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There remain a number of patients presenting with 5-fluorouracil (5-FU) toxicity despite normal dihydropyrimidine dehydrogenase (DPD) enzyme activity, suggesting possible deficiencies in the two enzymes downstream of DPD in the uracil catabolic pathway: dihydropyrimidinase (DHP), encoded by the DPYS gene, or [beta]-ureidopropionase (BUP-1), encoded by the UPB1 gene. Unlike DPD, which is expressed in peripheral blood mononuclear cells (PBMCs), DHP and BUP-1 enzyme activity is detected in only the liver and kidney, thus requiring an invasive biopsy for determination of activity. This dissertation, therefore, focuses on the role of the genetic and epigenetic regulation of DPYS and UPB1. The genetic variations present in DPYS and UPB1 were identified in 219 healthy volunteers with known DPD enzyme activity and [2-13C]-uracil breath test (UraBT) profiles and 50 cancer patients who experienced 5-FU toxicity despite normal DPD enzyme activity. In the volunteers with a deficient UraBT and normal DPD activity, three non-conservative DPYS mutations were detected, two of which caused a decrease in DHP activity. In the 50 cancer patients, two inactivating UPB1 non-conservative mutations were detected. Epigenetic regulation, mainly methylation, was examined in 43 liver samples (a tissue where DPYS and UPB1 are highly expressed) and in 20 PBMC samples (where neither gene is expressed) to determine if methylation is a possible mechanism of down-regulation/silencing in these genes. Low levels of methylation weredetected in the DPYS gene in the liver samples and PBMCs, suggesting that methylation may not be a key element in the regulation of DPYS. However, varying levels of methylation were detected in the CpG islands of the UPB1 gene in the liver samples and almost complete methylation was detected in the UPB1 CpG islands of the PBMCs, suggesting that methylation may be involved in the regulation of the UPB1 gene. This study was designed to expand our understanding of the molecular mechanisms governing the regulation of the uracil catabolic pathway and will highlight the importance of screening for the integrity of the entire pathway including DPD, DHP and BUP-1 in cancer patients prior to the administration of flouropyrimidine drugs to effectively lower the incidence of 5-fluorouracil toxicity.

xvi, 199 p. : ill., digital, PDF file

Pharmacology and Toxicology

Joint Health Sciences;

5-Fluorouracil Dihydropyrimidinase [Beta]-Ureidopropionase Uracil Catabolism Dihydropyrimidine Dehydrogenase Denaturing High Performance Liquid Chromatography

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Advisors/Committee Members: Johnson, Martin, Diasio, Robert B. <br>, El Kouni, Mahmoud <br>, Ezzeldin, Hany <br>, Zhang, Ruiwen.

▼ The small G-proteins, RalA and RalB, are important mediators of cellular responses to viral infection and nutrient availability. Prior work has demonstrated that the exocyst complex is an important effector of Ral G-protein signaling. The eight member exocyst contains two Ral effector proteins, Exo84 and Sec5, which contribute to distinct cellular responses. During viral infection, RalB promotes the activation of the innate immunity signaling kinase, TBK1, through direct assembly on a Sec5-containing subcomplex of the exocyst. Macroautophagy is an important cellular process which facilitates cellular adaptation to nutrient deprivation as well as the clearance of intracellular pathogens. The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. I report that RalB is localized to nascent autophagosomes. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB-Exo84 signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery. Conversely, I find that Sec5 associates with mTORC1, a key inhibitor of autophagy. Intriguingly, I find that the Ral–Sec5 activated kinase, TBK1, is necessary for amino acid stimulation of mTORC1 activity. Thus, distinct Ral-dependent subcomplexes of the exocyst mediate the cellular response to nutrient availability. Advisors/Committee Members: White, Michael A..

