Cell signaling is a list of chemical reactions that are used for intercellular and intracellular communication. Signaling pathways denote these chemical reactions in a systematic manner. Today, many signaling pathways are constructed by several experimental methods. However there are still many communication skills of cells that are needed to be discovered. RNAi system allows us to see the phenotypes when some genes are removed from living cells. By observing these phenotypes, we can build signaling pathways. However it is costly in terms of time and space complexity. Furthermore, there are some interactions RNAi data cannot distinguish that results in many different signaling pathways all of which are consistent with the RNAi data. In this thesis, we combine genetic algorithms with some greedy approaches to find the topologies that fit the Boolean single knock-down RNAi experiments. Our algorithm finds nearly all of the results for small inputs in a few minutes. It can also find a significant number of results for larger inputs. Then we eliminate isomorphic topologies from the output set of this algorithm. This process fairly reduces the number of topologies. Afterwards we offer a simple scheme for suggesting new wet-lab RNAi experiments which is necessary to have a complete approach to find the actual network. Also we describe a new activation and deactivation model for pathways when the activation of the phenotype after RNAi knock-downs are given as weighted variables. We adapt the first genetic algorithm approach to this model for directly finding the most possible network.

Reconstructing the topology of a signaling network by means of RNA interference (RNAi) technology is an underdetermined problem especially when a single gene in the network is knocked down or observed. In addition, the exponential search space limits the existing methods to small signaling networks of size 10-15 genes. In this thesis, we propose integrating RNAi data with a reference physical interaction network. We formulate the problem of signaling network reconstruction as finding the minimum number of edit operations on a given reference network. The edit operations transform the reference network to a network that satisfy the RNAi observations. We show that using a reference network does not simplify the computational complexity of the problem. Therefore, we propose an approach that provides near optimal results and can scale well for reconstructing networks up to hundreds of components. We validate the proposed method on synthetic and real datasets. Comparison with the state of the art on real signaling networks shows that the proposed methodology can scale better and generates biologically significant results.

High mobility group box 1 (HMGB-1) protein is a non-histone chromosomal protein. As a DNA binding protein, HMGB-1 is involved in the maintenance of nucleosome structure, regulation of gene transcription and it is active in DNA recombination and repair. It has been known that HMGB-1 is a late mediator of endotoxemia and sepsis. HMGB-1 is released from activated macrophages, induces the release of other proinflammatory mediators, and mediates cell death when overexpressed. We speculated that the course of sepsis maybe different without the involvement of HMGB-1. The aims of this study are to investigate the role of HMGB-1 in mediating sepsis and to observe the effects by using RNAi to affect the production of HMGB-1. Lipopolysaccharide (LPS) was used to simulate sepsis in culture as well as stimulate the release of HMGB-1 from RAW 264.7 cells. Levels of HMGB-1 in the culture medium were subsequently measured by Western blot. Other proinflammatory cytokines (TNF-£\, IL-6 and TGF-£]) were measured by ELISA. HMGB-1 could not be detected in the culture medium in the absence of LPS stimuli, but after 0.5 £gg/ml LPS treatment HMGB-1 release could be detected. HMGB-1 the amount of released from LPS activated RAW 264.7 cells was in a time- and dose-dependent manner. The present study demonstrated that RNAi in the treatment of LPS-stimulated RAW264.7 cells resulted in the blockade of HMGB-1 and decreased LPS-induced inflammatory response. The results demonstrated that HMGB-1 plays a pivotal role in macrophage inflammatory responses by modulating the production of inflammatory mediators.

