Frankfurt (Main), Univ., Diss., 2001.

Universiẗat, Diss., 2002--Frankfurt am Main.

Friedreich ataxia (FRDA) is a lethal autosomal recessive neurodegenerative disorder caused by expanded GAA repeats in the FXN gene, resulting in local epigenetic changes and reduced expression of the mitochondrial protein frataxin. The disease is characterised by neurodegeneration of large sensory neurons of the dorsal root ganglia and spinocerebellar tracts. It has been recently reported that a novel frataxin antisense transcript, FAST-1, is overexpressed in FRDA patient derived fibroblasts. However, the lack of fundamental information about FAST-1 gene such as size, sequence, length and its origin has hindered the understanding of its interactions with FXN gene. Therefore, I proposed to investigate these characteristics of FAST-1 in a panel of FRDA cells and mouse models. Firstly, using Northern blot hybridisation with small and large riboprobes, I identified two bands with different sizes (~500 bp and 9 kb size), representing potential FAST-1 transcripts. Then to confirm the exact size and the location of the FAST-1 gene, I performed 5’- and 3’ RACE experiments, followed by cloning and sequencing. This analysis resulted in identification of the 5’- and 3’-ends of FAST-1, which mapped to nucleotide positions ‘-359’ and ‘164’ of the FXN gene, giving the total length of FAST-1 as 523 bp size. Strikingly, the full-length 523 bp FAST-1 transcript also corresponds to one of the Northern blotting results where I identified a band at approximately 500 bp size, indicating that the Northern blotting may have correctly identified the same full-length FAST-1 transcript. Subsequently, by optimising number of experimental parameters within our lab, I developed a robust qRT-PCR method to quantify FAST-1 expression levels. Using this technique, I analysed the expression pattern of this antisense transcript in various FRDA cell lines and mouse models. I confirmed the original finding of increased FAST-1 levels in human FRDA fibroblasts, and further quantified FAST-1 levels in FRDA mouse model cell lines and tissues. However, no consistently altered patterns of FAST-1 expression were identified in relation to FXN expression. Therefore, either they are not directly connected, as originally reported by De Biase et al., or their relationship varies between cell and tissue types. Lastly, improved understanding of epigenetic changes in FRDA and growing evidence on long-gene regulation led me to study the ‘neighbouring genes’ rather than just focusing on the FXN gene. Therefore, I studied a region of approximately 750 kb on both sides of the FXN and quantified the expression levels of two genes (PGM5 and PIP5K1β) on 5’- end and four genes (TJP2, FAM189A2, APBA1 and PTAR1) on 3’- end of FXN gene in human primary fibroblasts. I found that PGM5 and PIP5K1β genes, located at 5’- end of the FXN genes, were downregulated in FRDA fibroblasts and these findings coincide with the recent epigenetic changes identified in FRDA, where significant enrichment of gene repressive histone marks and increased DNA methylation were shown in upstream region of GAA repeats in intron 1 of the FXN gene. Out of four genes that were studied in the 3’- end of the FXN gene, only one gene (APBA1) was downregulated, which suggests that there are fewer repressive epigenetic marks downstream of the GAA repeat.