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Malignant gliomas are the most common and deadly form of primary brain cancer afflicting adults. Current treatment regimens, including surgical debulking, radiotherapy, and chemotherapy, have limited efficacy, and median patient survival remains only 14 months. Therefore, novel therapies must target different aspects of glioma biology. Two of the most striking features of this cancer are the unusual ability of glioma cells to robustly proliferate and migrate in the brain, and recent evidence suggests that ClC-3, a voltage-gated Cl- channel/transporter is implicated in both of these processes. We hypothesize that ClC-3 may facilitate proliferation and migration by promoting hydrodynamic shape and volume changes; as Cl- efflux occurs, water osmotically leaves the cytoplasm. These shape and volume changes are critical as, for example, a glioma cell divides into 2 daughter cells, or migrates through narrow extracellular spaces in the brain. In this dissertation, we assess upstream signaling to determine how ClC-3 is activated in the context of proliferation and migration. Using a combination of biophysical, biochemical, genetic, and imaging techniques, we identify several mechanisms suggesting that Ca2+/calmodulin-dependent protein kinase (CaMKII) regulates ClC-3 activity. We demonstrate that channels or ligands that increase [Ca2+]i also activate CaMKII, leading to downstream ClC-3 activation and promoting proliferation and migration. CaMKII regulation of ClC-3 is required for a critical cytoplasmic condensation checkpoint at the metaphase-anaphase transition, and inhibition of either protein leads to disrupted volume regulation and proliferation. Additionally, we found that bradykinin, a chemotactic peptide, increases glioma cell migration by activating CaMKII-dependent ClC-3 channels. Inhibition of ClC-3 or CaMKII completely blocked bradykinin-induced migration. We propose that CaMKII activation of ClC-3 is a critical mediator of cellular proliferation and migration and should be integrated into preexisting models. We speculate that [Ca2+]i may be a ""master regulator"" of both proliferation and migration by simultaneously controlling cytoskeletal proteins, kinases, and Ca2+-sensitive ion channels. Finally, our data suggest that targeting Cl- channels or bradykinin receptors on human glioma cells may be a novel therapeutic strategy for the management of malignant gliomas.

PhD

1 online resource (xi, 213 p.) :ill., digital, PDF file.

Neurobiology

Joint Health Sciences

bradykinin cell migration chloride channel ClC-3 Cl- channel

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Advisors/Committee Members: Harald Sontheimer, Bedwell,David Kirk,Kevin Nabors,Burt Wadiche,Jacques.

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Heme oxygenase-1 (HO-1) is a critical enzyme catalyzing the degradation of heme and generating carbon monoxide, iron, and biliverdin. In addition to heme degradation, HO-1 expression is known to protect against various cellular insults and disease states including acute kidney injury, atherosclerosis, vascular restenosis, and others. Human HO-1 gene expression is tightly regulated at the level of transcription. The main goal of this study is to explore the transcriptional regulation of the human HO-1 gene in renal epithelial cells. Chromosome Conformation Capture (3C) demonstrates that multiple regulatory regions within the HO-1 promoter and enhancer regions are physically interacting with each other by forming chromatin loops and this looping is required for the initiation of the human HO-1 gene transcription. ChIP-Loop assay reveals that these regions share several common transcription factors such as Sp1, USF-1, and JunB. Sp1 binds to the intronic enhancer which interacts with the -4.5kb promoter region by chromatin looping. Inhibition of Sp1 by siRNA abolishes this interaction and reduces HO-1 transcription. To study human HO-1 transcription in vivo, a HO-1 bacterial artificial chromosome (BAC) transgenic mouse was generated by integrating a 87 kb BAC DNA (a portion of the human chromosome 22 including HO-1 gene and its regulatory regions) into the mouse genome. Global overexpression of HO-1 mRNA and protein in tissues of HO-1 BAC transgenic mice were detected. Moreover, using this HO- 1 BAC transgenic mouse, a “humanized” HO-1 BAC transgenic mouse (hHO-1 BAC) was generated by crossing HO-1 BAC mice with HO-1 knockout (HO-1-/-) mice. In addition to the overexpression of human-specific HO-1 mRNA and protein expression, the human HO-1 gene in hHO-1 BAC mice rescued the abnormal phenotype observed in the HO-1-/- mice such as increased incidence of abortion and embryonic lethality, heightened sensitivity to acute kidney injury, iron overload, anemia, and splenomegaly. These studies provide not only functional insights for the regulation of the human HO-1 gene but also the molecular architecture of the human chromosome containing the HO-1 gene and promoter in renal epithelial cells.

1 online resource (xiv, 136 p.) : ill., digital, PDF file.

