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published_or_final_version / Medicine / Master / Master of Philosophy

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Nucleoside (S)-cEt-LNA a été synthétisé par trois voies différentes à partir de la 5- méthyluridine qui est commercialement disponible. Le chemin le plus court comprend une méthylation diastéréosélective d'un aldéhyde, et une cyclisation 5-exo-tet d'un éther par déplacement SN2. / (S)-cEt-LNA nucleoside was synthesized by three different routes starting from commercially available 5-methyluridine. The shortest route includes a diastereoselective methylation of an aldehyde, and a 5-exo-tet cylization of an ether via SN2 displacement.

Transcription lies at the centre of gene expression. In eukaryotes, transcription occurs not only at genes but also across the non-coding portion of the genome, an apparently pervasive process that gives rise to a wide array of different transcripts. In recent years, it has emerged that genes themselves are frequently subject to non-coding transcription of their antisense strand. This antisense transcription is evident in eukaryotes from yeast to mammals; however its general genome-wide role, if indeed it has one, remains elusive. Here, the nature of antisense transcription in the budding yeast Saccharomyces cerevisiae is explored on a genome-wide scale. Antisense transcription is ubiquitous and often abundant, and appears to be driven by a promoter architecture at the 3’ end of genes, one which shows evidence of regulation, and which mirrors that found at the 5’ end. Furthermore, antisense transcription shows evidence of changing gene behaviour. It is associated with a drastically altered chromatin environment at the 5’ promoter and across the gene body; however it is not associated with a change in the level of gene transcription itself. Rather, these chromatin changes appear to enforce a change in the mode of gene transcription, promoting rapid bursts of transcription re-initiation that result in noisier gene expression – a hitherto unknown role of antisense transcription. It is proposed that antisense transcription represents a fundamental layer of gene regulation, and that it should be considered a canonical feature of eukaryotic genes.

Duchenne muscular dystrophy (DMD) is a fatal, muscle-wasting disorder due to mutations/deletions in the dystrophin gene. Whilst improvements in palliative care have increased the life expectancy of patients, cardiomyopathy and respiratory complications are still the leading causes of death. A potential therapy for the treatment of DMD is antisense oligonucleotides (AOs), which modulate dystrophin pre-mRNA splicing to restore the dystrophin reading frame and generate a truncated functional protein. Conjugation of AOs to cell penetrating peptides (CPP), such as Pip5e-, significantly improves delivery to skeletal muscles and to the heart, which is imperative given the impact of cardiomyopathy to mortality. However, it should be noted that the contribution of skeletal muscles, such as the core respiratory muscle, the diaphragm, in dystrophic cardiopulmonary function is poorly understood. The specific aims of the work in this thesis were to (i) understand the effect of the diaphragm on cardiac function using magnetic resonance imaging (MRI), (ii) screen a number of derivatives of Pip5e (Pip6) in an effort to discover further promising peptides and define the properties integral to heart penetrating capacity, and (iii) assess whether Pip6-PMOs restore cardiac function (MRI) following a repeat, low dose regimen. In short, the specific restoration of dystrophin in the diaphragm of the dystrophic mouse model, the mdx mouse, did not improve cardiac function, highlighting the importance of a body-wide therapy. The screening of multiple Pip5e-PMO derivatives revealed 3 promising peptides with improved cardiac splicing capacity; however, serial deletions of amino acids from the central core resulted in the diminution of dystrophin restoration, possibly due to a reduction in hydrophobicity. Finally, the Pip6-PMO treatment regimen substantially restored dystrophin protein (28% in heart) and stabilised cardiac function, even with an increased work load. In conclusion, this study illustrates the importance of a body-wide treatment, such as the CPP strategy (Pip-PMO). These Pip-PMO conjugates demonstrate high dystrophin restoration in a number of muscles, including cardiac muscle, and have a beneficial effect on cardiac function.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Le cancer de la prostate (CaP) est actuellement le cancer le plus fréquent en France et constitue l'une des principales causes de décès par cancer chez l'homme dans les pays industrialisés. Un tiers des patients avec un CaP à priori localisé auront déjà des micro-métastases au moment du traitement local. Ces patients qui répondent dans un premier temps à la castration (hormonothérapie) seront cependant en échappement hormonal dans les 2 ans qui suivent. Récemment, plusieurs essais cliniques de phase III ont rapporté un gain de survie avec la chimiothérapie à base de docétaxel dans les CaPs métastatiques résistants à la castration. Néanmoins, la survie n'est prolongée que de 2 ou 3 mois et de nouvelles approches thérapeutiques ciblant des voies de signalisation spécifiques sont donc nécessaires. Les travaux réalisés au cours de cette Thèse ont permis tout d'abord de montrer, grâce à l'utilisation de TMAs, que la surexpression de TP53INP1, une protéine de réponse au stress, était un facteur de mauvais pronostic dans le CaP, prédictif notamment du risque de rechute biologique. Nous avons ensuite pu montrer grâce à des xénogreffes de cellules tumorales (LNCaP) que les taux d'ARNm de TP53INP1 diminuaient durant l'hormonothérapie et que TP53INP1 était de nouveau significativement surexprimée dans les tumeurs résistantes à la castration. Nous avons développé et déposé un brevet pour un oligonucléotide antisens (ASO) inhibant TP53INP1. Le traitement in vitro des lignées cellulaires hormonosensibles LNcaP et hormono-résistantes C4-2 par l'ASO induit une diminution d'expression de la protéine TP53INP1, inhibe la prolifération cellulaire et induit une augmentation de l'apoptose. / Prostate cancer (PC) is the most common malignancy in France and one of the most frequent leading causes of cancer-related death in men in industrialized countries. Even with aggressive screening, approximately one-third of patients believed to have localized PC will already have micro-metastatic disease at the time of definitive local therapy. These patients initially respond to androgen ablative therapy, but with time, their tumors ultimately become unresponsive and recur within 2 years as castration-resistant prostate cancer (CRPC). Recently, docetaxel-based regimens have shown improved survival in men with CRPC in phase III studies. However, the median overall survival was prolonged for only 2-3 months, and thus development of new therapeutic approaches that target relevant signaling pathways are essential to restore the androgen-sensitivity of CRPC. We showed, using tissue micro-array (TMA) analysis, that over-expression of Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1), a cell stress response protein, is a worse prognostic factor in PC, particularly predictive of biological cancer relapse. We also we found that TP53INP1 protein expression decreases during castration therapy (CT) and significantly increases in human CRPC. TP53INP1 mRNA was also significantly increased in castration-resistant (CR) tumors of LNCaP xenograft compared to the castration-sensitive (CS) taken before CT. We developed and world-wide patented one antisense oligonucleotide (ASO) targeting TP53INP1 (PCT/IB2011/054555). Treatment of LNCaP and C4-2 cells in vitro with TP53INP1 ASO downregulates TP53INP1 protein level, inhibits proliferation and induces apoptosis.

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.