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1
Seow, Choon Sheong. "Analysis of gene expression in tumour immortalisation." Thesis, University of Aberdeen, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251848.
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Cellular senescence is an irreversible growth-arrested state seen in primary human cells after a finite number of cell divisions both in culture and in vivo. The escape from senescence to immortalisation is often thought to be a prerequisite for carcinogenesis. Many changes in senescent cells are consistent with changes in tissue and organ function with age. I used serial analysis of gene expression (SAGE) to analyse global gene expression profiles in senescent and early passage human foetal fibroblasts (hff). A total of over 20,000 SAGE tags were sequenced and characterised, corresponding to 2,675 unique transcripts. Relative to the early passage hff, transcripts which were found to be markedly increased in senescent hff included those encoding p21WAF1, Cyclin D1, ferritin heavy chain, transforming growth factor-beta induced gene (BIGH3), skin collagenase and amyloid. Amongst them, genes such as skin collagenase and amyloid are known to be up-regulated in human ageing. Together with these known genes, a number of "unknown" genes were up-regulated. Seven differentially expressed genes, up-regulated in senescence (BIGH3, prion, dickkopf (Xenopus laevis) homolog 1 (dkk1), Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2), Rap1a and Cysteine knot superfamily 1, bone morphogenetic protein antagonist 1 and retinoic acid receptor, alpha (RARA)) were selected for validation with reverse transcriptase-polymerase chain reaction (RT-PCR). The roles of these genes are discussed in relation to cancer and neurodegenerative diseases. My study demonstrates that replicative senescence is a complex phenomenon involving genes from the wingless-type (Wnt), mitogen-activated protein (Map) kinase kinases extracellular signal-regulated (Erk) kinase, transforming growth factor beta signalling and epigenetic pathways. The challenges remaining now are to identify senescence-related tumour suppressor gene(s) and to elucidate further the biochemical pathways of senescence.
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Wen, Victoria Wei-Yu Women's & Children's Health Faculty of Medicine UNSW. "Molecular alterations during immortalisation of human endothelial cells." Awarded by:University of New South Wales. Women's & Children's Health, 2009. http://handle.unsw.edu.au/1959.4/44743.
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Replicative exhaustion of endothelial cells (ECs) contributes to the pathogenesis of age-related vascular disorders, including atherosclerosis and impaired wound healing. Conversely, abnormal proliferation of ECs underlies the development of EC-derived malignancies, such as haemangioblastoma and angiosarcoma. The central objective of this thesis was to delineate mechanisms that regulate the replicative lifespan of human ECs and molecular alterations that occur during immortalisation of ECs. The gradual shortening of telomeres (chromosome-end structures) is one mechanism that restricts the replicative lifespan of human ECs. Telomere shortening initiates an irreversible growth arrest or senescence through activation of a TP53-mediated DNA damage response. Expression of the cyclin-dependent kinase inhibitor, p16INK4a, is also increased and reinforces senescence via the retinoblastoma pathway. Overexpression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity and extends the lifespan of human ECs, but is not sufficient for immortalisation. The current study demonstrated that p16INK4a repression by promoter methylation was a frequent event during immortalisation of hTERT-transduced bone marrow ECs (BMECs), occurring in 5 of 12 clones. Repression of p16INK4a concurred with the development of recurring chromosomal aberrations, which appeared to be a consequence of telomere dysfunction and chromosome fusions. Loss of p16INK4a and the development of a complex karyotype were associated with a more transformed phenotype in hTERT-immortalised BMECs. The investigations described in this thesis were the first to associate loss of p16INK4a expression with the accumulation of chromosome aberrations. Repression of p16INK4a in only a subset of immortal BMECs provided impetus for investigating whether there was a functionally analogous defect in the hTERT-immortalised BMECs that retained p16INK4a expression. In normal human cells, oncogenic Ras upregulates p16INK4a and induces senescence independently of telomere shortening. This thesis demonstrates that the immortal BMECs that retained p16INK4a expression had a defective response to oncogenic Ras, which may have contributed to the immortalisation of these cells. Whole genome and proteome analyses identified additional alterations in gene copy number and protein expression specific to p16INK4a-positive or -negative immortal BMECs. Overall, these investigations provide new insight to the potential consequences of p16INK4a repression during carcinogenesis and describe novel molecular alterations that occur during immortalisation of human ECs.
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Grix, Nicola. "Conditional immortalisation of mouse auditory sensory epithelial cells." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414134.
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Linne, Hannah Louise. "Investigating telomerase regulation in human breast cancer cells : a search for telomerase repressor sequences localised to chromosome 3P." Thesis, Brunel University, 2015. http://bura.brunel.ac.uk/handle/2438/11620.
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Cellular immortality is one of the ten hallmarks of human cancer and has been shown to be an essential prerequisite for malignant progression (Hanahan and Weinberg., 2011, Newbold et al., 1982, Newbold and Overell., 1983). In contrast, normal human somatic cells proliferate for a limited number of population doublings before entering permanent growth arrest known as replicative senescence. This is thought to be due to the progressive shortening of telomeric sequences with each round of cell division. Over 90% of human tumours, but not the majority of human somatic cells, have been found to express telomerase activity (Kim et al., 1994). The rate-limiting component of the human telomerase enzyme is the telomerase reverse transcriptase subunit, which is encoded by the hTERT gene. Transfection of hTERT cDNA into normal human fibroblasts and epithelial cells may sometimes be sufficient to confer cellular immortality (Newbold., 2005, Stampfer and Yaswen., 2002). Therefore, de-repression of hTERT and telomerase re-activation are thought to be critical events in human carcinogenesis and is the predominant mechanism by which cancer cells maintain their proliferative capacity. Previously, our group has shown that introduction of a normal, intact copy of human chromosome 3 into the 21NT primary breast cancer cell line by microcell-mediated monochromosome transfer (MMCT), is associated with strong telomerase repression and induction of cell growth arrest within the majority of hybrid clones (Cuthbert et al., 1999). Structural mapping of chromosome 3 within telomerase-positive revertent clones revealed two regions of deletion: 3p21.3-p22 and 3p12-p21.1, thought to harbour the putative telomerase repressor sequence(s). Subsequent studies showed that the chromosome 3p-encoded telomerase repressor sequence(s) mediates its function by means of transcriptional silencing of hTERT, in part, through chromatin remodelling of two sites within intron 2 of the hTERT gene (Ducrest et al., 2001, Szutorisz et al., 2003). Attempts to achieve positional cloning of hTERT repressor sequences on chromosome 3p identified two interesting candidates; the histone methyltransferase SETD2 and an adjacent long non-coding RNA (lncRNA) sequence known as FLJ/KIF9-AS1 (Dr. T. Roberts, unpublished data). Through MMCT-mediated introduction of intact chromosomes 3 and 17 into the 21NT cell line, I have demonstrated that at least two as yet unidentified telomerase repressor sequences (one located on each of these two normal chromosomes) may function to repress telomerase activity within the same breast cancer cell line, which suggests that multiple, independent telomerase regulatory pathways may be inactivated within the same cancer type. Furthermore, by examining the consequences of forced SETD2 and FLJ expression within the 21NT cell line, together with siRNA-mediated knockdown of SETD2 within a single telomerase-repressed 21NT-chromosome 3 hybrid, I have provided evidence to show that neither of these two candidate genes may function as a regulator of hTERT transcription. Through interrogation of relevant literature, a set of four candidate 3 telomerase regulatory genes (BAP1, RASSF1A, PBRM1 and PARP-3) were selected for further investigation based on their location within the 3p21.1-p21.3 region together with their documented role in the epigenetic regulation of target gene expression. Using mammalian expression vectors containing candidate gene cDNA sequences, my colleague Dr. T. Roberts and I demonstrated that forced overexpression of BAP1 and PARP-3 within the 21NT cell line is associated with consistent, but not always sustained, repression of hTERT transcriptional activity and telomerase activity. It is therefore possible that at least two sequences may exist on chromosome 3p that function collectively to regulate hTERT expression within breast cancer cells. Finally, using an in vitro model of human mammary epithelial cell (HMEC) immortalization, involving the targeted abrogation of two pathologically relevant genes, p16 and p53 to generate a series of variant clones at different stages of immortal transformation (developed by my colleague Dr. H. Yasaei), I have shown that single copy deletions on chromosome 3p are a frequent, clonal event, specifically associated with hTERT de-repression and immortal transformation. Subsequent high-density single nucleotide polymorphism (SNP) array analysis of immortal variants carried out by Dr. H. Yasaei, identified a minimal common region of deletion localized to 3p14.2-p22. Together, these findings provide additional evidence to show that chromosome 3p may harbour critical hTERT repressor sequences, that are lost as an early event during breast carcinogenesis.
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Kan, Chin-Yi. "Human Papillomavirus in human breast cancer and cellular immortalisation." Sydney : University of New South Wales. Biotechnology and Biomolecular Sciences, 2007. http://www.library.unsw.edu.au/~thesis/adt-NUN/public/adt-NUN20071004.080541/.
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McDonald, Jacqueline. "Conditional immortalisation of myeloid-precursors to model innate immunity." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/45729/.
