Das Harnblasenkarzinom (BCa) ist die zweithäufigste bösartige urologische Tumorerkrankung sowie die siebthäufigste tumorbedingte Todesursache bei Männern. Zur Senkung des erheblichen Rezidiv- und Progressionsrisikos oberflächlicher BCa kommen lokale Immun- oder Chemotherapeutika zum Einsatz, die jedoch starke Nebenwirkungen verursachen können bzw. ungenügende langfristige Effekte bewirken. Eine neuartige Therapieoption besteht in der gezielten Expressionshemmung von Genen, die den Tumorzellen einen Wachstumsvorteil vermitteln. Hierfür eignen sich besonders synthetische Nukleinsäuren wie Antisense-Oligodesoxynukleotide (AS-ODN) und small interfering RNAs (siRNAs). In der vorliegenden Arbeit wurde die Expressionshemmung des potenziellen Targetgens hTERT (humane Telomerase Reverse Transkriptase) mit AS-ODN und siRNAs in BCa-Zellen untersucht. Die Tumorspezifität der hTERT-mRNA-Expression konnte zunächst an tumor- und tumorfreien Gewebeproben von BCa-Patienten gezeigt werden. Die verwendeten AS-ODN reduzierten die hTERT-mRNA-Expression auf bis zu 40%, womit eine Verringerung der Telomeraseaktivität einherging. Die AS-ODN-Behandlung bewirkte des Weiteren eine konzentrationsabhängige Viabilitätsreduktion verschiedener BCa-Zelllinien sowie eine verminderte Zellkoloniebildungsrate. Diese antiproliferativen Effekte waren auf eine Apoptoseinduktion zurückzuführen. Durch eine Vorbehandlung von vier BCa-Zelllinien mit hTERT-AS-ODN konnten die zytotoxischen Effekte der für das BCa relevanten Chemotherapeutika Cisplatin, Mitomycin C und Gemcitabin signifikant verstärkt werden. Nach Untersuchung der AS-ODN-Wirkung in vitro erfolgte die Etablierung eines subkutanen Xenotransplantantmodells der Nacktmaus. Die Eignung einer intraperitonealen Applikation wurde mit fluoreszenzmarkierten AS-ODN belegt. In weiteren Zellkulturexperimenten kamen hTERT-siRNAs, als alternative Methode der Geninhibition, zum Einsatz. Die Reduktion der hTERT-mRNA-Expression auf 50% war mit der durch AS-ODN bewirkten Inhibition vergleichbar. Im Gegensatz zur AS-ODN-Behandlung induzierten siRNAs keine unmittelbare Apoptose. Eine Kombination der siRNAs mit Cisplatin und Mitomycin C bewirkte jedoch eine Verdopplung der Apoptoserate. Um die molekularen Mechanismen der Wirkung der nukleinsäurebasierten hTERT-Inhibitoren und den Einfluss targetunabhängiger Effekte zu untersuchen, wurden transkriptomweite Expressionsanalysen mittels Oligonukleotid-Microarrays durchgeführt. Hierbei zeigte sich, dass die AS-ODN-Behandlung vorwiegend zu einer gesteigerten Expression von Genen führte, die mit einer zellulären Stressantwort assoziiert sind (u.a. ATF3, EGR1, GADD45). Diese Expressionsmuster stimmten in hohem Maße mit denen überein, die durch Transfektion mit AS-ODN gegen andere Targets erhalten wurden. Diese Ergebnisse deuten auf eine, zumindest teilweise, durch off-Targeteffekte ausgelöste Wachstumshemmung hin. Die siRNA-Behandlungen gegen unterschiedliche Targets zeigten relativ geringe Übereinstimmungen in den Expressionsmustern und somit eine höhere Spezifität. Außerdem wurde erstmalig gezeigt, dass eine hTERT-Inhibition mit siRNAs zur trankriptionellen Hemmung der Onkogene EGFR und FOSL1 führt. Diese Daten sowie die Ergebnisse anderer Arbeitsgruppen deuten auf einen wechselseitigen Zusammenhang zwischen hTERT und EGFR in der Regulation der EGFR-stimulierten Proliferation von BCa-Zellen hin. Zusammenfassend lässt sich feststellen, dass hTERT als tumorspezifisch exprimierter und funktionell relevanter Faktor ein hervorragendes Target für eine nukleinsäurebasierte BCa-Therapieoption darstellt. Im Vergleich zu AS-ODN wirken siRNAs grundsätzlich targetspezifischer. Die therapeutische Wertigkeit der lokal applizierten Inhibitoren, insbesondere in Kombination mit herkömmlichen Chemotherapeutika, sollte in nachfolgenden Experimenten im Rahmen eines orthotopen BCa-Xenotransplantatmodells untersucht werden.

