by Lai Mei Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 130-136). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abstract --- p.ii / 論文撮耍 --- p.v / List of Figures --- p.viii / List of Tables --- p.xi / Abbreviations --- p.xii / Table of content --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Facilitative glucose transporters --- p.1 / Chapter 1.1.1 --- Predicted Secondary structure of Glutl --- p.1 / Chapter 1.1.2 --- The tissue-specific distribution of glucose transporters --- p.2 / Chapter 1.2 --- Increase of glucose uptake in cancer cells --- p.5 / Chapter 1.3 --- Antisense oligonucleotide therapeutics --- p.7 / Chapter 1.3.1 --- Chemical modifications of oligonucleotides --- p.7 / Chapter 1.3.2 --- Cellular Uptake of Oligonucleotide --- p.11 / Chapter 1.3.3 --- Mechanism of action --- p.13 / Antisense-mediated RNA Cleavage --- p.14 / """Occupancy-only"" mediated mechanism" --- p.15 / Chapter 1.3.4 --- Antisense treatment in vivo --- p.17 / Chapter 1.4.5 --- Human Studies of Antisense Treatment --- p.18 / Chapter Chapter 2 --- Materials & Methods --- p.20 / Chapter 2.1 --- Materials --- p.20 / Chapter 2.2 --- Cell Culture --- p.21 / Chapter 2.2.1 --- Human colon adenocarcinoma cell Line (Caco-2) --- p.21 / Chapter 2.3 --- General Methodology for treatment of cells with antisense oligonucleotides --- p.22 / Chapter 2.3.1 --- Treatment of cells with oligonucleotides --- p.22 / Chapter 2.4 --- Cytotoxicity Assay --- p.23 / Chapter 2.4.1 --- MTT assay --- p.23 / Chapter 2.4.2 --- 3H-thymidine incorporation --- p.23 / Chapter 2.5 --- RNA extraction --- p.24 / Chapter 2.6 --- Competitive Reverse-transcription polymerase chain reaction (RT-PCR) of glucose transporters --- p.25 / Chapter 2.7 --- Measurement of 2-deoxy-D-glucose and Fructose transport --- p.27 / Chapter 2.8 --- Western blotting --- p.28 / Chapter 2.9 --- Flow cytometry --- p.30 / Chapter 2.9.1 --- Measurement of cellular accumulation of fluorophore-labeled oligonucleotide --- p.30 / Chapter 2.10 --- Design of antisense oligonucleotide --- p.31 / Chapter 2.11 --- ATP assay --- p.34 / Chapter 2.12 --- Animals studies --- p.35 / Chapter Chapter 3 --- Optimization of phosphorothioate antisense oligonucleotide delivery by Lipofectin --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Measurement of oligonucleotide uptake --- p.38 / Chapter 3.2.1 --- Lipofectin as a delivery system for the oligonucleotide uptake --- p.39 / Chapter 3.2.2 --- Effect of Lipofectin ratio on the oligonucleotide uptake --- p.41 / Chapter 3.2.3 --- Effect of oligonucleotide concentration on the oligonucleotide uptake --- p.41 / Chapter 3.2.4 --- Effect of incubation time on the oligonucleotide uptake --- p.44 / Chapter 3.2.5 --- Effect of oligonucleotide length on cellular uptake --- p.44 / Chapter 3.3 --- Effect of Lipofectin on cell viability --- p.47 / Chapter Chapter 4 --- In vitro effect of Antisense Oligonucleotides against glucose transporters on Caco-2 Cell --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Design of Antisense Oligonucleotides against Glucose Transporters gene --- p.50 / Chapter 4.3. --- Antisense effect of different regions of antisense oligonucleotide --- p.52 / Chapter 4.4 --- Antisense and Sense effect of oligonucleotide against start codon (G5 7015) on Caco-2 cells --- p.59 / Chapter 4.4.1 --- Effect of oligonucleotide to Lipofectin ratio on cell viability --- p.59 / Chapter 4.4.2 --- Dose-Response Study: effect of concentration of antisense - oligonucleotide on cell viability --- p.61 / Chapter 4.4.3 --- Effect of length´ؤof oligonucleotide on cell viability --- p.61 / Chapter 4.4.4 --- Time-Response Study: effect of antisense oligonucleotide on cell viability --- p.66 / Chapter 4.5 --- "The effect of antisense oligonucleotide against Glut1, Glut3 and Glut5 on cell viability of Caco-2 cells" --- p.70 / Chapter 4.6 --- Analysis of ATP content in Caco-2 cells by using antisense oligonucleotide