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The mammalian genome is under constant threat from invasion by mobile genetic elements including transposons and viruses. To defend the genome, cells recognize incoming DNA and limit its transcription through repressive chromatin modifications. The human silencing hub (HUSH) complex transcriptionally represses long interspersed element-1 retrotransposons (L1s) and retroviruses through histone H3 Lys9 trimethylation (H3K9me3). How HUSH recognizes and initiates silencing of these invading genetic elements is unknown. By monitoring transcription from L1 transgenes, I found that incoming L1s are recognized by HUSH independently of their integration sites, a critical difference with previously studied viral silencing. By studying sequence determinants of L1 repression by HUSH, I discovered that HUSH is able to recognize and transcriptionally repress a broad range of sequence-diverse, intronless invading DNAs, despite no prior exposure to such DNAs. Sequence length and high adenine (A) content in the sense strand are important additional determinants of susceptibility to HUSH-targeting. My further work demonstrated that Periphilin binds transcripts from the target locus, prior to and independent of, H3K9me3 deposition, which explains why target transcription is essential for both initiation and propagation of HUSH-mediated H3K9me3. Whilst diverse intronless transgenes are susceptible to HUSH-repression, I found that the presence of an intron counteracts repression, even in the absence of intron splicing. A subset of endogenous intronless loci are silenced by HUSH, and Periphilin specifically binds transcripts from endogenous intronless loci. Therefore, intronless DNA, the product of reverse transcription, provides a versatile means to distinguish invading RNA-derived retroelements from intron-containing host genes and provides a mechanism by which HUSH protects the genome from 'non-self' DNA. I propose that HUSH is a component of the innate immune system and intronless DNA the molecular pattern recognized by HUSH. By silencing cDNA, the product of reverse transcription, HUSH controls the reverse flow of genetic information (i.e. from RNA to DNA) in the human genome.

X chromosome inactivation (XCI) is a process by which one X chromosome in female mammals is silenced to equalise the dosage of X-linked gene expression between XX females and XY males. XCI is initiated by a long non-coding RNA (lncRNA), Xist, expressed from the future inactive X chromosome (Xi) during embryonic development. Xist spreads in cis to coat the chromosome and recruits molecular pathways which modify the underlying chromatin from an active to a repressive state, leading to complete transcriptional silencing of almost all genes on Xi. XCI is an important paradigm for lncRNA-directed gene repression and its study can inform our understanding of mechanisms of chromatin regulation more widely. This thesis presents an experimental characterisation of iXist-ChrX, a cellular model that recapitulates the establishment of XCI during early mouse development. I perform quantitative, high-resolution and allele-specific genomic analyses of Xist-mediated changes to chromatin over time courses of Xist induction, providing novel insights into the cis-regulatory features that influence variable silencing dynamics on a gene-by-gene basis. A key finding from these analyses is that slow-silencing genes and genes which escape complete inactivation are marked by binding motifs for the transcription factor YY1. I also document a pilot scRNA-seq experiment to address questions of cellular heterogeneity in Xist-mediated gene silencing and lay the foundations for future investigations at single-cell resolution. In further experiments, I use CRISPR-Cas9 genome editing to interrogate two key molecular pathways acting downstream of Xist. By disrupting SPEN and Polycomb pathways individually and in combination in iXist-ChrX cells, I dissect the relative contributions of each pathway and demonstrate that they act in parallel, through distinct mechanisms of chromatin modification, and additively to silence X-linked genes. These experiments also reveal that both SPEN and PCGF3/5-PRC1 have secondary roles in ensuring correct localisation of Xist RNA over Xi, and highlight an interplay with cellular differentiation important for the complete establishment of silencing during the later stages of XCI.

Saprolegnia parasitica and Aphanomyces invadans are oomycetes that infect fish. S. parasitica is responsible for devastating infections in fresh water fish and in particular salmonids. It is estimated that at least 10% of all hatched salmon die due to Saprolegniosis in fish farms, which represents significant financial losses and is a serious animal welfare concern. A. invadans causes Epizootic Ulcerative Syndrome (EUS) in fish living in fresh and brackish water mainly and is mostly found in Asian aquaculture. With the completion of the genome sequence for S. parasitica and A. invadans, the interest from sequencing has shifted to the understanding of gene function and regulation. In order to functionally characterize genes from these organisms an efficient mutagenesis or silencing method needs to be in place. However, within the group of oomycetes, gene transformation technology has been developed but its efficiency is, at present, limited to a restricted number of oomycete species. For that reason, genome editing systems for fish pathogenic oomycetes involving stable and transient silencing were attempted in this study. In Chapter 2 we describe an attempt for a stable transformation protocol using cyanamide as a positive selection marker. Four main transformation protocols were tried: PEG-mediated protoplast, heat shock, electroporation and Turbofectin. Electroporation was selected for further optimization. The optimum conditions chosen were: L15 as electroporation buffer, 0.2mm cuvette gap and multiple pulses at 200V with 20s intervals with incubation temperature at 18°C. Transformations were also conducted using protoplasts and zoospores, however, although a few protoplasts survived following cyanamide selection, the cyanamide gene was not detected in their genome which suggested that only false positives were obtained. A different approach of stable transformation was attempted with GFP as a selection marker in Chapter 3, together with the development of CRISPR/Cas9 genome editing system for fish pathogenic oomycetes. In order to achieve this, sgRNAs were designed to target selected genes. Four target genes HlyE (sprg_03140), CotH (sprg_04414), SpHtp1 (sprg_04985) and SpHtp3 (sprg_03573) were selected initially and cloned into pYF2.3G-ribo-sg RNA. Also, the Cas9 vector that was originally designed for Phytophthora sojae, a plant pathogenic oomycete, was optimized in terms of the nuclear localization signal (NLS) and the resistance marker. The nuclear localization signal is important for the import of Cas9 enzyme into the cell nucleus where it modifies the target gene, while the resistance marker enables the selection of antibiotic-resistant transformants of S. parasitica. Following transformation, it was revealed that Cas9 was expressed in the S. parasitica protoplast and was detected in the DNA of transformants. However, it also seems that the Cas9 enzyme is degraded in Saprolegnia. In Chapter 4 and Chapter 5, transient silencing using RNA interference (RNAi) was conducted but using different species of fish pathogenic oomycetes, S. parasitica and A. invadans, respectively. In Chapter 4, a hemolysin gene of S. parasitica SpHlyE1 (SPRG_03140) was functionally investigated by RNAi silencing and a subsequent infection assay with Galleria mellonella was performed. The dsRNA-treated group showed reduced levels of mRNA of hemolysin compare to the untreated group. The hemolysin-silenced lines showed a delayed and decreased mortality rate in the infection assay with G. mellonella. Given the simple applicability of RNAi-silencing combined with the infection assay, this is a relatively fast strategy for future functional characterisation of genes in S. parasitica. In Chapter 5, a silencing experiment using RNA interference was conducted to reduce the mRNA level of a target gene, AiLhs1 followed by an infection assay with Galleria mellonella. The silencing resulted in the phenotypic impairment of sporulation. The dsRNA-treated group showed reduced levels of mRNA and high survival rate in the infection assay with G. mellonella. Phenotypic observations in AiLhs1-RNAi silenced lines revealed reduced size of spores in the hyphae, fewer spores in clusters and lower number of swimming spores in the medium with slightly delayed sporulation. Our results suggest that AiLhs1 is required for successful sporulation which is critical for host colonising. Based on the studies that have been conducted for this thesis, a better understanding of two genes predicted to be involved in virulence was achieved. However, a protocol for stable transformation was not achieved and this is urgently needed to functionally characterize genes or to express other genes, such as reporter genes, in these animal pathogenic oomycetes. The present work has provided important preliminary data that will help to generate a stable transformation method for animal pathogenic oomycetes and possibly a gene editing system.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are incurable diseases. For OA there are no treatments aimed at halting disease progression or promoting a regenerative response while in RA, despite the availability of drugs targeting the immune system, joint damage is not reversed. Thus, the targeting of other cells involved in the progression of these diseases is pursued. Fibroblast-like synoviocytes/mesenchymal stromal/stem cells (FLS/MSCs) have been demonstrated to actively contribute to synovial hyperplasia and produce degradative enzymes in OA, and to acquire an aggressive phenotype characterized by invasion and degradation of surrounding joint structures in RA. Thus, the identification of cellular pathways mediating FLS/MSC pathological behaviours might open novel therapeutic perspectives. Yap is a transcriptional co-factor which was demonstrated to be a positive regulator of proliferation and negative regulator of chondrogenesis in MSCs. Conditional ablation of Yap in synovial MSCs prevented synovial hyperplasia following cartilage injury. Here, Yap ablation was demonstrated to reduce proliferation and invasiveness of FLS/MSCs in vitro. Therefore, I aimed to design nanoparticle-based (DsiRNA/chitosan) and nanoparticle-free (gapmers) anti-Yap RNAi approaches. These were extensively validated in vitro and tested in mouse models of RA and OA. Despite the observation of DsiRNA/chitosan nanoparticle accumulation and Yap silencing in synovium, particles led to triggering of synovial hyperplasia in untreated legs and its aggravation in adjuvant-induce arthritis (AIA). Intra-articular injection of naked gapmers in mice with joint surface injury (JSI) resulted in no effects on synovial hyperplasia and proteoglycan loss. To overcome these issues, in vitro work has been started for the optimisation of novel gapmer delivery strategies based on MSC-derived nanoparticles and CaCO3 microparticles, which favour suppression of the immune system or sustained release of gapmers, respectively. Overall, we provided further insights into the role of Yap in controlling FLS/MSC behaviour and developed novel RNAi approaches.

