Dissertations / Theses on the topic 'Cell and Molecular Bioscience'
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Sargeant, Timothy John. "The effect of opiates on developing cerebral cortex : a thesis submitted to the Victoria University of Wellington in fulfilment of the requirements for the degree of Doctor of Philosophy in Cell and Molecular Bioscience /." ResearchArchive@Victoria e-Thesis, 2008. http://hdl.handle.net/10063/414.
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Reader, Karen Lee. "A quantitative ultrastructural study of oocytes during the early stages of ovarian follicular development in Booroola and wild-type sheep : a thesis submitted to the Victoria University of Wellington in fulfilment of the requirements for the degree of Master of Science in Cell and Molecular Bioscience /." ResearchArchive@Victoria e-Thesis, 2007. http://hdl.handle.net/10063/270.
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Speh, Michel Jonathan. "Transcriptional changes in stem cell-derived cardiomyocytes during extended cell cultures." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-18781.
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The recent advancement in the field of stem cell research gave rise to high expectations from both general and scientific audiences, and indeed, stem cells and stem cell-derived cells have an enormous potential to forward the fields of medical research and regenerative medicine. Nevertheless, there are still many obstacles and limitations associated with stem cell technology. One of those limitations is that currently no method to derive fully mature cardiomyocytes from human pluripotent stem cell is available. Instead, stem cell-derived cardiomyocytes only show basic characteristics of their adult counterparts and are thus only of limited use. However, there is experimental evidence that those cells may increase in maturity during extended culture times. To improve the understanding of those processes, a characterisation of the transcriptional changes in human embryonic stem cell-derived cardiomyocytes over two weeks was made. Using standard bioinformatics methods, including differential and enrichment analysis as well as basic machine learning technologies, changes in mRNA and miRNA transcription and several functions related to those changes could be identified. The analyses indicated increasing structural organisation in the cell cultures, increasing expression of cardiac ion channels and decreasing proliferative capacity in the cardiomyocyte cultures. Furthermore, potential dysregulations of important signalling pathways were observed. The results of this project may aid in developing protocols for differentiating stem cells into cardiomyocytes with features that are more mature than those of the currently available stem cell derived-cardiomyocytes.
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Patel, Angana Heet. "Unravelingthe molecular mechanism behind metabolic reprogramming caused by alterations of the enzyme PI3-kinase." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17410.
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Oncogenes and tumor suppressor genes play a key role in cancer induction and progression. They directly or indirectly regulate critical metabolic pathways, phosphatidylinositol-3 kinase pathway being frequently activated pathway in cancer. The catalytic subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), p110α, is the most frequently mutated kinase in human cancer, E542K, E545K, and H1047R mutations being the most common. Expression of hepatic E545K and H1047R p110α mutants in vivo shows marked and rapid increase in hepatic lipid and glycogen accumulation in mice with developmental (chronic) liver-specific deletion of p110α, which was not seen in mice when wildtype p110α is overexpressed. To investigate the logical pathways that could explain the lipid accumulation in mutant expressing mice, RNA sequencing from wildtype, knockout and mutated mouse livers was performed. Read alignment and count quantification was done using the Rsubread package and the statistical analyses are performed using the DeSeq2 package. Differentially expressed genes were identified with adjusted p-value of 0.05. Gene ontology analysis was performed on the differentially expressed genes using clusterProfiler, an R package to identify several key pathways which were upregulated and downregulated among the different sample groups. Signaling pathways related to cell cycle processes were mainly upregulated in the mutated samples when compared with the wildtype as well as knockout samples while signaling pathways related to many metabolic processes seem to be downregulated in mutated samples, even though these mutants showed upregulated metabolism by accumulation of lipids and glycogen physiologically. To confirm the results of gene expression data the results have to be cross validated with the gold standard quantitative Real Time Polymerase Chain Reaction.
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Babena, Omar. "Expression of the chloride channel CLCC1 is downregulated after 24 hours in LPS-primed THP-1 monocyte-like cell line." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-20377.
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Inflammation is the body's response to infection or injury and is mediated by the innate immune system. The NLRP3 inflammasome is a multi-protein complex that is a major contributor to many inflammatory disorders. Emerging evidence suggests the involvement of the Endoplasmic reticulum stress with the NLRP3 inflammasome. The endoplasmic reticulum stress is a series of stress signals that can activate the unfolded protein response and usually accompanies inflammation and eventually causes cell death. Recently, a localized endoplasmic reticulum micro-protein called the chloride clic like-1 channel was found to be involved in the endoplasmic reticulum homeostasis. Recent evidence suggests the involvement of endoplasmic reticulum stress in the inflammation pathways of the NLRP3 inflammasome. The relationship between the ER and the NLRP3 inflammasome has not been clearly described. This study aimed at investigating the expression levels of the microprotein CLCC1 to shed a light on the relationship between the endoplasmic reticulum stress and the NLRP3 inflammasome. The expression levels of CLCC1 were analyzed by qPCR in cultured monocytes under different time points of Lipopolysachaaride immuno-stimulation. The stability of expression in candidate reference genes was investigated for normalization purposes. This study reported the regulation of CLCC1 as a novel finding under prolonged LPS exposure of monocytes and stable reference genes such as GUSB and ACTB were identified. The relationship between CLCC1 and NLRP3 inflammasome priming by LPS indicated that CLCC1 is regulated and may be involved in the inflammatory mechanisms of endoplasmic reticulum stress and NLRP3 inflammasome inflammatory diseases, contributing to a potential therapeutic target in the endoplasmic reticulum and inflammasome related diseases.
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Holscher, Maxwell Arthur. "Molecular analysis of NK cell - target cell interactions." Thesis, University of Newcastle Upon Tyne, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287147.