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The prevalence of fungal infections is on the rise due to the increase of immune suppressed individuals. Neutrophils are key immune cells in the fight against fungal infections. The study of neutrophil biology is hampered by the short lived nature of the cells and the fact that they cannot be easily genetically modified. In this thesis, I generate and characterise myeloid precursor cell lines that can be genetically manipulated and differentiated into functional neutrophils. These in vitro generated neutrophils were adoptively transferred into live animals and tracked during inflammatory responses. Clec7a, a cell surface β-glucan receptor found on myeloid cells, and its role in immune response to fungal infections has been well characterised in macrophages and dendritic cells but less so on neutrophils. In this thesis, a model for elucidating the role of Clec7a on neutrophils was developed using primary cells and was able to show that Clec7a deficiency on neutrophils impairs recognition of zymosan and C. albicans but that this impairment was largely overcome by serum opsonisation of the particles. The in vitro generated neutrophils were comparably tested and, although the cells have their limitations, they largely supported the conclusions found using primary cells.
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Flanagan, James Michael. "The immortalisation of B-lymphocytes with Epstein-Barr virus /." St. Lucia, Qld, 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16501.pdf.
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Kohli, Jaskaren Singh. "Senescence and immortalisation in melanoma progression and multiple primary melanoma." Thesis, St George's, University of London, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.706529.
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Multiple primary melanoma is defined as the gain of at least one additional independent melanoma and occurs in approximately 5% of melanoma patients. Germline mutations can be identified in genes in these patients, which are known to, or are predicted to result in an extension of melanocyte lifespan e.g. pl6, CDK4, and components of the telomere shelterin cap. We therefore hypothesised that 'normal' melanocytes from pl6 and CDK4 wild-type multiple primary melanoma patients have a statistically longer lifespan compared to those from single primary melanoma patients. Melanocytes from multiple primary melanoma patients did display a significantly extended culture lifespan, independently of donor age. Multiple primary melanoma is therefore commonly associated with a delay in normal melanocyte senescence. There is currently a shortage of diagnostic markers for melanoma and novel ones are needed for more accurate diagnosis and prognosis. TERT (the enzymatic component of telomerase) expression is the commonest route to telomere maintenance, required for melanoma immortality. TERT expression was tested via immunohistochemistry in a series of melanoma precursor and melanoma lesions, to analyse at which point in progression its expression is activated. The protein was found to be localised in either the nucleolus, the nucleoplasm (designated non-nucleolarTERT), or both. Only non-nucleolarTERT expression significantly increased with melanoma progression, suggesting this location is associated with immortality. As senescence likely needs to be bypassed for advanced melanoma development, microarrays were previously carried out comparing growing and senescent wild-type and pl6-null melanocyte lines to evaluate significantly up- or downregulated genes which could be used as future markers. In the present study, potential novel markers were authenticated using PCR and immunoblotting and validated genes were analysed via immunohistochemistry in a series of melanoma precursor and melanoma lesions. ETS1 was tested owing to recent findings that it can bind to and activate the mutant TERT promoter found commonly in melanomas. ETS1 was expressed at all stages from benign nevi onwards, perhaps owing to its link with the MAPK pathway.
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Zwermann, Birgit. "Die Rolle der Telomerase in der Immortalisation und malignen Transformation von Nebennierenrindenzellen." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-145498.
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Whitaker, Noel James. "Involvement of p53 and RB-1 in the immortalisation of human cells." Thesis, The University of Sydney, 1995. https://hdl.handle.net/2123/27505.
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Normal diploid mammalian cells undergo a finite number of population doublings in culture before they undergo senescence [Hayflick & Moorhead, 1961]. In contrast, tumours often contain "immortalised" cells that exhibit an apparently unlimited in vitro and in vivo proliferative potential. Fusion of normal and immortalised cells usually results in hybrids with limited proliferative potential [Bunn & Tarrant, 1980; Muggleton-Harris & DeSimone, 1980] indicating that immortalisation is probably due to loss of normal gene function. Similarly, fusion of different immortalised human cell lines with each other often results in mortal hybrids, indicating that the cell lines have become immortalised via different genetic events. Such studies have identified at least four complementation groups for immortalisation, referred to as groups A, B, C and D (Pereira—Smith & Smith, 1988).
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Keough, Rebecca Anne. "An investigation of hair follicle cell immortalisation and hair keratin gene regulation /." Title page, table of contents and summary only, 1995. http://web4.library.adelaide.edu.au/theses/09PH/09phk373.pdf.
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Parouchev, Alexandre. "Immortalisation et transplantation de cellules hépatiques simiennes : modèles de thérapie cellulaire hépatique." Paris 7, 2010. http://www.theses.fr/2010PA077099.
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La transplantation orthotopique de foie est le seul traitement curatif de certains déficits métaboliques. Mais, le manque de donneurs et les complications liées à l’ immunosuppression incitent au développement d'alternatives thérapeutiques, dont la transplantation d'hépatocytes. Cependant, à ce jour les essais avec hépatocytes adultes sont peu fructueux. Nous avons exploré deux voies thérapeutiques. D'une part dans la recherche de sources alternatives de cellules, nous avons caractérisé une lignée de cellules hépatiques fœtales simiennes (IPFLS), immortalisées par l'antigène T de SV40, à long terme. In vitro ces cellules conservent leur capacité de prolifération et leur phénotype de progéniteurs hépatiques bipotents. Leur immortalisation est réversible après excision de Foncogène par Cre. In vivoy elles sont non tumorigènes et se différencient en hépatocytes, sans proliférer in situ. Toutefois, leur importante instabilité du caryotype souligne la nécessité d'évaluer d'autres gènes d'immortalisation. D'autre part, la thérapie génique ex vivo est une alternative à l’allotransplantation d'hépatocytes. Nous avons mis au point la transduction d'hépatocytes simiens adultes, fraîchement isolés et cryopréservés, en suspension par des lentivirus recombinants dérivés de VTH-1, exprimant GFP sous contrôle du promoteur hépatospécifique du gène de l’apolipoprotéine A-II humain. La fonctionnalité in vivo, des hépatocytes cryopréservés est attestée par la présence d'hépatocytes transduits trois mois après transplantation dans le foie de souris immunodéficientes. Le modèle du primate non humain est un bon modèle préclinique pour des approches de thérapie génique ex vivo à visée hépatiqueOrthotopic liver transplantation is the only available cure for certain metabolic deficiencies. But, lack of donors and complications related to immunosuppression urge the development of alternatives therapies such as hepatocyte transplantation. However, trials with adult hepatocytes have been so far disappointing. We have explored two directions. As a model for an alternative source of hépatocytes, we have characterized a line of simian fetal hepatic cells (IPFLS), immortalized by SV40-T antigen. In vitro these cells conserve their proliferative capacity and their hepatic bipotent progenitor phenotype. The immortalization is reversible after Cre-mediated excision of the oncogene. In vivo, they are non tumorigenic and differentiate into hepatocytes, with no in situ proliferation. However, the important karyotype instability underlines the need for new immortali2ation strategies. Moreover, ex vivo gene therapy is considered as an alternative to hepatocyte allotransplantation. We have set up conditions for efficient transduction of freshly-isolated and cryopreserved, adult, simian hepatocytes in suspension by HIV-1-derived recombinant lentiviruses, expressing GFP under transcriptional control of the hepatospecific promoter of the human apolipoprotein A-II gene. The in vivo persistence and fonction of cryopreserved cells is demonstrated by the presence of transduced hepatocytes three months after transplantation into the liver of immunodeficient mice. Thus the nonhuman primate appears as a good preclinical model for the development of liver-directed ex vivo gene therapy strategies
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Muntoni, Alessandra. "Molecular changes involved in oral cancer progression and their relevance to keratinocyte immortalisation." Thesis, University of Glasgow, 2002. http://theses.gla.ac.uk/1710/.
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Oral cancer has caused great concern in all of the western countries over the past two decades because of its progressively increasing incidence, mainly in young males, and its consistently low 5-year survival rate. Oral premalignant lesions (dysplasias) are thought to precede the development of cancer, but no clinical or histological criteria is at present available to predict their potential for malignant transformation. Therefore, it would be of diagnostic and therapeutic relevance to understand the molecular event, involved at different stages of oral cancer. Using a unique series of primary cultures from biopsies of normal oral mucosa, dysplasias and squamous cell carcinomas we have identified molecular changes characteristic of early oral cancer progression. Our group reported previously that acquisition of the immortal phenotype is an early event in oral cancer development (McGregor et al. 1997): data obtained during the course of this thesis project indicate that about half of oral dysplasia cultures are immortal and this is associated with loss of expression of retinoic acid receptor (RAR)-b and the cell cycle inhibitor, p16INK4a, p53 mutations and increased levels of telomerase/hTERT mRNA. In contrast, increased expression of the EGF receptor (EGF-R), known to be a characteristic of oral cancer, does not occur until after the dysplasia stage in squamous cell carcinomas (SCCs). Interestingly, one atypical mortal dysplasia with a considerably extended lifespan has lost expression of RAR-b and p16INK4a, but it still expresses wild-type p53 (albeit at higher level than normal) and has not activated telomerase. Further experiments demonstrate that retroviral transduction of hTERT immortalises D17 and this is associated with telomere lengthening but p53 remains wild-type. In contrast, transduction of hTERT does not extend the lifespan of two other typical mortal dyplasia cultures (that retain RAR-b and p16INK4a expression), even though telomeres are lengthened. Thus, telomerase activation/telomere maintenance is not sufficient per se to immortalise mortal dysplasias without concomitant loss of RAR-b and/or p16INK4a, but p53 mutation is not required if telomerase is activated exogenously. However, further work is required to clarify whether the molecular changes associated with the acquisition of an immortal phenotype in culture have any clinical or prognostic significance.
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Berrington, Mary. "Investigation of the role of chromosome 7 in human cell immortalisation and cancer." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301836.
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Tzellos, Stelios. "Mechanism of superior B cell immortalisation activity of type 1 Epstein-Barr virus." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/23223.