Barley powdery mildew (Blumeria graminis f.sp. hordei) is a prevalent fungal pathogen of barley in the UK. As an obligate pathogen, methods for gene knockouts or silencing have been limited, with no stable transformation method available. A transient assay using a biolisitic delivery system has been previously applied for Host Induced Gene Silencing (HIGS) of Blumeria effector candidates (BECs). However, HIGS requires cloning of a silencing cassette and since only a small proportion of cells are transformed, it forbids downstream whole-tissue analyses. The aim of the project was to overcome these caveats by developing a new silencing strategy of genes from barley and its powdery mildew in order to investigate their roles during infection. The method required the delivery of phosphorothioate-modified, short, antisense oligonucleotides (PTOs) via the vascular tissue of excised leaves. The method was validated by silencing the known Blumeria effectors BEC1011, BEC1019 and BEC1054, and the barley susceptibility factors Mlo and Blufensin1. While the disease phenotype was monitored microscopically by measuring the infection rate in term of successful secondary hyphae formation, relative mRNA and protein amounts of the cognate silenced gene were measured by qRT-PCR and multiple reaction monitoring mass spectrometry. Targeting the aforementioned effectors or susceptibility genes resulted in 40-60% reduction of secondary hyphae formation and up to 30% reduction in transcript. The technology was then applied to silence putative Blumeria Candidate Secreted Effector Proteins (CSEPs) AVRa1 and AVRa13, resulting in 22% and 31% reduction in secondary hyphae formation, respectively. A barley pathogenesis related protein 5 (PR5) was also targeted. Surprisingly, PR5 silencing led to a drastic decrease in Blumeria infection, suggesting that PR5 is a susceptibility factor. To investigate BEC1011 or PR5 roles in compromising plant immune responses, production of reactive oxygen species such as H2O2 was monitored after Blumeria infection in control, BEC1011, and PR5 silenced plants, showing that BEC1011 and PR5 are preventing a sustained oxidative burst response.

DNA methylation is essential for mammalian development and transcriptional repression of genes and retrotransposons during embryo development and in somatic cells. The patterns of DNA methylation are established by de novo DNA methyltransferases, which are regulated by developmental signalling and require access to chromatin. Besides DNA methyltransferases, other proteins have recently been implicated in DNA methylation, such as the ATP-dependent chromatin remodeler LSH. The absence of LSH in mouse embryos leads to defects in DNA methylation and development. In relation to this, mutations in LSH have been found to cause Immunodeficiency-Centromeric instability-Facial anomalies (ICF) syndrome. This syndrome is characterized by centromeric instability and CpG hypomethylation of centromeric satellite repeats, and is most often caused by mutations in the catalytic domain of the DNA methyltransferase DNMT3B. LSH is essential for developmentally programmed de novo DNA methylation of large chromosomal domains including promoters of protein coding genes and repetitive sequences. Importantly, fibroblasts derived from chromatin remodeling ATPase LSH-null mouse embryos, which lack DNA methylation at transposons and specific gene promoters, are capable of re-establishing normal patterns of DNA methylation and transcriptional silencing of misregulated genes upon re-expression of LSH. The ATP hydrolysis by LSH is essential for its function in gene silencing and de novo DNA methylation. However, the molecular mechanisms of LSH-dependent gene silencing and de novo DNA methylation are yet unclear. Here we use an inducible system that enables controlled expression of LSH in Lsh-null mouse embryonic fibroblasts (MEFs) to follow chromatin dynamics, transcriptional silencing and establishment of de novo DNA methylation. This conditionally reversible Lsh knockout cellular system allowed us to study the order of events occurring immediately after LSH restoration in MEF cell lines in order to elucidate the molecular mechanism of LSH-dependent gene silencing. We have demonstrated that LSH upon its restoration localises to the promoters of LSH-dependent loci leading to a mild decrease in the occupancy of H3, which reinforces the previously shown role of LSH as a chromatin remodeler. Simultaneously, there is removal of acetyl groups from H3 tails when LSH is bound to these target regions, which might be facilitated by the interaction of HDACs with LSH. The removal of H3Ac marks is followed by deposition of H3K9me2 by G9a/GLP histone methylases at the same time point when misregulated genes are silenced. This suggests that LSH creates a suitable substrate for G9a/GLP promoting gene silencing. Surprisingly, transcriptional repression occurs without acquisition of DNA methylation at the promoters of these loci. This order of events implies that LSH plays a role as a chromatin remodeler leading to changes in chromatin structure and modifications that facilitate epigenetic gene silencing without DNA methylation in the initial period when LSH is restored in MEF cell lines. Furthermore, deposition of H3K9me2 by the G9a/GLP complex is critical for silencing of specific genes, but not for repetitive elements such as IAPs. The histone modification H3K27me3 seems to play a transitory role in the silencing of IAP retrotransposons in the absence of G9a/GLP activity. In conclusion, this work has demonstrated that changes in chromatin modifications leading to a transcriptionally repressive chromatin state can be established in somatic cells by the chromatin remodeler LSH without acquisition of DNA methylation. This suggests that the primary role of LSH is to promote changes in chromatin structure and modifications that lead to gene silencing and not DNA methylation, which most likely occurs as a consequence of transcriptional silencing.

The rapid postharvest deterioration of the roots, known as postharvest physiological deterioration (PPD), has been a major problem to the utilisation and development of cassava (Manihot esculenta Crantz) as a food and industrial crop. PPD usually occurs within two to three days after harvest and it is characterised by a blue-black discoloration of the roots, which renders the roots unpalatable and unmarketable. Scopoletin, which is synthesised de novo and released from its glucoside, scopolin, during PPD, plays a central role in this discoloration response. Interconversion of scopoletin and scopolin, which is catalysed by scopoletin-glucosyltransferase (scopoletin-GT) and scopolin-beta-glucosidase (scopolin-BG), regulates homeostasis of scopoletin in the cells. However, how this interconversion contributes to root discoloration development is poorly understood. In the present study, we identified and characterised cassava genes for the enzymes that are responsible for the interconversion of scopoletin and scopolin, subsequently manipulated their expression in transgenic cassava through scopoletin-GT RNAi gene silencing and scopolin-BG overexpression constructs. These approaches would potentially alter scopoletin and scopolin content in the root, thereby affecting PPD response. A BLAST search for homologous cassava genes revealed that scopoletin-GT and scopolin-BG are encoded by multiple genes, most of which belong to glucosyltransferase family-1 (GT1) and glycosyl hydrolase family-1 (GH1), respectively. Scopoletin-GT-down-regulated and scopolin-BG overexpressed transgenic cassava lines showed reduced not only scopolin but surprisingly also scopoletin, and delayed PPD. Additionally, other coumarins esculetin and esculin were also identified and both scopoletin-GT, MeSGT1, and scopolin-BG, BGLU23, were up-regulated during PPD development at day 4 and day 2, respectively. Our study reveals that disrupting the interconversion of scopoletin and scopolin by inhibiting scopoletin-GT and overexpressing scopolin-BG led to the decrease of both scopoletin and scopolin content and delayed PPD in cassava. These findings provide useful insights into the role of interconversion of scopoletin and scopolin in cassava PPD response and may suggest alternative ways to tackle PPD.

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.

Improving the repopulation potential of human umbilical cord blood (UCB) haemopoietic stem cells (HSCs) remains a paramount goal in HSC transplantation (HSCT) therapy. This implies enhancing the homing and engraftment potential of UCB-CD34+CD133+ cells to the bone marrow (BM). Although an array of molecules continues to be identified as ‘key’ homing molecules, the molecular mechanisms controlling HSC homing are still not fully understood. The regulatory implications of hypoxia in the BM, with the concomitant stabilisation of hypoxia inducible transcription factor-1α (HIF-1α), are becoming more apparent, yet at the commencement of this thesis no study had explored whether hypoxia induced signalling can be adopted to regulate the homing and engraftment of transplanted HSCs. The aim of this DPhil project was thus to investigate whether hypoxic conditions as detected in the BM influence the adhesion of UBC-CD133+ cells to osteoblasts, BM stromal cells and BM endothelial cells-60 (BMEC-60), as well as their transmigration towards chemokine SDF-1α across BMEC-60. Increasing the exposure of UCB-CD133+ cells to 1.5% O2 doubled the percentage of transmigrating cells (p<0.05), and while hypoxia stimulated UCB-CD133+ cells preferentially adhered to IL-1β stimulated BMEC-60, their adhesion to non-stimulated (BMEC-60) was significantly improved (p<0.001). To help unravel the underlying molecular mechanisms, we attempted to examine the potential involvement of hypoxia regulated scaffolding protein HEF-1/NEDD9/Cas-L (HEF-1) in the increased percentage of migrating UCB-CD133+ cells after hypoxia pre-conditioning. The role of HEF-1 in HSCs is unexplored, and its multifunctional contribution in a variety of processes including cell migration, attachment and invasion make HEF-1 a prime candidate as a contributing homing molecule. After identifying a suitable short-hairpin RNA (shRNA) sequence to knockdown HEF-1, generating lentiviral (LV)-particles in house and optimising transduction protocols, HEF-1 knockdown was achieved in haemopoietic model cell lines KG-1 and KG-1A (KG-1/KG-1A–HEF1). Significantly decreased KG-1A–HEF1 cell adhesion to non-stimulated BMEC-60 was detected. Together, these studies provide a promising platform to further explore the role of HEF-1 in hypoxia induced UCB-CD133+ cell transmigration towards the key homing molecule SDF-1α.