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Zannettino, Andrew Christopher William. "Molecular definition of stromal cell-stem cell interactions /." Title page, contents and summary only, 1996. http://web4.library.adelaide.edu.au/theses/09PH/09phz32.pdf.
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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.
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Lee, Soo Young. "Dissecting Molecular Mechanisms of Shigella flexneri Cell-to-cell Spread." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13065011.
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Shigella is a causative agent of bacillary dysentery in humans. The ability of Shigella to disseminate in the intestinal epithelium is crucial for disease establishment. This process of cell-to-cell spread involves actin-based motility, which allows movement of Shigella through the cytoplasm, and the ability of Shigella to form filopodia-like membrane protrusions that are engulfed by adjacent cells.
Compared to the process of Shigella actin tail assembly, which requires recruitment and activation of host actin modulators such as N-WASP and Arp2/3, the mechanism of how Shigella moves from an infected cell into neighboring cells and what host factors are involved remain poorly characterized. In this dissertation, I investigate whether members of the Ena/VASP family, as key actin regulators, or Inverse-BAR (I-BAR) family proteins, as coordinators of membrane curvature and actin dynamics, are required in dissemination of S. flexneri in a cell monolayer.
Ena/VASP family proteins regulate cell migration, adhesion, shape, and cell-cell interaction. The members of the family include Vasodilator-Stimulated Phosphoprotein (VASP), Ena-VASP-like (Evl), and Mammalian enabled (Mena). We have previously shown that Mena, despite its localization to the actin tail, has no role in S. flexneri actin-based motility. Here, I investigate the role of Mena, Evl, and VASP in S. flexneri dissemination. I determine that the presence of VASP or Evl restricts cell-to-cell spread of S. flexneri. I further show evidence that the conserved EVH1 domain and phosphorylation of VASP regulate the ability of Shigella to spread.
I-BAR proteins, including IRSp53 and IRTKS, contain a conserved domain that directly binds to membrane lipids and induces convex membrane deformation. This unique property and the ability of these proteins to bind F-actin and actin modulators are necessary for the formation of actin pedestals by pathogenic E. coli and filopodia. Using cells with reduced levels of IRTKS or IRSp53, I examine the role of these proteins in cell-to-cell spread and show that neither IRTKS nor IRSp53 is required for S. flexneri spread.
Collectively, these results advance our understanding of host proteins that participate in S. flexneri dissemination.
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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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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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Clements, Sarah L. "Molecular studies of Guard Cell Function." Thesis, University of Sheffield, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521890.
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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.
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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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Schnyder, T. "Molecular mechanisms of B cell activation." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1379268/.
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Our body is under constant attack by pathogens such as viruses or bacteria. The immune system has evolved to efficiently counteract these attacks using an array of cells and soluble proteins. In particular, B cells support the immune system by producing high-affinity antibodies, which target pathogens for destruction by recognition of antigenic structures. B cells furthermore contribute to immunological memory to quickly mount responses to secondary challenges by the same pathogen. In order to produce antibodies, B cells need to get activated in secondary lymphoid organs by a two-step process: First, B cells sense antigen molecules on the surface of antigen-presenting cells using their B cell receptor (BCR), which leads to intracellular signalling and antigen acquisition. B cells then present processed antigen molecules to specific helper T cells. Both BCR ligation and adequate T cell help lead to full B cell activation and the production of antigenspecific antibodies. This thesis investigates the early molecular events of B cell activation. Using a combination of genetics, lipid bilayers and high-resolution microscopy we identify a role for the adaptors Grb2 and Dok-3 alongside the ubiquitin ligase Cbl in antigen gathering by B cells. Moreover, we show recruitment of Grb2, Dok-3 and Cbl to the signalling BCR and establish Grb2 as a central downstream adaptor. We further assess the role of several motor proteins in antigen gathering and find that dynein is recruited to the signalling BCR driving antigen gathering by the movement of antigen-containing BCR microclusters on the underlying microtubule network. Interestingly, recruitment of dynein to BCR microclusters is impaired in B cells lacking Grb2, Dok-3 or Cbl. As the amount of antigen acquired by B cells directly correlates with the extent of T cell help, the data presented significantly contributes to our understanding of the early molecular events underlying B cell activation.
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Halldorsson, Steinar. "Molecular determinants of phleboviral cell entry." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:56c5ef37-b023-4a8f-bdf2-8388226dc3b3.
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Phleboviruses are emerging zoonotic pathogens which constitute a global threat to human and animal health. The mosquito-borne Rift Valley fever virus (RVFV) is a widespread problem across the African continent and causes regular deadly outbreaks in ruminants. The recently emerged severe fever with thrombocytopenia syndrome virus (SFTSV) is a serious human public health concern in China which has rapidly spread to Japan and Korea with fatality rates as high as 16-30%. Phleboviral cell entry is mediated by two viral glycoproteins: the class II fusion protein Gc and the lesser known Gn. Initial cell attachment is glycan dependent and the penetration into the cell cytoplasm is mediated by the Gc fusion protein which catalyses viral and cell membrane merger. The entry mechanism is not well understood from a structural perspective which limits mechanistic insights. The purpose of this thesis is to further our understanding of the cell entry process by filling in the missing structural information on the phleboviral glycoprotein layer. To this end, an integrated structural approach using cryo-EM and X-ray crystallography was adopted. The crystal structure of the Gn ectodomain is presented which reveals an unprecedented structural relationship with seemingly unrelated viruses. Single-particle cryo-EM and localized reconstruction reveal the glycoprotein layer of the RVFV and a pseudo-atomic model of the RVFV is presented. The assembly shows the shielding of the Gc fusion protein and suggests that the Gn functions as a fusion chaperone. The post-fusion crystal structure of the Gc protein from SFTSV further consolidates a mechanism of membrane fusion by class II fusion proteins. Finally, preliminary data on receptor binding and mechanism of antibody mediated neutralization are presented. The work presented herein provides a novel platform for studying and understanding entry and assembly of phleboviruses as well as the design of novel therapeutics.
