▼ The quinic acid (qa) cluster of Neurospora crassa is subject to two levels of control. The primary mechanism is dependent on the presence of the inducer, quinic acid: in the absence of inducer, the genes are expressed at low basal levels. The cluster is also subject to catabolite repression – the genes are repressed in the presence of a preferred carbon source, such as dextrose, even in the presence of inducer. To focus on this second level of control, I utilized a constitutive mutant of the qa gene cluster which contains a deletion of the qa-1S repressor gene. This deletion removes the primary control mechanism, ensuring that any observed effect is due to catabolite repression. Northern blot analysis indicates that transcription of the qa-y gene, which encodes a quinate permease, is repressed by the presence of dextrose even in the absence of the qa 1S repressor, demonstrating that catabolite repression of the qa cluster may be occurring by inducer exclusion. Transcription of the remainder of the qa genes does not appear to be repressed to a significant degree in the constitutive mutant by the presence of dextrose. Additionally, I used 2-dimensional protein gel electrophoresis to examine the expression of the qa proteins in samples grown under different growth conditions. Preliminary results show changes in protein expression in repressor mutants when compared to wild-type. Gene regulation mechanisms can be revealed by comparing homologous sequences in related species. I sequenced the permease and repressor genes, along with the associated regulatory regions in the homothallic Neurospora species N. africana and N. terricola. The coding regions of both species share a high degree of homology to N. crassa: the permease genes are 93% identical and the repressor 91-92% identical. The noncoding region upstream of the repressor is less conserved, with N. africana showing 41% identity and N. terricola 59% identity to N. crassa. In contrast, the region upstream of the permease gene is highly conserved, with both species 83% identical to N. crassa. This highly conserved region could contain binding sites needed to affect catabolite repression of the permease gene. Advisors/Committee Members: Asch, David K.

▼ Bile acids, synthesized from precursor cholesterol molecules, serve as an important mechanism for elimination of excess cholesterol from the body. Bile acid synthesis is tightly regulated since excess bile acids are toxic and can cause liver damage. Bile acids inhibit their own synthesis by inhibiting the bile acid biosynthetic genes. Cholesterol 7α- hydroxylase (CYP7A1) is the rate-limiting enzyme of classic bile acid biosynthesis, which synthesizes two primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA) in the liver. Sterol 12α-hydroxylase (CYP8B1) catalyzes CA synthesis in the liver and is feedback inhibited by bile acids. In addition to activating farnesoid X receptor (FXR, nuclear receptor subfamily 1H4, NR1H4), bile acids also induce the release of inflammatory cytokines, like interleukin 1β (IL-1β) from Kupffer cells in the liver. The objective of this study was to investigate the mechanism by which inflammatory cytokines inhibit human CYP8B1 gene transcription. Real time PCR assays revealed that both CDCA and IL-1β markedly reduced CYP8B1, CYP7A1, and nuclear receptor hepatocyte nuclear factor 4α (HNF4α, NR2A1) mRNA expression levels in human primary hepatocytes. However, CDCA induced, but IL-1β reduced the negative nuclear receptor, small heterodimer partner (SHP, NR0B2) mRNA expression. IL-1β inhibited human CYP8B1 reporter activity only in liver cells, and a c-Jun N-terminus kinase(JNK)-specific inhibitor blocked IL-1β inhibition. Activated JNK1 or c-Jun inhibited, whereas their dominant negative forms blocked IL-1β inhibition of CYP8B1 transcription. Mutagenesis analyses mapped an IL-1β response element to a previously identified bile acid response element, which contains an HNF4α binding site. Furthermore, IL-1β inhibited HNF4α gene transcription, protein expression and binding to the CYP8B1 gene. JNK1 phosphorylated HNF4α and a JNK-specific inhibitor blocked the IL-1β inhibition of HNF4α expression. Expression of c-Jun, a downstream target of the JNK pathway, was induced by both IL-1β and CDCA in primary human hepatocytes. c-Jun inhibited the HNF4α and coactivator peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) mediated trans-activation of CYP8B1 reporter activity. Coimmunoprecipitation revealed an interaction between c-Jun and HNF4α, which was confirmed by GST pull down assay. IL-1β increased the ratio of c-Jun or phosphorylated c-Jun bound to HNF4α in HepG2 cells. Chromatin immunoprecipitation (ChIP) assay revealed that c-Jun did not affect HNF4α binding but blocked HNF4α recruitment of PGC-1α to the CYP8B1 chromatin. This study also showed that HNF4α stimulated the gene expression of SHP by binding to the promoter of the latter. The results suggest that IL-1β inhibits CYP8B1 gene transcription, in a SHP-independent manner via the JNK pathway that inhibits HNF4α gene expression and its DNA-binding ability. IL-1β also induces c-Jun, which blocks HNF4α recruitment of PGC-1α to CYP8B1 chromatin. This mechanism may play an important role in the adaptive response to inflammatory cytokines and in the protection of the liver during cholestasis. Advisors/Committee Members: Chiang, John.

