RNA interference (RNAi) is a conserved pathway that plays key roles in heterochromatin formation, gene regulation and genome surveillance across a wide range of eukaryotes. One of the most utilised model organisms for studying the RNAi pathway is the fission yeast Schizosaccharomyces pombe. However, this species is somewhat atypical, in that it has not retained the ancestral role for RNAi in the silencing of mobile genetic elements. In contrast, the related fission yeast S. japonicus has a large and diverse retrotransposon complement that appears to give rise to abundant siRNAs. For this reason, we believe that S. japonicus may be a more suitable model for studying the role of RNAi in silencing mobile genetic elements, a function that is conserved in many higher eukaryotes. Functional analysis of the S. japonicus RNAi pathway proved more challenging than expected, as it was generally not possible to recover strains bearing deletions of core RNAi components (Ago1/Clr4/Rdp1/Arb1/Arb2). This suggests that a functional RNAi pathway may be required for viability in S. japonicus, unlike in S. pombe. However, disruption mutants were isolated for the sole Dicer ribonuclease Dcr1, at very low frequency. Analysis of these mutants revealed that disruption of Dcr1 impaired the generation of retrotransposon derived siRNAs, and caused de-repression of retroelement transcript accumulation and mobilisation in an element dependent manner. Surprisingly however, Dcr1 appeared dispensable for the maintenance of H3K9me2 at transposons, suggesting that, in contrast to S. pombe, silencing may occur principally at the post-transcriptional level. It is also possible that the isolated Dcr1 mutants represent rare survivors that are viable due to the presence of suppressor mutations elsewhere in the genome. I utilised my genome wide RNA sequencing data to help improve the annotation of the S. japonicus genome, with a specific focus on the retrotransposon complement. From this, I identified 12 new families of LTR retrotransposon, which increased the annotated retrotransposon complement by around 40% in S. japonicus. Finally, I characterised the integrative preference of the S. japonicus retrotransposon Tj1, and found that it shares characteristics associated with the S. cerevisiae retrotransposons Ty1 and Ty3, mostly integrating upstream of RNA PolIII transcribed tRNA genes. The findings of this work highlight some potentially key differences in the way the RNAi pathway functions across the fission yeast clade, both in terms of its importance for viability and its mode of action. The work undertaken here also contributes to the establishment of S. japonicus as a model for the study of RNA interference and genome regulation.