flanking start codon (G5 7015) --- p.72 / Chapter 4.7 --- Effect of G5 7015 on HepG2 cells --- p.72 / Chapter Chapter 5 --- Effect of antisense oligonucleotides against Glut5 on mRNA and Protein levels of Glut5 gene --- p.76 / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.2 --- RT-PCR of Glut isoform in Caco-2 cells --- p.77 / Chapter 5.3 --- Effect of antisense oligonucleotides against Glut 5 on mRNA level in Caco-2 cells --- p.77 / Chapter 5.3.1 --- Effect of oligonucleotides targeted different region of Glut5 gene on Glut5 message level --- p.77 / Chapter 5.3.2 --- Reduction in expression of mRNA level of Glut5 by using antisense oligonucleotides targeting start codon (G5 7015) --- p.81 / Chapter 5.3.3 --- Study of the dose and time dependence on inhibition of mRNA expression in G5 7015 treated Caco-2 cells --- p.83 / Chapter 5.3.4 --- Cross-Inhibition of antisense targeting glucose transporter isoforms --- p.83 / Chapter 5.4 --- Reduction in Glut5 protein level using G5 7015 antisense oligonucleotide --- p.86 / Chapter 5.5 --- Inhibition of Glut5 activity using G57015 oligonucleotide --- p.88 / Chapter 5.6 --- Inhibition of Glut5 mRNA level in vivo --- p.93 / Chapter Chapter 6 --- The possible role for Glucose Transporters in the Modification of Multidrug Resistance in Tumor cells --- p.95 / Chapter 6.1 --- Introduction --- p.95 / Chapter 6.2 --- Materials & Methods --- p.97 / Chapter 6.2.1 --- Cell culture --- p.97 / Chapter 6.2.2 --- Chemicals --- p.98 / Chapter 6.2.3 --- Measurement of doxorubicin uptake --- p.99 / Chapter 6.3 --- The expression of P-glycoprotein and Doxorubicin resistance of R-HepG2 cells --- p.99 / Chapter 6.4 --- Comparison of H3-2-deoxyglucose uptake between HepG2 and R-HepG2 cells --- p.99 / Chapter 6.5 --- Quantification of Glut1 and Glut3 expression by RT-PCR --- p.102 / Chapter 6.6 --- Comparison of doxorubicin between HepG2 and R-HepG2 cells cultured accumulation in glucose free medium --- p.104 / Chapter 6.7 --- The time course of doxorubicin accumulation in R-HepG2 cells culturing in glucose free medium --- p.106 / Chapter 6.8 --- "Cell viability of R-HepG2 cells after treatment of glucose transporter inhibitors, phloretin (PT), cytochalasin B (CB) and mitochondrial inhibitor，2,4-Dinitrophenol (DNP)" --- p.106 / Chapter 6.9 --- "Effect of glucose transporter inhibitors (PT, CB) and mitochondrial inhibitor (DNP) on doxorubicin accumulationin R-HepG2" --- p.110 / Chapter 6.10 --- Effect of antisense oligonucleotide against Glutl on doxorubicin accumulation in R-HepG2 cell --- p.113 / Chapter 6.11 --- "Analysis of ATP content and 3H-2-deoxy-D-glucose uptakein R-HepG2 after treatments of PT, CB and DNP" --- p.115 / Chapter Chapter 7 --- Discussion --- p.117 / Chapter 7.1 --- Antisense oligonucleotide against glucose transportersin Caco-2 cell --- p.117 / Chapter 7.2 --- Cellular uptake of oligonucleotide --- p.119 / Chapter 7.3 --- In vitro study of using antisense oligonucleotide against Glut5 --- p.121 / Chapter 7.4 --- In vivo study of using antisense oligonucleotide against Glut5 --- p.126 / Chapter 7.5 --- Possible role of inhibition of glucose transport in reversing P- gp --- p.127 / Chapter Chapter 8 --- References --- p.130

Keung Yim Mei. / "October 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 187-195). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Many diseases are caused due to abnormalities in production of specific protein. Across this protein the conventional lock and key mechanism shows binding at the specific cites of protein. However use of antisense technology can prevent formation of protein. It does so by binding to mRNA and prevents transcription. The structural modifications lead to synthetic molecules with 18-mer units which show significant improvement in binding properties, this gives birth to a new class of oligomers called Peptide Nucleic Acid (PNA). We herein report cysteine based PNA called CPNA.