X-chromosome inactivation achieves dosage compensation of X-linked genes between male (XY) and female (XX) mammals. This process involves the down-regulation of most, but not all genes on one of the two X-chromosomes in the nucleus of each female somatic cell. The mechanism of X-inactivation has yet to be elucidated in full, but is known to involve the noncoding transcript of theXist gene, DNA methylation, histone hypo-acetylation and the condensation of higher order chromatin. Recent studies have established mechanisms linking methylation to repressive chromatin structures through methyl-binding proteins and histone deacetylase complexes. In order to better understand the role of methylation in X-inactivation, the promoters of the human Pyruvate dehydrogenase El a (PDHA1) and the human and murine Norrie disease protein (NDP/Ndp) genes were subjected to direct methylation sequencing, allowing the definition of methylation profiles at nucleotide resolution. The promoter of the PDHA1 gene was found to be hyper-methylated on the inactive X-chromosome and hypo-methylated on the active X-chromosome in agreement with studies at the promoters of other X-linked housekeeping genes. Methylation at the promoters of the NDP/Ndp genes was extensively investigated in a range of primary tissues and cell lines. The Ndp promoter was found to be methylated on both active and inactive X-chromosomes, but hypo-methylated in the proximal promoter exclusively in tissues that expressed the Ndp gene. The NDP promoter was found to be unmethylated on the active X-chromosome and hyper-methylated across the proximal promoter on the inactive X-chromosome in expressing cell lines and human retinal tissues. The novel promoter sequences of the human and murine SMCX/Smcx genes were isolated for comparative analysis and to provide a future resource for studying methylation at the promoters of genes which escape the X-inactivation process. Promoter sequences of the PDHA1, NDPI Ndp and SMCX/Smcx genes were screened for putative transcription factor binding sites and for conserved CpG-dinucleotide content. Promoter-reporter gene constructs for these genes were transfected into mammalian cells establishing that the sequences studied were functional promoters. Artificial methylation of these constructs was shown to repress their promoter activities.

DNA methylation has been studied for decades and is necessary to maintain cellular homeostasis from humans to plants. However, there remains a dearth of information regarding the function of proteins that bind DNA methylation. It remains unclear if plant DNA methylation binding proteins bind DNA methylation, which methylation sequence context (CG, CHG, or CHH) they prefer, or how these proteins interpret DNA methylation to regulate gene or transposable element (TE) expression. Therefore, we studied the methyl-CpG-binding domain (MBD) protein family in Arabidopsis thaliana to expand on the mechanisms of DNA methylation-mediated gene regulation. We discovered that Arabidopsis MBD proteins MBD5 and MBD6 redundantly regulate the expression of promoter-methylated genes and TEs by recruiting chaperone proteins ACD15, ACD21, and SLN. We further discovered that the MBD5/6 complex co-opts the oligomerization capacity of small heat shock proteins (sHSPs), ACD15 and ACD21, to accumulate the MBD5/6 complex at sites of high-density methylation creating nuclear foci which are further regulated by SLN. Although chaperone proteins often interact with bound complexes, the surprising specificity with which ACD15, ACD21, and SLN directly regulate the MBD5/6 complex is novel and unprecedented for DNA methylation binding proteins. Furthermore, proper accumulation is necessary for gene regulation by the MBD5/6 complex suggesting protein accumulation at DNA methylation sites is an integral step in DNA methylation-mediated gene regulation in plants. Our results suggest this chaperone-mediated accumulation is not specific to Arabidopsis proteins as protein accumulation was caused by sHSPs from other organisms including humans, archaea, bacteria, protists, and other plant species. Using a chimeric protein approach, we show that MBD6 foci can be replicated using these sHSPs, in an ACD15/ACD21 independent manner, leading to subtle gene silencing. We further demonstrate chaperone-mediated accumulation using these sHSPs by targeting specific genomic loci using the SunTag, dCa9 targeting system. These results support the role of protein accumulation in DNA methylation pathways while providing novel mechanisms for protein localization in chromatin environments. Evidence is growing that efficient protein accumulation and localization are important for the function of chromatin complexes. The discovery of phase separation events and membrane-less compartments has led to a reevaluation of how protein dynamics can impact chromatin function through novel and potentially overlooked mechanisms. Our work has added to this evidence by demonstrating that plants utilize chaperone proteins to efficiently and specifically accumulate DNA methylation-binding proteins, forming an organized and functional accumulation.

High-risk neuroblastoma is an aggressive childhood cancer, for which 5-year survival remains less than 50%. Current therapies are non-specific and can lead to acute and long-term side effects. Improved therapies that directly target tumour associated genes are required. Polo-like kinase 1 (PLK1) regulates cell cycle progression, and its over expression in tumours encourages growth. Gene silencing using short interfering RNA (siRNA), is a selective method to target PLK1, with reduced off-target effects. However, efficient delivery of siRNA to tumours requires improved delivery vehicles. Whilst lipid nanoparticles (LNPs) are the most clinically advanced RNA delivery vehicle, clinical siRNA-LNPs exhibit rapid accumulation in liver tissue. I hypothesised that changes in LNP formulation, and active targeting of LNPs to antigens such as epidermal growth factor receptor (EGFR) and disialoganglioside GD2 would improve siRNA delivery to neuroblastoma. siRNA-LNPs were formulated with PEG-lipids that have different detachment kinetics from the LNP surface; DMG-PEG (fast detachment), DSG-PEG and DSPE-PEG (slow detachment). Bispecific antibodies (BsAbs) that recognise either EGFR or GD2 and PEG were used to create targeted LNPs. EGFR targeting improved uptake of siRNA-LNPs in high EGFR-expressing SH-SY5Y neuroblastoma cells in vitro. Similarly, GD2 targeting improved uptake of siRNA-LNPs in GD2-expressing CHP-134 cells. BsAb targeting of LNPs containing PLK1 siRNA (siPLK1-LNPs) resulted in improved PLK1 silencing efficiency compared to untargeted siPLK1-LNPs. Additionally, GD2 targeting reduced the IC50 of siPLK1-LNPs up to 13-fold in CHP-134 cells. Next, the biodistribution of siPLK1-LNPS with and without BsAbs was compared in a neuroblastoma xenograft model. We found that DSG-PEG or DSPE-PEG had higher tumour accumulation compared to DMG-PEG siPLK1-LNPs. However, the presence of BsAb did not significantly impact tumour accumulation. Finally, efficacy studies with untargeted and GD2-targeted siPLK1-LNPs demonstrated that siPLK1 has anti-tumour effects in vivo, although further work is required to optimise therapeutic delivery. In conclusion, this study demonstrates the potential of BsAb targeting and increased PEG-lipid stabilities in improving cell specific uptake and PLK1 gene silencing in neuroblastoma.

Compaction of the eukaryotic genome into regions of open euchromatin and dense heterochromatin serves as a fundamental regulator of gene expression in cells. In the budding yeast Saccharomyces cerevisiae, the Silent information regulator (Sir) protein complex regulates a heterochromatin-like structure at the silent mating type loci, HML and HMR, and at the telomeres. In addition to the Sir proteins, Repressor activator protein 1 (Rap1) functions in establishing and maintaining silent chromatin at HML and HMR by binding to nucleation sites known as silencers (silent enhancers) and recruiting silencing machinery. As its name suggests, Rap1 is simultaneously an essential transcription factor that activates hundreds of genes across the genome, including many ribosomal protein genes and the MATα1 and α2 genes whose expression determine the mating-type of α cells. Sequence identity between the mating-type locus MAT and the auxiliary HML allows a unique opportunity to study the role of Rap1 in both silent and expressed contexts, and how local chromatin state affects function. Chapter two focuses on discerning the context-dependent functions of Rap1. Using ChIP-seq I established that Rap1 accessed its binding site at the promoter of HML, even in the silent context. My findings supported and extended the view that pre-initiation complex machinery is unable to act in silent chromatin, thus narrowing the mechanism of silencing to a step between recruitment of the native activator, Rap1, and occlusion of the pre-initiation complex. Surprisingly, Rap1 enrichment at its three binding sites across the silent locus was enhanced by the presence of Sir proteins, despite binding of this transcription factor preceding Sir protein recruitment. As Rap1 was bound in the silent locus but was not functionally recruiting transcription machinery, I tested whether the presence of this enigmatic protein could instead be enhancing silencing. Utilizing a highly sensitive assay that monitors loss-of-silencing events, I established a novel and specific contribution of promoter-bound Rap1, which was previously seen as having potential only for activation, to silencing. Furthermore, I investigated the mechanism by which Rap1 promotes transcription when acting as an activator and have evidence that it may be aiding in the transition from transcription initiation to elongation. ChIP offers a static view of protein-DNA interactions rather than a dynamic readout. To assess whether in vivo dwell time of Rap1 might contribute to its function as an activator or repressor, I coupled a nuclear depletion strategy with a ChIP-seq time course to measure enrichment at and decay of Rap1 from its binding sites genome wide. The apparent Rap1 dwell time did not differ between silenced HML and expressed MAT. However, genome-wide Rap1 residence time correlated with transcriptional output and nucleosome positioning. These results point toward a model in which the duality of Rap1 function is mediated by local chromatin environment rather than binding-site availability. I have conducted a number of experiments attempting to pinpoint the mechanism by which Rap1 switches function, following up mainly on post-translational modifications. Some of this work is described in the appendix. While study of Sir silencing at the HM loci has been integral in understanding the establishment and maintenance of heterochromatin in eukaryotes, the effects of these same proteins at telomeres has been less clear. Chapter three focuses on an incomplete set of experiments designed to better understand the functional similarities and differences in Sir-mediated silencing at telomeres and the HM loci. Early studies of telomere position effect in yeast led to a hypothesis that distance from telomere dictated the amount of gene repression imparted by Sir proteins in a gradated manner. At the time I began working on the project, there was mounting evidence against this hypothesis. I designed and performed preliminary experiments testing whether silencing was a function of distance from telomere utilizing fluorescent reporters for high throughput analysis. Furthermore, I investigated whether telomere length affected silencing of HML and HMR by titrating available Sir proteins away from these loci. I also pioneered usage of a novel form of single molecule RNA FISH in our lab and I planned to use this technique to assess whether subtelomeric genes, which are enriched for metabolic function, were spatially co-regulated in response to environmental stimuli.

