The homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis binds oxygen cooperatively and thus offers a simple model system for studying communication between two chemically identical sites. Although the individual subunits of HbI have the same myoglobin-fold as mammalian hemoglobins, the quaternary assemblage is radically different. Upon oxygen binding by HbI, only small tertiary changes are seen at the subunit interface in contrast to the relatively large quaternary changes observed with mammalian hemoglobins. Analysis of structures of this hemoglobin in the liganded (02or CO) and unliganded states has provided a framework for understanding the role of individual amino acid side-chains in mediating cooperativity. The work presented in this dissertation has directly tested the central tenets of the proposed structural mechanism for cooperativity in HbI, illuminating the key roles played by residue Phe 97 and interface water molecules in intersubunit communication. Heterologous expression of Scapharca dimeric hemoglobin: A synthetic gene has been utilized to express recombinant RbI in Escherichia coli. The HbI apoprotein constitutes 5-10% of the total bacterial protein in this system. Addition of the heme precursor δ-aminolevulinic acid to the expression culture results in a ~3-fold increase in the production of soluble hemoglobin. Recombinant HbI has been successfully purified to homogeneity, resulting in a final yield of 80-100 mg of pure holoprotein from a 12 L expression culture. Analysis of recombinant HbI reveals its oxygen binding properties to be indistinguishable from native HbI. It was necessary to correct a protein sequence error by mutating residue Asn 56 to aspartate in order to obtain diffraction quality crystals, that are isomorphous to native HbI crystals. These recombinant HbI crystals diffract to high resolution, permitting the functional effects of mutant HbI proteins to be correlated with detailed structural analysis.

The questions outlined in this thesis dissertation were proposed in order to provide insight regarding the mechanism by which the Drosophila Son of sevenless (dSOS) protein activates Ras. Ras proteins are GTP-binding proteins which bind guanine nucleotides very tightly and cycle between the inactive GDP-bound state and the active GTP-bound state. To address the mechanism by which the dSOS proteins activates Ras, a structure-function analysis of the dSOS protein was performed using truncation and deletion mutants of dSOS. In vivo Ras activation experiments using transiently transfected cells revealed that the NH2-terminal domain of dSOS is required in order for the catalytic domain of dSOS to exhibit exchange activity in cultured mammalian cells. The COOH-terminal GRB2 (Growth Factor Receptor Binding Protein) binding domain on the otherhand was insufficient to confer Ras exchange activity to the dSOS catalytic domain. Further analysis of the NH2-terminal domain of the dSOS protein demonstrated that the function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin (PH) and DBL (Diffuse B-cell Lymphoma) homology sequences. Fractionation studies of cells transiently transfected with various dSOS mutant proteins demonstrated that the NH2-terminus of dSOS is also necessary for membrane association. These findings suggested that the model proposing that the recruitment of SOS via the adaptor protein GRB2 to the membrane is the main mechanism by which SOS activates Ras is unlikely to be the only mechanism by which SOS can activate Ras. From our data, a model can be proposed which postulates that SOS can activate Ras as a consequence of at least two steps. One step involves the SOS/GRB2 interaction and the second step involves the NH2-terminal domain of SOS associating with unidentified cellular elements.

Necroptosis, a type of regulated necrotic cell death, involves cell membrane permeabilization and has been implicated in various acute and chronic pro-inflammatory diseases, including ischemia-reperfusion injury and neurodegenerative diseases. By using in vitro reconstitution studies and a chemical inhibitor, the kinase activity of the serine/threonine kinase RIPK1 had been shown to regulate necroptotic signaling downstream of TNF and Toll-like receptors (TLRs). To investigate the contribution of RIPK1 kinase activity to inflammation and necroptosis in vivo, we generated kinase inactive RIPK1 knock-in mice. Utilizing fibroblasts and macrophages from these mice, we demonstrate that RIPK1 kinase activity is required for necroptotic complex formation and death induction downstream of TNFR1 and TLRs 3 and 4. We show that RIPK1 kinase inactive mice are resistant to TNF-induced shock and exhibit impaired upregulation of TNF-induced cytokines and chemokines in vitro and in vivo. By using bone marrow reconstitution experiments, we demonstrate that RIPK1 kinase activity in a non-hematopoietic lineage drives TNF-induced lethality. We establish that RIPK1 kinase activity is required for TNF-induced increases in intestinal and vascular permeability and clotting, and implicate endothelial cell necroptosis as an underlying factor contributing to TNF/zVAD-induced shock. Thus, work in this thesis reveals that RIPK1 kinase inhibitors may have promise in treating shock and sepsis.

