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1
Kuleszewicz, Katarzyna. "Cell cycle regulation in mammalian oocytes." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/26148.
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An unusual feature of mammalian female germ cells is that they are arrested in meiotic prophase, equivalent to mitotic G2-phase, for an extended period of time. In this thesis I have investigated two aspects of this arrest. First, I examined whether cohesin replenishment is required for the maintenance of chromosome cohesion during protracted meiotic prophase arrest. Nipbl, an evolutionarily conserved protein, is a component of protein complex called kollerin, whose activity in loading cohesin onto chromosomes is necessary for accurate chromosome segregation during mitosis. However, until now its function in mammalian meiosis was unknown. I have showed that Nipbl is present on meiotic chromosomes throughout meiotic prophase in mouse spermatocytes and oocytes and it accumulates at chromosomal axes where it co-localises with cohesin. I employed conditional knockout strategy to inactivate Nipbl gene in mouse oocytes arrested in meiotic prophase. Although functional Nipbl transcripts were efficiently depleted, these oocytes underwent meiotic maturation with unaffected chiasmata and cohesion. Surprisingly, Nipbl-deleted eggs were fertile and the loading of mitotic cohesin containing Rad21 was unaffected in fertilized eggs. Aditionally, these eggs could develop into blastocysts upon parthenogenetic activation, however harbouring a high proportion of cells with misaligned chromosomes. These results suggest that Nipbl is very stable in the oocyte. In the second project we conceived that the maintenance of the cell cycle arrest in primordial oocytes is an important aspect of follicular survival. Previously proposed involvement of the anaphase promoting complex/cyclosome (APC/C), a cell cycle ubiquitin ligase complex in down-regulating the cyclin-dependent kinase activity in fully-grown oocyte led me to inactivate APC/C in dormant oocytes using conditional knockout system. I found that upon APC/C inactivation, primordial follicles were completely depleted before adulthood, within 5 weeks of birth, suggesting that the APC/C activity is required for the survival of primordial oocytes. These results propose the presence of previously unknown mechanism involving APC/C, essential for primordial follicle survival.
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Engstrom, Julia U. "Mammalian cell cycle regulates oligonucleotide-mediated repair." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 219 p, 2008. http://proquest.umi.com/pqdweb?did=1481658501&sid=9&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Weis, Michael Christian. "Computational Models of the Mammalian Cell Cycle." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1323278159.
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Sauvé, Gordon John. "Genetic complementation of a mammalian cell cycle mutant." Thesis, McGill University, 1986. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74037.
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Calegari, Federico, and Julieta Aprea. "Bioelectric State and Cell Cycle Control of Mammalian Neural Stem Cells." Sage-Hindawi, 2012. https://tud.qucosa.de/id/qucosa%3A27972.
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The concerted action of ion channels and pumps establishing a resting membrane potential has been most thoroughly studied in the context of excitable cells, most notably neurons, but emerging evidences indicate that they are also involved in controlling proliferation and differentiation of nonexcitable somatic stem cells. The importance of understanding stem cell contribution to tissue formation during embryonic development, adult homeostasis, and regeneration in disease has prompted many groups to study and manipulate the membrane potential of stem cells in a variety of systems. In this paper we aimed at summarizing the current knowledge on the role of ion channels and pumps in the context of mammalian corticogenesis with particular emphasis on their contribution to the switch of neural stem cells from proliferation to differentiation and generation of more committed progenitors and neurons, whose lineage during brain development has been recently elucidated.
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Calegari, Federico, and Julieta Aprea. "Bioelectric State and Cell Cycle Control of Mammalian Neural Stem Cells." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-185623.
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The concerted action of ion channels and pumps establishing a resting membrane potential has been most thoroughly studied in the context of excitable cells, most notably neurons, but emerging evidences indicate that they are also involved in controlling proliferation and differentiation of nonexcitable somatic stem cells. The importance of understanding stem cell contribution to tissue formation during embryonic development, adult homeostasis, and regeneration in disease has prompted many groups to study and manipulate the membrane potential of stem cells in a variety of systems. In this paper we aimed at summarizing the current knowledge on the role of ion channels and pumps in the context of mammalian corticogenesis with particular emphasis on their contribution to the switch of neural stem cells from proliferation to differentiation and generation of more committed progenitors and neurons, whose lineage during brain development has been recently elucidated.
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Greggains, Gareth David. "Cell cycle regulation and nuclear reprogramming in mammalian oocytes." Thesis, University of Newcastle Upon Tyne, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.538926.
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Swanton, Robert Charles. "Viral cyclin disruption of mammalian cell cycle control mechanisms." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286205.
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Lundgren, Andreas. "The ABC of the cell cycle: roles of the mammalian Cdc25 isoforms /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-639-5/.
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Avva, Jayant. "Complex Systems Biology of Mammalian Cell Cycle Signaling in Cancer." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1295625781.
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Wood, Christopher David. "Real-time imaging of gene expression during the cell cycle in single mammalian cells." Thesis, University of Liverpool, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366483.
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Drews-Elger, Katherine. "Role of the transcription factor NFAT5 in mammalian cell cycle regulation." Doctoral thesis, Universitat Pompeu Fabra, 2008. http://hdl.handle.net/10803/7177.
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The transcription factor NFAT5/TonEBP belongs to the Rel family, which also comprises NF ÛB and NFATc proteins. NFAT5 only shares structural and functional homology with other Rel family members at the level of the DNA binding domain, and differs from them considerably in other regions. NFAT5 enables mammalian cells to adapt to and withstand hypertonicity by orchestrating an osmoprotective gene expression program whose products include chaperones as well as ransporters and enzymes that increase the intracellular concentration of compatible osmolytes. NFAT5-null mice suffer severe embryonic and perinatal lethality, and surviving adults manifest growth defects, pronounced renal atrophy and lymphocyte dysfunction associated with ineffective responses to hypertonicity. To circumvent the lethality of these mice and study the function of NFAT5 in specific cell types without the possible side effects of generalized defects in the organism, we have produced conditional knockout mice that allow the deletion of NFAT5 in specific cell types. Here we have investigated the hypertonic stress response in wild-type and NFAT5-/- lymphocytes. Proliferating lymphocytes exposed to hypertonic conditions exhibited an early, NFAT5- independent, genotoxic stress-like response with induction of p53, p21 and GADD45, downregulation of cyclins E1, A2 and B1 mRNA, and arrest in S and G2/M. This was followed by an NFAT5-dependent adaptive phase in wild-type cells, which induced osmoprotective gene products, downregulated stress markers, and resumed cyclin expression and cell cycle progression. NFAT5-/- cells, however, failed to induce osmoprotective genes and though they downregulated genotoxic stress markers, they displayed defective cell cycle progression associated with reduced expression of cyclins E1, A2, B1, and aurora B kinase. Finally, T cell receptor-induced expression of cyclins, aurora B kinase, and cell cycle progression were inhibited in NFAT5-/- lymphocytes exposed to hypertonicity levels in the range reported in plasma in patients and animal models of osmoregulatory disorders. Our results support the conclusion that the activation of an osmoprotective gene expression program by NFAT5 enables cells to proliferate under hypertonic stress conditions by maintaining the expression of S and G2/M cyclins and cell cycle progression.
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Kim, Ju-Hwan. "Analysis of the function of Mad2B in the mammalian cell cycle." Thesis, University of Leicester, 2012. http://hdl.handle.net/2381/27771.
