The generation of site-directed mutants within the actin binding domain of the EGF receptor modulates receptor function in internalization and ligand binding. In addition, truncation of the EGFr at residue 996 results in a loss of high affinity ligand binding, inhibited internalization and reduced signaling capacity. Mutation of tyrosine 992 to phenylalanine (Y992F) and glutamate 991 to glutamine (E991Q) increases the rate at which receptors are internalized. The presence of a phenylalanine residue eliminates EGFr-mediated phosphorylation at Tyr992 while the E991Q mutation might also eliminate phosphorylation at this position due to a disruption of the kinase recognition motif. Thus, phosphorylation of Tyr992 appears to function in the regulation of receptor internalization. The mutation of tyrosine 992 to a glutamate residue (Y992E) causes a three-fold increase in receptor affinity for its ligand and demonstrates the existence of novel third and potentially fourth affinity states for the EGFr. A very high affinity EGFr state with a K$\sb{\rm d}$ of approximately 10 pM has been identified as has an intermediate state of 1.5 nM. The deletion of the C-terminal 190 amino acids of the EGFr causes a complete abolition of the previously observed high affinity state of the EGFr and also causes a significant reduction in the affinity of the low affinity state of the EGFr. Phorbol ester treatment of wild type and mutant EGFr causes a loss of the high affinity receptors, and also a decrease in the overall affinity of the receptor for its ligand which is similar to the loss seen in the deletion mutant. This suggests that control of the affinity state of the EGFr is mediated through the C-terminal 190 amino acids of the receptor. In addition, the C-terminal 190 amino acids of the receptor have been identified as containing a domain which regulates the phorbol ester induced conversion of receptor affinity. The amino acid composition in the vicinity of tyrosine 992 has been shown to play a role in the internalization of the EGF receptor and in the regulation of receptor affinity for its ligand.

Long nanotubes of fluid-bilayers were used to create templates for photochemical polymerization into solid-phase conduits and networks. Micromechanical methods were developed which allowed each nanotube to be pulled from a micropipette-held feeder vesicle by mechanical retraction of the vesicle after molecular bonding to a rigid substrate. The caliber of the tube was controlled precisely in a range from 20 to 200 nanometers by setting the suction pressure in the micropipette. Branched conduits were formed by coalescing separate nanotubes drawn serially from the feeder vesicle surface. Single nanotubes and nanotube junctions could be linked together between bonding sites on a surface to create a functionalized network. After assembly, the templates were stabilized by photoinitiated radical cross-linking of hydrophilic monomer contained in the aqueous solution confined by the lipid bilayer boundary. Nanometer-sized vesicles that were prepared by extrusion were also used as templates for photopolymerization. Results from dynamic light scattering and electron microscopy experiments suggest that UV initiated, free-radical polymerization of vesicle-encapsulated monomer resulted in the formation of cross-linked polymer networks that were surrounded by a bilayer lipid membrane. Using fluorescence spectroscopy to monitor the release of an initially entrapped marker, it was determined that lumenal polymerization neither disrupted the semi-permeable membrane, nor did it effect the osmotic release of encapsulated solutes. The addition of detergent to a suspension of polymerized vesicles completely dissolved the bilayer membrane, leaving behind a rigid gel replica of the vesicular template. Two polymerizable amphiphiles with reactive headgroups were prepared and incorporated into the phospholipid bilayer, so as to provide a means to copolymerize the bilayer sheath with the liposome-encapsulated monomer. By monitoring the release of entrapped solutes, it was discovered that photopolymerization of vesicles constructed with either one of the amphiphilic monomers in combination with encapsulated monomer resulted in membrane destabilization, and the complete release of the entrapped solutes. This represented a new approach to the photoinduced release of vesicle contents. In fact, vesicles that contained the polymerizable lipid but not the entrapped monomer also exhibited contents release upon polymerization. During polymerization, the propagating membrane-bound polymer destabilized the membrane. Two simple models were put forth to explain the photoinduced release from polymerized vesicles. One takes into consideration the mechanical stresses that develop in the membrane asymmetric polymerization; the second suggests that release of contents was caused by formation of a membrane-bound, polymeric surfactant which forms “pores” in the membrane.