Post-transcriptional gene silencing (PTGS), also termed as RNA interference (RNAi) possess a great therapeutic potential. Three siRNA-based products have been recently approved that have developed a renewed interest in the scientific community to explore this alternative therapeutic area rigorously. However, several challenges are limiting their use as a widespread therapeutic agent. The limitations include their membrane impermeability, short half-life, elimination by the kidney, entrapment by the reticuloendothelial system, sequestration by plasma proteins, endosomal trap, and off-target effects. Several delivery systems and transfection agents have been investigated to address these challenges. Still, most of the systems suffer from toxicity and low efficiency. Hence, there is a critical need for developing non-toxic and more efficient siRNA delivery tools. For the last two decades, it has been found that cell-penetrating peptide (CPP)-based delivery systems show enhanced uptake of siRNA. This dissertation aimed to investigate fatty acyl cell-penetrating peptides (CPPs) as siRNA delivery tools. We hypothesized that conjugation of hydrophobic molecules to the (HR)n peptide will enhance hydrophobicity, modulate cytotoxicity, and provide an efficient delivery system for siRNA. Therefore, we synthesized peptide conjugates with the sequence X-(R)n-(HR)4, and X-RnH4 (where X was a flexible hydrophobic chain (oleic acid) or a conformationally constrained steroidal hydrophobic molecule (cholesterol) and n = 0, 3, 4 or 5). Varying the arginine residues at the N-terminal helped us explore the structure-activity relationship of the peptide conjugate. Also, we synthesized another series of peptides with the sequence X-(HR)4-CGKRK, where X was either XI cholesterol, oleic acid or polyethylene glycol (PEG). Among the studied peptides, Olyel-R4-(HR)4, Oleyl-R5-(HR)4, Oleyl-K-(HR)4-CGKRK, and Cholesteryl-K-(HR)4-CGKRK demonstrated convincing data for siRNA delivery in MDA-MB-231 cells. However, X-RnH4 peptides were cytotoxic to all the studied cell lines, such as MDA-MB-231, MCF-7 and MCF-10A, even at the 5 μM range. We tested the cellular uptake of some of the conjugates for siRNA uptake, but these did not show significant uptake, so we stopped pursuing this series of peptides for further studies. Oleyl conjugated histidine-arginine peptides with variable arginine residues proved to be non-cytotoxic at N/P 40 ( ~20 μM) in MCF-7, MCF-10A, and MDA-MB231 cells. Oleyl conjugates formed nanocomplexes with siRNA, possibly due to the presence of fatty acyl chain. Also, an increasing number of arginine residues in oleyl conjugated (HR)4 increased cellular uptake with subsequent silencing efficiency of the siRNA. Visual confocal images further strengthened the quantitative cellular internalization data obtained from flow cytometry. Oleyl-R5-(HR)4 resulted in ~80% inhibition of STAT-3 protein comparable to the commercially available transfection agent Lipofectamine 2000. Furthermore, X-(HR)4-CGKRK conjugates were successfully synthesized and purified that formed nanocomplexes with siRNA with ~ diameter of/P ≥40. Cholesteryl-(HR)4-CGKRK caused even more silencing of STAT-3 in MDA-MB-231 (~ 89%) than oleyl-(HR)4-CGKRK (~82%) and lipofectamine (82%). Pegylated derivative of (HR)4-CGKRK caused ~60% inhibition of STAT-3. Further in-vivo exploration of cholesteryl and oleyl-CPPs may provide promising results in siRNA delivery.

Dendroctonus bark beetles are among the most economically and ecologically significant forest pests in North America and play a critical role in the overall health of conifer forest ecosystems. Dendroctonus bark beetles influence ecosystem benefits and biodiversity and drive forest succession, and adversely affect timber production, forest management, and recreation. As temperatures surge and climatic fluctuations become more extreme, catastrophic bark beetle outbreaks are increasing in frequency, escalating pressures on highly vulnerable conifer forests already compromised by heat and drought. Eruptive outbreaks of Dendroctonus beetles are largely unhindered by traditional silvicultural management; these practices further disrupt forest ecosystem services, including the capacity to sequester carbon, thereby compromising their ability to temper our changing climate. The limitations of current management approaches for Dendroctonus beetles makes them prime targets for innovative and aggressive management strategies that are less taxing ecologically. RNA interference is one such innovative approach touted as a next generation pest control tactic. RNA interference (RNAi) is a naturally occurring eukaryotic immune response that can be artificially manipulated through the introduction of carefully designed double stranded RNAs (dsRNAs) to silence gene function. Gene silencing via RNAi is a powerful tool used to elucidate gene function and expose inter-individual epigenetic differences and can also be applied in pest management as a biopesticide. I investigated the use of RNAi technology in Dendroctonus bark beetles. Chapter one offers an overview of Dendroctonus systems. In Chapters Two and Three I demonstrate the efficacy of RNAi to silence gene function in southern and mountain pine beetles, Dendroctonus frontalis and D. ponderosae, respectively. I show that triggering the RNAi pathway in each species halts the production of critical proteins and results in significant mortality in a species-specific manner. I then investigated the specificity of RNAi to target in Chapter Four and found that the technology has congeneric effects. In Chapter Five I demonstrate a marked sensitivity to epigenetic changes spurred by differences in climate and host in southern pine beetle. I then investigated potential deployment methods of RNAi technology for forest-wide southern pine beetle suppression, beginning with the transformation of southern pine beetle associated fungi to act as a delivery mechanism for RNAi inducing dsRNAs in Chapter six, followed by the identification and validation of olfactory receptor genes and the specific pheromones to which they correspond in Chapter Seven. Chapter Eight highlights that, collectively, my work offers new insights into Dendroctonus bark beetle biology and is a foundational step to adapting RNAi technology to broadscale forest protection.

The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is a highly invasive phloem-feeding insect native to Asia. It has killed millions of ash trees (Fraxinus spp.) since its accidental introduction into North America, causing profound economic and ecological impacts. RNA interference (RNAi) or double-stranded RNA (dsRNA)-mediated gene silencing is an emerging biotechnology that is developing as an innovative tool for pest management. Exogenous dsRNA triggers the RNAi pathway, silences genes, and disrupts protein function, causing insect mortality. RNAi has proven effective in reducing target gene expression and causing mortality in EAB; however, two aspects stand as barriers to the deployment of RNAi as a viable pest management approach: its specificity and practical delivery. I based my work in Chapter 2 on the Environmental Protection Agency risk assessment framework for genetically engineered crops. I designed feeding bioassays followed by gene expression studies to evaluate the spectrum of activity of dsRNAs targeting the EAB genes hsp, shi and sn-rnp in model insects representing five feeding guilds including herbivore, predator, detritivore, pollinator, and parasitoid; the last represented by the classical biological control agents currently deployed for EAB management in North America. Bioassays demonstrated no lethal effects on the model insects, suggesting a narrow spectrum of activity for the three EAB-specific dsRNAs evaluated. The gene expression analyses suggest potential sublethal effects on the model pollinator; however, I found no effects on insect survival. In Chapter 3, I evaluated dsRNA delivery using fluorescently tagged dsRNA and confocal microscopy to provide a proof of concept of uptake, movement, and bioactivity of dsRNA in green, F. pennsylvanica, and tropical ash, F. uhdei, through root and/or petiole absorption. Chapter 4 describes my greenhouse study exposing 2-year old green ash seedlings to dsRNAs as a foliar spray; I demonstrate in planta spread and persistence of topically applied dsRNA up to 21 days with an accompanying reduction in EAB target gene expression and reduced EAB gallery areas, demonstrating insecticidal activity of dsRNA applied as a foliar spray. In Chapter 5, I describe a parallel study using a root soak application; uptake, translocation, and persistence is demonstrated for up to 30 days, but no bioassays were conducted. Overall, my data demonstrate the high specificity of dsRNAs designed to suppress EAB and provide proof of concept of topical application feasibility to deliver dsRNA to EAB through the host plant.

