There has been much interest in the use of therapeutics based on ribonucleic acid interference(RNAi) to inhibit synthesis of mutant proteins ever since Elbashir et al. (Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411, 494-498.) found that synthetic double stranded small interfering ribonucleic acids (siRNAs) can initiate this evolutionarily conserved process in mammalian cells. Since RNAi is able to target single genes and therefore mitigate the underlying molecular pathology of diseases, RNAi-based therapeutics will most likely benefit monogenic neurodegenerative diseases such as Huntington’s disease. It is however particularly difficult to deliver exogenous materials such as siRNAs into neurons in vivo as the blood-brain barrier (BBB) isolates the brain from the vascular system and prevents permeation of most materials. Neurons also do not take up exogenous materials readily. Therefore, effective delivery of siRNAs into the brain remains one of the biggest challenges impeding their use as a potential neurotherapeutic. Layered double hydroxide (LDH) nanoparticles are a class of anionic clay materials that have demonstrated great potential as a DNA (deoxyribonucleic acid) delivery system for a variety of mammalian cell lines due to their unique physiochemical properties. This thesis examined the feasibility of LDH as a siRNA delivery system for cultured neurons and demonstrated that the delivered siRNAs are able to effectively down-regulate synthesis of a target protein with minimal toxicity. Experiments were conducted using double stranded DNAs (dsDNAs) initially, and siRNAs were then used to verify these results. It was shown that nucleic acids(dsDNAs and siRNAs) could successfully intercalate into pristine LDHs to form nucleic acid-LDH complexes that had properties suitable for use as a delivery system in mammalian cells. These studies established that LDHs and nucleic acid-LDH complexes were biocompatible with neurons isolated from embryonic day 17.5 mouse cerebral cortex, suggesting that LDH can be used for nucleic acid delivery into cultured neurons. LDHs were also shown to successfully deliver nucleic acids into a non-neural mammalian cell line (NIH 3T3 cells). Finally, this thesis demonstrated for the first time that LDHs were able to deliver siRNAs into neurons, providing encouraging preliminary evidence that sequence specific gene silencing of the Mus Musculus Deleted in Colorectal Cancer (DCC) gene had occurred. However, down-regulation of the DCC protein did not occur consistently, suggesting that further optimisation is needed to improve the efficacy of siRNA-LDH complexes to inhibit expression of target protein in neurons. In future, LDHs should be further developed as an efficient siRNA delivery system for therapeutic gene silencing in the central nervous system using a neurodegenerative disease model such as the Huntington’s disease mouse model, which closely phenocopies the human disease. This model will allow the in vivo efficacy of these nanoparticles to be tested and subsequently improved in order to deliver siRNAs locally and systematically into the brain.

Made available in DSpace on 2016-06-02T20:20:35Z (GMT). No. of bitstreams: 1 5217.pdf: 18042962 bytes, checksum: 6c55d259639b3b244326b14d21f608f1 (MD5) Previous issue date: 2012-02-29 / Universidade Federal de Minas Gerais / The sugarcane weevil, Sphenophorus levis, is an insect that feeds on the rhizome of sugarcane in its larval stage, boring channels that cause damage and death to the plant. Conventional methods of insect control have not been efficient. The aim of the present study was to generate knowledge on the biology of this insect on the molecular level using a transcriptomic approach to determine potential targets genes for the engineering of insect-resistant plants. After sequence processing and assembly using the dCAS program, 3804 sequences were grouped into 201 contigs and 1363 singlets, which were manually annotated. Several plant cell wall degrading enzymes were identified, including