Cheng Chin Hung. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 138-145). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.i / ACKNOWLEDGMENT --- p.iv / ABBREVIATIONS --- p.v / ABSTRACT --- p.vi / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1 --- Endogenous Antisense RNAs --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Class --- p.2 / Chapter 1.3 --- Natural Antisense RNAs in Prokaryotes and Viruses --- p.3 / Chapter 1.4 --- Endogenous Antisense RNAs in Eukaryotes --- p.8 / Chapter 1.4.1 --- Distribution --- p.8 / Chapter 1.4.2 --- Conserved Pattern of Antisense Transcription --- p.10 / Chapter 1.5 --- Potential Functions of Antisense RNAs --- p.10 / Chapter 1.5.1 --- Template for Translation --- p.11 / Chapter 1.5.2 --- Regulation of Sense Gene Expression --- p.12 / Chapter 1.5.2.1 --- Nucleus --- p.13 / Chapter 1.5.2.1.1 --- Transcriptional Regulation --- p.13 / Chapter 1.5.2.1.2 --- Post-transcriptional Nuclear Regulation --- p.14 / Chapter 1.5.2.2 --- Cytoplasm --- p.16 / Chapter 1.5.2.2.1 --- Messenger Stability --- p.16 / Chapter 1.5.2.2.2 --- Translation --- p.17 / Chapter 1.6 --- Possible Mechanism of Antisense-mediated Regulation --- p.18 / Chapter 1.6.1 --- Two Possible Mechanisms --- p.18 / Chapter 1.7 --- Novel Endogenous Antisense RNA Against Cation Channel --- p.23 / Chapter 2 --- CNG1 Cation Channel --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Classification and Distribution of CNG Channels --- p.25 / Chapter 2.3 --- Structure of CNG Channels Gene Gamily --- p.27 / Chapter 2.4 --- Interactions Between CNG Channels and Ca2+ --- p.29 / Chapter 2.5 --- Distribution of CNG Channels in the Central Nervous System --- p.30 / Chapter 2.6 --- CNG Channels Function in CNS --- p.31 / Chapter 3 --- Aim of Study --- p.33 / Chapter Chapter Two: --- Materials and Methods --- p.35 / Chapter 4 --- Materials --- p.35 / Chapter 4.1 --- Library --- p.35 / Chapter 4.2 --- Multiple Tissue Blots --- p.35 / Chapter 4.3 --- Paraffin Sections --- p.35 / Chapter 5 --- Library Screening of Human Brain cDNA Library --- p.37 / Chapter 5.1 --- Amplification of Human Brain cDNA Library Stock --- p.38 / Chapter 5.2 --- Primary Screening --- p.38 / Chapter 5.3 --- Hybridization --- p.39 / Chapter 5.4 --- Secondary Screening --- p.40 / Chapter 5.5 --- Tertiary Screening --- p.40 / Chapter 6 --- Clones confirmation by Manual Sequencing --- p.41 / Chapter 6.1 --- Plasmid DNA Preparation --- p.41 / Chapter 6.2 --- DNA Sequencing --- p.41 / Chapter 6.3 --- Primer Walking Strategy --- p.44 / Chapter 7 --- Probe Preparation for Northern Blot and In-Situ Hybridization --- p.45 / Chapter 7.1 --- Probe for Anti-CNGl --- p.45 / Chapter 7.1.1 --- Enzyme Digestion --- p.45 / Chapter 7.1.2 --- Self-ligation --- p.47 / Chapter 7.1.3 --- Transformation --- p.47 / Chapter 7.1.4 --- Insert Confirmation --- p.48 / Chapter 7.1.5 --- Second Round Modification of cDNA Clone --- p.48 / Chapter 7.2 --- Probe for Sense CNG1 Gene --- p.49 / Chapter 7.2.1 --- RT-PCR Amplification from Cultured human Epithelial Cell Line ECV304 --- p.49 / Chapter 7.2.2 --- Automatic Sequencing --- p.49 / Chapter 7.2.3 --- Cloning of PCR Product --- p.50 / Chapter 7.2.4 --- Transformation --- p.50 / Chapter 7.2.5 --- Clone Confirmation --- p.50 / Chapter 8 --- Northern Hybridization --- p.51 / Chapter 8.1 --- Probe Linealization --- p.51 / Chapter 8.2 --- Labeling of Riboprobe with Radioisotope 32P --- p.53 / Chapter 8.3 --- Prehybridization and Hybridization with Radiolabeled RNA Probes --- p.54 / Chapter 9 --- In Situ Hybridization --- p.56 / Chapter 9.1 --- Preparation of Anti-CNGl Probe --- p.56 / Chapter 9.2 --- Preparation of Sense CNG1 Probe --- p.59 / Chapter 9.3 --- Testing of DIG-labeled RNA Probe --- p.61 / Chapter 9.4 --- Pre treatment --- p.61 / Chapter 9.5 --- "Prehybridization, Hybridization and Posthybridization" --- p.62 / Chapter 9.6 --- Colorimetric Detection of DIG Label --- p.63 / Chapter Chapter Three: --- Results --- p.64 / Chapter 10 --- Isolation and Sequence Analysis of cDNA Clones --- p.64 / Chapter 11 --- Northern Blot Analysis of anti-CNGl RNA in Human Brain Multiple Tissues --- p.72 / Chapter 11.1 --- Human Brain Blot IV --- p.72 / Chapter 11.2 --- Human Brain Blot II --- p.75 / Chapter 11.3 --- Human Multiple Tissues Blot --- p.77 / Chapter 12 --- In Situ Hybridization Analysis of anti-CNGl RNA Expression in Human Embryonic and Adult Brain Regions --- p.80 / Chapter 12.1 --- Expression of Anti-CNGl RNA in Human Embryonic Brain Regions… --- p.80 / Chapter 12.1.1 --- Hippocampus --- p.80 / Chapter 12.1.2 --- Frontal Cortex --- p.84 / Chapter 12.1.3 --- Visual Cortex --- p.88 / Chapter 12.2 --- Expression of Anti-CNGl RNA in Human Adult Brain Regions --- p.91 / Chapter 12.2.1 --- Occipital Cortex --- p.91 / Chapter 12.2.2 --- Frontal Cortex --- p.95 / Chapter 12.2.3 --- Hippocampus --- p.99 / Chapter 13 --- Expression of Sense CNG1 mRNA in Human Embryonic and Adult Brain Regions --- p.102 / Chapter 13.1 --- Expression of Sense CNG1 mRNA in Human Embryonic Brain Regions --- p.102 / Chapter 13.1.1 --- Frontal Cortex --- p.102 / Chapter 13.1.2 --- Visual Cortex --- p.107 / Chapter 13.1.3 --- Parahippocampus --- p.111 / Chapter 13.2 --- Expression of CNG1 mRNA in Human Adult Brain Region --- p.113 / Chapter 13.2.1 --- Frontal Cortex --- p.113 / Chapter Chapter Four: --- Discussion --- p.117 / Chapter 14.1 --- Cloning of Endogenous Anti-CNGl Transcript --- p.117 / Chapter 14.2 --- Neuron-specific Coexpression of Anti-CNGl and CNG1 Transcriptsin Central Nervous System --- p.124 / Chapter 15 --- Implications --- p.128 / Chapter 15.1 --- Endogenous Anti-CNGl Down-regulate Expression of CNG1 Channel --- p.128 / Chapter 15.2 --- Coordinated Co-expression of Sense and Antisense CNG1 Transcripts --- p.129 / Chapter 15.3 --- CNG1 Channel Functions in Human Nervous System --- p.130 / Chapter 15.3.1 --- CNG1 Channel Provides a Novel Ca2+ Entry Mode --- p.130 / Chapter 15.3.2 --- Activation of CNG1 Channel Through G-protein-linked Receptors --- p.130 / Chapter 15.3.3 --- Activation of CNG1 Channel Through Nitric Oxide --- p.131 / Chapter 15.3.4 --- Synaptic Plasticity and CNG1 channel --- p.131 / Chapter 15.3.5 --- A Role of CNG1 Channel in Development --- p.135 / Chapter 16 --- Conclusion --- p.136 / Chapter 17 --- Future Studies --- p.137 / References --- p.138

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 239-251).