Cytokinesis, the process of physically separating cells for division, requires the precise orchestration of numerous physical, mechanical, chemical and biological processes. For these processes to function well, complex coordination of various proteins, with crosstalk between them, either as signaling molecules or as just plain structural components that contribute to the physical separation must exist. Actin, a structural polymer and myosin, a motor are two proteins that contribute to this process significantly. Both proteins are assembled in the contractile ring and together are responsible for the process of constriction. A thorough understanding of the behavior of these proteins, in the contractile ring as well as outside in a cell undergoing cytokinesis is therefore important to prevent possible defects that might lead to deleterious diseases. In this dissertation research, a combination of techniques are made use of, that involve live imaging of fluorescently labeled proteins in cells undergoing cytokinesis along with the use of drugs that either disrupt the structure (integrity) or function of cytokinetic proteins. I generated two LLCPK1 (pig epithelial cell lines); one that stably expresses GFP-actin and the other that stably expresses Tandemn Dimer RFP-myosin regulatory light chain (TDRFP-MRLC). Live imaging and analysis of cells expressing GFP-actin shows that actin in the contractile is highly dynamic and need to be dynamic. Evidence is presented for new roles of Myosin II, in addition to generating the force for cytokinesis. Myosin not only contributes to disassembly of actin in the contractile ring but is also required to maintain actin in the equatorial region. Live imaging of the cell lines that expresses TDRFP-MRLC or GFP-actin helped in the better understanding of the role of microtubules in simultaneously regulating actin and myosin dynamics, not only in the contractile ring to allow ingression, but also in preventing contractile activity outside in the contractile ring. Cytokinesis involves other proteins besides actin and myosin, which help in their recruitment, assembly, ingression and subsequent disassembly. Decreasing the accumulation of actin in the contractile ring, by treatment with Latrunculin B facilitated the examination of spatial and temporal events involved in building the ring. Actin, myosin and other proteins organized as nodes that coalesce during ingression, similar to the fission yeast. We conclude that this mode of cytokinesis a highly conserved feature of cytokinesis.

At the adult frog neuromuscular junctions (Rana pipien and Rana catesbiana), F-actin microfilaments are enriched in the nonrelease domains of nerve terminals, outside the vesicle-rich release sites. The development of this defined F-actin cytoskeleton may be critical for nerve terminal function as microfilaments may play a role in synaptic vesicle release and recycling and/or synaptic maintenance. I used cutaneous pectoris muscles of adult frogs (Rana pipiens) and bullfrog larvae (Rana catesbiana) stained with markers of synaptic and cytoskeletal components to ask how elements of the neuronal cytoskeleton, microfilaments, microtubules, and neurofilaments, are organized at the frog neuromuscular junction and how they organize during development of presynaptic motor nerve terminals. The presynaptic actin cytoskeleton stained by β-actin antibody extended the length of the nerve terminal in a series of interconnected rings that surrounded clusters of synaptic vesicles. At developing neuromuscular junctions β-actin stain is initially concentrated at growth cones and intermittently along the lateral surfaces of the nerve terminal. The assembly of the β-actin cytoskeleton appeared secondary to clustering of synaptic vesicles. I compared the stability of the presynaptic actin cytoskeleton of developing and adult neuromuscular junctions after treatment with latrunculin A. The β-actin cytoskeleton was noticeably less stable at larval neuromuscular junctions than at adult synapses. These data support a role for the actin cytoskeleton in presynaptic maturation and stability. I tested whether the perisynaptic Schwann cells (PSCs) have a role in maintaining the actin cytoskeleton of the nerve terminal using complement-mediated cell lysis to selectively ablate PSCs in vivo. At various time points after ablation, I examined the actin organization at denuded motor nerve terminals. I report here that the stability and long-term maintenance of the nerve terminal actin cytoskeleton is dependent at least in part on the presence of PSCs. Following ablation of PSCs, a significant decrease in the intensity of presynaptic actin stain was observed and remained altered for several weeks. After PSC ablation, terminals also displayed reduced staining for synaptic vesicles.