Joint Health Sciences;

Heme oxygenase-1 Chromatin looping Transgenic mice Acute kidney injury rhabdomyolysis chromosome conformation capture

UNRESTRICTED

Advisors/Committee Members: McDonald, Jay M., Agarwal, Anupam<br>, Chugh, Sumant S.<br>, Hardy, Robert W.<br>, Ponnazhagan, Selvaragan<br>, Ryan, Thomas.

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The ubiquitin proteasome system (UPS) is essential for regulated protein degrada-tion, a requirement for numerous neuronal process, including vesicle cycling, neuro-transmitter release, spine morphology, and synaptic plasticity. Better understanding of UPS function in neurons will increase our knowledge of neurological diseases caused by alterations in ubiquitin signaling. I utilized the ataxia (axJ) mice as a tool to study the UPS in the nervous system. The axJ mice have lowered expression of ubiquitin-specific protease 14 (Usp14) in all tissues; this decreased expression results from an intracisternal-A particle insertion into Usp14. The axJ mice are phenotypically indistinguishable from wild type littermates at birth. By 2 weeks, axJ mice are smaller in size and display a resting tremor. Muscle wasting and tremor become more pronounced until 6 weeks of age, and death occurs by 8 weeks. It was suggested that the axJ phenotypes are caused by dystrophic Purkinje cell axons. However, the global loss of Usp14 indicates that the axJ phenotypes result from multiple organ system failure. To understand Usp14’s function in neurons, I characterized the expression and ac-tivity of Usp14 gene products. Loss of Usp14 results in a decrease in steady state levels of monomeric ubiquitin. Because Usp14’s catalytic activity is stimulated by proteasome binding, loss of Usp14 on the proteasome implies altered proteasome function; evidence of which is strengthened by ubiquitin loss in axJ mice. Furthermore, I specifically expressed Usp14 in axJ neurons to test the hypothesis that the axJ phenotype results from loss of Usp14 expression in these cells. Neuronal-specific expression of Usp14 rescued the premature death, muscle wasting, and tremor independently of Purkinje cells. Here we demonstrate an essential requirement for Usp14 in the mammalian nerv-ous system. Surprisingly, Usp14 appears dispensable in all tissues except neurons, indi-cating the UPS may have nervous system specific functions not required in other cell types. Our data also provide the first evidence for a primary defect in proteasome func-tion as the underlying cause of a neurological disease in mammals. Through increasing our understanding of Usp14 in neurons, we can better understand UPS function in other diseases; illuminating new therapeutic targets for neurological disorders.

ix, 105 p. : ill., digital, PDF file.

Neurobiology

Joint Health Sciences

Ataxia Ubiquitin Mutation Proteasome Synapse Transgenic

UNRESTRICTED

Advisors/Committee Members: Wilson, Scott, Brenner, Michael <br>, Crawford, David <br>, Voll, Gail Johnson <br>, Theibert, Anne.

▼ Human African Trypanosomiasis (HAT), also known as Sleeping Sickness, is a disease caused by the protozoan parasite Trypanosoma brucei. About 70 million people are at risk of infection in sub-Saharan Africa. While current treatments have efficacy, they are difficult to administer and serious toxicity is associated with one of the key compounds, emphasizing the need for novel therapeutic approaches. The polyamine biosynthetic pathway is vital for parasite viability and the first committed step catalyzed by ornithine decarboxylase is the target of Eflornithine, which is used to treat late stage disease. Our lab has investigated this pathway to identify other potential targets for drug development. During this work I also demonstrated that the parasite employs unique regulatory strategies to control metabolism through this pathway. Among them, I found T. brucei S-adenosylmethionine decarboxylase (TbAdoMetDC), which is a homodimer in other eukaryotes, is a heterodimer in the trypanosomatids. The active enzyme is composed of two paralogs, one with limited activity (AdoMetDC) and the other inactive, which I termed prozyme. Prozyme activates trypanosomatid AdoMetDC through an allosteric mechanism that involves relief of autoinhibition through a conformational change. Prozyme is found in limited quantities relative to TbAdoMetDC and upregulated upon knockdown or chemical inhibition of TbAdoMetDC, providing a mechanism to regulate polyamine flux in the cell. The cellular mechanism of regulation is not fully understood, as gene expression in T. brucei differs significantly from that of mammals. Herein, I show prozyme is regulated at the level of protein translation. I found that TbAdoMetDC suppresses prozyme expression at the protein level in an enzyme activity-independent manner. Upon its loss, prozyme is expressed constitutively in an unregulated manner. I also show that the enzymatic product of TbAdoMetDC, dcAdoMet, acts as a metabolic signal for prozyme upregulation. Under chemical inhibition of AdoMetDC by Genz-644131 I show dcAdoMet is the only significant metabolite to correlate with prozyme protein upregulation. I further support this hypothesis by characterizing the effects of knockdown of S-adenosylmethionine synthetase by RNAi and through methionine starvation to correlate dcAdoMet depletion with prozyme upregulation independent of AdoMetDC manipulation. Through this work I also demonstrated that TbAdoMetSyn is an essential enzyme and validate the activity of its allele. Taken together, I expand upon our mechanistic understanding of this complex regulatory paradigm between an enzyme and pseudoenzyme in T. brucei. Advisors/Committee Members: Conrad, Nicholas, Phillips, Margaret A., Goodman, Joel M., Reese, Michael L., Tu, Benjamin.

