Genome regulation is an extremely complex phenomenon. There are various mechanisms in place to ensure smooth performance of the organism. Post-transcriptional regulation of gene expression is one such mechanism. Many proteins bind to mRNAs and regulate their translation. In this thesis, I have focused on the Cytoplasmic Polyadenylation Element Binding family of proteins (CPEB1-4); a group of sequence specific RNA binding proteins important for cell cycle progression, senescence, neuronal function and plasticity. CPEB protein binds mRNAs containing a short Cytoplasmic Polyadenylation Element (CPE) in 3’ untranslated Region (UTR) and regulates the polyadenylation of these mRNAs and thereby controls translation. In Chapter II, I have presented my work on the regulation of mitochondrial function by CPEB. CPEB knockout mice have brain specific defects in mitochondrial function owing to a reduction in Electron transport chain complex I component protein NDUFV2. CPEB controls the translation of this NDUFV2 mRNA and thus affects mitochondrial function. A consequence of this reduced bioenergetics is reduced growth and branching of neurons, again emphasizing the importance of this pathway. Chapter III focuses on the role of CPEB4 in neuronal survival and protection against apoptosis. CPEB4 shuttles between nucleus and cytoplasm and becomes nuclear in response to stimulation with ionotropic glutamate receptors, focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose; nuclear CPEB4 affords protection against apoptosis in ischemia model. The underlying cause for nuclear translocation is reduction in Endoplasmic Reticulum calcium levels. These studies give an insight into the function and dynamics of these two RNA binding proteins and provide a better understanding of cellular biology.

The cells and molecules that comprise the immune system are essential for mounting an effective response against microbes. A successful immune response limits pathology within the host while simultaneously eliminating the pathogen. The key to this delicate balance is the correct recognition of the pathogen and the appropriate response of immune cells. Cellular activation originates through receptors that relay information about the state of the microenvironment to different compartments within the cell. The rapid relay of information is called signal transduction and employs a network of signaling mediators such as kinases, phosphatases, adaptor molecules, and transcription factors. IL-2 inducible T cell kinase (Itk) is a non-receptor tyrosine kinase that is an integral component of signal transduction downstream of many immunoreceptors. This dissertation describes two distinct pathways that utilize Itk in both phases of the immune response. T cells use the TCR to sense a multitude of peptide-based ligands and to transmit signals inside the cell to activate cellular function. In this regard, the diversity of ligands the T cells encounter can be portrayed as analog inputs. Once a critical threshold is met, signaling events transpire in close proximity to the plasma membrane to activate major downstream pathways in the cell. The majority of these pathways are digital in nature resulting in the on or off activation of T cells. We find, however, that altering the TCR signal strength that a T cell receives can result in an analog-based response. Here, the graded expression of a transcription factor, IRF4, is modulated through the activity of Itk. We link this graded response to an NFAT-mediated pathway in which the digital vs. analog nature has been previously uncharacterized. Finally, we demonstrate that the repercussions of an analog signaling pathway is the altered expression of a second transcription factor, Eomes, which is important in the differentiation and function of T cells. These results suggest that Itk is crucial in the modulation of TCR signal strength. Mast cells primarily rely on the IgE-bound FcεR1 for pathogen recognition. Crosslinking this receptor activates mast cells and results in degranulation and cytokine production via an expansive signaling cascade. Upon stimulation, Itk is recruited to the plasma membrane and phosphorylated. Little else is known about how Itk operates inside of mast cells. We find that mast cells lacking Itk are hyperresponsive to FcεR1-mediated activation. This is most apparent in the amount of IL-4 and IL-13 produced in comparison to wild-type mast cells. Increased cytokine production was accompanied by elevated and sustained signaling downstream of the FcεR1. Finally, biochemical evidence demonstrates that Itk is part of an inhibitory complex containing the phosphatase SHIP-1. These results indicate a novel function for Itk as a negative regulator in FcεR1- mediated mast cell activation.