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Feige, Peter. "Molecular Regulation of Satellite Cell Fate." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/40804.
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Muscle homeostasis and regeneration are complex cellular processes orchestrated by muscle stem cells and their interaction with their stem cell microenvironment. The fate of a muscle stem cell is influenced by different conditions such as muscle injury, cold stress, or disease. During extensive muscle repair and in the context of muscular dystrophy, we identified the critical function of the Epidermal Growth Factor Receptor (EGFR) in establishing cell polarity and in turn the efficient formation of myogenic progeny able to repair muscle. Using a novel drug screen, we identified the p53 protein to regulate muscle stem cell fate decision to repress the formation of brown adipose tissue as a means to regulate whole-body metabolism. To increase the impact of our research we also optimized protocols evaluating mouse satellite cell transplantation to delineate stem cell hierarchy and developed a new paradigm to model human muscle stem cell fate to better translate our findings into the clinical arena. These findings reveal the tunable nature of stem cell fate decisions and highlight the development of research tools to accelerate the translation of research findings to improve human health.
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Harrington, Elizabeth Anne. "Analysis of the molecular regulation of cell proliferation and cell death." Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283286.
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Law, Bic-fai Fian. "Molecular genetics of esophageal squamous cell carcinoma." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B3660446X.
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Knezevich, Stevan Robert. "Molecular characterization of pediatric spindle cell tumors." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0021/NQ48668.pdf.
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Sun, Li. "Molecular cytogenetics of oral squamous cell carcinoma." Click to view the E-thesis via HKUTO, 2002. http://sunzi.lib.hku.hk/HKUTO/record/B38627887.
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Rytkönen, Anne. "Molecular studies of Neisseria - host cell interactions /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-018-4/.
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James, John Robert. "Molecular organisation of the T cell surface." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437053.
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Brodie, Douglas William. "Molecular analysis of T cell costimulatory pathways." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249517.
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Sims, Stuart. "Molecular profiling CD8+ T-cell memory inflation." Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598035.
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Infection with murine cytomegalovirus (MCMV) induces a large population of virusspecific CDS· T-cells that is maintained at a high frequency in the peripheral organs for the lifetime of the host. This striking response to MCMV is termed "memory inflation". It has also been shown that a similar response occurs after immunization with a non-repl icating adenovirus expressing a transgene. The key features which distinguish "infiating" CDS· T-cell memory responses from classica l "non-infiating" CDS· T-cell memory responses have not been fully defined. To determine the molecular signature of memory inflation I sorted antigen-specific ce lls ex vivo and compared the gene expression profiles of infiationary CDS· T-cells to those of central memory and effector CDS+ T-cells. This data showed that memory inflation has a distinct expression profile, with high expression of KLR receptors, specific chemokine receptors such as CX3CR1, transcription factors such as T-bet and Foxk1, and survival factors, such as Bcl-XL and down regu lation of inhibitory molecules such as BTLA. These inflationary cells are functional - expressing an array of cytokines, granzymes, IFNy, and TNFa, and lack transcriptional features of T-cell exhaustion. Similar features were identified in CDS· T-cells undergoing memory inflation using both the MCMV and adenovirus models. To address the turnover of these "inflationary" CDS+ T ce lls and their role in vivo, I created tetramers bound to the toxin saporin to specifically knock out inflationary cells. I found that they contribute to the control of vira l repl ication, since depletion was followed by a rise in viral load and a subsequent rebound increase in the tetrarner+ popul ation. In the final chapter I addressed the ro le of one specific cel l surface molecule, CD73, during CDS· T-cell memory. I found that although CD73 is thought to playa role in immune regu lation, CD73-1 - mice showed normal memory inflation and class ical noninflationary memory development. Overall the data in this thesis define for the first time the transcriptional profile associated with CDS· T-cell memory and show the distinct nature of this T-cell programme in vivo. Future work using further genetically modified mouse strains should allow definition of the critical molecules and pathways involved in the development of these important memory pools.
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Hu, Yingchuan, and 胡穎川. "Molecular pathogenesis of oesophageal squamous cell carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31241797.
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Sun, Li, and 孫莉. "Molecular cytogenetics of oral squamous cell carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B36544267.
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Law, Bic-fai Fian, and 羅璧輝. "Molecular genetics of esophageal squamous cell carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B3660446X.
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Morrissey, Catherine. "The molecular pathology of renal cell carcinoma." Thesis, University of Birmingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420407.
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Yap, Lee Fah. "Molecular characterization of oral squamous cell carcinoma." Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435716.
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Lambert, Sally Ruth. "Molecular profiling of cutaneous squamous cell carcinoma." Thesis, Queen Mary, University of London, 2010. http://qmro.qmul.ac.uk/xmlui/handle/123456789/564.
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Cutaneous squamous cell carcinoma (cSCC) is the second most common form of non-melanoma skin cancer and accounts for the majority of deaths from this disease. Its incidence is increasing rapidly, contributing significant morbidity to patients and a burden on healthcare resources. The molecular events underlying cSCC development remain largely uncharacterised, despite the well established role of ultraviolet radiation as a principal carcinogen. Genomewide analyses of the genetic changes underlying cSCC development have shown they are subject to large chromosomal aberrations, which often involve whole chromosome arms. Many of these events occur in a high proportion of tumours, yet the genes they target are unknown. In this study, genomewide expression microarray data has been obtained from a series of cSCC and integrated with single nucleotide polymorphism (SNP) microarray data, to provide a comprehensive analysis of the events associated with tumour development. In total, 222 genes were identified as differentially expressed in cSCC, of which, 21% were concordant with copy number changes. Previous genomewide SNP data of cSCC had identified microdeletions within the PTPRD gene in a subset of tumours (Purdie et al., 2009). This was investigated in further detail and revealed microdeletions in this gene were significantly associated with metastatic cSCC. Sequencing analysis showed 37% of cSCC had a mutation at this locus, which suggests PTPRD is aberrant in a significant proportion of tumours. Decreased expression levels of PTPRD were correspondingly found in moderately and poorly differentiated tumours. The role of PTPRD in skin biology is not known and further functional work is required to elucidate its role in skin cancer. Taken together, these data provide a valuable insight into the genetic background against which cSCC develop. Furthermore, the association of PTPRD disruption with aggressive tumours may potentially be of future benefit as a prognostic biomarker and therapeutic target.