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Epstein-Barr virus (EBV) establishes lifelong latent infections in humans. EBV isolates worldwide are classified as type 1 or type 2 based on their EBNA-2 gene sequence. Type 1 EBV is more efficient at B cell transformation, a property previously mapped to the EBNA-2 locus. Previous work using EREB2.5 cells in a trans-complementation assay showed that the superior ability of type 1 EBNA-2 to sustain B cell proliferation is mostly determined by its C-terminal region. In this study, conversion of a single amino acid in the transactivation domain (TAD), from serine in type 2 EBNA-2 to the aspartate residue in the type 1 protein (S442D), was remarkably found to confer the type 1 growth-promoting phenotype in the EREB2.5 growth assay. The mechanism of the greater transformation efficiency of type 1 EBV appears to involve differential regulation of EBNA-2 target genes. The superior growth properties of type 1 EBNA-2 correlate with the greater induction and activation of viral LMP-1 and cellular CXCR7, compared to type 2 EBNA-2. 5' RACE was used to identify the transcription start site (TSS) of the CXCR7 promoter transcribed in response to EBNA-2. In chromatin immunoprecipitation (ChIP) assays, type 1 EBNA-2 was found to associate more strongly than the type 2 protein with EBNA-2 binding sites near the LMP-1 and CXCR7 genes. D442 was shown to increase binding of type 2 EBNA-2 to some sites at differentially regulated genes. Unbiased motif searching identified an ETS-interferon regulatory factor (IRF) composite element (EICE) that closely resembles the sequence known to mediate EBNA-2 regulation of the LMP-1 promoter. This element may therefore confer the differential effects of type 1 and type 2 EBNA-2 on both LMP-1 and cell gene activation resulting in superior immortalisation by type 1 EBV.
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Boolay, Sihaam. "Immortalisation, characterisation and differentiation of temperature sensitive cell lines from the Olfactory Neuroepithelium." Doctoral thesis, University of Cape Town, 1999. http://hdl.handle.net/11427/22552.
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Bibliography: p. 194-210.Embryonic olfactory neuroepithelium provides a useful experimental system for the study of olfactory neurogenesis. As a substrate for experimental neural cell biology, olfactory neuroepithelium is of unique interest since, unlike other neural cells, olfactory neurons are continually replaced - a feature that is dictated by their direct exposure to the damaging external environment. Basal cells in the olfactory placode are the source of this replacement. Each olfactory neuron expresses only one or a few of the many olfactory receptors that are encoded by the large array of olfactory genes. Despite this limited cellular display of receptors, vertebrates are able to distinguish many thousands of different odorants, implying a complicated need for perceptive neurological processing of signals coming from individual olfactory neurons. To study the events that take place during the differentiation of neuronal precursors - a process that sustains a diverse receptor repertoire - I felt that lines of conditionally immortalised cells that could be induced to differentiate would provide useful reagents. In this thesis I describe my successful attempts to immortalise olfactory cell lines from the neuroepithelium of E 10.5 mouse embryos. I used a conditionally immortalising retrovirus that included the coding sequence for the temperature-sensitive SY 40 large T antigen. Integration of this retrovirus into the genome of cells allowed continuous proliferation at the permissive temperature of 33°C. A shift to the nonpermissive temperature of 39°C inactivated the SV40 large T antigen, the cells ceased proliferation and differentiation commenced. Sixty cell lines were derived of which four were chosen for further characterisation. These four cell lines (OP6, OP27, OP47 and OP55) were clonally derived and were immortalised rather than transformed. They continued to express the SV40 large T antigen at 33°C but lost expression at 39°C concomitant with cessation of proliferation. When the OP cells were shifted to 39°C in the absence or presence of the morphogen, retinoic acid, morphological changes ensued that were consistent with the development of neuronal characteristics. The OP6, OP27 and the OP47 cells became phase-bright with neuritic extensions. The OP55 cells were the exception in that they did not develop extensions but instead differentiated to form compact epithelial islands when grown in DM-10 medium but not in RA medium. Differentiation of the OP cells at 39°C was further documented by the induced expression of a number of markers demonstrated by RT-PCR and/or immunocytochemistry. The OP cells differentiated at 39°C in DM-10 and in retinoic acid-containing medium to express olfactory receptor transcripts. Cloning and sequencing showed that each cell line expressed a single receptor type but that different receptors were expressed by different cell lines. Sequencing revealed that the receptors cloned from the OP27 cells were 98% homologous to the mouse-M65 olfactory receptor whereas OP55 had greatest homology to rat-Olf3 olfactory receptor. The transcripts induced in OP6 and the OP47 cells showed greatest homology with Gus58 - a taste receptor homologous to olfactory receptors. Sequences obtained from OP6, OP47 and OP55 cells were not 100% identical to published receptors and could thus represent members of different subfamilies. Interestingly, induced OP55 cells also expressed mRNA for clusterin - a molecule that has no homology with olfactory receptor transcripts but is involved in differentiation during embryogenesis.
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Bayad, Jamal. "Immortalisation de lignées cellulaires hépatocytaires : expression et régulation des enzymes du métabolisme des xenobiotiques." Nancy 1, 1991. http://www.theses.fr/1991NAN10450.
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Aure, Karine. "Physiopathologie moléculaire et cellulaire des maladies mitochondriales à présentation neurologique." Paris 6, 2007. http://www.theses.fr/2007PA066281.
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Les maladies mitochondriales sont liées à un déficit de la chaîne respiratoire mitochondriale. Ces maladies posent des problèmes de diagnostic, dans leurs relations phénotype/génotype et dans leurs mécanismes physiopathologiques. L'étude longitudinale d'un cas de déficit en ubiquinone a démontré les limites de la supplémentation thérapeutique. L'analyse de l’histoire naturelle et des caractéristiques moléculaires de 69 patients porteurs de délétion de l’ADN mitochondrial a permis d'établir une nouvelle classification clinique et des facteurs pronostiques. L’efficacité de l’immortalisation par le gène de la télomérase a été démontrée dans les fibroblastes déficitaires. Nous avons montré la présence d’apoptose dans le muscle de patients avec mutations de l'ADN mitochondrial. Les difficultés de la démonstration du pouvoir délétère des mutations homoplasmiques de l’ADN mitochondrial ont été abordées à travers l'étude de familles portant la même mutation de l'ARN de transfert de la proline.
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Powell, Andrew Jonathan. "Studies on the immortalisation of rodent embryo fibroblasts by simian virus 40 large tumour antigen." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307774.
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Bernard, Rozenn. "Transfert de gènes dans des précurseurs gliaux : immortalisation cellulaire, prolifération et différenciation des lignées établies." Paris 11, 1994. http://www.theses.fr/1994PA11T009.
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Allain, Jean-Etienne. "Immortalisation et transplantation de cellules foetales hépatiques simiennes et humaines : modèles de thérapie cellulaire hépatique." Paris 7, 2002. http://www.theses.fr/2002PA077004.
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Perrem, Kilian Thomas. "Molecular Studies of an alternative lengthening of telomeres (ALT) mechanism." Thesis, The University of Sydney, 2001. http://hdl.handle.net/2123/793.
