The instructive signals received by T cells during the programming stages of activation will determine the fate of effector and memory populations generated during an immune response. Members of the tumor necrosis factor (TNF) superfamily play an essential role in influencing numerous aspects of T cell adaptive immune responses including cell activation, differentiation, proliferation, survival, and apoptosis. My thesis dissertation describes the involvement of two such members of the TNF superfamily, TNF and FasL, and their influence on the fate of T cells early during responses to viral infections and to the induction of transplantation tolerance. TNF is a pleiotropic pro-inflammatory cytokine that has an immunoregulatory role in limiting the magnitude of T cell responses during a viral infection. Our laboratory discovered that one hallmark of naïve T cells in secondary lymphoid organs is their unique ability to rapidly produce TNF after activation and prior to acquiring other effector functions. I hypothesized that T cell-derived TNF will limit the magnitude of T cell responses. The co-adoptive transfer of wild type (WT) P14 and TNF-deficient P14 TCR transgenic CD8+ T cells, that recognize the GP33 peptide of lymphocytic choriomeningitis virus (LCMV), into either WT or TNF-deficient hosts demonstrated that the donor TNF-deficient P14 TCR transgenic CD8+ T cells accumulate to higher frequencies after LCMV infection. Moreover, these co-adoptive transfer experiments suggested that the effect of T cell-derived TNF is localized in the microenvironment, since the TNF produced by WT P14 TCR transgenic CD8+ T cells did not prevent the accumulation of TNF-deficient P14 TCR transgenic CD8+ T cells. To determine if T cell-produced TNF is acting on professional APC to suppress the generation of virus-specific T cell responses, I performed co-adoptive transfer experiments with WT P14 TCR transgenic CD8+ and TNF-deficient P14 TCR transgenic CD8+ T cells into TNFR1/2 (1 and 2) deficient mice. These experiments demonstrated that the absence of TNFR1/2 signaling pathway in the host cells resulted in a greater accumulation of WT P14 TCR transgenic CD8+ T cells, thereby considerably diminishing the differences between donor WT P14 TCR transgenic CD8+ and donor TNF-deficient P14 TCR transgenic CD8+ T cells. The increased frequency and absolute numbers of WT P14 TCR transgenic CD8+ T cells in TNFR1/R2 deficient recipients suggests that one mechanism for the suppressive effect of T cell-derived TNF on antigen-specific T cells occurs as a result of TNFR signaling in the host cells. However, the donor TNF-deficient P14 TCR transgenic CD8+T cells still accumulated to higher frequency and numbers compared to their donor WT transgenic counterparts. Together, these findings indicate that T cell-produced TNF can function both in an autocrine and a paracrine fashion to limit the magnitude of anti-viral T cell responses. Given the immunoregulatory role of TNF and the ability of peripheral naïve T cells to produce this cytokine, I questioned at what stage of development do T cells become licensed to produce this cytokine. The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. I hypothesized that naïve T cells emigrating from the thymus will be competent to produce TNF only after undergoing a maturation process in the periphery. To test this hypothesis, I compared cytokine profiles of CD4+ and CD8+single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics with respect to the upregulation of CD25 and CD69 following stimulation. The reduced ability of SP thymocytes to produce TNF correlated with a decreased level of detectable TNF message following stimulation when compared to splenic counterparts. Stimulation of SP thymocytes in the context of a splenic environment did not fully enable TNF production, suggesting an intrinsic defect in their ability to produce TNF as opposed to a defect in antigen presentation. Using a thymocyte adoptive transfer model, I demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs identified by the expression of green fluorescent protein (GFP) (NG-BAC transgenic mice), showed a significantly enhanced ability to express TNF relative to SP thymocytes, but not to the extent of MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs. This highlights the functional heterogeneity of the naïve T cell pool (with respect to varying degrees of TNF production) during early T cell activation that can contribute to the many subsequent events that shape the course of an immune response. The productive activation of naïve T cells requires at least initial two signals; the first being through the TCR and the second is the engagement of co-stimulatory molecules on the surface of the T cells. T cells activated in the absence of co-stimulation become anergic or undergo cell death. Agents that