The interferon (lFN) signalling and RNA interference (RNAi) pathways are the major antiviral pathways in animals and plants, respectively. Although the mechanism of RNAi remains to be completely characterised, the genes that encode the proteins involved in this process have been identified in the plant, fungi and animal kingdoms (Fagard et al., 2000, Grishok et aI., 2000, Hall et al., 2003, Kanellopoulou et al., 2005, Kolb et al., 2005); with comparative analyses indicating that RNAi is an evolutionarily conserved mechanism. Several studies have identified RNAi suppressors encoded by animal viruses, suggesting an antiviral role for the RNAi pathway in animals as well as plants (Andersson et al., 2005, Bennasser et al., 2006, Garcia et al., 2006, Li et al., 2004, Lichner et al., 2003, Lingel et al., 2005, Lu & Cullen, 2004, Wang et al., 2006). However, most of these studies were performed in non-mammalian systems and as yet, there is no direct evidence indicating that the RNAi pathway plays a significant antiviral role during the infection of mammalian cells. Interestingly, several viruses have now been shown to express their own microRNA (miRNA) in infected cells (Grey et al., 2005, Pfeffer et al., 2005, Pfeffer et al., 2004, Samols et al., 2005, Sullivan et al., 2005). Further, in the case of hepatitis C virus (HCV), there is evidence that the virus usurps the host cell miRNAs to enhance viral replication (Jopling et al., 2005). The principal aim of this project was to investigate the role of RNAi in mammalian cells during viral infection, particularly infection with the influenza A virus. This thesis is divided into six major chapters followed by a brief general discussion. Chapter 1 contains a general introduction to the RNAi pathway. It describes the history of the discovery of RNAi and summarizes the known and proposed antiviral roles of the RNAi pathway in plants and mammalian cells. Chapter 2 describes the general materials and methods used for this project. There are four main result chapters, each dealing with a specific experimental system. Each chapter is divided into a brief introduction, specific materials and methods used, followed by presentation of the experimental results and a brief discussion. Chapter 3 describes the development of an in vitro Dicer activity assay to study the effect of viral proteins on the activity of the mammalian Dicer protein. It was demonstrated that crude cell lysates derived from influenza A virus infected cells impaired the activity of Dicer and this observation was not due to degradation of the Dicer protein by virally-induced proteases. Chapter 4 describes the use of a GFP reporter assay for screening potential RNAi suppressors. This assay is suitable for studying viral proteins in isolation. The effect of the influenza NS1 protein on the RNAi pathway in HEK293 cells was investigated and it was shown that NS1 could exert modest, but nevertheless significant, suppression of the RNAi pathway. Northern studies, performed to examine the processing of shRNA in the presence of NS1, demonstrated that NSI suppressed the RNAi mechanism through interfering with the maturation ofshRNA into siRNA. Chapter 5 describes the effect of over-expressing components of the RNAi pathway on influenza A virus infection. In these experiments, Exportin 5, which encodes a protein involved in the transport of pre-miRNA/shRNA into the cytoplasm, was over-expressed during influenza A virus infection. Reduced viral infection was observed in cells over-expressing Exportin 5, suggesting that this treatment protects cells from virus infection. Chapter 6 describes the expressed small RNA profile during influenza A virus infection in MDCK cells. Novel canine miRNA homologues were identified through cloning and sequencing. No definitive evidence for virally-derived siRNA/miRNA was found but a general reduction of endogenous miRNA expression was detected.

RNA interference (RNAi) is a diverse, conserved regulatory mechanism in eukaryotic cells, which silences the target gene expression in a homology-dependent manner. Although it has been well documented that RNAi is an antiviral mechanism in plants and insects, it is still unclear whether RNAi naturally limits viral infections in vertebrates. Viruses are masters of adopting strategies to subvert cellular defense mechanisms. Not only can viruses use elaborate strategies to suppress the effects of defensive RNAi, but they can also redirect or interfere with cellular functions orchestrated by endogenous small RNAs. In our work we have focused on studying the relationship of human adenovirus type 5 (Ad5) infection and the RNAi/miRNA pathways. We show that Ad5 infection inhibits RNAi by blocking the activity of Dicer and the RNA-induced silencing complex (RISC). For Dicer inhibition, the virus-associated RNAs, VA RNAI and VA RNAII bind Dicer through their terminal stems and are cleaved by Dicer into functional small RNAs that are incorporated into active RISC. Furthermore, by cloning small RNAs, we found that approximately 80% of Ago2-containing RISC immunopurified from late infected cells was associated with VA RNA-derived small RNAs (mivaRNAs). Interestingly, the small RNAs derived from VA RNAII, the minor VA RNA species, appear to be the major mivaRNAs occupying RISC and associate with polyribosomes, which indicates their potential roles as miRNAs regulating translation of cellular mRNAs. During our previous work, we observed that the strand bias of VA RNAI derived small RNA (mivaRI) incorporating into active RISC varied in the different viable Ad5 mutant viruses infected cells. It has been reported that Ad5 VA RNAI had two transcription initiation sites, which produced two clusters of VA RNAI with 3 nt difference at their 5’ end. Our data show that this heterogeneity resulted in a dramatic difference in mivaRI guide strand selection. Collectively, our data contributes to understanding the interplay between virus and host. This study would be beneficial in designing optimal adenovirus vectors for therapeutic RNAi application.