This thesis proposes a mechanism for the induction of peripheral tolerance to protein antigens. I have investigated the mechanism of tolerance induction to soluble protein antigens by targeting an antigen to small, resting B cells. For this purpose I have used a rabbit antibody directed at the IgD molecule found on the surface of most small, resting B cells but missing or lowered on activated B cells. Intravenous injection of normal mice with 100 μg of an ultracentrifuged Fab fragment of rabbit anti-mouse IgD (Fab anti-δ) makes these mice profoundly tolerant to challenge with nonimmune rabbit Fab (Fab NRG) fragments. This tolerance is antigen specific since treated mice make normal responses to an irrelevant antigen, chicken immunoglobulin (Ig). Fab fragments of rabbit Ig (rabbit Fab) not targeted to B cells do not induce tolerance as well as Fab anti-δ. Evidence suggests that the B cells must remain in a resting state for tolerance to be induced, since injection of F(ab)'2 anti-δ does not induce tolerance. Investigation of the mechanisms of the tolerance, by adoptive transfer, have shown that rabbit Fab specific B cell function has been impaired. The major effect however is in helper T cell function, as shown by adoptive transfer and lack of help for a hapten response. In vitro proliferation experiments show that the T cell response has not been shifted toward activation of different T cell subsets which do not help Ig production, nor is there any change in the Ig isotypes produced. Suppression does not appear to be the major cause of the helper T cell defect as shown by cell mixing experiments. This work shows that an antigen targeted to small B cells can induce tolerance to a soluble protein antigen, and suggests a role for small B cells in tolerance to self-proteins not presented in the thymus.

Members of the Argonaute family of proteins, which interact with small RNAs, are the key players of RNAi and other related pathways. The C. elegans genome encodes 27 members of the Argonaute family. During this thesis research, we sought to understand the functions of the members of this gene family in C. elegans. Among the Argonaute family members, rde-1 and alg-1/2have previously been shown to be essential for RNAi and development, respectively. In this work, we wanted to assign functions to the remaining members of this large family of proteins. Here, we describe the phenotype of 31 deletion alleles representing all of the previously uncharacterized Argonaute members. In addition to rde-1, our analysis revealed that two other Argonaute members csr-1 and prg-1 are also essential for development. csr-1 is partially required for RNAi, and essential for proper chromosome segregation. prg-1, a member of PIWI subfamily of Argonaute genes, exhibits reduced brood size and temperature-sensitive sterile phenotype, implicating that it is required for germline maintenance. Additionally, we showed that RDE-1 interacts with trigger-derived sense and antisense siRNAs (primary siRNAs) to initiate RNAi, while several other Argonaute proteins, SAGO-1, SAGO-2, and perhaps others, functioning redundantly, interact with amplified siRNAs (secondary siRNAs) to mediate downstream silencing. Moreover, our analysis uncovered that another member of Argonaute gene family, ergo-1, is essential for the endogenous RNAi pathway. Furthermore, we built an eight-fold Argonaute mutant, MAGO8, and analyzed its developmental phenotype and sensitivity to RNAi. Our analysis revealed that the genes deleted in the MAGO8 mutant function redundantly with each other, and are required for RNAi and the maintenance of the stem cell totipotency.