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Mad2B (also called Rev7 or Mad2L2) is a regulatory subunit of DNA polymerase ζ that is involved in translesion DNA synthesis (TLS). Previous studies have implicated Mad2B in several cell functions including cell cycle regulation where it was shown to inhibit the Anaphase Promoting Complex (APC)/cyclosome by binding to Cdc20/Cdh1. As Mad2B shares 26% identity and 48% similarity at the amino acid level with the human mitotic checkpoint protein Mad2 (also called Mad2A or Mad2L1), it has been suggested that Mad2B may also function as a mitotic checkpoint protein. However, recent studies have demonstrated that Mad2B has an important function in the DNA damage response in mammalian cells. Hence, it has been suggested that Mad2B may play a role in the DNA damage signaling pathway rather than in the spindle assembly checkpoint (SAC). The aim of this project was to investigate the function of Mad2B (Rev7) in mammalian cells. The results demonstrate that Mad2B is not a mitotic checkpoint protein. Instead the data indicate that Mad2B is involved in the DNA damage response. Interestingly, Mad2B binds to Cdc20 following DNA damage. Mad2B was also found to associate with Cdc27 (a component of the Anaphase Promoting Complex or APC/cyclosome) both before and after activation of the DNA damage checkpoint. In addition, the results of the in vitro ubiquitylation assay indicated that Mad2B does not inhibit APC/C activity. Taken together the results in this project suggest a model where binding of Cdc20 to the Mad2B (Rev3/Rev1)-APC/C complex following DNA damage may alter APC/C substrate specificity of as yet unidentified substrates.
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Shorter, James Gordon. "Molecular mechanisms regulating Golgi architecture during the mammalian cell division cycle." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313395.
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Cutts, Timothy John Rogers. "Checkpoint controls in the latter half of the mammalian cell cycle." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627147.
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Hau, Pok Man. "Polyploidization increases the sensitivity to DNA-damaging agents in mammalian cells /." View abstract or full-text, 2007. http://library.ust.hk/cgi/db/thesis.pl?BICH%202007%20HAU.
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Xu, Naihan. "Regulation of the metaphase-anaphase transition in mitosis in mammalian cells /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202003%20XU.
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Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003.Includes bibliographical references (leaves 242-266). Also available in electronic version. Access restricted to campus users.
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Kyei, Foster. "Id proteins in the regulation of mammalian cell cycle, growth and survival." Thesis, University of Essex, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.571481.
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Members of the Id family of helix-loop-helix protein function as antagonists of several classes of transcription factor. Numerous studies have documented how Id proteins affect mammalian cell growth and survival. However, it is not known how these effects are influenced by cell lineage and tumourigenic conversion. This research aimed to investigate Id proteins on growth and survival by gain- and loss-of-function analysis in a diverse range of mammalian cell lines representing both primary and immortalised/tumourigenic cell lines of different lineages. By using automated image analysis software (CellProfiler Analyst), cell cycle and apoptosis profiles of cell populations transfected with either Id protein or an Id protein antagonist including siRNA down-regulation of Id proteins have been investigated. Also, cell viability analysis with MTT assay was evaluated. The study also employed flow cytometric analysis using a mitochondrial membrane-potential marker of viable cells (DiOC6) and Propidium Iodide (PI) as a marker of non- viable necrotic cells. The data showed that Id3 is pro-apoptotic in normal and tumourigenic cells of embryonic and epithelial lineages. Over-expression of Id3 also arrested growth in non-tumourigenic embryonic fibroblasts and tumourigenic colon cancer cells. Analyses of loss-or gain-of- function mutants of Id proteins suggest that Id2 Ala5 may be loss-of-function mutant in non- tumourigenic cell lines of embryonic lineage. The study revealed that Id2 Asp5 may be a loss-of-function mutant in tumourigenic cell lines of both embryonic and epithelial lineages. Id2 HE also behaved as a null mutant in both non-tumourigenic and tumourigenic cells representing embryonic and epithelial lineages. Data obtained also implicate Id3 Asp5 as a gain-of-function mutant whilst Id3 Ala5 may represent a loss-of-function mutant in non- tumourigenic and tumourigenic cells of different lineages. Another important finding was that Id protein antagonist (aHLH) induced apoptosis and impaired cell growth irrespective of the cell lineage and tumourigenic conversion. The effects on cell growth and survival following down-regulation of Idl, Id2 and Id3 differ in embryonic, epithelial and endothelial cells. The study also suggests that cell death as a result of Id2 knock-down may occur via a different mechanism. The results reveal distinct functions of Id proteins in normal, tumourigenic and primary cells of different lineages, suggesting that Id protein functions are dependent on cell lineage and tumourigenic properties.
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Xiao, Kang. "Investigating the functional roles of Mcl-1 in apoptosis in mammalian cells /." View abstract or full-text, 2009. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202009%20XIAO.
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He, Enuo. "Stochastic modelling of the cell cycle." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:04185cde-85af-4e24-8d06-94b865771cf1.
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Precise regulation of cell cycle events by the Cdk-control network is essential for cell proliferation and the perpetuation of life. The unidirectionality of cell cycle progression is governed by several critical irreversible transitions: the G1-to-S transition, the G2-to-M transition, and the M-to-G1 transition. Recent experimental and theoretical evidence has pulled into question the consensus view that irreversible protein degradation causes the irreversibility of those transitions. A new view has started to emerge, which explains the irreversibility of cell cycle transitions as a consequence of systems-level feedback rather than of proteolysis. This thesis applies mathematical modelling approaches to test this proposal for the Mto- G1 transition, which consists of two consecutive irreversible substeps: the metaphase-to-anaphase transition, and mitotic exit. The main objectives of the present work were: (i) to develop deterministic models to identify the essential molecular feedback loops and to examine their roles in the irreversibility of the M-to-G1 transition; (ii) to present a straightforward and reliable workflow to translate deterministic models of reaction networks into stochastic models; (iii) to explore the effects of noise on the cell cycle transitions using stochastic models, and to compare the deterministic and the stochastic approaches. In the first part of this thesis, I constructed a simplified deterministic model of the metaphase-to-anaphase transition, which is mainly regulated by the spindle assembly checkpoint (the SAC). Based on the essential feedback loops causing the bistability of the transition, this deterministic model provides explanations for three open questions regarding the SAC: Why is the SAC not reactivated when the kinetochore tension decreases to zero at anaphase onset? How can a single unattached kinetochore keep the SAC active? How is the synchronized and abrupt destruction of cohesin triggered? This deterministic model was then translated into a stochastic model of the SAC by treating the kinetochore microtubule attachment at prometaphase as a noisy process. The stochastic model was analyzed and simulation results were compared to the experimental data, with the aim of explaining the mitotic timing regulation by the SAC. Our model works remarkably well in qualitatively explaining experimental key findings and also makes testable predictions for different cell lines with very different number of chromosomes. The noise generated from the chemical interactions was found to only perturb the transit timing of the mitotic events, but not their ultimate outcomes: all cells eventually undergo anaphase, however, the time required to satisfy the SAC differs between cells due to stochastic effects. In the second part of the thesis, stochastic models of mitotic exit were created for two model organisms, budding yeast and mammalian cells. I analyzed the role of noise in mitotic exit at both the single-cell and the population level. Stochastic time series simulations of the models are able to explain the phenomenon of reversible mitotic exit, which is observed under specific experimental conditions in both model organisms. In spite of the fact that the detailed molecular networks of mitotic exit are very different in budding yeast and mammalian cells, their dynamic properties are similar. Importantly, bistability of the transitions is successfully captured also in the stochastic models. This work strongly supports the hypothesis that uni-directional cell cycle progression is a consequence of systems-level feedback in the cell cycle control system. Systems-level feedback creates alternative steady states, which allows cells to accomplish irreversible transitions, such as the M-to-G1 transition studied here. We demonstrate that stochastic models can serve as powerful tools to capture and study the heterogeneity of dynamical features among individual cells. In this way, stochastic simulations not only complement the deterministic approach, but also help to obtain a better understanding of mechanistic aspects. We argue that the effects of noise and the potential needs for stochastic simulations should not be overlooked in studying dynamic features of biological systems.