Chung Ka Wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 151-162). / Abstracts in English and Chinese. / Contents --- p.i / Acknowledgements --- p.v / Abstract --- p.vi / 論文摘要 --- p.ix / List of Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Breast Cancer --- p.2 / Chapter 1.1.1 --- Incidence Rate of Breast Cancer --- p.2 / Chapter 1.1.2 --- Risk Factors Lead to Breast Cancer --- p.5 / Chapter 1.1.3 --- Conventional Treatments --- p.5 / Chapter 1.2 --- Relationship between Breast Cancer and Glucose Transporters --- p.7 / Chapter 1.2.1 --- Importance of Glucose and Fructose --- p.7 / Chapter 1.2.2 --- Facilitative Glucose Transporters (Gluts) and The Relationship with Breast Cancer --- p.7 / Chapter 1.3 --- Antisense Oligonucleotides --- p.13 / Chapter 1.3.1 --- Characteristics of Antisense Oligonucleotides --- p.13 / Chapter 1.3.2 --- Action Mechanism of Antisense Oligonucleotides --- p.15 / Chapter 1.3.3 --- Sequence Selection --- p.19 / Chapter 1.3.4 --- Chemical Modifications of Antisense Oligonucleotides --- p.20 / Chapter 1.3.5 --- Uptake and Delivery Means of Antisense Oligonucleotides --- p.24 / Chapter 1.4 --- Objectives of Present Study --- p.26 / Chapter Chapter 2 --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Cell Lines and Culture Medium --- p.32 / Chapter 2.1.2 --- Buffers and Reagents --- p.33 / Chapter 2.1.3 --- Reagents for Transfection --- p.34 / Chapter 2.1.4 --- Reagents for D-[U14C]-Fructose and 2-Deoxy-D-[l-3H] Glucose Uptake Assay --- p.35 / Chapter 2.1.5 --- Reagents for ATP Assay --- p.35 / Chapter 2.1.6 --- Reagents for RT-PCR --- p.36 / Chapter 2.1.6.1 --- Reagents for RNA Extraction --- p.36 / Chapter 2.1.6.2 --- Reagents for Reverse Transcription --- p.36 / Chapter 2.1.6.3 --- Reagents for Gel Electrophoresis --- p.37 / Chapter 2.1.7 --- Reagents for Real Time-PCR --- p.38 / Chapter 2.1.8 --- Reagents and Chemicals for Western Blotting --- p.39 / Chapter 2.1.8.1 --- Reagents for Protein Extraction --- p.39 / Chapter 2.1.8.2 --- Reagents for SDS-PAGE --- p.39 / Chapter 2.1.9 --- Reagents for Flow Cytometry --- p.42 / Chapter 2.1.10 --- In Vivo Study --- p.43 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Oligonucleotide Design --- p.44 / Chapter 2.2.2 --- Trypan Blue Exclusion Assay --- p.47 / Chapter 2.2.3 --- Transfection --- p.47 / Chapter 2.2.4 --- MTT Assay --- p.47 / Chapter 2.2.5 --- D-[U14C]-fructose and 2-deoxy-D-[l-3H] Glucose Uptake Assay --- p.48 / Chapter 2.2.6 --- Detection of Intracellular ATP Concentration --- p.49 / Chapter 2.2.7 --- Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.51 / Chapter 2.2.7.1 --- RNA Extraction by TRIzol Reagent --- p.51 / Chapter 2.2.7.2 --- Determination of RNA Concentration --- p.51 / Chapter 2.2.7.3 --- Reverse Transcription --- p.52 / Chapter 2.2.7.4 --- Polymerase Chain Reaction (PCR) --- p.52 / Chapter 2.2.8 --- Real-Time PCR --- p.55 / Chapter 2.2.8.1 --- Analysis of the Real-Time PCR Data --- p.57 / Chapter 2.2.9 --- Western Blot Analysis --- p.58 / Chapter 2.2.9.1 --- Protein Extraction --- p.58 / Chapter 2.2.9.2 --- Protein Concentration Determination --- p.58 / Chapter 2.2.9.3 --- Western Blotting --- p.60 / Chapter 2.2.10 --- Flow Cytometry --- p.62 / Chapter 2.2.10.1 --- Detection of Cell Cycle Pattern with PI --- p.62 / Chapter 2.2.10.2 --- Detection of Apoptosis with Annexin V/PI --- p.62 / Chapter 2.2.11 --- In Vivo Study --- p.63 / Chapter 2.2.11.1 --- Establishment of Tumor-Bearing Animal Model --- p.63 / Chapter 2.2.11.2 --- Treatment Schedule --- p.63 / Chapter 2.2.11.3 --- Toxicity of Antisense Oligonucleotides --- p.64 / Chapter Chapter 3 --- Results --- p.66 / Chapter 3.1 --- In Vitro Study --- p.67 / Chapter 3.1.1 --- Effect of Tamoxifen on MCF-7 cells and MDA-MB-231 cells --- p.67 / Chapter 3.1.2 --- Cytotoxicity of Antisense Oligonucleotides against Glut 5 on MCF-7 cells and MDA-MB-231 cells by MTT Assay --- p.69 / Chapter 3.1.3 --- Effect of Antisense Oligonucleotides against Glut 5 on Fructose and Glucose Uptake of MCF-7 cells and MDA-MB-231 cells by D-[U14C]-Fructose & 2-Deoxy-D-[l-3H] Glucose Uptake Assay --- p.77 / Chapter 3.1.4 --- Effect of Antisense Oligonucleotides against Glut 5 on Intracellular ATP Content of MCF-7 cells and MDA-MB-231 cells by ATP Assay --- p.81 / Chapter 3.1.5 --- Effect of Antisense Oligonucleotides against Glut 5 on Glut 5 RNA Expression of MCF-7 cells and MDA-MB-231 cells by RT-PCR and Real-Time PCR --- p.83 / Chapter 3.1.5.1 --- RT-PCR --- p.83 / Chapter 3.1.5.2 --- Real-Time PCR --- p.87 / Chapter 3.1.6 --- Effect of Antisense Oligonucleotides against Glut 5 on Glut 5 Protein Expression of MCF-7 cells and MDA-MB-231 cells by Western Blot Analysis --- p.89 / Chapter 3.1.7 --- "Effect of Antisense Oligonucleotides against Glut 5 on Change in Cell Cycle Pattern of MCF-7 cells and MDA-MB-231 cells by Flow Cytometry, Using PI Stainning" --- p.93 / Chapter 3.1.8 --- "Effect of Antisense Oligonucleotides against Glut 5 on Induction of Apoptosis of MCF-7 cells and MDA-MB-231 cells by Flow Cytometry, Using Annexin V-FITC Stainning" --- p.98 / Chapter 3.2 --- In Vivo Study --- p.101 / Chapter 3.2.1 --- Animal Model: Nude Mice --- p.101 / Chapter 3.2.2 --- Effect of Antisense Oligonucleotides against Glut 5 on the MCF-7 cells-Bearing Nude Mice --- p.101 / Chapter 3.2.2.1 --- Change of Weight of the Tumor-Bearing Nude Mice --- p.101 / Chapter 3.2.2.2 --- Tumor Growth Rate --- p.105 / Chapter 3.2.2.3 --- Glut 5 RNA Expression by Real-Time PCR --- p.109 / Chapter 3.2.2.4 --- Glut 5 RNA Expression by Western Blotting --- p.111 / Chapter 3.2.3 --- "Assessment of Side Effects of Antisense Oligonucleotides against Glut 5, by Measuring the Plasma Enzyme Level" --- p.113 / Chapter Chapter 4 --- Discussion --- p.118 / Chapter 4.1 --- Antisense Oligonucleotides against Glut 5 on Human Breast Cancer --- p.119 / Chapter 4.1.1 --- Antisense Oligonucleotides Strategy --- p.119 / Chapter 4.1.2 --- Role of Glut 5 in Breast Cancer --- p.123 / Chapter 4.1.3 --- Effects of Tamoxifen on MCF-7 and MDA-MB-231 --- p.126 / Chapter 4.2 --- In Vitro Study of Antisense Oligonucleotides against Glucose Transporter 5 on Breast Cancer Cells --- p.127 / Chapter 4.3 --- In Vivo Study of Antisense Oligonucleotides against Glucose Transporter 5 on Breast Cancer Cells --- p.135 / Chapter 4.3.1 --- Effects of Antisense Oligonucleotides against Glut 5 on Body Weight and Tumor Size --- p.137 / Chapter 4.3.2 --- Expression Level of Glut 5 of the Tumor --- p.138 / Chapter 4.3.3 --- Assessment of Side Effects of Antisense Oligonucleotides against Glut 5，by Measuring the Plasma Enzymes Level --- p.140 / Chapter 4.4 --- Possible Mechanism of Antisense Oligonucleotides against Glut 5 on Breast Cancer --- p.141 / Chapter Chapter 5 --- Future Prospectus and Conclusions --- p.143 / Chapter 5.1 --- Future Prospectus of Antisense Oligonucleotides --- p.144 / Chapter 5.1.1 --- Antisense Oligonucleotides and Treatment of Breast Cancer --- p.144 / Chapter 5.1.2 --- Role of Glut 5 in Breast Cancer --- p.147 / Chapter 5.2 --- Conclusions and Remarks --- p.148 / References --- p.151