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Matrix metalloprotinases are zinc-dependent endopeptidases with broad substrates from extracellular matrix proteins to bioactive molecules. Physiologically, they regulate tissue remodeling and immune responses. However, in cancers and inflammatory diseases, MMP expression is increased and exacerbates disease development. Aberrant upregulation of one MMP, MMP-9, is closely related to disease progression. Understanding positive and negative regulation of MMP-9 will shed light on the rational design of MMP-9 inhibitors. First, I investigated the roles of transcriptional coactivators in MMP-9 gene expression. Histone acetyltransferases (HAT) CBP/p300 and PCAF can activate MMP-9 promoter activity and this requires the HAT activity of PCAF. CARM1 acts as coactivator through its protein arginine methyltransferase activity. GRIP1, a p160 protein, has only a modest effect alone on MMP-9 transcription, but shows significant synergy with p300 and CARM1. Furthermore, this synergy relies on the interaction of p300 with the AD1 domain of GRIP1 and CARM1 with the AD2 domain of GRIP1. In addition, all these coactivators are present at the endogenous MMP-9 promoter as demonstrated by chromatin immunoprecipitation (ChIP) assays. Knockdown of p300, CARM1 and GRIP1 greatly reduces the expression of endogenous MMP-9. This study identifies the critical function of coactivators in activation of MMP-9 expression. Next, I studied the mechanism of negative regulation of PMA-induced MMP-9 expression by IFN-[Beta]. Gene activation by IFN-[Beta] is well studied and it is mediated mainly through activation of the JAK-STAT pathway. This pathway activates two potent transcriptional activation complexes, GAF (STAT-1 homodimer) and ISGF3 (STAT-1, STAT-2 and IRF-9). My studies show that STAT-1, STAT-2 and IRF-9 are required for IFN-[Beta] inhibition of MMP-9 expression in cells that lack functional STAT-1, STAT-2, and IRF-9. However, the ISGF3 components are not associated with the MMP-9 promoter and IFN-[Beta] does not inhibit the signaling pathways responsible for MMP-9 induction. Lastly, ChIP assays illustrate that IFN-[Beta] reduces association of NF-[Kappa]B p65, Sp1, CBP, p300 and RNA Pol II, and decreases permissive acetylation of histones H3 and H4 on the MMP-9 promoter. This work demonstrates for the first time that IFN-[Beta] activated ISGF3 complex not only activates but suppresses gene expression.

viii, 129 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

Gene Regulation Interferon MMP Coactivator Signal Transduction Transcription Factors

UNRESTRICTED

Advisors/Committee Members: Benveniste, Etty N., Brenner, Michael<br>, Collawn, James<br>, Lin, Fang-Tsyr<br>, Nabors, L. Burt.

