiateur lors des rechutes associés à la maladie. Toutefois, ils peuvent aussi induire les réactions inflammatoires compartimentées dans le SNC qui semblent être à la base des stades chroniques-progressifs de la maladie. La persistance des plasmocytes dans le SNC et les agrégations cellulaires riche en cellules B, cellules T et en cellules ressemblant aux CDFs dans les méninges des patients suggèrent que le SNC inflammé lors de la SP fonctionne comme étant un environnement favorisant les cellules B. Les facteurs qui contribuent à cet environnement permissif sont restés faiblement compris.Nous démontrons que les cellules gliales et leurs produits solubles peuvent supporter la survie des cellules B ainsi que les fonctions pertinentes à la SP, incluant l'expression des molécules co-stimulatrices et l'activation des cellules T, la sécrétion des cytokines effectrices et la production des immunoglobulines. Les produits solubles gliaux sont anormalement élevés dans la FCS des patients, nous avons donc supposé que le FCS de la SP pourrait supporter la survie des cellules B. Nous démontrons que le FCS seul, en isolement de l'environnement cellulaire complexe du SNC de la SP inflammé, n'est pas capable de supporter la survie des cellules B in-vitro. Nous démontrons aussi que les produits solubles sécrétés par les CEs de la BHE et des méninges peuvent augmenter ou modérer la survie des cellules B et peuvent aussi augmenter l'expression de la molécule co-stimulatrice CD86.Nos observations suggèrent que les cellules gliales et les CEs résidants dans le SNC ainsi que leur produits solubles peuvent significativement contribuer à un environnement permissif pour les cellules B et peuvent aussi supporter leurs fonctions effectrices pertinentes à la SP dans le SNC inflammé de patients.
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Wredle, Ulla. "Autophagic programmed cell death in the suspensor and endosperm of Vicia faba : An ultrastructural study." Doctoral thesis, Stockholm : Botaniska institutionen, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-123.
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Marques, Graça Susete Costa de Carvalho. "Establishing a cell biology platform: isolation and preservation of human blood products." Master's thesis, Faculdade de Ciências e Tecnologia, 2014. http://hdl.handle.net/10362/11009.
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Dissertação para obtenção do Grau de Mestre em
Genética Molecular e BiomedicinaThe use of human primary cells provide researchers in different areas with irrefutable more biologically relevant data than using cell lines or animal blood cells. The work was performed in the scope of the Cell Biology Services @ CEDOC, aiming to provide viable and trustful human primary cells and products. We had three main objectives: protocol optimizations for blood cell isolation, culture and cryopreservation; cost estimation and divulgation of the services. We have reviewed standard protocols and compared different strategies for blood cell isolation. The impact of those methodologies was evaluated regarding cell yield and purity, cell functional characteristics and cost. We also developed a method for serum isolation from human plasma in blood buffy coats. The resultant sera were sterile and suitable to be used in leukocyte cultures. Different protocols for T cells isolation were compared: positive versus negative immunomagnetic selection and isolation using nylon wool fiber columns. Positive selection provided the highest isolation yield (32.35%), while negatively selected cells had the highest purity (92.81%). Although nylon wool fiber column was the fastest and cheapest method, unlike the immunomagnetic methods, it did not allow complete separation of T from B lymphocytes. Positive selection of monocytes was compared using two widely used commercial kits. Miltenyi’s kit provided the highest isolation yield (25.92%), recovery rate (86.70%) and purity (95.01%). Monocytes isolated with StemCell kit presented a higher cell complexity, and when differentiated into dendritic cells (DCs), showed a more mature phenotype. Differences between both kits are probably caused by the nature of the magnetic beads, suggesting caution when choosing one or other kit, as it may have an impact on DCs’ function. Overall, although dealing with apparently straight forward methodologies, our results show that testing commercial products and optimizing protocols is very important and contribute for a better quality of products and services.
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Belbaraka, Loubaba. "Growth, differentiation and cell-cell coupling in the human neuroblastoma cell line SH-SY5Y." Thesis, University of Ottawa (Canada), 2002. http://hdl.handle.net/10393/6112.