block co-stimulation of antigen-specific T cells are emerging as an alternative to immunosuppressive drugs to prolong allograft survival in transplant recipients. Targeted blockade of CD154-CD40 interactions using a αCD154 monoclonal antibody (MR1) with a simultaneous transfusion of allogeneic splenocytes (donor specific transfusion or DST) efficiently induces tolerance to allografts. This co-stimulation blockade-induced tolerance is characterized by the deletion of host alloreactive T cells within 24 hours of treatment. Toll-like receptor (TLR) agonists abrogate tolerance induced by co-stimulation blockade by impairing the deletion of host alloreactive T cells and resulting in allograft rejection. The goal of my study was to determine the underlying molecular mechanisms that protect host alloreactive T cells from early deletion after exposure to TLR agonists. I hypothesized that TLR ligands administered during co-stimulation blockade regimen differentially regulate the expression of pro- and anti-apoptotic molecules in alloreactive T cells, during the initial stages of activation thereby preventing deletion. To test this hypothesis, I used syngeneic bone marrow chimeric mice containing a trace population of alloreactive KB5 TCR transgenic CD8+ T cells (KB5 Tg CD8+ T cells) that recognize H-2Kb as an alloantigen. I show here that KB5-CD8+ T cells downregulate CD127 (IL-7R!) and become apoptotic as early as 12 hrs after co-stimulation blockade. In contrast, KB5 Tg CD8+ T cells from mice treated with bacterial lipopolysaccaride (LPS) during co-stimulation blockade failed to become apoptotic, although CD127 was downregulated. Examination of the mRNA expression profiles of several apoptotic genes in purified KB5 CD8+ T cells from mice treated with DST+anti-CD154 for 12 hrs revealed a significant upregulation of FasL mRNA expression compared to the untreated counterparts. However, in vitro FasL blockade or in vivo cytotoxicity experiments with mice deficient in Fas or FasL indicated that the Fas-FasL pathway might not be crucial for tolerance induction. Another pro-apoptotic molecule BIM was upregulated in alloreactive T cells during co-stimulation blockade. This suggests that both the Fas pathway and BIM may be playing complementary roles in inducing deletional tolerance. Although FasL expression was diminished in alloreactive T cells in the presence of LPS, BIM expression was not diminished, suggesting that alloreactive T cells may still be vulnerable to undergo apoptosis. Concomitantly, I also found that LPS treatment during co-stimulation blockade resulted in non-specific upregulation of Fas expression in alloreactive T cells and non-transgenic T cells (CD4+ and CD8+). I demonstrate here that treatment with Fas agonistic antibody in vitrofor 4 hours can selectively induce apoptosis of alloreactive T cells that were believed to be refractory to apoptosis during LPS treatment. I speculate that under these conditions, deletion may be occurring due to the involvement of both Fas and BIM. Further, the mRNA expression profile revealed interleukin-10 (IL-10) as a molecule induced in alloreactive T cells during LPS treatment. Analysis of serum confirmed the systemic expression of IL-10 protein in mice treated with LPS during co-stimulation blockade. I hypothesized that LPS-induced IL-10 can have an anti-apoptotic role in preventing the deletion of alloreactive T cells and mediating allograft rejection. Contrary to my hypothesis, I found that IL-10 KO mice rejected allogeneic target cells similar to their WT counterparts, suggesting that IL-10 may not be required for LPS-mediated abrogation of tolerance induction. In addition to the systemic induction of IL-10, LPS also induced cytokines such as interleukin-6 (IL-6), TNF and interferon-γ (IFN-γ). These findings suggest that both Fas-FasL and BIM mediated apoptotic pathways may play complementary roles in inducing the early deletion of activated alloreactive T cells during tolerance induction. On the other hand, the mechanism of LPS mediated abrogation of tolerance induction can not be attributed to IL-10 alone as it may be playing a synergistic role along with other proinflammatory cytokines that may in turn result in the prevention of alloreactive T cell death during this process. Most importantly, these findings indicate that despite emerging from a pro-inflammatory cytokine milieu, alloreactive T cells are still susceptible to undergo Fas-mediated apoptosis during the first 24 hours after co-stimulation blockade and LPS treatment. Therefore, targeting the Fas-FasL pathway to induce deletion of alloreactive T cells during the peri-transplant period may still be a potential strategy to improve the efficacy of co-stimulation blockade induced transplantation tolerance during an environmental perturbation such as inflammation or infection.