The nervous system is composed of neurons and glia. Glial cells have been neglected and thought to have only a supportive role in the nervous system, even though ~60% of the mammalian brain is composed of glia. Yet, in recent years, it has been shown that glial cells have several important functions during the development, maintenance and function of the nervous system. Glial cells regulate both pre and post mitotic neuronal survival during normal development and maintenance of the nervous system as well as after injury, are necessary for axon guidance, proper axon fasciculation, and myelination during development, promote synapse formation, regulate ion balance in the extracellular space, are required for normal synaptic function, and have immune functions in the brain. Although glia have crucial roles in nervous system development and function, there are still much unknown about the underlying molecular mechanisms in glial development, function and glial-neuronal communication. Drosophila offers great opportunity to study glial biology, with its simple yet sophisticated and stereotypic nervous system. Glial cells in flies show great complexity similar to the mammalian nervous system, and many cellular and molecular functions are conserved between flies and mammals. In this study, I use Drosophila as a model organism to study the function of one subtype of glia: astrocytes. The role of astrocytes in synapse formation, function and maintenance has been a focus of study. However, their role in engulfment and clearance of neuronal debris during development remains unexplored. I generated a driver line that enables the study of astrocytes in Drosophila.In chapter two of this thesis, I characterize astrocytes during metamorphosis, when extensive neuronal remodeling takes place. I found that astrocytes turn into phagocytes in a cell-autonomous, steroid-dependent manner, by upregulating the phagocytic receptor Draper and forming acidic phagolysosomal structures. I show that astrocytes clear neuronal debris during nervous system remodeling and that this is a novel function for astrocytes during the development of nervous system. I analyzed two different neuronal populations: MB γ neurons that prune their neurites and vCrz+ neurons that undergo apoptosis. I discovered that MB γ axons are engulfed by astrocytes using the Draper and Crk/Mbc/dCed-12 pathways in a partially redundant way. Interestingly, Draper is required for clearance of vCrz+ cell bodies, while Crk/Mbc/dCed-12, but not Draper, are required for clearance of vCrz+ neurites. Surprisingly, I also found that loss of Draper delayed vCrz+ neurite degeneration, suggesting that glia facilitate neurite destruction through engulfment signaling. Taken together, my work identifies a novel function for astrocytes in the clearance of synaptic and neuronal debris during developmental remodeling of the nervous system. Additionally, I show that Crk/Mbc/dCed-12 act as a new glial signaling pathway required for pruning, and surprisingly, that glia use different engulfment pathways to clear neuronal debris generated by cell death versus local pruning.

This thesis comprises two separate studies that focus on the consequences of cellular damage. The first investigates the effects of DNA damage on centriole behavior and the second characterizes phototoxicity during live-cell imaging. Cancer treatments such as ionizing radiation and/or chemotherapeutic DNA damaging agents are intended to kill tumor cells, but they also damage normal proliferating cells. Although centrosome amplification after DNA damage is a well-established phenomenon for transformed cells, it is not fully understood in untransformed cells. The presence of extra centrosomes in normal cell populations raises the chances of genomic instability, thus posing additional threats to patients undergoing these therapies. I characterized centriole behavior after DNA damage in synchronized untransformed (RPE1) human cells. Treatment with the radiomimetic drug, Doxorubicin, prolongs G2 phase by at least 72hrs, where 52% of cells display disengaged centrioles and 10% contain extra centrioles. This disengagement is mediated by Plk and APC/C activities both singly and in combination. Disengaged centrioles are associated with maturation markers suggesting they are capable of organizing spindle poles. Despite the high incidence of centriole disengagement, only a small percentage of centrioles reduplicate due to p53/p21 dependent inhibition of Cdk2 activity. Although all cells become prolonged in G2 phase, 14% eventually go through mitosis, of which 26% contain disengaged or extra centrioles. In addition to cancer treatments, cellular damage can be acquired from various external conditions. Short wavelengths of light are known to be toxic to living cells, but are commonly used during live-cell microscopy to excite fluorescent proteins. I characterized the phototoxic effects of blue (488nm) and green (546nm) light on cell cycle progression in RPE1. For unlabeled cells, I found that exposure to green light is far less toxic than blue light, but is not benign. However, the presence of fluorescent proteins led to increased sensitivity to both blue and green light. For 488nm irradiations, spreading the total irradiation durations out into a series of 10s pulses or conducting single longer, but lower intensity, exposures made no significant changes in phototoxicity. However, reducing oxidative stress by culturing cells at physiological (~3%) oxygen, or treatment with a water-soluble antioxidant, Trolox, greatly improved the cells tolerance to blue light. Collectively, my work offers an explanation for centrosome amplification after DNA damage and demonstrates the importance of proper centriole regulation in untransformed human cells. Further, it provides a practical assessment of photodamage during live-cell imaging.