▼ Altered oxytocin cell development and function are associated with several neuropsychiatric disorders, including autism and Prader-Willi Syndrome. However, the molecular control of oxytocin cell development is poorly understood. Zebrafish have been shown to be a powerful model for identifying and analyzing regulatory genes that control brain development. The objective of this dissertation was to establish the zebrafish as a model system to study the molecular genetic control of development of isotocin, homolog of oxytocin, producing cells. The central hypothesis of this proposal is that an evolutionarily conserved regulatory gene network consisting of Sim1, Otp, Arnt2 and Pou3f2, control the development of isotocin cells in the developing zebrafish hypothalamus. The following four specific aims were accomplished: Aim 1 characterized the zebrafish hypothalamo-neurohypophysial system (HNS) during development. This aim identified and characterized isotocin and vasotocin cells in the zebrafish hypothalamus. Aim 2 identified and characterized transcriptional regulatory genes controlling zebrafish isotocin cell development. Potential zebrafish orthologs of the mammalian oxytocin cell regulatory genes Sim1, Otp, Arnt2 and Pou3f2 were identified and screened for influencing isotocin cell development. Aim 3 confirmed the requirement of sim1 and otp in isotocin cell development and evaluated the genetic interactions between these genes. This study demonstrated sim1 and otp act in parallel pathways to control differentiation of isotocin cells. Aim 4 evaluated the role of two potential, equally related, homologs of mammalian Pou3f2 in zebrafish isotocin cell development, pou47 and brn1.2. Pou47 and brn1.2 were both found to be required for isotocin cell development and do not genetically interact to specify isotocin cellular identity. Aim 5 evaluated the genetic interaction of these Pou3f2 homologs with sim1 and otp in their control of zebrafish isotocin cell development. The data in this aim, along with a re-evaluation of the mouse literature, suggests these genes act combinatorially to specify isotocin cell development. Therefore, a new genetic model of this regulation is proposed. The rational for this proposal is that an elucidation of the molecular control of oxytocin (isotocin) cell development will contribute to an understanding of the neurodevelopmental causes of several neuropsychiatric diseases, such as autism and Prader-Willi Syndrome. Advisors/Committee Members: Glasgow, Eric.

▼ Resveratrol has been considered a chemopreventive and therapeutic candidate against carcinogenesis because of its potential to modulate multiple molecular pathways associated with the development and progression of cancer. Novel resveratrol analogs were developed, tested and compared with resveratrol. Anti-proliferatory activity of resveratrol, KSA and KST compounds were demonstrated. Amount them, KST201 was the most selective compound against cancer cell growth according to its cytotoxicity selective index value. Resveratrol blocked cell cycle of hormone-independent prostate cancer DU145 cells in the G0/G1 phase, and a subG0/G1 peak was present. KST201 caused DU145 cell cycle arrest in the G0/G1 phase. In ovarian cancer MDAH cells and bladder cancer T24 cells, KST201 blocked cell cycle in the S phase. KST201 induced apoptotic subG0/G1 cell population in DU145, MDAH and T24 cells. Resveratrol and KST201 produced cytoplasmic DNA fragments, induced caspase-3 activity and caused morphological changes in DU145 cells. These findings confirmed the pro-apoptotic activity of these two compounds. Activation of NF-kappa B was inhibited in resveratrol- and KST201-treated DU145. Both resveratrol and KST201 attenuated cyclooxygenase enzymatic activity but were shown more specific to cyclooxygenase-1. Antioxidant activity of resveratrol was shown in solutions containing free radicals; however, KST201 was less effective on free radical scavenging. Resveratrol inhibited 2'7'-dichlorofluorescin oxidation mediated by hydrogen peroxide, whereas KST201 increased it. Depletion of hydrogen peroxide inhibited anti-proliferatory activity of KST201 but not resveratrol. Contrary to resveratrol, KST201 behaved as a pro-oxidant rather than an antioxidant suggesting a different role it played in the biological system examined in this study. Compared to resveratrol-treated DU145, a broader spectrum of genes were regulated in KST201-treated cells. These genes encode proteins involved in various signaling pathways controlling cancer formation. 57% of resveratrol-regulated genes were induced or suppressed by KST201 indicating the overlap of signaling molecules targeted by these two compounds. Advisors/Committee Members: Tsai, Chun-Che.

