Wnt ligands are a family of highly conserved glycoproteins that act as morphogens to regulate development in many organisms. Drosophila Wnt-1 (Wingless) is involved in directing cell fate decisions and pattern formation during differentiation. Wnt signaling are of high interest of many developmental biologists due to their important functions, yet little is known about how these ligands function on a biochemical level. Previously it was found that Porcupine, an ER-membrane-bound acyltransferase is required for Wingless secretion. But it is unclear how a secreted morphogen requires an acyltransferase to function. Studies reported here demonstrated that Wingless undergoes a hydrophobic modification, in which a lipid moiety containing a palmitate group is covalently attached to the polypeptide through an ester linkage. And it partitions with the specialized detergent insoluble lipid raft microdomains in the plasma membrane. Porcupine is required for the modification and the raft targeting of Wingless. Blocking Wingless modification with a specific inhibitor results in the loss of rafts-association as well as loss of protein secretion. Disrupting raft microstructures by cholesterol depletion reagents also impaired Wingless secretion, indicating that the ligand secretion is dependent on its specific association with the plasma membrane. This work provided the first insight on the function of Porcupine and the important biochemical evidence on the role of specialized membrane microdomains in Wnt signaling.

Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP) to CD8 + single positive (SP) transition, but regulation of Notch activation is not well understood. In this thesis, I explored the potential of p53, endocytosis, and Cbl-b to regulate Notch activation. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. One aim of this thesis was to determine if p53 regulates Notch activity during T cell development. I found that Notch1 expression and activation were negatively regulated by p53 in several thymoma lines. Additionally, NIC was elevated in Trp53 −/− thymocytes as compared to Trp53 +/+ thymocytes. To determine if elevated Notch1 activation in Trp53−/− thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP:CD8+ SP T cell ratio was decreased in Trp53 −/− splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8 + SP T cells in Trp53−/− mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, I suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation. In the second aim of my thesis I present preliminary data showing that endocytosis does not appear to be involved in mammalian Notch activation although there is evidence in Drosophila for a positive endocytic role in Notch activation. The ability of Cbl-b to regulate Notch activation was the final aim of this thesis. Cbl-b, like Notch, has been shown to play a role in regulating the T cell signaling threshold. With this aim, I wanted to address the possibility of Cbl-b regulating T cell signaling via regulating Notch activation. Due to technical difficulties I was only able to obtain preliminary data suggesting that Cbl-b does positively regulate Notch activation in peripheral T cells. In this dissertation I have shown that Notch activation is regulated by controlling the expression of cellular components needed for cleavage, not just by encountering ligand on neighboring cells.

The advent of nanobiotechnology has accelerated the application of nanoparticle in biological systems. The unique chemical and physical properties of nanoparticles have established their utility in the sensing and visualization of biological processes. Our goal, however, was to use nanoparticles to control biological activities, specifically through binding protein surfaces. Protein surface binding provides a powerful tool for evaluating and controlling biological processes beyond the scope of small molecules. Monolayer protected nanoparticles are versatile macromolcular scaffolds for protein surface binding. We have demonstrated the use of nanoparticles for efficient binding and inhibition of a model enzyme, chymotrypsin. Nanoparticles featuring a carboxylate-terminated alkanethiol monolayer elicited inhibition by a two-step process; initial binding via electrostatic complementarity followed by slow denaturation. By integrating poly(ethylene glycol) between the recognition unit and the alkane monolayer, inhibition was achieved with no change in chymotrypsin conformation, demonstrating the ability to control the mechanism of enzyme inhibition. Further control of the inhibition can be mediated through modification of the nanoparticle monolayer using cationic surfactants. These strategies for protein surface binding using nanoparticles as a scaffold have been extended to a more complex protein system by decorating the nanoparticles with target-specific peptides. In this manner, the interaction between HDM2 and p53 was successfully disrupted.