La localisation des ARNm dans différents compartiments subcellulaires est conservée dans un large éventail d'espèces et de divers types cellulaires. Le trafic est médié par l'interaction entre les protéines de liaison à l'ARN (RBP) et l'ARNm. Les RBP reconnaissent les éléments cis-régulateurs de l'ARNm, également appelés éléments de localisation. Ceux-ci sont définis par leur séquence et/ou leurs caractéristiques structurelles résidant dans la molécule d'ARNm. La localisation des ARNm est essentielle pour la résolution subcellulaire et temporelle. De plus, les ARNm se sont avérés enrichis dans de nombreux compartiments cellulaires, notamment les mitochondries, l'appareil mitotique, et le réticulum endoplasmique. En outre, des études ont démontré que les RBP et les ARNm sont associés aux structures de l'appareil mitotique. Cependant, le rôle que joue la localisation de l'ARNm au cours de la mitose reste largement inexploré. Ma thèse de doctorat vise à comprendre comment le trafic d'ARNm est impliqué lors de la mitose. La première partie de cette thèse porte sur l'interaction post-transcriptionnelle qui se produit entre les deux ARNm, cen et ik2. Les gènes qui se chevauchent sont une caractéristique frappante de la plupart des génomes. En fait, il a été constaté que le chevauchement des séquences génomiques module différents aspects de la régulation des gènes tels que l'empreinte génomique, la transcription, l'édition et la traduction de l'ARN. Cependant, la mesure dans laquelle cette organisation influence les événements réglementaires opérant au niveau post-transcriptionnel reste incertaine. En étudiant les gènes cen et ik2 de Drosophila melanogaster, qui sont transcrits de manière convergente avec des régions 3' non traduites qui se chevauchent, nous avons constaté que la liaison physique de ces gènes est un déterminant clé dans la co-localisation de leurs ARNm aux centrosomes cytoplasmiques. Le ciblage du transcrit ik2 dépend de la présence et de l'association physique avec l'ARNm de cen, qui est le principal moteur de la co-localisation centrosomale. En interrogeant les ensembles de données de séquençage de fractionnement, nous constatons que les ARNm codés par des gènes qui se chevauchent en 3' sont plus souvent co-localisés par rapport aux paires de transcrits aléatoires. Ce travail suggère que les interactions post-transcriptionnelles des ARNm avec des séquences complémentaires peuvent dicter leur destin de localisation dans le cytoplasme. La deuxième partie de cette thèse consiste à étudier le rôle que jouent les RBP au cours de la mitose. Auparavant, les RBP se sont avérés être associés au fuseau et aux centrosomes. Cependant, leur rôle fonctionnel au niveau de ces structures reste à étudier. Grâce à un criblage par imagerie avec plus de 300 anticorps, nous avons identifié 30 RBP localisés dans les structures mitotiques des cellules HeLa. Ensuite, pour évaluer les rôles fonctionnels de ces RBP, nous avons utilisé l'interférence ARN (ARNi) pour évaluer si la fidélité du cycle cellulaire était compromise dans les cellules HeLa et les embryons de Drosophila melanogaster. Fait intéressant, nous avons identifié plusieurs candidats RBP pour lesquels le knockdown perturbe la mitose et la localisation de l'ARNm dans les cellules HeLa. De plus, la perte des orthologues a entraîné des défauts de développement chez l'embryon de mouche. Grâce à ce travail, nous avons démontré que les RBP sont impliquées pour assurer une mitose sans erreur. En résumé, les travaux que j'ai menés mettent en lumière l'implication de la régulation post-transcriptionnelle au cours de la mitose. En définissant les fonctions et le mécanisme de localisation des ARNm en mitose, ce travail permettra de définir de nouvelles voies moléculaires impliquées dans la régulation de la mitose. Puisque la division cellulaire non contrôlée peut mener à des maladies tel le cancer, étudier le contrôle du cycle cellulaire sous cet angle « centré sur l'ARN » peut aider à développer de nouvelles approches thérapeutiques pour trouver des solutions aux problèmes de santé. / The localization of mRNAs to different subcellular compartments is conserved in a wide range of species and diverse cell types. Trafficking is mediated by the interaction between RNA binding proteins (RBPs) and mRNA. RBPs recognize mRNA cis regulatory motifs, otherwise known as localization elements. These are defined by their sequence and/or structural features residing within the mRNA molecule. Localization of mRNAs is essential for subcellular and temporal resolution. Furthermore, mRNAs have been found to be enriched in many cellular compartments including the mitochondria, mitotic apparatus, and endoplasmic reticulum. Moreover, studies have demonstrated that RBPs and mRNAs are associated with mitotic apparatus structures. However, the role that mRNA localization plays during mitosis remains largely unexplored. My PhD thesis aims to understand how the trafficking of mRNAs is implicated during mitosis. The first part of this thesis