▼ The nuclear receptor pregnane X receptor (PXR) is activated by bile acids, steroids and drugs and regulates a network of genes in lipid and drug mechanisms. The goal of this study is to investigate the role of PXR in the coordinate regulation of bile acid synthetic and detoxification genes and its implications in cholestatic liver diseases and treatments. Cholesterol 7alpha hydroxylase (CYP7A1) catalyzes the rate-limiting step in the classic bile acids synthetic pathway and plays a key role in controlling bile acids homeostasis. Quantitative real-time PCR (Q-PCR) showed human PXR agonist rifampicin inhibited CYP7A1 mRNA expression in primary human hepatocytes. Mammalian two-hybrid assays, co-immunoprecipitation (co-IP) assays and chromatin immunoprecipitation (ChIP) assay revealed that ligand-activated PXR strongly interacted with HNF4alpha, the key activator of human CYP7A1, and blocked HNF4alpha interaction with co-activator PGC-1alpha, thus resulted in inhibition of CYP7A1. CYP3A4 is the most abundant cytochrome P450 monooxygenase expressed in human liver and intestine. Bile acids and drugs-activated PXR induces CYP3A4, which converts toxic bile acids to non-toxic metabolites for excretion. Studies using Q-PCR, reporter assays, GST pull-down assays and ChIP assays revealed that PXR strongly induced CYP3A4 gene transcription by interacting with HNF4alpha, SRC-1 and PGC-1alpha. SHP, a negative nuclear receptor, reduced PXR recruitment of HNF4alpha and SRC-1 to the CYP3A4 chromatin and inhibited CYP3A4. Interestingly, PXR concomitantly inhibited SHP gene transcription and maximized the PXR induction of CYP3A4. Taken together, PXR inhibits CYP7A1 to reduce bile acid synthesis and induces CYP3A4 to detoxify bile acid. Thus, PXR may play a protective role against cholestasis. Drugs targeted to PXR may be developed for treating cholestatic liver diseases induced by bile acids and drugs. Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyzes the side-chain cleavage reaction in bile acid synthetic pathways and 27-hydroxylation of cholesterol mainly in the peripheral tissues. Q-PCR revealed that rifampicin induced CYP27A1 mRNA expression in the intestine-derived Caco2 cells, but not in primary human hepatocytes and HepG2 cells. Rifampicin stimulated CYP27A1 gene transcription in cholesterol laden Caco2 cells and increased intracellular 27HOC, which stimulates cholesterol efflux by induction of cholesterol efflux transporters ABCA1 and ABCG1. Mutagenesis analysis, electrophoretic mobility shift assay and ChIP assays identified a functional PXR binding site in the human CYP27A1 gene. These data suggest that 27-hydroxycholesterol is an endogenous LXRalpha ligand and the PXR/CYP27A1/LXRalpha signaling pathway regulate cholesterol efflux in intestine cells. Results revealed an intestine-specific regulation of human CYP27A1 gene by PXR and suggested a novel role for PXR in cholesterol metabolism and detoxification in the intestine. Advisors/Committee Members: Chiang, John Y.L.

▼ Cardiac fibroblasts (CFs) are the major non-contractile cells present in the myocardium, and are primary regulators of synthesis and secretion of extracellular matrix (ECM) proteins. Both proliferation and differentiation of CFs can potentially result in excess ECM protein production and cardiac fibrosis, a condition characterized by a stiffening of the myocardium. This condition is common after myocardial infarction and develops during heart failure, resulting in compromised cardiac function. Hormonal input, as well as input from the surrounding ECM can affect CF proliferation and/or differentiation, and an increase in either one of these parameters will result in elevated ECM production. Consequently, limiting prolonged fibroblast activation and the subsequent detrimental ECM production after myocardial infarction or heart failure might help to preserve left ventricular function. The specific ECM composition in the myocardium likely imparts significant effects on CF function. However, to date little is known about the effect of the ECM on CF function or the signaling pathways utilized by ECM molecules. In the adult, the myocardium is primarily composed of types I and III collagen, in addition to lower levels of types IV, V, and VI collagen. Extensive remodeling of the ECM occurs following myocardial infarction, and the resulting ECM composition can influence cardiac fibroblast activation in addition to affecting cardiac performance. My goals are to determine the mechanism of Gq/Gs cross-talk and the functional consequences in CFs, to determine the functional effects of specific types of collagen on CF differentiation and proliferation, and to identify the collagen composition and myofibroblast content post-myocardial infarction. Advisors/Committee Members: Meszaros, J Gary.