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Petersen, Birgit Otzen. "Regulation of mammalian CDC6 by CDK phosphorylation and proteasome dependent degradation." Thesis, Open University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298212.
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Andrews, Anton Oguzhan Alford. "A study of cyclin D1 and its role in the mammalian cell cycle." Thesis, King's College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362382.
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Kawesa, Sarah. "A Differential Response to Newt Regeneration Extract by C2C12 and Primary Mammalian Muscle Cells." Thesis, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/26059.
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Tissue regeneration in mammals does not occur via the process of dedifferentiation, a process whereby differentiated cells lose their specialized characteristics and revert to a less differentiated state. McGann et al. (2001) showed that mouse C2C12 myotubes treated with newt extract derived from regenerating limbs can re-enter the cell cycle, fragment and proliferate ( a characteristic of muscle dedifferentiation). However, the validity of these studies has been called into question since others have been unable to repeat them. My research attempts to replicate the results of McGann et al, and to carry them further. I examined several strategies for tracking the extract-treated cells and I also repeated the studies in a primary muscle culture system. Furthermore, I examined the effect of the extract on myoblast differentiation.
The most effective dedifferentiation assay that I developed involved the microinjection of myotubes with extract and with a GFP plasmid that allowed tracking of the injected cells. Cells were then examined for cell cycle re-entry using BrdU incorporation or Ki-67 immunostaining. In addition, immunocytochemistry and RT-PCR analysis were used to examine the expression or down-regulation of muscle-specific markers. Finally, a preliminary GeneChip analysis was conducted to examine which genes were up or down regulated following extract treatment.
The results show that newt extract is able to block the differentiation of confluent myoblasts, resulting in fewer multinucleated, myosin heavy chain expressing myotubes. However, when myoblasts were differentiated into myotubes and subsequently treated with newt extract, the results suggest that cell cycle re-entry and down-regulation of differentiation markers can occur in C2C12 myotubes, but not in primary myotubes. Fragmentation though, was seen in both C2C12 and primary myotubes following treatment or injection with newt extract. Moreover, the fragmented cells appeared to be viable. Transcriptional profiling indicated that newt extract affects genes implicated in cell cycle, transcription, stress, chromatin modification, growth, cell adhesion, extracellular matrix, wound healing and microtubule binding. These findings confirm that mammalian myotubes can be induced to dedifferentiate following treatment with newt extract; however, a differential response was observed between C2C12 and primary muscle cells.
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Ford, Matthew Jonathan. "Design and implementation of transgenic tools to visualise cell cycle progression in mammalian development." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/23658.
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Cell cycle progression is the series of steps a cell has to take in order to duplicate its DNA and produce two daughter cells. Correct spatial and temporal coordination of the cell cycle is key for the normal development of any organ or tissue and is stringently controlled during embryogenesis and homeostasis. Misregulation of cell cycle progression is causal in many developmental disorders and diseases such as microcephaly and cancer. Fucci (Fluorescent Ubiquitination based Cell Cycle Indicator) is a system that allows for the visualisation of cell cycle progression by the use of two differently coloured fluorescent probes whose abundance is regulated reciprocally during the cell cycle. The probes contain the E3 ligase recognition domains of Cdt1 and Geminin fused to the fluorophores mCherry (red fluorescence) and mVenus (yellow fluorescence) respectively. Cells are therefore labelled red during G1, yellow in the G1/S transition and green during late S/G2 and M phases of the cell cycle. In order to study development and tissue homoeostasis a Fucci expressing mouse line was developed however this has several key limitations: First, the two Fucci probes are expressed from separate loci complicating mouse colony maintenance. Second, the constructs were not inducible, making it impossible to follow cell cycle progression in specific cell lineages and third the mice were generated by random transgenesis which is prone to silencing and can exhibit variation in expression between different tissues. Here I have characterised an improved version of the original Fucci system known as Fucci2a designed by Dr Richard Mort (University of Edinburgh) to overcome these limitations. The Fucci2a genetic construct contains both Fucci probes fused with the Thosea asigna virus self-cleaving peptide sequence T2A. This allows expression of both probes as a single bicistronic mRNA with subsequent cleavage by ribosomal ‘skipping’ during translation to yield separate proteins. A Fucci2a mouse (R26Fucc2aR) was generated by homologous recombination into the ROSA26 locus using the strong, ubiquitous CAG promoter to drive expression and incorporating a floxed-Neo stop cassette. This allows tissue specific activation by Cre recombinase when combined with a second Cre expressing mouse line. Building on the bicistronic Fucci2a technology I have gone on to develop and characterise four new tricistronic reporter constructs which allow for the dual visualisation of cell cycle progression with apoptosis, cytokinesis and ciliogenesis. In each case an additional fluorescent probe was added to the original Fucci2a construct separated by the self-cleaving peptide P2A and the construct characterised in 3T3 stable cell lines. The combination of a dual cilia and cell cycle reporter construct proved fruitful and I have gone on to investigate the relationship between cell cycle progression and ciliogenesis in 3T3 cells and have generated and characterised the R26Arl13b-Fucci2aR mouse line. I have also illustrated the utility of the R26Fucci2aR mouse for generating quantitative data in development research in two development situations; melanocyte development and lung branching morphogenesis. Melanocytes are specialised melanin producing cells responsible for the pigmentation of the hair, skin and eyes. Their precursors, melanoblasts, are derived from the neural crest where they migrate and proliferate before becoming localised to hair follicles and their study provides a good model for understanding the development of other neural crest derived lineages such as the peripheral nervous system. Using time-lapse imaging of ex vivo skin cultures in which melanoblasts are labelled with the Fucci probes I have characterised melanoblast migration and proliferation. In addition, I have shown that Kit signalling, which is necessary for melanoblast migration and survival, controls melanoblast proliferation in a density dependent manner and that melanoblast migration is more persistent in S/G2/M phases of the cell cycle. Lung branching morphogenesis requires constant proliferation at the apical tip of a growing epithelial branch. Loss of epithelial symmetry through an unidentified mechanism (requiring BMP, FgF10, Shh and Wnt signalling) within a branch is required to initiate branching either latterly from the side of a elongating branch by domain branching or by bifurcation of the tip. In the final section of this thesis I performed a comparative analysis of the behaviour of the developing lung epithelium using proliferative status (Fucci2a expression) to categorise each cell. Using a combination of live imaging and immunohistochemistry I have identified a transition zone 100-150μm from the tip of the branching lung epithelium where epithelial cells become stationary and drop out of the cell cycle corresponding with the onset of proximal bronchial progenitor marker Sox2. A comparative gene expression analysis of the proliferating and non-proliferating regions using Fucci2a to distinguish them has eluded to several interesting genes which could influence branching morphogenesis during lung development.