encompasses the post-transcriptional interaction that occurs between the two mRNAs, cen and ik2. Overlapping genes are a striking feature of most genomes. In fact, genomic sequence overlap has been found to modulate different aspects of gene regulation such as genomic imprinting, transcription, RNA editing and translation. However, the extent to which this organization influences regulatory events operating at the post-transcriptional level remains unclear. By studying the cen and ik2 genes of Drosophila melanogaster, which are convergently transcribed with overlapping 3’untranslated regions, we found that the physical linkage of these genes is a key determinant in co-localizing their mRNAs to cytoplasmic centrosomes. Targeting of the ik2 transcript is dependent on the presence and physical association with cen mRNA, which serves as the main driver of centrosomal colocalization. By interrogating global fractionation-sequencing datasets, we find that mRNAs encoded by 3’overlapping genes are more often co-localized as compared to random transcript pairs. This work suggests that post-transcriptional interactions of mRNAs with complementary sequences can dictate their localization fate in the cytoplasm. The second part of this thesis involves investigating the role that RBPs play during mitosis. Previously, RBPs have been found to be associated with the spindle and centrosomes. However, their functional role at these structures was yet to be investigated. Through an imaging screen with >300 antibodies, we identified 30 RBPs localized to mitotic structures in HeLa cells. Then, to assess the functional roles of these RBPs, we used RNA interference (RNAi) to assess whether cell cycle fidelity was compromised in HeLa cells and Drosophila melanogaster embryos. Interestingly, we identified several RBP candidates for which the knockdown disrupted mitosis and mRNA localization in HeLa cells. Furthermore, loss of the orthologs led to developmental defects in the fly embryo. Through this work, we demonstrated that RBPs are involved in ensuring an error-free mitosis. In summary, the work that I have conducted sheds light on the involvement of post-transcriptional regulation during mitosis. By defining the functions and mechanism of mRNA localization in mitosis, this work will help define new molecular pathways involved in mitosis regulation. As uncontrolled cell division can lead to diseases such as cancer, studying cell cycle control from this ‘RNA-centric’ angle may help to develop new therapeutic approaches to find solutions to health problems.

Au cours d'une étude sur le contrôle transcriptionnel du gène de l'alpha-foetoprotéine, le laboratoire s'était intéressé aux facteurs de transcription de la famille SP/KLF et S. Scohy avait découvert une séquence définissant un nouveau membre: SP6. Afin de déterminer la structure du gène chez la souris, nous avons isolé un fragment génomique contenant la totalité du gène et nous l'avons séquencé. Une analyse informatique de cette séquence, l'isolement d'ESTs ainsi qu'une expérience d'extension d'amorce, nous ont permis d'affirmer que le gène Sp6 murin possède deux exons, générant une protéine de 376 acides aminés à partir d'un ATG repéré au début de l'exon 2. En même temps, des travaux réalisés sur un gène nommé epiprofin ont été publiés (Nakamura et al. 2004). Ce gène s'est avéré correspondre au gène Sp6 car il code pour la même protéine. Les exons 2 sont en effet identiques, seuls les exons 1 diffèrent. Nos études sur l'expression du gène Sp6 ont indiqué qu'elle est ubiquiste mais que c'est durant le développement embryonnaire, et surtout pendant les stades les plus tardifs de celui-ci, qu'il est le plus exprimé. Cette expression se localise surtout au niveau des dents, de l'épithélium olfactif, du cerveau, des bourgeons de membres et des follicules pileux de l'embryon. A l'état adulte, l'expression de Sp6 se réduit fortement dans tous les tissus; seuls les poumons présentent un taux d'expression relativement important. Ce travail a également permis de mettre en évidence l'existence d'un transcrit non-codant issu d'une transcription antisens du locus Sp6 et dont le premier exon inclu la totalité de l'exon 2 du gène Sp6. Nous l'avons appelé Sp6os et avons montré que son expression est absente dans de nombreux tissus et est très faible dans les tissus où on détecte le transcrit. Une comparaison de l'expression des transcrits Sp6 et Sp6os nous a permis d'imaginer un rôle pour Sp6 dans le développement et une possible modulation de son activité, par Sp6os, dans certains tissus. Afin de préciser la fonction du locus Sp6, nous l'avons invalidé chez la souris. Les mutants Sp6-/- se sont avérés viables mais présentent des anomalies dans tous les tissus où Sp6 est le plus fortement exprimé. En effet, ils n'ont ni pelage, ni vibrisse et montrent des anomalies des dents, des membres et des poumons. Nous avons également noté une dérégulation importante de l'apoptose (et parfois aussi de la prolifération cellulaire) chez ces souris Sp6-/-.