The process of transcription is quite complicated and involves many more steps than the classic textbook view of: Bind, melt, transcribe, release. We have studied the complex nature of transcription from the initiation of transcription through the transition from an unstable initiation complex to a stable, processive, elongation complex. We show that promoter release is not required to make the transition to a stable elongation complex. We show conclusively that a proposed model of transient excursion is not adequate for explaining the abortive cycling process that occurs in all known polymerases. We have proposed and tested a model that unites the processes of promoter release, collapse of the initially melted bubble, and proper RNA displacement. Additionally, we have challenged the existing model for the mechanism of inhibition by T7 lysozyme on T7 RNA polymerase.

DNA replication forks are frequently detained during their progression from oriC to the replication terminus due to encounters with DNA lesions or proteins blocking the way. For survival, cells deploy a number of enzymes catalyzing replication restart at or near the damaged site. The preprimosomal protein, PriA, along with several accessory proteins, directs DnaB helicase to the lagging-strand template during assembly of the replisome. This helicase loading mechanism is analogous to that catalyzed by DnaA at oriC, except that PriA-directed fork assembly does not require oriC or any specific initiation sequence. Instead, it recognizes and binds with high specificity to various recombinational intermediates such as D loops that form during the recombination-mediated repair of replication forks. In this manner, PriA effectively links the processes of homologous recombination and DNA replication. PriA also possesses a 3′ to 5′ helicase activity that might be important for restarting stalled forks directly in the absence of homologous recombination. This lab uses a genetic approach to understand the in vivo functions of PriA and its accessory proteins, PriB, PriC, DnaT, DnaC, and Rep. Extensive phenotypic analyses of mutants of priA, priB, priC, dnaC and rep have clearly indicated that replication restart occurs by multiple pathways (Sandler 2000). The work presented in this dissertation continues to explore and test the multiple pathways model for replication restart. Three lines of investigation were pursued: (1) Can genetic evidence support a role for dnaT in replication restart? If so, then for which pathway(s) is dnaT important? (2) It has been proposed that priA mutants accumulate recombinational intermediates. Can this be detected in individual cells using a DNA stain to examine nucleoid organization? (3) PriA mutants are chronically SOS induced. Microscopy indicates that two kinds of cells are present in log phase cultures. Are SOS genes turned on in both populations of cells or only in a subpopulation?

The expression of genetic information carried in the DNA of living cells involves two major steps, transcription and translation. In eukaryotes, the initial transcription products need to be further processed before they can serve as templates for protein synthesis. One such process is splicing, which selectively removes the introns and connects the coding sequences (exons). Splicing also deposits a multiprotein complex, the exon junction complex (EJC), at a position approximately 20 nucleotides upstream of exon exon junctions of spliced mRNA to facilitate post-splicing RNA processing. Among eight identified EJC components, transcription factor Aly/REF and splicing factor UAP56 have been shown to stimulate the export of both spliced and unspliced mRNA. They provide links between these cellular events. During the export, two other components, Y14 and Mago will accompany mRNA into the cytoplasm and maintain association with mRNA until the first round of translation. They are considered core proteins in EJC. During the period of my study, I first crystallized and determined the structure of UAP56(Δ1–44) at 1.95 Å resolution by molecular replacement. The structure reveals that UAP56 belongs to the DEXD RNA helicase family and contains minimal helicase domains in an elongated shape similar to the well-studied protein eIF4A. I then crystallized and determined the crystal structure of Drosophila Y14 (DmY14) RRM domain (a.a. 47–156) in complex with full-length Mago nashi (Mago) at 1.85 Å resolution. The structure shows that the strong interaction maintained between these two proteins is the result of hydrophobic interaction between the RNP motifs of Y14 and Mago. The structure implies that Y14 can not interact with RNA in a manner similar to other RRM-containing proteins. The stable association between Y14/Mago and mRNA is either bridged by unknown protein factors or requires a folding rearrangement.