Biological processes depend upon the structural and functional quality of the molecules that comprise living organisms. The integrity of molecules such as DNA, RNA, proteins, carbohydrates and lipids is crucial and the precise three-dimensional shape and the detailed chemistry of these molecules orchestrate the biochemical processes vital for life. Within a cell, each protein must be present at a specific concentration during certain specific conditions. To maintain cellular homeostasis and the ability to respond to the environment the proteome is in a dynamic state of synthesis and degradation. In eukaryotic cells the ubiquitin-proteasome pathway is the principal mechanism for regulated protein turnover in both the cytoplasm and the nucleus. The 20S proteasome is a cylindrical multi-subunit protease. Proteasomes play an essential role in the targeted and timely ordered degradation of key regulatory proteins and their inhibitors. The 26S proteasome is a 2.500 kDa complex composed of the 20S proteasome sandwiched between two 19S regulators. This is the enzymatic complex responsible for ATP-dependent ubiquitin mediated protein degradation. A polyubiquitin chain attached to a protein serves as a general recognition signal for destruction via the 26S proteasome. It is known that the 19S regulator confers ubiquitin recognition and substrate unfolding to the 20S proteasome, however, the specific functions for many of the different subunits within the 19S complex are not known. We have used RNA interference to study the S13/Rpn11 and S5a/Rpn10 subunits of Drosophila melanogatser proteasomes. We have produced stable cell lines with the human S13 gene under inducible promoters that was used to rescue the knockdown phenotype after RNA interference. The rescue was successful in demonstrating that the human protein is a functional homologue to the Drosophila protein. We call the technique RNAi+c (RNA interference + complementation). This procedure enabled us to also test different mutants of the human S13 protein for their ability to function in the proteasome. Using RNA interference to a Drosophila proteasome subunit in combination with complementation with a corresponding human protein we have been able to study residues important for the deubiquitinating activity of this subunit (Paper I). Interestingly, upon a decrease of either S13 or S5a we see an induction in the levels of active 20S proteasomes. Increase in the levels of the non-targeted 19S subunit can be detected when RNAi treatment is carried out on either S13 or S5a. We have used RNA interference and proteasomal inhibition together with whole genome microarray analysis to reveal a co-regulated network of proteasome genes. This network likely contributes to an overall regulatory system that maintains proper proteasome levels in the cell. Initial studies of the mechanism of transcriptional co-regulation of proteins involved in the 26S proteasome pathway were also performed (Paper II). Finally, the biological function of the proteasome regulator PA28g/REGg is not known. We have studied this regulator in Drosophila using RNA interference and promoter mapping (Paper III).

The use of molecular biology as a means to advance agriculture has proven beneficial in many fields. However, the development lentiviral vectors that utilize a livestock promoter to express short hairpin RNA (shRNA) has been limited to date. The goal of this research project was to develop and characterize lentiviral bovine Pol III mir30 shRNA expression vectors for future use in livestock research. The bovine Pol III promoter (7sk, U6-2, or H1) was inserted directly upstream of a mir30 shRNA expression sequence in the lentiviral vector pNef-GT. A transient luciferase knockdown assay in human embryonic kidney (HEK) 293T cells was used to compare the functionality of these vectors. The bPol III mir30 shRNA expression vector was co-transfected with the pGL3 luciferase expression vector and the renilla expression vector pLB at a ratio of 5:10:1 respectively. The vectors were allowed 48 hrs to produce their respective products before luciferase activity was measured with the Stop-n-Glo Assay (Promega). Each bPol III promoter was able to express a functional shRNA resulting in a reduction of luciferase activity greater than 68 percent. The bH1 and bU6-2 Luc shRNA vectors were the most effective vectors when transfected with >76 percent (p-value <0.05) reduced luciferase activity. To confirm that these promoters were functional after integration into a bovine genome, recombinant lentivirus was made from these vectors. These particles were then used to transduce a bovine kidney (MDBK) cell line that expressed luciferase. After transduction, transgenic cells were selected by the addition of the antibiotic, Geneticin to the culture media until a population of 100 percent bPol III expression cells were observed for two passages and luciferase activity was measured. The 7sk promoter was the most effective bPol III promoter that reduced luciferase activity in these cells by 72 percent (p-value <0.05), while the bU6-2 and bH1 were moderately effective at reducing luciferase levels (37 percent, 46 percent respectively). These experiments were the first to quantify the bovine Pol III promoter function after integration into a bovine genome. While variability was observed, for livestock based research, the b7sk lentiviral vector appears to be the best choice to express a shRNA from the genome of a bovine genome.