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Stott, Francesca Joanne. "Analysis of the INK4 family of cyclin dependent kinase inhibitors, in the mammalian cell cycle." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322001.
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Miles, Lauren E. C. "Mammalian cell growth and proliferation mediated by the gonadotropin-releasing hormone (GnRH) receptor : role of novel interacting protein partners." University of Western Australia. Centre for Medical Research, 2005. http://theses.library.uwa.edu.au/adt-WU2005.0090.
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[Truncated abstract] It is becoming increasingly obvious that cell signalling pathways are more complicated than we originally perceived. Research is revealing that, not only is there a multitude of new proteins involved in signalling cascades, but also that previously identified proteins may have additional, alternate roles in intracellular trafficking. Gonadotropin-releasing hormone (GnRH) in conjunction with its receptor (GnRHR), the primary regulator of reproduction in all species, is no exception. In the past few years it has become readily accepted that the classic linear GnRHR-Gαq/11 signalling pathway is not universal and that this receptor is involved in a far greater range of cellular activities than was previously considered. In particular, it is widely accepted that continuous administration of GnRH analogs results in an inhibition of growth of a number of reproductive-derived tumours and that this may, in part, be mediated by direct activation of GnRHs expressed on these cells. However, it is not fully understood how the GnRHR mediates these growth effects or whether such effects are unique to reproductive-derived cancer cells. Research within this thesis aimed to determine how the presence or absence of this receptor in different cell types might affect the ability of GnRH to directly mediate growth effects. We demonstrate that continuous treatment with a GnRH agonist (GnRHA) induces an anti-proliferative effect in a gonadotropederived cell line (LβT2) and also in HEK293 cells stably expressing either the rat or human GnRHR. The anti-proliferative effect was time- and dose-dependent and was specifically mediated via the GnRHR, as co-treatment of the GnRHRexpressing cell lines with a GnRH antagonist blocked the growth suppressive effect induced by GnRHA treatment. Cell cycle analysis revealed that the GnRHA treated HEK/GnRHR cell lines induced an accumulation of cells in the G2/M phase while a G0/G1 arrest was observed in LβT2 cells. Previous identification by our group of a potential interaction between the GnRHR and the transcription factor E2F4, an integral cell cycle regulatory protein, prompted further investigation as to the nature of this interaction. Bioluminescence energy transfer (BRET) was utilised to demonstrate that the GnRHR also interacts with E2F5, another member of the E2F family of cell cycle proteins that shares a high level of homology to E2F4. In addition, it was determined that the interaction between human GnRHR and E2F4, detected using BRET, was influenced by cell density.
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Pelttari, Danielsson Jeanette. "Molecular analysis of protein complexes involved in pairing of mammalian chromosomes during meiosis /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-682-0.
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Singhania, Rajat. "Modeling Protein Regulatory Networks that Control Mammalian Cell Cycle Progression and that Exhibit Near-Perfect Adaptive Responses." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/37722.
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Protein regulatory networks are the hallmark of many important biological functionalities. Two of these functionalities are mammalian cell cycle progression and near-perfect adaptive responses. Modeling and simulating these functionalities are crucial stages to understanding and predicting them as systems-level properties of cells.
In the context of the mammalian cell cycle, the timing of DNA synthesis, mitosis and cell division is regulated by a complex network of biochemical reactions that control the activities of a family of cyclin-dependent kinases. The temporal dynamics of this reaction network is typically modeled by nonlinear differential equations describing the rates of the component reactions. This approach provides exquisite details about molecular regulatory processes but is hampered by the need to estimate realistic values for the many kinetic constants that determine the reaction rates. To avoid this problem, modelers often resort to â qualitativeâ modeling strategies, such as Boolean switching networks, but these models describe only the coarsest features of cell cycle regulation. In this work, we describe a hybrid approach that combines features of continuous and discrete networks. The model is evaluated in terms of flow cytometry measurements of cyclin proteins in asynchronous populations of human cell lines. Using our hybrid approach, modelers can quickly create quantitatively accurate, computational models of protein regulatory networks found in various contexts within cells.
Large-scale protein regulatory networks, such as the one that controls the progression of the mammalian cell cycle, also contain small-scale motifs or modules that carry out specific dynamical functions. Systematic characterization of smaller, interacting, network motifs whose individual behavior is well known under certain conditions is therefore of great interest to systems biologists. We model and simulate various 3-node network motifs to find near-perfect adaptation behavior. This behavior entails that a system responds to a change in its environmental cues, or signals, by coming back nearly to its pre-signal state even in the continued presence of the signal. We let various topologies evolve in their parameter space such that they eventually stumble upon a region where they score well under a pre-defined scoring metric. We find many such parameter sample sets across various classes of topologies.Ph. D.
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Khan, Imtiaz Ali. "Novel bioinformatics approach for encoding and interrogating the progression and modulation of the mammalian cell cycle." Thesis, Cardiff University, 2008. http://orca.cf.ac.uk/55787/.
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The cell cycle, with its highly conserved features, is a fundamental driver for the temporal control of cell growth and proliferation in tissues - while abnormal control and modulation of the cell cycle are characteristic of cancer cells, particularly in response to therapy. A central theme in cancer biology is to resolve and understand the origin and nature of innate and induced heterogeneity at the cell population level. Cellular heterogeneity - comprising structural, temporal and functional dimensions - is a confounding factor in the analysis of cell population dynamics and has implications at physiological, pathological and therapeutic levels. There is an exceptional advancement in the applications of imaging and cell tracking technologies dedicated to the area of cytometric research, that demand an integrated bioinformatics environment for high-content data extraction and interrogation. Image-derived cell-based analyses, where time is the quality parameter also demand unique solutions with the aim of enabling image encoding of spatiotemporal cellular events within complex cell populations. The perspective for this thesis is the complex yet poorly understood nature of cancer and the opportunities offered by rapidly evolving cytometric technologies. The research addresses the intellectual aspects of a bioinformatics framework for cellular informatics that encompass integrated data encoding, archiving, mining and analysis tools and methods capable of producing in silico cellular fingerprints for the responses of cell populations to perturbing influences. The overall goal is to understand the effects of anti-cancer drugs in complex and potentially heterogeneous neoplastic cellular systems by providing hypothesis testing opportunities. Cell lineage maps encoded from timelapse microscopy image sequences sit at the core of the proposed bioinformatics infrastructure developed in the current work. Through a number of data mining, analysis and visualisation tools the interactions and relationships within and between lineages have provided dynamic patterns for the modulation of the cell cycle in disease and under stress. The lineage data, accessible through databases implemented during the current study, has provided a rich repository for pharmacodynamic (PD) modelling and validation and has thus laid the foundation for fabricating a comprehensive knowledge base for linking both cellular and molecular behaviour patterns. These provide the foundation for meeting the aspirations of systems biology and drug discovery.
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Myer, David. "Role of the Mammalian Polo-Like Kinase 3(Plk3) in Cell Cycle Regulation and DNA Damage Checkpoints." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1141395911.
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Yarmishyn, Aliaksandr. "Analysis of the effects of the cyclin encoded by murine g-herpesvirus-68 on mammalian cell cycle control." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443805.
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Chen, Hui-Zi. "Mammalian Atypical E2Fs Link Endocycle Control to Cancer." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1316540844.