Influenza is an important human pathogen that causes fatal disease in 250,000-500,000 people worldwide each year. Because of high levels of variation between influenza strains, vaccines are not always effective and must be administered annually. Influenza virus, which replicates primarily in the lung epithelium, encodes only 10 proteins and relies heavily on host products to replicate. Determining which cellular factors are important for influenza replication represents an important area of virology and cellular biology research, and could elucidate proteins or pathways to target for antiviral therapies. We developed a high throughput screening method in primary human bronchial epithelium (HBECs) to identify novel regulators of influenza replication. We first used this method to functionally examine 1745 genes that were identified as potential influenza regulators due to transcriptional regulation by virus or viral products, direct interaction with viral proteins via yeast two-hybrid, or through computational analysis. This screen confirmed some known regulators of influenza replication while identifying novel viral interactors as influenza regulators (e.g. USHBP1, ZMAT4). We also found that the WNT, p53, and ER stress pathways, among others, affect viral replication and interferon production. The life cycle of influenza involves extensive intracellular trafficking of viral components. We again used RNAi to systematically examine the roles of vesicle, RNA, and protein trafficking genes in the production of infectious influenza A virus in primary lung cells. Among the factors that significantly impact viral infection, we identify a set of five genes with strong antiviral effects that are mutated in patients with Hermansky-Pudlak syndrome (HPS). Depletion of HPS genes leads to elevated viral RNA at an early stage of influenza infection prior to transcription. In contrast, depletion of these genes does not alter the innate immune response to virus or interferon. Using an HPS-1 patient cell line, we find an increase in viral fusion to endosomal compartments but no change in viral binding to the cell surface or entry into the early endosome. Our studies uncover a potential role for many trafficking factors in the influenza life cycle, and point to an HPS1-dependent process that inhibits viral entry prior to viral membrane fusion.

The innate immune system is of central importance to the early containment of infection. When receptors of innate immunity recognize molecular patterns on pathogens, they initiate an immediate immune response by inducing the expression of cytokines and other host defense genes. Altered expression or function of the receptors, the molecules that mediate the signal transduction cascade, or the cytokines themselves can predispose individuals to infectious or autoimmune diseases. Here we used genomic approaches to uncover novel components underlying the innate immune response to cytosolic DNA and to characterize variation in the innate immune responses of human dendritic cells to bacterial and viral ligands. In order to identify novel genes involved in the cytosolic DNA sensing pathway, we first identified candidate proteins that interact with known signaling molecules or with dsDNA in the cytoplasm. We then knocked down 809 proteomic, genomic, or domain-based candidates in a high-throughput siRNA screen and measured cytokine production after DNA stimulation. We identified ABCF1 as a critical protein that associates with DNA and the known DNA-sensing components, HMGB2 and IFI16. We also found that CDC37 regulates stability of the signaling molecule, TBK1, and that chemical inhibition of CDC37 as well as of several other pathway regulators (HSP90, PPP6C, PTPN1, and TBK1) potently modulates the innate immune response to DNA and to retroviral infection. These proteins represent potential therapeutics targets for infectious and autoimmune diseases that are associated with the cytosolic DNA response. We also developed a high-throughput functional assay to assess variation in responses of human monocyte-derived dendritic cellsto LPS (receptor: TLR4) or influenza (receptors: RIG-I and TLR3), with the goal to ultimately map genetic variants that influence expression levels of pathogen-responsive genes. We compared the variation in expression between the dendritic cells of 30 different individuals, and within paired samples from 9 of these individuals collected several months later. We found genes that have significant inter- vs. intra-individual ariation in response to the stimuli, suggesting that there is a substantial genetic component underlying variation in these responses. Such variants may help to explain differences between individuals’ risk for infectious, autoimmune, or other inflammatory diseases.

RNA interference (RNAi) potently and specifically induces gene knockdown, and its potential for reverse genetics in Caenorhabditis elegans is enormous. However, even in these nematodes, RNAi can be induced more effectively via enhanced RNAi (Eri) mutant backgrounds. With advances in small RNA sequencing, evidence has suggested that the eri pathway plays an endogenous gene regulatory role, which competes with experimentally introduced RNAi triggers for limiting resources. However, the nature, cellular location, and physiological consequences of this small RNA pathways competition remain unclear. To answer these questions, I first fully characterized the genetic phenotypes of all known Eri mutants. I discovered that different components of the eri pathway have subtle differences upon mutation, which affects more than exogenous RNAi. I then attempted to screen for novel enhanced RNAi mutants, guided by hypothetical mechanisms or tissues of expression not associated with known mutants. After these attempts, I fully characterized the genetic mechanisms that account for enhanced RNAi. Surprisingly, I discovered that the nuclear Argonaute nrde-3 and the peri-nuclear P-granule component pgl-1 are necessary and sufficient for an Eri response. Finally, I examined the impact of the competition among microRNA, endogenous siRNA, and exogenous RNAi pathways. I discovered that C. elegans develops slower upon perturbations to its normal flux of small RNA pathways. Insights from these phenotypes and genetic mechanisms shed light on the importance of small RNA biology and offer a novel suite of tools for sensitizing RNAi in broader contexts, especially given the deep evolutionary conservation of most eri-associated genes.

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17β-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 → S phase progression. Surprisingly, TCDD alone induced G0/G1 → S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 → S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 → S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.