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Mathur, Divya Ph D. Massachusetts Institute of Technology. "Molecular control of embryonic stem cell identity." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/46786.
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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.
Embryonic Stem (ES) cells are the in vitro derivatives of the inner cell mass of a developing embryo, and exhibit the property of pluripotency, which is the ability of a cell to give rise to all cell lineages of an organism. Therefore, these cells hold great promise in the treatment of several degenerative diseases through patientspecific cell-based therapy. Consequently, a detailed knowledge of the factors regulating ES cell identity is required in order to exploit this therapeutic potential. In order to address this subject, genome-wide location analysis (or ChIP-chip) has been used to identify downstream genes that are bound, and potentially regulated by the key pluripotency transcription factors, Oct4 and Nanog. The data from this study have also been compared and integrated with Oct4 and Nanog DNA binding data obtained in a different study using the ChIP-PET technology. In order to gain further insight into the mechanisms by which the transcription factor Nanog regulates its downstream targets, an attempt at identifying proteins interacting with Nanog has also been described. Research on ES cells has been plagued with ethical controversies since the creation of these cells requires the destruction of embryos. Recent studies have reported the reprogramming of somatic fibroblasts into an ES cell-like induced pluripotent state (iPS) by virus-mediated transduction of four transcription factors-- Oct4, Sox2, c-Myc and Klf4, thereby circumventing the use of embryos in producing pluripotent cells.In these studies, selection for the activation of the markers Oct4 or Nanog led to completely reprogrammed cells, but selection for fbx15, a downstream target of Oct4, resulted in partially reprogrammed intermediates. An unresolved issue in the field was whether these intermediates were obtained due to early drug selection in the case of fbx15 selection, or because Fbx15 expression is not relevant to pluripotency. Drug selection for fbx15 activation at later time-points, and an examination of the methylation status of the Oct4 locus of Fbx15-iPS cells suggests that the intermediates were obtained due to early drug selection and not due to selection for fbx15. Therefore, these studies have begun to elucidate a framework that governs ES cell identity, and the mechanism by which a differentiated cell can be reprogrammed into a pluripotent state.
by Divya Mathur.
Ph.D.
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Noordin, Liza. "Molecular mechanisms of cell proliferation in endometriosis." Thesis, University of Strathclyde, 2011. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=16808.
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Griffiths, Dean Stuart. "Molecular characterisation of embryonic stem cell neurogenesis." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/13958.
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One of the biggest challenges currently facing ES cell biology is to understand the mechanisms involved in the differentiation of ES cells to specific lineages. Pure populations of a specific cell lineage cannot be achieved without selection, such as fluorescence activated cell sorting (FACS), immunopanning or growing cells in a selective environment following genetic manipulation. However, such techniques do not address why ES cells do and do not differentiate to particular lineages and cell types. To achieve greater understanding of the mechanism of neuronal differentiation from ES cells, an ES cell line was generated with eGFP driven by the neuronal specific gene tau. Tau is expressed exclusively in all neurons from the earliest stages of neuronal commitment, we find that a neural differentiation of this line results in eGFP expressing neurons. Using FACS, a pure population of neurons can be obtained from a heterogeneous population of differentiated cells. Neuronal differentiation can be quantified either by fluorescent microscopy or flow cytometry. These ES cells have been used to analyse the effect that density and the addition of exogenous factors have on neuronal differentiation, a transcriptome analysis experiment was performed by microarray analysis. Genes already known to be important during mammalian neural development were analysed for their involvement in ES cell neurogenesis. This comparison revealed a strong correlation between events of ES cells differentiation and normal embryonic development. The microarray analysis of ES cell neurogenesis also identified genes with an expression profile suggestive of a role in ES cell neurogenesis and development of the murine nervous system.
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Laird, Alexander. "Molecular prognostic markers in renal cell carcinoma." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17873.
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Renal cell carcinoma (RCC) is the most deadly of urological malignancies. While metastatic disease affects one third of patients at diagnosis, a further third of patients who undergo extirpative surgery with curative intent subsequently develop metastatic disease. Inconsistency in the clinical course ensures predicting subsequent metastasis is notoriously difficult, despite the routine use of prognostic clinico-pathological parameters in risk stratification. With greater understanding of pathways involved in disease pathogenesis, a number of biomarkers have been proposed to be of prognostic significance; however there are currently no molecular prognostic markers in clinical use. Genetic intra-tumoural heterogeneity (genetic ITH) has been described in clear cell RCC (ccRCC) and may limit the clinical translation of biomarkers. There has been no assessment of ITH at other molecular levels. The aim of this work was to define and compare proteomic, transcriptomic and DNA methylation ITH in ccRCC, and identify potential prognostic biomarkers. Using reverse phase protein arrays to study protein expression in multiple spatially separate regions of primary and metastatic ccRCC, proteomic ITH was demonstrated for the first time. Interestingly there was no significant difference in proteomic ITH in metastatic ccRCC tumour deposits compared to primary tumours. However, on analysis of differential protein expression between primary and metastatic ccRCC tissue using a tissue microarray and automated analysis of immunofluorescence, there was significantly greater expression of Ki67, p53, VEGFR1, SLUG and SNAIL in the metastases compared to the primary tumours. Subsequent profiling of gene expression and DNA methylation in multiple areas of the same primary tumours confirmed transcriptomic and methylomic ITH. On comparison of this multimolecular ITH, significantly greater proteomic ITH was seen compared to gene expression and DNA promoter methylation heterogeneity. Recent evidence suggests DNA methylation may be prognostically important in RCC and given the lower methylomic ITH in ccRCC, the identification of prognostic DNA methylation changes in ccRCC were pursued using the Infinium HumanMethylation450K Beadchip. Following development of an analysis pipeline, identification and validation of prognostic differentially methylated regions (DMR) was performed on an experimental cohort and published dataset respectively. Five DMRs, which were associated with disease recurrence in ccRCC, were identified. NEFM gene promoter methylation was the only DMR associated with cancer specific survival, independent of TNM stage and nuclear grade on multivariate analysis, which was confirmed on a third independent published dataset. This thesis therefore demonstrates multi-molecular ITH in ccRCC for the first time. Despite this, NEFM promoter methylation may be a useful independent prognostic marker of cancer specific survival.
