The root of the Tibetan plant Rhodiola crenulata is part of eastern traditional medicine. Studies have suggested that members of the Rhodiola genus display anticancer properties. In this study we examine the effect of R. crenulata in a cellular model of invasive breast cancer, this disease being the second cause of cancer death among women in the US. Deregulation of the Wnt/β-catenin pathway has been frequently observed in breast cancers and appears to have a key role in the transformation of benign cells to a malignant form. Although mutations of the Wnt growth factor are rarely observed in cancer, the Wnt signaling pathway is often up-regulated by either mutations that result in stabilization of β-catenin or by hypermethylation and subsequent loss of expression of Wnt signaling antagonists like secreted Frizzled-Related Protein 1 (SFRP1) (Hanahan and Weinberg 2000; Miyoshi, Rosner et al. 2002; Reya and Clevers 2005; Suzuki, Toyota et al. 2008) (Hanahan and Weinberg 2000; Miyoshi, Rosner et al. 2002; Reya and Clevers 2005; Suzuki, Toyota et al. 2008) (Hanahan and Weinberg 2000; Miyoshi, Rosner et al. 2002; Reya and Clevers 2005; Suzuki, Toyota et al. 2008). We used an engineered cell line in which SFRP1 expression has been knocked down. These cells were derived from 76NTert cell line, an immortalized human mammary epithelium cell line. The resulting 76NTert-siSFRP1 cells display a mesenchymal-like phenotype, invasive behavior and are more resistant to apoptosis triggered by anchorage independent conditions, or anoikis. Additionally we used a highly invasive estrogen receptor negative (ER-), progesterone receptor negative (PR-), Her2/neu negative (triple negative) breast cancer cells line MDA-MB-231. Treatment of MDA-MB-231 and 76NTert-siSFRP1 cells with an extract of R. crenulata inhibited migration and invasion of both cell types, as compared to untreated cells. Furthermore, R. crenulata sensitizes cells to anoikis but does not increase γ-irradiation induced cell death. We provide evidence that death induced by R. crenulata does not occur through the inhibition of an epithelial-to-mesenchymal transition (EMT). Taken together, our initial results suggest R. crenulata as a potential therapeutic agent for breast cancer patients with mutations in the Wnt/β-Catenin signaling pathway. Additionally, we present evidence that R. crenulata exerts its anti-metastasis effect by inhibiting cell migration and increasing cell attachment to a substrate. We demonstrate that this effect occurs by R. crenulata's modulation of the Rho GTPase effector ROCK1. We further show evidence that R. crenulata's effect on ROCK is not limited to cancer cells (in vitro), but also affects isolated and intact embryonic tissues. We discovered that treatment of embryos with R. crenulata can cause a spina bifida phenotype, suggesting (1) that compounds in R. crenulata may prove detrimental to a developing embryo, and (2) the active compounds in R. crenulata may prove useful in the study of developmental anomalies that lead to conditions such as spina bifida. More importantly, our results suggest that pregnant women should avoid taking R. crenulata-containing supplements during pregnancy. Compounds in R. crenulata may be contraindicative to the pregnancy and cause injury to a developing fetus. The information provided may help health providers offer better advice on natural supplements to expecting mothers. We also characterized and identified multiple R. crenulata compounds and report predicted protein targets for these compounds. Finally, we provide evidence that R. crenulata affects cancer cell metabolism and suggest potential protein targets of the chemical components of this extract. This study provides new information that will help dissect the mechanisms of action of the R. crenulata compounds and possible synergies amongst them.