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Fuge, Grischa [Verfasser], and An-Ping [Akademischer Betreuer] Zeng. "New approaches for characterizing and monitoring mammalian cell cycle and specific growth rate in production cell lines / Grischa Fuge ; Betreuer: An-Ping Zeng." Hamburg : Universitätsbibliothek der Technischen Universität Hamburg-Harburg, 2018. http://d-nb.info/1155237811/34.
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Dall\'Orto, Clarissa Campo. "Avaliação das interações das células endoteliais e das células musculares lisas arteriais com os inibidores do mammalian target of rapamycin (mTOR) na presença de soro rico em plaquetas." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/5/5167/tde-05122018-115750/.
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INTRODUÇÃO: O sucesso a longo prazo da intervenção coronária percutânea, inicialmente realizada apenas com balão, era limitado pelo recolhimento elástico da artéria e pela hiperplasia neointimal. Com o advento dos stents convencionais (BMS) houve melhora nesse cenário e diminuição da reestenose, que é resultante de uma complexa cadeia de eventos iniciada após a injúria causada na parede vascular pela insuflação de balões e da aposição das hastes do stent. A proliferação excessiva de células musculares lisas (VSMC) tem papel fundamental na formação da neoíntima no contexto da reestenose intra-stent com a consequente redução da luz arterial. Com o advento dos stents farmacológicos (DES) houve diminuição importante da hiperplasia neointimal e um dos fármacos que se mostrou efetivo nesse papel é o sirolimo, que atua se ligando à proteína de ligação 12 e o heterodímero resultante se liga à mTOR impedindo sua ativação e causando parada do ciclo celular entre as fases G1 e S, desse modo inibindo a proliferação e migração de VSMC e das células endoteliais (HUVEC). Portanto a intervenção coronária acaba interferindo diretamente no endotélio, interferindo na produção das HUVEC não apenas no aspecto quantitativo, mas também na função das mesmas, e a qualidade funcional do endotélio é tão fundamental quanto à sua presença. Após o implante dos DES, principalmente os de primeira geração, ocorre disfunção endotelial cujo principal marcador é a perda da capacidade do relaxamento do vaso. Há correlação também entre cobertura das hastes incompleta e ocorrência de trombose dos stents. Consequentemente há espaço para o aprimoramento dos DES, para que se tornem dispositivos com eficácia já alcançada na prevenção da reestenose porém com um perfil de segurança maior. O presente trabalho tem como objetivo avaliar as alterações causadas pelos DES nas HUVEC e nas VSMC em cocultura na presença e na ausência do soro rico em plaquetas. MATERIAIS E MÉTODOS: Utilizamos células HUVEC e VSMC em modelos de monocultura e cocultura, na presença e na ausência de soro rico em plaquetas, tratadas com BMS ou DES. Realizamos a determinação da IC50 do inibidor da mTOR, avaliação da citotoxicidade pelo método colorimétrico do MTT, determinação da formação de peróxidos lipídicos, avaliação das fases do ciclo celular e da expressão de marcadores de controle de proliferação e inflamação. RESULTADOS: Na avaliação da citotoxicidade pelo método colorimétrico do MTT e determinação da IC50 as VSMC foram menos sensíveis ao sirolimo que as HUVEC (IC50 em 24/48 horas 14,85/10,47uM e 9,48/22,24 uM, respectivamente para HUVEC e VSMC). As plaquetas e fatores solúveis potencializam o estresse oxidativo gerado pela presença dos stents possivelmente por ampliar o ambiente inflamatório. Houve parada do ciclo celular na fase G0/G1 causada pelos DES somente com adição das plaquetas ao meio de cultura. Nos modelos de cultura celular sem as plaquetas a parada do ciclo celular foi em G2/M. Não houveaumento das células na fase DNA fragmentado (sub-G0) evidenciando que não houve indução de morte celular. CONCLUSÃO: As VSMC foram menos sensíveis ao sirolimo que as HUVEC. Nos modelos de cocultura com adição das plaquetas os DES eluídores de sirolimo causaram parada do ciclo celular na fase G0/G1 sem indução de morte celular, sugerindo que o sirolimo exerce seus efeitos anti-inflamatórios nessas populações celulares e consequentemente reduz a hiperplasia neointimal por um mecanismo citostáticoINTRODUCTION: The long-term success of percutaneous coronary intervention, initially performed only with a balloon, was limited by the elastic recoil of the artery and by neointimal hyperplasia. There was improvement in this scenario with the advent of bare metal stents (BMS), because they decrease in restenosis, that resulting from a complex network of events initiated after the injury caused in the vascular wall by insufflation of balloons and apposition of the stent struts. Excessive proliferation of smooth muscle cells (VSMC) plays a key role in neointimal hyperplasia in the context of intrastent restenosis with consequent reduction of arterial lumen. With the advent of drug-eluting stents (DES) there was a significant decrease in neointimal hyperplasia and one of the drugs that proved effective in this role is sirolimus, which acts by binding to the binding protein 12 and the resulting heterodimer binds to mTOR preventing its activation and causing cell cycle arrest between G1 and S phases and thereby inhibiting the proliferation and migration of VSMC and also inhibiting endothelial cells (HUVEC). Therefore, coronary intervention interferes directly in the endothelium, interfering in the production of endothelial cells, not only in the quantitative aspect, but also in their function, and the functional quality of the endothelium is as fundamental as its presence. After the implantation of DES, especially those of the first generation, endothelial dysfunction occurs, whose main marker is the loss of the capacity of the vessel relaxation. There is also correlation between incomplete stem coverage and stent thrombosis. Consequently, it is possible to improve of the DES, so that they become devices with already achieved effectiveness in the prevention of restenosis but with a greater safety profile. The present study aims to evaluate the changes caused by DES in human HUVEC and VSMC in co-culture in the presence and absence of platelet-rich serum. MATERIALS AND METHODS: We used HUVEC and VSMC in monoculture and co-culture models in the presence and absence of platelet rich serum treated with BMS or DES. We performed the determination of IC50 for mTOR inhibitor, cytotoxicity evaluation by the colorimetric method of MTT, determination of lipid peroxide formation, cell cycle and expression of necrosis and inflammation markers. RESULTS: In the assessment of cytotoxicity by the MTT colorimetric method and determination of the IC50, VSMC were less sensitive to sirolimus than HUVEC (IC50 in 24/48 hours 14.85 uM/10.47uM and 9.48 uM/ 22.24 uM, respectively for HUVEC and VSMC). Platelets potentiate the oxidative stress generated by the presence of stents, possibly by increasing the inflammatory environment. Drug-eluting stents arrested VSMC and HUVEC in the G0/G1 phase of the cell cycle only with the addition of platelets to the culture medium. In cell culture models without platelets the cell cycle arrest was in G2/M. There was no increase of the cells in the fragmented DNA phase (sub-G0) evidencing that there was no induction of apoptosis. CONCLUSION: Human aorta smooth muscle cells of the were less sensitive to sirolimus than HUVEC. In coculture models with platelet addition, DES with sirolimus caused cell cycle arrest in the G0/G1 phase without induction of apoptosis, suggesting that sirolimus exerts its antiinflammatory effects in these cellular populations and consequently reduces neointimal hyperplasia via a cytostatic mechanism
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Saddick, Salina Yahya. "Effect of the reproductive cycle on morphology and activity of the ovarian surface epithelium in mammals." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4713.