In insects, ecdysis is an important feature of growth and development and is tightly controlled by a variety of neuropeptides. In holometabolous insects, crustacean cardioactive peptide (CCAP) is one of many factors that regulate ecdysis behaviours; however, not much is known about the control of ecdysis in hemimetabolous insects. In this thesis, the CCAP-signaling pathway is shown to be essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The cDNA sequence of the CCAP gene has been cloned from the R. prolixus central nervous system (CNS) and the functional role of CCAP as a neuromodulator/neurotransmitter demonstrated. Specifically, the expression of RhoprCCAP in CNS neurons producing extensive CCAP-like immunoreactive processes within the neuropile indicates that CCAP plays central roles in coordination of other neurons. RhoprCCAP also acts as a neurohomone/neuromodulator released peripherally to coordinate many tissues. Thus, CCAP-like immunoreactive processes are found in neurohemal sites and also on peripheral tissues. The RhoprCCAP receptor (RhoprCCAPR) has been cloned and shown to be a G-protein coupled receptor (GPCR). RhoprCCAPR expression is observed in the CNS and certain peripheral tissues of R. prolixus. Also, CCAP stimulates hindgut contractions and increases the heartbeat rate in a dose-dependent manner. The involvement of CCAP in R. prolixus ecdysis has been investigated. Up-regulation of the RhoprCCAP transcript in the CNS and the RhoprCCAP receptor (RhoprCCAPR) transcript in the CNS and specific peripheral tissues was observed immediately prior to ecdysis. Also, decreasing staining intensity of CCAP-like immunoreactivity in neurons immediately following ecdysis indicates the release of CCAP during ecdysis. The critical importance of the CCAP-signalling pathway was further demonstrated by knockdown of the RhoprCCAP and RhoprCCAPR transcripts utilizing double stranded RNA interference. Insects with these transcripts knocked down have high mortality (up to 84%), typically at the expected time of ecdysis, or have ecdysis extremely delayed. Taken together, this thesis demonstrates that RhoprCCAP plays a crucial role in regulating ecdysis behaviours in R. prolixus, and clearly shows the conserved nature of the CCAP-signaling pathway in ecdysis for both holometabolous and hemimetabolous insects.

In insects, ecdysis is an important feature of growth and development and is tightly controlled by a variety of neuropeptides. In holometabolous insects, crustacean cardioactive peptide (CCAP) is one of many factors that regulate ecdysis behaviours; however, not much is known about the control of ecdysis in hemimetabolous insects. In this thesis, the CCAP-signaling pathway is shown to be essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The cDNA sequence of the CCAP gene has been cloned from the R. prolixus central nervous system (CNS) and the functional role of CCAP as a neuromodulator/neurotransmitter demonstrated. Specifically, the expression of RhoprCCAP in CNS neurons producing extensive CCAP-like immunoreactive processes within the neuropile indicates that CCAP plays central roles in coordination of other neurons. RhoprCCAP also acts as a neurohomone/neuromodulator released peripherally to coordinate many tissues. Thus, CCAP-like immunoreactive processes are found in neurohemal sites and also on peripheral tissues. The RhoprCCAP receptor (RhoprCCAPR) has been cloned and shown to be a G-protein coupled receptor (GPCR). RhoprCCAPR expression is observed in the CNS and certain peripheral tissues of R. prolixus. Also, CCAP stimulates hindgut contractions and increases the heartbeat rate in a dose-dependent manner. The involvement of CCAP in R. prolixus ecdysis has been investigated. Up-regulation of the RhoprCCAP transcript in the CNS and the RhoprCCAP receptor (RhoprCCAPR) transcript in the CNS and specific peripheral tissues was observed immediately prior to ecdysis. Also, decreasing staining intensity of CCAP-like immunoreactivity in neurons immediately following ecdysis indicates the release of CCAP during ecdysis. The critical importance of the CCAP-signalling pathway was further demonstrated by knockdown of the RhoprCCAP and RhoprCCAPR transcripts utilizing double stranded RNA interference. Insects with these transcripts knocked down have high mortality (up to 84%), typically at the expected time of ecdysis, or have ecdysis extremely delayed. Taken together, this thesis demonstrates that RhoprCCAP plays a crucial role in regulating ecdysis behaviours in R. prolixus, and clearly shows the conserved nature of the CCAP-signaling pathway in ecdysis for both holometabolous and hemimetabolous insects.

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