pectinases and cellulases. An invertasecontaining contig was identified for the first time in a coleopteran. Total probable digestive enzymes accounted for 19.3% and unknown genes accounted for 28.8% of the total number of expressed sequence tags. Considering the predominance of cathepsin L enzymes among the digestive enzymes found in the transcriptome (54.3%) and their importance to the breakdown of proteins in the insect digestive process, a cDNA clone encoding a cathepsin L enzyme, denominated Sl-CathL, was chosen for recombinant expression in Pichia pastoris cells, characterization and in vitro inhibition by the sugarcane cystatin CaneCPI-4 (Ki = 0.196 nM). Immunolocalization assays demonstrated the production of Sl-CathL in the midgut epithelium and secretion into the gut lumen from vesicles containing the enzyme. S. levis demonstrated sensitivity in triggering RNA interference machinery induced by dsRNA injections in the body cavity and gene-specific knockdown was confirmed by qRT-PCR. Larvae injected with V-ATPase E dsRNA died within three weeks after injection and serpin 1-silenced larvae either exhibited delayed development, arresting in the pupal stage, or died as pharate adults. In conclusion, transcriptome analyses, together with the inhibition of the main digestive enzyme by Cane-CPI-4 and gene silencing using RNAi, are promising procedures for the development of transgenic sugarcane plants to enhance resistance to Sphenophorus levis. / O bicudo da cana-de-açúcar, Sphenophorus levis, é um inseto que na fase larval se alimenta do rizoma da cana-de-açúcar cavando galerias que causam danos e levam à morte das plantas. Os métodos convencionais de controle disponíveis não tem sido eficientes contra esse inseto. O objetivo desse estudo foi gerar conhecimento sobre a biologia desse inseto em nível molecular utilizando uma abordagem transcriptômica para a identificação de possíveis genes alvo, visando futuros estudos para desenvolvimento de plantas transgênicas resistentes ao ataque deste inseto. Após o processamento e montagem das sequências utilizando o programa dCAS, 3804 sequências foram agrupadas em 201 contigs e 1363 singlets, os quais foram manualmente anotados. Foram identificados diversos genes de degradação de parede celular, incluindo pectinases e celulases. Pela primeira vez um contig contendo uma sequência que codifica uma invertase foi identificado em um coleóptero. As prováveis enzimas digestivas totalizam 19,3% e os genes desconhecidos representam 28,8% do total de sequências expressas. Considerando a predominância de catepsinas L dentre as enzimas digestivas identificadas no transcriptoma (54,3%) e a sua importância para a hidrólise de proteínas nos processos digestivos, um clone de cDNA codificando uma catepsina L, denominado Sl-CathL, foi escolhido para a expressão recombinante em células de Pichia pastoris, caracterização e inibição in vitro utilizando a cistatina da cana-de-açúcar CaneCPI-4 (Ki = 0,196 nM). Os ensaios de imunolocalização evidenciaram a produção da Sl-CathL no epitélio do intestino médio e a secreção de vesículas, contendo a enzima, no lúmen do intestino. Larvas do inseto S. levis também apresentaram sensibilidade para disparar a maquinaria de RNA de interferência induzida por injeções de dsRNA na cavidade corpórea e o silenciamento gênico específico foi confirmado por qRT-PCR. As larvas que receberam injeções com dsRNA da V-ATPase E morreram dentro de três semanas, enquanto as larvas que tiveram a serpina 1 silenciada exibiram atraso no desenvolvimento, aprisionamento na fase pupal, ou morreram como adultos farados. Desse modo, concluímos neste trabalho iniciado a partir da análise do transcriptoma, que a inibição da principal enzima digestiva pela Cane-CPI-4 e o silenciamento gênico via RNAi são alternativas promissoras para o estabelecimento de plantas resistentes ao inseto e podem ser aplicadas no desenvolvimento de plantas de cana-de-açúcar transgênicas com o objetivo de aumentar sua resistência ao Sphenophorus levis.