▼ The aim of this study was to develop noninvasive measurement procedures to identify the passive and active contributions to stiffness during ankle dorsiflexion in individuals with chronic stroke. Joint stiffness was defined as the change in joint moment per unit change in joint angle. Stiffness was compared between the hemiplegic and nonhemiplegic ankles of 24 subjects with stroke, as well as the ankle of 24 age-/sex-matched control subjects. Total stiffness was measured using gait analysis. Passive stiffness was measured using the KinCom dynamometer. By subtracting passive stiffness from total stiffness, an active stiffness component was obtained. The ankle was also passively dorsiflexed at three speeds: slow (3°/sec), high (60°/sec), and at each participant’s walking speed (mean = 35°/sec) to determine the presence of hyperreflexia, as reflected by EMG activity, or velocity dependent viscoelastic changes in stiffness. There was no statistically significant difference in passive or active stiffness between the hemiplegic, nonhemiplegic, or control ankles. However, the hemiplegic ankle was significantly (p < .05) more variable in stiffness than the control ankle. Reflex activity was noted only in a small number of KinCom stretching trials (< 10%) in the hemiplegic and control ankles, and did not correlate with the presence of clonus. Yet, when reflex activity was present, ankle stiffness was significantly greater (p < .05) in the hemiplegic ankle as compared to the nonhemiplegic ankle. Also, stiffness was significantly greater (p < .05) in the hemiplegic ankles that demonstrated reflex activity, than in those that did not. In KinCom stretching trials without evidence of reflex activity, there was a significant difference (p < .05) in stiffness in control and hemiplegic ankles, but not nonhemiplegic ankles, between low and gait velocity trials, reflecting a velocity dependent component to passive ankle stiffness. There was no correlation between clinical measures of spasticity or motor recovery with stiffness. Advisors/Committee Members: Verstraete, Mary.

▼ The significant variation within wildtype animals is an underutilized resource for studying skeletal development. In the mammalian metatarsal one end forms a growth plate and secondary center of ossification as in other long bones, however the opposite end undergoes direct ossification in a manner more similar to short bones. Thus, the metatarsal can serve as a model to explore the signals and factors that specify growth plate formation in the vertebrate skeleton. In pursuit of this goal I first compared the patterns of chondrocyte differentiation and proliferation during growth plate formation and direct ossification of an age series of mouse metatarsals. As in the human, both growth plate formation and direct ossification are easily discernable in the mouse metatarsal. In addition, growth plate formation is characterized by a region of peak of proliferation corresponding to reserve zone chondrocytes that distinguishes it from both established growth plates and direct ossification. Second, immunohistochemisty reveals that patterns of PTHrP and PTH/PTHrP-receptor are indistinguishable at each end of the metatarsal suggesting PTHrP signaling is not specific to growth plates. In contrast, the distribution of Patched, the Ihh receptor, is highly variable during growth plate formation and appears to co-localize to regions of increased proliferation, suggesting a potential role for Ihh signaling in specifying growth plate formation. Third, a comparative analysis of metapodial ossification between mice and alligators (Alligator mississippiensis) reveals similar proliferative patterns and protein expression during growth plate formation and within established growth plates, indicating that both species share common mechanisms of chondrocyte regulation. However, unlike mice, alligators establish growth plates at both ends of their metapodials. Phylogenetic analysis reveals that the direct ossification of one epiphysis and reliance on a single growth plate is restricted to eutherians and marsupials and thus is a shared derived character (synapomorphy) of placental mammals. Finally, mouse metatarsals raised in culture continue to undergo growth plate formation and direct ossification at its respective ends, demonstrating that chondrocytes themselves have the intrinsic capacity to dictate these processes in the absence of normal systemic (hormonal) signals and mechanical environment. Advisors/Committee Members: Lovejoy, C. Owen.