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The layer of cells lining the outer surface of the mammalian ovary, the ovarian surface epithelium (OSE), is a constant feature throughout the dynamic tissue remodeling that occurs throughout the reproductive cycle (follicle growth, ovulation, corpora lutea formation and pregnancy). Abnormal development of these cells is responsible for 90% of all epithelial ovarian cancers in women and epidemiological studies have shown that susceptibility to ovarian cancer is negatively correlated with increasing pregnancy. Little is known about how OSE cells are affected at each stage of the cycle, so the main aim of this study was to determine how the reproductive cycle affected proliferation and degeneration of OSE cells. This study utilised three animal models each with a different type of reproductive cycle: a mono-ovular seasonal breeder (Sheep), a mono-ovular polyoestrous breeder (Cow) and a poly-ovular non human primate (marmoset) to allow comparisons to be made. Comparison of OSE proliferative activity was made in sheep and marmoset at each stage of the cycle including pregnancy and anoestrous. The bovine model was used to investigate apoptotic cell death. Proliferative activity of somatic cells within the sheep ovary was monitored throughout the reproductive cycle by detection of cell cycle markers PCNA and Ki67 using immunohistochemistry. The pattern of OSE proliferation was correlated with the pattern of follicle development at each stage (sheep and marmoset). During pregnancy cell proliferation was significantly lower in OSE and in granulosa cells, reflecting a suppression of mature follicle development during these stages whereas in cycling animals proliferation was increased. Differences in OSE proliferation were observed in relation to the local underlying tissue environment in both sheep and marmoset. Epithelial cell rupture and regeneration enhanced the hormonal mitogenic action on epithelial cells, which showed highest proliferation over corpora lutea in each animal model. To test the hypothesis that these changes are mediated by hormones or growth factors ovine OSE cells were cultured and proliferative activity monitored after treatment with several factors: fetal calf serum (FCS), follicular fluid from follicles of varying sizes, corpora lutea extracts, recombinant human IGF-1, oestradiol and progesterone. IGF alone was demonstrated to have an affect on increasing proliferation of cultured OSE cells. Levels of FSHr and LHr were monitored by quantitative real- time PCR and it was demonstrated that the concentration of gonadotrophin receptors in OSE, increased prior to and after ovulation, at which time the in vivo OSE proliferation also peaked. The in situ apoptosis index was determined in bovine tissue using TUNEL throughout the regular cycle, and at mid and late-pregnancy stages. The results showed that pregnancy induced apoptotic activity in OSE cells and up regulated the tumour suppressor gene p53. Cultured bovine OSE cells also exhibited an increased level of apoptosis following progesterone treatment. Since p53/p53 gene expression in OSE over the corpora lutea producing progesterone also increased, this progesterone-mediated apoptosis may be mediated through an up-regulation of p53 synthesis. The effect of pregnancy and low production of gonadotrophins in the regulation of OSE cell morphology and activity was further investigated in the marmoset monkey (a non-human primate) treated with GnRH antagonist and infused with BrdU to monitor proliferative activity. OSE proliferation was correlated to ovarian events (follicular growth, ovulation and luteinization) and this was suppressed during pregnancy. Inhibition of gonadotrophin secretion by treatment with a GnRH antagonist also markedly inhibited OSE proliferation. Taken together these studies support the hypothesis that pregnancy and periods of anovulation reduce proliferation of OSE cells and alter the pattern of apoptotic cell death and that this effect is independent of species and reproductive pattern. Suppression of gonadotrophins and other growth factors during pregnancy could enhance p53-mediated apoptosis of damaged and mitogenic cells arising from repeated ovulations. This effect may partly explain why increasing number of pregnancies in woman reduces the chance of epithelial ovarian cancers.
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Grinfeld, Simone. "Etude du blocage en phase G du premier cycle cellulaire, induit par les rayons X dans l'oeuf de souris." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb376057177.
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Cohen, Edith. "Etude cytologique de la cellule neuro-epitheliale, chez l'embryon de souris, au stade initial de la differenciation neuronale." Paris 6, 1987. http://www.theses.fr/1987PA066155.
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Soubigou, Pascal. "Cycle du recepteur de l'insuline et devenir de l'hormone chez l'hepatocyte foetal en culture : relation avec la reponse glycogenique a l'insuline." Paris 6, 1987. http://www.theses.fr/1987PA066214.
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Moris, Gilbert. "Le diadenosine tetraphosphate : roles dans la regulation de la croissance et du cycle cellulaires et dans la reponse cellulaire a un stress a l'ethanol." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13118.
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Berrou, Eliane. "Synthese des proteoglycannes par les cellules musculaires lisses d'aorte de porc en culture : etude en fonction du cycle cellulaire, effet de l'insuline et de facteur(s) secrete(s) par les cellules endotheliales." Paris 7, 1988. http://www.theses.fr/1988PA077013.
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Orfanoudakis, Georges. "Diadenosine tetraphosphate : implication dans l'activite mitotique, la replication et la reparation du dna." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13121.
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Le diadenosine tetraphosphate (ap**(4)a) est le principal produit de la reaction d'aminoacylation catalysee par certaines aminoacyl-trna synthetases. L'ap**(4)a est une molecule "signal" s'accumulant a l'interphase g1/5 du cycle cellulaire des cellules eucarydes declenchant ainsi la synthese du dna precedant la division cellulaire. Quantification du contenu cellulaire en ap**(4)a et en atp apres synchronisation des cellules (hepertome de rat, fibroblaste de souris) en culture par l'aphidicoline agent bloquant les cellules en phase s et par privation de serum qui arrete la croissance en mi-phase g. Mise au point d'un modele de reparation, dans les ovocytes ou les oeufs non fecondes de xenopus laevis, du plasmute pbr322 modifie par l'acetylaminofluorene. L'effet de l'ap**(4)a sur la reparation est etudie
42
Husson, Annie. "Contrôle multi-hormonal des enzymes cytosoliques du cycle de l'urée pendant la période périnatale chez le rat." Rouen, 1986. http://www.theses.fr/1986ROUES024.
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Millet, Pascal. "Développement, en culture cellulaire, de quelques Coccidiomorphes (Isopora, Plasmodium) : mise au point d'un modèle expérimental pour la chimiothérapie du paludisme." Paris 6, 1986. http://www.theses.fr/1986PA066272.
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Tan, Xiao-Tong, and 陳筱彤. "The role of hHR23A function in mammalian cell cycle progression." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/11363471089685662878.
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碩士國立中興大學
生物醫學研究所
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Rad23 is an evolutionarily conserved protein from yeast to human. Human cells express two isoforms of Rad23, named hHR23A and hHR23B. Rad23 plays a critical role in nuclear excision repair (NER) and ubiquitin-proteasome system (UPS). However, the signaling regulation of Rad23 function is still unclear. In the previous experiments showed that ubiquitin chain binding ability of hHR23 might be used for degradation of cell cycle regulator protein, which consequently affect the cell cycle progression. hHR23 proteins has been shown a key role to regulate cell cycle progression in yeast but seldom investigated in mammalian cells. Based on it, the goal of our study is to distinguish the different role of hHR23 homologs in mammalian cell cycle progression. Therefore, we further knocked down hHR23 by siRNA and shRNA and found that knockdown of hHR23 didnt affect cell cycle progression. Interestingly, depletion of hHR23 expression led to significantly enhance cisplatin induced cell cycle delay. Depended on these results, we suggest that hHR23A is important for cell cycle progression but hHR23B plays a different role in it. Moreover, from cell synchronize assay, we showed that knockdown of hHR23A alone delay cell cycle progression and cell division. Based on these results, we screened cell cycle regulator proteins and tested which protein was effected when depletion of hHR23A. In collectively, we found that Checkpoint kinase 1 (Chk1) was being upregulated with or without DNA damage agent in knockdown hHR23A cell lines. Moreover, immunoprecipitated analysis showed that Chk1 and hHR23A interaction in nucleus and hHR23A responsible for Chk1 degradation. In sum, the molecular mechanism of hHR23A and Chk1 regulates cell cycle progression by signaling will be further investigated.