Au cours de la dernière décennie, les avancées du séquençage à haut débit ont permis de caractériser un grand nombre d’ARN non codant et d’établir l’existence de transcrits “antisens” pour de nombreux gènes chez les mammifères. Cependant leur rôle dans le contrôle de l’expression des gènes “sens” auxquels ils sont associés est encore très mal connu. Mes travaux ont porté sur la caractérisation de certains aspects du mécanisme d’action des longs ARN non codants. Ils reposent sur le développement d’une approche de constructions indicatrices fluorescentes dont l’expression est suivie par cytométrie en flux en présence ou non d’ARN “antisens”. Cette approche a le potentiel de mettre en évidence une régulation même si elle n’est présente que dans une sous population cellulaire. Une première série d’expériences a été réalisée en expression transitoire pour bénéficier d’un contexte chromatinien simplifié. Mais dans ce cas les silencing observés sont aussi actifs sur une construction contrôle, indiquant la mise en place d’une réponse non spécifique de séquence qui évoque la réponse de type interféron. Cependant, l’expression globale des gènes cellulaire n’est pas significativement affectée, indiquant une certaine spécificité de la réponse. Parmi les voies testées ni la kinase PKR, ni la RNaseL ou la voie de l’interférence par l’ARN ne peuvent rendre compte du silencing observé. Une des caractéristiques de cette régulation est qu’elle n’affecte pas les gènes intégrés au génome mais uniquement les gènes exprimés à partir d’une construction épisomale ce qui évoque des caractéristiques souhaitables pour un mécanisme antiviral. Cependant l’ampleur de cette réponse non spécifique empêche toute étude plus approfondie d’un mécanisme spécifique s’il existe. Mes travaux se sont alors portés sur l’étude de ces mêmes constructions en clone stable dans deux contextes différents pour l’expression de l’ARN antisens ; en cis ou en trans. Dans le cas de l’expression en trans, un ARN antisens sans séquence régulatrice particulière ne permet pas la mise en place d’un silencing. Cette observation est en accord avec le faible nombre de longs ARN antisens connus pour agir en trans dans la nature. Par contre l’expression en cis d’un ARN antisens peut conduire à un silencing spécifique. Cette organisation dans laquelle les gènes « sens » et « antisens » sont situés sur le même fragment d’ADN correspond à celle majoritairement observée pour les longs ARNs antisens dans la nature (cisNAT, cis Natural Antisense Transcripts). Cependant, mes travaux montrent que le silencing observé n’est pas stable dans le temps et disparaît dès lors que la transcription antisens cesse, indiquant l’absence d’une mémoire épigénétique. Un tel mode de régulation est compatible avec une interférence transcriptionnelle dans laquelle la transcription et non le produit ARN est la cause du silencing. Par ailleurs, j’ai observé un certain nombre de cas de co-régulation du transcrit sens et antisens ce qui traduit la possibilité d’activer en cis la transcription du gène cible par le promoteur de son ARN antisens. Ce phénomène est probablement facilité par la petite taille de nos constructions, mais cette dualité de réponse est en accord avec l’absence de corrélation (positive ou négative) entre l’expression des gènes et de leur transcrits antisens. L’ensemble de mes travaux montrent la faible capacité d’un ARN antisens à induire un silencing. L’approche développée doit donc permettre de rechercher des co-activateurs du silencing, par exemple en introduisant des sites de recrutement