Calmodulin (CaM) is a major intracellular calcium receptor. There is abundant calmodulin (CaM) in the oocytes and eggs of B. germanica during vitellogenesis and early embryogenesis. The accumulation of CaM in oocytes may be for immediate use in the oocytes and/or in preparation for later stages of their development. Previous investigation from this laboratory suggested that maternal follicle cells are the most likely source of this CaM. Tissue culture labeling with $\sp{35}$S methionine showed a 13-fold higher rate of synthesis of CaM in the follicle cells than in oocyte preparations (Zhang & Kunkel, 1994). The high rate of biosynthesis of CaM in the follicle cells, and the absence of extracellular CaM in transit in the hemolymph suggested that CaM is made in the follicle cells and transferred to the oocytes. In order to obtain more information about the site of CaM synthesis I isolated total RNA from different tissues that could potentially contribute to the high amounts of follicular CaM and measured the amounts of CaM transcripts during development. I show that isolated whole follicles accumulate more transcripts for CaM than the fat body. The steady state levels of CaM transcripts increases 150 fold during the 4 day developmental period under study. This is in addition to a 32 fold increase in total follicle RNA during the period. Steady state levels of CaM transcripts in whole follicles also show a pattern of increase disproportionate to the increase in volume of the whole follicle. In comparison steady state levels of actin transcripts increase 35 fold during the same developmental period. At 96 hr post feeding, in a given amount of total RNA, follicle cell total RNA contains 3 times more CaM transcripts than whole follicle total RNA, and 70 times more CaM transcripts than the fat body tissue. The oocyte total RNA collected from material expelled from the whole follicle contains less than 10% of the amount of CaM transcripts available in the follicle cells. The fat body tissue preparation shows little developmental increase in steady state levels of CaM transcripts despite a 4 fold increase in total RNA. In my investigation into the control of the accumulation of this transcript I found that deprivation of JH, by head ligation, not only causes atresia of the follicles, but also reduces CaM transcript accumulation. Reconstituting JH titer by injection allows a selected population of follicles to develop to full size and also reinstates steady state CaM transcript levels above that of unligated controls. The results of my study makes the CaM gene a potentially important target for the study of JH action in follicle cells during oogenesis.

Isolated presynaptic nerve terminals (synaptosomes) prepared from rat brain were used to evaluate the action of a classic T-syndrome (cismethrin) and CS-syndrome (deltamethrin) pyrethroid on voltage-sensitive calcium channels by measuring calcium influx and neurotransmitter release with multiple fluorescent assays. Both cismethrin and deltamethrin stimulated calcium influx in a stereospecific manner; however, they did so by different mechanisms. Neurotransmitter released occurred only with synaptosomes treated with deltamethrin. This release was stereospecific, stimulated by depolarization, unaltered by tetrodotoxin, but blocked by ω-conotoxin MVIIC. Electrophysiological experiments with Cav 2.2 expressed in Xenopus oocytes validated the interaction of deltamethrin with this N-type calcium channel. Thus, cismethrin and deltamethrin elicit different actions at presynaptic nerve terminals. The modification of Cav 2.2 voltage-sensitive calcium channels by deltamethrin is consistent with enhanced neurotransmitter release, a physiological response that has been observed during acute neurotoxicity of CS syndrome pyrethroids. Rat brain synaptosomes were also used to evaluate the action of 11 pyrethroids (bifenthrin, bioallethrin, cismethrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin, permethrin, and tefluthrin) by measuring calcium uptake, membrane depolarization, and neurotransmitter release as before. Our results indicate that only a subset of the commercially available pyrethroids act as voltage-sensitive calcium channels agonists (permethrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, and esfenvalerate). Pyrethroids that significantly enhanced calcium influx were also more potent in releasing neurotransmitter. Increased glutamate release appears to be due to an agonistic action of these compounds on Cav 2.1 and Cav 2.2 voltage-sensitive calcium channels, most associated with neurotransmitter release. Biochemical and electrophysiological experiments provide functional evidence for a distinct mechanism of action for some of the pyrethroids on voltage-sensitive calcium channels at the presynaptic nerve terminal that results in enhanced neurotransmitter release, a response that is consistent with the observed symptomology of CS-syndrome pyrethroids. Such results will allow a more complete understanding of the molecular and cellular nature of pyrethroid-induced neurotoxicity and expand our knowledge of the structure-activity relationships of pyrethroids in regards to the voltage-sensitive calcium channel.