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Neuroblastoma is one of the most common paediatric solid tumours, frequently occurring in infancy with the primary lesion in the adrenal gland and sympathetic nervous system. It originates from primitive neural crest cells. In rare cases, this tumour regresses spontaneously to a more benign ganglioneuroma probably by neuronal differentiation or apoptosis. We investigated the role of induction of neuronal differentiation and apoptosis in vitro in SH-SY5Y neuroblastoma cells. A variety of agents that are known to induce neuronal differentiation including retinoic acid, nerve growth factor (NGF), protein kinase C (PKC) inhibitors and a cAMP-dependent protein kinase A (PKA) activator were tested solely or in combination for their capacity to induce terminal differentiation. The cells were characterised for markers of differentiation as well as their ability to withdraw from the cell cycle. We found that the combination of 8-Br-cAMP (PKA activator) with NGF in the presence of the cell cycle inhibitor aphidicolin was the best treatment to induce terminal differentiation in SH-SY5Y cells. Treated cells showed long neurites resembling those of neurones. They expressed markers characteristic of the cytoskeleton (NF200, NF68) and of neuronal function (tyrosine hydroxylase, choline acetyltransferase and the neurone-specific enolase) and showed an increase in the expression of TrkA, the receptor for NGF. Furthermore, the expression of the N-myc oncogene that is normally overexpressed in these cells decreased. Since neurones are dependent on NGF for survival, we tested the ability of terminally differentiated SH-SY5Y cells to survive in the absence of NGF. We found that the cells became NGF-survival dependent and that deprivation from this neurotrophic factor induced programmed cell death. We also tested the effect of PKC specific and non-specific inhibitors on cell proliferation, differentiation and apoptosis in SH-SY5Y cells. We found that only the non-specific PKC inhibitors staurosporine and H7 induced morphological and molecular differentiation as well as decreased N-myc amplification followed by apoptosis. The effect of differentiation induction on p53 sub-localisation was also investigated in undifferentiated and differentiated SH-SY5Y cells. p53 immunostaining showed that p53 was sequestered in the cytoplasm in undifferentiated SH-SY5Y cells and that the induction of differentiation resulted in the partial transfer of p53 to the nucleus where it probably became able to regulate the cell cycle and apoptosis since p53 is not mutated in neuroblastoma. Gap junction intercellular communication (GJIC) is known to be involved in the regulation of cell growth and homeostasis and has been shown to contribute to many diseases including cancer. We investigated the role of GJIC in neuroblastoma. We found that SH-SY5Y cells have altered GJIC due to an aberrant localisation in the perinuclear region of connexin 43 (Cx43), one of the proteins of GJIC normally present at the plasma membrane. GJ channel formation and gating is regulated by PKC, PKA and/or MAPK. We found that induction of differentiation using the PKA activator 8-Br-cAMP relocalised Cx43 to the plasma membrane region and restored GJIC. Furthermore, inhibition of p38 MAPK induced a block of cell proliferation associated with GJIC proficiency while inhibition of the subfamily of MAPK, Erk1/Erk2, likely promoted the degradation of Cx43.
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Eastwood, Deborah Jane. "An investigation into the biology of seminoma." Thesis, University of Sheffield, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324438.
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Delorme, Marilyne. "Downregulation of ATRX disrupts cell proliferation and cell cycle progression." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27627.
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ATRX is a chromatin remodelling protein of the SNF2 family of chromatin remodelling proteins. Mutations in the ATRX gene have been shown to cause the ATR-X syndrome, an X-linked mental retardation disorder. ATRX is part of a chromatin-remodelling complex with Daxx that localizes to PML nuclear bodies or pericentromeric heterochromatin and is thought to regulate gene expression. In mice, Atrx inactivation results in embryonic lethality whereas conditional forebrain specific Atrx ablation showed impaired development and disorganization of the cortex. Furthermore, ATRX phosphorylation was shown to be cell cycle dependant, suggesting an important role for ATRX in cell cycle regulation. In this study we investigated the effects of ATRX downregulation in cell culture models, using siRNA transient transfection, a clone expressing an shRNA targeted to ATRX, and Atrxnull MEFs. ATRX downregulated cells showed reduced growth rates and cell cycle defects at the G1 and S phases of the cell cycle. Moreover, ATRX ablation was associated with an altered Rb phosphorylation status and decreased expression of the cyclin A and E2F-1 proteins. Taken together our results suggest that ATRX may play a significant role in cell cycle progression that is pertinent for proper development.