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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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Oliveira, Marta Isabel Abreu. "Molecular interactions at the T cell surface." Doctoral thesis, Instituto de Ciências Biomédicas Abel Salazar, 2007. http://hdl.handle.net/10216/7220.
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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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Godec, Jernej. "Molecular Mechanisms of CD8+ T Cell Differentiation." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493424.
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CD8+ T cells are a crucial component of the adaptive immune system and are required for optimal protection from most pathogens and cancer. They function by secreting pro-inflammatory cytokines and by directly eliminating infected and malignant cells. In order to be effective, CD8+ T cells must be activated through a complex sequence of steps involving engagement of the antigen-specific T cell receptor (TCR) and other receptors, which orchestrate transcriptional, epigenetic, and metabolic changes that direct the differentiation of the responding cells. Following optimal activation, naive CD8+ T cells rapidly undergo clonal expansion and effector differentiation that enables prompt resolution of infection. Following pathogen clearance, a fraction of effector CD8+ T cells differentiate into long-lived memory CD8+ T cells that provide robust protection from re-challenge with the same microbe. However, in the context of persistent abundance of antigen and inflammation, such as in chronic infections and in cancer, the T cells instead become gradually more dysfunctional – a state known as T cell exhaustion.
The overarching goal of this thesis is to identify the cardinal features and molecular mechanisms associated with three main states in which CD8+ T cells exist: T cell memory, T cell exhaustion, and T cell effector differentiation. I used two complementary approaches to examine CD8+ T cells at the different states in vivo. First, I used classical immunology techniques including knockout mice and cellular phenotypic analyses to examine the role of cell surface molecules PD-1 and CD39 on CD8+ T cells in the context of memory and exhaustion, respectively. Secondly, I developed a novel experimental platform that enables gene perturbation in naive CD8+ T cells in vivo during their differentiation. I used this approach to systematically interrogate the transcriptional programming of activated CD8+ T cells and to identify novel regulators of effector differentiation. In a proof of concept study, I used this system to further define how the transcription factor BATF regulates CD8+ T cell activation. Additionally, I used this experimental platform to systematically interrogate the functional role of a set of ~80 transcription factors in CD8+ T cell differentiation, and identified TGIF1 as a novel regulator of this process.
The role of the co-inhibitory receptor PD-1 on CD8+ T was examined in mice using an acute respiratory infection model. PD-1 is a co-inhibitory receptor that is up-regulated on T cells following activation and recruits SHP1/2 phosphatases to directly antagonize signals through the TCR and this way inhibit the activation of T cells. It is down-regulated following the resolution of an acute infection but remains persistently expressed on CD8+ T cells in chronic infections and cancer. As such, PD-1 has been exhaustively studied for its contribution to the functional exhaustion of T cells. However, its role in acute infections remains less defined. We found that this receptor prevents over-activation and over-expansion of CD8+ T cells following initial differentiation, and is crucial for optimal differentiation of effector CD8+ T cells into functional memory cells.
Exhausted CD8+ T cells express several markers distinctive of the state. Some, like PD-1, Tim-3, and Lag-3 are well known. However, genome-wide transcriptional studies identified numerous additional genes that are differentially expressed in the exhausted state. Thus, we hypothesized that additional markers may provide characteristic features of the exhausted cell state and may function in chronic infections. We investigated one such gene – ENTPD1 – that encodes for CD39. This cell surface molecule is an ectonucleotidase that hydrolyzes extracellular ATP into ADP and AMP, which can be further broken down to immunosuppressive adenosine by CD73. In the context of the immune system, CD39 has largely been studied on CD4+ regulatory T cells, which use CD39 as a mechanism to suppress immune responses. However, surprisingly, we found that CD8+ T cells can also express CD39, but its expression is largely restricted to terminally exhausted CD8+ T cells. These cells are most dysfunctional as measured by the most limited proliferative capacity and ability to produce pro-inflammatory cytokines. We have observed this biology in both human and mouse chronic viral infections. Additional studies further demonstrated the importance of CD39 and the purinergic pathway in regulating lethal immunopathology associated with chronic LCMV infection in mice.
In addition to interrogating memory and exhaustion fates of CD8+ T cells, we also examined the initial regulatory programs involved in CD8+ T cell differentiation in vivo through gene silencing. Gene perturbation in naive T cells without prior cellular stimulation has been a continuous challenge in the field. To circumvent this limitation, we engineered a novel experimental platform that enables inducible gene knock-down in any immune cell in mice in vivo without prior manipulation of these cells. Initially, I validated this system by knocking down BATF and confirmed its essential role in CD8+ T cell responses to acute LCMV infection. Additionally, leveraging the inducible nature of the platform, I showed that BATF functions in the early stages of T cell activation but becomes dispensable once its transcriptional program is initiated.
