Although much is known about the role of serotonin (5-HT) in the pathophysiology of depression, little is known about the temporal and regional brain alterations in 5-HT as they relate to the treatment of depression and anxiety. This study aimed to evaluate the acute effects of the selective serotonin reuptake inhibitor (SSRI), citalopram, on neuronal activation elicited during an emotional task using functional MRI (fMRI) in healthy subjects. Eight healthy men completed the double-blind placebo-controlled crossover study of citalopram (20 mg infused over 30 min) and normal saline. Subjects performed the emotional task once before drug/placebo infusion (Faces 1) and twice during drug/placebo infusion, once early in the infusion (Faces 2) and once at the end of infusion (Faces 3). A main effect of task was found in the L and R amygdala. A cluster in the right amygdala had increased activation for the Faces 2 task during the citalopram infusion, compared to the baseline Faces 1 task. An even greater bilateral amygdala response to citalopram was found at the end of infusion (Faces 3), when the citalopram concentrations approach their maxima, compared to the baseline Faces 1 task. This suggests that acute citalopram administration potentiates the amygdala response to emotional stimuli. An exploratory analysis was done using serotonin transporter genotype as a covariate. S allele carriers (2 s/s and 3 s/l) had a greater baseline amygdala response than l/l (n=3) homozygotes. However l/l homozygotes had a greater response to citalopram, comparing the Faces 3 to the Faces 1 task. This study generated the first in vivo human data regarding the regional effects of acute intravenous SSRI administration on affective task-related neuronal activation. An understanding of the regional effects of SSRIs may aid in understanding the mechanism by which these agents produce their therapeutic effects. By including 5-HTTLPR genotype in the analyses, we may account for some of the variability in response to citalopram and other SSRIs. These efforts contribute to the identification of biological mechanisms and pathways that mediate response to SSRIs, and contribute to our understanding of individual differences in complex behaviors and vulnerability to psychiatric illnesses.

Mice deficient in oxytocin (OT) (OT KO), those that do not synthesize or release OT at any point in the life cycle, have shown significantly enhanced ingestion of sweet (sucrose and saccharin) and non-sweet carbohydrate-containing solutions (Polycose and cornstarch) as compared to wildtype (WT) mice of the same C57BL/6 background. The purpose of this work is two-fold: to determine if the effects observed with sweet and non-sweet carbohydrate-containing solutions extend to a palatable non-carbohydrate containing emulsified fat solution (Intralipid), and to further elucidate the drinking behavior of WT and OT KO mice exposed to sucrose solutions. Mice were exposed to a variety of two-bottle access tests to compare ingestion of Intralipid, a fat-containing emulsion, as well as sucrose and aversive (sodium chloride (NaCl) and citric acid) solutions mixed with sucrose. On the first day of exposure to Intralipid OT KO animals consumed significantly greater volumes of Intralipid as compared to WT mice. Animals showed preference for the Intralipid emulsion over water at a variety of concentrations, however, no genotypic differences were observed in Intralipid ingestion beyond the first exposure day, suggesting that the increased ingestion of palatable liquids observed in OT KO mice does not extend to Intralipid solutions. OT KO mice continually consumed more 10% sucrose than WT mice during repeated four-day trials and during a two-week trial. Sucrose bottle placement did not have a significant effect on sucrose consumption. OT KO mice also consumed significantly more sucrose at concentrations of 0.625%, 1.25%, 2.5%, and 5% during four-day trials. Similar drinking patterns were observed in male and female animals and across generations. When exposed to two-bottle access to 0.5M NaCl or 30mM citric acid mixed with 10% sucrose and water, OT KO animals consumed more of the aversive solution mixed with 10% sucrose than WT counterparts. The results of this thesis suggest that OT plays a role in the ingestion of sweet carbohydrate-containing solutions. However, it appears that these effects do not extend to the ingestion of Intralipid, a palatable fat-containing emulsion. The exact function of OT in these ingestion behaviors has yet to be determined.