Autoreactive, immature T cells (thymocytes) are deleted from the thymus during development by the process of negative selection. This mechanism occurs when the thymocyte, T cell receptor (TCR) recognizes self-antigen, causing the cell to die by an apoptotic pathway. This mechanism results in the deletion of autoreactive T cells. Thymocyte development proceeds through several stages, determined by the differential expression of the T cell co-receptor molecules CD4 and CD8. The developmental stage where negative selection occurs is one in which thymocytes are expressing a functional TCR on the cell surface along with both CD4 and CD8, termed the double positive stage. These thymocytes are in a quiescent, G0 state and make up greater than 80% of the total population. The demonstration that cell cycle progression plays a role in the apoptotic process of several quiescent cell types, as well as the requirement for mature T cells to be in the late G1 phase during activation induced cell death, suggested that thymocytes may also advance to the G1 stage of the cell cycle prior to apoptosis. It has been established that the early cell cycle genes c-fos, c-jun, and c-myc are induced in thymocytes after stimulation, indicating possible entry into the cell cycle. The hypothesis that thymocytes enter the cell cycle before undergoing apoptosis was tested by examining expression levels of the various G1 cyclins and cyclin dependent kinase inhibitors at the mRNA and protein levels. Several indications of an early G1 cell cycle transition occurring during thymocyte apoptosis were observed, such as the downregulation of p27KIP1 and p130, the upregulation of cyclin D3, and the phosphorylation of the retinoblastoma protein. Finally, the requirement for the activation of the cyclin-dependent kinase 2 (CDK2) in negative selection was examined. It was shown that the phosphorylation and expression of the TCR-mediated apoptosis-related transcription factors Nur77 and Egr1 are downstream of CDK2 activation. In addition, a protein associated with the transcription factor Egr-1 was identified as a possible target of CDK2 kinase activity.

The ultimate means of limiting the influence of an individual cell on the physiology of a multicellular organism is to induce the death of that cell. Apoptosis is a genetically regulated form of cell death that removes cells that are malfunctioning, unnecessary or damaged. During development, cells are produced in excess and those that are not optimal in form, location or function are removed via apoptosis. In the adult organism, apoptosis allows for the turnover of cells that have carried out specialized functions and maintains tissue homeostasis. Negative selection is the developmental process by which immature T cells that have inappropriate reactivity to self antigen are induced to undergo apoptosis. During work in the lab confirming the requirement for the orphan nuclear hormone receptor, Nur77, for thymocyte apoptosis, an upregulation of the early growth response 1 gene (egr-1) was observed. This thesis investigates the requirement for transcriptional activation mediated by EGR-1 during the apoptosis of DO11.10, a cell line model of thymocyte negative selection. A dominant negative form of EGR-1, WT1EGR1, was expressed in DO11.10. The ability of these transfectants to undergo apoptosis in response to a variety of stimuli was measured. Another important function of apoptosis is to limit the life span of activated immune cells. The inception of the second part of this work was the clinical observation that exposure of non-healing wounds to hyperbaric oxygen (HBO), 100% oxygen at elevated atmospheric pressures, aids in the healing of these wounds. The hypothesis tested here is that HBO enhances the apoptosis of immune cells. Such an enhancement would promote the resolution of chronic inflammation and aid in wound healing. It is demonstrated that HBO enhances apoptosis of immune cells in response to stimuli relevant to both the regulation of the immune system and the application of HBO as an adjuvant to anti-cancer therapy. This study provides a new approach for studying the role of oxygen and its derivatives in apoptosis. The findings also support the continued investigation of expanding the clinical application of HBO.