Made available in DSpace on 2016-06-02T19:22:52Z (GMT). No. of bitstreams: 1 2981.pdf: 6989540 bytes, checksum: 48d65263f0c7e6493fadd24c1378e4d0 (MD5) Previous issue date: 2009-10-14 / Financiadora de Estudos e Projetos / ADAMs is a term used to describe the presence of disintegrin and metalloprotease domains(A Disintegrin And Metalloprotease) in a certain class of multi-functional membrane proteins,expressed in several animal species such as mammals and insects. They play important rolesin many physiological processes as in fertilization, myoblast fusion, migration, proliferationand cell survival, as well in diseases including breast, prostate, and pancreas cancer. ADAM9is involved in cellular processes such as adhesion, migration and signaling of tumor cells andit is involved in the metastatic spreading. Aims: To analyze the expression of ADAM9 intumor cell lines (MDA-MB-231 and DU-145), no tumors (FH and C2C12), and to generateknockout clones for ADAM9 expression using silencing RNA techniques for the study ofADAM9 effects on invasion, proliferation and gene expression of MDA-MB-231 cells.Methods: Cells were cultured and plated (2x106 cells/plate of 6cm) for 24 hours in DMEM(10% FBS) and lysed for western blotting and zymography. To check for inhibition ofadhesion to immobilized collagen I, MDA-MB-231 cells and FH (5x106 cells/ml) werelabeled with CMFDA, and then incubated with anti-ADAM9D and anti-RP3ADAM9 indifferent concentrations. For ADAM9 silencing, it was used a kit (Silencer ® siRNA StarterKit - Ambion), 2x105 cells (MDA-MB-231) and the transfection agent (Lipofectamine 2000 -Invitrogen). The cells were plated in 5ml of culture medium DMEM/plate. On the third daycells were treated with the transfection agent and RNA silencing primers. Total RNA wasisolated for RT-PCR, and proliferation and invasion in matrigel assays. Results: All the celllines studied expressed ADAM9 in the tested conditions. MMP-2 (Gelatinase-A) activity wasdetected in the cell extracts of all studied cell lines. Silencing of ADAM9 did not affect therate of MDA-MB-231 proliferation, at 4, 5, 6, 7, 8, 9 and 10 days after silencing (24 or 48hours of incubation in 96-well plates). Silencing of human ADAM9 inhibited tumor cellinvasion in matrigel (71,51±8,02%) when compared to control. Conclusion: The generation ofMDA-MB-231 knockout clones lacking ADAM9 expression using siRNA technique did notaffect the rate of cell proliferation but inhibited tumor cell matrigel invasion, suggesting thatADAM9 is an important molecule in the processes of invasion and metastasis. / ADAMs é um termo usado para descrever a presença de domínios desintegrina emetaloprotease (A Disintegrin And Metalloprotease) em uma determinada classe de proteínasde membrana, multifuncionais, expressas em diferentes espécies animais como mamíferos einsetos. Elas possuem funções importantes em muitos processos fisiológicos como nafertilização, fusão de mioblastos, migração, proliferação e sobrevivência celular, entre outros,bem como em processos patológicos tais como muitos tipos de tumores humanos, incluindomama, próstata, pâncreas, fígado, rins e pele. A ADAM9 está envolvida em diversosprocessos celulares, tais como adesão celular, migração e sinalização de células tumorais,contribuindo para o desenvolvimento de metástases. Objetivos: Analisar a expressão daADAM9 em linhagens de células tumorais (MDA-MB-231 e DU-145) e não tumorais (FH eC2C12), gerar clones com o gene que codifica para a ADAM9 silenciados e verificar o efeitoda ausência desta proteína na invasão, proliferação e expressão gênica das células MDA-MB-231. Métodos: As células foram cultivadas e posteriormente plaqueadas (2x106 células/placade 6cm) por 24 horas e em 5ml de meio de cultura DMEM (10% de FBS) e lisadas para oensaio de western blotting e zimografia. Para o ensaio de inibição de adesão as células MDAMB-231 e FH (5x106 células/ml) foram marcadas com CMFDA (clorometil diacetatofluoresceína) e posteriormente incubadas com os anticorpos anti-ADAM9D e anti-RP3ADAM9 em diferentes concentrações. Para a técnica de RNAi foi utilizado um kit(Silencer® siRNA Starter Kit Ambion), 2,0x105 de células (MDA-MB-231) e agente detransfecção (Lipofectamina 2000 - Invitrogen). As células foram plaqueadas em 5ml de meiode cultura DMEM/placa. No terceiro dia de plaqueamento as células foram tratadas comagente de transfecção e primer de silenciamento do RNA, para serem utilizadas na RT-PCR,ensaio de proliferação e invasão em matrigel. Resultados: Todas as linhagens estudadasexpressaram a ADAM9 nas condições de estudo. A atividade MMP-2 (gelatinase-A)intermediária estava presente em todos os tipos celulares testados. O silenciamento deADAM9 não afetou a taxa de proliferação das células MDA-MB-231 após 4, 5, 6, 7, 8, 9 e 10dias do silenciamento (24 ou 48 horas de incubação). O silenciamento da ADAM9 humanainibiu a invasão celular em células de câncer de mama (MDA-MB-231) em matrigel (71,51 ±8,02%) quando comparados com o controle. Conclusão: A geração de clones knockout sem aexpressão da ADAM9 utilizando a técnica de silenciamento de RNA em células de câncer demama (MDA-MB-231), não afetou a taxa de proliferação celular. No entanto, a invasão dascélulas tumorais em matrigel foi inibida em aproximadamente 70% quando comparada com ocontrole, demonstrando que ADAM9 é uma importante molécula envolvida no processo deinvasão e metástase.

