Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

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2.1 Radial Glia Introduction Radial glia are by no means a uniform cell population. In fact, they are found within the cortex and also throughout the developing brain and spinal cord. This spatial and temporal heterogeneity likely generates the diversity of cellular phenotypes within the nervous system. For example, region-specific expression of transcription factors in radial glia is likely to determine the fate of progeny towards one of the lineages of the CNS [249]. Furthermore, there are circumstances in which the radial glia phenotype is reacquired, such as after injury, during reprogramming and dedifferentiation in vitro, and following epigenetic disruptions in tumorigenesis [400]. Anatomically, a ventricular system is present within the cerebrum 26 that is composed of four communicating compartments, or ventricles, filled by CSF [387]. The SVZ is a paired brain structure situated in the lining of the two lateral ventricles and is one of the major germinal layers during embryogene- sis [18,41] together with the sub-granular zone (SGZ 27 ), as well as the largest district in which NS cells with the characteristics of astrocytes persist after birth in the mammalian adult brain [18,332]. Several studies have demonstrated that radial glia not only give rise to multiple classes of brain cells, but also generate adult SVZ stem cells that maintain the neurogenic lineage in the adult brain [293,332,413], with a similar relationship proposed also between radial glia and hippocampal progenitors [223,449]. Specifically, these adult SVZ stem cells have been shown to arise from a subpopulation of radial glia present within the developing striatum and display characteristics intermediate between normal astrocytes and radial glia, hinting that NEP cells, radial glia and adult SVZ stem cells are the components of a continuous lineage with multipotent neural differentiation potential [107,332]. Although these in vitro studies have demonstrated the shared molecular and morphological characteristics between radial glia and adult SVZ stem cells (Fig 2.2) [107], several aspects of in vivo biology cannot be accounted for. For example, the fact that radial glia exist only transiently during fetal development makes it harder experimentally to validate whether they function as self-renewing stem cells. Also, the artificial environment of cultures may result in a unique synthetic cell state, and the combination of transcription factors expressed in cultured SVZ stem cells is not found in vivo. Therefore, it is fairest to term these precursor 26Largest structure in the mammalian and human brain composed of the white and grey matter in the cranial cavity. 27Adult mammalian neural stem cells have also been isolated from the sub-granular zone (SGZ) of the dentate gyrus in the hippocampus, and the subcortical white matter [442]. 36
2.2 Neural Stem Cells Introduction cells "radial glia-like NS cells", although in the rest of this thesis they will be referred to simply as NS cells [400]. Radial glia can be obtained from the Figure 2.2: Surface markers of radial glia are expressed by NS cell lines, indicating that these cells may provide the biological context to work with progenitors of the CNS. For example, the GFAP is a type III Intermediate filament; GLAST is an astrocyte-specific glutamate transporter; Prominin, also known as CD133, is a glycoprotein that is a neural and hematopoietic stem cell marker. Adapted from Conti et al 2005 [107]. dissociation of fetal CNS tissues and the subsequent establishment of primary cultures. The heterogeneity linked to these primary cultures was overcome by the development of cell type specific monoclonal antibodies like RC1, which reduces the presence of multiple immature and differentiated cell types and distinct radial glia subtypes. Fluorescent activated cell sorting (FACS) can also be used with cell surface markers like CD15 and CD133, which, however, enable only the enrichment and not the isolation of radial glia subpopula- tions. Another technique uses reporter mice in which an endogenous radial glia promoter drives the expression of fluorescent reporters, and cells with activated reporter expression are then isolated using FACS. Although primary cell cultures are a useful tool to isolate and characterise radial glia cells isolated directly from neural tissues, the mitogen-driven expansion of cells in vitro leads to the formation of NS cell lines that are an invaluable tool for molecular and biochemical studies on nervous system pathological models amongst others [400]. 2.2 Neural Stem Cells NS cells are defined as clonogenic cells capable of self-renewal and multipotent differentiation into the three main cell types of the CNS: neurons, astrocytes 37
Page 1 and 2: Transcriptional Characterization of
Page 3 and 4: This dissertation is my own work an
Page 5 and 6: Acknowledgements I would like to th
Page 7 and 8: 5.9 External Dataset Expression Cor
Page 9 and 10: Introduction 1
Page 11 and 12: 1.1 Glial Cells in the Central Nerv
Page 13 and 14: 1.2 Glioblastoma Multiforme Introdu
Page 19 and 20: 1.3 Primary and Secondary Glioblast
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Page 39 and 40: 1.5 Pathway Crosstalk Introduction
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4.1 MicroRNA regulation Introductio
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4.1 MicroRNA regulation Introductio
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4.2 Target Prediction and Validatio
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Methods 93
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5.1 Tag-sequencing Data Processing
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5.1 Tag-sequencing Data Processing
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5.2 Array Comparative Genomic Hybri
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5.4 Quantitative Real Time-PCR Vali
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5.5 Literature Mining Methods Figur
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5.5 Literature Mining Methods How m
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5.6 Differential Isoform Expression
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5.9 External Dataset Expression Cor
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5.10 Glioblastoma Pathway Construct
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5.11 MicroRNA Target Prediction Ana
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Results 119
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6.2 Tag mapping Results Table 6.1:
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6.2 Tag mapping Results 123 Figure
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6.3 Copy Number Aberrations Results
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6.3 Copy Number Aberrations Results
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6.4 Core Differentially Expressed G
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6.5 Large-scale qRT-PCR Validation
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6.6 Literature Mining for Different
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6.7 Isoform Differential Expression
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6.8 Long ncRNA Differential Express
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6.8 Long ncRNA Differential Express
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Chapter 7 Dataset Correlation Analy
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7.1 Enrichment Analysis Results Tab
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7.1 Enrichment Analysis Results Fig
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7.1 Enrichment Analysis Results Tab
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7.2 Glioblastoma Expression Signatu
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7.3 Tumour Expression Correlation R
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7.4 Survival Analysis Results Table
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7.4 Survival Analysis Results 867 g
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7.5 Glioblastoma Pathway Analysis R
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8.1 Principles Results say, this is
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8.3 Databases Results Figure 8.1: O
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8.3 Databases Results Figure 8.3: W
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8.4 Filters Results Bayesian method
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8.5 Target Prediction Ensemble Anal
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9.1. Digital Profiling of GNS Cell
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9.2. MicroRNA Target Prediction Ana
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9.3. Concluding Remarks Appendix pr
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A.1 Differential Expression Appendi
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A.2 Classified Differential Express
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A.3 Quantitative RT-PCR Appendix Ta
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A.3 Quantitative RT-PCR Appendix We
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A.3 Quantitative RT-PCR Appendix Ta
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A.3 Quantitative RT-PCR Appendix We
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A.4 Tag-seq vs qRT-PCR Correlation
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Appendix "BEX5", "DIAPH2", "GBP3",
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End Select End If End Sub Appendix
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If NameStr.ToLower().Equals("query"
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Appendix C Long ncRNAs We detected
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C. Long ncRNAs Appendix Tag Accessi
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D.1 Pathway Interactions Appendix F
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D.2 Pathway Images Appendix Figure
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D.2 Pathway Images Appendix Figure
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E. Exon Array Data Appendix Average
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F. MicroRNA Array Data Appendix Tab
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F. MicroRNA Array Data Appendix GNS
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F. MicroRNA Array Data Appendix GNS
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Ln Natural logarithm LOH Loss of He
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3.3 Schematisation of cell cycle ph
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8.3 Workflows at the core of the pr
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7.1 Selected Gene Ontology terms an
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