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.

N-linked glycans serve as quality control tags in the eukaryotic secretory pathway. The endoplasmic reticulum (ER) protein UDP-glucose: glycoprotein glucosyltransferase 1 (GT1) is the main enzyme that modifies carbohydrate tags based upon the folding state of the maturing substrate. GT1 adds glucoses to non-glucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate as well as its ability to differentiate between native or aberrant secretory cargo is poorly understood. Additionally, due to inherent difficulties in studying GT1 activity in the cell, identification of endogenous substrates and the necessity of reglucosylation remain unknown. Here, we analyzed the role of GT1 in glycoprotein maturation in the intact mammalian ER. GT1 post-translationally reglucosylates N-linked glycans in slow-folding regions of substrate glycoproteins. Maturation mutants that disrupt oxidation or oligomerization also support regio-specific reglucosylation by GT1. Our studies have also revealed an abundant endogenous substrate of GT1, identified as prosaposin. GT1 is critical for the maturation of endogenous prosaposin. In the absence of GT1, the endogenous protein is mislocalized to large intracellular juxtanuclear aggregates. Together, these results propose that GT1 acts as an ER quality control sensor by post-translationally targeting glycans on slow folding or non-native domains to recruit chaperones specifically to critical unstable regions. GT1 plays a vital role in endogenous protein folding and trafficking, since in its absence misfolded proteins accumulate intracellularly. This investigation provides new insight into the integral role of GT1 in glycoprotein maturation.

A pollen tube is the cell that navigates sperm cells through the style and delivers them to the ovum, in a rapid and highly-polarized fashion. Underlying the process of pollen tube growth are a dynamic actin cytoskeleton and oscillating gradients and fluxes of ions. The work presented in this dissertation focuses on visualzing the actin cytoskeleton and gauging calcium dynamics in growing pollen tubes. Three different GFP-labeled actin-binding domains are used to probe the actin cytoskeleton of growing Lilium and Nicotiana pollen tubes. Each marker highlights different aspects of the actin cytoskeleton, whereas no single marker reveals the entire spectrum of actin present. Thus, GFP-ADF and GFP-talin reveal elements of the cortical fringe of actin, whereas GFP-fimbrin generously labels actin filaments in the shank of the pollen tube. Because high levels of expression inhibit pollen tube growth and cause structural aberrations of the actin cytoskeleton, I urge caution in their use. A steep tip-focused gradient of calcium that oscillates with the same periodicity as growth rate, is a consistent feature of growing pollen tubes. Conventional methods for monitoring calcium in growing pollen tubes rely on microinjection of fluorescent dyes, a procedure that is both invasive and technically challenging in the small pollen tubes of the model species Nicotiana tabacum and Arabidopsis thalaiana . Here, two transformable calcium indicators, yellow cameleon YC2.1 and ratiometric pericam, are introduced as effective indicators of cytoplasmic calcium in growing pollen tubes. Finally, mag-fura 2 is employed to measure the calcium concentration in the lumen of the endoplasmic reticulum ([Ca 2+]L) of growing Lilium formosanum pollen tubes. I provide evidence that calcium ions are periodically withdrawn from the apex and funneled into the endoplasmic reticulum. I thus support the hypothesis that the endoplasmic reticulum sequesters calcium in the clear zone and thereby confines the tip-focused gradient to the apex. The endoplasmic reticulum therefore emerges as a potential key player in the process of pollen tube growth.

Programmed cell death is essential for normal development and adult tissue homeostasis in almost all multicelluar organisms. Acheron gene was first isolated from the intersegmental muscles (ISMs) in Manduca sexta as a death-associated gene. Subsequently, we cloned human and mouse homolog of Acheron. Acheron encodes a novel protein that has not been previously characterized. Protein structure analysis revealed that Acheron proteins are structurally related to La proteins, but define a novel subfamily. Tissue expression analysis showed that mAcheron is widely expressed in most tissues at both the RNA and protein levels, with brain and heart displaying the highest levels. In mouse C2C12 cells, endogenous Acheron is constitutively expressed in cycling myoblasts and myotubes. Despite the presence of a putative nuclear localization site, the protein is localized predominantly in the cytoplasm. Analyses of the different Acheron transfected C2C12 cells suggested that Acheron is implicated in mediating differentiation and apoptosis in C2C12 cells by differentially regulating the expression of MyoD, Myf5 and Bcl-2. Acheron expression allows C2 C12 cells to up-regulate MyoD and differentiate into myotubes when the cells are induced to undergo differentiation. However, it does not support the myoblast self-renewal by specifically inhibiting the expression of Bcl-2, a key survival factor for ‘reserve’ cells in DM. Inhibition of Acheron activity by tAch (a putative dominant negative regulatory factor of Acheron) or antisense Acheron results in greatly increased ‘reserve’ cell population and decreased differentiation under differentiation condition. The mediation of differentiation and survival by Acheron may be achieved through its regulation on integrin—FAK signaling. To help determine how Acheron functions, we performed a yeast 2-hybrid screen with Acheron as the bait. A clone that contains partial cDNA of Ariadne was isolated from the screen. Ariadne contains RING finger domain and is known to bind to ubiquitin E2 conjugase. In vitro ubiquitination assay revealed that Ariadne has ubiquitin E3 ligase activity. We speculate that Ariadne may function as an E3 to target Acheron for ubiquitination and subsequent proteasome-dependent degradation.