de complexes modificateurs de la chromatine. / During the last decade next generation sequencing has allowed the characterization of a large number of non-coding RNA and to establish that a majority of mammalian genes were also transcribed in the opposite orientation. However the functional significance of this antisense transcription is currently unclear.My work focused on the characterization of the regulatory potential of long non-coding RNA. It relied on the use of fluorescent reporter constructs, the expression of which in the presence or absence of antisense RNA is analyzed by flow cytometry. . This approach has the potential to uncover a regulation mechanism even if it takes place only in a subpopulation of cells.A first series of experiments has been realized by transient expression assays in order to benefit from a simplified chromatin context. However in this case the silencing associated with antisense transcripts is also active on control constructs, indicating that at least part of the response is not sequence specific suggesting the involvement of an interferon-type response. However, cellular gene expression is not significantly affected indicating some level of specificity. Among the investigated pathways, neither the PKR kinase, nor RNaseL or RNA interference pathway can account for the observed silencing. One of this regulation attributes is that it does not affect genes integrated in the genome but only genes expressed from episomes, a selectivity which would seem appropriate for an antiviral mechanism. Nevertheless the extent of this non-specific response impedes any further study on a specific mechanism, if it operates.My work then focused on the study of these reporter constructs after integration in the genome, antisense RNA being expressed in cis or in trans.In the case of trans expression, an antisense RNA devoid of any specific regulatory sequence does not allow the setting of a silencing. This observation is consistent with the low number of long antisense RNA known to act in trans in nature.On the other side, the cis expression of an antisense RNA can lead to a specific silencing. This organization in which “sense” and “antisense” genes are located in the same DNA fragment matches with the ones mostly observed for long antisense RNA in nature (cisNAT, cis Natural Antisense Transcripts). However, my work shows that the observed silencing is not stable over time and the effects terminate once antisense transcription stops, which indicates the absence of an epigenetic memory. This mode of regulation is compatible with a transcriptional interference in which transcription – and not its RNA product - is causing the silencing. Besides, I observed a certain number of sense and antisense transcript co-regulation cases highlighting the possibility to activate the transcription of the target gene by the promoter of its antisense RNA. This phenomenon is probably facilitated by the small size of our constructs, but this duality of response is in agreement with the lack of correlation (either positive or negative) between the expression of genes and their antisense transcripts.This study shows the limited capacity of an antisense RNA to induce a silencing. The developed approach should allow the search for silencing co-activators, for instance by introducing chromatin remodeling complexes recruitment sites.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Yau, Pak Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 150-167). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / List of abbreviations --- p.vi / List of figures --- p.viii / List of tables --- p.xi / Table of content --- p.xii / Chapter Chapter One: --- General introduction --- p.1 / Chapter 1.1. --- Introduction / Chapter 1.1.1. --- Tumor hypoxia --- p.2 / Chapter 1.1.2. --- Non-coding RNA --- p.6 / Chapter 1.1.3. --- Long non-coding RNAs: regulation and related diseases --- p.7 / Chapter 1.1.3.1. --- aHIF and cancer --- p.11 / Chapter 1.1.4. --- Objective --- p.11 / Chapter Chapter Two: --- Regulation of HIF-lα by aHIF --- p.13 / Chapter 2.1. --- Introduction / Chapter 2.1.1. --- aHIF: a natural antisense long non-coding RNAs --- p.14 / Chapter 2.1.2. --- The relationship between aHIF and HIF-lα --- p.15 / Chapter 2.1.3. --- HIF-lα regulation --- p.19 / Chapter 2.2. --- Materials and Methods / Chapter 2.2.1. --- Cell culture --- p.22 / Chapter 2.2.2. --- Western blot analysis --- p.22 / Chapter 2.2.3. --- RNA isolation and reverse transcription --- p.23 / Chapter 2.2.4. --- Quantitative Real-time PCR --- p.23 / Chapter 2.2.5. --- Plasmids construction --- p.24 / Chapter 2.2.5.1. --- aHIF over-expression clone --- p.24 / Chapter 2.2.5.2. --- Luciferase reporter with HIF-lα 3'UTR --- p.25 / Chapter 2.2.5.3. --- HIF-lα and PTB over-expression vector --- p.25 / Chapter 2.2.5.4. --- PTB knock-down vector --- p.30 / Chapter 2.2.6. --- Stable Clone --- p.30 / Chapter 2.2.7. --- Transfection --- p.31 / Chapter 2.2.8. --- Luciferase reporter assay --- p.31 / Chapter 2.2.9. --- Statistical analysis --- p.32 / Chapter 2.3. --- Results / Chapter 2.3.1. --- Effect of aHIF (FL) on HIF-lα expression --- p.33 / Chapter 2.3.2. --- Effect of aHIF (FL) on HIF-lα 3，UTR --- p.33 / Chapter 2.3.3. --- Effects of aHIF (OL) and aHIF (NOL) on HIF-lα level --- p.37 / Chapter 2.3.4. --- Effects of aHIF (NOL) and aHIF (OL) on HIF-lα 3，UTR --- p.39 / Chapter 2.3.5. --- Effect of aHIF (FL) on HIF-lα 3' UTR in PTBi cells --- p.41 / Chapter 2.3.6. --- Effect of aHIF (NOL) on HIF-lα 3，UTR in PTBi cells --- p.43 / Chapter 2.3.7. --- Effect of aHIF (OL) on HIF-lα 3' UTR in PTBi cells --- p.45 / Chapter 2.4 --- Discussion / Chapter 2.4.1. --- aHIF regulates HIF-la through HIF-la 3' UTR (FL) --- p.47 / Chapter 2.4.2. --- Factors involved in aHIF- HIF-lα interaction --- p.53 / Chapter Chapter Three: --- aHIF regulates drug sensitivity through BNIP3 --- p.58 / Chapter 3.1 --- Introduction / Chapter 3.1.1. --- aHIF and drug sensitivity --- p.59 / Chapter 3.1.2. --- BNIP3: its regulation and functions --- p.61 / Chapter 3.1.3. --- Taxol and its action mechanism --- p.67 / Chapter 3.1.4. --- Objective --- p.69 / Chapter 3.2. --- Materials and Methods / Chapter 3.2.1. --- Cell culture --- p.70 / Chapter 3.2.2. --- Cell viability assay --- p.70 / Chapter 3.2.3. --- Western blot analysis --- p.70 / Chapter 3.2.4. --- Plasmid construction --- p.71 / Chapter 3.2.5. --- Transfection --- p.71 / Chapter 3.2.6. --- Stable clone formation --- p.71 / Chapter 3.2.7. --- Quantitative real-time PCR --- p.71 / Chapter 3.2.8. --- Annexin V binding assay --- p.72 / Chapter 3.2.9. --- DNA fragmentation assay --- p.72 / Chapter 3.2.10. --- Detection of mitochondrial membrane potential by flow cytometry --- p.73 / Chapter 3.2.11. --- Cytochrome c and AIF translocation assay --- p.73 / Chapter 3.2.12. --- Statistical analysis --- p.74 / Chapter 3.3 --- Results / Chapter 3.3.1. --- Effect of aHIF on Taxol and vincristine sensitivity in HepG2 cells --- p.75 / Chapter 3.3.2. --- Effect of HIF-lαi on Taxol and vincristine sensitivity in HepG2 cells --- p.75 / Chapter 3.3.3. --- Effect of aHIF on Taxol-induced