The mammalian immune system has evolved a complex and diverse set of mechanisms to detect and respond to pathogens by recognizing conserved molecular structures and inducing protective immune responses. While many of these mechanisms are capable of sensing diverse molecular structures, a large fraction of pathogen sensors recognize nucleic acids. Pathogen-derived nucleic acids trigger nucleic acid sensors that typically induce anti-viral or anti-microbial immunity, however host-derived nucleic acids may also activate these sensors and lead to increased risk of inflammatory or autoimmune disease. Animal models and humans lacking key DNA nucleases, such as Trex1/Dnase3, accumulate intracellular DNA and develop progressive autoimmunity marked by increased Type-I Interferon (IFN) expression and inflammatory signatures. Double-stranded DNA (dsDNA) is a potent inducer of the Type-I IFN response. Many of the sensors and signaling components that drive the IFN signature following simulation with transfected dsDNA (also called 'Interferon Stimulatory DNA' or 'ISD') remain unknown. We set out to identify novel components of the ISD pathway by developing a large-scale loss-of-function genetic perturbation screen of 1003 candidate genes. We interrogated multiple human and murine primary and immortalized cells, tested several Type-I IFN reporters, and considered multiple loss-of-function strategies before proceeding with an RNAi screen whereby mouse embryonic fibroblasts were stimulated with ISD and Type-IFN pathway activation was assessed by measuring Cxcl10 protein by ELISA. Candidate genes for testing in the RNAi screen were curated from quantitative proteomic screens, IFN-beta and ISD stimulated mRNA expression profiles, and a selection of domain-based proteins including helicases, cytoplasmically located DNA- binding proteins and a set of potential negative regulators including phosphatases, deubiquitinases and known signaling proteins. We identified a number of novel ISD pathway components including Abcf1, Ptpn1 and Hells. We validated hits through siRNA-resistant cDNA rescue, chemical inhibition or targeted knockout. Additionally, we evaluated protein-protein interactions of our strongest validated hits to develop a network model of the ISD pathway. In addition to the identification of novel ISD pathway components, our enriched screening data set may provide a useful resource of candidate genes involved in the response to cytosolic DNA.

Hemangioma is a type of tumor commonly found in infants that is characterized by heavy vascularization and a disfiguring appearance. Hemangioma, though benign, can sometimes proliferate and be threatening to infants. Current treatments for infantile hemangioma include surgical removal as well as the use of topical and oral medication. However, current therapies are often ineffective at treating lesions and are commonly accompanied by dangerous side effects, creating the need for a new, safer treatment. This study targets the Yes-Associated Protein-1 (YAP-1), which has been described as an oncogene, by use of an interfering RNA technique in attempts to mediate tumor growth and progression. Western blotting of treatment and control BEND3 murine cells reveals that YAP-1 is knocked-down in treatment groups which have been infected with shYAP-1 siRNA genes. By successfully knocking down the YAP-1 protein, the potential for developing a novel targeted therapy for infantile hemangioma has been established.