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

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

The use of DNA-based therapeutics requires efficient delivery systems to transport the DNA to their place of action within the cell. To accomplish this, we investigated multiwalled carbon nanotubes (pristine MWCNT, p-MWCNT) functionalized with hydroxyl groups via 1,3-dipolar cycloaddition. In this way, we have obtained MWCNT-f-OH with improved stability in aqueous dispersions which is an advantageous property for their use in cellular environments. Afterwards, a carrier strand oligodeoxynucleotide (CS-ODN) was adsorbed to MWCNT-f-OH followed by hybridization with a therapeutic antisense oligodeoxynucleotide (AS-ODN). The amount of adsorbed CS-ODN, as well as the complementary AS-ODN and a non-complementary oligodeoxynucleotide (NS-ODN) as reference, was directly measured by radionuclide labeling of ODNs. We show that subsequent release of AS-ODNs and NS-ODNs was possible for MWCNT-f-OH above the melting temperature of AS-ODNs at 80 °C and under physiological conditions at different pH values at 37 °C. We also show a very low influence of p-MWCNT and MWCNT-f-OH on the cell viability of the bladder carcinoma (BCa) cell line EJ28 and that both MWCNT types were internalized by EJ28. Therefore, MWCNT-f-OH represents a promising carrier able to transport and release AS-ODNs inside cells.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.