Autophagy delivers cytoplasmic material to the lysosome for degradation, and has been implicated in many cellular processes, including stress, infection, survival, and death. Although the regulation and role that autophagy plays in stress, infection, and survival is apparent, its involvement during cell death remains relatively unclear. In this thesis I summarize what is known about the roles autophagy can play in cell death, and the differences between the utilization of autophagy during nutrient deprivation and cell death. Utilizing Drosophila melanogaster as a model system, the roles autophagy plays in both of these contexts can be studied. The goal of this thesis is to provide a better understanding of the regulatory mechanisms that distinguish between autophagy as a survival mechanism and autophagy as a cell death mechanism. From my studies I was able to determine that microRNAs can regulate autophagy in vivo, and that the microRNA miR-14 controls autophagy specifically during the destruction of the larval salivary glands of Drosophila melanogaster. I found that miR-14 regulates autophagy through modulation of IP3 and calcium signaling, and this miR-14 control of IP3 and calcium signaling does not influence the induction of autophagy during nutrient deprivation. Therefore, this knowledge demonstrates how autophagy can be regulated to distinguish its use during cell survival and death providing insight into how autophagy can used to treat diseases.

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Using a forward genetic screen in Drosophila, we identified that loss of the Toll receptor adaptor dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway. This death signaling pathway can be activated without injury by loss of the N-terminal self-inhibitory domain, resulting in spontaneous neurodegeneration. To investigate the role of axon self-destruction in disease, we assessed the effects of Sarm1 loss on neurodegeneration in the SOD1-G93A model of amyotrophic lateral sclerosis (ALS), a lethal condition resulting in progressive motor neuron death and paralysis. Loss of Sarm1 potently protects motor axons and synapses from degeneration, but only extends animal survival by 10%. Thus, there appears to be at least two driving forces in place during ALS disease progression: (1) Sarm1 mediated axon death, and (2) cell body destruction via some unknown mechanism.

All living creatures change their gene expression program in response to nutrient availability and metabolic demands. Nutrients and metabolites can directly control transcription and activate second-­‐messenger systems. In bacteria, metabolites also affect post-­‐transcriptional regulatory mechanisms, but there are only a few isolated examples of this regulation in eukaryotes. Here, I present evidence that RNA-­‐binding by the stem cell translation regulator Musashi-­‐1 (MSI1) is allosterically inhibited by 18-­‐22 carbon ω-­‐9 monounsaturated fatty acids. The fatty acid binds to the N-­‐terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. I identify stearoyl-­‐CoA desaturase-­‐1 as a MSI1 target, revealing a feedback loop between ω-­‐9 fatty acid biosynthesis and MSI1 activity. To my knowledge, this is the first example of an RNA-­‐binding protein directly regulated by fatty acid. This finding may represent one of the first examples of a potentially broad network connecting metabolism with post-­‐transcriptional regulation.

