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The role of the translational regulator p97 in mammalian cellsNousch, Marco, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2008 (has links)
Members of the eukaryotic initiation factor 4G (eIF4G) family play a central role in the translation initiation process. One member of this family is p97 (also called DAP5 and NAT1), a protein that is highly homologous to the C-terminal two thirds of eIF4G. Overexpression studies suggested that p97 is a pure translational repressor that has to be cleaved into a shorter form called p86, in order to show translational activity. In this study a series of experiments indicated that full length p97 has a number elF property such as association with active translating ribosomes, stimulatory effects in the Direct Initiation Factor assay and accumulation in stress granules. Additionally the endogenous p97 complex was isolated from HeLa cells and mRNA as well as the protein components were characterized. P97 associated mRNAs were described by a custom made 5'UTR focus array, showing that the protein binds to a broad range of mRNA. The relative lack of mRNA specificity argues for a general role of p97 in translation, which does not seems to be essential in unchallenged cells, because a down regulation of p97 protein levels has no effect on the translational status of the bulk of mRNAs. Mass spectrometry analysis revealed a novel protein-protein interaction between p97 and DNA methyltransferase 1 (Dnmt1), which does not rely on a nucleic acid. For this interaction the C- and N-terminus of p97 play a critical role. Further, Dnmt1 has the ability to interact with elF4G and the small ribosomal subunit, which might provide evidence for a novel function of Dnmt1 in RNA metabolism.
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Regulation of nitric oxide synthase expression in mammalian cells張婓怡, Cheung, Filly. January 2001 (has links)
published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy
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RNA/protein interactions during group II intron splicing and toward group II intron targeting in mammalian cellsCui, Xiaoxia 28 August 2008 (has links)
Not available / text
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Regulation of ubiquitin-mediated proteolysis in Xenopus laevis and mammalian cellsRoark, Ryan Leigh January 2011 (has links)
No description available.
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Studies on the RNA interference pathway in mammalian cellsJagannath, Aarti January 2009 (has links)
No description available.
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A Novel Analytical Framework for Regulatory Network Analysis of Single-Cell Transcriptomic DataVlahos, Lukas January 2023 (has links)
While single-cell RNA sequencing provides a remarkable window on pathophysiologic tissue biology and heterogeneity, its high gene-dropout rate and low signal-to-noise ratio challenge quantitative analyses and mechanistic understanding. This thesis addresses this issue by developing PISCES, a pipeline for regulatory network-based single-cell analysis of mammalian tissues. PISCES accurately estimates the mechanistic contribution of regulatory and signaling proteins to cell state implementation and maintenance based on the expression of their lineage-specific transcriptional targets, inferring protein activity for a putative set of transcriptional regulators and cell-state markers. Experimental validation assays – including technical analysis via downsampling of high depth data and biological analysis by assessing concordance with CITE-Seq-based measurements – show a significant improvement in the ability to identify rare subpopulations and to elucidate key lineage markers compared to gene expression analysis.
The improved ability to identify biologically meaningful cellular subpopulations makes PISCES an ideal tool to deconvolute heterogeneity in a wide variety of biological contexts. A systematic analysis of single-cell gene expression profiles in the Human Protein Atlas (HPA) by PISCES generated tissue-specific clustering and master regulator analyses across 26 human tissues, as well as a publicly available repository of ready-to-use regulatory networks specific to cell-lineages in each tissue. This resource will allow researchers to access the algorithmic advantages of PISCES without requiring prohibitively expensive or technically challenging computational resources.
Additionally, PISCES is able to unravel the heterogeneous stromal environment of Pancreatic Ductal Adenocarcinoma, a malignancy defined by a large and complicated stromal compartment. This analysis reveals several novel candidate subpopulations, including a fibroblast subtype that has never been observed in humans, a potential pro-metastatic population of endothelial cells, and a population of immune-suppressing stellate cells.
PISCES is also able to deconvolute more continuous forms of heterogeneity, as demonstrated by an analysis of epithelial cells in the developing murine lung. Here, PISCES is able to computationally reconstruct a developmental trajectory between Sox9+ distal cells and Sox2+ proximal cells, which is then leveraged to identify several novel markers of the critical intermediate population. Subsequent analysis suggests that these transition zone cells may share programs similar to those seen in injury repair and identifies a candidate therapeutic target that can drive cells into or out of this transition state.
Finally, protein activity measured by PISCES is used to refine faulty experimental labels through differential density analysis. This analysis lead to the development of a machine learning classifier that accurately predicted increased degrees of stemness in experimentally transduced populations. Additionally, the density analysis paradigm has been extended to unsupervised settings, allowing for the detection of stable cellular populations and transitory trajectories.