The rapid development of proteomic techniques in the post-genomics era has allowed much attention to be paid to the understanding of molecular mechanisms of functional regulatory proteins. This amazingly fast transformation from know-how to know-why has allowed the determination of if functional regulation is based on up- or down-regulation, or based on any particular chemical modification on the amino acid residues of protein targets. Here, different biologically important regulatory proteomes or protein targets were investigated using comprehensive combinations of proteomics techniques. A large emphasis was placed on expression techniques suitable for protein and protein binding partner affinity purification for targeted analysis of phospho-proteins. The main protein of interest in these particular studies was an important transcriptional factor in cardiac development, serum response factor-1. Several proteomics methods were examined and, along with the difficulties encountered, the results are presented. Small molecule-protein interaction was examined with the cell cycle regulatory protein Cdc25B incubated with an inhibitor. Post-translational modification-related functional proteomics were also studied, first in a rodent early hemorrhage model wherein S-nitrosylation of plasma proteins were determined, then the phosphorylation of serum response factor-1. Finally, top-down functional proteome mapping was represented by a detailed and very successful analysis of the proteins found enriched in brain tumor cell pseudopodia. This dissertation illustrates the vast range of potential of proteomics technologies in studying function-related biological systems. Meanwhile, some pioneering work using targeted functional proteomics strategies was achieved, and the results will help in part to advance the field of targeted functional proteomics, the combination of cell and molecular biological techniques with chemical, affinity and mass spectrometric approaches to study regulation of biological systems.

The N-end rule pathway relates the in vivo half-life of a protein to the identity of its N terminal residue. In this pathway, a substrate bearing N-degron is recognized and ubiquitylated by a family of E3 ubiquitin ligases named UBR proteins. The N-end rule pathway is implicated in various physiological and pathological processes including cardiac development and angiogenesis. It has been previously shown that mice lacking ATE1, which mediates N-terminal arginylation, die during embryogenesis associated with various defects in cardiovascular development. The goal of my graduate research was to understand the function of the N-end rule pathway in cardiovascular development, signaling, and homeostasis. In my first project, I employed a genome-wide functional proteomic approach to identify physiological substrates of ATE1, that potentially underlie the above cardiovascular phenotypes. I found that RGS4, RGS5, and RGS16 are in vivo substrates of the N-end rule pathway, the first to be identified in mammals. These RGS proteins, emerging regulators for cardiovascular G protein signaling, were degraded through sequential N-terminal modifications including N-terminal exposure of their Cys 2, its oxidation, and arginylation. In the second project, to understand the physiological meaning of ATE1-mediated RGS proteolysis in cardiac development and signaling, I characterized ATE1-/- mice and embryonic cardiomyocytes with an emphasis on GPCR signaling. I found that cell-autonomous function of ATE1 regulates the proliferation of cardiomyocytes and the homeostasis of Gq-dependent cardiac signaling. In the third project, I explored a model of heterovalent interaction by developing RF-C11, a small molecule inhibitor of the N-end rule pathway. Its two heterovalent ligands were designed to cooperatively target two cognate sites of N-recognins. RF-C11 showed higher inhibitory efficiency than its homovalent controls, providing molecular basis of designing multivalent inhibitors for specific intracellular pathways. Moreover, the treatment of RF-C11 reduced cardiac proliferation and hypertrophy in cardiomyocytes, unveiling a previously unknown function of the pathway in cardiac proliferation and signaling. In summary, my graduate research contributes to comprehensive understanding of the function of the N-end rule pathway in the cardiovascular system.