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Stimson, Krista Marie. "Cell-Cell Interactions in the Development of the Vascular System of Xenopus." W&M ScholarWorks, 1998. https://scholarworks.wm.edu/etd/1539626164.
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Serpente, Norberto. "Cells from icons to symbols : molecularising cell biology in the 1980’s." Thesis, University College London (University of London), 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.563093.
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Cornforth, Terri Victoria. "Characterising the cell biology of leukemic stem cells in acute myeloid leukemia." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:654b2176-fd50-427e-86f2-74e928054bef.
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Acute Myeloid leukemia (AML) is an aggressive haematological malignancy that mainly affects the elderly. Relapse is common and is thought to be due to the presence of chemotherapy resistant leukemic stem cells (LSC). Within the CD34+ disease (>5% of the blast cells expressing CD34) , two subtypes have been identified; an LMPP/GMPlike expanded type and a MPP/CMP-like expanded type, the former is the most common, accounting for around 80% of CD34+ AML. Both the GMP-like and LMPPlike expanded populations show LSC activity. To improve our understanding of the disease and gain better insight in to how to develop treatments, the molecular basis of the disease needs to be investigated. I investigated miRNAs in the GMP/LMPP-like expanded AML. miRNAs are small non-coding RNAs involved in the regulation of mRNA. In recent years miRNAs have been shown to be implicated in many different diseases. To investigate the role miRNAs play in AML, miRNA expression was profiled in leukemic and normal bone marrow. Bioinformatic analysis was then used to examine the different miRNA expression profiles between normal and leukemic marrow. Our study showed that miRNAs are dysregulated in AML. miRNAs from the miR-17-92 and its paralogous cluster miR-106b-92 were amongst the miRNAs to be found down regulated in AML As had been seen previously at an mRNA level, on an miRNA level the LSC populations more closely resembled more mature progenitor populations than HSC and MPP populations, however the LSC populations did display an aberrant stem cell-like miRNA signature.
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Xie, Yuan. "Modeling glioblastoma heterogeneity to decipher its biology." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-278529.
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Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumor that mainly affects adults. GBM displays remarkable intra- and inter-tumoral heterogeneity and contains a subpopulation of cells named glioma stem cells that is believed to be responsible for tumor maintenance, progression and recurrence. We have established and characterized a biobank of 48 cell lines derived from GBM patients. The cells were explanted and maintained as adherent cultures in serum-free, defined neural stem cell medium. These GBM cells (GCs) displayed NSC marker expression in vitro, had orthotopic tumor initiating capability in vivo, harboured genomic alterations characteristic of GBM and represented all four TCGA molecular subtypes. Our newly established biobank is also connected with a database (www.hgcc.se) that provides all molecular and clinical data. This resource provides a valuable platform of valid in vitro and in vivo models for basic GBM research and drug discovery. By using RCAS/tv-a mouse models for glioma, we found that GBMs originating from a putative NSC origin caused more tumorigenic GCs that had higher self-renewal abilities than those originating from putative glial precursor cell origin. By transcriptome analysis a mouse cell origin (MCO) gene signature was generated to cluster human GCs and GBM tissue samples and a functional relationship between the differentiation state of the initially transformed cell and the phenotype of GCs was discovered, which provides the basis for a new predictive MCO-based patient classification. LGR5 was found to be highly expressed in the most malignant mouse GC lines of putative NSC origin and also enriched in proneural GBMs characterized by PDGFRA alterations and OLIG2 up-regulation. By overexpressing or depleting LGR5 we discovered that high LGR5 expression in proneural GC lines increased the tumorigenicity, self-renewal and invasive capacities of the cells and could potentiate WNT signalling through its ligand RSPO1. Through transcriptome analysis we identified the candidate genes CCND2, PDGFRA, OLIG2, DKK1 that were found to be regulated by LGR5. In the last study, we found that mouse OPCs could initiate both astrocytic and oligdendroglial gliomas, which indicated that oncogenic signalling is dominant to cell of origin in affecting the histology of gliomas.