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Telomeres are specialised structures, consisting of TTAGGG DNA repeats and binding proteins, that cap the ends of human chromosomes and maintain chromosome integrity. It has been shown that telomeres shorten with each round of cell division in most normal human somatic cells. It has become generally accepted that this shortening is due, in part, to the inability of DNA polymerases to replicate the extreme ends of chromosomes which is a phenomenon known as the 'end replication problem'. An intriguing hypothesis that has emerged from these observations is that critically shortened telomeres trigger growth arrest and senescence. This is regarded as a key determining factor in the limited lifespan of normal cells in culture and is commonly known as the 'Telomere Hypothesis of Senescence'. In support of this hypothesis it has been demonstrated that immortalised human cells, that have an unlimited lifespan in culture, maintain stable telomere lengths and do not undergo progressive telomere shortening. In most cases this is due to the ribonucleoprotein enzyme telomerase, the activation of which is as a key step in the immortalisation process. Telomerase compensates for sequential telomere shortening by utilising an RNA template to catalyse the addition of repeat sequences by reverse transcription. It is absent from most normal tissue but is present in the germline and is presumably downregulated during development. Significantly, analysis of human tumour cells has shown that a majority also have active telomerase, which supports the importance of immortalisation in tumourigenesis. Previous work in this laboratory has shown that, although the majority of in vitro immortalised cells and tumour cells that have been studied maintain telomeres by reactivation of telomerase, a proportion do not have detectable telomerase activity. These telomerase-negative cells still maintain telomeres, however, and this is via a mechanism(s) yet to be fully elucidated known as Alternative Lengthening of Telomeres (ALT). ALT is characterised, in addition to lack of telomerase activity, by extreme telomere length heterogeneity with telomere lengths ranging from over 50 kilobases (kb) of DNA to almost undetectable. This phenotype is evident, by Southern analysis and fluorescent in situ hybridisation (FISH), in all ALT cells. Alternative mechanisms of telomere maintenance, via retrotransposition and recombination, had already been characterised in lower eukaryotes. It has been shown in this laboratory that ALT cell lines and tumours contain a novel type of PML body, referred to as ALT-associated PML bodies (APBs). APBs have been found in all of the ALT cell lines so far tested and also in archival tumour sections, and contain a number of factors which co-localise. These include PML, TTAGGG repeats, TRF 1 & TRF 2 telomere binding proteins and proteins involved in homologous recombination: RAD51 & RAD52. More recently, it has been shown that the RAD50/Mre11/Nbs1 complex, which is involved in cell cycle checkpoint control and repair of DNA damage, is also present in APBs. The presence of these RAD proteins in APBs is of great interest as a recombination between telomeres has been proposed as the central mechanism by which ALT lengthens telomeres. Studies in yeast have identified such a mechanism and it was proposed that a similar process occurred in human immortal cells that utilise ALT. It has now been shown by this laboratory that a recombination mechanism is indeed evident at the telomeres of ALT cells. To date all in vitro immortalised cell lines and most tumour cell types that have been studied have a telomere maintenance mechanism either via telomerase or ALT. Targeting telomerase has become a major focus of anti-cancer research as inhibitors have the potential to treat a wide variety of different tumour types. An understanding of ALT and its regulation is likely to be important in such therapeutic strategies, as selective pressure due to telomerase inhibition may result in ALT revertants within the tumour mass. Development of inhibitors of both telomerase and ALT may therefore be required when targeting telomere maintenance. The main focus of this thesis is the understanding of ALT repression in the SV40 immortalised skin fibroblast cell line GM847, as a means to further understanding the mechanism of ALT. The data presented provide new insights into the repression of ALT and also the relationship between telomerase and ALT which is important for our understanding of telomere maintenance in human cancer. Hybrids formed by fusion of normal cells and ALT cells underwent rapid telomere loss followed by senescence, indicating that normal cells contain factors that repress ALT. This strongly suggests that ALT is recessive and is activated in part by loss or mutation of repressors. Similar experiments were performed with ALT cells and telomerasepositive cells, and the resulting hybrids were all telomerase-positive and ALT repressed. It is possible that the same negative regulators are involved as additional data show that telomerase does not act as an ALT inhibitor. Exogenous expression of telomerase in ALT cells did not repress ALT, but both mechanisms co-existed in these transfected cells. This result provides a further argument for targeting both ALT and telomerase in any future treatments of tumours as it demonstrates in principle that these mechanisms are not mutually exclusive. A serendipitous finding was that a dominant-negative telomerase catalytic subunit caused telomere shortening in ALT cells, had not been reported elsewhere, and indeed was in contrast to previous findings. This provided further evidence for a link between telomerase and ALT as it suggested that there were essential components that were common to both pathways. As a further means to understanding ALT repression, a series of experiments was performed to determine the chromosomal localisation of ALT repressor(s) by microcell mediated chromosome transfer. This was done to facilitate the eventual isolation of repressors. A repressor of ALT in the chemically immortalised fibroblast cell line SUSM-1, had been reported to be localised to chromosome 7. This result could not be repeated in the GM847 cell line, but ALT repression was evident in GM847 cells upon transfer of chromosome 6. Another important question regarding the nature of ALT is the structure and sequence of the long heterogeneous telomeres generated by ALT specific recombination, which is the focus of the final series of data that is presented. ALT telomere length heterogeneity was detected under denaturing conditions, ruling out the possibility that it was an artefact of electrophoresis conditions due to novel secondary structure. Although the hybridisation signal intensity of TTAGGG increases at the onset of immortalisation in ALT cells, it had been demonstrated by restriction digests that degenerate repeats did exist at the telomeres of some ALT cell lines. Sequences containing telomere repeats were cloned from the ALT cell line WI38 VA13/2RA (SV40 immortalised fibroblasts) and these were found to be interspersed with a number of other sequence fragments. The significance of these sequences in relation to the mechanism of ALT is discussed.
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Perrem, Kilian Thomas. "Molecular Studies of an alternative lengthening of telomeres (ALT) mechanism." University of Sydney. Children's Medical Research Institute, 2001. http://hdl.handle.net/2123/793.
Full textAbstract:
Telomeres are specialised structures, consisting of TTAGGG DNA repeats and binding proteins, that cap the ends of human chromosomes and maintain chromosome integrity. It has been shown that telomeres shorten with each round of cell division in most normal human somatic cells. It has become generally accepted that this shortening is due, in part, to the inability of DNA polymerases to replicate the extreme ends of chromosomes which is a phenomenon known as the �end replication problem�. An intriguing hypothesis that has emerged from these observations is that critically shortened telomeres trigger growth arrest and senescence. This is regarded as a key determining factor in the limited lifespan of normal cells in culture and is commonly known as the �Telomere Hypothesis of Senescence�. In support of this hypothesis it has been demonstrated that immortalised human cells, that have an unlimited lifespan in culture, maintain stable telomere lengths and do not undergo progressive telomere shortening. In most cases this is due to the ribonucleoprotein enzyme telomerase, the activation of which is as a key step in the immortalisation process. Telomerase compensates for sequential telomere shortening by utilising an RNA template to catalyse the addition of repeat sequences by reverse transcription. It is absent from most normal tissue but is present in the germline and is presumably downregulated during development. Significantly, analysis of human tumour cells has shown that a majority also have active telomerase, which supports the importance of immortalisation in tumourigenesis. Previous work in this laboratory has shown that, although the majority of in vitro immortalised cells and tumour cells that have been studied maintain telomeres by reactivation of telomerase, a proportion do not have detectable telomerase activity. These telomerase-negative cells still maintain telomeres, however, and this is via a mechanism(s) yet to be fully elucidated known as Alternative Lengthening of Telomeres (ALT). ALT is characterised, in addition to lack of telomerase activity, by extreme telomere length heterogeneity with telomere lengths ranging from over 50 kilobases (kb) of DNA to almost undetectable. This phenotype is evident, by Southern analysis and fluorescent in situ hybridisation (FISH), in all ALT cells. Alternative mechanisms of telomere maintenance, via retrotransposition and recombination, had already been characterised in lower eukaryotes. It has been shown in this laboratory that ALT cell lines and tumours contain a novel type of PML body, referred to as ALT-associated PML bodies (APBs). APBs have been found in all of the ALT cell lines so far tested and also in archival tumour sections, and contain a number of factors which co-localise. These include PML, TTAGGG repeats, TRF 1 & TRF 2 telomere binding proteins and proteins involved in homologous recombination: RAD51 & RAD52. More recently, it has been shown that the RAD50/Mre11/Nbs1 complex, which is involved in cell cycle checkpoint control and repair of DNA damage, is also present in APBs. The presence of these RAD proteins in APBs is of great interest as a recombination between telomeres has been proposed as the central mechanism by which ALT lengthens telomeres. Studies in yeast have identified such a mechanism and it was proposed that a similar process occurred in human immortal cells that utilise ALT. It has now been shown by this laboratory that a recombination mechanism is indeed evident at the telomeres of ALT cells. To date all in vitro immortalised cell lines and most tumour cell types that have been studied have a telomere maintenance mechanism either via telomerase or ALT. Targeting telomerase has become a major focus of anti-cancer research as inhibitors have the potential to treat a wide variety of different tumour types. An understanding of ALT and its regulation is likely to be important in such therapeutic strategies, as selective pressure due to telomerase inhibition may result in ALT revertants within the tumour mass. Development of inhibitors of both telomerase and ALT may therefore be required when targeting telomere maintenance. The main focus of this thesis is the understanding of ALT repression in the SV40 immortalised skin fibroblast cell line GM847, as a means to further understanding the mechanism of ALT. The data presented provide new insights into the repression of ALT and also the relationship between telomerase and ALT which is important for our understanding of telomere maintenance in human cancer. Hybrids formed by fusion of normal cells and ALT cells underwent rapid telomere loss followed by senescence, indicating that normal cells contain factors that repress ALT. This strongly suggests that ALT is recessive and is activated in part by loss or mutation of repressors. Similar experiments were performed with ALT cells and telomerasepositive cells, and the resulting hybrids were all telomerase-positive and ALT repressed. It is possible that the same negative regulators are involved as additional data show that telomerase does not act as an ALT inhibitor. Exogenous expression of telomerase in ALT cells did not repress ALT, but both mechanisms co-existed in these transfected cells. This result provides a further argument for targeting both ALT and telomerase in any future treatments of tumours as it demonstrates in principle that these mechanisms are not mutually exclusive. A serendipitous finding was that a dominant-negative telomerase catalytic subunit caused telomere shortening in ALT cells, had not been reported elsewhere, and indeed was in contrast to previous findings. This provided further evidence for a link between telomerase and ALT as it suggested that there were essential components that were common to both pathways. As a further means to understanding ALT repression, a series of experiments was performed to determine the chromosomal localisation of ALT repressor(s) by microcell mediated chromosome transfer. This was done to facilitate the eventual isolation of repressors. A repressor of ALT in the chemically immortalised fibroblast cell line SUSM-1, had been reported to be localised to chromosome 7. This result could not be repeated in the GM847 cell line, but ALT repression was evident in GM847 cells upon transfer of chromosome 6. Another important question regarding the nature of ALT is the structure and sequence of the long heterogeneous telomeres generated by ALT specific recombination, which is the focus of the final series of data that is presented. ALT telomere length heterogeneity was detected under denaturing conditions, ruling out the possibility that it was an artefact of electrophoresis conditions due to novel secondary structure. Although the hybridisation signal intensity of TTAGGG increases at the onset of immortalisation in ALT cells, it had been demonstrated by restriction digests that degenerate repeats did exist at the telomeres of some ALT cell lines. Sequences containing telomere repeats were cloned from the ALT cell line WI38 VA13/2RA (SV40 immortalised fibroblasts) and these were found to be interspersed with a number of other sequence fragments. The significance of these sequences in relation to the mechanism of ALT is discussed.
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Horton, Sarah Jayne. "Establishment of an in vitro model to identify the molecular mechanism of immortalisation by MLL-ENL." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1444423/.