A significant portion of the cognitive decline seen in older adults may be due to anticholinergic medications (i.e., muscarinic receptor antagonists) which are known to cause memory loss, confusion, and delirium. A competitive radioligand binding assay has been used in the research setting to measure the cumulative level of muscarinic receptor binding present in an individuals serum, referred to as serum anticholinergic activity (AA). Serum AA is the measure of binding of all compounds present in a persons serum (e.g., medications, metabolites, and possibly endogenous substances) to muscarinic receptors. Multiples studies have shown that even low serum AA levels are associated with impaired cognitive performance, impaired self-care capacity, and the presence of delirium in nondemented or mildly demented elderly. Serum AA has the potential to be a useful tool for clinicians. However, there are multiple items which first need to be addressed to enhance the reliability and clinical applicability of this assay. One concern is that the muscarinic receptor binding profiles of most medications and their metabolites have never been examined. Thus, even if a clinician decides that a patient is suffering from anticholinergic-induced toxicity, he/she has little guidance on which medication(s) to adjust. To address this issue, we investigated the in vitro AA of 106 commonly used medications and estimated the relationship between dose and AA in older adults. The change in serum AA over time in the absence of medication adjustments is not known. Another limitation is that serum AA is a peripheral measure, while the central anticholinergic effects of a medication are dependent on its distribution into the CNS. An optimal tool to predict medication-induced cognitive impairment would be one which better estimates drug distribution into the CNS. To address these issues, we conducted a pilot study investigating the utility of using centrally mediated pupillary oscillationsin conjunction with serum AA as a possible predictor of cognitive performance. Serum AA levels and ocular response were measured in a double-blind, cross-over study across an 8 hour time period following administration of placebo or the anticholinergic medication, oxybutynin.

Human papillomavirus (HPV) oncoproteins E6 and E7 which are constitutively expressed in cervical cancer cells are ideal targets for developing immunotherapies for treatment of existing HPV-associated carcinoma. In this project, we developed a simple, safe, and efficient, peptide-based therapeutic cancer vaccine, DOTAP/E7 complex, which comprises only two molecules: a DOTAP cationic lipid and an MHC class I-restricted peptide antigen derived from HPV-16 E7 protein. TC-1 cell line which is HPV-16 E7+ was used as a tumor model in an H-2b murine system. Tumor-bearing mice showed significant tumor inhibition following a single injection of DOTAP/E7 at the optimal lipid dose, suggesting that DOTAP liposome alone can be a potent adjuvant. E7 peptide formulated with DOTAP was taken up by dendritic cells (DC) and induced DC activation and migration to the draining lymph node (DLN), eliciting antigen-specific CD8+ cytotoxic T lymphocyte (CTL) responses. The mechanism of DOTAP as a vaccine adjuvant was revealed by DOTAP-mediated reactive oxygen species (ROS) production in DC. At the optimal lipid dose, DOTAP/E7 generates an adequate level of ROS for the initiation of the vaccine mechanism. In addition, we have improved the vaccine formulation by incorporation of E7-lipopeptide instead of the water-soluble native E7 peptide. The improved DOTAP/E7-lipopeptide vaccine showed a significantly enhanced therapeutic effect, including CTL response and anti-tumor activity. The vaccine was also effective for suppression of tumor growth in later stages of tumor progression, suggesting applications for advanced cancer treatment. Furthermore, we extended the studies of the vaccine efficacy observed in the mouse model to human cells in vitro. Instead of H-2Db-restricted peptide, an HLA-A2-restricted E7 peptide epitope (hE7) was formulated into the liposome. In vitro stimulation of naïve HLA-A2+ human T lymphocytes by DOTAP/hE7-activated autologous DC elicited a stronger clonal T cell proliferation and higher HPV-specific CTL response compared to those stimulated with DC pulsed with hE7 peptide alone. The in vivo CTL and anti-tumor activity induced by DOTAP/hE7 vaccine were demonstrated in an HLA-A2 transgenic mouse model. Overall, our data suggest that DOTAP/hE7 is a potent therapeutic cancer vaccine formulation with potential for clinical applications for the treatment of HPV-related neoplasia.