RNA-based therapeutics is a rapidly expanding field due to its enormous potential for treatment of diseases through knockdown of genes or expression of therapeutic proteins. However, due to the overall negative-charge of an RNA molecule, RNA-based therapeutics must rely on delivery systems to overcome the various biological barriers for ultimate release of an RNA payload into the cytosol. Over the past three decades, the development of lipid-based RNA delivery systems, especially lipid nanoparticles (LNPs), have been comprehensively studied due to their unique properties. LNPs represent the most widely used delivery systems for RNA-based therapeutics, as evidenced by the clinical approvals of three LNP-RNA formulations: patisiran (ONPATTRO®), and BNT162b2 and mRNA-1273, which are LNP-encapsulated mRNA-based vaccines for prevention of COVID-19. Cationic lipids are also considered suitable candidates for RNAi therapeutics delivery. To this end, the present thesis work centers on examining liposome formulations of oxime ether (OELs) containing hydroxylated head groups in animal studies to validate their suitability as in vivo RNA carriers. This thesis work also examines the effects of adding a boron-containing lipid to OEL4-based formulations. Conjugation chemistry, particularly oximation, is a critical component for the development of these lipids. Oximation involves the reaction of an aminooxy group with a carbonyl group of an aldehyde or ketone to form an oxime ether. The versatility of oximation is highlighted and exploited in this thesis. In addition, as part of my initial studies on micelles, the synthesis and synthetic applications of a novel, environmentally benign surfactant developed to form micelles containing polar cores is also described. This thesis is divided into two parts. Part I focuses on the synthesis and applications of PS-750-M, a surfactant developed to enable organic synthesis transformations in water. Accordingly, Chapter 1 reviews the historical development of micellar catalytic processes. Chapter 2 details the synthesis of the surfactant PS-750-M and its application in Suzuki-Miyaura couplings of unactivated quinoline systems. PS-750-M contains a proline-based linker between its hydrophobic and hydrophilic regions that imparts polarity to the micellar core, which is presumed to play a significant metal-coordinating role in the micellar Suzuki-Miyaura coupling reactions. PS-750-M functions as a green solvent that mimics toxic dipolar-aprotic solvents, such as DMF, DMAc, NMP, and 1, 4-dioxane. The micellar reaction medium derived from PS-750-M is demonstrated to be recyclable. Part II focuses on the use of hydroxylated oxime ether lipids (OELs) as delivery agents for RNAi therapeutics. We first explore the in vivo utility of OELs. We found that OEL4 liposomes formulated using 3 mol% DSPE-PEG350 accumulate in tumors at highest efficiency as compared to other formulations and exhibited significant increases in tumor to liver ratios, indicative of improved delivery selectivity. The PEGylated liposomes also showed a statistically significant luciferase signal reduction in tumors as compared to an untreated mice control group. Chapter 3 reviews the structural features of cationic lipids in siRNA/DNA therapy and contrasts it with novel features of cationic OELs. A modified synthesis of OELs that proceeds in 7 steps in overall 13% yield, constituting a 4-fold improvement over the previously reported synthesis, is also presented. The formulation and results of in vitro and in vivo transfection studies using OEL4 are presented in Chapter 4. Our studies show that the surface modification of OEL4 was essential for tumor accumulation in mice bearing human lung cancer xenografts. The surface-modified OEL4 formulations were developed by inclusion of distearoylphosphatidylethanolamine (DSPE) bound to varying lengths of poly(ethylene glycol) (PEG) polymer. The results of in vivo studies on mice bearing human lung cancer (A549-luc2) determined the optimal OEL4 formulation for delivery of an RNA payload to be the formulation OEL4:DOPE:DSPE-PEG350 (48.5:48.5:3). Chapter 5 discusses the synthesis of boron-containing lipids and preliminary results of boron-assisted RNA transfection studies using both oxime ether- and boron lipid-derived formulations. The results of in vitro transfection studies using oxime ether- and boron lipid-derived formulations established that inclusion of the novel boronic acid-containing cationic lipid DMDBH to an OEL4 lipoplex formulation enhances the gene silencing of capability of OEL4 by about 20%. The action of DMDBH in this regard remains unknown; however, nmr studies of OEL4 and DMDBH suggest dimerization of DMDBH and OEL4, which could increase the strength of the interaction with RNA and increase lipoplex stability. Chapter 6 presents detailed experimental procedures of the chemistry and biology described in Chapters 2, 3, 4 and 5. Supporting spectroscopic data of synthesized compounds are also included in Chapter 6.

Unique among metazoan repressive histone methyltransferases, G9a and GLP, which target histone 3 lysine 9 (H3K9), require dimerization for productive H3K9 mono (me1)- and dimethylation (me2) in vivo. Intriguingly, even though each enzyme can independently methylate H3K9, the predominant active form in vivo is a heterodimer of G9a and GLP. How dimerization influences the central H3K9 methyl binding (“reading”) and deposition (“writing”) activity of G9a and GLP, and why heterodimerization is essential in vivo remains opaque. Here, we examine the H3K9me “reading” and “writing” activities of defined, recombinantly produced homo- and heterodimers of G9a and GLP. We find that both reading and writing are significantly enhanced in the heterodimer. Compared to the homodimers, the heterodimer has higher recognition of H3K9me2, and a striking ~ 10-fold increased kcat for nucleosomal substrates under multiple turnover conditions, which is not evident on histone tail peptide substrates. This however is not encoded by altered nucleosome affinity, which is dominated by the G9a protomer and comparable across the homo- and heterodimer. Our results indicate that heterodimerization may be required to relieve autoinhibition of H3K9me reading and chromatin methylation evident in G9a and GLP homodimers. Relieving this inhibition may be particularly important in early differentiation when large tracts of H3K9me2 are deposited by G9a-GLP, which may require a more active form of the enzyme.

Abstract: Research in the field of nucleic acids and molecular genetics has enormously enhanced our knowledge about the genetic origins of a myriad of disease. Currently from this knowledge, various gene-targeted therapies and biomarker detection and diagnostic assays are being developed, which are discussed in Chapter 1, as targeting sequences of interest is deemed a simpler task compared to protein targeting. The treatment and understanding of Acute Myeloid Leukemia (AML) development has greatly benefited from the advances in molecular characterization and the World Health Organization (WHO) recognizes certain recurrent genetic abnormalities as diagnosis for the disease. In this thesis, we first demonstrate how we can develop gene silencing therapeutic options targeting messenger RNA (mRNA) in AML cells by delivering small interfering RNAs (siRNA) using polyethylenimine (PEI) carriers. Next, we show how we can target the aberrant formation of DNA adducts in the sequence of the KRAS oncogene using synthetic nucleobase analogs. More specifically, in Chapter 2, we explored the use of RNA silencing as a promising candidate to be used in conjunction with traditional treatments as it has the potential to act in synergy to lower doses, length of treatments and side effects. We identified molecular targets using lipid modified PEI delivery systems that could be used as the basis of therapy during AML progression using the stem-cell like KG1a cell line and primary cells. We identified linoleic acid and lauric acid modified PEI as effective and versatile carriers and showed how they can effectively downregulate the mRNA levels of genes including BCR5 (survivin), and BCL2L12 leading to a reduction in proliferation of the cells. Moreover, we highlight the versality of the synthetic delivery systems and how gene silencing allows for personalized and targeted patient treatment. In Chapter 3, we showed the versatility of incorporating DNA base analogs to oligonucleotide sequences for the detection of O6-methylguanine (O6-meG), a DNA adduct that is highly mutagenic. The base analogs were synthesized by the Sturla group in ETH Zurich to have higher affinity to the methylated guanine in comparison to the non-methylated form. Our main objective was to develop a templated-ligation based detection assay and identify parameters that enhance the selectivity of the synthetic base for downstream sequence amplification. The templated ligation reaction would simplify previously reported detection methods for the same adduct and also give sequence specificity of the location of interest which a lot of previous methods lacked. We examined different parameters of the ligation reaction, modified the reacting probes, and reaction conditions to increase the detection limit of our assay in the presence of non-methylated background DNA. By enhancing the selectivity of the ligation reaction, we aimed to increase the selectivity of the O6-meG detection retaining sequence specificity so that in the future we can design a fast screening test for adduct formation that can be used as a biomarker test for hotspot mutation sites.

Nucleic Acids are the most important macromolecules in living systems. They directly participate in storing, transferring and expressing the genetic information, thereby controlling and regulating the function of living systems. Therefore, nucleic acid research will give us molecular insights into lives. Meanwhile, many novel research methods based on nucleic acids have been developed. Moreover, nucleic acid-based therapeutics have been developed rapidly over the past several years, leading to a revolution on drug discovery and disease study at the molecular level and the genetic level. Consequently, the structure and function studies on nucleic acids and protein-nucleic acid complexes have attracted tremendous research attention, especially in medical science and biological chemistry. Since oxygen and selenium are in the same main group in the periodic table, we anticipate oxygen atoms on nucleic acids can be replaced by selenium atoms without significant perturbations on structure and function. Previously, we successfully completed the chemical and enzymatic synthesis of Se-modified nucleic acids (SeNA) with selenium substitutions at various positions. Indeed, we have demonstrated that the Se-modifications would not lead to significant changes in the structure. In addition, SeNA can largely facilitate nucleic acid-protein X-ray crystallography, and its unique properties may lead to many other potential applications, such as gene silencing, therapeutics and cellular imaging. This dissertation has explored potential applications of SeNA, including therapeutic and biochemical research, via structural and functional studies. We demonstrated that SeNA has great potential for antisense drug discovery because of its stronger nuclease resistance, better compatibility with RNase H and more efficient gene silencing in cells. SeNA could also be applied in cellular imaging via micro-X-ray fluorescence (microXRF), which allowed us to establish a novel approach to image mRNA transportation in cells. With this approach, we have successfully monitored the GFP mRNA in HeLa cell. Furthermore, we found that SeNA doesn’t perturb the structure of DNA/DNA polymerase complex, which allows an accurate structural determination of protein-nucleic acid complexes and provides a bright future on X-ray crystallography of nucleic acids and their protein complexes.