Changes in the morphological character of appendages are essential to arthropod diversification and adaptation to a variety of living conditions. For instance, the fruit fly Drosophila melanogaster possesses cylindrical, uniramous (unbranched) walking legs that are well suited for terrestrial life, while Triops longicaudatus possesses paddle-like, multiramous (multibranched) limbs adapted to aquatic life. Comparative studies of limb patterning between different species of arthropods suggest that most animals utilize a conserved set of genes to construct a limb. How is this common set of genes used to produce morphological divergence? This question is addressed here by examining the spatiotemporal expression patterns of genes shown to establish distinct domains along the proximodistal (P/D) axis in arthropod species with legs morphologically distinct from Drosophila legs.In this dissertation, I investigate the role of the limb patterning genes, dachshund (dac) and homothorax (hth), in patterning the appendages of the crustacean Triops longicaudatus. I examine the spatiotemporal relationships of the expression of these two essential limb-patterning genes individually and simultaneously with two previously reported leg patterning genes, extradenticle (exd) and Distal-less (Dll). I discovered that Triops dac and hth, as expected, are expressed during leg development. I verified a cell-to-cell association between HTH and nuclear-EXD (n-EXD), a spatial relationship that had only been conjectured to exist outside of Drosophila. This spatial relationship represents an ancient unchangeable constraint on limb patterning. HTH expression reported here in addition to previously reported EXD and DLL expressions suggests a common, early subdivision of the leg into broad proximal and distal domains. However, the reiterated stripes of DAC expression found along the ventral axis do not support establishment of an intermediate leg domain but instead suggest that the ventral branches of the Triops limb are generated by a mechanism of segmentation not previously observed in other arthropod limbs. Additionally, I present a record of my attempts aimed at functional determination of genes believed to specify, pattern, or modify branchiopod appendages. Finally, I demonstrated that dac functions in the dung beetle leg to properly segment the tarsus as well as producing a structural modification, such as spiked protrusions.

Allatostatins (ASTs) are neuropeptides that inhibit the biosynthesis and release of juvenile hormone from the corpora allata (CA) of various insects including the cockroach Diploptera punctata. We hypothesized that a similar allatostatin receptor would exist in the cockroach D. punctata that may regulate the numerous physiological effects that this family of peptides exerts on a range of target tissues. Using polymerase chain reaction (PCR) strategies successful in isolating other insect allatostatin receptor sequences utilized primers designed to known mammalian somatostatin receptors and Drosophila allatostatin receptor (AlstR). Once an internal PCR fragment was proven to be consistent with the sequence of an allatostatin receptor (AstR) then the sequence was completed by rapid amplification of cDNA ends (RACE). The putative allatostatin-like receptor sequence encoding 425 amino acid residues was isolated from a cDNA library prepared from corpora allata of D. punctata. We show that dsRNA targeting the allatostatin receptor gene of D. punctata injected into freshly moulted adult cockroaches produced a long-lasting reduction in the mRNA levels in midgut tissues. The effect lasted up to 6 days. Following dsRNA injection, the juvenile hormone (JH) titers in the corpora allata were clearly raised suggest that the putative inhibition of receptor RNA expression may increase JH production. The receptor is expressed in brains, corpora allata, abdominal ganglion, midguts, ovaries, and testes. We have examined these same tissues with regard to changes in expression levels of Dippu-AstR. JH biosynthesis peaks on day 5 post-emergence in mated females. In mated females, Dippu-AstR mRNA is expressed at the highest levels on day 6 post-emergence in brain and corpora allata and day 2 post-emergence in midgut. Dippu-AstR is likely responsible for the decline in JH biosynthesis after day 5 post-emergence. Virgin females midgut and CA Dippu-AstR mRNA expression dramatically elevated on days 6 and 7, respectively. Expression of Dippu-AstR was similar in the abdominal ganglia of mated or virgin females. Ovarian Dippu-AstR expression declines to low levels by day 4. Testes also express peaks of Dippu-AstR expression on days 4 and 7. A role for Dippu-AST in testes is yet unknown. / Thesis (Ph.D, Biology) -- Queen's University, 2008-04-22 15:21:55.107