Full textAbstract:
The t (l I 19)(p22 q23) translocation, which gives rise to the MLL-ENL fusion protein is commonly found in infant acute leukaemias of both the myeloid and lymphoid lineage. In order to study the molecular mechanism of haematopoietic progenitor cell (HPC) immortalisation by MLL-ENL, a conditional system of MLL- ENL expression was established in primary murine HPCs. This was achieved by delivering the Tet-Off inducible expression system to primary cells using two retroviral expression constructs. Several conditional immortalised myeloid cell lines were generated in vitro which were dependent on continued MLL-ENL expression for their survival and proliferation. The immortalised cells either terminally differentiated or died when MLL-ENL expression was turned off with doxycycline. Since several Hox genes are targets of MLL, the expression profile of all 39 murine Hox genes was analysed in the MLL-ENL immortalised cell lines by real-time quantitative PCR. Loss of MLL-ENL expression resulted in a decrease in the expression of multiple Hoxa genes. By comparing these changes with Hox gene expression in cells induced to differentiate with granulocyte-colony-stimulating factor, we found that reduced Hox expression was specific to loss of MLL-ENL expression and was not a consequence of differentiation. Affymetrix microarray analysis revealed that MLL-ENL may maintain or activate the expression of the transcription factor Sdccag33 and the serine / threonine kinase Pim-2, which confers protection from apoptosis. The analysis also demonstrated that MLL-ENL may repress the expression of apoptosis promoting genes such as Ddb2 and Aatk. In summary, MLL-ENL is required to initiate and maintain the immortalisation of myeloid progenitors and may contribute to immortalisation by aberrantly maintaining the expression of multiple Hoxa genes. The pathways regulated by multiple Hoxa genes and the MLL-ENL target genes identified by Affymetrix analysis represent new possibilities for therapies which may combat these aggressive leukaemias.
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BERTHON, PHILIPPE. "Immortalisation et transformation tumorale de l'epithelium mammaire humain normal : caracterisation et reponse aux facteurs de croissance." Paris 6, 1992. http://www.theses.fr/1992PA066043.
Full textAbstract:
En utilisant un vecteur plasmidique recombine avec le grand t de sv40 (t-sv40) nous avons immortalise des cellules epitheliales mammaires humaines normales (cemh) etablies en culture primaire a long terme. La transfection genomique et fonctionnelle des cemh par t-sv40 a permis l'etablissement des lignees immortalisees s2t2, s1t3 et s3t3. La lignee s2t2 est a l'origine de plusieurs clones ns2t2 de cellules transformees. Les cemh proliferent pendant 32 generations avec un temps de doublement de 446 heures. Etudiee pendant 2 ans, la proliferation des lignees mammaires immortalisees et transformees est augmentee de plus de 25%. Le caryotype des cellules transfectees est diploide et presente des anomalies clonales couramment observees dans les cancers du sein (trisomie 1q pour s1t3 et s3t3, trisomie 8q pour s2t2). Les caracteristiques epitheliales et les determinants specifiques des cellules mammaires normales, immortalisees et transformees sont confirmes en microscopie optique et electronique, en immuno-histochimie par l'expression d'antigenes specifiques de la glande mammaire, des cytokeratines 18 et 19 specifiques des cellules luminales. Ces cellules mammaires normales, immortalisees et tumorales possedent un recepteur de l'egf fonctionnel sur la proliferation cellulaire, un recepteur vipergique couple au systeme adenylate cyclase. La presence eventuelle de recepteurs aux strogenes n'a pu etre confirmee dans les cemh et les cellules transfectees (immuno-histochimie et dosage radio-immunologique). Par contre, nous detectons une faible expression des recepteurs a la progesterone. L'stradiol n'est pas mitogene sur les cellules normales mais augmenterait la sensibilite des cemh a l'egf. La transfection des cemh par t-sv40 a permis d'obtenir, lors d'une etape unique, leur immortalisation puis leur transformation. Ces modeles permettent une analyse sequentielle et relativement simplifiee des mecanismes genetiques et moleculaires impliques dans la progression de l'epithelium mammaire vers la transformation tumorale. Les lignees immortalisees s2t2, s1t3 et s3t3 peuvent etre egalement le support de transfections secondaires par les oncogenes cellulaires representatifs des cancers du sein, i. E. C-erbb-2 et ras
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Pickles, Jessica Chiara. "Mechanisms of senescience bypass in cells derived from the Syrian hamster embryo cell transformation assay." Thesis, Brunel University, 2014. http://bura.brunel.ac.uk/handle/2438/10526.
Full textAbstract:
Recent European legislation has enforced a reduction in the use of animal models for safety assessment purposes and carcinogenicity testing. The Syrian hamster embryo cell transformation assay (SHE CTA) has been proposed as a suitable animal alternative, but its implementation into test batteries has been delayed. This is due to concerns regarding the assay’s endpoint subjectivity and, moreover, the model’s relevance to carcinogenicity remains mostly unexplored. Senescence is an essential barrier against uncontrolled cell proliferation and its evasion is necessary for clonal evolution and tumour development. Carcinogenesis can be modelled by reproducing underlying mechanisms leading to senescence bypass. In this project, the SHE CTA was performed using the known mutagen and human carcinogen, benzo(a)pyrene, and the resulting SHE colonies were analysed. It was found that morphological transformation (MT) does not guarantee senescence bypass and cell immortalisation, but increases the likelihood of MT-derived cells subsequently acquiring unlimited growth potential. A limited number (between 10 and 20 %) of MT colonies produced cell clones capable of sustained proliferation and in most cases secondary events were necessary for the evasion of senescence barriers. With regard to mechanisms, p53 point mutations were present in 30 % of immortal B(a)P-induced MT colony-derived cells and located within the protein’s DNA binding domain. No p16 mutations were identified. Expression of p16 mRNA was commonly silenced or markedly reduced by a combination of mechanisms including monoallelic deletion, promoter methylation and BMI-1 overexpression. Taking advantage of the recently available Syrian hamster genomic sequence information generated by the Broad Institute, the coding regions of the Syrian hamster CDKN2A/B locus were shown to have good homology to human nucleotide sequences and confirmed the exonic structures of SH p16, ARF and p15. The findings further implicate the importance of p16 in regulating senescence while providing a molecular evaluation of SHE CTA-derived MT clones.
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Zwermann, Birgit Verfasser], and Felix [Akademischer Betreuer] [Beuschlein. "Die Rolle der Telomerase in der Immortalisation und malignen Transformation von Nebennierenrindenzellen / Birgit Zwermann. Betreuer: Felix Beuschlein." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1024483878/34.
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Löfqvist, Madelaine. "The role of Epstein-Barr virus nuclear antigen 3C in the immortalisation process of primary human B-lymphocytes." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-23230.
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Bikkul, Mehmet Ural. "Using drug treatments to control genome behaviour in normal and Hutchinson-Gilford Progeria Syndrome fibroblasts, with and without hTERT immortalisation." Thesis, Brunel University, 2016. http://bura.brunel.ac.uk/handle/2438/12774.
Full textAbstract:
Hutchinson-Gilford Progeria Syndrome (HGPS) is an exceedingly rare genetic condition with striking features reminiscent of marked premature ageing. HGPS is commonly caused by a ‘classic’ mutation in the A-type lamin gene, LMNA (G608G). This leads to the expression of an aberrant truncated lamin A protein, progerin. The nuclear lamina is known to anchor chromosomes, stabilising and regulating the genome. Interphase chromosomes are non-randomly positioned in the nuclei of cells and they occupy specific locations with respect to a radial distribution, gene-poor chromosomes are positioned at the nuclear periphery and gene-rich chromosomes are positioned towards the nuclear interior. The findings indicated that FTI-277, pravastatin, Zoledronic acid, N-acetyl-L-cysteine and all three combination treatments; FG, PZ, FPZ, have a positive effect on anchoring the genome to the nuclear matrix in AG01972 cell line. Furthermore, it was shown that in terms of positiong of chromosomes 18 and X, treatment of AG01972 HGPS cells with FTI + GGTI and FTI-277 + pravastatin + zoledronic acid drug combinations greatly restored the chromosomal organisation as well as the chromosome repositioning. The data in this thesis indicated that HP1α was found affected in T08 cells and upon lovastatin treatment T08 cells exhibited increased HP1α staining which is the good indication of rescue of heterochromatin organization. Whole exome sequencing data obtained from AG08466 atypical HGPS cells revealed that 2457589 position of promoter region is missing on LMNB2 gene located on chromosome 19. Intriguinly, the radial positions of chromosomes 10, 13, 18, and X results revealed that first time ever in our lab chromosome X found occupying the nuclear interior in atypical T08 cells. Colocalisation analysis of chromosome and fibrillarin findings confirmed chromosome positioning results since chromosomes X colocalised with fibrillarin with higher percentages in T08 cells than other cells suggesting the situation appears to be due to elongated telomeres with more chromosome territories being in the nuclear interior associated with nucleoli. M-FISH karyotyping analysis results confirmed unequivocally metaphase chromosome findings that immortalised T08 cell line has aneuoploidy including deletions and translocations. Histone modification marks H3K9me3, H3K27me3, H4K20me3 and HP1α results indicated that proteins were severely affected in T06 cells, suggesting that expression of truncuated progerin protein alters chromatin organization in T06 cells and also the structure of histone marks are severely affected in atypical T08 cells. The structure of nucleolus is affected in both typical and atypical immortalised HGPS cells, suggesting epigenetic regulation to have a crucial role in HGPS. Subsequently, the telomere lengths of immortalised normal and atypical T08 HGPS cell lines were assessed using IQ-FISH. The results indicated that both immortalised cells had chromosomes with relatively longer telomeric repeats in comparison to the control NB1 cells. The small non-peptidic, non-nucleosidic synthetic compounds (BIBR1532) was utilised to target the telomerase/telomere complex of our immortalised cell lines to shorten telomeres in order to test the hypothesis that elongated telomeres had mislocalised chromosomes. Furthermore, the effect of BIBR1532 on telomeres position in cells was assessed. Remarkably, results demonstrated that BIBR1532 is capable of shortening telomere length of immortalised cells to the level of normal NB1 fibroblasts and immortalised cell lines were capable of proliferating after drug treatment. Furher, results revealed that BIBR1532 treatment can restore the position of chromosome X towards the nuclear periphery within the NB1T and T08 nucleus. Furthermore, although BIBR1532 treatment can restore the position of chromosome 18 towards the nuclear periphery in NB1T cells, treatment did not alter the position of chromosome 18 in T08 cells. Finally, the telomere dysfunction-induced foci (TIF) assay was performed to detect DNA damage in NB1T and T08 cells using an antibody against DNA damage marker γ-H2AX and a synthetic PNA probe for telomeres. Surprisingly, results indicated that BIBR1532 treatment of cells did not give rise to high DNA damage response in both NB1T and T08 cells.