ABSTRACT Background: UC781, a tight-binding non-nucleotide reverse transcriptase inhibitor (NNRTI) of HIV-1, is a thiocarboxanilide that has been identified as a potential microbicide agent. UC781 prevents HIV-1 infection by potently inhibiting HIV-1 replication (EC50l8nM) with a broad therapeutic index (>62,000). However, its extremely poor water solubility leads to a great challenge for its formulation development. A beta-cyclodextrin (beta-CD) based drug delivery system was developed for UC781 to overcome this issue. Method: The complex of UC781: beta-CD was assessed with UV, FTIR, DSC, and NMR. An HPLC method was used to investigate the thermodynamic behavior of the UC781 complex. Complexation of UC781 with either hydroxypropyl -beta-Cyclodextrin (HP-beta-CD) or methyl-beta-cyclodextrin (M-beta-CD) was optimized by evaluation of four processing methods (autoclave, lyophilization, shaking, and kneading), incorporation of four water-soluble polymers (HPMC, HEC, PVA, and PVP K30), and utilization of three buffering systems (pH 7.0, 9.0 and 11.0). Finally, three formulations¡Xmethylcellulose (MC) gel, hydroxyethylcellulose (HEC) gel, and polyvinyl alcohol (PVA) film¡Xwere developed for UC781. The physical properties, toxicity, and anti-HIV activity of UC781 containing formulations were evaluated with in vitro and ex vivo models. Results: Complexation of UC781 with beta-CDs was confirmed and characterized with UV, FTIR, DSC, and NMR. UC781¡¦s complexation was found to be an enthalpy driven process. The solubility of UC781 was increased from almost none to 35 ug/ml in 15% HP-beta- CD and 180 ug/ml in 15% M-beta- CD solutions after optimization. Complexation technique significantly improved the release of UC781 from all three formulations. The complexation of UC781 with HP-beta- CD or M-beta- CD greatly increased the osmolality and decreased the viscosity of MC and HEC gel; shortened the disintegration time of PVA film; and reduced IC50 for UC781 in all three formulations. No observed toxicity was found in all complexed UC781 containing formulations. Conclusion:beta-CD complexation technique provided an effective method to overcome the aqueous solubility challenge for UC781. UC781 complexation can be used as a safe and effective drug delivery system for UC781. Of the formulations tested, PVA film with complexed UC781 provided the most promising option for microbicide product development.

The ubiquitin (Ub)-dependent N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. A substrate with destabilizing N-terminal residue is recognized by a family of mammalian E3 ubiquitin ligases of the N-end rule pathway, including UBR1 and UBR2. However, little is known about their roles in biological processes. The aim of this dissertation is to understand the physiological functions and underlying molecular mechanisms of the E3 ubiquitin ligases in the N-end rule pathway. First, I find that UBR2, a recognition E3 component of the N-end rule pathway, localizes to the meiotic chromatin, where it mediates the transcriptional silencing through histone ubiquitylation in a spatiotemporal manner. UBR2-lacking spermatocytes show impaired global ubiquitylation, including ubiquitylation of histone H2A, a histone modification often associated with transcriptional inactivation. HR6B, an E2 conjugating enzyme of the N-end rule pathway, interacts with UBR2 as an E2-E3 complex and cooperatively mediate H2A monoubiquitylation in vitro. Impaired H2A ubiquitylation in UBR2-deficient spermatocytes correlates to defects in transcriptional silencing. Furthermore, I provide evidences that UBR2 is involved in DNA damage response (DDR) pathway through protein ubiquitylation presumably to maintain genome integrity. UBR2 is exclusively associated with non-heterochromatin throughout nucleus and responds to genotoxic stress via post-translational modification. Ubiquitylation induced by DNA damage are significantly impaired in UBR2-deficient cells. UBR2-/- mouse embryonic fibroblasts (MEFs) show increased vulnerability to the genotoxic agents and abnormality of chromosomes. Moreover, I show that divergent and cooperative functions of the E3 ligases of N-end rule pathway. UBR1 and UBR2 are 46% identical, and appear to be indistinguishable in their recognition of N degrons, yet show different physiological implications in mutant mice. UBR1-/-UBR2-/- embryos die at midgestation, with defects in neurogenesis and cardiovascular development. These defects include reduced proliferation as well as precocious migration and differentiation of neural progenitor cells. The expression of regulators such as D type cyclins and Notch1 is also altered in UBR1-/-UBR2-/- embryos. Overall, my dissertation suggests that the E3 Ub-ligases of the N-end rule pathway are required in meiosis, DDR pathway, and embryogenesis.