During development, axons respond to a variety of guidance cues in the environment to navigate to the proper targets. Sonic hedgehog (Shh), a classical morphogen, has been shown to function as a guidance factor that directly acts on the growth cones of various types of axons. We previously found that Shh affects retinal ganglion cell (RGC) axonal growth and navigation in a concentration-dependent manner. However, the signaling pathways that mediate such events are still unclear. In this thesis, we show that high concentrations of Shh induce growth cone collapse and repulsive turning of the chick RGC through rapid increase of Ca2+ in the growth cone, and specific activation of PKCα and Rho signaling pathways. We further found that integrin linked kinase (ILK) acts as an immediate downstream effector of PKCα. PKCα directly phosphorylates ILK in vitro at two previously unidentified sites threonine-173 and -181. Inhibition of PKCα, Rho, and ILK by pharmacological inhibitors and/or dominant-negative approaches abolished the negative effects of high-concentration of Shh. We provide evidence that Rho likely functions downstream of PKC and suggest that PKC, Rho and ILK may cooperatively mediate the negative effects of high concentrations of Shh. Furthermore, retroviral expression of dominant-negative constructs of PKCα (DN-PKCα) and ILK-double mutants (ILK-DM) resulted in misguidance of RGC axons at the optic chiasm in vivo. These results demonstrate that new signaling pathways composed of PKCα, Rho, and ILK play an important role in Shh-induced axonal chemorepulsion. In contrast, we show that attractive axonal turning in response to low concentrations of Shh is independent of PKCα, but requires the activity of cyclic nucleotides cAMP. Taken together, our results suggest that the opposing effects of Shh on axon guidance are mediated by different signaling pathways.

Post-transcriptional regulation of gene expression sits at the core of proteomic complexity; trans-acting factors that regulate RNA localization and translation capacity are thus indispensible. In this thesis, I present studies of the cytoplasmic polyadenylation element binding protein (CPEB), a sequence specific RNA-binding protein important for cell cycle progression and neural synaptic plasticity. I focus on CPEB because the activity of RNA-binding proteins affects the destiny of their mRNA substrates. As presented in Chapter II, CPEB, though mostly cytoplasmic at steady state, shuttles between the nucleus and the cytoplasm. Surprisingly, the RNA recognition motifs are essential for the nuclear localization. CPEB associates with the polyadenylation machinery in both compartments, suggesting it is involved in both nuclear mRNA processing and cytoplasmic translational regulation. Moreover, the nuclear translocalization is critical to relay a tight translation repression on CPE-containing mRNAs. Chapter III focuses on the regulation of CPEB dimerization. CPEB dimerizes through the RNA-binding domains to inhibit its own RNA binding ability in a cell cycle-dependent manner. By dimerizing, CPEB has enhanced binding to protein destruction factors so that robust active degradation occurs in the later cell cycle. The degradation of CPEB is required for translation activation of a subset of mRNAs and cell cycle progression. In addition, dimerization protects cells from being overloaded with excess CPEB. In sum, the localization and dimerization status of CPEB is dynamic and highly regulated; they in turn regulate the activity of CPEB, which results in responsive translation control. These studies provide a strong foundation to decipher CPEB-mediated gene expression.

Newcastle disease virus (NDV) belongs to the Paramyxoviridae, a family of enveloped RNA viruses that includes many important human and animal pathogens. Although many aspects of the paramyxovirus life cycle are known in detail, our understanding of the mechanisms regulating paramyxovirus assembly and release are poorly understood. For many enveloped RNA viruses, it has recently become apparent that both viral and host cellular determinants coordinate the proper and efficient assembly of infectious progeny virions. Utilizing NDV as a model system to explore viral and cellular determinants of paramyxovirus assembly, we have shown that host cell membrane lipid raft domains serve as platforms of NDV assembly and release. This conclusion was supported by several key experimental results, including the exclusive incorporation of host cell membrane raftassociated molecules into virions, the association of structural components of the NDV particle with membrane lipid raft domains in infected cells and the strong correlation between the kinetics of viral protein dissociation from membrane lipid raft domains and incorporation into virions. Moreover, perturbation of infected cell membrane raft domains during virus assembly resulted in the disordered assembly of abnormal virions with reduced infectivity. These results further established membrane raft domains as sites of virus assembly and showed the integrity of these domains to be critical for the proper assembly of infectious virions. Although specific viral protein-protein interactions are thought to occur during paramyxovirus assembly, our understanding of how these interactions are coordinated is incomplete. While exploring the mechanisms underlying the disordered assembly of non-infectious virions in membrane raft-perturbed cells, we determined that the integrity of membrane raft domains was critical in the formation and virion incorporation of a complex consisting of the NDV attachment (HN) and fusion (F) proteins. The reduced virus-to-cell membrane fusion capacity of particles released from membrane raft-perturbed cells was attributed to an absence of the HN – F glycoprotein-containing complex within the virion envelope. This result also correlated with a reduction of these glycoprotein complexes in membrane lipid raft fractions of membrane raft-perturbed cells. Specifically, it was determined that the formation of newly synthesized HN and F polypeptides into the glycoprotein complex destined for virion incorporation was dependent on membrane lipid raft integrity. Finally, a novel virion complex between the ribonucleoprotein (RNP) structure and the HN attachment protein was identified and characterized. Unlike the glycoprotein complex, the detection of the RNP – HN protein-containing complex was not affected by membrane raft perturbation during virus assembly in the cell. The biological importance of this novel complex for the proper assembly of an infectious progeny virion is currently under investigation. The results presented in this thesis outline the role of host cell membrane lipid raft domains in the assembly and release processes of a model paramyxovirus. Furthermore, the present work extends our understanding of how these particular host cell domains mechanistically facilitate the ordered assembly and release of an enveloped RNA virus.