Although over 20 different neuron ally-expressed genes have been implicated in the regulation of body weight, the transcriptional mechanisms controlling expression of these genes remain unclear. Evidence from our laboratory suggested that the basic helix-loop-helix transcription factor Nhlh2, plays a key role in the regulation of these body weight control genes. Nhlh2 knockout mice are obese after maturation. We found that Nhlh2 is expressed in hypothalamic areas that control body weight such as arcuate nucleus (ARC), paraventricular nucleus (PVN), dorsal and ventral medial hypothalamus (DMH and VMH), and lateral hypothalamus (LHA). In ARC, we showed that pro-opiomelanocortin (POMC) neurons co-expressed Nhlh2 mRNA in a rostral-caudal pattern. In these neurons, the POMC pro-peptide is processed into several bioactive signaling peptides that regulate food intake and energy expenditure. We found that while POMC mRNA levels are comparable in normal and Nhlh2 knockout mice, levels of hypothalamic POMC derived peptides such as β-endorphin and α-MSH were dramatically reduced in the preobese KO mice. These findings suggested that Nhlh2 KO animals could have a defect that prevents production of bio-active POMC derived peptides post-transcriptionally. Since POMC is processed by prohormone convertases such as pro-hormone convertase I (PC1) and prohormone convertase II (PC2), we set out to investigate if differential expression of POMC derived peptides in wild type and Nhlh2 KO mice are regulated via regulation of PC1 and PC2 expression. Indeed, levels of PC1 and PC2 mRNAs were reduced over 50% in the KO animals. Other signaling molecules such as TRH showed the similar expression change to POMC expression that confirms our hypothesis that PC1 and/or PC2 are regulation target candidates of Nhlh2. We identified motifs in the PC1 promoters that could act as direct binding sites for Nhlh2. Thus it is likely that these genes are direct regulatory targets of the Nhlh2 transcription factor, and that decreased expression of PC1 and PC2 contribute to the maturity onset obesity in Nhlh2 KO mice.

Cuticular collagen is a major structural protein of nematode cuticles encoded by a large multigene family. The cuticle of nematode parasites provides a barrier between the worm and its hosts. This study is focused on the collagen genes expressed in the third larval (L3) and the molting third larval (3M) developmental stages of a human filarial parasite, Onchocerca volvulus , the major causative agent of onchocerciasis, or African River Blindness, one of the four leading causes of blindness worldwide. Based on the River Blindness Genome Project launched in Steven Williams' laboratory, EST analysis has identified 45 distinct cuticular collagen clusters derived from L3 and 3M stages, showing differential expression patterns through the life cycle stages. The collagens appear to be the most abundant gene family in the O. volvulus genome, reflecting their critical role in parasite development. The genomic structures of the three most highly expressed cuticular collagen genes (Ov-col-1, Ov-col-9, and Ov-col-10) have been characterized. These three genes are similar in the size of the genomic copy of the gene, transcripts and open reading frame, but belong to three distinct subfamilies based upon the feature of conserved cysteine residues. Due to its extremely high expression level and 3M-stage upregulation, Ov-col-9 is considered a putative candidate for vaccine design. The gene is 2.6 kb long, has 4 introns, a 1017 by transcript, and encodes a protein 289 amino acids in length. Western blot analyses identified the native COL-9 protein at a mass between 64 kDa and 80 kDa, about 3-fold the predicted size, indicating a nonreducible convalent crosslink between the COL-9 and other polypeptides under reducing conditions. Immunogold EM localized the COL-9 to the L3 body channel, to the 3M hypodermis, and to uterine microfilariae in the adult female. Analysis of the human IgG and IgG subclass responses to COL-9 demonstrated statistically similar levels between putatively immune (PI) and infected (INF) group except slightly elevated in IgG1 by the INF group.