Genome transcription is much more widespread than has been traditionally thought because our view of a "gene" or "transcription unit" has changed dramatically over the past 4 to 5 years with the identification of many different non-coding ribonucleic acids. In the yeast, Saccharomyces cerevisiae, meiosis and sporulation are an important part of the life cycle and IME4 gene expression is required for these processes. IME4 sense transcript levels of expression are influenced by the level of its complementary non-coding antisense strand by mechanisms that are currently unknown. The a1-alpha2 heterodimer binding in the downstream 3' region of IME4 is one component required for repression of IME4 antisense transcription. However, this thesis shows that the general regulatory protein Reb1 is also required in this system. Reb1 involvement is most likely to create a nucleosome-free zone in the promoter region of the IME4 antisense strand therefore contributing to transcription.

O splicing alternativo do pré-mRNA de BCL-X produz duas isoformas de mRNAs com funções antagônicas, a pró-apoptótica BCL-XS e a anti-apoptótica BCL-XL, cujo balanço regula a homeostasia celular. Entretanto, o mecanismo que regula esse processamento ainda é desconhecido. Nesse trabalho, nós identificamos e caracterizamos um longo RNA não codificador de proteínas (lncRNA) nomeado INXS, que é transcrito a partir da fita oposta do locus genômico de BCL-X, sendo menos abundante em linhagens celulares tumorais e tecidos tumorais de pacientes quando comparados com os respectivos pares não tumorais. INXS é um RNA unspliced de 1903 nts, é transcrito pela RNA Polimerase II, possui cap 5\', está enriquecido na fração nuclear das células e se liga à proteína Sam68 do complexo modulador de splicing. O tratamento de células tumorais 786-O com cada um de três agentes indutores de apoptose aumentou a expressão endógena do INXS, levando ao aumento expressivo da proporção entre os mRNAs de BCL-XS / BCL-XL, e ativação das caspases 3, 7 e 9. Estes efeitos foram anulados na presença do knockdown do INXS. Da mesma forma, a superexpressão ectópica do INXS causou uma mudança no splicing favorecendo a isoforma BCL-XS e ativação das caspases, aumentando os níveis da proteína BCL-XS e conduzindo as células à apoptose. Utilizando um modelo in vivo, cinco injeções intra-tumorais do INXS durante 15 dias causaram uma regressão acentuada no volume dos xenotumores. Portanto, INXS é um lncRNA que induz a apoptose, sugerindo que essa molécula seja um possível alvo a ser explorado na terapia contra o câncer. / BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL, whose balance regulates cell homeostasis. However, the mechanism that regulates the splice shifting is incompletely understood. Here, we identified and characterized a long noncoding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was less abundant in tumor cell lines and patient tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA Polymerase II, 5\'-capped, nuclear enriched and binds Sam68 splicing-modulator. The treatment of tumor cell line 786-O with each of three apoptosis-inducing agents increased endogenous INXS lncRNA, increased BCL-XS / BCL-XL mRNA ratio, and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing towards BCL-XS and activation of caspases, increasing the levels of BCL-XS protein and then leading the cells to apoptosis. In a mouse xenograft model, five intra-tumor injections of INXS along 15 days caused a marked regression in tumor volume. INXS is an lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies.