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Electrogenic Na/bicarbonate cotransporters (NBCes) are important regulators of intracellular pH in many tissues including the kidney, brain, and pancreas, and are members of a superfamily of HCO3 – transporters including Anion Exchangers (AEs) and Nadriven, Cl–bicarbonate exchangers (NDCBEs). There are three NBCe1 splice variants (NBCe1-A, -B, and -C) that only differ in their cytoplasmic N and C termini. The first study is a systematic comparison of the three variants expressed in Xenopus oocytes, and examination of the role of the N and C termini. NBC function of the constructs was assessed using the 2-electrode, voltage-clamp technique or the macropatch technique with NBC-expressing, voltage-clamped oocytes. The three variants had similar ion- and voltage- dependencies, but the A variant displayed a ~4-fold higher current in whole-cell experiments due to its unique N terminus. Data from truncation mutants indicate that the cytoplasmic C termini influence function and the N termini can influence regulation of NBC activity. Whereas, the unique N terminus of the A variant stimulates NBC function and the different N termini of the B/C variants inhibit function. This finding was also observed in macropatch experiments. In the second study, I performed cysteine-scanning mutagenesis and Substituted-Cysteine Accessibility Method (SCAM) on NBCe1 TMD8, because positions in the homolgous region of AE1 are involved in ion translocation. 21 residues of TMD8 were each replaced with cysteine. Function and/or sulfhydryl sensitivity of these mutants were assessed using the 2-electrode, voltage-clamp technique with NBC-expressing oocytes clamped at -60 mV. Eight mutants are mildly sensitive to one or both sulfhydryl reagents, MTSEA and pCMBS. The L750C mutant is inhibited ≥ 85% by both reagents. pCMBS accessibility of L750C is 1) reduced in the presence of both Na+ and HCO3 – when the transporter is active, 2) reduced in the presence of an NBC inhibitor, and 3) stimulated at more positive holding potentials. In summary, TMD8 forms part of the anion translocation pathway, and accessibility of L750C is affected by the state of the transporter.

xi, 211 p. : ill., digital, PDF file

Physiology and Biophysics

Joint Health Sciences

UNRESTRICTED

Advisors/Committee Members: Bevensee, Mark O., Fuller, Catherine M.<br>Romero, Michael F.<br>Schafer, James A.<br>Smith, Peter R.<br>Weiss, David S..

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O-GlcNAcylation is an abundant and dynamic post-translational modification on serine and threonine residues of nuclear and cytoplasmic proteins. O-GlcNAc Transferase (OGT) and Nuclear Cytoplasmic O-GlcNAcase and Acetyltransferase (NCOAT) are the only two enzymes and major regulators in this process. In the past two decades, many studies have demonstrated its important modulation roles in nutrient sensing, protein turnover, cell cycle progression, transcription, translation as well as stress resistance. Among these biological functions, the understanding of O-GlcNAcylation dependent oxidative stress is relatively unknown. This study aims to examine the effects of OGlcNAcylation on JNK1 and FOXO4 transcription factors under acute oxidative stress treatment. Oxidative stress is an imbalance between production and clearance of reactive oxygen species (ROS) in cells. Overload oxidative stress may cause many aging-related diseases including Type II diabetes, neurodegenerative disorders and muscle atrophy. MAPK death signal and PI3 kinase survival signal are two of the major evolutionary conserved phosphorylation cascade pathways involved in oxidative stress mediated cellular response. In this study, we show that OGT overexpression in cells have a high survival rate and a low cell death under acute hydrogen peroxide. Further studies found that OGT specifically associates with, and O-GlcNAcylates, JNK1 but not ERK1 and p38-MAPK. Oxidative stress induced JNK1 O-GlcNAcylation also prevented phosphorylation-dependent death signal transduction. On the other hand, hydrogen peroxide induced OGT interaction with FOXO4 and enhanced its transcriptional activity on survival target gene regulation. Overall, the current work demonstrates OGT and O-GlcNAcylation can positively regulate the FOXO4 survival signal, and can negatively modulate the JNK1 death signal to protect cells from acute oxidative stress. These data provide one molecular mechanism to explain O-GlcNAcylation-dependent stress resistance and cell survival upon acute oxidative stress as well as other stress stimuli.

1 online resource (xv, 84 p.) : ill., digital, PDF file.

Cell Biology

Joint Health Sciences

O-GlcNAc Transferase Oxidative stress JNK1 FOXO4

UNRESTRICTED

Advisors/Committee Members: Paterson, Andrew J., Collawn, James F.<br>, Frank, Stuart J.<br>, Lin, Fang-Tsyr<br>, Townes, Tim M..

