Fetal alcohol exposure is the leading known cause of mental retardation in the western world. However, the mechanisms underlying alcohol-induced damage in fetal brain are largely unknown. The goal of this dissertation is to identify ethanol-responsive genes during brain development to provide more insights into the mechanisms. I chose a well-established animal model for all the studies in this dissertation. First, I demonstrated that this ethanol paradigm increased the mRNA of cellular retinol binding protein I (CRBP-1) in gestational day 13 (G13) brain and the incidence of apoptosis in G16 brain. Second, I identified 12 putative ethanol-responsive genes using mRNA differential display. After the quantitative analysis by Northern blot, in situ hyridization, western blot and relative quantitative RT-PCR, ribosomal protein S6 (rpS6), neuroendocrine-specific protein-A (NSP-A) and a novel gene were verified as ethanol-responsive genes. Third, I isolated 32 putative ethanol-responsive genes using cDNA microarray analysis. They encode proteins engaged in cell signaling, cell cycle regulation, metabolism, stress response and cell structure. Among all the putative genes, alcohol dehydrogenase 3 (Adh3) and glutathione S transferase pi 2 (GST pi 2) are previously known ethanol-responsive genes. Additionally, bone morphogenetic protein receptor type IA (BMPR-IA) showed the largest change induced by ethanol, 2.1-fold, and the ethanol effect on its expression was confirmed by relative quantitative RT-PCR. Fourth, because NSP-A is also a thyroid hormone-regulated gene, I analyzed the expression of two other thyroid hormone-regulated genes, Oct-1 and Hes-1, in my ethanol-treated animal model. However, ethanol did not affect thyroid hormone regulation of these two genes in G16 cerebral cortex. Fifth, I examined the effects of ethanol on protein expression and phosphoralytion using two-dimensional (2D) electrophoresis and western blotting. Ethanol was shown not to robustly change the abundance of individual proteins, but may change the post translation modifications of some proteins during brain development, such as glycosylation. In conclusion, these studies systematically and thoroughly examined the effects of fetal alcohol exposure on gene expression during brain development. They provide useful insights for analyzing the complex pathways leading to CNS damage in the children born to mothers who drank heavily during pregnancy.

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.

Signal transduction in bacterial chemotaxis system is mediated by a family of transmembrane proteins that are clustered in the membrane and form noncovalent complexes with cytoplasmic signaling proteins. Recent evidence suggests that receptor clusters play an important role in mediating the signaling in bacterial chemotaxis. To understand the functional role of receptor clusters in kinase regulation and the mechanism of cluster formation, we employed a strategy to engineer an intrasubunit disulfide bond between the two methylation regions in the cytoplasmic domain of E. coli aspartate receptor (Tar CF). We assumed the potential stabilizing effect of an internal disulfide bond might change receptor's propensity to cluster. Furthermore, the intrasubunit disulfide bond effectively prevents domain swapping, thus providing a tool to test the recently proposed "domain swapping model" as a mechanism that generates cluster formation and CheA activation. Our results showed one disulfide-bonded CF, termed CFSP3, exhibited a slight increase in protein stability together with a dramatic increase in CheA activation, high cooperativity and a strong propensity to form large receptor clusters in solution. Conversely, we did not observe notable cluster formation in any CFs in solution that failed to activate CheA. Yet when these assembled into clusters by templating methods, some measure of CheA activation was achieved. We also found that addition of CheW significantly decreased the cluster size, suggesting CheW might bind to the same region as where Us make contact to oligomerize. The results demonstrate that domain swapping is not required for the formation of active receptor clusters. We also propose a clustering model in which a receptor's propensity to form large clusters correlate with the receptor-coupled kinase activity in solution. Tar CF is highly dynamic, and the regulation of dynamics has been proposed to be involved in signaling. Therefore, HDX properties of the engineered CFs were studied to gauge dynamics. Overall Us showed near complete exchange, although a detectable difference in HDX protection was observed between the reduced and oxidized forms of CFSP3, which may be functionally significant. Future experiments conducted with ternary complexes should provide invaluable information to clarify the functional role of receptor dynamics in signaling.