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Pon, Robert A. "The generation, immortalisation, and characterisation of human gammadelta T cells derived from the blood and cerebrospinal fluid of MS patients." Thesis, University of Ottawa (Canada), 2001. http://hdl.handle.net/10393/9045.
Full textAbstract:
Multiple sclerosis (MS) is believed to be an autoimmune, inflammatory, demyelinating disease of the central nervous system (CNS), resulting in myelin degradation, loss of the myelin forming cell, the oligodendrocyte (ODC), and axonal degeneration. The hypothesis underlying this work is that gammadelta T cells play a distinct role in MS pathogenesis by initiating, perpetuating, or regulating the immune response directed against the myelin/ODC unit. Initial comparative experiments utilising short term gammadelta T cell lines from peripheral blood (PB) and cerebrospinal fluid (CSF) of MS and other neurological disease (OND) patients, indicated no significant gammadelta subtype or cytotoxicity differences between cells derived from PB and CSF compartments or between MS and OND patients. During the course of these studies, it became apparent that the variably short in vitro lifespans of PB and CSF gammadelta T cells represented a major limitation that hampered further comprehensive studies. Efforts to increase their culture lifespans through restimulation regimens were only marginally successful, so an alternate immortalisation strategy using Herpesvirus saimiri (HVS) was employed. This study reports for the first time, the successful HVS growth transformation of human gammadelta T cells derived from both MS PB and CSF. Multiple HVS transformed PB and CSF gammadelta T cell (t -gammadelta T cell) lines and clones were generated, and have existed in IL-2 dependent culture for periods in excess of 2.5 years without the need for additional stimulation. Comparative analysis of a series of t-gammadelta T cell lines and their personal non-transformed lines indicated the transformation process did not alter their surface molecule expression, cytokine, or cytotoxicity responses. Cell surface characterisation of the t-gammadelta cell lines and clones demonstrated HVS was capable of immortalising a wide spectrum of gammadelta TcR subtypes, along with identifying t -gammadelta T cell clones displaying rare gammadelta T cell phenotypes, not commonly studied. MS t-gammadelta T cells uniformly expressed proinflammatory cytokine profiles (IFN-gamma, TNF-alpha), but only minimal IL-2 or anti-inflammatory IL-4 and IL-10. All gammadelta TcR subtypes displayed identical cytokine pattern expression, suggesting that cytokine expression is independent of gammadelta T cell subtype. t-gammadelta T cell lines demonstrated a graded cytotoxicity towards a panel of tumour cell targets, ranging from high (U937, Jurkat) to moderate (KG-1) to poor (Colo-205). Similar killing patterns of tumour targets were observed for subtype specific t-gammadelta T cell clones, and antigen recognition studies indicated a graded recognition of tumour cell ligands, together suggesting that both cytolytic function and tumour cell recognition, as was seen with cytokine profiles, are independent of gammadelta T cell subtype. In contrast, only Vgamma9Vdelta2 positive t-gammadelta T cells responded to candidate non-peptidic phospholigand and alkylamine antigens. No MS t-gammadelta T cell reactivity was observed to putative MS antigens myelin basic protein, alphaB-crystallin, or Chlamydia pneumoniae. (Abstract shortened by UMI.)
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Thenet, Sophie. "Immortalisation de chondrocytes articulaires de lapin par les fonctions precoces du virus sv40. Etude de la modulation des fonctions differenciees." Paris 7, 1992. http://www.theses.fr/1992PA077196.
Full textAbstract:
Nous avons etabli des lignees de chondrocytes articulaires de lapin en utilisant le pouvoir immortalisant de certains oncogenes, puis, nous avons etudie la possibilite chez les chondrocytes immortalises d'exprimer ou de reexprimer les fonctions specifiques du cartilage. La transfection de chondrocytes articulaires de lapin en primoculture par un plasmide portant les fonctions precoces de sv40 nous a permis d'isoler plusieurs clones de chondrocytes immortalises. Ces cellules s'expriment l'antigene grand t de sv40, presentent un caryotype hypotetraploide et une tumorigenicite faible chez la souris nude. Elles expriment un phenotype dedifferencie caracterise par l'absence de synthese de matrice colorable par le bleu alcian et de collagene de type ii, et par la synthese de collagenes genetiquement distincts. L'analyse par northern blot montre que l'inhibition du collagene de type ii se situe au niveau transcriptionnel. Les conditions de culture qui permettent a des chondrocytes normaux dedifferencies de reexprimer le collagene de type ii de facon majoritaire n'entraine pas de reexpression de collagene de type ii par les chondrocytes immortalises. La transfection de chondrocytes fraichement isoles du cartilage avec les fonctions precoces de sv40, et la selection des cellules immortalisees en culture en agregats, a conduit a des resultats identiques. Nous montrons donc que l'immortalisation des chondrocytes articulaires de lapin par les fonctions precoces de sv40 entraine une perte apparemment irreversible de l'expression du phenotype differencie des chondrocytes, qui ne necessite pas la transformation complete des cellules
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DJELLOUL, SIHAM. "Immortalisation des cellules epitheliales digestives par l'oncogene grand t de sv40 : consequences sur la transformation, la differenciation et l'effet antiproliferatif du tgf1." Paris 6, 1997. http://www.theses.fr/1997PA066066.
Full textAbstract:
Nous demontrons ici que l'immortalisation de cellules epitheliales isolees de l'estomac ou de l'intestin chez le rat (modeles rgc et eski), apres infection par des retrovirus recombinants pour le sv40lt, est associee a l'emergence de cellules epitheliales 1) relativement indifferenciees et possedant certains determinants specifiques de l'epithelium digestif 2) capables de resister aux effets antiproliferatifs du tgf. Cependant, la differenciation enterocytaire est compatible avec le transfert de sv40lt et la perte de l'anti-oncogene p53 dans les cellules coliques humaines caco-2, suggerant ainsi que cet oncogene viral a un effet permissif quand il est introduit dans des cellules epitheliales digestives deja differenciees et un effet represseur de la differenciation quand il est introduit dans un compartiment de cellules normales, germinatives et indifferenciees. Le phenotype dominant de la differenciation enterocytaire dans les cellules caco-2 peut egalement s'expliquer par surexpression des anti-oncoproteines prb1 et prb2 qui forment des complexes moleculaires avec l'antigene sv40lt, et l'independance de ces cellules aux facteurs de croissance exogenes. La perte des effets antiproliferatifs du tgf engendres par le sv40lt et l'immortalisation cellulaire peut s'expliquer en partie par la reduction selective du nombre de recepteurs du tgf de type i, mais n'implique pas la perte de leur fonctionalite. Les recepteurs du tgf (types i et ii) retiennent la capacite d'activer : 1) la serine/threonine kinase p78, impliquee dans les effets antiproliferatifs du tgf, 2) les gtpases de la famille rho et la voie de jnk qui sont des elements intermediaires dans la transmission du signal transcriptionnel du tgf et de l'apoptose. Le sv40lt interfere avec la signalisation du tgf au niveau de l'element de reponse de l'inhibiteur de l'activateur du plasminogene pai-1 qui intervient dans les processus de remodelage de la matrice extracellulaire et l'invasion. L'ensemble de ces travaux ouvre de nouvelles perspectives sur l'elucidation et le controle des mecanismes impliques dans l'independance des tumeurs humaines aux facteurs de croissance et dans leur resistance aux effets antiproliferatifs du tgf.
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BORDE, ISABELLE. "Transfert de genes et immortalisation de precurseurs de cellules nerveuses murines. Etude de la proliferation et de la differenciation des lignees immortalisees." Paris 7, 1994. http://www.theses.fr/1994PA077012.
