The development and maintenance of multicellular organisms depends fundamentally on cell division, a series of events largely mediated by the mitotic spindle. Errors in spindle formation and/or function are often associated with severe consequences, most notably cancer. In order to elucidate the cause of such errors and the potential for therapeutic intervention, it is imperative to attain a clear understanding of how cell division normally operates. In this regard, this dissertation focuses on the activity of two microtubule-based motor proteins, Eg5 and dynein, prior to and immediately following nuclear envelope breakdown during mitosis. I show that prophase microtubules are remarkably more dynamic than their metaphase counterparts, moving both toward and away from centrosomes across a wide distribution of rates. Inhibition of Eg5, dynein and Kif2a revealed that a subset of this motion is consistent with microtubule flux, a well-established phenomenon temporally limited to metaphase and anaphase spindles by the preceding literature. My data indicates that flux is operational throughout all of mitosis, possibly functioning at early stages to collect centrosomal components. Immediately following prophase, cells begin assembling bipolar spindles. While the establishment of spindle bipolarity fails in the physical or functional absence of Eg5, I show that co-inhibition of dynein restores a cell's ability to organize microtubules into a bipolar structure. Despite inhibition of both Eg5 and dynein, these spindles are morphologically and functionally equivalent to controls. Together, these data suggest that Eg5 and dynein share an antagonistic relationship and that a balance of forces, rather than a definitive set of players, is important for spindle assembly and function. To determine how Eg5- and dynein-mediated forces functionally coordinate to bring about antagonism during spindle assembly, I utilize a nocodazole washout assay. I show, via in vivo imaging and in silico modeling, that spindle collapse in the absence of functional Eg5 requires dynein activity and an initial intercentrosomal distance of less than 5.5μm. These data are consistent with a model in which dynein antagonizes Eg5 by crosslinking and sliding antiparallel microtubules, a novel role for dynein within the framework of spindle assembly.

Chloroplast biogenesis relies on the import of thousands of nuclear encoded proteins into the organelle and proper sorting to their sub-organellar compartment. The majority of nucleus-encoded chloroplast proteins are synthesized in the cytoplasm and imported into the organelle via the Toc-Tic translocation systems of the chloroplast envelope. In many cases, these proteins are further targeted to subcompartments of the organelle (e.g. the thylakoid membrane and lumen or inner envelope membrane) by additional targeting systems that function downstream of the import apparatus. The inner envelope membrane (IEM) plays key roles in controlling metabolite transport between the organelle and cytoplasm, and is the major site of lipid and membrane biogenesis within the organelle. In contrast to the protein import and thylakoid targeting systems, our knowledge of the pathways and molecular mechanisms of protein targeting and integration at the IEM are very limited. Previous reports have led to the conclusion that IEM proteins are transferred to the IEM during protein import via a stop-transfer mechanism. Recent studies have shown that at least two components of the Tic machinery (AtTic40 and AtTic110) are completely imported into the stroma and then re-inserted into the IEM in a post-import mechanism. This led me to investigate the mechanisms and pathways involved in the integration of chloroplast IEM proteins in more detail. I selected candidates (AtTic40 for post-import and IEP37 for stop-transfer) that are predicted to have only one membrane-spanning helix and adopt the same IEM topology to facilitate my analysis. My studies confirm the existence of both stop-transfer and post-import mechanisms of IEM protein targeting. Furthermore, I conclude that the IEP37 transmembrane domain (TMD) is a stop-transfer signal and is able of diverting AtTic40 to this pathway in the absence of AtTic40 IEM targeting information. Moreover, the IEP37 TMD also functions as a topology determinant. I also show that the AtTic40 targeting signals are context dependent, with evidence that in the absence of specific information in the appropriate context, the AtTic40 TMD behaves as a stop-transfer signal. This is an indication that the stop-transfer pathway is the default mechanism of protein insertion in the IEM.

U14 small nucleolar RNA (snoRNA) is required for processing of 18S ribosomal RNA. It was hypothesized that U14 might base pair with 18S RNA through two highly conserved U14 sequence elements known as domains A and B. Using Saccharomyces cerevisiae as the experimental system, I showed that: (1) the domain A and B elements are functionally interdependent, and (2) single-point mutations in domain A combined with complete substitution of domain B causes lethality while either mutation alone does not. Direct interaction of U14 with 18S RNA was shown by demonstrating that a lethal mutation in U14 domain A can be suppressed with a mutation which restores complementarity in the corresponding region of 18S RNA. Y-domain in yeast U14 was postulated to serve as a recognition element for vital intermolecular or intramolecular interactions. Consistent with this assumption, mutations in several conserved nucleotides of the loop cause growth defects. In contrast, alterations to the stem have little or no effect. Using a lethal mutation in the loop, three different intragenic suppressor mutations were mapped to three positions adjacent to the primary mutation, and are predicted to influence the structure of the loop. An extragenic suppressors (UF1) able to rescue a cold-sensitive mutation in the loop encodes an essential putative ATP-dependent RNA helicase. Loss of UF1 gene expression caused a reduction in 18S rRNA production, without affecting accumulation of 25S rRNA or U14 snoRNA. Pulse-chase analysis showed that depletion of UF1 protein impaired pre-18S rRNA processing. Finally, an effort was made to define minimum pre-rRNA substrates that can be used to produce functional 18S and 25S rRNAs in vivo. The rDNA operon was split either between the 18S RNA and 5.8S/25S coding units, or between the 18S/5.8S RNA and 25S RNA coding units. The test fragments were expressed from GAL7 promoters. The results showed that functional rRNAs could be produced in trans, but only when the operon was divided between the 18S RNA and 5.8S/25S RNA coding sequence.