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"Role of the transcription factor NFAT5 in mammalian cell cycle regulation." Universitat Pompeu Fabra, 2008. http://www.tesisenxarxa.net/TDX-1102109-130647/.
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Liu, Chenshu. "Cytoskeletal Regulation of Centromere Maintenance and Function in the Mammalian Cell Cycle." Thesis, 2016. https://doi.org/10.7916/D89S1RBQ.
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Equal partitioning of genetic materials of the chromosomes is key to the mitotic cell cycle, as unequal segregation of chromosomes during mitosis leads to aneuploidy, a hall mark of human cancer. Accurate chromosome segregation is directed by the kinetochore, a proteinaceous structure on each sister chromosome that physically connects the chromosome to the spindle microtubules. Kinetochore assembles at the centromere, a specialized chromosome region epigenetically defined by the histone H3 variant centromere protein A (CENP-A) in higher eukaryotes including mammals. In order to maintain centromere identity against CENP-A dilution caused by S phase genome replication, new CENP-A molecules are loaded at preexisting centromeres in G1 phase of the cell cycle. Despite of the several important stages and molecular components identified in CENP-A replenishment, little is known about how new CENP-A proteins become stably incorporated into centromeric nucleosomes. Here by using quantitative imaging, pulse-chase labeling, mutant analysis, cellular fractionation and computational simulations, I have identified the cytoskeleton protein diaphanous formin mDia2 to be essential for the essential for the stable incorporation of newly synthesized CENP-A at the centromere. The novel function of mDia2 depends on its nuclear localization and its actin nucleation activity. Furthermore, mDia2 functions downstream of a small GTPase molecular switch during CENP-A loading, and is responsible for the formation of dynamic and short actin filaments observed in early G1 nuclei. Importantly, the maintenance of centromeric CENP-A levels requires a pool of polymerizable actin inside the nucleus. Single particle tracking and quantitative analysis revealed that centromere movement in early G1 nuclei is relatively confined over the time scale of initial CENP-A loading, and the subdiffusive behavior was significantly altered upon mDia2 knockdown. Finally, knocking down mDia2 results in prolonged centromere association of Holliday junction recognition protein (HJURP), a chaperone required to undergo timely turnover to allow for new CENP-A loading at the centromere. Our findings suggest that diaphanous formin mDia2 forms a link between the upstream small GTPase signaling and the downstream confined viscoelastic nuclear environment, and therefore regulates the stable assembly of new CENP-A containing nucleosomes to mark centromeres’ epigenetic identity (Chapter 2 and 3).
While centromere identity is essential for kinetochore assembly, once kinetochores are assembled, fine-tuned interactions between kinetochores and microtubules become important for a fully functioning mitotic spindle during chromosome segregation. It has been previously found that another diaphanous formin protein mDia3 and its interaction with EB1, a microtubule plus-end tracking protein, are essential for accurate chromosome segregation1. In Chapter 4 of this thesis, I found that knocking down mDia3 caused a compositional change at the microtubule plus-end attached to the kinetochores, marked by a loss of EB1 and a gain of CLIP-170 and the dynein light chain protein Tctex-1. Interestingly, this compositional change does not affect the release of cytoplasmic dynein from aligned kinetochores, suggesting a population of Tctex-1 can be recruited to the kinetochores without dynein. During mitosis, Tctex-1 associates with unattached kinetochores and is required for accurate chromosome segregation. Tctex-1 knockdown in cells does not affect the localization and function of dynein at the kinetochore, but produces a prolonged mitotic arrest with a few misaligned chromosomes, which are subsequently missegregated during anaphase. This function is independent of Tctex-1’s association with dynein. The kinetochore localization of Tctex-1 is independent of the ZW10-dynein pathway, but requires the Ndc80 complex. Thus, our findings reveal a dynein independent role of Tctex-1 at the kinetochore to enhance the stability of kinetochore-microtubule attachment.
Together, these work suggest novel regulatory roles of the cytoskeletal systems in the maintenance as well as subsequent functions of the centromere/kinetochore, and provide mechanistic insights into the complex control principles of accurate chromosome segregation. Our findings provide a new model in understanding the epigenetic maintenance of genome integrity, and will have implications with regard to how aberrant cell divisions underlying aneuploidy can be targeted in the treatment of cancer.
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Pacal, Marek. "Coordinating Cell Cycle Exit and Differentiation in the Mammalian Retina and its Dependence on Rb." Thesis, 2012. http://hdl.handle.net/1807/33857.
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Cell cycle exit (“birth”) of retinal progenitor cells (RPCs) is considered a watershed that is preceded by changing levels of cell cycle regulators, and followed rapidly by induction of a post M-phase differentiation cascade. Yet the actual dynamics of these events are largely unclear, thus whether mitosis separates pre- and post- birth differentiation cascades is unproven. We characterized the regulation of many division and differentiation markers relative to each other and final mitosis. Unexpectedly, classic “cell cycle” markers were present well beyond exit (e.g. Ki67, Pcna), early embryonic RPCs expressed “differentiation” markers that later labeled post-mitotic neurons
exclusively (e.g. Brn3b, Tubb3, Ptf1a), and factors detected just after cell birth in the
embryo were induced well beyond M-phase post-natally (e.g. Nrl, Crx). Thus, the dynamics of birth-associated events shift dramatically during development, even to either side of mitosis. Instead of mitosis behaving as a cog that activates post-exit
differentation events we suggest that a common trigger induces both the exit and
differentiation programs in RPCs, precisely coordinating their startpoints, but that each
subsequent cascade unfolds independently. This model explains the convergence of birth
and differentiation but also their temporal maliability. This view fits with our
observation that in the absence of the Rb tumor suppressor, differentiation still initiates even without cell cycle exit. Finally, neoplastic transformation in the mouse retina requires loss of Rb and its relative p107, and emerging tumor features suggest an amacrine cell-of-origin. We studied Rb/p107 null clones, and noted two striking features. First, despite initial expansion of aberrantly dividing differentiating cells, apoptosis
pruned clones precisely to wild type sizes. “Cell competition” maintains tissue size by selecting fitter over weaker progenitors; our data provide a unique example of competition among differentiating cells. Second, despite normal numbers of amacrine cells per Rb/p107 null clone, more clones contained amacrine cells and fewer had bipolar cells. Both this effect and ectopic division were E2f1-dependent. Thus, the oncogenic initiation event in mouse retinoblastoma triggers a very early fate switch, even before neoplastic transformation, broadening the possibilities for the cell-of-origin of retinoblastoma, and arguing that even very early stage tumors cannot be used to define cancer origin.
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Kremer, Brandon Edward. "Mammalian septins regulate microtubules, actin, and the DNA damage checkpoint response." 2007. http://wwwlib.umi.com/dissertations/fullcit/3300246.