Repairing a chromosomal DNA double strand break is essential for survival and maintenance of genomic integrity of a eukaryotic organism. The eukaryotic cell has therefore evolved intricate mechanisms to counteract all sorts of genomic insults in the context of chromatin structure. Modulating chromatin structure has been crucial and integral in regulating a number of conserved repair processes along with other fundamental genomic processes like replication and transcription. The work in this dissertation has focused on understanding the role of chromatin remodeling enzymes in the repair of a chromosomal DNA double strand break by homologous recombination. This has been approached by recapitulating the biochemical formation of recombination intermediates on chromatin in vitro. In this study, we have demonstrated that the mere packaging of DNA into nucleosomal structure does not present a barrier for successful capture of homologous DNA sequences, a central step of the biochemical pathway of recombinational repair. It is only the assembly of heterochromatin-like more complex nucleo-protein structure that presents additional constraints to this key step. And, this additional constraint can be overcome by the activities of ATP-dependent chromatin remodeling enzymes. These findings have great implications for our perception of the mechanism of the recombinational repair process of a chromosomal DNA double strand break within the eukaryotic genome.

Understanding the mechanisms of memory is vital in making sense of the continuity of the self, our experience of time and of the relation between mind and body. The invertebrate Drosophila melanogaster offers us an opportunity to study and comprehend the overwhelming complexity of memory on a smaller scale. The work presented here investigates the neural circuitry in the fly brain required for olfactory memory processing. Our observation that Dorsal Paired Medial (DPM) neurons, which project only to mushroom body (MB) neurons, are required during memory storage but not for acquisition or retrieval, led us to revisit the role of MB neurons in memory processing. We show that neurotransmission from the α'β' subset of MB neurons is required to acquire and stabilize aversive and appetitive odor memory but is dispensable during memory retrieval. In contrast neurotransmission from MB αβ neurons is only required for memory retrieval. These data suggest a dynamic requirement for the different subsets of MB neurons in memory and are consistent with the notion that recurrent activity in a MB α'β' neuron-DPM neuron loop is required to consolidate memories formed in the MB αβ neurons. Furthermore, we show that a single two-minute training session pairing odor with an ethologically relevant sugar reinforcement forms long-term appetitive memory that lasts for days. This robust, stable LTM is protein-synthesis-, Creb- and radish-dependent and relies on the activity in the DPM neuron and mushroom body α'β' neuron circuit during the first hour after training and mushroom body αβ neuron output during retrieval. Lastly, experiments feeding and/or starving flies after training reveals a critical motivational drive that enables memory retrieval. Neural correlates of motivational states are poorly understood, but using our assay we found a neural mechanism that accounts for this motivation-state-dependence. We demonstrate a role for the Neuropeptide F (dNPF) circuitry, which led to the identification of six dopaminergic MB-MP neurons that innervate the mushroom bodies as being critical for appetitive memory performance. Directly blocking the MB-MP neurons releases memory performance in fed flies whereas stimulating them suppresses memory performance in hungry flies. These studies provide us with an enhanced knowledge of systems level memory processing in Drosophila.