Several other transcription factors such as T-bet, Eomes, Bcl6, and Blimp-1 have been described to regulate CD8+ T cell differentiation. However, numerous additional transcription factors may function in this process based on their rapid up-regulation following T cell activation. I used the novel platform to systematically test the functional relevance of ~80 additional transcription factors in a pooled setting. These experiments identified several novel candidate regulators of this process. We validated one such gene – Tgif1 – to confirm its role in the effector CD8+ T cell differentiation following acute LCMV infection and provide clues to the potential mechanism in which it may function.
The above projects have benefited significantly from genome-wide transcriptional datasets of cells at various states or of different genotypes that we generated or that originate from published studies. One particularly powerful approach to examine differences between different groups is gene set enrichment analysis (GSEA) that examines coordinate up- or down-regulation of sets of genes rather than assessing differential expression of specific genes. This is particularly important because changes in biological processes are often guided by relative small changes of groups of genes that act in concert rather than by a robust expression change of a single gene. This approach, however, is only informative if a relevant gene-set collection is used to analyze the data. Existing collections are largely centered around cancer biology and general biological processes but no extensive gene-set collection existed that contained information describing immune processes. Thus, we created ImmuneSigDB – the largest collection of immunology-focused gene sets to date by identifying, annotating, and reanalyzing ~400 published immunology studies. To show its broad use, we used it to examine the cross-species conservation of transcriptional responses in the immune system. We focused on analyzing transcriptional data from systemic responses to sepsis using GSEA and a novel approach, called leading edge metagene analysis. Using these approaches, we uncovered shared and species-specific biology in mouse and human transcriptional responses to sepsis.
Deciphering CD8+ T cell biology is key for conceptualizing new medical interventions that may boost their activation, memory development, and rejuvenation from functional exhaustion. We have determined that PD-1 is essential for optimal CD8+ T cell memory responses, and that BATF is a key transcription factor initiating effector T cell transcriptional programming. We also identified CD39 as a new marker of terminally exhausted CD8+ T cells and uncovered a key role for purinergic signaling in regulating lethal immunopathology in LCMV Clone 13 infection in mice. Furthermore, we developed a new experimental platform that enables systematic interrogation of gene function in any hematopoietic cell type by inducible knock-down of genes and identified TGIF1 as a novel negative regulator of CD8+ T cell responses. We have also developed a new computational resource to improve analyses of transcriptional profiles in the immune system. Together, the body of work presented in this thesis advances our knowledge of major states of CD8+ T cell biology, uncovering both novel mechanisms underlying CD8+ T cell function, as well as highlighting potential novel therapeutic targets that may be transformative in creating better vaccines, treating infections, or fighting cancer.Medical Sciences
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Rai, Varun. "Molecular modeling of PEM fuel cell electrochemistry /." May be available electronically:, 2008. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Oliveira, Marta Isabel Abreu. "Molecular interactions at the T cell surface." Tese, Instituto de Ciências Biomédicas Abel Salazar, 2007. http://hdl.handle.net/10216/7220.
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Lantela, Daniel. "CUX1 and the Cell Cycle." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119509.
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CUX1 is a transcription factor implicated in the control of cell proliferation. Over-expression of CUX1 is observed in many human tumors and cancer cell lines. Cells constitutively over-expressing the p110 isoform of CUX1 proliferate faster and spend less time in G1 following quiescence. We studied three aspects of CUX1 function and regulation during the cell cycle. In the first part of this study we investigated how over-expression of p110 CUX1 results in a shortened G1. Using quantitative PCR we showed that over-expression of p110 CUX1 caused an increase in the transcription of DDK genes (cdc7 and Dbf4) upon exit from quiescence. Western blot analysis revealed that p110 CUX1 cells display elevated phosphorylation of MCM2 serine 5 (pS5-MCM2) during quiescence, indicating an increased activity of DDK. This was associated with a faster loading of MCM2 onto the chromatin after re-entry into the cell cycle and a shortening of G1 as shown by FACS. In a set of experiments, we investigated how the phosphorylation of CUX1 by cyclinD1-CDK4 contributes to cell cycle regulation. Electrophoretic mobility shift assay (EMSA) analysis revealed that phosphorylation of a recombinant CUX1 protein (1125-1505) by cyclinD1-CDK4 inhibited its DNA binding while PKA activated it. Another recombinant CUX1 protein (1125-1308), missing the C-terminal repression domains, is activated by cyclinD1-CDK4 and inhibited by PKA. Autoradiography and western blot analysis revealed that cyclinD1-CDK4 phosphorylates CUX1 on S1216 while PKA phosphorylates S1215 and S1216. FACS analyses showed that cells expressing mutant p110 CUX1S1215/1216A decrease in size with extended passages in culture and eventually die by apoptosis, indicating the importance of cyclinD1-CDK4 regulation in maintaining cell size. Thirdly, during mitosis CUX1 appears ~15kDa larger when observed by SDS-PAGE. We wanted to search for any large post translational modifications such as ubiquitin. No such modifications were identified, however, using mass spectrometry we demonstrated that during mitosis CUX1 is phosphorylated on at least twelve residues compared to six during G2.CUX1 est un facteur de transcription impliqué dans la régulation de la prolifération cellulaire. La surexpression de CUX1 est observée dans de nombreuses tumeurs humaines et lignées de cellules cancéreuses. Les cellules qui surexpriment constitutivement l'isoforme p110 de CUX1 prolifèrent plus rapidement et passent moins de temps en G1 après quiescence. Nous avons