<p><p>Nociceptors are specialized primary sensory neurons that represent the first line of defense against potentially tissue damaging environmental stimuli, and are involved in pathological pain states caused by nerve damage, inflammation and many chronic diseases. In nociception, these neurons detect harmful stimuli and contribute to the reactions to avoid them. Nociceptors transduce noxious stimuli into membrane depolarization, which in turn, triggers action potentials. These action potentials are conducted to synapses in the central nervous system (CNS), resulting in release of neurotransmitters at the presynaptic terminal. The unifying factor in the progression of nociceptive signaling i.e. transduction, action potential propagation, and neurotransmitter release, is the contribution of ion channels. </p><p><p>In this study, I use <italic>Drosophila melanogaster</italic> larvae as a model system to study the contribution of ion channels to nociception. Larvae stimulated with a noxious thermal or mechanical stimulus perform a stereotyped and quantifiable escape behavior. Larvae exhibiting this nocifensive behavior rotate around their long body axis in a corkscrew-like manner thus escaping the damage of the noxious stimulus. This behavior is triggered by the Class IV multidendritic (md) neurons, which are the main larval nociceptors. I describe here, the results of my systematic screen for ion channels required for larval thermal nociception. To perform this screen, I utilized RNAi to knock down the expression of 98% of the predicted ion channels in the <italic>Drosophila</italic> genome. I observed the effects of ion channel knockdown in the thermal nociception behavioral assay. </p><p><p>In addition, I present detailed characterization of an ion channel that I found to be critical for inhibition of nociceptor excitability, the small conductance calcium-activated potassium channel, SK. This channel inhibits both thermal and mechanical nociception. Results of calcium imaging studies show enhanced excitability of larval nociceptors in <italic>SK</italic> mutant animals. My findings support a role for SK function at the sensory afferents, cell body, and axon. </p><p><p>Another candidate ion channel gene, <italic>shadrach</italic>, encodes a Degenerin/Epithelial Na+ channel (DEG/ENaC) that I found to be required for thermal nociception. DEG/ENaCs are conserved in flies, nematodes, and several vertebrates including humans. These channels are expressed in a variety of tissues including kidney epithelia, muscle, and neurons. Members of this superfamily play a role in a host of biological processes including salt homeostasis, neurodegeneration, proprioception, touch transduction, and nociception. RNAi knockdown of <italic>shadrach</italic> results in increased thermal nociceptive threshold. Optogenetic experiments suggest that shadrach functions downstream of transduction. </p><p><p>Furthermore, I identified seven ion channel genes in the thermal nociception screen, which affect nociceptor dendrite morphology. It is possible that thermal nociception behavioral phenotypes in these RNAi mutants are a consequence of the altered dendritic field. Reduction in segmental coverage by the nociceptors may influence the ability to detect noxious stimuli. Future research in our laboratory will establish the relationship between these ion channels, nociceptor development, and nociceptive behavioral output. </p><p><p> <italic>Drosophila melanogaster</italic> is emerging as a powerful model for the study of pain signaling. I have uncovered several candidate ion channel genes that contribute to thermal nociception; of these, <italic>SK</italic> and <italic>shadrach</italic> are required for the response to noxious heat. I have shown that dendritic field coverage is important for the detection of noxious stimuli, and I have identified many candidate genes that are required for normal dendrite morphology.</p> / Dissertation

Two Tomato bushy stunt virus (TBSV) proteins, P19 and P22, have been found to interact with the Nicotiana benthamiana host proteins Hin19 and HFi22 in yeast two,hybrid assays. To determine functional roles of these interacting host proteins, viral induced gene silencing (VIGS) was employed to knock,down their expression. TBSV has been demonstrated to activate a virus,specific antiviral response pathway in N. benthamiana. To characterize this pathway, the antiviral RNAi induced silencing complex (RISC) was isolated from TBSV-infected plants. Additionally, putative RISC-associated proteins were identified in silico and suggested roles for these have been identified through literature and database searches. A further aim was the identification of proteins that coimmunoprecipitate with the TBSV-induced RISC following RISC isolation. A primary aim of this investigation was to identify functional roles for host proteins that interact with the two TBSV 3-terminal encoded proteins, P22 and P19. Each of these has functional roles in viral movement and pathogenicity. In yeast two-hybrid assays, P22 has been shown to interact with HFi22 while P19 interacts with Hin19. VIGS was utilized in attempts to silence the expression of these two host proteins in order to determine their functional roles. VIGS-mediated suppression of the TBSV-interacting proteins Hin19 and HFi22 has not been accomplished. Despite multiple attempts and multiple approaches, these proteins have not been amenable to silencing. In light of this finding, it is proposed that rather than utilizing VIGS to down-regulate protein levels for Hin19 and HFi22, other approaches should be utilized. To characterize the TBSV-mediated RNAi pathway, functionally active antiviral RISC was purified from TBSV-infected N. benthamiana plants using ion-exchange chromatography. This RISC was found to be active only in the degradation of TBSV transcripts, indicating the specificity expected from a programmed RISC. Characterization and identification of proteins that copurify with RISC has not yet been accomplished, though in silico analysis has yielded over 150 putative RISC-associated proteins. Of these, a subset has been identified as highly likely candidates based upon function and/or homology to RISC-associated proteins in non-plant organisms, and a model for the TBSV-induced antiviral pathway has been proposed.