Dynamic interactions between the amino acid acceptor end of tRNA and the ribosome underlie the synthesis of successive peptide bonds at the peptidyl transferase center (PTC) of the 50S ribosomal subunit. Photo-crosslinking of the 3′-terminal nucleotide of tRNA, which is adjacent to the attached amino acid or peptide, to components of the 50S subunit has proven to be a sensitive means for identifying specific protein and RNA segments in close proximity to the site of peptide bond formation. I have used this approach to follow changes in the position of the tRNA during peptide bond formation using several photoreactive tRNA-derived ligands. Three new photoreactive tRNA derivatives have been synthesized for use as probes of the PTC of the ribosome. In two of these derivatives, the 3′ adenosine in position 76 of yeast tRNAPhe has been replaced by either 2-azidodeoxyadenosine or 2-azido-2′-O-methyladenosine, while in a third the 3′-terminal 2-azidodeoxyadenosine of the tRNA is joined to puromycin via a phosphoramidate linkage to generate a photoreactive transition-state analog. All three derivatives bind to the P site of 70S ribosomes with affinities similar to that of unmodified tRNA Phe and can be crosslinked to components of the 50S ribosomal subunit by irradiation with near UV light. Yeast tRNAPhe containing 2-azidoadenosine, [2N3A76]tRNAPhe, typically crosslinks to the N-terminal sequence of protein L27 as well as to nucleotides U2506 and U2585 of the 23S rRNA. While the photoreactive transition-state analog, [2N3dA76]tRNAPhe-p-Puro, crosslinked the same components as [2N3A76]tRNAPhe, the distribution of crosslinks is altered significantly. The crosslinking to nucleotide U2506 is strongly reduced, and two new crosslinked nucleotides A2450 and A2602 were detected. Characteristic differences in the crosslinking patterns suggest that these tRNA derivatives can be used to follow subtle changes in the position of the tRNA relative to the components of the PTC.

During the course of DNA replication, replication forks often stall or collapse as they proceed from oriC to the terminus due to housekeeping types of DNA damage or proteins bound to DNA. If the DNA is not repaired or if the replication forks do not restart, viability of the cell then becomes compromised. In Escherichia coli, if DNA damage is detected, approximately 40 genes are expressed to repair the offending DNA lesion. This is known the SOS response. Two proteins RecA and LexA regulate the SOS response, where RecA (when bound to ssDNA), serves as the sensor for DNA damage and LexA serves the repressor for SOS expression. When the RecA nucleoprotein filament forms, this complex will accelerate autocleavage of LexA inducing the response. Recently it has been observed that in a population of cells approximately 15% of the population had RecA bound to DNA, however at any given time approximately 0.3% of the population is induced for SOS expression suggesting that the cell can decide whether induce the SOS response or not. The aim of this work is to understand how the cell decides whether or not to express the SOS response at housekeeping types of DNA damage. Regulation is important because the cell would not want to express the SOS response every time replication forks encounter housekeeping types of DNA damage. The first component of this work looks at SOS expression in populations of cells during log phase growth using the fluorecense microscopy and the transcriptional fusion sulA-gfp. Results show that a SOS expression is stochastic and occurs in a small population of wild type cells. The second component of my work focuses on how the cell decides when to express the SOS response by using recA constitutive mutants that are defective in this regulation. Results show that the concentration and conformation of the RecA nucleoprotein filament is crucial for this to occur. Lastly novel recA mutants were created and examined for their role in suppressing constitutive SOS expression. It is observed that suppression of constitutive SOS expression could be seen when these mutations were supplied in cis and in trans, suggesting multiple levels of SOS regulation.

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.