▼ Angiotensin (Ang) II is well known for its classic role in the renin-angiotensin system. However, it also plays a central role in the remodeling of the vascular wall associated with hypertension, atherosclerosis and restenosis via the activation of AT1 receptors on vascular smooth muscle cells (VSMC). Upon activation by Ang II, AT1 receptors stimulate the cytosolic phospholipase A2 (cPLA2)-dependant release of arachidonic acid (ArAc) in VSMC. ArAc release mediates reactive oxygen species (ROS) production and transactivation of the epidermal growth factor receptor, leading to the activation of downstream kinases resulting in VSMC growth. To determine the involvement of Akt in this mitogenic pathway, I used cultured rat VSMC to link Ang II-induced ArAc release to ROS production, Akt phosphorylation, Akt kinase activity, and VSMC growth. Using western analysis, I observed that Ang II (100nM), ArAc (20uM), or H2O2 (200uM) increased Akt phosphorylation by 45, 46 or 39%, respectively, while increasing Akt activity by 324, 250 or 249%, respectively. We also observed that Ang II, ArAc, or H2O2 increased 3H-thymidine incorporation into DNA by 210, 150 or 140%, respectively. The Akt inhibitor SH6 (10uM) effectively blocked Ang II-, ArAc-, or H2O2-induced Akt phosphorylation, Akt kinase activity, and VSMC growth. The inhibition of phosphoinositide 3-kinase (PI3K) by10uM LY294002 decreased Akt phosphorylation, Akt kinase activity, and VSMC growth by 95, 91, or 95%, respectively, indicating this pathway is PI3K-dependant. Inhibition of cPLA2 by 10uM AACOCF3 blocked Ang II-induced Akt phosphorylation, Akt kinase activity, and VSMC growth by 94, 76 or 100%, respectively. Finally, the ROS scavenger NaC (10mM) decreased Ang II-induced Akt phosphorylation and Akt kinase activity by 61 or 75%, respectively, and ArAc-induced Akt phosphorylation and Akt kinase activity by 91 or 60%, respectively. Thus it appears that AT1 receptor activation, subsequent ArAc release and ROS production is necessary for PI3K-dependant phosphorylation/activation of Akt and VSMC growth to occur. Advisors/Committee Members: Freeman, Ernest J.

▼ Bcl-2 is an anti-apoptotic protein that has recently been shown to regulate other cellular functions. We previously reported the novel function of Bcl-2 that regulates chondrocyte matrix gene expression, independent of its anti-apoptotic function. The first hypothesis was that Bcl-2 regulates chondrocyte phenotype through the specific pathways. The role of three intracellular signaling pathways likely to be associated with Bcl-2 function, namely, NFkappaB, PKCalpha, and ERK1/2, was examined. The NFkappaB and PKCalpha signaling pathways were not involved in Bcl-2 regulated matrix expression, even thought these are known to regulate Sox9. The ERK1/2 signaling pathway was activated in Bcl-2 deficient cells that lost the chondrocyte phenotype by decreasing chondrocyte matrix protein expression and increasing fibroblastic collagen expression. The inhibition of phopspho-ERK1/2 reversed cells to have chondrocyte phenotype. Moreover, the MEK-ERK1/2 pathway limits the gene expression of matrix protein in wild type chondrocytes. These data indicate that Bcl-2 regulates chondrocyte phenotype through the MEK-ERK1/2 pathway. The second hypothesis was that Bcl-2 regulates chondrocyte phenotype in vitro as well as in vivo specifically, in human osteoarthritis. Osteoarthritis (OA) is an age-related degenerative cartilage disease and is known that the chondrocyte phenotype is altered. However, the significance of the altered phenotype in OA is unclear due to the use of non-age match samples, different source of samples, and lack of precise determination of the stage of OA progression. We developed an intrajoint comparison model using human OA samples to control for patient age and genetic background effects. The advanced OA cartilage was taken from within 1cm of overt lesions. In contrast, minimal OA cartilage was taken from areas without any obvious lesions. The chondrocyte matrix protein and Bcl-2 mRNA expression was decreased in advanced OA cartilage compared with minimal OA cartilage in most of patients studied. In contrast, osteopontin mRNA expression was up-regulated in advanced OA cartilage compared with minimal OA cartilage. A correlation was observed between the steady state mRNA coding for aggrecan and Bcl-2, and Bcl-2 and Sox9. These results support the hypothesis that Bcl-2 regulates chondrocyte phenotype in vivo as well as in vitro. Advisors/Committee Members: Horton, Walter E.