Les oligonucleotides sont utilisés pour de nombreuses applications dans le domaine du diagnostic et ils peuvent également être utilisés comme traitement pour de nombreuses maladies. Les oligonucléotides sont des polyanions qui viennent s'hybrider sur leurs séquences complémentaires elles aussi anioniques. Les répulsions électrostatiques impliquent que l'addition de charges positives sur les oligonucléotides serait bénéfique pour diminuer les répulsions et améliorer l'hybridation. Dans le but de diminuer ces répulsions électrostatiques, des conjugués oligonucléotide-oligocation sur lesquels sont greffées des unités spermines ont été développés. Les conjugués oligonucléotideoligocation sont synthétisés sur un synthétiseur automatique d'oligonucléotide en utilisant la chimie des phosphoramidites. Les« Zip Nucleic Acid »ou ZNAs sont des oligonucléotides portant une queue cationique de quelques unités de spermine et sont de charge globale négative. Les modifications apportées permettent d'améliorer l'hybridation en accélérant la reconnaissance de la séquence cible et en augmentant la température de fusion linéairement avec le nombre de spermines greffées sans altérer la spécificité. Les ZNAs se révèlent être efficaces utilisés comme amorces ou sondes en PCR et ils apparaissent comme de nouveaux outils intéressants pour la biologie moléculaire. Les petits ARNintérferents (si RNA) induisant l'extinction d'un gène par la voie d' ARN interférence ont suscités un grand engouement ces dernières années, cependant leur très faible pénétration cellulaire est un frein majeur à leur utilisation. C'est pour cela que les conjugués oligonucléotide- oligospermine ont un réel intérêt pour le domaine de la thérapie in vivo. Les duplexes SIRNAPLUS cationiques sont des siRNAs ciblantspécifiquement un ARN messager. Ils sont constitués d'un brin sens ARN-oligospermine de charge globale positive hybridé à un brin antisens. Les résultats ont montré que lesSIRNAPLUS pouvaient entrer seuls dans les cellules sans agent de transfert pour induire l'extinction d'un gène cible et les premières expériences montrent qu'ils sont actifs in vivo. Mes travaux de thèse ont porté sur le développement des conjugués oligonucléotide oligospermine et démontrent des applications potentielles dans le domaine du diagnostic et de la thérapie. / Oligonucleotides are finding an extremely large number of applications in molecular diagnostics and might become a very selective class of drugs for the treatment of a vast palette of diseases. Oligonucleotides are polyanions that exert their specifie activity following hybridization to a complementary sequence borne by another polyanionic nucleic acid. Simple electrostatic considerations imply that hybridization energy and cell binding couId benefit from addition of cationic groups to the oligonucleotide structure. Towards the aim of improving hybridization by decreasing electrostatic repulsions between the negatively charged strands, oligonucleotide-oligocation conjugates whose global charge is modulated by the number of cationic spermine moieties grafted on the oligonucleotides have been developed. Oligonucleotide-oligospermine conjugates are produced using an automated solid-phase synthesis of conjugates that are entirely based on the phosphoramidite coupling chemistry. Zip Nucleic Acids {ZNAs} are oligonucleotides with a short polycationic tail, composed of relatively few spermine units, leading to molecules overall negative in charge. The modification improves hybridisation by accelerating the target recognition and increases the melting temperature linearly with the number of grafted spermines on the oligonucleotide without altering the specificity. ZNAs have been shown to be potent primers and probes for PCR and are new interesting tools for molecular biology and diagnostics applications. Small interfering RNA (siRNA}-mediated gene silencing has become a drug development paradigm. As drug candidates, they must aIso be able to cross the anionic cell membrane. However, still one major limitation of the use of siRNA remains their inability to penetrate efficiently into cells of a particular tissue or tumour. That gives to oligonucleotide-oligospermine conjugates a real interest in this domain and more generally in vivo therapies. Cationic SIRNAPLUS are duplexes of small RNAs targeting a specifie mRNA. They are produced as an oligospermine-RNA sense strand, with positive global charge, associated to an antisense RNA strand. Results have shown that cationic siRNAs are able to enter cells efficiently without vector and to display silencing activity at nanomolar concentration. To have positive global charge, the number of spermine moieties has been increased. Purification and characterization methods have been developed to have cationic siRNAs compatible with in vivo experiments. My thesis will describe the synthesis of oligonucleotide-oligospermine conjugates as well as their applications.

Psoroptic mange, caused by the Astigmatid mite Psoroptes ovis, is an ectoparasitic disease of significant economic importance to agriculture on a global scale and poses a serious welfare concern. With the current chemotherapeutic controls considered unsustainable, there is pressing need for novel control strategies. RNA interference has been proposed as a potential high throughput approach for the identification of novel therapeutic targets with high specificity, speed and at a relatively low cost compared to the existing methods. The presence of the components of the RNA interference (RNAi) pathway in P. ovis was first confirmed through in silico analyses of the P. ovis transcriptome and, following development of a non-invasive immersion method of double stranded RNA (dsRNA) delivery, gene silencing by RNAi was demonstrated in P. ovis. Statistically-significant reduction of transcript level was measured for the three genes targeted: P. ovis mite group 2 allergen (Pso o 2), P. ovis mu class glutathione S-transferase (PoGST-mu1) and P. ovis beta tubulin (Poβtub). This is the first demonstration of gene silencing by RNAi in P. ovis and provides a key mechanism for mining transcriptomic and genomic datasets in the future for novel targets of intervention against P. ovis. The first assessment of gene silencing was also performed in two related Astigmatid mites of high medical importance; the European house dust mite Dermatophagoides pteronyssinus and the scabies mite Sarcoptes scabiei. A statistically-significant reduction in expression of a D. pteronyssinus mu class glutathione S-transferase (DpGST-mu1) transcript was observed. No significant reduction in expression of a S. scabiei mu class glutathione S-transferase (SsGST-mu1) transcript was observed. Additionally, microRNAs (miRNAs) from the related miRNA pathway were identified in a P. ovis small RNA sample and were sequenced and annotated.