RNA was originally considered to be a molecule whose primary function was to encode proteins. When it was discovered during the human genome project that the number of protein coding genes was radically lower than previously estimated, the idea was posited that non-coding RNA (ncRNA) could contribute to increasing complexity and account for the observed similarity of number of protein-coding genes between humans and other less complex organisms. Over the last decade, experimental observations have been used to categorise non-coding RNAs by function, which increasingly points towards them being involved in performing a role in regulating the transcriptional and epigenetic states of gene expression. In this thesis, a number of ncRNA species have been studied to ascertain a greater understanding of the mechanisms involved in transcriptional and epigenetic regulation. I examine short antisense RNA directed transcriptional gene silencing of Periostin, resulting in a loss of cellular metastasis. Periostin is involved in altering the extracellular matrix to favour increased invasive potential. I investigate the mechanisms involved in the complex PTEN regulatory network, involving RNA:RNA hybrid interactions between an lncRNA pseudogene transcript and a 5’-UTR ncRNA transcript the PTEN promoter which regulate gene expression of PTEN via transcriptional and epigenetic regulation. I also studied the deamination DNA repair pathways, resulting in the arisal of cytosine to thymine SNP mutations at loci targeted by shRNAs, antisense lncRNAs, and small antisense RNAs when MBD4 and SMUG repair enzymes have been repressed. Collectively, these data suggest a far more complex role for ncRNAs in the cell has been understood previously, as they appear to play a large part in directing epigenetic regulation and modification of the human genome.

The transferrin (TfR) and epidermal growth factor receptors (EGFR) are known to be overexpressed on the surface of a wide variety of tumor cells. Therefore, the peptides B6 (TfR specific) and GE11 (targeted to the EGFR) were linked to the PAMAM (polyamidoamine) structure via a polyethylenglycol (PEG) 2 kDa chain with the aim of improving the silencing capacity of the PAMAM-based dendriplexes. The complexes showed an excellent binding capacity to the siRNA with a maximal condensation at nitrogen/phosphate (N/P) 2. The nanoparticles formed exhibited hydrodynamic diameters below 200 nm. The zeta potential was always positive, despite the complexes containing the PEG chain in the structure showing a drop of the values due to the shielding effect. The gene silencing capacity was assayed in HeLa and LS174T cells stably transfected with the eGFPLuc cassette. The dendriplexes containing a specific anti luciferase siRNA, assayed at different N/P ratios, were able to mediate a mean decrease of the luciferase expression values of 14% for HeLa and 20% in LS174T cells, compared to an unspecific siRNA-control. (p < 0.05). In all the conditions assayed, dendriplexes resulted to be non-toxic and viability was always above 75%.

Small RNAs (sRNAs), ~20-30 nucleotides in length, are non-coding RNAs that play essential roles in the regulation of gene expression in eukaryotes. They lead to inactivation of cognate sequences at the post-transcriptional level via a variety of mechanisms involved in translation inhibition and/or RNA degradation. In the Chlorophyta Chlamydomonas reinhardtii, however, the molecular machinery responsible for sRNA-mediated translational repression remains unclear. To address the mechanisms of translation inhibition by sRNA, we have isolated an RNAi defective mutant (Mut26), which contains a deletion of the gene encoding the homolog of CCR4 in Chlamydomonas. We investigated the expression of both an exogenous siRNA target and endogenous miRNA target. Additionally, the pattern of poly(A) tailing in diagnostic mRNAs was examined with the G/I tailing assay and CCR4 partner proteins were identified through affinity purification. Our overall results are consistent with the role of CCR4 in sRNA-dependent translational repression without target mRNA degradation in Chlamydomonas. The biological function(s) of miRNAs in responses to nutrient deprivation in Chlamydomonas reinhardtii were also explored. Transcriptome analysis using cells grown under various trophic conditions revealed that several miRNAs were differentially expressed, but their predicted targets showed no changes in transcript abundance. Collective evidence suggests that miRNAs may not play an essential role in endogenous gene regulation in Chlamydomonas. Advisor: Heriberto Cerutti

Silencing phenomena have been observed in plants for decades. Despite our detailed knowledge of plant silencing pathways, it is unclear what initially targets silencing pathways and how different pathways are connected. Through the introduction of transposable element (TE) transgenes, I provide strong evidence for two independent mechanisms that can initiate silencing and establish chromatin modification: 1. a mechanism dependent on RNA polymerase II (Pol II) transcription, which I call expression-dependent RNA-directed DNA methylation (RdDM) and 2. a mechanism dependent on sequence homology to endogenous siRNA, which I call identity-dependent RdDM. Using endogenous TE copies, endogenous TE transgenes, and exogenous TE transgenes, I demonstrate that silencing pathways are linked in a linear progression from initiation by Pol II expression, establishment of chromatin modification by expression-dependent RdDM pathways, and transition to maintenance methylation via Pol IV-RdDM. Once later pathways are activated, earlier pathways are no longer required to maintain silencing. This work has implications for transgene usage in crop improvement and in research.

Haemoglobin consists of two α- and two β-like globin chains, responsible for oxygen transport through the body. During development, different haemoglobin types are produced, giving rise to first embryonic haemoglobin followed by foetal and finally adult haemoglobin. Understanding this switch in expression and the mechanisms regulating β-like globin genes is of interest, not only as a model system for understanding gene regulation, but also since research has shown that beneficial mutations causing the persistent activation of foetal haemoglobin are able to ameliorate symptoms of defective adult globin genes. Transcription factors Kruppel-like Factor 3 (KLF3), Kruppel-like Factor 8 (KLF8), and Sry-related HMG box 6 (SOX6) are expressed in erythroid cells. In Klf3 and Klf8 double knockout mice, moderate de-repression of embryonic β-like globin genes is seen. Sox6 null mice show persistent expression of embryonic β-like globin. KLF3, KLF8, and SOX6 all contain a PIDLS motif which enables binding of the transcriptional co-repressor C-terminal Binding Protein (CtBP). CtBP has an important role in many transcriptional pathways. To investigate the role of KLF3, KLF8, SOX6, and CtBP in globin gene expression, we used CRISPR/Cas9 technology to generate, Klf3-/-, Klf8-/-, CtBP1-/-, CtBP2-/-, CtBP1CtBP2-/- and Sox6-/- erythroid cell lines. We also examined the importance of the PIDLS motif in β-like globin gene regulation by mutating the PIDLS motif to a PIASS motif in Klf3 and Sox6. We found that β-like globin expression was deregulated by the ablation of KLF3, KLF8 and SOX6 with good correlation to previous studies. Our major novel finding was of the importance of CtBP1 and CtBP2 in regulating β-like globin expression. Undifferentiated CtBP1CtBP2-/- MEL exhibited de-repression of the adult β-like globin genes. It is possible that the deregulation of β-like globin gene expression in the CtBP1CtBP2-/- MEL was due in part to the concurrent de-repression of Klf8 and Sox6 and their role in silencing embryonic β-like globin genes. Microarray and further analysis of CtBP1CtBP2-/- MEL revealed deregulation of erythropoiesis regulatory gene, Id2. Overall, our data suggests that CtBP plays an interesting role in haematopoiesis; in normal undifferentiated erythroid progenitor cells, CtBP co-represses the expression of key erythroid regulatory transcription factors, and regulates the β-like globin gene locus.

Cytosine methylation is a key epigenetic mark that mediates diverse biological phenomena by regulating chromatin states. In plants, it is predominantly found in transposons and repeats, and contributes to genome defense through transcriptional gene silencing (TGS). In my thesis research, two genes and their molecular mechanisms in DNA methylation and TGS are uncovered in the model plant Arabidopsis thaliana. First, the LUCH line containing a luciferase (LUC) transgene is characterized as a suitable system for forward genetic screens that aim to isolate genes in TGS. RNA-directed DNA methylation (RdDM), a mechanism that establishes DNA methylation, and active demethylation influence LUC expression by affecting its DNA methylation levels. The moderate LUC expression in LUCH enables genetic screens to isolate both enhancers and suppressors of TGS. Second, TATA-binding proteins-associated factor6 (TAF6) is identified as a positive factor of RdDM and TGS. TAF6 is required for the TGS of the LUCH reporter and several endogenous RdDM targets. Genome-wide methylation assays have uncovered TAF6-dependent loci for proper DNA methylation. TAF6 is well known as a transcription factor of RNA polymerase II (Pol II), however, I find that a TAF6 mutation compromises the transcription by a Pol II homolog Pol V. These findings indicate that Pol V adopts the Pol II regulatory mechanism to generate long non-coding RNAs that guide RdDM. Third, a heat shock protein 20 (HSP20) is uncovered as a negative regulator of DNA methylation and TGS. HSP20 prevents the silencing of TGS reporters as well as of several endogenously methylated targets. Mutations in HSP20 result in hypermethylation and mislocalization of the RdDM effector protein ARGONAUTE4 in the nucleus. Moreover, HSP20 is found to interact with three methyl CpG-binding domain (MBD) proteins that may link cytosine methylation to histone modifications. Mutation in MBD5 or MBD6 also hypermethylated several HSP20-dependent loci. Based on these findings, I propose that HSP20 acts with MBDs to recognize loci that undergo DNA methylation and antagonize TGS at these loci. While HSP20-MBDs likely act downstream of DNA methylation, they also affect the DNA methylation status through direct or indirect effects, such as AGO4 recruitment or histone modifications.