L'orientation du fuseau mitotique joue un rôle essentiel dans le choix du destin cellulaire et dans l'homéostasie des tissus. Dans certains contextes, l'orientation du fuseau est contrôlée par le complexe moléculaire LGN, dont la localisation sous-corticale détermine le site de recrutement du moteur dyneine, lequel exerce des forces sur les microtubules astraux pour orienter le fuseau. Chez les vertébrés la régulation moléculaire de ce processus est cependant peu caractérisée. Nous avons décidé de chercher de nouveaux régulateurs de l'orientation du fuseau chez les vertébrés. Avec cet objectif, j'ai développé un modèle d'orientation du fuseau spécifiquement contrôlé par le complexe LGN. Avec ce modèle, j'ai réalisé un crible RNAi en évaluant 110 candidats incluant des moteurs moléculaires pour leur fonction dans l'orientation du fuseau. Notamment, ce crible a révélé que les régulateurs de la dyneine sont inégalement requis pour orienter le fuseau. De plus, entre les sous-unités de la dynactine, j'ai trouvé que la protéine du capping de l'actine, CAPZ-B, est un régulateur majeur de l'orientation du fuseau. La caractérisation de la fonction de CAPZ-B in vitro a révélé que CAPZ-B contrôle l'orientation du fuseau en régulant les complexes dyneine et dynactine ainsi que la dynamique des microtubules du fuseau, indépendamment de son rôle comme modulateur de l'actine. Finalement, nous avons démontré que CAPZ-B régule l'orientation planaire du fuseau in vivo dans le neuroépithelium. Je pense que mes travaux vont contribuer à la compréhension de la fonction de la dyneine dans l'orientation du fuseau chez les vertébrés, ouvrant la voie pour de nouvelles recherches dans le domaine. / Mitotic spindle orientation is involved in cell fate decisions, tissue homeostasis and morphogenesis. In many contexts, spindle orientation is controlled by the LGN molecular complex, whose subcortical localization determines the site of recruitment of the dynein motor which exerts forces on astral microtubules orienting the spindle. In vertebrates, there is missing information about the molecules regulating the formation of the complex and those working downstream of it. This prompted us to screen for new regulators of vertebrate spindle orientation. For this, I developed a novel model of spindle orientation specifically controlled by the LGN complex. Using this model, I performed a live siRNA screen testing 110 candidates including molecular motors for their function in spindle orientation. Remarkably, this screen revealed that specific dynein regulators contribute differentially to spindle orientation. Moreover, I found that an uncharacterized member of the dynactin complex, the actin capping protein CAPZ-B, is a strong regulator of spindle orientation. Analyses of CAPZ-B function in cultured cells showed that CAPZ-B regulates spindle orientation independently of its classical role in modulating actin dynamics. Instead, CAPZ-B controls spindle orientation by modulating the localization/activity of the dynein/dynactin complexes and the dynamics of spindle microtubules. Finally, we demonstrated that CAPZ-B regulates planar spindle orientation in vivo in the chick embryonic neuroepithelium. I expect that my work will contribute to the understanding of dynein function during vertebrate spindle orientation and will open the path for new investigations in the field.