Full textAbstract:
Nous avons transferre de facon stable, dans des cellules nerveuses murines, des genes immortalisants: les oncogenes grand t de polyome et grand t de sv40. Nous avons ainsi obtenu des lignees cellulaires variees issues de differentes regions du cerveau (cortex, mesencephale et striatum) qui correspondent a differents types de precurseurs de cellules nerveuses. D'autres lignees ont ete etabli a partir de souris transgeniques portant le gene grand t du virus polyome. Certaines de ces lignees sont des precurseurs astrogliaux qui montrent une correlation entre le controle de la division cellulaire et leur differenciation terminale. Des lignees etablies apres transduction retrovirale du grand t de sv40, presentent des caracteristiques de precurseurs astrocyte-neurone, et expriment un ensemble de recepteurs de type adrenergique, dopaminergique, muscarinique et serotoninergique, caracteristique de cette region du cerveau. Nous avons precise les differentes sous-classes de recepteurs dopaminergiques exprimees par ces cellules. Des vecteurs retroviraux recombinants dans lesquels l'oncogene immortalisant grand t de polyome est place entre les ltr du genome d'un retrovirus neurogliotrope le cas-br-e mulv, nous ont permis d'etablir plusieurs lignees de cellules nerveuses, dont certaines expriment des marqueurs de differenciation de cellules gliales. Un clone immortalise apres transfection des sequences e1a, qui est un precurseur bipotent oligodendrocyte-astrocyte, peut entrer en apoptose dans certaines conditions de culture restrictives. Ces lignees sont appropriees a des etudes pharmacologiques. Elles presentent egalement un interet particulier pour des etudes concernant l'effet des facteurs de croissance sur la division, la differenciation et l'apoptose ainsi que pour analyser des genes impliques dans ces processus
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Thorley, Matthew. "Analysis of the dystrophin interactome." Electronic Thesis or Diss., Paris 6, 2016. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2016PA066619.pdf.
Full textAbstract:
Le but de ce projet était d'identifier de manière méthodique et standardisée les partenaires interagissant avec la protéine dystrophine dans les cellules musculaires squelettiques humaines différenciées et découvrir de nouveaux rôles de la dystrophine. Des cellules immortalisées ont été obtenue en sur-exprimant de manière stable hTERT / CDK4. Nous avons réalisé une analyse transcriptomique comparant des lignées immortalisées avec leurs populations primaires correspondantes, à l’état de prolifération et de différentiation. Nous avons constaté que l'immortalisation n'a pas d'effet mesurable sur le programme myogénique ou sur tout autre processus cellulaires, et qu'elle avait un effet protecteur contre le processus de sénescence. Les lignées de cellules musculaires humaines constituent donc de bon model in vitro pour l’étude de l’interactome de la dystrophine. Nous avons déterminé l’interactome de la dystrophine en utilisant l’approche proteomique ‘QUICK’. Nous avons identifié 18 nouveaux partenaires directs de la dystrophine, partenaires étant impliqués dans le transport vésiculaire ou étant des protéines d'adhésion. Ces résultats renforcent les données précédemment publiées suggérant un lien entre la dystrophine et le trafic vésiculaire, ainsi que dystrophine et adhesion cellulaire. Ces nouveaux partenaires ont été ajoutés à l’interactome de la dystrophine, interactome accessible sur le Web: sys-myo.rhcloud.com/dystrophin-interactome. Ce site web est dédié à être un outil facile d’utilisation permettant d’explorer et de visualiser l’interactome de la dystrophine du muscle squelettiqueThe aim of this project was to systematically identify new interaction partners of the dystrophin protein within differentiated human skeletal muscle cells in order to uncover new roles in which dystrophin is involved, and to better understand how the global interactome is affected by the absence of dystrophin. hTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research however, disruption of the cell cycle has the potential to affect many other cellular processes to which it also linked. A transcriptome-wide analysis of healthy and diseased lines comparing immortalized lines with their parent primary populations in both differentiated and undifferentiated states testing their myogenic character by comparison with non-myogenic cells found that immortalization has no measurable effect on the myogenic cascade or on any other cellular processes, and that it was protective against the senescence. In this context the human muscle cell lines are a good in vitro model to study the dystrophin interactome. We investigated dystrophin’s interactors using the high-sensitivity proteomics ‘QUICK’ approach. We identified 18 new physical interactors of dystrophin which displayed a high proportion of vesicle transport related proteins and adhesion proteins, strengthening the link between dystrophin and these roles. The proteins determined through previously published data together with the newly identified interactors were incorporated into a web-based data exploration tool: sys-myo.rhcloud.com/dystrophin-interactome, intended to provide an easily accessible and informative view of dystrophins interactions in skeletal muscle
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Thorley, Matthew. "Analysis of the dystrophin interactome." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066619/document.
Full textAbstract:
Le but de ce projet était d'identifier de manière méthodique et standardisée les partenaires interagissant avec la protéine dystrophine dans les cellules musculaires squelettiques humaines différenciées et découvrir de nouveaux rôles de la dystrophine. Des cellules immortalisées ont été obtenue en sur-exprimant de manière stable hTERT / CDK4. Nous avons réalisé une analyse transcriptomique comparant des lignées immortalisées avec leurs populations primaires correspondantes, à l’état de prolifération et de différentiation. Nous avons constaté que l'immortalisation n'a pas d'effet mesurable sur le programme myogénique ou sur tout autre processus cellulaires, et qu'elle avait un effet protecteur contre le processus de sénescence. Les lignées de cellules musculaires humaines constituent donc de bon model in vitro pour l’étude de l’interactome de la dystrophine. Nous avons déterminé l’interactome de la dystrophine en utilisant l’approche proteomique ‘QUICK’. Nous avons identifié 18 nouveaux partenaires directs de la dystrophine, partenaires étant impliqués dans le transport vésiculaire ou étant des protéines d'adhésion. Ces résultats renforcent les données précédemment publiées suggérant un lien entre la dystrophine et le trafic vésiculaire, ainsi que dystrophine et adhesion cellulaire. Ces nouveaux partenaires ont été ajoutés à l’interactome de la dystrophine, interactome accessible sur le Web: sys-myo.rhcloud.com/dystrophin-interactome. Ce site web est dédié à être un outil facile d’utilisation permettant d’explorer et de visualiser l’interactome de la dystrophine du muscle squelettiqueThe aim of this project was to systematically identify new interaction partners of the dystrophin protein within differentiated human skeletal muscle cells in order to uncover new roles in which dystrophin is involved, and to better understand how the global interactome is affected by the absence of dystrophin. hTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research however, disruption of the cell cycle has the potential to affect many other cellular processes to which it also linked. A transcriptome-wide analysis of healthy and diseased lines comparing immortalized lines with their parent primary populations in both differentiated and undifferentiated states testing their myogenic character by comparison with non-myogenic cells found that immortalization has no measurable effect on the myogenic cascade or on any other cellular processes, and that it was protective against the senescence. In this context the human muscle cell lines are a good in vitro model to study the dystrophin interactome. We investigated dystrophin’s interactors using the high-sensitivity proteomics ‘QUICK’ approach. We identified 18 new physical interactors of dystrophin which displayed a high proportion of vesicle transport related proteins and adhesion proteins, strengthening the link between dystrophin and these roles. The proteins determined through previously published data together with the newly identified interactors were incorporated into a web-based data exploration tool: sys-myo.rhcloud.com/dystrophin-interactome, intended to provide an easily accessible and informative view of dystrophins interactions in skeletal muscle
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Ait, Mebarek Mazhoura. "Nouvelles approches méthodologiques pour l'obtention d'anticorps humains monoclonaux." Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00829106.
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Les anticorps monoclonaux représentent aujourd'hui un outil de choix en thérapeutique et en diagnostic. Les anticorps thérapeutiques sont des biomédicaments en plein essor depuis les années 1970 et représentent 10% du marché des produits pharmaceutiques. Les anticorps monoclonaux sont utilisés dans divers domaines : en cancérologie, pour lutter contre les maladies auto-immunes ou en infectiologie. Le nombre des anticorps monoclonaux en développement ne cesse d'augmenter. Les premiers anticorps monoclonaux utilisés en thérapie étaient d'origine murine et leur administration à l'Homme est susceptible de déclencher des effets secondaires. De nouveaux anticorps visant à limiter voir faire disparaitre ces effets indésirables tels que d'abord les anticorps chimériques, puis les anticorps humanisés et enfin les anticorps totalement humains ont été développés. 9 anticorps totalement humains sont actuellement sur le marché et d'autres sont en cours de développement. Le phage display, les souris transgéniques et l'utilisation de lymphocytes B humains sont les trois stratégies mises en œuvre pour produire des anticorps totalement humains. L'utilisation des lymphocytes B humains, peu étudiée à cause d'un faible rendement et de problèmes de stabilité, a connu ces dernières années un regain d'intérêt grâce à l'immortalisation virale par le virus Epstein-Barr et à la découverte de myélomes humains. Dans ce contexte, l'objectif de mon projet de thèse a été la production d'anticorps monoclonaux humains à partir de lymphocytes B humains. Pour ce faire, deux approches basées sur l'immortalisation virale par le virus Epstein-Barr couplée ou non à une immortalisation cellulaire par des myélomes ont été mises en œuvre. La première approche utilise des lymphocytes B mémoires isolés de sang périphérique de donneurs infectés ou vaccinés. L'entérotoxine B de Staphylococcus aureus (SEB) a été utilisée comme modèle.La deuxième approche implique une immunisation in vitro de lymphocytes B naïfs extraits de sang périphérique. Cette stratégie pourrait permettre la production d'anticorps humains contre des antigènes pour lesquels il n'existe pas de donneurs infectés ou vaccinés. Deux modèles, le peptide N-terminal de la neurotoxine A de Clostridium Botulinium A (BoNT/A) et la protéine de fusion ZZTat101, comportant le domaine ZZ de Staphylococcus aureus lié covalemment à la protéine transactivatrice Tat du virus de l'immunodéficience humaine VIH-1, ont été employés. Nous avons réussi à obtenir des IgMs dirigés contre la neurotoxine Clostridium Botulinium A, ainsi que des IgMs (et peut-être des IgGs) dirigés contre la protéine Tat. L'immortalisation par Epstein-Barr, nous a permis d'isoler 7 lignées de lymphocytes immortalisés sécrétant des anticorps IgMs anti-TBA-Nter humains. L'immunisation in vitro produisant essentiellement des IgMs, la possible production d'IgGs après stimulation par la protéine ZZTat101 se révèle un résultat très intéressant. Nous avons montré que la production d'anticorps par ZZTat101 impliquait les 7 cystéines, la région 22-57 et la liaison aux héparanes sulfates de Tat.