The mdx mouse is widely used as a model for Duchenne Muscular Dystrophy, a fatal X-linked disease caused by a deficiency of the sub-sarcolemmal protein, dystrophin. This dissertation reports characterisation of the features of dystrophy in the mdx mouse, including parameters such as electrophysiological and contractile properties of dystrophic cardiac tissue, quantitative evaluation of kyphosis throughout the mdx lifespan, and contractile properties of respiratory and paraspinal muscles. Following these characterisation studies, the efficacy of antisense oligonucleotides (AOs) to induce alternative mRNA splicing in mdx skeletal muscles (diaphragm and paraspinal muscles) was evaluated. The left atria of younger (<6 weeks) and older (>15 months) mdx mice showed consistently lower basal forces and responsiveness to increased calcium, while action potential duration was significantly shorter in young mice (3 weeks) and older mice (9 and 12 months) (P<0.05). Cardiac fibrosis increased with age in mdx atria and ventricles and was elevated in young (6-8 weeks) and old (15 months) mdx compared to control mice (P<0.01). This study provided insights into DMD cardiomyopathy, and suggested that very young or old mdx mice provide the most useful models. Mdx mice show thoracolumbar kyphosis like boys with Duchenne Muscular Dystrophy. A novel radiographic index, the Kyphotic Index (KI), was developed and showed that mdx mice are significantly more kyphotic from 9 months of age, an effect maintained until 17 months (P<0.05). At 17 months, the paraspinal and respiratory muscles (latissimus dorsi, diaphragm and intercostal muscles) are significantly weaker and more fibrotic (P<0.05). Administration of AOs at four sites within the diaphragm at 4 and 5 months of age significantly increased twitch and tetanic forces compared to sham treated mdx (P<0.05). However, no difference in collagen was evident and dystrophin was not detected, possibly due to the low concentration of AO utilised. This study suggested that AOs can provide functional improvement in treated skeletal muscles. Monthly injections with AOs into the paraspinal muscles from 2 months to 18 months of age alleviated kyphosis, without significantly altering twitch and tetanic forces of latissimus dorsi, diaphragm and intercostal muscles. There was evidence of less fibrosis in diaphragm and latissimus dorsi muscles (P<0.05) and reduced central nucleation of the latissimus dorsi and intercostal muscles (P<0.05). Again, dystrophin was not detected by immunoblot. These studies indicate that very young and old mdx mice display previously uncharacterised dystrophic features, and are useful models for testing new therapies such as AOs. Low doses of AOs were shown to be safe and efficacious for long-term use, however there remains a need for testing higher concentrations and improved delivery strategies.