Philosophiae Doctor - PhD / The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene. / South Africa

Interés del estudio: Los viroides son patógenos exclusivos de plantas que infectan un gran número de especies de interés agronómico. Sin capacidad descrita para codificar proteínas, todas las fases de su ciclo vital son estrictamente dependientes de su interacción con factores del huésped. Históricamente se ha asumido que la patogénesis es consecuencia de la competencia huésped-patógeno por factores celulares implicados en el ciclo vital del viroide. En los últimos años se ha propuesto que la respuesta de silenciamiento de RNA (RNAi) del huésped frente a estos patógenos es la responsable de la respuesta patogénica de los viroides. En el presente trabajo se ha tratado de estudiar en profundidad la relación entre el mecanismo de silenciamiento de RNA y la patogénesis viroidal (en concreto del viroide del enanismo del lúpulo, HSVd). Objetivos: 1- Estudiar el proceso de patogénesis inducido por HSVd y la maquinaria de RNAi 2- Caracterizar mediante secuenciación masiva los sRNAs derivados de HSVd (vd-sRNAs). 4- Analizar la distribución diferencial de vd-sRNAs en distintos tejidos (hoja y floema). 5- Determinar las alteraciones inducidas por la infección en las vías endógenas de RNAi. Elementos de la metodología a destacar: - Uso de un sistema transgénico para estudiar el fenómeno de la patogénesis. - Uso de técnicas de secuenciación masiva para la caracterización de sRNAs. - Análisis bioinformático de los datos generados por la secuenciación masiva. Resultados logrados: - Primera publicación en la literatura de la relación de un componente de las rutas de RNAi en la patogénesis viroidal ocasionada por HSVd. - Publicación de una de las primeras secuenciaciones masivas de sRNAs derivados del HSVd. - Primera caracterización de las poblaciones de sRNAs endógenos de C.sativus, incluyendo la primera caracterización de microRNAs en esta especie. - Identificación y detección de nuevos microRNAs en C.sativus. / Martínez Arias, GE. (2011). Relación entre el silenciamiento de RNA y la patogénesis inducida por un viroide con replicación nuclear [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11302 / Palancia

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplantation, with no approved therapeutics available. Despite years of research, the exact molecular mechanism of NASH progression is not well understood, but fat accumulation is believed to be the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long term gene silencing with single subcutaneous administration. Here we identified a hyper-functional, fully chemically stabilized GalNAc conjugated siRNA targeting DGAT2 (Dgat2-1473) that upon injection elicits up to three months of DGAT2 silencing (>80-90%, p<0.0001) in wild-type and NSG-PiZ “humanized” mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (> 50%, p:0.0008), resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat didn’t translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19542号 / 理博第4202号 / 新制||理||1603(附属図書館) / 32578 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 阿形 清和, 教授 平野 丈夫, 教授 森 和俊 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Il devient de plus en plus clair que pour traiter efficacement les tumeurs solides, nous devons également nous intéresser au microenvironnement tumoral. Physiologiquement, les zones intratumorales peuvent présenter une disponibilité anormale en nutriments, un pH altéré ou encore des niveaux d’oxygène bas (hypoxie). Il est connu que l’adaptation hypoxique engendre des cellules tumorales qui sont plus difficiles à traiter indépendamment de l’approche thérapeutique. De plus, l’adaptation hypoxique est nécessaire pour la progression tumorale puisque cette dernière favorise des processus tels que: la survie cellulaire, la motilité, l’angiogenèse, le métabolisme du glucose, l’immunomodulation ainsi que la résistance aux médicaments. Ces phénotypes passent par la régulation des ARN messager (ARNm) et des micro ARN (miARN). Pour ces raisons, des efforts importants ont été déployés pour comprendre l’adaptation hypoxique et les interventions thérapeutiques potentielles pouvant la contrer. À l’heure actuelle, il y a un manque de cohérence et de variété dans les protocoles de traitement hypoxique in vitro qui ne tient pas compte des aspects importants de l’hypoxie in vivo, comme la réduction de la disponibilité en éléments nutritifs, la durée de l’exposition hypoxique ainsi que le degré d’hypoxie. Pour mieux simuler le microenvironnement hypoxique in vivo, nous avons développé de nouveaux protocoles hypoxiques in vitro qui visent à simuler ces aspects. Tout d’abord, en utilisant une lignée cellulaire B16-HIF1a-eGFP, nous avons optimisé le stress métabolique à court terme en conjonction avec