Attenuated Salmonella typhimurium, a motile, nonpathogenic facultative anaerobic bacterium, has been demonstrated experimentally as a novel anticancer agent because of its favored growth within tumors. Specificity towards tumors allows for its potential use as an adjunctive approach to pharmacologic, radiation, and ablative therapies in the treatment of solid malignancies. However, limitations in its use as a tumor-specific vector may be present due to preferential colonization within nutrient-rich and hypoxic microenvironments of necrosis. Using a syngeneic murine model of mammary carcinoma, experiments were designed to study the spatiotemporal dynamics of bacterial proliferation within tumor microenvironments. With time, bacteria accumulate in the tumor transition zone, a region of quiescence between viable tumor and necrosis. An increase in tumor apoptosis and a decrease in tumor growth were noted at two days aftera single systemic treatment; this response was abrogated by four days after treatment. Bacterial specificity in colonization was noted towards subcutaneous tumors compared to liver, as well as hepatic metastases compared to normal liver. Using knowledge of observed patterns in bacterial accumulation it was determined that by two days adequate colonization in viable tumor and optimal effect in tumor apoptosis was achieved. Subsequently, strains were electroporated with radiation-inducible prokaryotic-expression plasmids to allow for tumor-specific production of either a green fluorescent protein (control) or murine tumor necrosis factor related apoptosis-inducing ligand (TRAIL). The effect of systemic infection of mice with subcutaneous mammary tumors was examined, comparing the administration of bacterial vectors with or without addition of 2Gy gamma radiation at two days after colonization. The combination of systemic infection with attenuated S. typhimurium and gamma-irradiation conferred a significant increase in tumor doubling time. The expression vector for TRAIL under a radiation-inducible promoter conferred a significant improvement in survival, with flattening of tumor growth curves after induction by radiation. Repeated dosing and irradiation after one week limited tumor growth from baseline, with a significant survival benefit. By capitalizing on the intrinsic motility of bacteria and their preferred microenvironments within tumors in time, the therapeutic utility of targeted therapy using attenuated Salmonella typhimurium as a TRAIL expression vector has been demonstrated in vivo.

Mutations in zebrafish mosaic eyes result in the disrupted retina lamination and other abnormalities. The moe locus encodes a FERM protein. In this study I sought to determine in which molecular pathway moe acts. We propose that Moe forms a complex with the Crumbs (Crb) proteins which are key determinants of the apical cell polarity. I identified zebrafish crb genes and found that expression of crb2a resembles the moe expression. Injection of crb2a antisense morpholinos phenocopies the moe mutations. Moe and Crumbs proteins colocalize in the photoreceptors. I showed Moe and Crumbs proteins, Pals1, and aPKCλ form a complex by pull-down assays and coimmunoprecipitation. I demonstrated that Moe can directly interact with the Crumbs proteins. Using genetic mosaic analyses, I showed that moe is required for rod morphogenesis and moe- rods have greatly expanded apical structures, suggesting that Moe is a negative regulator of Crumbs protein function in photoreceptors. Next I sought to determine the function of each domain of Crb2a/b proteins in rod morphogenesis. I constructed nine Crb2a constructs and made stable fish lines to express each of them specifically in rods. I also made lines that overexpress a Moe peptide that contains the predicted Crumbs proteins binding motif. I showed that Crb2aΔFBD , Crb2aΔFBDΔPBD, Crb2aIntraDD, Crb2aIntraAA, and Crb2aTM-Extra proteins mostly go to the outer segment. Crb2aIntraWT, Crb2aFL, and Crb2aΔPBD localize mostly to the inner segment and cell body. Binding assays showed that GST-Crb2aΔFBD, GST-Crb2aIntraDD, and GST-Crb2aIntraAA do not bind HIS-Moe_FERM as well as GST-Crb2aIntraWT. Overexpression of Crb2aFL and Crb2aΔPBD causes Rhodopsin mislocalization. Crb2aIntra expression causes mislocalization of endogenous Crumbs proteins, indicating a dominant effect of transgene expression. I also showed that Crb2aIntra expression causes an increase in the size of the outer segment by over 50%, and Crb2aIntraAA produces the largest increase. These data suggest that targeting of transgene products to the outer segment is likely due to the impaired binding ability to Moe and that the apical membrane adding activity of Crb2aIntra proteins can be inhibited by Moe. Further, my data show that the interaction of Moe and Crumbs proteins depends on the phosphorylation state of Crumbs proteins.