Over 60 million people have been infected with HIV since the beginning of the epidemic. Currently available methods for prevention have not been sufficient to stop the progression of this pandemic. Considering that many women are unable to negotiate condom use with their partners and are more susceptible to HIV, strategies to prevent heterosexual transmission of HIV must include female-controlled methods. A promising strategy is the development of a topical microbicide to prophylactically inhibit transmission of sexually transmitted infections, including HIV. The work presented makes significant contributions to the microbicide research field, focusing on product development, preformulation strategies, stability in biological fluids, and drug targeting. We investigated two biomolecule (protein/peptide) microbicide candidates: PSC-RANTES, a chemokine analog of RANTES; and RC-101, a circular theta-defensin analog. We hypothesized that the use of a drug delivery system will protect the microbicide candidate against degradation before administration, and in biological fluids after administration, while maintaining drug activity. Further, the interaction of the microbicide candidates with human vaginal fluids can result in chemical modification of the drug. Identification of degradation pathways for PSC-RANTES and RC-101 was conducted by performing preformulation studies under selected conditions of temperature, pH, and oxidative conditions. Analytical methods used included HPLC, MALDI-TOF MS, CD, and SDS-PAGE. Chemical modifications of RC-101 were evaluated in the presence of human vaginal fluid collected from healthy female volunteers and detected by LC-MS/MS. RC-101 was formulated in a quick-dissolving vaginal film and showed short term stability, efficacy in vitro and safety in vivo in an animal model. Tissue localization of RC-101 was evaluated using excised human (ectocervical and endometrium) and monkey (vaginal and endometrium) tissues. Major findings from this work show that: RC-101 formulated in a film drug delivery system protected the peptide from degradation prior to administration; anti-HIV activity of RC-101 was maintained in the formulation; RC-101 was stable at least for 48 h in the presence of human vaginal fluid; and penetration of RC-101 into epithelial tissue was demonstrated. These results contribute to the development of RC-101 into a successful microbicide product and provide a systematic tool for the development of other microbicide molecules.

The aryl hydrocarbon receptor (AhR) is a PAS domain transcriptional factor also known as the ¡§dioxin receptor¡¨ or ¡§xenobiotic receptor.¡¨ My thesis work has uncovered an endobiotic role for AhR in hepatic steatosis and other metabolic functions. Activation of AhR induced spontaneous hepatic steatosis, which is characterized by the accumulation of triglycerides. The steatotic effect of AhR was likely due to the combined upregulation of fatty acid translocase CD36/FAT, suppression of fatty acid oxidation, inhibition of hepatic export of triglycerides, and an increase in the mobilization of peripheral fat. Promoter analysis established CD36 as a novel transcriptional target of AhR. Moreover, the steatotic effect of an AhR agonist was inhibited in mice deficient of CD36. Results from this study may help to establish AhR and its target fatty acid translocase CD36 as attractive targets for intervention in fatty liver disease. The liver X receptors (LXRs), both the Ñ and Ò isoforms, are nuclear receptors identified as sterol sensors that modulate cholesterol and lipid metabolism and homeostasis. In the second part of my thesis research, I report a novel LXR-mediated mechanism of androgen deprivation. Genetic or pharmacological activation of the liver X receptor (LXR) in vivo lowered androgenic activity by inducing the hydroxysteroid sulfotransferase 2A1 (SULT2A1), an enzyme essential for the metabolic deactivation of androgens. Activation of LXR also inhibited the expression of steroid sulfatase (STS) in the prostate, which may have helped to prevent the local conversion of sulfonated androgens back to active metabolites. At the physiological level, activation of LXR in mice inhibited androgen-dependent prostate regeneration in castrated mice. Treatment with LXR agonists inhibited androgen-dependent proliferation of prostate cancer cells in LXR- and SULT2A1-dependent manner. The ability of LXRs to regulate androgen metabolism makes them novel therapeutic targets for the treatment and prevention of hormone-dependent prostate cancer. Taken together, my work has revealed novel functions of AhR in lipid metabolism and LXR in androgen deprivation. It is hoped that understandings of the endobiotic functions of AhR and LXR may establish these two receptors as therapeutic targets for the management of metabolic disease in humans.