by Chan Ka Kui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 104-113). / Abstracts in English and Chinese. / ABSTRACT --- p.1 / Chapter 1 --- INTRODUCTION --- p.5 / Chapter 1.1 --- Incidence rate of breast cancer in Hong Kong --- p.5 / Chapter 1.2 --- Estrogen and breast cancer --- p.6 / Chapter 1.3 --- The relation between glucose transporters and breast cancer --- p.7 / Chapter 1.4 --- Antisense oligonucleotide --- p.10 / Chapter 1.5 --- Action mechanisms of antisense oligonucleotide --- p.11 / Chapter 1.6 --- Modification of the oligonucleotide --- p.13 / Chapter 1.7 --- Length --- p.16 / Chapter 1.8 --- Sequence selection of the antisense oligonucleotide --- p.16 / Chapter 1.9 --- Delivery means in antisense oligonucleotide --- p.18 / Chapter 1.10 --- The therapeutic role of antisense oligonucleotide --- p.19 / Chapter 1.11 --- Objective of the project --- p.21 / Chapter 2 --- MATERIAL AND METHODS --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.2 --- Methods --- p.26 / Chapter 3 --- RESULTS --- p.37 / Chapter 3.1 --- The characteristics of MCF-7 and MDA-MB-231 cells --- p.37 / Chapter 3.2 --- Trend of uptake of antisense oligonucleotides in MCF-7 and MDA- MB-231 cells --- p.41 / Chapter 3.3 --- The integrity of the oligonucleotide in serum-free medium during transfection --- p.48 / Chapter 3.4 --- Detection of effects of Glut5 antisense oligonucleotides of breast tumor cells-MTT assay --- p.50 / Chapter 3.5 --- Detection of the antiproliferative effect by trypan blue exclusion assay and thymidine incorporation --- p.56 / Chapter 3.6 --- Cell cycle analysis and DNA extraction --- p.61 / Chapter 3.7 --- Suppression of Glut5 mRNA detected by RT-PCR --- p.66 / Chapter 3.8 --- Suppression of translation of Glut5 proteins as indicated by Western blotting --- p.73 / Chapter 3.9 --- Measurement of the fructose and glucose uptake in MCF-7 and MDA -MB-231 cells after antisense treatment --- p.76 / Chapter 3.10 --- Change of the phosphofructokinase-1 (PFK-1) activities in MDA- MB-231 cells --- p.82 / Chapter 3.11 --- Measurement of the change in the intracellular pH of the breast tumor cells --- p.84 / Chapter 4 --- DISCUSSION --- p.89 / Chapter 4.1 --- The insights of Glut5 antisense oligonucleotide into cancer therapy --- p.89 / Chapter 4.2 --- The uptake pattern of Glut5 antisense oligonucleotides in breast tumor cells --- p.90 / Chapter 4.3 --- Stability of antisense oligonucleotide during transfection --- p.92 / Chapter 4.4 --- Effects of Glut5 antisense oligonucleotide on MCF-7 and MDA-MB- 231cells --- p.93 / Chapter 4.5 --- Proofs of undergoing antisense action mechanism --- p.95 / Chapter 4.6 --- Physiological changes in breast tumor cells after antisense treatment --- p.97 / Chapter 5 --- CONCLUSION --- p.103 / Chapter 6 --- References --- p.104

Lam Mei Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-181). / Abstracts in English and Chinese. / Abstract --- p.i / 論文撮要 --- p.iv / Acknowledgement --- p.vii / Table of contents --- p.viii / List of tables --- p.xi / List of figures --- p.xii / Abbreviations --- p.xvii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- The facilitative glucose transporter family --- p.2 / Chapter 1.2 --- Overexpression of glucose transporters in tumor cells --- p.5 / Chapter 1.3 --- Antisense strategy --- p.8 / Chapter 1.3.1 --- Modifications of oligonucleotides --- p.9 / Chapter 1.3.2 --- Delivery system for oligonucleotides --- p.13 / Chapter 1.3.3 --- Factors influencing antisense activity --- p.16 / Chapter 1.3.4 --- Mechanism of action of antisense oligonucleotides --- p.17 / Chapter 1.3.5 --- Clinical trials of antisense treatment --- p.21 / Chapter 1.4 --- Objective of present study --- p.23 / Chapter Chapter 2: --- Materials and Methods --- p.24 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Reagents and buffers --- p.25 / Chapter 2.1.2 --- Reagents for Western blot analysis --- p.26 / Chapter 2.1.3 --- Culture medium --- p.28 / Chapter 2.1.4 --- Chemicals --- p.29 / Chapter 2.1.5 --- Culture of cells --- p.31 / Chapter 2.1.5.1 --- Differentiated Human Hepatoblastoma cell line (HepG2) --- p.31 / Chapter 2.1.5.2 --- "Multi-drug resistant hepatoma cell line, R-HepG2 cells" --- p.32 / Chapter 2.1.6 --- Animal Studies --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- In vitro studies --- p.34 / Chapter 2.2.1.1 --- Design of oligonucleotide sequence --- p.34 / Chapter 2.2.1.2 --- Transfection --- p.35 / Chapter 2.2.1.3 --- MTT assay --- p.36 / Chapter 2.2.1.4 --- Flow cytometry --- p.37 / Chapter 2.2.1.5 --- H-thymidine incorporation assay --- p.45 / Chapter 2.2.1.6 --- 2-Deoxy-D-[l-3H] glucose uptake assay --- p.46 / Chapter 2.2.1.7 --- Adenosine-5'-triphosphate (ATP) assay --- p.47 / Chapter 2.2.1.8 --- Western blot analysis --- p.50 / Chapter 2.2.2 --- In vivo studies --- p.55 / Chapter 2.2.2.1 --- Animal studies --- p.55 / Chapter (i) --- Lactate dehydrogenase (LDH) assay --- p.58 / Chapter (ii) --- Creatine kinase (CK) assay --- p.60 / Chapter (iii) --- Aspartate transaminase (AST) assay --- p.62 / Chapter (iv) --- Alanine transaminase (ALT) assay --- p.64 / Chapter Chapter 3: --- Results --- p.67 / Chapter 3.1 --- In vitro studies --- p.68 / Chapter 3.1.1 --- Characteristics of the multi-drug resistant cell line (R-HepG2) developed in our laboratory --- p.68 / Chapter 3.1.2 --- Effect of lipofectin on cell viability --- p.77 / Chapter 3.1.3 --- Cellular uptake of antisense oligonucleotide --- p.82 / Chapter 3.1.4 --- Effect of Glut 2 antisense oligonucleotides on human hepatoma HepG2 and its multidrug resistant (R-HepG2) cells by MTT assay --- p.87 / Chapter 3.1.5 --- Suppression of Glut 2 protein expression by antisense oligonucleotides as revealed by Western blot analysis --- p.96 / Chapter 3.1.6 --- Uptake of glucose in HepG2 and R-HepG2 after Glut 2 antisense treatment --- p.100 / Chapter 3.1.7 --- ATP content in HepG2 and R-HepG2 was lowered after treating the cells with antisense oligonucleotides --- p.108 / Chapter 3.1.8 --- Antisense oligonucleotides against Glut 2 exhibited antiproliferative effect on HepG2 and R-HepG2 cells --- p.117 / Chapter 3.1.9 --- Change in cell cycle pattern after antisense treatment --- p.125 / Chapter 3.1.10 --- Glut 2 antisense oligonucleotides did not induce apoptosis --- p.131 / Chapter 3.2 --- In vivo studies --- p.135 / Chapter 3.2.1 --- Effect of antisense oligonucleotides on the tumor weight in nude mice bearing HepG2 cells or R-HepG2 cells --- p.135 / Chapter 3.2.2 --- Assessment of any side effect of antisense drug done on normal tissues of nude mice --- p.139 / Chapter 3.2.2.1 --- Treatment on tumor bearing nude mice with Glut 2 antisense or sense oligonucleotides did not cause myocardial injury --- p.139 / Chapter 3.2.2.2 --- Liver injury was not detected in Glut 2 antisense or sense oligonucleotides treated tumor bearing nude mice --- p.147 / Chapter Chapter 4: --- Discussion --- p.151 / Chapter 4.1 --- In vitro study of the effect of antisense oligonucleotides against Glut 2 on HepG2 and its multi-drug resistant R-HepG2 cell lines --- p.152 / Chapter 4.1.1 --- Design of antisense oligonucleotides against Glut 2 --- p.154 / Chapter 4.1.2 --- Conditions for antisense inhibition by oligonucleotides --- p.155 / Chapter 4.1.3 --- Biological effects of antisense oligonucleotides --- p.158 / Chapter 4.2 --- In vivo study of the effect of antisense oligonucleotides against Glut 2 on HepG2 or R-HepG2 cells bearing nude mice --- p.166 / Chapter 4.2.1 --- Effect of Glut 2 antisense oligonucleotides on tumor weight --- p.167 / Chapter 4.2.2 --- In vivo side effects of oligonucleotides --- p.168 / Chapter 4.3 --- Conclusion --- p.169 / Bibliography --- p.172

O gene, uma seqüência de nucleotídeos necessária para a síntese de moléculas funcionais, é transcrito e regulado por um conjunto de processos e fatores extremamente complexos. Entender o momento e o tecido em que os genes são expressos, as isoformas funcionais, as regiões controladoras e os fatores envolvidos na regulação da expressão de cada gene é um dos grandes desafios da biologia molecular moderna. Hoje, com a enorme quantidade de informações de seqüências genômicas e de transcriptomas, aliado ao desenvolvimento de métodos computacionais para agrupar e analisar estes dados em larga escala, o estudo dos fenômenos relacionados à transcrição e regulação gênica está passando por uma revolução. Por exemplo, é possível medir, concomitantemente, a expressão gênica de milhares de genes em diferentes tecidos, assim como identificar diversos fenômenos que atuam nestes genes. Neste trabalho nós desenvolvemos e aplicamos métodos computacionais no estudo de quatro temas envolvendo aspectos chave da transcrição e regulação gênica. No primeiro trabalho, nós abordamos a expressão gênica tecido-específica através do estudo dos genes expressos no cérebro e em dez regiões cerebrais de camundongo. No segundo trabalho, nós identificamos seqüências potencialmente envolvidas no controle da transcrição gênica através do estudo de motivos sobre representados na região promotora dos genes de receptores olfativos. No terceiro trabalho, analisamos o transcriptoma humano quanto a presença de eventos de retenção de intron, um tipo de splicing alternativo. No quarto trabalho, nós abordamos a complexidade do