A functional central nervous system (CNS) is composed of numerous types of neurons. Neurons are derived from a limited number of multipotent neural stem cells. Previous studies have suggested three major strategies nature uses to diversify neurons: lineage identity specification that gives an individual neural stem cell distinct identity based on its position in the developing CNS; temporal identity specification that gives neurons derived from a neural stem cell distinct identities based on their birth-order within the lineage; and binary cell fate specification that gives different identities to the two sister postmitotic neurons derived from the terminal division of a common precursor. Through the combination of the three strategies, almost unlimited neuron types can be generated. To understand neuronal diversification, we have to understand the underlying molecular mechanisms of each of the three strategies. The fruit fly Drosophila melanogaster, has been an excellent model for studying neuronal diversity, mainly due to its easily traceable nervous system and an impressive collection of genetic tools. Studies in fly have provided us fundamental insights into lineage identity, temporal identity, and binary cell fate specifications. Nevertheless, previous studies mostly centered on the embryonic ventral nerve cord (VNC) because of its simpler organization. Our understanding of the generation of neuronal diversity in the fly brain is still rudimentary. In this thesis work, I focused on the mushroom body (MB) and three antennal lobe neuronal lineages, studying their neuronal diversification during postembryonic brain development. In Chapter I, I reviewed the previous studies that have built our current understanding of the neuronal diversification. In Chapter II, I showed that MB temporal identity changes are instructed by environmental cues. In Chapter III, to search for the potential factors that mediate the environmental control of the MB temporal identity changes, I silenced each of the 18 nuclear receptors (NRs) in the fly genome using RNA interference. Although I did not identify any NR important for the regulation of MB temporal identities, I found that unfulfilled is required for regulating axon guidance and for the MB neurons to acquire all major subtype-specific identities. In Chapter IV, I demonstrated that the Notch pathway and its antagonist Numb mediate binary cell fate determination in the three classical antennal lobe neuronal lineages— anterodorsal projection neuron (adPN), lateral antennal lobe (lAL), and ventral projection neuron (vPN)—in a context-dependent manner. Finally, in Chapter V, I did detailed lineage analysis for the lAL lineage, and identified four classes of local interneurons (LNs) with multiple subtypes innervating only the AL, and 44 types projection neurons (PNs) contributing to olfactory, gustatory, and auditory neural circuits. The PNs and LNs were generated simultaneously but with different tempos of temporal identity specification. I also showed that in the lAL lineage the Notch pathway not only specifies binary cell fates, but is also involved in the temporal identity specification.

Emerging evidence in both vertebrates and invertebrates is redefining glia as active and mobile players in synapse formation, maturation and function. However, the molecular mechanisms through which neurons and glia interact with each other to regulate these processes is not well known. My thesis work begins to understand how glia use secreted factors to modulate synaptic function. We use Drosophila melanogaster, a simple and genetically tractable model system, to understand the molecular mechanisms by which glia communicate with neurons at glutamatergic neuromuscular junctions (NMJs). We previously showed that a specific subtype of glia, subperineurial peripheral glia cells (SPGs), establish dynamic transient interactions with synaptic boutons of the NMJ and is required for synaptic growth. I identified a number of potential functional targets of the glial transcription factor, reverse polarity (repo) using ChIP-chip. I found that one novel target of Repo, Wg, is expressed in SPGs and is regulated by repo in vivo. Wnt/Wg signaling plays a pivotal role during synapse development and plasticity, including the coordinated development of the molecular architecture of the synapse. While previous studies demonstrated that Wg is secreted by motor neurons, herein I provide evidence that a significant amount of Wg at the NMJ is additionally provided by glia. I found that Wg derived from SPGs is required for proper GluR distribution and electrophysiological responses at the NMJ. In summary, my results show that Wg expression is regulated by Repo in SPGs and that glial-derived Wg, together with motor neuron-derived Wg, orchestrate different aspects of synapse development. My thesis work identifies synapse stabilization and/or assembly as a new role for SPGs and demonstrates that glial secreted factors such as Wg regulate synaptic function at the Drosophila NMJ.

The minimal element of the nervous system, the synapse, is a plastic structure that has the ability to change in response to various internal and external factors. This property of the synapse underlies complex behaviors such as learning and memory. However, the exact molecular and cellular mechanisms involved in this process are not fully understood. To understand the mechanisms that regulate synapse development and plasticity I took advantage of a powerful model system, the Drosophila larval neuromuscular junction (NMJ). In this system, both anterograde and retrograde signaling pathways critical for coordinated synapse development and plasticity have been documented. An anterograde WNT/Wingless (Wg) signaling pathway plays a crucial role in both developmental and activity-dependent synaptic plasticity at the NMJ. Presynaptic motor neuron terminals secrete highly hydrophobic Wg, which travels to relatively distant postsynaptic sites where it activates a signal transduction pathway required for postsynaptic development. In the first half of my thesis I unraveled a previously unrecognized cellular mechanism by which Wg is shuttled to postsynaptic sites. In this mechanism Wg rides on secreted microvesicles or exosomes that contain a dedicated WNT secretion factor, the WNT-binding transmembrane protein, Evenness Interrupted/Wntless/Sprinter (Evi/Wls/Srt). To our knowledge, this was the first in vivo study demonstrating that neurons release exosomes, which are involved in trans-synaptic communication. Moreover, this was the first study showing that hydrophobic WNT signals are transported to the extracellular space on exosomes to reach WNT-receptor containing target cells. Retrograde signals are also critical during development and plasticity of synaptic connections. These signals function to adjust the activity of presynaptic cells according to postsynaptic cell outputs, to maintain synaptic function within a dynamic range. However, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are not clear. In the second half of my thesis, I provided evidence that a crucial component of retrograde signaling at the fly NMJ, Synaptotagmin-4 (Syt4), is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Drosophila Syt4 is known to reside on postsynaptic vesicles at the NMJ and function as a calcium sensor to release a retrograde signal upon synaptic activity. This event is required for coordinated maturation of the presynaptic terminal. We demonstrated that retrograde Syt4 function in postsynaptic muscle is required for activity-dependent presynaptic growth. However, surprisingly, Syt4 protein was not synthesized in postsynaptic muscles. Instead, Syt4 was produced in motorneurons and transferred to postsynaptic muscle cells via exosome secretion by presynaptic cells. The above study provided evidence for a presynaptic control of postsynaptic retrograde signaling through exosomal transfer of an essential retrograde signaling component. In summary, this body of work reveals a novel mechanism of trans-synaptic communication through exosomes. While intercellular communication through exosomes had been demonstrated during antigen presentation in the immune system, our studies were the first to substantiate this mode of communication in the nervous system. Thus, these studies provide a significantly deeper and novel understanding of the mechanisms underlying synapse development and plasticity.