A major contributor to mortality in patients with plasma cell dyscrasias (PCDs); i.e., multiple myeloma, light chain deposition disease and AL amyloidosis is the deposition as insoluble aggregates of monoclonal immunoglobulin light chain proteins (LC) in the kidneys and other organs. Currently anti-plasma cell chemotherapies are used to reduce LC synthesis, and slow deposition. While effective, these treatments are toxic, non-specific, expensive, and might not be appropriate in all cases, making the identification of an alternate means of reducing toxic LC species desirable. To this end, we have investigated whether RNA interference (RNAi) could achieve these goals. Human (RPMI 8226, Bur) and transfected mouse myeloma (SP2/O-lambda 6) cells which produce measureable quantities of human LC protein were used as model systems for testing the efficacy of both synthetic small interfering RNAs (siRNAs) and short hairpin RNA (shRNA) expression vectors in reducing LC synthesis. Sequencing of LC genes provided the basis for design of siRNA duplexes targeting either the variable (V) or joining (J) regions of individual LCs, or the constant (C) region of either kappa or lambda LC isotypes. Myeloma lines were transfected with siRNAs using lipid-based transfection media. Cells receiving non-silencing siRNAs served as controls. Exposure of myeloma lines to siRNAs was well tolerated and no cytotoxicity was observed. LC mRNA expression was shown to be reduced ≥40% in 8226 and SP2/O- lambda 6 cell lines receiving siRNA treatment as compared with untreated controls. Exposure to siRNAs was also effective in significantly reducing both intracellular and secreted LC protein levels in cell lines tested as evidenced by flow cytometry or enzyme-linked immunosorbent assays (ELISAs). Effective siRNA nucleotide sequences were used to generate shRNA cassettes which were ligated into lentiviral expression vectors under the control of the RNA polymerase III promoter, U6. These expression systems were used to generate replication incompetent lentiviral particles. Exposure of 8226 to lentiviral particles resulted in significant knockdown of LC mRNA and protein both in vitro and in xenograft tumor bearing immune compromised mice. These results provide positive evidence for the ability of RNAi based approaches to reduce LC secretion in models of PCD.

Over the past several decades, gene therapy technologies have been developed for a diverse number of applications ranging from DNA-based vaccines to gene silencing with RNAi. While all are powerful tools, a common limitation for these technologies is the need for effective and safe delivery to target sites within the body. Such delivery vectors are necessary for retention of bioactivity and stability, while also providing a method of cellular and tissue uptake and distribution, which may require endosomal escape. Although, viral and lipid-based technologies have shown promise as nucleic acid delivery vectors, both have inherent issues such as cytoxicity, oncogenicity, and immunogenicity. Thus, the development of polymer-based non-viral vectors has been an area of great focus over the past decade. While many polymeric vectors have been developed for plasmid DNA (pDNA) delivery, very few have shown effective delivery of short interfering RNA (siRNA), a powerful tool for gene silencing via the RNA interference mechanism. Furthermore, very few prospective delivery vectors have shown versatility for the administration of siRNA through multiple routes of administration. The overall goal of this research was to develop a biocompatible non-viral delivery system for the delivery of plasmid DNA, siRNA, and contrast agents through the modification of the natural biopolymer chitosan. We have synthesized an imidazole modified chitosan (chitosan-IAA) by conjugation of imidazole acetic acid to chitosan. Extensive evaluation and characterization of the modified polymer demonstrates enhanced solubility and buffering capacity within the physiological and endosomal pHs, thus providing enhanced endosomal escape by exploiting the "proton sponge" effect. We have demonstrated effective in vitro gene expression and gene silencing with chitosan-IAA mediated delivery of pDNA and siRNA, respectively. Furthermore, we have demonstrated in vivo gene silencing by delivery of siRNA through both intranasal and intravenous routes of delivery with chitosan-IAA/siRNA nanocomplexes. We have also demonstrated delivery of contrast agents up to 45 nm in size through mucosal tissue following treatment with chitosan and no contrast agent modification in both human and animal tissue. In conclusion, we have successfully developed a versatile and highly effective delivery vector for both nucleic acids and contrast agents.

Gene regulation is controlled by multiple mechanisms that function at levels ranging from genome-wide to gene-specific. Understanding the mechanisms of gene regulation at all levels is important for discerning proper organ development and function as well as interpreting the changes that occur in mutant organs, which lead to defects and diseases. To better understand the mechanisms functioning at different levels of gene regulation in organ development and function, I studied the role of the environment, chromatin remodeling, and gene-specific transcriptional regulation on the C. elegans excretory system, which is similar to mammalian kidneys.Class III POU-homeobox transcription factors are expressed in the renal organs of species from C. elegans to mammals. However, the functional role for these factors in renal organs is not well characterized. In this dissertation, I studied the role of the C. elegans POU-III transcription factor, CEH-6, in the excretory cell to address this question. I performed molecular and biochemical studies, which show CEH-6 regulates a subset of genes expressed in the excretory cell. This work suggests that a role for POU-III factors in renal organs may be to modulate the expression of functionally related genes.Chromatin structure also influences the accessibility of transcription factors to target genes, and thereby is important for gene expression. In this study, I show LEX-1 and TAM-1 function to attenuate transgene silencing in somatic tissue by acting regionally as part of a complex that broadly regulates expression of genes in repetitive DNA.Additionally, environmental cues can play a role in regulating gene expression. Previous work by other labs has shown dauer larvae exhibit an increased osmotic tolerance, suggesting the excretory system may be altered. I was interested in understanding the biological changes responsible so I characterized the excretory system anatomy in dauer larvae. I found an altered morphology and the positional alignment of the excretory duct cell and excretory cell in dauer larvae, which may be related to the increased osmotic tolerance.Together, the results from these studies support the idea that organisms integrate information from multiple levels and mechanisms of regulation in order to direct their development and function.

People experience the irritating tearing and burning sensation of lachrymatory factor (LF, propanthial S-oxide) when cutting or chopping onion bulbs. LF is produced by lachrymatory factor synthase (LFS) specifically from 1-propenyl sulfenic acid, a breakdown product of trans-1-propenyl-L-cysteine sulfoxide (1-PRENCSO) by alliinase. This thesis describes strategies to produce a tearless onion by using RNA interference (RNAi) silencing. To determine whether a gene silencing cassette can silence lfs gene transcripts from onion (Allium cepa L.), a crop recalcitrant to genetic transformation, a gene silencing assessment system was developed by using a model plant as a host for the gene of interest. Tobacco (Nicotiana tabacum) plants transgenic for LFS enzyme activity from onion were first produced by introducing a CaMV 35S-onion-lfs gene construct. These plants were then subjected to a second transformation with an RNAi construct directed against the lfs gene sequence. LFS enzyme activity assay showed that the transgenic plants, containing both the lfs gene and the RNAi construct, had significantly reduced LFS activity. This observation was supported by Western analysis for the LFS protein and further validated by quantitative RT-PCR analysis that demonstrated a significant reduction in the lfs transcript level in the dual transformants. This work demonstrated that the RNAi construct is a suitable candidate for the development of a tearless onion. This model plant RNAi system has wide reaching applications for assessment and targeting of plant secondary pathway genes, from poorly studied or recalcitrant plant species, that are important in pharmacological, food and process industries. The functional RNAi vector identified in the model system was transformed into onion. Endogenous lfs transcript levels were successfully reduced by up to 43-fold in six transgenic lines. In consequence, LFS enzyme activity was decreased by up to 1573-fold and this observation was supported by Western analysis for the LFS protein. Furthermore, the production of the deterrent LF upon tissue disruption was reduced up to 67-fold. Subjective olfactory assessment of silenced lines indicated that the pungent odour given off by the leaf and bulb material was much reduced compared with that of non-transgenic counterparts, and that this was replaced by a sweeter milder onion odour. A novel colorimetric assay demonstrated that this silencing had shifted the 1-PRENCSO breakdown pathway so that by reducing LFS protein, more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of the raised thiosulfinates levels was a marked increase in the downstream production of a non-enzymatically produced zwiebelane isomer that has never previously been identified, and other volatile compounds, di-1-propenyl disulfides and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophenes, which had previously been reported either in small amounts or had not been detected in onions. These raised volatile sulfur compounds provide an explanation for the unique flavour notes of the LF reduced onion and are predicted to have health benefits akin to those found in garlic. These results demonstrated that silencing of LFS enzyme activity by introducing an RNAi construct directed against the lfs gene sequence simultaneously reduced levels of the deterrent LF and increased the desirable thiosulfinates in onions.

S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.