Eukaryotic cells contain scores of small nucleolar RNAs (snoRNAs), which are required for maturation of pre-rRNA. Two large snoRNA families exist defined by vital box C/D and box H/ACA motifs. The goal of the present study was to gain new insights into the structure and biogenesis of the box C/D snoRNAs; the knowledge developed from this effort was then recruited for practical applications. The investigation was conducted with the phylogenetically conserved U14 and U3 box C/D snoRNAs, from the yeast Saccharomyces cerevisiae. The specific aims included: (1) identification of cis-elements sufficient for biogenesis of the U14 snoRNA; (2) development of a functional map for the U3 snoRNA, and; (3) development of a U3-based model ribozyme system for in vivo studies. Conclusions derived from the U14 biogenesis studies are: (1) production of U14 involves ordered folding of the precursor RNA, and this step is required for formation of the vital box C/D structure motif, and; (2) the active box C/D motif, which is now predicted to consist solely of the box C and D elements, is necessary and sufficient for both accumulation and targeting RNA to the nucleolus. A general model for box C/D snoRNA biogenesis is proposed. Functional mapping of U3 revealed that: (1) boxes C$\sp\prime$ and D and flanking helices are critical for U3 accumulation; (2) boxes B and C are not essential for U3 production, but are important for function, due most likely to binding of a trans-acting factor(s); (3) the 5$\sp\prime$ portion of U3 is required for function, but not stability, and; (4) the non-conserved hairpins, which account for 50% of the molecule, are not required for accumulation or function. Based on the knowledge obtained with U14 and U3, a model ribozyme system featuring chimeric U3:ribozyme RNAs, or "snorbozymes", was developed and tested in vivo. Remarkably, the cleavage efficiency by a hammerhead ribozyme, both in cis- and in trans-configurations, appears quantitative! Other advantages of the system are: (1) a final product is stable, and; (2) authentic in vivo cleavage can be easily distinguished from artifactual cleavages. Snorbozymes are predicted to be useful for targeting natural transcripts in any eukaryotes, for fundamental research or practical applications.

RAC/ROPs are a unique group of RAS-related monomeric G proteins (small G proteins) that constitute the sole family of Rho GTPases in plants. RAC/ROPs, like their counterpart Rho GTPases from mammalian and fungal systems, can interconvert between an active GTP and an inactive GDP bound state. These powerful signaling molecules lie upstream in many diverse signal transduction pathways. Their controlled regulation is critical to overall plant fitness, growth, development, and responses to abiotic and biotic stress. RAC/ROP activation is regulated by guanine nucleotide exchange factors (GEFs). The work in this dissertation initiated from the characterization of the expression and functions of RopGEF1, a broadly expressed GEF that localizes to the sites of root hair formation and regulates polarized cell growth. The focus of this dissertation is on FERONIA (FER), an upstream regulator of RopGEF1 that was initially identified as a RopGEF1 interacting protein in a yeast two-hybrid screen. FERONIA (FER) was found to regulate RAC/ROP-mediated signal transduction for auxin-regulated root hair growth and a number of other auxin-dependent responses, consistent with RAC/ROPs playing an important role in auxin signaling. ^ In flowering plants, pollen tubes deliver sperm to fertilize the female gametophytes located inside the ovules. Once a pollen tube enters it subsequently bursts releasing its sperm, which enables fertilization. Mechanisms are in place to coordinate tube rupture as well as repel late arriving pollen tubes from an already visited ovule. In this way not yet visited ovules have higher chances of fertilization and fertilized ovules avoid polyspermy. In this work, FER is demonstrated to mediate NADPH oxidase-dependent reactive oxygen species production required for pollen tube rupture. In addition, FER is also required for de-esterified pectin deposition outside the female gametophyte, which correlates with the ability of ovules to divert late arriving pollen tubes. Furthermore, the extracellular domain of FER, which contains predicted carbohydrate-binding malectin-like domains, interacts directly with pectin. This finding establishes FER as a cell wall-binding receptor kinase in plants and illuminates unprecedented mechanisms of pollen tube reception. ^ The presence of carbohydrate binding motifs in the extracellular domain of FER and its direct interaction with pectin prompted my investigation of its role in sugar sensing and signaling pathways. My work shows that feronia (fer) mutants are hypersensitive to sucrose, while over-expressing FER suppresses sugar signaling. Moreover, fer mutants accumulate elevated levels of starch, which demonstrates defects in their distribution of carbohydrate resources in source (sugar producing) and sink (sugar consuming) tissues. The fer mutants also display sucrose-induced cell wall defects, alterations to cellular morphology, and enhanced production of the stress-associated pigment, anthocyanin. These results suggest that FER functions as a negative regulator of sugar sensing and signaling pathways. Additional support for this model stems from the finding that FER is highly expressed in tissues involved in sucrose transport and its expression is stimulated by sucrose. Furthermore, and consistent with the hormone abscisic acid being part of the overall sugar sensing network, fer mutant seedlings are hypersensitive to ABA and display enhanced ABA response gene expression. Taken together, the data presented in this dissertation reveal the regulation of widespread and essential plant functions including RAC/ROP signaling, pollen tube reception, cell wall integrity, and sugar signaling by a single cell surface receptor kinase.^