l’hypoxie pour augmenter la stabilisation de l’HIF1a. Pour déterminer comment le programme HIF1 adapte les cellules à ces différentes conditions, nous avons analysé les données de séquencage d’ARN qui démontrent que le stress métabolique induit un programme transcriptionnel HIF1 plus robuste et diversifié dans les cellules hypoxiques, et que ce dernier est représentatif du stress hypoxique in vivo. Nous avons également identifié de nouveaux miARN induits par l’hypoxie et démontré que notre protocole d’incubation régule davantage les miRNA associés au pronostic négatif du patient. Nous avons aussi étudié l’adaptation hypoxique à long terme et extrême in vitro. Nous avons observé que l’incubation hypoxique à long terme induit une transition épithéliale à mésenchymateuse (TME), indépendante de l’expression différentielle des facteurs de transcription du TME canonique. Ce changement se produit à des niveaux spécifiques d’oxygène, et nécessite une pré-incubation à des niveaux hypoxiques plus faible. Avec ce protocole, nous avons découvert une nouvelle isoforme de WT1 (tWT1), un moteur potentiel du TME. tWT1 commence la transcription dans l’intron 5 du gène WT1, une région avec plusieurs séquences d’ADN contenant des éléments de réponse à l’hypoxie. La protéine tWT1 a une fonctionnalité limitée : elle est localisée au niveau du noyau, et conserve la liaison de l’ADN aux régions précédemment connues. Nous avons aussi identifié l’expression de tWT1 dans les échantillons de patients atteints de leucémie ainsi qu’une isoforme tWT1 potentiellement plus fonctionnelle grâce à des analyses par kmer. Pour cibler ces phénotypes identifiés dans nos expériences d’adaptation hypoxiques, nous avons développé une nouvelle catégorie d’ARN intérférent (ARNi) thérapeutique : le microARN synthétique (synmiR). Les synmiR sont des molécules de RNAi avec des multiples cibles. En utilisant des expériences in vivo, nous avons établi de nouveaux principes de RNAi qui élargissent considérablement l’espace de conception pour les synmiR. Nous avons mis au point deux algorithmes de conception de synmiR distincts et avons testé leur efficacité dans le contrôle de l’activité transcriptionnelle du génome du VIH in vivo. En conclusion, nous avons montré que l’inclusion de facteurs physiologiques supplémentaires associés à l’hypoxie in vitro entraîne un engagement plus robuste de l’adaptation de l’hypoxie. À ce jour, aucun de nos protocoles d’hypoxie n’a été reproduit dans la littérature. Nous contribuons aux connaissances dans le domaine en décrivant les nouveaux ARNm/miARN induits par l’hypoxie, ainsi que la méthode d’induction fiable de l’EMT par l’hypoxie seulement. Nous faisons également état de l’existence de nouveaux isoformes de WT1 et de leurs liens avec le cancer et l’hypoxie. La connaissance de ces isoformes est importante pour l’avenir de la recherche sur WT1, car elle pourrait faire la lumière sur des résultats auparavant inexplicables. Notre travail dans les synmiR ouvre une nouvelle voie d’investigation pour le traitement de certaines maladies, et fournit un mécanisme d’action testable pour les miRNA endogènes. Une fois suffisamment développés, les synmiR offrent une occasion thérapeutique unique d’exploiter leur multi-ciblage pour avoir un impact spectaculaire sur une seule voie, ou affecter plusieurs voies par le ciblage simultané de gènes clés. / It is becoming increasingly clear that in order to effectively treat solid tumours, we must also address the tumour microenvironment. Physiologically, intratumoral areas may have abnormal nutrient availability, pH, or lower oxygen levels (hypoxia). It is known that hypoxic adaptation results in tumour cells which are harder to treat regardless of therapeutic approach, and hypoxic adaptation is necessary for disease progression due to the induction of tumour promoting phenotypes such as, but not limited to: cell survival, motility, angiogenesis, glucose metabolism, immunomodulation, and drug resistance. This is accomplished through the regulation of both mRNAs and miRNAs. For these reasons, significant effort has been applied to understanding hypoxic adaptation and potential therapeutic interventions. Currently, there is a lack of consistency and protocol variety in in vitro hypoxic treatments that leaves out important aspects of in vivo hypoxia, such as reduced nutrient availability, length of hypoxic exposure, and degree of hypoxia. To better simulate the in vivo hypoxic microenvironment, we have developed new in vitro hypoxic protocols which aim to simulate these aspects. First, using a B16-HIF1α-eGFP hypoxia reporter cell line, we optimized short-term metabolic stress in conjunction with hypoxia to enhance HIF1α stabilization. To ascertain how the HIF1 program adapts the cells to these different conditions, deep