<p>During photosynthesis, two multi-protein complexes, photosystems (PS) I and II work in tandem to convert the light-energy absorbed by the light-harvesting antennae into chemical energy, which is subsequently used to assimilate atmospheric carbon dioxide into organic carbon compounds. This is the main nutritional basis for life on Earth.</p><p>The photosynthetic antenna of higher plants comprises at least ten different pigment-binding proteins (LHC), which play important roles in photosynthesis. Chlorophyll and carotenoid molecules associated with the LHC proteins are organised into an array, which can be modulated, thereby optimising light-harvesting processes and protection against oxidative damage under conditions of excessive light absorption. All ten LHC proteins have been conserved through eons of evolution, suggesting that there are strong evolutionary pressures to retain all ten proteins, and hence that each protein has a unique function.</p><p>The light-harvesting antenna of higher plant PSI consists of at least four proteins, Lhca1-4, collectively called LHCI. By constructing transgenic Arabidopsis thaliana plants in which each Lhca gene has been individually repressed or knocked-out, a collection of plants with different Lhca protein contents was obtained. The objective was to use these plants to study the structure, function and regulation of the Lhca proteins in vivo. The major findings of this work are as follows.</p><p>Removing single Lhca proteins influenced the stability of the other Lhca proteins, showing that there is a high degree of inter-dependency between the polypeptides in LHCI, and hence that a full set of Lhca proteins is important for maintaining the structural integrity of LHCI. This has provided insight into the organisation of LHCI by revealing clues about the relative positions of each Lhca protein in the antenna complex. The physiological consequences of removing individual Lhca proteins were dependent on the degree of antenna depletion. Plants with relatively small antenna changes could compensate, to some extent, for the loss of LHCI, while larger depletions had profound effects on whole plant resulting in growth reductions.</p><p>The fitness of each Lhca plant was assessed by measuring their seed production in the harsh conditions in the field. We found that all Lhca-deficient plants produced fewer seeds under some conditions, with seed-production compared to wild type varying between 10-80% depending on the extent of LHCI reduction. Therefore, we conclude that each Lhca protein is important for plant fitness, and hence for the survival of the species.</p><p>PSI is characterised by a pool of pigments absorbing light in the red end of the solar visible spectrum, thought to be especially important for plants in dense vegetation systems where the incident light is enriched in wavelengths higher than 690 nm. A majority of these pigments are situated on LHCI and, based on in-vitro studies, were thought to be mainly associated with Lhca4. Using our plants, we have established that red pigments are indeed present on all Lhca proteins and that these pigments become even more red upon association with PSI.</p>

<p>Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. </p><p>In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using <i>in vivo</i> reporter gene fusion expression and different <i>in vitro </i>methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays.</p><p>Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn.</p><p>Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.</p>

<p>The Escherichia coli small RNA (sRNA) MicA was identified recently in a genomewide search for sRNAs. It is encoded between the genes <i>gshA</i> and <i>luxS</i> in E. coli and its close relatives. The function of sRNAs in bacteria is generally believed to be in maintenance of homeostasis via stress-induced modulation of gene expression. Our studies on MicA have been aimed at attributing function(s) to this molecule.</p><p>We carried out high throughput assays aimed at identifying genes that are differentially regulated upon knocking out or overexpressing MicA. Among the protein candidates identified was the outer membrane protein, OmpA. Subsequent analysis allowed us to show this regulation to be antisense in nature with MicA binding within the translation initiation region of <i>ompA</i> mRNA. Furthermore, blocking the ribosome from loading caused a translational decoupling that instigates degradation of the mRNA. The regulation was apparent in early stationary phase and seen to be dependent on the RNA chaperone Hfq. </p><p>We went on to characterize the regulation of MicA, looking at its own transcription. Testing various stress conditions, we were able to identify putative promoter elements that we confirmed using transcriptional fusions. The results showed MicA to be dependent on the extracytoplasmic function ECF sigma E (σ^E) and could not detect MicA in mutants deleted for this factor.</p><p>Lastly, we identified an additional target for MicA being the adjacently encoded <i>luxS</i> mRNA. The LuxS protein is essential for the synthesis of the quorum sensing AI-2 molecule. Transcription of the <i>luxS </i>mRNA is commences within the <i>gshA</i> gene, on the other side of MicA coding region. We were able to show that MicA interacts with <i>luxS </i>mRNA and is recognized by RNase III which processes this complex leading to a shorter <i>luxS</i> mRNA isoform. The significance of this processing event is as yet undetermined. Our data elucidated a new promoter driving transcription of <i>luxS,</i> and we demonstrated this promoter to be stationary phase responsive.</p><p>In summary, the work presented here characterizes the sRNA MicA as a dual regulatory sRNA molecule, moonlighting between its cis-encoded target and its trans-encoded target. .</p>

Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using in vivo reporter gene fusion expression and different in vitro methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays. Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn. Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.