Hearing impairment is the most common sensory deficit worldwide, affecting at least one child in every one thousand born. Gene therapy targeting the inner ear offers promise for treatment of genetic forms of hearing loss. Many genetic forms of deafness are congenital and gene therapies in these cases would require treatment prior to inner ear maturation. Included in this category is the dominant-negative R75W mutation in GJB2 which encodes connexin 26, a gap junction protein expressed in the supporting cells of the organ of Corti. RNA interference (RNAi)-based therapeutics offer promise for treating dominant-negative diseases. Our goal has been the in vivo application of RNAi-therapy to the GJB2-R75W transgenic mouse, a model of severe-to-profound dominant-negative hearing loss. Here we describe our efforts to identify a therapeutic, a suitable delivery route, and an optimal delivery vector. We have designed and optimized siRNA to achieve robust silencing of the mutant transgene in vitro and have prepared artificial miRNA constructs for in vivo application. We have determined to use the embryonic otocyst microinjection technique as the route for therapeutic delivery and have successfully utilized this technique to study the tropism and safety of several viral vector (adeno-associated virus 2/1, early- and late-generation adenoviruses, and bovine adeno-associated virus). For the first time we have characterized viral tropism for cochlear supporting cells following in utero delivery to their progenitor cells in the developing cochlea and identified bovine adeno-associated virus as a safe vector for gene delivery to the supporting cells of the cochlea. We have also described two previously unreported phenotypes in the GJB2-R75W transgenic mouse model: skin disease and cataracts. Both can be caused by dominant connexin mutations in humans. Our work shows that although gene therapy is not simple, powerful tools are in place for treating dominant forms of hereditary hearing loss.

Angiotensin II signals cardiovascular disease through metalloproteinases including MMP-2, MMP-7 and ADAM-17/TACE. We hypothesized that these metalloproteinases regulate each other at the transcriptional level. Further, MMP-2, being a major gelatinase in cardiac and vascular tissue, could mediate angiotensin II-induced cardiovascular disease. We studied the development of hypertension (by tail cuff plethysmography), cardiac hypertrophy (by M-mode echocardiography and qRT-PCR analysis of hypertrophy marker genes) and fibrosis (by collagen staining and qRT-PCR analysis of fibrosis marker genes) in mice receiving angiotensin II. Angiotensin II induced hypertension, cardiac hypertrophy and fibrosis which correlated with an upregulation of MMP-2. Downregulation of MMP-2 by pharmacological inhibition and RNA interference attenuated hypertension but not cardiac hypertrophy or fibrosis. Downregulation of MMP-7 or ADAM-17/TACE by RNA interference attenuated angiotensin II-induced MMP-2 upregulation as well as hypertension, cardiac hypertrophy and fibrosis. We conclude that MMP-2 selectively mediates angiotensin II-induced hypertension under the transcriptional control of MMP-7 and ADAM-17/TACE.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 90-99).

Variability of transgene expression levels resulting from gene silencing is considered as ahindrance to the successful application of plant genetic engineering. Towards alleviatinggene silencing, I decided to screen for novel genes involved in transgene silencing and toinvestigate how these genes regulate plant development. Genes encoding putative chromatinremodeling factors, especially those including a SET domain, were selected as candidatetargets. A bioinformatic analysis of the Arabidopsis SET genes (AtSET) was performed andthese genes were classified into 6 groups based on the domain architecture. RNA interference (RNAi) vectors were constructed for ~ 20 AtSET genes and wereintroduced into both wild type lines and transgenic lines silenced for a GFP reporter gene.Surprisingly, altered developmental phenotypes were only observed for three constructs,raising questions as to the effectiveness of the RNAi approach for the chosen Arabidopsissystem. To assess this situation, I targeted a phytoene desaturase (PDS) gene using the sameRNAi approach. Inactivation of PDS renders plant a readily identifiable phenotype. Whereasthe RNAi penetrance in Arabidopsis can be very high, the expressivity of RNAi in varioustissues and among different plants can vary dramatically. Contradictory to previous reports,I found that there is correlation between transcript level and silencing phenotype. Possiblereasons for this discrepancy are discussed. No apparent correlation between transgene copynumber and RNAi phenotypes was observed. Among the three RNAi constructs that caused an abnormal development inArabidopsis, K-23 which targets SuvR3 has the highest expressivity and could reactivate asilenced GFP locus. SuvR3 RNAi lines were selfed for six generations and were screenedfor morphological phenotypes. Abnormal number of flower organs, loss of viability of malegametophytes, and decreased seedling germination percentage were found in SuvR3 RNAilines. A progressive increase in both severity and frequency of abnormal phenotypes wereseen in subsequent generations, suggesting an epigenetic regulatory mechanism involvedwith SuvR3. Alternative splicing of SuvR3 was also observed in most of Arabidopsis tissues.One of the protein isoforms, SuvR3, lacks 16 amino acids within the highly conserved SETdomain. Possible effects of isoform interaction are proposed.