Hedgehog (Hh) signaling is important for patterning and cell differentiation within many embryonic tissues. Hh is expressed in the notochord and the floor plate and acts as a morphogen in patterning of the ventral central nervous system (CNS), pituitary gland, somites, pancreas, and many other tissues. Gli transcription factors act as main mediators of Hh signaling in vertebrates. Their unique and overlapping functions lead to differential outcome of Hh signaling in different cells and tissues. The goal of my dissertation is to determine how zebrafish Gli proteins work together to transduce Hh signals and to activate or repress the transcription of Hh target genes. I have shown that Gli1 acts only as an activator of Hh signaling similar to what is known from other species, while Gli2 and Gli3 act as both activators and repressors. Gli2 functions as a co-activator of Gli1 in subset of ventral cells in the posterior diencephalon and in the adaxial cells in the embryonic trunk. In contrast, activator role of Gli3 overlaps with Gli1 throughout the ventral CNS during early development of the embryo. Later, GO represses Hh targets in the dorsal spinal cord, but not in the forebrain. In contrast, Gli2 represses genes in both, the dorsal telencephalon and the spinal cord. I also demonstrate that Gli3 repressor function is temporally regulated by active Hh signaling, unlike Hh independent Gli2 repressor function. Thus, my detailed analysis of zebrafish Gli functions reveals complex interactions between Gli proteins in embryonic patterning. I also studied a novel zebrafish mutation umleitung (uml) that was identified because of defects in axon guidance and neural patterning in the ventral forebrain. My detailed phenotypic analysis showed that the uml mutation disrupts Hh signaling and forebrain patterning. I have genetically mapped uml on zebrafish chromosome 24 near the Zmarker z10372. My linkage analysis indicates there are no known components of the Hh signaling cascade in the uml genetic region. This leads to the exciting possibility that uml may encode a previously undefined regulator of Hh signaling. I have initiated a genomic walk toward finding a gene that encodes uml.

Mutation and loss of p53 function are common features among human breast cancers. We use BALB/c-Trp53+/- mice as a model to examine the sequence of events leading to mammary tumors. Mammary epithelium proliferation rates were similar in both BALB/c-Trp53+/- mice and wild type controls. Among the 28 mammary tumors collected from BALB/c- Trp53+/- mice, loss of heterozygosity for Trp53 was detected in more than 90% of invasive mammary tumors. Transplantation of Trp53+/- ductal hyperplasias indicated an association between loss of the wild type allele of Trp53 and progression to invasive carcinomas. Expression of biomarkers such as ERα, PR, Her2/Neu and activated Notch1 varied among the tumors suggesting that multiple oncogenic events collaborate with loss of p53 function. The majority of the tumors expressed both luminal and basal cytokeratins (59%). Gene expression analysis showed ligands and receptors of stem cell related pathways, such as Notch and Wnt, were increased in the tumors. These results indicate that mammary tumors in BALB/c Trp53+/- mice might initiate from bipotent mammary progenitor cells. Using magnetic nanoparticles for cancer thermotherapy. Alternating magnetic field (AMF) heating of magnetic nanomaterials provides a promising method for executing therapeutic thermal treatment for cancer patients. In order to explore the potential of magnetic nanoparticles (MNPs) for hyperthermia treatment, we synthesized iron oxide MNPs with various passivation by citric acid, folate, trimethylamine carboxylic acid, or albumin. The albumin passivated MNP (MNP-A) surpassed other MNPs, showing efficient heating with very low inherent cytotoxicity. Confocal microscopy located MNP-A (FITC tagged) accumulation in both cell nucleus and cytosol after 24hr incubation with HeLa cells. The quantity of cell bound MNP-A (including internalized and cell membrane bound MNP-A) was positively associated with MNP-A concentration and incubation time with cells. The MNP-A bound to cells was sufficient to increase the temperature in the cell pellet Δ7°C after 8min exposure to AMF. No significant temperature increase or cell death was detected in control groups. Our data demonstrate that MNP-A provides a selective tool for AMF-induced thermal treatment, as well as useful dosing information for future preclinical animal studies.