Throughout human history, societies have always had the quest to harness the energy sources nature has provided. The discovery of fire, by humans was the most significant step into human civilization. Fire provided light and heat, to heat dwellings and cook food. Thus, the burning of wood provided energy. As human civilization advanced the drive to explore other sources of energy became a necessity which eventually led to the discovery of fossil fuels such as petroleum by the end of the 19th century. Petroleum is the cradle of our civilization and it is one of the primary energy sources for modern societies. Today, many industrialized and developing nations obtain their energy from the world's petroleum reserves. This demand has led to depletion of some of the reserves such as those that contain "light" sweet crude oil. To meet this never ending demand, petroleum reserves that contain "heavy" conventional and unconventional crudes are being harnessed at present. Crude oil is arguably one of the most complex mixtures in the world, and its composition varies widely, depending upon origin and age. Comprehensive compositional knowledge of crude oils from different origin is a necessity as it improves their production and processing, and ultimately determines their market value. Detailed characterization of the elemental composition of "heavy" conventional and unconventional crudes is imperative as these crudes are rich in polar heteroatoms (nitrogen, sulfur and oxygen containing compounds). The polar heteroatomic species in crude oils have some detrimental effects that can result in economic loss during production and processing (e.g., catalyst deactivation, corrosion and storage instabilliy). In addition, combustion of such crude oils results in harmful environmental implications such as atmospheric pollution from nitrogen oxides (NxOy) and sulfur oxides (OySz). A better tool to facilitate detailed characterization of crude oils is mass spectrometry. Mass spectrometry has surpassed many analytical tools in crude oil characterization. The advent of ultrahigh resolution FT-ICR mass spectrometry revolutionized crude oil characterization by its abililty to afford high mass resolving power and mass accuracy, crucial parameters necessary for detailed elemental composition of a complex mixture such as crude oil. Chapter 1 provides a brief introduction to FT-ICR principles, instrumentation and data analysis. Figures of merit (e.g., high mass resolution and mass accuracy) that make FT-ICR MS an outstanding tool for complex mixture analysis are also discussed. I also highlight the breakthrough and significance of electrospray ionization (ESI) in characterization of polar molecules in fossil fuels such as petroleum and petroleum derived materials. In Chapter 2, a detailed description of naphthenic acids and naphthenate deposits is presented. The challenges of naphthenic acids characterization, their role in naphthenate deposition and economic significance are discussed. Chapter 3 highlights naphthenic acids that form calcium and sodium naphthenate deposits. The solid deposits and emulsions are formed by the interaction of naphthenic acids with divalent (Ca2+, Mg2+) or monovalent (Na+, K+) ions in produced waters. Calcium naphthenate formation, an interfacial phenomenon, is thought to depend largely on tetraprotic naphthenic acids known as "ARN" acids with C80 hydrocarbon skeleton whereas sodium naphthenates originate from lower molecular weight (C15 to C35) monoprotic saturated naphthenic acids. In Chapter 4, the characterization of naphthenic acids isolated from calcium and sodium naphthenates by collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) is discussed. IRMPD and CAD experiments reveal structural differences of the naphthenic acids found in calcium and sodium naphthenate deposits. IRMPD fragmentation of ARN acids results in dehydration and decarboxylation of the carboxylic acid groups without dealkylation whereas CAD fragmentation gives similar results to IRMPD with extensive fragmentation that leads to dealkylation of the hydrocarbon skeleton. In Chapter 5, we present our first attempts to preconcentrate and determine the broadband limit of detection (LOD), and consequently quantify ARN acids in whole crude oils. We highlight the significance of the preconcentration step as a method to enhance the detection of ARN acids and consequently yield good quantitation. Chapter 6 highlights the extraction and isolation of naphthenic acids from calcium naphthenate deposits of different geographical origin, by use of ammonia in a custom built deposition reaction cell. The acid extracts were characterized by negative-ion ESI FT-ICR MS. The ammonia extraction method effectively extracts and isolates tetraprotic naphthenic acids known as ARN acids, with a C80 hydrocarbon skeleton from all the calcium naphthenate deposits. Low molecular weight ARN acids with C53-59 and C60-79 hydrocarbon skeletons were also identified in some of the calcium naphthenate deposits. The ammonia extraction method further confirms that ARN acids are the main constituent of and a prerequisite for calcium naphthenate deposition. In Chapter 7, we discuss and compare naphthenic acid extraction in crude oils by two different extraction methods. The efficiency of three basic alcoholic solutions of increasing base strength in the extraction of naphthenic acids in crude oils by a liquid-liquid extraction method is evaluated. The three different basic alcoholic solutions of increasing basic strength were; ammonium hydroxide (NH4OH) / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2010. / April 30, 2010. / Ion Cyclotron Resonance, Petroleum, FT-ICR, Naphthenic Acids, Naphthenates, Electrospray / Includes bibliographical references. / Alan G. Marshall, Professor Directing Dissertation; Philip N. Froelich, University Representative; Albert E. Stiegman, Committee Member; William T. Cooper, Committee Member; Ryan P. Rodgers, Committee Member.