transcriptoma e a regulação da expressão gênica através do estudo de pares de genes senso-antisenso em humanos e camundongos. Em todos os trabalhos, obtivemos resultados que nos permitiram tirar conclusões específicas sobre cada fenômeno estudado e nos mostraram a importância de estudá-los através de uma abordagem em larga escala. Adicionalmente, verificamos que os nossos métodos computacionais foram eficientes e adequados para o estudo da transcrição e regulação gênica em Homo sapiens e Mus musculus. / Genes, nucleotide sequences necessary for the synthesis of functional molecules, are transcribed and regulated by extremely complex cellular and molecular processes. To understand when and in which tissues the genes are expressed, their functional isoforms, control regions and the factors involved in gene regulation is one of major challenges of modern molecular biology. Today, the availability of complete genome sequences and transcriptomes, together with the development of new computational methods allows the study of phenomena related to the transcription and gene regulation in a large scale. For example, it is possible to quantify, concomitantly, gene expression of thousands of genes in different tissues and analyze different aspects of their regulation. In this work we developed and applied computational methods to the study of four key aspects of gene transcription and regulation. In the first study, we addressed tissue specific gene expression through the study of genes that are preferentially expressed in the brain and ten different mouse brain regions. In the second study, we identified sequences that are potentially involved in the control of gene transcription through the study of motifs that are over represented in the promoter region of olfactory receptor genes. In the third study, we browsed the human for the presence of intron retention, a type of alternative splicing. In the fourth study, we addressed the transcriptoma complexity and gene expression regulation through the study of pair of sense-antisense genes in human and mouse. In all studies, our results allowed us to make specific conclusions about each phenomenon analyzed which showed us the importance of a large scale approach. In addition, we verified that our computational methods can be efficiently applied to the study of transcription and gene regulation in Homo sapiens and Mus musculus.

La protoporphyrie érythropoïétique (PPE) est une maladie héréditaire rare caractérisée par un déficit en activité FECH responsable d’une accumulation de PPIX. Elle se manifeste par une photosensibilité très invalidante. Il n’existe pas de traitement efficace pour la PPE. 95 % des malades présentent un allèle FECH hypomorphe (c.315-48C) en trans d'une mutation FECH délétère, ce qui entraine une diminution de l'activité FECH résiduelle dans les érythroblastes en dessous d'un seuil critique d'environ 35 % de l'activité normale. L’allèle hypomorphe (c.315-48C) favorise l'utilisation d'un site cryptique d'épissage situé en -63 de l’intron 3 générant un ARNm FECH incluant une partie de l’intron 3 et possédant un codon stop prématuré. L’ARN est alors dégradé par NMD pendant sa maturation. Nous avons déjà identifié un oligonucléotide antisens (ASO-V1) qui redirige l'épissage vers le site accepteur physiologique de l’intron 3 et augmente la production d’ARN FECH WT. Nous avons développé par ce travail une nouvelle stratégie d’adressage d’ASO-V1 en utilisant des peptides ciblant le récepteur de la transferrine (RTf1) qui est exprimé à un niveau très élevé dans les progéniteurs érythroïdes en différenciation concomitamment à la FECH. Nous avons développé des peptides bifonctionnels à partir des séquences peptidiques ciblant le RTf1 tout en les couplant à des séquences Cell Penetrating Peptide (CPP) qui facilitent la sortie de l’ASO-V1 de la vésicule endosomale. Après la transfection des lignées lymphoblastoïdes de malades PPE par différents nanocomplexes RTf1-CPP/ASO-V1, nous avons pu montrer que plusieurs des peptides bifonctionnels utilisés permettaient une redirection efficace et prolongée de l’épissage cryptique vers l’épissage physiologique exon3-exon4 et que cela permettait une correction des taux d’ARN FECH WT. Nous avons ensuite testé l’effet des nanocomplexes RTf1-CPP/ASO-V1, ex vivo, dans les progéniteurs érythroïdes en différenciation de différents sujets atteints de PPE et nous sommes arrivés à augmenter l’ARN FECH WT et diminuer significativement l’accumulation de la PPIX dans ces cellules par rapport à celles transfectées par des nanocomplexes RTf1-CPP/ASO-Mock. La prochaine étape de notre étude serait d’apporter la