Post-transcriptional regulation of gene expression is a mechanism that governs developmental and cellular events in metazoans. In early embryogenesis, transcriptionally quiescent cells depend upon maternally supplied factors such as RNA binding proteins and RNA that control key decisions. Morphogen gradients form and in turn pattern the early embryo generating different cell types and spatial order. In the nematode Caenorhabditis elegans, the early embryo relies upon several RNA binding proteins that control mRNA stability, translation efficiency, and/or mRNA localization of cell fate determinants essential for proper development. MEX-5 and MEX-3 are two conserved RNA-binding proteins required to pattern the anterior/posterior axis and early embryo. Mutation of either gene results in a maternal effect lethal phenotype with proliferating posterior muscle into the anterior blastomeres (Muscle EXcess). Several cell-fate determinants are aberrantly expressed in mex-5 and mex-3 embryos. Both proteins are thought to interact with cis-regulatory elements present in 3’-UTRs of target RNAs controlling their metabolism. However, previous studies failed to demonstrate that these proteins regulate maternal transcripts directly. This dissertation presents a thorough assessment of the RNA binding properties of MEX-5 and MEX-3. Quantitative biochemical approaches were used to determine the RNA binding specificity of both proteins. MEX-5 has a relaxed specificity, binding with high affinity to linear RNA containing a tract of six or more uridines within an eight-nucleotide window. This is very different from its mammalian homologs Tristetraprolin (TTP) and ERF-2. I was able to identify two amino acids present within the MEX-5 RNA binding domain that are required for the differential RNA recognition observed between MEX-5 and TTP. MEX-3 on the other hand is a specific RNA binding protein, recognizing a bipartite element with flexible spacing between two four-nucleotide half-sites. I demonstrate that this element is required for MEX-3 dependent regulation in vivo. Previous studies only identify a small number of candidate regulatory targets of MEX-5 and MEX-3. The defined sequence specificity of both proteins is used to predict new putative targets that may be regulated by either protein. Collectively, this study examines the RNA binding properties of MEX-5 and MEX-3 to clarify their role as post-transcriptional regulators in nematode development.

KCNE peptides are a class of type I transmembrane ß-subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K+ channels. Accordingly, mutations that affect the assembly and trafficking of K+ channel/KCNE complexes give rise to disease. The cellular mechanisms that oversee KCNE peptide assembly with voltage-gated K+ channels have yet to be elucidated. In Chapter II, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with KCNQ1 K+ channel subunits. Co-assembly with KCNQ1 channel subunits mediates efficient forward trafficking of KCNE1 peptides through the biosynthetic pathway and results in cell surface expression. KCNE1 peptides possess two N-linked glycosylation sites on their extracellular N-termini. Progression of KCNE1 peptides through the secretory pathway can be visualized through maturation of N-glycans attached to KCNE1. In Chapter III, we examine the kinetics and efficiency of N-linked glycan addition to KCNE1 peptides. Mutations that prevent glycosylation of KCNE1 give rise to the disorders of arrhythmia and deafness. We show that KCNE1 acquires N-glycans co- and post-translationally. Mutations that prevent N-glycosylation at the co-translational site have a long range effect on the disruption of post-translational glycosylation and suggest a novel biogenic mechanism for disease. In Chapter IV, we determine the presence of an additional post-translational modification on KCNE1 peptides. We define specific residues as sites of attachment of this modification identified as sialylated O-glycans and show that it occurs in native cardiac tissues where KCNE1 plays a role in the maintenance of cardiac rhythm. Taken together, these observations demonstrate the importance of having correctly assembled K+ channel/KCNE complexes at the cell surface for their proper physiological function and define a role for the posttranslational modifications of KCNE peptides in the proper assembly and trafficking of K+ channel/KCNE complexes.