étudié trois aspects de la régulation de CUX1 au cours du cycle cellulaire. Dans la première partie de cette étude, nous avons analysé le mécanisme par lequel la surexpression de p110 CUX1 conduit à une réduction dans la durée de la phase G1. En utilisant la méthode de PCR quantitative, nous avons montré que la surexpression de p110 CUX1 a augmenté la transcription de gènes DDK (Cdc7 et Dbf4) à la sortie de quiescence. L'analyse par immuno-buvardagea révélé que les cellules p110 CUX1 montrent une phosphorylation élevée de pS5-MCM2 pendant la quiescence, ce qui indique une augmentation de l'activité de DDK. Cette phosphorylation élevée est associée à un chargement plus rapide de MCM2 sur la chromatine après entrée dans le cycle cellulaire et un raccourcissement de la phase G1 tel que mesuré par FACS. Dans un deuxième projet, nous avons étudié l'effet de la phosphorylation de CUX1 par le complexe cyclin D1/CDK4 sur la régulation du cycle cellulaire. Des test de liaison à l'AND ont révélé que la phosphorylation d'une protéine recombinante CUX1 (1125-1505) par cyclinD1-CDK4 inhibeé sa liaison à l'ADN tandis que la PKA l'active. À l'inverse, une autre protéine recombinante CUX1 (1125-1308), qui ne contient pas les domaines répression en C-terminaux, est activée par cyclinD1-CDK4 et inhibée par la PKA. L'autoradiographie et l'analyse par immuno-buvardage ont révélé que cyclinD1-CDK4 phosphoryle la sérine 1216, alors que PKA phosphoryle sur les sérines 1215 et 1216. Les analyses par FACS ont montré que les cellules exprimant un mutant p110 CUX1S1215/1216A passent moins de temps en G1, deviennent progressivement plus petites et finissent par mourir par apoptose. Ces résultats suggèrent que la phosphorylation de CUX1 par le complexe cyclinD1-CDK4 sert à contrôler la taille des cellules. Finalement, au cours de la mitose, CUX1 semble ~ 15 kDa plus grands quand on l'observe par SDS-PAGE. Nous avons vérifié si cette différence de poids moléculaire résultait de modifications post-traductionnelles telles que l'ajout d'un peptide de la famille des ubiquitines. Aucune de ces modifications n'a été identifiée, mais en utilisant la spectrométrie de masse, nous avons démontré que, durant la mitose, CUX1 est phosphorylé sur au moins douze résidus par rapport à six au cours de G2.
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Banerjee, Sangeeta. "Host cell response to coronavirus infection." Access restricted to users with UT Austin EID Full text (PDF) from UMI/Dissertation Abstracts Internataional, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3025137.
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Minhas, Raman. "Cytokine-dependent hemopoietic cell linker : role in immune-cell receptor signaling." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79052.
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Adaptor proteins provide mechanisms by which extracellular signals are amplified and downstream effector proteins are regulated. Cytokine-dependent hemopoietic cell linker (Clnk), a third member of the SLP-76 family of adaptor molecules, was previously shown to be a positive regulator of TCR signaling. To better understand the role of Clnk in the immune cell signaling network, we first sought to identify additional, unknown, Clnk-binding partners, ultimately confirming the known partners, namely HPK1 and Fyb. Further studies surrounding the Clnk-HPK1 interaction illustrated important binding regions between the two proteins. Stable transfection studies of Clnk in the SLP-76 deficient cell line, J14, revealed the ability of Clnk to rescue SLP-76 function, particularly in reference to optimal tyrosine phosphorylation and activation of Phospholipase C-gammal and MAP kinase. Transient transfections of the parental Jurkat T-cell line illustrated TCR-inducible, Clnk-dependent, gene promoter activation, including identification of novel downstream transcriptional targets such as the pro-apoptotic Nur77.
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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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凌明達 and Ming-tat Patrick Ling. "A study of molecular and cell biology of prostate tumorigenesis in cell culture." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31223102.
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Annunen-Rasila, J. (Johanna). "Molecular and cell phenotype changes in mitochondrial diseases." Doctoral thesis, University of Oulu, 2007. http://urn.fi/urn:isbn:9789514284427.
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Abstract
The mitochondrial oxidative phosphorylation system (OXPHOS) generates energy but also deleterious reactive oxygen species (ROS). Changes in the cytoskeleton, composed mainly of microfilaments, microtubules and intermediate filaments, have been observed in OXPHOS deficiency. The 3243A>G point mutation in mitochondrial DNA (mtDNA) leads to mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), which is the most common mitochondrial disease. Interestingly, mitochondrial aberrations have been demonstrated in patients with a mutation in NOTCH3, the genetic cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).
Randomization of vimentin intermediate filament direction and length together with slower population growth was observed in myoblasts with 3243A>G, with no difference in the amount of apoptotic cell death. Upon complex IV inhibition (with or without the microtubule-depolymerizing compound nocodazole) or a lack of mtDNA (ρ0) in osteosarcoma cells the vimentin network collapsed perinuclearly, forming thick bundles, whereas complex I inhibition led to thinner vimentin network bundles. Furthermore, the amount of vimentin was increased in ρ0 cells. Mitochondria accumulated around the nucleus upon complex IV inhibition and in ρ0 cells. Analysis of the total proteome revealed that specific OXPHOS deficiencies led to changes in the expression of cytoskeletal proteins and proteins involved in apoptosis, OXPHOS, glycolysis and oxidative stress response. Muscle histochemical and genetic analysis showed ragged red fibres and cytochrome c oxidase-negative fibres to be associated with 5650G>A in a patient with R133C in NOTCH3 and 5650G>A in MTTA. Immunolabelling of cells with R133C and 5650G>A revealed a sparse tubulin network with asters and less abundant mitochondria by comparison with control cell lines. Comparison of nucleotide diversity between CADASIL pedigrees and controls showed increased mtDNA sequence variation in the CADASIL patients. Also maternal relatives in two CADASIL pedigrees differed from each other in their mtDNA.