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STEIMBERG, NATHALIE. "Immortalisation de chondrocytes articulaires de lapin par l'antigene grand t de sv40 place sous le controle des sequences regulatrices du gene du collagene de type ii." Paris 7, 1997. http://www.theses.fr/1997PA077297.
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Le chondrocyte, unique type cellulaire du cartilage, est responsable de la synthese de la matrice cartilagineuse, essentiellement composee de collagene de type ii et d'agregats d'agrecanne. Au cours de la culture, l'expression de ces fonctions differenciees est rapidement perdue. Plusieurs equipes se sont donc interessees a l'immortalisation de chondrocytes afin d'etablir des lignees immortalisees et differenciees. L'expression du phenotype differencie des lignees obtenues est tres variable, particulierement pour le collagene de type ii, l'un des marqueurs principaux de la differenciation du chondrocyte,. Au cours de ce travail, nous avons montre que contrairement a l'expression de l'oncogene src, l'expression de l'antigene t de sv40 ou de l'oncogene c-myc n'etait pas incompatible avec l'activite des sequences regulatrices du collagene de type ii. Dans un deuxieme temps, une nouvelle strategie d'immortalisation a ete elaboree, qui repose sur l'utilisation d'un vecteur dont l'expression devrait etre ciblee dans des chondrocytes differencies. Pour cela, nous avons construit un plasmide dans lequel l'antigene t de sv40 a ete place sous le controle des sequences regulatrices du gene du collagene de type ii de rat. La transfection de chondrocytes articulaires de lapin avec ce vecteur a permis d'isoler plusieurs clones de chondrocytes immortalises, exprimant tous l'antigene t de sv40, et dont l'expression du collagene de type ii est restreinte a une faible proportion de cellules. Ces chondrocytes immortalises et des chondrocytes normaux dedifferencies par subcultures successives, ont ete cultives en 3 d. La culture en billes d'alginate ne permettant pas de restaurer totalement l'expression du phenotype differencie dans les chondrocytes immortalises, un modele de culture en agregats a ete developpe. Ce mode de culture permet de restaurer l'expression du phenotype differencie dans des chondrocytes normaux dedifferencies par subculture. Pour les chondrocytes immortalises, la culture en agregats semble favoriser l'expression du collagene de type ii, qui reste cependant minoritaire. De plus, l'expression des proteoglycannes specifiques est induite, mais est reduite comparee a celle observee dans les agregats de chondrocytes normaux.
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Delgado, Charris Jean-Paul. "Caractérisation phénotypique et moléculaire des cellules progénitrices foetales hépatiques simiennes et humaines." Paris 11, 2006. http://www.theses.fr/2006PA114825.
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Le foie est un organe de cible pour les thérapies cellulaires. Les perspectives de ces approches avec les hépatocytes humains adultes sont limitées : manque de données, absence de prolifération, et inefficacité de greffe. Nous avons caractérisé phénotypiquement à différents doublements de population (DP) une lignée d’hépatoblastes bipotents de singe immortalisés (IPFLS) dans le laboratoire à l’aide de l’oncogène T de SV40 flanqué de sites LoxP. Nous avons montré que l’immortalisation était réversible après excision du transgène par transduction rétrovirale du gène codant la Cre recombinase. Nous avons aussi montré qu’aux passages tardifs (120DP) l’activité télomérase était réduite, les télomères raccourcis et les remaniements chromosomiques importants. Après transplantation dans des souris immunodéficientes, les cellules IPFLS se sont différenciées en hépatocytes mais n’ont pas proliféré. Nous avons contribué à l’isolement et la caractérisation des hépatoblastes/hépatocytes fœtaux humains précoces (10-12 semaines). Après transplantation, ces cellules repeuplent plus efficacement le foie des souris transplantées que les IPFLS. In vitro elles ont un potentiel de migration et d’invasion, au contraire des hépatocytes adultes, qui est accru après stimulation par HGF. Ce processus est corrélé à une augmentation de la sécrétion et l’activation des métalloprotéases 2 et 9 et à une activation de la voie ERK. La transplantation d’hépatoblases stimulés par HGF dans des souris nouveau-nées améliore la dispersion de ces cellules dans le parenchyme hépatique. Ces résultats suggèrent que les hépatoblastes humains ont des propriétés spécifiques qui permettront d’améliorer la prise de greffeThe liver is a target organ for cell-based therapies. The use of human adult hepatocytes for such approaches is limited by the lack of donors, the absence of proliferation and low cell engraftment. We characterized a simian bipotent hepatoblast line (IPFLS) immortalized in our lab by using the Sv40 virus T oncogene flanked by LoxP sites, at different population doublings (PD). We showed that immortalization process was reversible after Cre recombinase mediated retroviral gene transfer. We also showed a telomerase activity reduction correlated with telomere shortening and chromosomal rearrangements at 120 PD. After transplantation in the liver of immunocompromised mice, IPFLS cells were differentiated into hepatocytes but did not proliferate. We contributed to the isolation and characterization of early fetal human heptoblasts/hepatocytes (10-12 weeks). After transplantation, these cells repopulated more efficiently the liver of transplanted mice than IPFLS cells. Human hepatoblasts, contrary tu adult hepatocytes, have an in vitro migration and invasion potential which is enhanced by the Hepatocyte Growth Factor (HGF). This process is correlated with an up regulation in the secretion and activation of matrix metalloproteinases 2 and 9 and the activation of the ERK pathway. Transplantation of hepatoblasts stimulated with HGF into new born mice improves cell spreading in the liver parenchyma. These results suggest that human hepatoblasts have specific properties which allow the improvement of cell engraftement
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Kan, Chin Yi Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Human Papillomavirus in human breast cancer and cellular immortalisation." 2007. http://handle.unsw.edu.au/1959.4/40593.
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Human Papillomavirus (HPV) is a small, double stranded DNA tumour virus. Infection with HPV normally results in formation of warts. Certain types of HPV, such as type -16 and -18, are shown to have a causal role in the development of uterine cervical cancer, and are so called high risk type HPV. Recently, a role of HPV in breast cancer has been suggested, although a causal role for HPVs in human breast cancer is yet to be demonstrated. The first part of this study investigates the association of HPV with human breast cancer. The results demonstrate that 48% of breast cancers that occurred in Australian women are HPV positive and they are mainly variants of HPV-18. Further analysis shows that HPV positive breast cancer patients are significantly younger than HPV negative patients, suggesting infection with HPV increases the risk of breast cancer development. This is coincidental with increased risk of HPV infection in sexually active young women and provides evidence that HPV has a role in breast cancer development. The second part of this project investigates the mechanisms by which high risk type HPV oncogenic protein E6, transforms primary human foreskin keratinocytes (natural host cells of HPV). HPV E6 is always expressed in HPV positive cervical carcinoma and results in the degradation of the cellular tumour suppressor protein p53. It is generally believed that HPV E6 contributes to HPV transformation by degradation of p53 protein which leads to cellular immortalisation ? an early step in tumorigenic transformation. Subsequent studies, however, indicate that HPV E6 possesses other functions (such as induction of telomerase activity) which may also be involved in cellular immortalisation. The results of my investigations demonstrate: 1) that degradation of p53 protein is required but is insufficient to immortalise primary cells; 2) that HPV E6 induced telomerase activity is coincidental with an increase in cell culture passage number; 3) that multiple functions of high risk type HPV E6 protein are required for cellular immortalisation. This finding suggests HPV infection is associated with early onset of breast cancer and that multiple functions of high risk type HPV E6 protein are involved in cellular immortalisation. Further study in both of these areas should provide alternative diagnostic markers, leading to prevention and treatment strategies for HPV positive breast cancer and other cancers.
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Keough, Rebecca Anne. "An investigation of hair follicle cell immortalisation and hair keratin gene regulation / Rebbeca Anne Keough." Thesis, 1995. http://hdl.handle.net/2440/18632.
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Bibliography: leaves 87-113.xi, 113, [72] leaves, [42] leaves of plates : ill. (some col.) ; 30 cm.
Presents results from an investigation into the regulation of hair-specific gene expression, including attempts to produce an immortalised hair follicle cortical cell line for this purpose and the use of mouse transgenesis and invitro gel mobility shift assays.
Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1995
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Veillette, François. "Caractérisation de HTDE et de son potentiel oncogénique in vitro et in vivo." Thèse, 2004. http://hdl.handle.net/1866/15541.
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Rodier, Francis. "Étude moléculaire des événements associés à la transformation par l'antigène grand T du virus de polyome (PyLT-Ag) = An analysis of molecular events associated with transformation by polyomavirus large T antigen (PyLT-Ag)." Thèse, 2004. http://hdl.handle.net/1866/15532.
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Löfqvist, Madelaine [Verfasser]. "The role of Epstein-Barr virus nuclear antigen 3C in the immortalisation process of primary human B-lymphocytes / von Madelaine Löfqvist." 2004. http://d-nb.info/972056904/34.
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Hajj, Hassan Houssein. "Établissement d'une lignée cellulaire pro-érythroïde de souris : outil d'étude de la régulation transcriptionnelle des gènes de globine." Thèse, 2004. http://hdl.handle.net/1866/14663.
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