preuve de concept, in vivo, dans un modèle murin humanisé de PPE après l'administration de nanocomplexes RTf1-CPP/ASOV1 / Erythropoietic protoporphyria (EPP) is a rare hereditary disease characterized by a deficiency in FECH activity responsible for the accumulation of PPIX. EPP is manifested by a very disabling photosensitivity. There is no effective treatment for EPP. 95% of the patients present a hypomorphic FECH allele (c.315-48C) in trans of a deleterious FECH mutation, resulting in a decrease in residual FECH activity in erythroblasts below a critical threshold of about 35% of normal activity. The hypomorphic allele (c.315-48C) promotes the use of a cryptic splicing site located at -63 of the intron 3 generating a FECH mRNA including a part of the intron 3 and possessing a premature stop codon. The RNA is then degraded by NMD during its maturation. We have previously identified an antisense oligonucleotide (ASO-V1) that redirects splicing to the physiological acceptor site of intron 3 and increases the production of WT FECH mRNA. Here, we developed a new ASO-V1 addressing strategy using transferrin receptor (TRf1) targeted peptides. TfR1 is expressed at a very high level in differentiating erythroid progenitors concomitantly with FECH. We developed bifunctional peptides from peptide sequences targeting TfR1 while coupling them to Cell Penetrating Peptide (CPP) sequences that facilitate the release of ASO-V1 from the endosomal vesicle. We transfected the lymphoblastoid cell lines from EPP patients by different TfR1-CPP/ASO-V1 nanocomplexes and we demonstated that several of the bifunctional peptides allowed an efficient and prolonged redirection of the cryptic splicing towards the exon3-exon4 physiological splicing and the correction of the WT FECH mRNA levels. Then, we tested the effect of TfR1-CPP/ASO-V1 nanocomplexes, ex vivo, in differentiating erythroid progenitors of different EPP subjects and we were able to increase WT FECH mRNA and decrease significantly the accumulation of the PPIX in these cells compared to those transfected by TfR1-CPP/ASO-Scr nanocomplexes. The next step of our study would be to provide a proof of concept, in vivo, in a humanized murine model of EPP after the administration of TfR1-CPP/ASOV-1 nanocomplexes

This report mainly discusses the development of the cysteine based PNA (CPNA), which is an analogue of PNAs. Peptide nucleic acids (PNA), a pseudopeptide DNA mimic, was discovered by Nielsen and his coworker in 1991. PNA is proved to sequence-specifically form a very stable duplex with complementary DNA and RNA strands through Watson-Crick base paring, and it is also capable of binding to duplex DNA by helix invasion. These intriguing properties of PNA implicated great potential for medical and biotechnical applications. Therefore, PNA has attracted many scientists in the fields of chemistry, biology, medicine including drug discovery and genetic diagnostics, molecular recognition. Due to its acyclic, achiral and neutral nature of the backbone, PNA has shown problems such as its poor aqueous solubility, poor cell permeability and instability of PNA-DNA duplexes and triplexes. Accordingly, many synthetic approaches have been directed toward developing modified backbones of PNA. Among those PNA analogs, only few examples including lysine-based monomers, guanidine-based peptide nucleic acids (GPNA) and the aminoethylprolyl PNA (aep-PNA) showed noticeable enhancements with regards to the daunting challenges mentioned above. Reported herein is the summary of our research endeavor to develop the CPNA oligomers with the great water-solubility and cell permeability. Chapter one briefly summarizs the background and history of the PNA as the front-runner of the antisense therapeutic agents. Chapter two discusses the novel protocols that enabled synthesis of the various versions of CPNA monomers for both Fmoc and Boc solid phase synthesis strategies. Chapter three includes the experimental procedures for solution phase preparation of the CPNA monomers. Chapter four starts with the introduction of solid phase synthesis strategy. After the brief review, our efforts on solid phase based synthesis of CPNA oligomers are discussed. Detailed procedures for the solid phase synthesis are summarized in Chapter five. Disclosed In the final chapter is a methodology which enables regioselective mono-acylation of hydrazines. Remarkably, this new protocol gives the mono-acylation on the less-reactive nitrogens of the hydrazines. Carbon disulfide takes the key role for this unique transformation. At the end of the dissertaion, selected NMR and Mass spectra are attached.