Human lysosomal α-N-acetylgalactosaminidase (α-NAGAL) is responsible for the break down of glycolipids and glycopeptides that contain a terminal α-linked N-acetylgalactosamine residues. Deficiency of α-NAGAL results in Schindler and Kanzaki diseases. α-NAGAL is closely related to another lysosomal enzyme, α-galactosidase (α-GAL), which breaks down glycolipids and glycopeptides with a terminal α-linked galactose residues. Fabry disease results from a deficiency of α-galactosidase activity. We studied the reaction mechanism of both enzymes using biochemistry and X-ray crystallography, and found that α-GAL and α-NAGAL use an identical reaction mechanism, and differ only in substrate specificity. We solved the first structure of human α-NAGAL, allowing us to examine the disease-causing patient mutations in the context of a high-resolution 3D atomic structure, moving Schindler and Kanzaki disease into the realm of personalized molecular medicine. We then developed the first ever proof-of-principle treatment of Schindler and Kanzaki disease, by developing and characterizing 2 pharmacological chaperones that show promise to treat Schindler and Kanzaki diseases, which currently have no treatment options.

Comparative cytological and molecular phylogenetic data suggest that amitochondriate trichomonads (Archaeprotista: Parabasalia) are an early branching lineage of eukaryotes. Prominent in their cells is the karyomastigont, a cytoskeletal organellar system that includes a nucleus, 4–5 kinetosomes, an axostyle, a parabasal body (Golgi complex), and associated fibers. The karyomastigont is an ancestral character of eukaryotes. Genera of the trichomonad family Calonymphidae (Calonympha, Coronympha, Metacoronympha, Stephanonympha and Snyderella) are polymastigont: multinucleate cells with many karyo- or akaryomastigonts (karyomastigonts that lack nuclei). Termite hindgut trichomonads were examined for their susceptibility to antibiotics, pattern of cell division, and to test the hypothesis that DNA is associated with centriole-kinetosomes. Several types of bacterial symbionts are harbored on the surface membranes, in the cytoplasm and nuclei of these anaerobic protists rendering them susceptible to antibacterial antibiotics. Caduceia sp., a devescovinid trichomonad with four types of bacterial symbionts, was removed within 14 days from the Cryptotermes cavifrons (Florida) gut when penicillin and streptomycin were fed to the termite. Snyderella tabogae was far less susceptible to the antibiotic treatment, which also enhanced formation of calcium-rich crystals in the intestine. Metacoronympha divides asymmetrically. M. senta from Incisitermes nr. incisus (Trinidad) also showed a bimodal distribution of cell size. Approximately one third of the population averaged 100 μm in length with as many as 1000 karyomastigonts, unlike the previously described 50 μm mean length with up to 350 karyomastigonts. The cytoskeleton of Snyderella tabogae, the only calonymphid whose complete set of nuclei are not attached to centriole-kinetosomes, consists solely of 500 or more akaryomastigonts (>2000 undulipodia and their kinetosomes). In Snyderella tabogae, groups of 20–50 akaryomastigonts simultaneously beat and form locally organized regions of the cortex. These polymastigont cells are ideal to seek centriole-kinetosome-associated DNA. Fluorescent cytological (DAPI, SYTOX, acridine orange, ethidium bromide, propidium iodide) as well as Feulgen stains were negative: no DNA was present in the centriole-kinetosomes of S. tabogae. However, DAPI accumulates in the parabasal bodies (Golgi complex).

Viral infection of cells often requires many host cell factors. Despite the identification of numerous viral receptor molecules relatively little is known about the roles of these molecules in viral entry, and intracellular targeting. Major histocompatibility complex (MHC) class I proteins are components of the Simian Virus 40 (SV40) cell surface receptor. This interaction between virus and host cell is of interest since MHC class I proteins have typically been studied for their importance in cellular immune responses. MHC class I proteins acquire antigenic peptides in the endoplasmic reticulum (ER). SV40 is unique in its ability to target the ER from the cell surface. Thus, the possibility exists that surface MHC class I proteins may recycle and target the ER. A major question is do MHC class I proteins internalize and target SV40 to the ER. The goal of this thesis was to better characterize SV40 entry, and determine if MHC class I proteins are directly involved in viral internalization. Basic studies were therefore conducted to determine the fate of MHC class I proteins on fibroblasts. SV40 entry into cells was found to occur rather slowly. The majority of preadsorbed virions remain at the cell surface for up to 2.5 hours. Furthermore, three hours were required for preadsorbed SV40 to internalize and escape antiserum neutralization. This suggests that SV40 infectious entry is relatively slow. Cytosol acidification greatly reduced clathrin-dependent endocytosis, but had no inhibitory effect on SV40 infectious entry. Therefore, SV40 infectious entry does not occur by receptor-mediated endocytosis. Treatment of cells with PMA, nystatin, or filipin prevents internalization via caveolae. All three treatments prevented SV40 infectious entry. Thus, caveolae may be the sites of SV40 penetration into cells. To evaluate the role of MHC class I proteins in SV40 endocytosis, cell surface proteins were labeled with $\sp{125}$I. Protease treatment of cells was used to distinguish proteins that have internalized from those remaining at the cell surface. Protease resistant $\sp{125}$I-MHC class I proteins were not detected in the absence, or presence of SV40. Furthermore, a $\sp{125}$I-MHC class I protein-specific monoclonal antibody did not internalize into cells. These results suggest that surface MNC class I proteins are not internalized into cells, and SV40 does not induce their internalization. Truncated $\sp{125}$I-MHC class I proteins were detected in the media. 1,10-phenanthroline prevented accumulation of $\sp{125}$I-MHC class I proteins in the media. Thus, metalloprotease activity is involved in shedding of surface MHC class I proteins from CV-1 cells. The loss of the B$\sb2$-microglobulin subunit from the MHC class I heavy chain was found to precede metalloprotease cleavage of the heavy chain. Collectively, these results suggest that the fate of MHC class I proteins on fibroblast is shedding into the media, and these molecules are probably not directly involved in SV40 entry.