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Kiewisz, Robert. "Ultrastructural characterization of mammalian k-fibers by large-scale electron tomography." 2021. https://tud.qucosa.de/id/qucosa%3A76019.
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Eukaryotic cells have to divide constantly in order to promote the growth of certain organs, to replace dying or damaged cells, or to set up an entire organism. These essential processes are called mitosis in the case of somatic cell division. Mitotic cell division is the process during which chromosomes, centrosomes, and microtubules (MTs) are involved to set up a bipolar structure called the “mitotic spindle”. This bipolar spindle is formed by MTs, which are presumably mainly organized from the centrosomes. However, more data are being published that suggest MTs nucleation can occur from other MTs or even a chromosome surface. These biopolymers are built from α/β-tubulin heterodimers and can dynamically grow and shrink to exert forces necessary for chromosome segregation. Previous studies of spindles during mitosis have allowed the identification of different MT classes based on their plus-ends interaction with different cellular target sites. One of the MT classes is the kinetochore microtubules (KMTs), which physically connect chromosomes and centrosomes (i.e. spindle pole) via a specialized protein structure termed the “kinetochore”. This kinetochore-to-spindle pole connection has been studied in many organisms. In budding yeast, this connection is established by only a single KMT. In contrast, multiple KMTs bind to each mammalian kinetochore and form an MT bundle also called “k-fiber”. The ultrastructural architecture of the mammalian k-fiber connection is not well documented. Currently, different models concerning the nature of the kinetochore-to-spindle pole connection via k-fibers are discussed in the literature, i.e. a direct, semi-direct or indirect connection. The widely accepted ‘direct’ model proposes that all k-fibers of the mammalian spindle are formed through tight bundles of up to 20 KMTs, with all MT minus ends associated with the centrosome. However, it is necessary to understand the k-fibers structure in order to interpret its role during chromosome segregation. Here the architecture of the k-fiber was studied in human HeLa, U2OS and RPE-1 cell lines, as these different types of cells have been widely used in studies of mitosis. This thesis aimed to systematically investigate the characteristics of mammalian k-fibers and their attachment to the kinetochore within mammalian metaphase spindles. For that, the ultrastructure of mitotic spindles and k-fibers were analyzed using serial-section electron tomography primarily in HeLa cells. Furthermore, the spindle ultrastructure was compared by electron tomography to metaphase spindles in both U2OS and RPE-1 cells. Electron tomographic analysis of the mitotic spindle in HeLa cells revealed that the kinetochore-to-spindle pole connection is formed by k-fibers consisting of ~9 KMTs. Moreover, the data revealed that not all KMTs in k-fibers are directly associated with one of the spindle poles. Instead, KMT ends were located along the length of k-fibers indicating strongly for a semi-direct connection between the kinetochores and the spindle poles. Unexpectedly, by correlating the k-fiber ultrastructure with its position in the mitotic spindle, it can be demonstrated that the k-fiber structure varied depending on the position on the metaphase plate. It can also be shown that k-fibers located in the center of the metaphase plate had a tendency to form straighter and more bundled k-fibers. In contrast, k-fibers associated with the periphery of the metaphase plate had a more loose and disorganized structure resembling a fusiform shape. Furthermore, additional analysis of U2OS and RPE-1 cells indicated ultrastructural differences between the different cell lines. Mainly, differences between HeLa and RPE-1 cells were observed. K-fibers observed in RPE-1 cells showed a lower curvature and overall a more bundled ultrastructure compared to HeLa or U2OS cells. However, due to the small sample size for U2OS and RPE-1 cells, the results have to be confirmed in future experiments to conclude that there are indeed functional and structural differences in the k-fiber organization in different mammalian cell lines. Taken together, this work presents the first detailed quantitative ultrastructural analysis of KMTs in whole spindles in three different human cell lines. The data revealed that the currently favored direct model of k-fiber ultrastructure is oversimplified and needs to be corrected in terms of the k-fibers interaction with the spindle pole and the surrounding MT network within the mitotic spindle. The data here will serve as a structural basis for further analyses of mutant situations and contribute to our understanding of the overall organization and function of MTs in mitotic spindles.:Summary 6
Zusammenfassung 8
List of figures 10
List of tables 13
List of abbreviations and symbols 14
1 Introduction 19
1.1 The morphology of the mitotic spindle 21
1.1.1 Centrosomes 22
1.1.2 Microtubules 23
1.2 Kinetochores, KMTs and k-fibers 28
1.2.1 A brief history of k-fiber formation in mammalian cells 30
1.2.2 Models of the k-fiber ultrastructure in mammalian cells 32
2 Aims of this thesis 35
3 Materials and methods 36
3.1 Materials 37
3.1.1 Mammalian cell lines 37
3.1.2 Chemicals 38
3.1.3 Instrumentation and materials 40
3.1.4 Solutions and buffers 44
3.1.5 Software 46
3.2 Methods 47
3.2.1 Handling of cell cultures 47
3.2.2 Custom-designed incubation chambers 49
3.2.3 Specimen preparation for electron microscopy 51
3.2.4 Quality assessment of samples, acquisition of the tomographic data, and the 3D reconstruction 59
3.2.5 Ultrastructural analysis of MTs in mitotic spindles 62
3.2.6 Ultrastructural analysis of the k-fiber organization 70
4 Results 76
4.1 Initial characterization of mammalian mitotic spindles 77
4.2 Ultrastructure of KMTs 84
4.3 Curvature and tortuosity of KMTs 91
4.4 Ultrastructure of k-fibers 98
4.5 Effect of metaphase position on the k-fiber ultrastructure 102
5 Discussion 110
5.1. Comparison of data sets from different cell lines 111
5.2. Establishing a data analysis pipeline for the analysis of KMTs 113
5.3 Ultrastructural characterization of KMTs and k-fibers in HeLa cells 114
5.3.1 K-fibers have an unexpectedly low number of KMTs 115
5.3.2 Semi-direct kinetochores-to-spindle pole connection 117
5.3.3 Shape of k-fibers 121
5.4 Positional effect on the k-fiber shape 124
5.5 Comparison of k-fiber ultrastructure in different mammalian cells 127
5.6 Outlook 130
References 133
Appendix 1 149
Appendix 2 150
Appendix 3 151
Appendix 4 152
Acknowledgments 153
50
Schwartz, Jeanette Marie. "First evidence of skelemin, a myosin-associated protein, in smooth muscle and its involvement in cell adhesion, and, The role of the cell cycle in cell type choice during mammalian development." Thesis, 1996. http://hdl.handle.net/1911/17130.
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Indirect immunofluorescence in conjunction with Northern and Western blot analysis were used to identify the presence of skelemin, a myosin-associated protein, in smooth muscle. Feline uterus was cryosectioned and triple-stained for skelemin, smooth muscle myosin, and desmin. I observed that skelemin antibodies colocalized with the myosin-II filaments as well as the desmin intermediate filament cytoskeleton. This is the first evidence of a myosin-associated protein within smooth muscle, and it raises the possibility that smooth muscle myosin may be more organized than has been assumed. Antisense oligonucleotide treatment was used to analyze the underexpression of skelemin protein in developing embryoid bodies. Skelemin underexpression was seen to cause loss of cell-cell as well as cell-substrate adhesion. Time lapse video microscopy was used to analyze the role of the cell cycle in cell type choice during mammalian development. Discussed are methods employed to improve resolution for accurate analysis.