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Newman, Jamie Jennifer. "Regulation of gene expression and cell state in embryonic stem cells." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/58526.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, June 2010."May 2010." Cataloged from PDF version of thesis.
Includes bibliographical references.
Cell state is established and maintained through the combined action of transcription factors, chromatin regulators and signaling pathways, which all contribute to a transcriptional regulatory circuitry. Embryonic stem (ES) cells are capable of self-renewal and can give rise to nearly all differentiated cell-types, making them an ideal system in which to address the challenges of understanding gene expression and cell state. Valuable insights into the control of cell state have been revealed by recent studies of the ES cell transcriptional regulatory circuitry. Here I present work contributing to the understanding of transcriptional regulatory mechanisms that control ES cell state, specifically signaling pathways and proteins that affect chromatin structure.
by Jamie J. Newman.
Ph.D.
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Hosseini, Shirazi Seyed Farshad. "Cell cycle dependency of cisplatin cytotoxicity on ovarian cancer cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0028/NQ36776.pdf.
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Wearing, Helen Jane. "Mathematical modelling of cell-cell signalling in developmental biology and wound healing." Thesis, Heriot-Watt University, 2001. http://hdl.handle.net/10399/1184.
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Matsiaka, Oleksii. "New mathematical models for cell biology assays incorporating realistic cell size dynamics." Thesis, Queensland University of Technology, 2020. https://eprints.qut.edu.au/198192/1/Oleksii_Matsiaka_Thesis.pdf.
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This thesis provides novel insights into several contemporary problems in the mathematical biology involving migration of living cells. Primarily, we focus on cell motility and how dynamic changes in cell size affect collective cell migration. Additionally, this thesis investigates the importance of cellular heterogeneity and how it might affect the choice of modelling techniques we use to describe in vitro cell cultures.
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Fernandez, Daniel. "Cell States and Cell Fate: Statistical and Computational Models in (Epi)Genomics." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226043.
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This dissertation develops and applies several statistical and computational methods to the analysis of Next Generation Sequencing (NGS) data in order to gain a better understanding of our biology. In the rest of the chapter we introduce key concepts in molecular biology, and recent technological developments that help us better understand this complex science, which, in turn, provide the foundation and motivation for the subsequent chapters.
In the second chapter we present the problem of estimating gene/isoform expression at the allelic level, and different models to solve this problem. First, we describe the observed data and the computational workflow to process the data. Next, we propose frequentist and bayesian models motivated by the central dogma of molecular biology and the data generating process (DGP) for RNA-Seq. We develop EM and Gibbs sampling approaches to estimate gene and transcript-specic expression from our proposed models. Finally, we present the performance of our models in simulations and we end with the analysis of experimental RNA-Seq data at the allelic level.
In the third chapter we present our paired factorial experimental design to study parentally biased gene/isoform expression in the mouse cerebellum, and dynamic changes of this pattern between young and adult stages of cerebellar development. We present a bayesian variable selection model to estimate the difference in expression between the paternal and maternal genes, while incorporating relevant factors and its interactions into the model. Next, we apply our model to our experimental data, and further on we validate our predictions using pyrosequencing follow-up experiments. We subsequently applied our model to the pyrosequencing data across multiple brain regions. Our method, combined with the validation experiments, allowed us to find novel imprinted genes, and investigate, for the first time, imprinting dynamics across brain regions and across development.
In the fourth chapter we move from the controlled-experiments in mouse isogenic lines to the highly variant world of human genetics in observational studies. In this chapter we introduce a Bayesian Regression Allelic Imbalance Model, BRAIM, that estimates the imbalance coming from two major sources: cis-regulation and imprinting. We model the cis-effect as an additive effect for the heterozygous group and we model the parent-of-origin detect with a latent variable that indicates to which parent a given allele belongs. Next, we show the performance of the model under simulation scenarios, and finally we apply the model to several experiments across multiple tissues and multiple individuals.