Body weight is controlled by gene regulation through the activation of signal transduction pathways which ultimately regulate transcription factors and their gene targets. Fluctuating leptin levels regulate hypothalamic pathways controlling the body’s response to energy availability fluctuations. The Nescient basic helix-loop-helix transcription factor 2 (Nhlh2) is a target of leptin stimulation in proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus. POMC is cleaved by prohormone convertase 1 (PC1) to α-melanocyte stimulating hormone (αMSH) regulating the body’s response to leptin signals. Nhlh2 knockout (N2KO) mice display adult-onset obesity starting at 12 weeks of age characterized not by hyperphagia, but by reduced activity levels. In this dissertation, studies examining the role of Nhlh2 during energy deficit show that N2KO mice have altered leptin, body weight and temperature responses. Nhlh2 likely regulates the transcription of many genes that lead to the development of obesity in N2KO mice. Using microarray technology, more than 7,000 genes that are differentially regulated between WT and N2KO mice in varying energy availability states are reported herein. Previous work in the lab showed that N2KO mice have a POMC processing defect caused by reduced PC1 levels leading to decreased αMSH and increased pro-forms of POMC. Here, new work shows that Nhlh2 binds to and transactivates the PC1 promoter through two putative E-box motifs. These E-box motifs are adjacent to two putative STAT3 transcription factor binding sites. In this work, STAT3 is shown to interact with Nhlh2 at these E-box motifs to regulate PC1. This research further characterizes the obesity phenotype of N2KO mice and the method by which Nhlh2 regulates PC1. This work has identified a new purpose for Nhlh2 in modulating leptin levels following changes in energy availability, and has identified a novel synergism between Nhlh2 and STAT3 to control basal and induced levels of PC1 in the hypothalamus. Finally, I have identified over 4000 potential targets of Nhlh2 downstream of leptin stimulation which can be analyzed in the future. In summary, work presented in this dissertation provides new insight into the role of Nhlh2-mediated gene regulation and the downstream effects on energy availability signals.

Estrogenic compounds can stimulate proliferation of the mammary epithelium, but also potentiate the activity of the p53 tumor suppressor protein. These contradictory activities of estrogenic compounds in mammary tissues may be mediated through activation of two estrogen receptor (ER) subtypes, ERα and ERβ. The following experiments were conducted to examine the roles of these receptors in regulating p53 activity in the mammary epithelium in vivo and in vitro. Selective agonist for ERα (PPT) and ERβ (DPN) were compared with 17β-estradiol to examine the roles of ERα and ERβ in potentiating p53 activity, radiation-induced apoptosis and proliferation in ovariectomized mice. DPN was sufficient to potentiate p53-dependent apoptosis in the mammary epithelium following irradiation without inducing proliferation. DPN was also 2.5-fold more potent in stimulating expression of Egr1 , a modulator of p53 activity. Introduction of ERβ into MCF-7 cells increased in the transcriptional activity of p53. As radiation-induced apoptosis was diminished in mice lacking ERβ (BERKO) mice, ERβ appears necessary for optimal activity of p53 in the mammary epithelium. The ability of DPN to maximally stimulate responsiveness of p53 to ionizing radiation in the absence of proliferation suggests that ERβ agonists may be an effective adjuvant therapy. Phytoestrogens are estrogenic compounds that are abundant in soy-based products, a key component in Asian diet associated with reduced breast cancer incidence in Asian women, and are preferential ligands for ERβ. However, the effects of soy differ greatly depending on the form and doses administered. Therefore, the effects of water-soluble extracts of non-fermented and fermented soy (NFSE and FSE, respectively) were compared. At physiological relevant doses both NFSE and FSE inhibited proliferation of cell lines from normal breast epithelium (76N-TERT) and breast cancers (21MT-1,MDA-MB-231). The FSE also increased the tumor-free survival of mice bearing xenografts of MDA-MB-231 cells. However, these effects of soy extracts were independent of both p53 and ERα. As both p53 and ERα are commonly lost in breast tumors, the pathways by which soy extracts antagonize tumor growth could provide valuable therapeutic targets for the treatment and prevention of breast tumors.