Glioblastoma multiforme (GBM) tumors are highly malignant in nature and despite an aggressive therapy regimen, long–term survival for glioma patients is uncommon as cells with intrinsic or acquired resistance to treatment repopulate the tumor. This creates the need to investigate new therapies for enhancing GBM treatment outside of the standard of care, which includes Temozolomide (TMZ). Our lab focused on two novel strategies to overcome resistance in GBMs. In our first approach, the cellular responses of GBM cell lines to two new TMZ analogues, DP68 and DP86, are reported. The efficacy of these compounds was independent of DNA repair mediated by Methyl Guanine Methyl Transferase (MGMT) and the mismatch repair (MMR) pathway. DP68 or DP86 treated cells do not give rise to secondary spheres, demonstrating that they are no longer capable of self-renewal. DP68-induced damage includes interstrand DNA crosslinks and exhibits a distinct S-phase accumulation before G2/M arrest; a profile that is not observed for TMZ-treated cells. DP68 induces a strong DNA damage response and suppression of FANCD2 expression or ATR expression/kinase activity enhanced the anti-GBM effects of DP68. Collectively, these data demonstrate that DP68, and to a lesser extent DP86, are potent anti-GBM compounds that circumvent TMZ resistance and inhibit recovery of cultures. Our second approach stems from a previous discovery in our lab which demonstrated that the combination of TMZ with Notch inhibition, using a gamma secretase inhibitor (GSI), enhances GBM therapy. Efficacy of TMZ + GSI treatment is partially due to GBM cells shifting into a permanent senescent state. We sought to identify a miR signature that mimics the effects of TMZ + GSI as an alternative vi approach to enhance GBM therapy. MiR-34a expression was highly upregulated in response to TMZ or TMZ + GSI treatment. Exogenous expression of miR-34a revealed that it functions as a tumor suppressor and mimicked the in vitro effects of TMZ + GSI treatment. Additionaly, miR-34a overexpression leads to the downregulation of Notch family members. Together these two studies contribute to our understanding of the complex mechanisms driving resistance in GBM tumors and suggest strategies to develop more effective therapies.

Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. Precursors of these cytokines are expressed downstream of TLR signaling and are then enzymatically processed into mature bioactive forms, typically by caspase-1 which is activated through a process dependent on multi-molecular structures called inflammasomes. Y. pestis evades immune detection in part by using a Type three secretion system (T3SS) to inject effector proteins (Yops) into host cells and suppress IL-1β and IL-18 production. We investigated the cooperation between two effectors, YopM and YopJ, in regulating inflammasome activation, and found that Y. pestis lacking both YopM and YopJ triggers robust caspase-1 activation and IL-1Β/IL-18 production in vitro. Furthermore, this strain is attenuated in a manner dependent upon caspase-1, IL-1β and IL-18 in vivo, yet neither effector appears essential for full virulence. We then demonstrate that YopM fails to inhibit NLRP3/NLRC4 mediated caspase-1 activation and is not a general caspase-1 inhibitor. Instead, YopM specifically prevents the activation of a Pyrin-dependent inflammasome by the Rho-GTPase inhibiting effector YopE. Mutations rendering Pyrin hyperactive are implicated in the autoinflammatory disease Familial Mediterranean Fever (FMF) in humans, and we discuss the potential significance of this disease in relation to plague. Altogether, the Y. pestis T3SS activates and inhibits several inflammasome pathways, and the fact that so many T3SS components are involved in manipulating IL-1β/IL-18 underscores the importance of these mechanisms in plague.

Key regulatory events take place at very early stages of human embryonic stem cell (hESC) differentiation to accommodate their ability to differentiate into different lineages; this work examines two separate regulatory events. To investigate precise mechanisms that link alterations in the cell cycle and early differentiation, we examined the initial stages of mesendodermal lineage commitment and observed a cell cycle pause that occurred concurrently with an increase in genes that regulate the G2/M transition, including WEE1. Inhibition of WEE1 prevented the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. These findings reveal a novel G2 cell cycle pause required for endodermal differentiation. A role for phenotypic transcription factors in very early differentiation is unknown. From a screen of candidate factors during early mesendodermal differentiation, we found that RUNX1 is selectively and transiently up-regulated. Transcriptome and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that RUNX1 knockdown impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion compromised TGFβ2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFβ2, but not TGFβ1. These findings identify novel roles for RUNX1-TGFβ2 signaling in mesendodermal lineage commitment.