The formation of the anterior-posterior axis requires a symmetry-breaking event that starts gastrulation. Ultimately, the morphogenetic movements of gastrulation reshape the embryo to its final tri-dimensional form. In mouse embryos, the identity of the molecule that breaks the bilateral symmetry and sets in motion gastrulation remains elusive. The Wnt signaling pathway plays a pivotal role during axial specification and gastrulation in metazoans. Loss-of-function experiments have demonstrated a requirement of Wnt3 for gastrulation in mice. But because Wnt3 is expressed sequentially in two tissues, the visceral endoderm and the epiblast, its tissue specific requirements remain uncertain. Here, we report that embryos lacking Wnt3 specifically in the visceral endoderm do not form a primitive streak, mesoderm, endoderm or any derivatives. Visceral endoderm-specific Wnt3 mutants also lack primordial germ cells. Moreover, we provide data demonstrating that Wnt3 carries out its actions in the epiblast via the canonical Wnt pathway. Together, these data suggest that the posterior visceral endoderm via Wnt3, regulates the development of mouse embryos in a similar fashion to the amphibian Nieuwkoop center. Next, we conditionally ablated Wnt3 locus in the epiblast to investigate whether Wnt3 expression is also required in that tissue. Embryos lacking Wnt3 expression in the epiblast, but retaining its expression in the visceral endoderm, show delayed but not absent gastrulation. We conclude that the expression of Wnt3 in the epiblast is required for maintenance but not initiation of gastrulation in mouse embryos. Furthermore, we used in vitro and in vivo approaches to demonstrate that the Wnt3-mediated activation of the canonical Wnt pathway leads to β-catenin occupancy followed by transcription of key loci, including the Wnt3 locus itself, during gastrulation in mice. Our data indicate the presence of an autoregulatory loop in which Wnt3 controls its own expression and orchestrates the process of gastrulation in the mouse embryo.

While substrate-borne vibrations are utilized by different reptile species, true conspecific communication via biotremors has not yet been demonstrated in reptiles. This study follows a preliminary report that the veiled chameleon (Chamaeleo calyptratus) could produce biotremors in communicative contexts. I tested chameleon behavioral sensitivity to vibrations by placing them on a dowel attached to a shaker emitting vibrations of 25, 50, 150, 300, and 600 Hz and then measured their changes in velocity before and after the stimulus. I then paired chameleons in various social contexts [anthropogenic disturbance (human disruption of animal); dominance (malemale; female-female C. calyptratus); courtship (male-female C. calyptratus); heterospecific (C. calyptratus + C. gracilis); and predator-prey (adult + juvenile C. calyptratus)] and used a video camera and accelerometers to record their behavior. This study demonstrates that chameleons produce biotremors and that receivers exhibit a freeze response when exposed to a simulated biotremor stimulus. Furthermore, veiled chameleons produce biotremors in anthropogenic disturbance, conspecific dominance and courtship contexts, and these biotremors are elicited by visual contact with another adult conspecific and heterospecifics. Overall, two classes of biotremor were identified, "hoots” and “rumbles,” which differ significantly in dominant frequency and waveform. No correlation was identified between animal size and dominant frequency of the biotremors they produced as biotremors originate from rapid muscle contractions. Juvenile chameleons of two months of age are able to produce biotremors, suggesting this behavior may have multiple functions. Overall, the data suggest that the veiled chameleon has the potential to utilize substrate-borne vibrational communication during conspecific and possibly heterospecific interactions.