This dissertation attempts to provide insight into the mixing, hydration and annealing of self-assembled, multilayered films based on precise counting of their doping ions and water content. Several practical applications are then explained in light of this theory. Polyelectrolyte multilayers are ideal mixes forming amorphous films. While the properties of ordered organic monomolecular layers such as liquid crystals or amphiphiles can easily be dominated by the nature of a few defects, in disordered systems the overall amorphous nature not only defines their properties but also makes the materials less sensitive to details of the preparation process. Films have been built by inverted dipped spin-coating and traditional pipetted spin-coating. Charged polymer chains are entropically driven to interact electrostatically based on the concentration of small ions in solution. Their hydration can be used to extrapolate a universal doping parameter which can be used to better characterize and classify any existing ionic dopants and films. These films can be used to control the electroluminescence of Ru(bpy)3 3+ based on its controlled diffusion to a coated platinum electrode. They can also inhibit corrosion of metals by shielding of harmful ions in solutions. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2008. / May 16, 2008. / polyelectrolyte multilayers, swelling, doping / Includes bibliographical references. / Joseph B. Schlenoff, Professor Directing Dissertation; Kenneth A. Goldsby, Committee Member; William Cooper, Committee Member; Irinel Chiorescu, Committee Member.

Signaling through the notch pathway originates upon interaction between the notch receptor and one of its two ligand proteins, delta or serrate. Extracellular binding of either ligand protein induces the release of the notch intracellular domain, effecting changes in gene expression and leading to cell fate determination. The receptor protein exists as one of two possible glycoforms. The two ligand proteins Delta and serrate differ in their ability to recognize the different glycoforms on notch epidermal growth factor-like domain 12 but the molecular basis for this differential binding is not well understood. The region of the ligand proteins that interact with notch encompasses roughly the first 230 amino acids of the extracellular domain. In this study, it is shown that a portion of the delta protein encompassing amino acids A132-K230, referred to as the delta DSLNT2 region, is capable of inducing release of the NICD. Biophysical characterization of this construct indicates that the construct adopts an unstructured form similar to that seen in the pre-molten globule family of intrinsically disordered proteins. This disordered structure may confer delta with structural plasticity that allows it to recognize and bind to either glycoform of the notch receptor. After conclusion of the biophysical characterization of the delta DSLNT2 protein, NMR characterization of domain 2 of the NS52a protein from hepatitis C virus was performed. The characterization of this protein sought to elucidate a possible mechanism for viral drug resistance exhibited by point mutants within domain 2 of NS5a. Hepatitis C virus is dependent on a number of host proteins, among these is the peptidyl-prolyl isomerase cyclophilin A (Cyp A). NS5a has been shown to be a substrate for Cyp A, and the interaction between the two has become a viable therapeutic target. Disrupting the interaction between Cyp A and NS5a, or knocking out expression of Cyp A in host cells has been shown to stop viral replication. Mutations at D317E and Y317N, which map to domain 2 of NS5a, allow for drug resistant viral replication or replication without a dependence on Cyp A. Through the use 13C NMR, structural consequences of these mutations, as well as a possible mechanism for drug resistance were investigated. / A Dissertation submitted to the Program in Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2011. / July 6, 2011. / Includes bibliographical references. / Timothy Logan, Professor Directing Dissertation; Richard Hyson, University Representative; Richard Bertram, Committee Member; P. Bryant Chase, Committee Member; Scott Stagg, Committee Member.