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Manipulation of mammalian cells by femtosecond laser irradiation. / 飛秒激光對哺乳動物細胞的操控 / CUHK electronic theses & dissertations collection / Fei miao ji guang dui bu ru dong wu xi bao de cao kongJanuary 2010 (has links)
1. Transfection is a key technique in cell and molecular biology with many important biochemical applications. We selected a fiber fs laser at 1554 nm, an instrument widely used in optical communication research, as the excitation source. Our results demonstrated that the fs laser could perforate the cell membrane and the hole would close in sub-second interval after the laser exposure. We determined the safe exposure duration by detecting if there was any sign of mitochondrial depolarization at 1.5 hours after photoporation. Furthermore, we had successfully transfected HepG2 cells with a plasmid DNA containing the OFP gene, whose fluorescence could still be detected 24 hours after exposure. The transfection efficiency was as high as 77.3%. We also observed the proliferation of the transfected cells after 48 hours. / 2. Cell-cell fusion is a powerful tool for the analysis of gene expression, chromosomal mapping, monoclonal antibody production, and cancer immunotherapy. One of the challenges of in vitro cell fusion is to improve the fusion efficiency without adding extra chemicals while maintaining the cells alive and healthy. We show here that targeted human cancer cells could be selected by an optical tweezer and fused by a finely focused fs laser beam at 1554 nm with a high fusion eftlciency. The result confirmed that human cells could be fused exclusively by fs laser pulses, and this is the first time human cells are fused together all-optically. Mixing of cytoplasm in the fused cells was subsequently observed, and cells from different cell lines were also fused. Based on these, we firstly developed the method of optical cell-cell fusion. / 3. Failure in the induction of apoptosis or programmed cell death is one of the major contributions to the development of cancer and autoimmune diseases. Here we used a fs laser as a novel method to provide a direct apoptosis trigger to observe dynamic changes at subcellular level during apoptosis. First, we examined the effect of fs laser irradiation on the creation of reactive oxygen species (ROS) in exposed cells, which could trigger programmed cell death. By controlling the mitochondria electron transport chain (ETC), we investigated the mechanism of ROS generation by the fs pulses, including thermal effect and direct free electron liberation. Second, we induced apoptosis to targeted cells by the fs laser and found that the nuclear envelope (NE) formed tubular or tunnel-like structures (nuclear tubules - NT) inside the nucleus. The average number of NTs in each cell with laser treatment was significantly larger than in the control. Besides, the development of a NT was observed since its inception and it eventually merged with another one to form a larger NT. Meanwhile, mitochondria and tubulin were found inside the NT, and the NT formation always occurred after an upsurge of cellular Ca2+ concentration. More DNA fragmentation were also found in the region around the NTs. Based on this, we propose that NTs are developed during apoptosis and mitochondria migrate into the nucleus through the NTs to release death signals to trigger DNA fragmentation. Third, we used the fs laser to induce Ca2+ in cells in the form of a slow release, and firstly discovered that most Ca2+ was stored in the cytoplasm, and could diffuse into the nucleus after the optical trigger. Using fast confocal scanning, we obtained the path way of Ca2+ diffusion after the trigger in different cases. Our findings thus provide a new method of regulating the rate of apoptosis. / Biophotonics is an exciting and fast-expanding frontier which involves a fusion of advanced photonics and biology. It has not only developed many novel methodologies for biomedical research, but also achieved significant results as an independent field. Aided with femtosecond (fs) laser technologies, important progresses have been made on manipulating, imaging, and engineering of biological samples from single molecules to tissues in the last 10 years. The laser beam of ultra-short pulses at near-infrared band enjoys a lot of advantages: high nonlinear efficiency, low absorption by biological samples, high spatial and temporal resolution with tight confinement, low photo-toxicity, non-invasive, and ease of control. In this thesis, we report new findings from cell manipulation by fs laser, including transfection, cell-cell fusion, and induction of apoptosis in cells, which are detailed as follows: / He, Hao. / Adviser: Kam Tai Chan. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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Optical sorting and photo-transfection of mammalian cellsMthunzi, Patience January 2010 (has links)
Recently, laser light sources of different regimes have emerged as an essential tool in the biophotonics research area. Classic applications include, for example: manipulating single cells and their subcellular organelles, sorting cells in microfluidic channels and the cytoplasmic delivery of both genetic and non-genetic matter of varying sizes into mammalian cells. In this thesis several new findings specifically in the optical cell sorting as well as in the photo-transfection study fields are presented. In my optical cell sorting and guiding investigations, a new technique for enhancing the dielectric contrast of mammalian cells, which is a result of cells naturally engulfing polymer microspheres from their environment, is introduced. I explore how these intracellular dielectric tags influence the scattering and gradient forces upon these cells from an externally applied optical field. I show that intracellular polymer microspheres can serve as highly directional optical scatterers and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human), embryonic kidney, Chinese hamster ovary as well as pluripotent stem cells using a tightly focused titanium sapphire femtosecond pulsed laser beam spot. These investigations permitted advanced biological studies in femtosecond laser transfection: firstly, the influence of cell passage number on the transfection efficiency; secondly, the possibility to enhance the transfection efficiency via whole culture treatments of cells thereby, synchronizing them at the mitotic (M phase) as well as the synthesis phases (S phase) of the cell cycle; thirdly, this methodology can activate the up-regulation of the protective heat shock protein 70 (hsp70). Finally, I show that this novel technology can also be used to transfect mouse embryonic stem (mES) cell colonies and the ability of differentiating these cells into the extraembryonic endoderm.