The complexity and dynamics of the developing nervous system challenges the traditional approaches of reverse genetics. Vector-driven RNA interference (RNAi) provides a viable alternative as a convenient, economic and versatile research tool to inhibit gene expression. RNAi is a type of post-transcriptional gene silencing mechanism, in which small RNAs of ∼19-23 nt bind to the target mRNA sequences with perfect or near-perfect complementarity and induce cleavage, leading to sequence specific degradation of mRNAs. A novel class of endogenous small RNAs called microRNAs (miRNAs) was recently discovered to be an important regulator of gene expression at the post-transcriptional level. Varying in size between 19 and 25 nucleotides, miRNAs either inhibit the translation of partially complementary mRNAs without affecting mRNA transcript abundance, or employ an RNAi mechanism to degrade target mRNAs with a higher degree of complementarity. To trigger highly efficient RNAi by utilizing the cellular machinery for biogenesis of natural miRNAs, we have developed new RNA polymerase II expression vectors for RNAi designated synthetic inhibitory BIC-derived RNA (SIBR) vectors. The SIBR vectors use a modified miR-155 microRNA (miRNA) precursor stem-loop and flanking sequences as a cassette to express synthetic miRNAs complementary to target RNAs. By placing the SIBR cassettes within an intron of a protein-coding gene, we can coexpress a marker protein with miRNAs in a tightly correlated temporal and spatial pattern. Furthermore, multimerization of SIBR cassettes in tandem enables us either to inhibit a protein more effectively, or inhibit multiple proteins simultaneously. We utilized the unique combination of features in SIBR vectors to study the biological function of the scaffold protein POSH (Plenty of SH3 domains) during neuronal differentiation. By inhibiting POSH protein in differentiating mouse primary cortical neurons, combined with time-lapse image analysis, we demonstrate that POSH down-regulates the extension phase of axon outgrowth in cortical progenitors, without affecting neuronal survival or apoptosis. In similar experiments, the F-actin binding protein Shroom, a newly identified interacting partner of POSH, also inhibits process elongation, presumably by regulating the neuronal cytoskeleton via its actin binding domain. Combined, these results suggest that POSH and Shroom work together to negatively regulate axon outgrowth in differentiating neurons, potentially through modulation of the neuronal cytoskeleton.

RNA interference with viral vectors that express short hairpin RNAs (shRNAs) has emerged as a powerful tool for functional genomics and therapeutic purposes. However, little is known about shRNA in vivo processing, accumulation, functional kinetics, and side effects related to shRNA saturation of the cellular gene silencing machinery. Therefore, we constructed first-generation recombinant adenoviruses encoding different shRNAs against murine ATP-binding cassette multidrug resistance protein 2 (Abcc2), which is involved in liver transport of bilirubin to bile, and analyzed Abcc2 silencing kinetics. C57/BL6 mice injected with these viruses showed significant impairment of Abcc2 function for up to 3 weeks, as reflected by increased serum bilirubin levels. The lack of Abcc2 function correlated with a specific reduction of Abcc2 mRNA and with high levels of processed shRNAs targeting Abcc2. Inhibition was lost at longer times postinfection, correlating with a decrease in the accumulation of processed shRNAs. This finding suggests that a minimal amount of processed shRNAs is required for efficient silencing in vivo. This system was also used to evaluate the effect of shRNA expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters the accumulation and functionality of endogenous microRNAs (miRNAs) and pre-miRNAs. However, expression of functional exogenous shRNAs did not change the levels of endogenous miRNAs or their precursors. In summary, this work shows that adenoviral vectors can deliver sufficient shRNAs to mediate inhibition of gene expression without saturating the silencing machinery.

Geminivirus AL2 and L2 proteins are pathogenicity determinants that cause enhanced susceptibility when expressed in transgenic plants. To understand how geminiviruses defeat host, we found that the viral proteins interact with and inactivate adenosine kinase (ADK), a nucleoside kinase catalyzing the salvage synthesis of 5'-AMP from adenosine and ATP. We show that the AL2 and L2 proteins inactivate ADK in vitro, in E. coli and yeast. We also demonstrate that ADK activity is reduced in transgenic plants expressing the viral proteins and in geminivirus infected plant. In contrast, ADK activity is increased in plants infected with diverse RNA viruses or a geminivirus lacking a functional L2 gene. We also demonstrate that AL2 is present in both the nucleus and the cytoplasm of infected plant cells. This is the first evidence that ADK is targeted by viral pathogens, and that this "housekeeping" enzyme might be a part of host defense responses. In previous work, we showed that AL2 and L2 also interact with and inactivate SNF1 kinase, a global regulator of metabolism that is activated by 5'-AMP. Together these observations suggest that metabolic alterations mediated by SNF1 are an important component of innate antiviral defenses and that inactivation of ADK and SNF1 by the geminivirus proteins represents a dual strategy for countering this defense. AL2 proteins have also been shown to act as suppressors of RNA silencing, an adaptive host defense response. By recycling adenosine, ADK plays a critical role in sustaining the methyl cycle and SAM-dependent methyltransferase activity. In this thesis, we confirm that the geminiviruses TGMV and BCTV induce and are targeted by RNA silencing, and that the AL2 and L2 proteins are RNA silencing suppressors. Remarkably, we found that inhibiting ADK activity at the RNA level by expression of a dsRNA construct directed against ADK, or at the protein level by using an ADK inhibitor, also results in silencing suppression. ADK activity in tissue showing silencing suppression following infiltration with AL2, L2, dsADK, or RBI was significantly reduced (>50%). These data provide strong evidence that AL2 and L2 suppress silencing by inhibiting ADK activity.

RNA-mediated gene silencing is a highly conserved cellular mechanism induced by the intracellular presence of double-stranded RNA (dsRNA). Small interfering RNA duplexes (siRNA) are produced after enzymatic cleavage of dsRNA and induce the degradation of homologous RNA. siRNA-mediated silencing appears to be an RNA based system for counteraction of virus infections in plants and it could be used in specific silencing of genes for genomic studies involving gene characterization. This thesis explores the use of multiple siRNAs for silencing of the mgfp5-ER gene in protoplasts expressing the Green Fluorescent Protein (GFP). To investigate this, hairy roots expressing GFP were produced by Agrobacterium-mediated transformation. Nicotiana tabacum cv. Petit Havana SR1 (tobacco), Solanum tuberosum cv. Iwa (potato), rapid-cycling Brassica oleracea, and B. campestris var. pekinensis cv. Wong Bok (Chinese cabbage) explants were co-cultivated with Agrobacterium strain A4T. Hairy roots were produced in tobacco and Chinese cabbage; the efficiency of transformation was 33% and 7% respectively. The hairy roots were selected on the basis of their uniform expression of GFP and their characteristic hairy root phenotype. PCR was used to confirm the presence of the GFP gene. Additionally, seeds were obtained from rapid-cycling B. oleracea plants stably expressing GFP. Chinese cabbage hairy roots and hypocotyls from etiolated rapid-cycling B. oleracea seedlings were subsequently used as source material for protoplast isolation. Yields of 3-5x10⁵ protoplasts per gram of Chinese cabbage hairy roots and a mean yield of 1.2x10⁶ protoplasts per gram of etiolated hypocotyls were obtained. A mixture of multiple siRNA was produced to target silencing of GFP in the protoplasts by transfection. Delivery of the siRNA was confirmed by observing the fate of control Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA labelled with Cy3 fluorescent dye. As performed in this study, transfection of siRNA using a combination of polyethylene glycol (PEG) and Lipofectamine™ 2000 was not successful in delivering the siRNA into the protoplasts. Image analysis was used to measure GFP levels in this experiment. Successful transfection was achieved using Lipofectamine™ 2000 alone. Control Cy3-labeled GAPDH siRNA was observed in the protoplasts transfected, which confirmed transfection. Approximately 30% of the protoplasts showed red-fluorescent spots consistent with fluorescence of the dye. However, Lipofectamine™ 2000 was toxic and protoplast viability was reduced to approximately 50% when measured 48 h after transfection. Silencing of GFP was measured by qualitative RT-PCR. Preliminary studies were carried out to partly optimise qualitative RT-PCR analysis for confirmation of GFP silencing. Much work needs to be done before gene silencing in protoplasts using transfection of siRNA becomes efficient but this study has set the basis for future research on transfection of siRNA into protoplasts to reduce gene expression.

Viral vectors carrying a host-derived sequence insert induce silencing of the corresponding gene in infected plants. This virus-induced gene silencing (VIGS) is a defense mechanism that is related to post-transcriptional gene silencing (PTGS) in transgenic plants. Here I describe modified vectors derived from two members of the Begomoviruses: Tomato Golden Mosaic Virus (TGMV) and Cabbage Leaf Curl Virus (CbLCV). In order to gain insight into the geminivirus-induced gene silencing (G-VIGS) mechanism, I tested six known silencing-deficient Arabidopsis mutants: sgs1, sgs2, sgs3, ago1, som and mom. Sgs1, sgs2, sgs3 and ago1 mutants are impaired for PTGS of transgenes and endogenous genes, whereas som and mom mutants release transcriptional gene silencing (TGS). SGS2 and AGO1 encode an RNA-dependent RNA polymerase (RdRP) and an eIF2C-like protein belonging to PAZ/PIWI family, respectively. SGS1 and SGS3 map to genes with unknown function. SOM and MOM are chromatin-associated proteins. All mutants were bombarded with CbLCV carrying a gene fragment homologous to an endogenous gene required for chlorophyll biosynthesis, magnesium chelatase (CH42). G-VIGS of CH42 endogenous gene occurred efficiently in the sgs1, ago1, som and mom mutants, but it was inhibited (not abolished) in sgs2 and sgs3 mutants. These results indicate that SGS2 and SGS3 are required for G-VIGS, whereas SGS1, AGO1, SOM and MOM have minimal or no effect. The findings that SOM and MOM are not required for G-VIGS support the ideas that G-VIGS is PTGS-specific or that geminiviruses counteract TGS. Considering these results, G-VIGS has unique requirements distinct from previously described silencing pathways in plants. I also showed that TGMV- and CbLCV-derived vectors triggered VIGS of endogenous genes in Nicotiana benthamiana and Arabidopsis thaliana, respectively. G-VIGS silencing of RdRP affected silencing while G-VIGS of a gene upregulated by AL1 negatively affected geminivirus replication or movement, suggesting that G-VIGS could be used as a preliminary screen for resistance genes. This research has provided an insight into genetic silencing mechanisms mediated by the Begomoviruses, and their potential tools as episomal vectors in reverse genetic studies.

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus i) location effect, ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.