In the fifth chapter we characterize the transcriptional regulation and gene expression of in-vitro Embryonic Stem Cells (ESCs), and two-related in-vivo cells; the Inner Cell Mass (ICM) tissue, and the embryonic tissue at day 6.5. Our objective is two fold. First we would like to understand the differences in gene expression between the ESCs and their in-vivo counterpart from where these cells were derived (ICM). Second, we want to characterize the active transcriptional regulatory regions using several histone modifications and to connect such regulatory activity with gene expression. In this chapter we used several statistical and computational methods to analyze and visualize the data, and it provides a good showcase of how combining several methods of analysis we can delve into interesting developmental biology.
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Raeiszadeh, Mohammad. "Reconstitution of CMV-specific T-cells following adoptive T-cell immunotherapy and haematopoietic stem cell transplantation." Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/6968/.
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This thesis investigated reconstitution of CMV-specific T-cells in two cohorts of HSCT patients and studied the potential role of Tumour Necrosis Factor Receptor 2 (TNFR2) in regulation of CMV-specific T-cell expansion post HSCT. The first cohort included patients of a randomized phase II trial of adoptive cellular therapy for CMV-specific CD8\(^+\) T-cells. Cellular therapy resulted in earlier and greater expansion of CMV-specific CD8\(^+\) T cells and also reconstitution of CMV-specific CD4\(^+\) and non-infused CMV-specific CD8\(^+\) T-cells. The number of infused therapeutic T-cells and circulating levels of Alemtuzumab were found to influence immunotherapy. Additionally, reconstitution of CMV-specific CD4\(^+\) T-cells was studied using HLA-class II tetramers. CMV-specific CD4\(^+\) T-cell count of >0.7x10\(^3\)/ml was found to protect from recurrent CMV reactivation. One third of specific CD4\(^+\) T-cells were perforin and granzyme-B positive indicating cytotoxic potential, whilst the majority expressed T-bet. Expression of CD57 molecule on CD4\(^+\) T-cells was demonstrated as a potential biomarker of immune response to CMV. Also, distinct cytokine receptor expression patterns in naïve versus memory T-cells were observed. The results showed rapid decrease in IL-6R and increase in expression of TNFR2 after T-cell differentiation from naïve to effector cells and engagement of TNFR2 led to the apoptosis of CMV-specific T-cells.
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Bumgarner, Stacie L. "Mechanisms underlying cell-to-cell diversity in clonal populations of yeast." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45150.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references.
The FLO promoters are among the largest promoters in yeast and receive a complex combination of signals from upstream signaling pathways through their association with downstream DNA binding factors and chromatin remodelers. The genes regulated by these promoters encode cell-surface glycoproteins that mediate a range of cell-to-cell and cell-to-surface adhesions. Phenotypic diversity in clonal populations of yeast cells is mediated in part by epigenetic silencing of the FLO10 and FLO11 promoters. Silencing of the FLO promoters is heterogeneous, or variegated, within a clonal population of cells. The variegated transcription of FLO10 and FLO11 results in a population of yeast cells that exhibits cell-to-cell variability in flocculation, adhesion to and invasion of inert surfaces, and filamentous growth. In this thesis, I discuss chromatin modifying proteins that localize to the FLO10 and FLO11 promoters and act in trans to affect transcription and silencing at these promoters. I describe the results of genome-wide screens to identify additional trans-acting chromatin modifying factors that play roles in the transcriptional regulation and silencing of the FLO10 and FLO11 promoters. Some of the candidates identified in these screens had effects on FLO transcription that initially seemed paradoxical in light of contemporary theories regarding the role of chromatin structure in regulating transcription. Given that histone deacetylases generally repress transcriptional activity, we were particularly surprised to find that mutations in components of the Rpd3L histone deacetylase complex reduce FLO promoter activity, indicating that Rpd3L plays a role in transcriptional activation of FLO genes. Careful analysis of these mutants, their phenotypes, the transcription of FLO11, and most importantly, the noncoding transcripts that we have detected in the promoter region of FLO11, have revealed the basis for this apparent paradox.
by Stacie L. Bumgarner.
Ph.D.
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Solomon, Jonathan M. (Jonathan Micah). "Cell-cell signaling and the regulation of development in Bacillus subtilis." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/43310.
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