Biology is the study of life and life systems. Until recently, biologists have concentrated in examining how cells proliferate, differentiate, and survive in biological systems. Although such studies reveal extremely interesting insights into the complexity of living organisms, there is much more to this story. Just as cells live, cells must also eventually die. The study of how and why cells die has become the focus much scientific research over the past decade. Because the regulation of the number and specificity of cells in the immune system is critical to the life of a mammalian organism, researchers began to investigate how this strict control was accomplished. It was found that large quantities of immature T cells die in the course of their development by a specific type of cell death process coined apoptosis. This Ph.D. dissertation was directed towards examining the mechanisms involved in the regulation of thymocyte apoptosis in a murine model system. The first portion of the project involved isolating differentially regulated genes using either a plus/minus or subtractive hybridization screening strategy The second component of this dissertation investigated possible roles molecular oxygen and/or free radicals play during thymocyte apoptosis. Results from these studies both identified numerous putative death transcripts as well as revealed the requirement for oxygen during cell death in thymocytes induced by specific stimuli.

Glioblastoma, previously known as Glioblastoma Multiforme (GBM), is a highly angiogenic tumor and is defined pathologically by its ability to create microvascular proliferations. Patients with glioblastoma have a high rate of morbidity and mortality because of the aggressive nature of this type of tumor. Despite current optimal treatments, glioblastoma is almost universally fatal with the current median survival being between 12–15 months. The current treatment for glioblastoma involves surgical resection followed by chemotherapy and radiation. However, recently a new anti-angiogenic treatment called Bevacizumab (trade name Avastin) has been able to prolong survival, significantly in some cases. Some patients on this therapy have demonstrated wonderful responses, but in most cases, this response is transitory and there is eventual recurrence, thus highlighting that there could be resistance to anti-angiogenic therapies inherent within glioblastoma. Here, it is proposed that this resistance is mediated by an alternative angiogenic pathway that is produced by glioblastoma stem-like cells. Through a trans-differentiation process, we have found that the vast majority of the vasculature (80%) acquires a mural cell phenotype that "mimics" the vasculature and creates a blood supply to the tumor. Although this process has been defined in other types of cancer as Vasculogenic Mimicry, this process has never been demonstrated to be due to mural cell involvement. We have also identified and more fully characterized this alternative form of vasculogenesis by its dependence on a molecular pathway through which it is created and maintained in glioblastoma. We have found that the glioblastoma stem-like cells transdifferentiated in vitro, and in vivo in a Flk-1 (VEGFR2) dependent manner when transplanted into SCID/beige mice. When this receptor was knocked down in transdifferentiated glioblastoma stem-like cells, it inhibited tumor growth and development almost completely. Since current angiogenic therapies for glioblastoma do not target this form of vasculogenesis, it is hoped that this work will in part fill a void in the current research and create additional knowledge to help better guide treatment for these patients.