apoptosis --- p.78 / Chapter 3.3.4. --- HIF-1α regulation of BNIP3 expression --- p.78 / Chapter 3.3.5. --- Effect of aHIF on BNIP3 expression --- p.81 / Chapter 3.3.6. --- BNIP3 expression in BNIP3i stable transfectant --- p.81 / Chapter 3.3.7. --- The response of BNIP3i cells towards Taxol and vincrisinte --- p.84 / Chapter 3.3.8. --- Effect of BNIP3 on Taxol and vincristine sensitivity in BNIP3i cells --- p.84 / Chapter 3.3.9. --- Taxol- or vincristine- induced apoptosis in BNIP3i cells --- p.87 / Chapter 3.3.10. --- "Effects of aHIF, HIF-lα and BNIP3 on Taxol-induced apoptosis in HepG2 cells" --- p.89 / Chapter 3.3.11. --- Caspases activation in Taxol - or vincristine - induced apoptosis in BNIP3i cells --- p.91 / Chapter 3.3.12. --- Mitochondrial membrane depolarization in Taxol - or vincristine - induced apoptosis in BNIP3i cells --- p.91 / Chapter 3.3.13. --- AIF and cytochrome c expressions in BNIP3i cells --- p.92 / Chapter 3.3.14. --- Effect of aHIF on other chemo- and radio-therapeutics in HepG2 cells --- p.96 / Chapter 3.3.15. --- Effect of HIF-lα on other chemo- and radio-therapeutics in HepG2 cells --- p.96 / Chapter 3.3.16. --- BNIP3i cells became more sensitivity to a number of drugs --- p.99 / Chapter 3.3.17. --- BNIP3i became more resistance to some drugs --- p.99 / Chapter 3.4 --- Discussion / Chapter 3.4.1. --- aHIF affected Taxol sensitivity through BNIP3 --- p.102 / Chapter 3.4.2. --- Mechanism of BNIP3 regulated Taxol or vincristine induced apoptosis --- p.106 / Chapter 3.4.3. --- Possible roles of BNIP3 in response to other therapeutics --- p.110 / Chapter Chapter Four: --- aHIF regulation of tumorigenesis --- p.116 / Chapter 4.1 --- Introduction / Chapter 4.1.1. --- aHIF in cancer biology --- p.117 / Chapter 4.1.2. --- Ras proteins --- p.118 / Chapter 4.1.3. --- K-Ras and cancers --- p.121 / Chapter 4.1.4. --- Regulation of Ras --- p.122 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1. --- Cell culture --- p.124 / Chapter 4.2.2. --- Western blot analysis --- p.124 / Chapter 4.2.3. --- Plasmids construction --- p.124 / Chapter 4.2.4. --- Transfection --- p.124 / Chapter 4.2.5. --- Cell growth assay --- p.124 / Chapter 4.2.6. --- Soft agar assay --- p.125 / Chapter 4.2.6. --- Statistical analysis --- p.125 / Chapter 4.3 --- Results / Chapter 4.3.1. --- Effect of aHIF and HIF-lα on cell proliferation --- p.127 / Chapter 4.3.2. --- Effect of aHIF and HIF-lα on anchorage-independent growth --- p.127 / Chapter 4.3.3. --- Effect of aHIF and HIF -lα on K-Ras expression --- p.130 / Chapter 4.3.4. --- Effect of FTS on cell transfected with aHIF or HIF-lα --- p.130 / Chapter 4.4 --- Discussion / Chapter 4.4.1. --- Role of aHIF in tumorigenesis --- p.133 / Chapter 4.4.2. --- Proposed pathways of aHIF-regulated tumorigenesis --- p.136 / Chapter Chapter Five: --- General discussion and conclusion --- p.140 / Chapter 5.1 --- General discussion --- p.141 / Chapter 5.2 --- Conclusion --- p.146 / Chapter 5.3 --- Future perspectives --- p.147 / Chapter 5.3.1 --- Role ofPTB in aHIF-HIF-lα interaction --- p.147 / Chapter 5.3.2 --- Effect of aHIF (OL) on HIF-lα mRNA 3' UTR --- p.147 / Chapter 5.3.3 --- Effect ofaHIF on AIF --- p.148 / Chapter 5.3.4 --- Confirmation of the involvement of K-Ras --- p.148 / Chapter Chapter Six --- References --- p.150 / Chapter 6.1 --- References --- p.151

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 2000. / Typescript. Bibliography: leaves 92-100.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: Lorson, Christian L. "December 2008" Includes bibliographical references