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Auto-organisation temporelle du réseau de kinases dépendantes de cyclines contrôlant le cycle cellulaire chez les mammifères / Temporal self-organization of the cyclin/Cdk network driving the mammalian cell cycleGérard, Claude 25 November 2009 (has links)
Au cours de ce travail de thèse, nous avons établi un modèle global pour le réseau de kinases dépendantes des cyclines (Cdks) qui contrôle la dynamique du cycle cellulaire chez les mammifères. Le modèle contient quatre modules Cdk régulés par phosphorylation-déphosphorylation, par des inhibiteurs de Cdk, et par la synthèse ou la dégradation de protéines. Les facteurs de croissance suscitent la transition d’un état stationnaire stable, état de quiescence, à un état de prolifération caractérisé par des oscillations entretenues du réseau de cyclines/Cdk. Ces oscillations correspondent à l’activation transitoire et répétitive des complexes cycline D/Cdk4-6 en phase G1, cycline E/Cdk2 à la transition G1/S, cycline A/Cdk2 en phase S et à la transition S/G2, et cycline B/Cdk1 à la transition G2/M. Le modèle rend compte de plusieurs propriétés majeures du cycle cellulaire des mammifères :(1) oscillations entretenues du réseau de Cdk en présence d’un niveau suffisant d’un facteur de croissance ;(2) contrôle de la progression dans le cycle cellulaire par la balance entre les effets antagonistes du suppresseur de tumeur pRB et du facteur de transcription E2F ;(3) existence d’un point de restriction situé dans la phase G1, au-delà duquel la cellule n’a plus besoin de la présence d’un facteur de croissance pour compléter son cycle de division cellulaire ;(4) entraînement du cycle cellulaire par l’horloge circadienne. Le modèle rend compte également du phénomène d’endoréplication qui correspond au découplage entre réplication de l’ADN et mitose :la cellule duplique à de multiples reprises son ADN sans entrer en phase de mitose. En incorporant des points de contrôle («checkpoints») dans le modèle pour le cycle cellulaire, et en particulier le point de contrôle de réplication de l’ADN régulé par les kinases ATR et Chk1, nous montrons comment ce point de contrôle ralentit la dynamique du cycle cellulaire et mène à une meilleure séparation des phases de réplication de l’ADN et de mitose. Le modèle pour le cycle cellulaire des cellules de mammifères montre comment la structure de régulation du réseau de cyclines/Cdk suscite son auto-organisation temporelle, menant à l’activation répétitive et séquentielle des quatre modules Cdk qui assurent la progression ordonnée dans les différentes phases du cycle cellulaire./We propose an integrated computational model for the network of cyclin-dependent kinases (Cdks) that controls the dynamics of the mammalian cell cycle. The model contains four Cdk modules regulated by reversible phosphorylation, Cdk inhibitors, and protein synthesis or degradation. Growth factors trigger the transition from a quiescent, stable steady state to self-sustained oscillations in the Cdk network. These oscillations correspond to the repetitive, transient activation of cyclin D/Cdk4-6 in G1, cyclin E/Cdk2 at the G1/S transition, cyclin A/Cdk2 in S and at the S/G2 transition, and cyclin B/Cdk1 at the G2/M transition. The model accounts for the following major properties of the mammalian cell cycle: (1) repetitive cell cycling in the presence of supra-threshold amounts of growth factor ;(2) control of cell cycle progression by the balance between antagonistic effects of the tumor suppressor pRB and the transcription factor E2F ;(3) existence of a restriction point in G1, beyond which completion of the cell cycle becomes independent of growth factor ;(4) entrainment of the cell cycle by the circadian clock. The model also accounts for endoreplication and for self-sustained oscillations in the presence of only Cdk1 or in the absence of pRB. Incorporating the DNA replication checkpoint mediated by kinases ATR and Chk1 slows down the dynamics of the cell cycle without altering its oscillatory nature and leads to better separation of the S and M phases. The model for the mammalian cell cycle shows how the regulatory structure of the Cdk network results in its temporal self-organization, leading to the repetitive, sequential activation of the four Cdk modules that brings about the orderly progression along cell cycle phases. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished
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