The Escherichia coli small RNA (sRNA) MicA was identified recently in a genomewide search for sRNAs. It is encoded between the genes gshA and luxS in E. coli and its close relatives. The function of sRNAs in bacteria is generally believed to be in maintenance of homeostasis via stress-induced modulation of gene expression. Our studies on MicA have been aimed at attributing function(s) to this molecule. We carried out high throughput assays aimed at identifying genes that are differentially regulated upon knocking out or overexpressing MicA. Among the protein candidates identified was the outer membrane protein, OmpA. Subsequent analysis allowed us to show this regulation to be antisense in nature with MicA binding within the translation initiation region of ompA mRNA. Furthermore, blocking the ribosome from loading caused a translational decoupling that instigates degradation of the mRNA. The regulation was apparent in early stationary phase and seen to be dependent on the RNA chaperone Hfq. We went on to characterize the regulation of MicA, looking at its own transcription. Testing various stress conditions, we were able to identify putative promoter elements that we confirmed using transcriptional fusions. The results showed MicA to be dependent on the extracytoplasmic function ECF sigma E (σE) and could not detect MicA in mutants deleted for this factor. Lastly, we identified an additional target for MicA being the adjacently encoded luxS mRNA. The LuxS protein is essential for the synthesis of the quorum sensing AI-2 molecule. Transcription of the luxS mRNA is commences within the gshA gene, on the other side of MicA coding region. We were able to show that MicA interacts with luxS mRNA and is recognized by RNase III which processes this complex leading to a shorter luxS mRNA isoform. The significance of this processing event is as yet undetermined. Our data elucidated a new promoter driving transcription of luxS, and we demonstrated this promoter to be stationary phase responsive. In summary, the work presented here characterizes the sRNA MicA as a dual regulatory sRNA molecule, moonlighting between its cis-encoded target and its trans-encoded target. .

Genomic imprinting is an epigenetic phenomenon by which a subset of genes is expressed in a parent of origin specific manner. Most of the imprinted genes are located in clusters. Genetic evidences suggest that genes in imprinted clusters are regulated by Imprinting Control Regions (ICRs). To elucidate the mechanisms by which the imprinting is maintained in clusters, we have chosen a well characterized cluster at the distal end of mouse chromosome 7. This cluster contains 15 imprinted genes and they have been shown to be regulated by H19 and Kcnq1 ICRs. The mouse H19 ICR, which is shown to have a chromatin insulator function, is implicated in the regulation of H19 and Igf2 genes by interacting with the CTCF protein. It has been documented that CTCF is also involved in the maintenance of differential methylation at the ICR. In this investigation we demonstrated that CTCF maintained differential methylation is lost when we subjected the ICR containing episomal plasmids to de novo methylation machinery of the human choriocarcinoma cell line, JEG3, suggesting that the H19 ICR looses its methylation privilege property under neoplastic conditions. The Kcnq1 ICR has been implicated in the regulation of 11 imprinted genes. The Kcnq1 ICR is methylated on the active maternal allele but unmethylated on the inactive paternal allele and overlaps an oppositely oriented and paternally expressed gene known as Kcnq1ot1. In this investigation, we documented that the Kcnq1 ICR controls the imprinting of neighboring genes by behaving as a bidirectional silencer and that this function is regulated by antisense RNA Kcnq1ot1. Furthermore, we have documented that duration of antisense transcription plays a critical role in the antisense RNA- mediated silencing. In conclusion, this thesis provides more insights into the complex mechanistic aspects by which ICRs, control imprinting of genes in clusters during development and neoplasia.