Lung cancer is the leading cause of cancer-related mortality worldwide. The main risk factor associated with lung cancer is cigarette smoking. Research through the years suggests that nicotine in cigarettes promotes lung cancer by activating signaling pathways that lead to cell proliferation, cell survival, angiogenesis, and metastasis. Nicotine’s cellular actions are mediated by its cognate receptors, nicotinic acetylcholine receptors (nAChRs). Here, I describe the expression levels of all known human nAChR subunit genes in both normal and lung cancer cells. Of note, the genes encoding the α5, α3, and β4 subunits (CHRNA5/A3/B4) are over-expressed in small cell lung carcinoma (SCLC), the most aggressive form of lung cancer. This over-expression is regulated by ASCL1, a transcription factor important in normal lung development and lung carcinogenesis. The CHRNA5/A3/B4 locus has recently been the focus of a series of genetic studies showing that polymorphisms in this region confer risk for both nicotine dependence and lung cancer. I show that CHRNA5/A3/B4 depletion results in decreased SCLC cell viability. Furthermore, while nicotine promotes SCLC cell viability and tumor growth, blockade of α3β4 nAChRs inhibits SCLC cell viability. These results suggest that increased expression and function of nAChRs, specifically the α3β4α5 subtype, potentiate the effects of nicotine in SCLC. This dual hit from the carcinogens in tobacco and the cancer-promoting effects of nicotine, may provide a possible mechanism for the increased aggressiveness of SCLC. In addition, nAChRs can be activated by the endogenous ligand, acetylcholine, which acts as an autocrine/paracrine growth factor in SCLC. Increased function of α3β4α5 nAChRs in SCLC could also potentiate acetylcholine’s mitogenic effects. This mechanism, combined with other known autocrine/paracrine growth loops in SCLC, may help explain the ineffectiveness of available therapies against SCLC. In an effort to add to the current arsenal against SCLC, I screened a 1280-compund library using a bioluminescence-based viability assay I developed for high-throughput applications. Primary screening, followed by secondary and tertiary verification, indicate that pharmacologically active compounds targeting neuroendocrine markers inhibit SCLC cell viability.

The nervous system is made up of two major cell types, neurons and glia. The major distinguishing feature between neuronal cells and glial cells is that neurons are capable of transmitting action potentials while glial cells are electrically incompetent. For over a century glial cells were neglected and it was thought they existed merely to provide trophic and structural support to neurons. However, in the past few decades it has become increasingly clear that glial cell functions underlie almost all aspects of nervous system development, maintenance, and health. During development, glia act as permissive substrates for axons, provide guidance cues, regulate axon bundling, facilitate synapse formation, refine synaptic connections, and promote neuronal survival. In the mature nervous system glial cells regulate adult neurogenesis through phagocytosis, act as the primary immune cell, and contribute to complex processes such as learning and memory. In recent years, glial cells have also become a primary focus in the study of neurodegenerative diseases. Mounting evidence shows that glial cells exert both beneficial as well as detrimental effects in the pathology of several nervous system disorders, and modulation of glial activity is emerging as a viable therapeutic strategy for many diseases. Although glial cells are critical to the proper development and functioning of the nervous system, there is still relatively little known about the molecular mechanisms used by glial cells, how they exert their effects on neurons, and how glia and neurons communicate. Despite the relative simplicity and small size of the Drosophila nervous system, glial cell organization and function in flies shows a remarkable complexity similar to vertebrate glial cells. In this study I use Drosophila as a model organism to study cellular and molecular mechanisms of glial clearance of axonal debris after acute axotomy. In chapter two of this thesis, I characterize three distinct subtypes of glial cells in the adult brain; cell body glia which ensheath neuronal cell bodies in the cortex region of the brain, astrocyte like glial cells which bear striking morphological similarity to mammalian astrocytes and share common molecular components, and ensheathing glial cells which I show act as the primary phagocytic cell type in the neuropil region of the brain. In addition, I identify dCed-6, the ortholog of mammalian GULP, as a necessary component of the glial phagocytic machinery. In chapter three of this thesis, I perform a candidate based, in vivo, RNAi screen to identify novel genes involved in the glial engulfment of degenerating axon material. The Gal4/UAS system was used to drive UAS-RNAi for approximately 300 candidate genes with the glial specific repo-Gal4 driver. Two assays were used as a readout in this screen, clearance of axon material five days after injury, and Draper upregulation one day after maxillary palp or antennal injury. Overall, I identified 20 genes which, when knocked down specifically in glial cells, result in axon clearance defects after injury. Finally, in chapter four I identify Stat92E as a novel glial gene required for glial phagocytic function. I show that Stat92E regulates both basal and injury induced Draper expression. Injury-induced Draper expression is transcriptionally regulated through a Stat92E dependent non-canonical signaling mechanism whereby signaling through the Draper receptor activates Stat92E which in turn transcriptionally activates draper through a binding site located in the first intron of Draper. Draper represents only the second receptor known to positively regulate Stat92E transcriptional activity under normal physiological conditions.