These findings suggest that defects in OXPHOS lead to selective changes in the vimentin network, which may have a role in the pathophysiology of mitochondrial diseases. They also suggest a relationship between NOTCH3 and mtDNA, and establish the pathogenicity of 5650G>A. The overall results emphasize that a deficiency in the energy converting system together with oxidative stress can lead to cytoskeletal changes.
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Song, Yu. "On the molecular bases of dictyostelium cell death." Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM4052/document.
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Des conditions de carence entrainent une mort cellulaire développementale chez le protiste Dictyostelium discoideum. Dans un système in vitro, des cellules de Dictyostelium sont mises en conditions de carence, puis l'addition des inducteurs DIF-1 ou c-di-GMP conduit à une mort cellulaire vacuolaire. DIF-1 est un polyketide produit par Dictyostelium et induisant la différenciation des cellules pré-tiges. Le dinucléotide cyclique c-di-GMP était connu comme un second messager chez les procaryotes, et comme un déclencheur de l'immunité innée dans des cellules de mammifères. Il a été montré par d'autres que des cellules de Dictyostelium peurent produire et détecter c-di-GMP.Pour analyser la signalisation par c-di-GMP chez Dictyostelium, nous avons utilisé la mutagénèse aléatoire et la mutagénèse ciblée. En utilisant des mutants inactivant stlB ou dmtA, nous avons démontré que DIF-1 endogène ou exogène est nécessaire pour la signalisation par c-di-GMP dans Dictyostelium. En conséquence, nous avons amélioré l'étape de sélection dans une mutagenèse aléatoire en utilisant c-di-GMP et un peu de DIF-1 comme inducteurs, ce qui a produit plusieurs mutants. Par ailleurs j’ai testé par mutagenèse ciblée des hypothèses basées sur les informations connues dans Dictyostelium ou d'autres types de mort cellulaire. Trois molécules ont été essayées, DDX41 comme récepteur putatif de c-di-GMP, l' uniport mitochondrial pour le Ca2+(MCU) et la Na+/K+ATPase (IonA).En résumé, au cours de ma thèse, nous avons démontré une relation entre la signalisation c-di-GMP et a signalisation DIF-1 dans Dictyostelium et identifié plusieurs nouvelles molécules de la mort cellulaire par mutagenèse aléatoireThe protist Dictyostelium discoideum undergoes development cell death when under starvation. To investigate the molecular mechanism of Dictyostelium cell death, an in vitro system has been used. Dictyostelium cells were starved and then cell death was induced by DIF-1 or c-di-GMP. About 40h after induction, cells underwent vacuolar cell death. DIF-1 is a polyketide, produced by Dictyostelium prespore cells, which induces prestalk cell differentiation. c-di-GMP was well known not only as a second messenger produced and sensed by bacteria but also as a trigger of innate immunity in mammalian cells. Dictyostelium was recently found by another laboratory to produce and sense c-di-GMP. To analyze c-di-GMP signaling in Dictyostelium cell death, we used random mutagenesis and targeted mutagenesis. By using the knockout mutants stlB- and dmtA-, we demonstrated that endogenous or exogenous DIF-1 is required for c-di-GMP signaling in Dictyostelium. In contrast, endogenous c-di-GMP is not necessary for exogenous DIF-1 signaling. As a consequence, we improved the selection step in random mutagenesis by using c-di-GMP and a little DIF-1 as inducers, which produced several mutants. Another part of my project was to test by targeted mutagenesis some hypotheses, based on known information in Dictyostelium or other similar cell death types. Three molecules have been tested, the c-di-GMP putative receptor DDX41, the mitochondrial Ca2+ uniporter (MCU) and the Na+/K+-ATPase (IonA).In summary, during my thesis, we have demonstrated a relation between c-di-GMP signaling and DIF-1 signaling in Dictyostelium and identified several new cell death molecules by random mutagenesis
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Child, Fiona Jane. "Molecular studies in primary cutaneous B-cell lymphoma." Thesis, King's College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408514.
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Hobman, Tom Cunningham. "Molecular cell biology of Rubella virus structural proteins." Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/30614.
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Rubella virus (RV) is a small, enveloped, positive-stranded RNA virus in the family Togaviridae, and bears striking similarities to the prototype alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SV) in terms of genome organization and structural protein expression strategy. However unlike alphaviruses, RV infection of cultured cells is characterized by relatively long latency periods, slow replication kinetics, limited cytopathology, and the ability to establish a persistent infection in virtually every cell line capable of supporting its growth.
RV virions contain three structural proteins C, E2, and El which are derived by post-translational processing of a precursor polyprotein pllO (NF₂-C-E2-El-COOH). Processing and intracellular transport of RV structural proteins has been studied by jn vitro and jn vivo expression of RV cDNAs. It was found that targeting of El and E2 into the endoplasmic reticulum was mediated by two independently functioning signal peptides. Coincident with translocation into the ER, both proteins underwent addition of N-linked glycans and proteolytic processing. C protein did not appear to play a role in the processing of pllO. Expression of the RV structural proteins in COS cells revealed that E2 exited the ER, and was transported through the Golgi to the cell surface in an El-independent manner, although coexpression of El seemed to increase the rate of transport. Conversely, El was retained in a Golgi-like region and was not found on the plasma membrane in the absence of E2.
Oligonucleotide-directed mutagenesis of El and E2 cDNAs showed that El andE2 both contain three N-linked glycans respectively. Lack of glycosylation did not appear to affect the intracellular localization of the RV glycoproteins in COS cells. A number of significant differences between RV and SFV/SV structural protein expression strategies were discovered, and their possible relationship to RV virion assembly are discussed.Medicine, Faculty of
Medical Genetics, Department of
Graduate