transcriptome profiling were performed and demonstrated metabolic stress induces a more robust and diversified HIF1 transcriptional program in cells under hypoxia, which was more representative of in vivo hypoxic stress. We identified novel hypoxia-induced miRNAs as well, and demonstrated our incubation protocol regulated more miRNAs associated with negative patient prognosis. We also investigated long-term and extreme hypoxic adaptation in vitro. Long term hypoxic incubation induced a epithelial to mesenchymal transition (EMT), independent of canonical EMT factor differential expression. This switch occurred at specific oxygen levels, and required pre-incubation at milder hypoxic levels, highlighting the relevance of simulating in vivo hypoxia development in vitro. Through this protocol, we discovered a novel isoform of WT1 (tWT1), a potential driver of our EMT. tWT1 begins transcription within intron 5 of the WT1 gene, a region with several Hypoxia Response Elements DNA sequences. tWT1 retains limited functionality: it is able to localize to the nucleus, and retains DNA binding to previously known gene promoter regions. We also identified the expression of tWT1 in leukemic patient samples as well as a potentially more functional tWT1 isoform through kmer-based analyses. To target these multiple phenotypes identified in our hypoxia adaptation experiments, we worked towards developing a new category of RNA-interference (RNAi) therapeutic, the synthetic microRNA (synmiR). SynmiRs are single-sequence, multi-targeted RNAi molecules. Using in vivo knock-down experiments, we established new RNAi principles which dramatically expand the design space for synmiRs. We developed two philosophically distinct synmiR design algorithms, and validated their efficacy in controlling HIV genome transcriptional activity in vivo. In conclusion, we have shown the inclusion of additional physiological factors associated with hypoxia in vitro results in a more robust engagement of hypoxia adaptation. To date, neither of our hypoxia protocols have been replicated in the literature. We contribute to the literature by describing novel hypoxia induced mRNAs/miRNAs, as well as methods for reliably inducing EMT through hypoxia alone. We also discovered the existence of novel WT1 isoforms and their links to cancer and hypoxia. Knowledge of these isoforms is important for WT1 research moving forward, as it may shed light on previously unexplainable results. Our work in synmiRs opens a new therapeutic avenue for multiple disease states, and provides a testable mechanism of action for endogenous miRNAs. Once sufficiently developed, synmiRs offer a unique therapeutic opportunity to harness their multi-targeting to dramatically impact a single pathway, or affect multiple pathways through simultaneous targeting of key genes.

Alzheimer’s disease (AD) is a neurodegenerative disease and the most common form of dementia associated with amyloid-beta peptide deposition and loss of mitochondrial function and regulation. Currently, there is no cure for AD, thus, there is a need to continuously develop therapeutic strategies that could address the complex multifactorial causes of AD development. Due to this necessity, this study has investigated the role of vitamin B2 as a disease modifying drug for AD by employingamyloid-beta and mitochondrial based AD therapeutic strategies. Using a transgenic C. elegans AD worm model expressing amyloid-beta (Aβ1-42) in muscle cells at temperature upshift to 25°C, we screened for protective effect of dose-dependent concentrations of active forms of vitamin B2, FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), against amyloid-beta mediated paralysis. Protective concentrations were then assayed for improvement of mitochondrial metabolic functions by performing ATP, oxygen consumption and reactive oxygen species (ROS) production assays. Consequently, we investigated for drug protective mechanisms of FMN and FAD using RNAi genetic screening technique. FMN and FAD significantly delayed amyloid-beta mediated paralysis and improved mitochondrial metabolic functions at final concentrations of 0.74mM and 0.74µM respectively. More so, both compounds induced activation of stress response FOXO transcription factor, daf-16. Specifically, FMN treatment induced mitochondrial unfolded protein response (UPRmt) pathway through ubiquitin-like protein (ubl-5) activation as well as other stress response pathway signature such as Activating Transcription Factor Associated with Stress (atfs-1). This study will be useful in understanding the importance of micronutrients such as vitamin B2 in normal cellular function as related to neurodegenerativediseases and aging. Therefore, vitamin B2 supplementation could be an important source of Alzheimer’s disease therapeutic strategy.