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.

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.

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.

Programmed cell death (PCD) is an essential developmental process in all multicellular organisms. It serves multiple functions including selected removal of unneeded and/or deleterious cells, and regulation of cell numbers (reviewed in Milligan and Schwartz, 1996). The intersegmental muscles (ISMs) of the hawkmoth Manduca sexta provide an ideal model for studying PCD (reviewed in Schwartz, 1992). In response to a decline in the circulating titer of the steroid hormone 20-hydroxyecdysone (20-HE), these cells initiate a death program which includes both the up- and down-regulation of specific genes. Following eclosion, the ISMs undergo PCD that results in the complete destruction of the muscles during the subsequent 30 hours (Finlayson, 1956). This dissertation examines in detail the repression of actin and myosin heavy chain expression that occurs when the muscles become committed to die. At the protein level, actin expression was reduced by 84% at the time the muscles were committed to die, which presumably plays a role in the rapid dissolution of the muscles. When the ISMs became committed to die, there were dramatic increases in proteolytic activity that are correlated with an approximately eightfold increase in the absolute amounts of multicatalytic proteinase (MCP). At the time of commitment, four new MCP subunits were observed to be associated with the complex. Correlated with the addition of these new subunits was a dramatic increase in the levels of immunodetectable MCP throughout the cytoplasm and within the nuclei of dying muscles. These changes in MCP were regulated by the same hormonal signals that mediate cell death. Cells dying by PCD often display a characteristic set of features termed apoptosis. These features include chromatin condensation, DNA fragmentation, membrane blebbing and phagocytic removal of the dying cells. However, dying ISMs display few characteristics of apoptosis. Interestingly, apoptotic cell death does occur in Manduca embryogenesis. Evidently Manduca possesses the necessary biochemical machinery to undergo apoptosis and does so in specific developmental circumstances. These data suggest that more than one cell death mechanism is used during development.

The pituitary gland is the major endocrine gland in the forebrain. The hormones secreted from this gland regulate vital processes such as reproduction, growth and stress response. Distinct endocrine cells arise from pituitary precursors cells. The endocrine cells are spatially organized along the anterior-posterior axis within the anterior lobe of the pituitary, the adenohypophysis. Several signaling molecules have been shown to play roles in the development of this endocrine gland. Previous work in our lab showed that Sonic Hedgehog (Shh) is required for induction and patterning of the adenohypophysis. Hedgehog (Hh) signaling is involved in many developmental processes including induction, patterning and differentiation of many tissues. In addition, independent studies show that Fibroblast growth factor (Fgf) signaling also plays a role in the development of zebrafish adenohypophysis. ^ One of the main aims of my dissertation was to determine how the Hh and Fgf signaling pathways specify the functional patterning of the adenohypophysis. Using small molecule inhibitors I show that high levels of Hh signaling are required for the formation of the anterior adenohypophysis, the pars distalis (PD) and high levels of Fgf signaling are required for the formation of the posterior adenohypophysis, the pars intermedia (PI). My dissertation work also shows that high Hh levels are required for differentiation of the endocrine cells in the PD, and in contrast high Fgf levels are required for differentiation of the endocrine cells in the PI. Using live-imaging of a transgenic zebrafish line, I show that the PD and PI originate from distinct regions. My analyses has revealed that graded Hh and Fgf signaling help pattern the adenohypophysis along anterior-posterior axis by guiding endocrine cell differentiation in a dose dependent manner. ^ A related aim of my research was to analyze the role of Hh signaling in zebrafish neural tube patterning. The transcriptional response to varying Hh levels is well characterized in chick and mouse neural tube, and this transcriptional response has been partially described in zebrafish. The analysis of the Hh transcriptional response in wild type, Hh mutant and Hh over-expressing embryos show that there is a conserved transcriptional response to Hh signaling in the zebrafish neural tube. My comprehensive analyses of the Hh transcriptional response in the zebrafish neural tube provides a useful tool for the characterization of Hh signaling in zebrafish. ^