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B lineage cells are major players in the adaptive immune system. Pax5 is essential for B lineage cell development and function. Pax5 controls B lineage cell developmental progression by regulating expression of many B lineage specific genes and the B lineage specific VH to DJH recombination; and the meantime, Pax5 also represses the transcription of lineage- and developmental stage- inappropriate genes to restrict the B lineage developmental pathway. It is not clear how Pax5-mediated function can be regulated to fulfill its role in different biological reactions. In this dissertation, we focused on the study of the molecular regulation of Pax5-mediated functions. In part I, our results showed that Pax5-mediated transcriptional activation can be dramatically elevated by overexpression of histone acetyltransferase p300. We found that p300 directly interacts with Pax5 and acetylates multiple lysine residues within the Nterminal regions of the Pax5 protein. Mutation of the lysine 67, 87, 89, 103, or 142 residues into alanine diminished p300 mediated enhancement of Pax5 function in activation of Luc-Cd19 reporter gene expression. Moreover, mutations of lysine 67, 87/89 in Pax5 impaired Pax5-mediated activation of endogenous Pax5 target genes in Pax5-/- mouse pro B cells, indicating that acetylation of Pax5 provides an important regulation for Pax5-mediated biological functions during B lineage cell development. In part II, to understand how Pax5 mediates global effects during B cell development, we analyzed the nuclear sub-localization of Pax5. It has long been hypothesized that transcription in cells occurs on the discrete loci associated with the nuclear matrix inside the nucleus. We found that most endogenous Pax5 in human or mouse B lineage cells associates with the nuclear matrix. In addition, Pax5 co-localizes with the basal transcriptional machinery. We found that the partial homeodomain and its flanking regions contain signals to target Pax5 to the nuclear matrix. Deletion of the partial homeodomain of Pax5 compromises Pax5-mediated transcriptional activation of many target genes. These results indicate that the association with the nuclear matrix is essential for Pax5 to fully activate endogenous B lineage specific genes. Taken together, the results presented in this dissertation uncovered two novel mechanisms that regulate Pax5-mediated transcription function.

x, 138 p. : ill., digital, PDF file

Microbiology

Joint Health Sciences

Pax5 p300 acetylation nuclear matrix

UNRESTRICTED

Advisors/Committee Members: Zhang, Zhixin, Justement, Louis B. <br>, Klug, Christopher A. <br>, Burrows, Peter B. <br>, Raman, Chander.

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It has been established that the environment, including the diet, plays a critical role in a woman’s risk of breast cancer. Two dietary polyphenols that have received attention due to their abilities to protect against breast cancer are genistein, the predominant isoflavone in soy, and resveratrol, a phytoalexin found in red grapes and wine. The purpose of this research was to investigate if resveratrol in the diet, alone and in combination with genistein, could protect against chemically-induced mammary cancer. Further, we wanted to look at possible toxicities associated with resveratrol and the mechanisms through which it may be exerting a mammary-protective effect. When administered in the diet, resveratrol along with combinations of resveratrol and genistein could protect the mammary gland against dimethylbenz(a)anthracene (DMBA)-induced mammary cancers in rats. For resveratrol, we observed a slight but significant increase in the more mature lobular structures in the mammary gland, and significantly less cell proliferation in all terminal ductal structures in the mammary gland. Resveratrol treatment in the diet had no significant effect on several toxicological parameters, including: body weight, food and drink consumption, uterine to body weight ratio, vaginal opening, and ovarian weight. As to the molecular mechanisms through which resveratrol may be protecting against mammary carcinogenesis and modulating mammary epithelial cell proliferation and apoptosis, we employed TaqMan low density arrays. We selected genes with a reported role in mammary gland proliferation, apoptosis, steroid and carcinogen metabolism, and growth factors involved in normal and malignant mammary gland development. Exposure to resveratrol in the diet resulted in a significant decrease in gene expression of COX-1 and tended to increase the message levels of PPAR-γ. Both of these genes can play a role in cell proliferation and apoptosis. We also observed a significant increase in the expression levels of caspase 2, 9, and 3, all of which are involved in the apoptotic cascade and may help to explain the increased apoptosis that we observed with resveratrol in the mammary gland. We conclude that resveratrol, alone and in combination with genistein, can protect against breast cancer by regulating epithelial cell proliferation and apoptosis in critical terminal ductal structures in the mammary gland.

x, 98 p. : ill., digital, PDF file

Pharmacology and Toxicology

Joint Health Sciences

Resveratrol Geristein Breast Cancer Chemoprevention

UNRESTRICTED

Advisors/Committee Members: Lamartiniere, Coral A., Barnes, Stephen<br>, Eltoum, Isam-Eldin<br>, Grubbs, Clinton J.<br>, Welch, Danny R..