When fractionated using an HPLC ion exchange column, three distinct peaks (peak I, II, and III) of phosphatase activity were observed in the supernatant of homogenized bovine adrenal medulla cells, suggesting the presence of at least three different phosphatases. These phosphatases showed different activities toward phosphocasein in the presence of Mg$\sp{2+}$ and Mn$\sp{2+}$. Peak III, which represents about 50% of the active enzyme activity when phosphocasein is used as substrate, showed a molecular weight of 140 KDa as determined by HPLC gel filtration and has been identified as a type 2A protein phosphatase. Okadaic acid, a phosphatase inhibitor (specific for type 2A and type 1) and tumor promoter, was employed to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. After 3 days cultured in the presence of 50 ng/ml NGF, 20-25% of the PC12 cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC$\sb{50}$ of about 7 nM. This inhibition was rapidly reversed after wash-out of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells indicating that continual phosphatase activity is required to maintain neurites. A 27-mer oligonucleotide was synthesized as a hybridization probe to isolate clones encoding the sequence of protein phosphatases from a bovine adrenal medulla cDNA library. A cDNA clone encoding the full length of the catalytic subunit of protein phosphatase type 2A has been isolated. The deduced protein sequence (309 residues, 35.63 KDa) is 99.7% identical to that of phosphatase 2A$\alpha$ form from rabbit skeletal muscle, human liver and porcine kidney and differs by only one amino acid (Arg-55 vs. Cys-55). At the nucleotide level, the clone showed 97% identity with that of the catalytic subunit of protein phosphatase type 2A$\alpha$ from human liver. Sequence comparison of bovine adrenal medulla clone with phosphatase type 1 from rabbit skeletal muscle and type 2B from mouse brain identifies six highly conserved domains in the three enzymes that are expected to be crucial for the catalytic activity of protein phosphatase.

The intersegmental muscles (ISMs) of the tobacco hawkmoth, Manduca sexta, undergo two periods of developmentally programmed cell death. The first period occurs during the larval/pupal transition where half of the ISMs die and the remainder persist throughout pupal development until death upon eclosion of the adult. Both periods of ISM death employ much of the same molecular machinery even though the endocrine cues for the degeneration process is different for each period of muscle death. A gene that is dramatically increased in its expression during both periods of ISM death is polyubiquitin. This gene has been isolated and characterized in this research. Despite a large amount of allelic heterogeneity in the population, it has been determined that not only is this gene increased in response to developmental cues, but that its transcript is also increased in response to stress. Therefore, polyubiquitin is multifaceted in its regulation. Due to the diverse transcriptional regulation of this gene, it was determined to be a good candidate in which to develop a method to introduce promoter/reporter constructs into the ISMs of the hawkmoth. Even though this research does not conclusively demonstrate that reporter activity after transfection of the promoter/reporter constructs is due to the polyubiquitin promoter, the research does demonstrate that DNA mediated transfection of insect muscle tissue can be achieved.

<p>Over the course of development, cells and tissues of the embryo must take on the correct fates and morphologies to produce a functioning organism. The patterning events and morphogenetic processes that accomplish this task have been the subject of decades of research, the consequence of which has been a detailed comprehension of the molecular mechanisms that regulate each. Equally important is an understanding of the mechanisms that coordinate patterning with morphogenesis, such that they occur with the correct relative spatiotemporal dynamics. My thesis work sought to characterize such co-regulation in the context of two developmental events in a vertebrate model, the African clawed frog Xenopus laevis: induction of bottle cell formation at the onset of gastrulation after germ layer induction, and regulation of the morphogenetic movements of neurulation in relation to neural plate patterning. Chapter 1 of this dissertation provides a general introduction to the patterning and morphogenetic events of early development relevant to my thesis. Chapter 2 presents a discussion of my work to characterize the function of two signaling pathways, namely Nodal signaling and Wnt/Planar Cell Polarity, in the induction of bottle cells. My experiments confirm the requirement for Nodal signaling in bottle cell induction, but do not support a role for the individual transcriptional targets of Nodal signaling tested here or for Wnt/PCP. Chapter 3 summarizes my work to address the function of two transcription factor-encoding genes, sall1 and sall4, in neural development, including their roles in anteroposterior neural patterning, neural tube morphogenesis, and neural differentiation. My work shows that both sall1 and sall4 are required for all three processes, and supports the hypothesis that their key role in this context is to transcriptionally repress stem cell factors of the pou5f3 family, allowing progression through neural development. As a whole, this work summarizes my research to characterize molecules that co-regulate early patterning and morphogenetic events in the X. laevis embryo.