The freshwater crayfish, Pacifastacus leniusculus, mounts a strong innate immune response against microbes such as viruses and bacteria. In this thesis, a novel RNA interference (RNAi) method mediated with histone H2A was developed and applied in crayfish hematopoietic tissue cell cultures for gene functional studies. Further, the interactions between host (crayfish) and pathogens (white spot syndrome virus and Aeromonas hydrophila, respectively) were studied using RNAi technology in live animals. An antilipopolysaccharide factor isolated from viral challenged crayfish by suppression subtractive hybridization was shown to interfere with the propagation of white spot syndrome virus both in vivo and in vitro in crayfish, suggesting an important role of this factor in antiviral defense. Besides, RNAi of phenoloxidase, a critical immune effector involved in melanization, revealed that phenoloxidase activity is necessary for crayfish immune defense against a highly pathogenic bacterial infection in crayfish. In addition, RNAi was also employed to study a marker protein gene involved in hemocyte maturation in crayfish. Taken together, these studies may provide more insights into the immune responses against pathogen invasion as well as hemocyte ontogenesis in crustaceans.

Melanocortins (MCs) play an important role in feeding, metabolism, and energy expenditure. While melanocortin receptor (MCR) mRNA has been found in the mesolimbic dopamine (DA) pathway, the ability of melanocortins to regulate feeding and other behaviors through actions on the mesolimbic DA system have not been examined. Short-hairpin RNAs (shRNAs) were created targeting MC3R and MC4R and were tested via in vitro studies for their ability to knockdown their target receptor. A total of three shRNAs were created targeting each receptor, and each shRNA caused successful knockdown. These shRNAs are tools that can be used for future in vivo studies to examine the various behavioral effects of melanocortins on the mesolimbic DA pathway.

Variability of transgene expression levels resulting from gene silencing is considered as ahindrance to the successful application of plant genetic engineering. Towards alleviatinggene silencing, I decided to screen for novel genes involved in transgene silencing and toinvestigate how these genes regulate plant development. Genes encoding putative chromatinremodeling factors, especially those including a SET domain, were selected as candidatetargets. A bioinformatic analysis of the Arabidopsis SET genes (AtSET) was performed andthese genes were classified into 6 groups based on the domain architecture. RNA interference (RNAi) vectors were constructed for ~ 20 AtSET genes and wereintroduced into both wild type lines and transgenic lines silenced for a GFP reporter gene.Surprisingly, altered developmental phenotypes were only observed for three constructs,raising questions as to the effectiveness of the RNAi approach for the chosen Arabidopsissystem. To assess this situation, I targeted a phytoene desaturase (PDS) gene using the sameRNAi approach. Inactivation of PDS renders plant a readily identifiable phenotype. Whereasthe RNAi penetrance in Arabidopsis can be very high, the expressivity of RNAi in varioustissues and among different plants can vary dramatically. Contradictory to previous reports,I found that there is correlation between transcript level and silencing phenotype. Possiblereasons for this discrepancy are discussed. No apparent correlation between transgene copynumber and RNAi phenotypes was observed. Among the three RNAi constructs that caused an abnormal development inArabidopsis, K-23 which targets SuvR3 has the highest expressivity and could reactivate asilenced GFP locus. SuvR3 RNAi lines were selfed for six generations and were screenedfor morphological phenotypes. Abnormal number of flower organs, loss of viability of malegametophytes, and decreased seedling germination percentage were found in SuvR3 RNAilines. A progressive increase in both severity and frequency of abnormal phenotypes wereseen in subsequent generations, suggesting an epigenetic regulatory mechanism involvedwith SuvR3. Alternative splicing of SuvR3 was also observed in most of Arabidopsis tissues.One of the protein isoforms, SuvR3, lacks 16 amino acids within the highly conserved SETdomain. Possible effects of isoform interaction are proposed.