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Syndecan-1 (CD138), a transmembrane heparan sulfate-bearing proteoglycan, is expressed at high levels on most myeloma cells and is shed into the microenvironment. In patients, high levels of serum syndecan-1 are indicative of poor prognosis and elevation of shed syndecan-1 in animal models dramatically enhances tumor growth, angiogenesis and metastasis. Because syndecan-1 is a key regulator of myeloma pathogenesis, we hypothesized that reduction of syndecan-1 levels expressed by the myeloma cells will block their growth and dissemination. Syndecan-1 knockout and knockdown variants of two human myeloma cell lines, CAG and RPMI-8226, were developed using short hairpin RNA (shRNA) technology. In vitro, knock out of syndecan-1 from the myeloma cell surface resulted in a loss of cell viability via initiation of apoptosis. In contrast, syndecan-1 knockdown cells expressing 14-28% normal syndecan-1 levels were phenotypically similar to the control cells and did not reveal significant growth differences when compared to the control cells in vitro. In contrast, when these knockdown cells were injected into severe combined immunodeficient (SCID) mice in vivo, they formed tumors poorly as compared to cells expressing wild-type levels of syndecan-1. Tumor immunohistology revealed that the VEGF levels were dramatically lower in the knockdown tumors as compared to the controls. Moreover, knockdown of syndecan-1 resulted in grossly underdeveloped vasculature in these tumors. Importantly, most syndecan-1 knockdown cells also failed to grow when injected intravenously into SCID mice, suggesting that post-intravasation steps of the metastatic cascade may be syndecan-1 dependent. Few tumors that arose from knockdown cells in animals injected intravenously formed at extra-osseous sites, further implicating syndecan-1 as a critical molecule for homing of myeloma cells to bone. Taken together, these results indicate that syndecan-1 expression is required for robust myeloma tumor development and growth in vivo via regulation of angiogenesis and metastasis, and that therapies aimed at reducing expression of this proteoglycan may benefit myeloma patients.

1 online resource (xi, 114 p. : ill., digital, PDF file)

Pathology

Joint Health Sciences

Multiple myeloma syndecan-1 angiogenesis heparan sulfate proteoglycan metastasis

UNRESTRICTED

Advisors/Committee Members: Sanderson, Ralph D., Lopez, Richard<br>, Welch, Danny R.<br>, Woods, Anne<br>, Zayzafoon, Majd<br>, Zinn, Kurt R..

▼  In order to generate healthy daughter cells, nuclear division and cytokinesis need to be coordinated. Premature division of the cytoplasm in the absence of chromosome segregation or nuclear proliferation without cytokinesis might lead to aneuploidy and cancer. The cyclin dependent kinases, CDKs, are a main regulator of the cell cycle. Timely increase and decrease in their activity is required for cell cycle progression. To enter mitosis, mitotic CDK activity needs to rise. CDK activity stays elevated until chromosome segregation is completed and exit from mitosis requires decrease in CDK activity. Observations in several experimental systems suggest that coordination of cytokinesis with the nuclear cycle is regulated through CDK activity. Prolonged high CDK activity, as it occurs when chromosome segregation is delayed, was found to oppose cytokinesis. Prevention of cytokinesis through high CDK activity may therefore provide a mechanism to prevent precocious cell division in the absence of chromosome segregation. To prevent polyploidy when cell division is delayed, progression through the next nuclear cycle should be inhibited until cytokinesis is completed, presumably by the inhibition of CDK activity. In the fission yeast Schizosaccharomyces pombe, a signaling cascade called Septation Initiation Network (SIN) is required for the coordination of cytokinesis with the nuclear cycle. The SIN is essential for cytokinesis, triggering the execution of cell division through constriction of the actomyosin ring. The activation of the SIN signaling cascade, and thus cytokinesis, is opposed by high CDK activity, preventing precocious cytokinesis. S. pombe delay entry into the next nuclear division in response to delayed cytokinesis due to defects in the contractile ring until cytokinesis is completed thereby preventing the accumulation of multinucleate, non viable cells. This safeguard against multinucleate cells is termed the cytokinesis checkpoint. The cytokinesis checkpoint keeps CDK activity low, preventing nuclear cycle progression. The SIN is required for the cytokinesis checkpoint and therefore is a key coordinator between nuclear cycle and cytokinesis. How the SIN functions in the cytokinesis checkpoint was not known. Cdc14-family phosphatases are highly conserved from yeast to humans, but were only characterized in Saccharomyces cerevisiae at the time this thesis was initiated. Cdc14 had been identified as the effector of a signaling cascade homologous to the SIN, called the mitotic exit network (MEN), which is required for exit from mitosis. This thesis describes the identification of the S. pombe Cdc14-like phosphatase Clp1p as a component of the cytokinesis checkpoint. Clp1p opposes CDK activity, and Clp1p and the SIN activate each other in a positive feedback loop. This maintains an active cytokinesis checkpoint and delays mitotic entry. We further found that Clp1p regulates chromosome segregation. Concluding, this thesis describes discoveries adding to the… Advisors/Committee Members: Dannel McCollum, Ph.D..

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Interleukin-8 (IL-8) is a potent chemoattractant of numerous cells, particularly neutrophils, in the innate immune response. In addition to immune functions, IL-8 is known to contribute to the pathogenesis of a number of diseases, including cancer. Interferon-[beta] (IFN-[beta]), a Type I interferon, inhibits the expression of IL-8, but the details of this effect are not known. In this study we investigate transcriptional control of the IL-8 gene and the mechanism by which IFN-[beta] exerts inhibitory effects on IL-8. Herein we show that stimulation of U87-MG glioma cells with phorbol 12-myristate 13-acetate (PMA) results in a rapid recruitment of NF-[kappa]B p65 to the IL-8 promoter. Additionally, we show that the IL-8 promoter is constitutively acetylated on histones 3 and 4, indicating that the gene is accessible to transcription factors. Positive regulators of gene transcription such as the histone acetyltransferase (HAT) p300 are rapidly recruited to the IL-8 promoter along with RNA polymerase II. At the same time, negative regulators such as the histone deacetylases (HDACs) 1 and 3 that are constitutively present at the IL-8 promoter are dismissed. Upon treatment with IFN-[beta], however, there are rapid decreases in acetylation of histones 3 and 4 along with decreased levels of NF-[kappa]B p65 and RNA polymerase II present at the IL-8 promoter. We show that these promoter effects result in decreased IL-8 promoter activity, mRNA levels and protein levels when cells are treated with IFN-[beta]. We also demonstrate that all three components of the Interferon Stimulated Gene Factor 3 (ISGF3) complex, namely the Signal Transducer and Activator of Transcription (STAT) proteins 1 and 2 and the accessory factor Interferon Regulatory Factor-9 (IRF-9), are required for IFN-[beta] to mediate inhibitory effects on IL-8. In contrast to gene induction by this complex, however, STAT-1 and -2 transactivation domains were not required for inhibition by IFN-[beta]. This work defines the promoter-specific events necessary for induction of IL-8 expression in malignant asctrocytoma cells, and the mechanistic requirements for inhibition of IL-8 by IFN-[beta] in these cells.

xiii, 109 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

Interferon NF-kappaB Interleukin-8 Gene regulation Glioma

UNRESTRICTED

Advisors/Committee Members: Benveniste, Etty N., Chen, Xinbin <br>, Frank, Stuart J.<br>, Nabors, Louis B.<br>, Wilson, Scott M..

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The benefits of calorie restriction (CR) have historically been attributed to a reduction in food intake. More recently, reduced fat stores have been proposed as an important mediator of CR, spurring interest into the potential for changes in energy expenditure and body composition as a CR mimetic. In order to better understand the relationship of body composition, energetics, cancer and longevity, four separate studies were performed. In Experimental Aim 1, a mouse model of prostate cancer (TRAMP) was used to determine if leanness rather than reduced food intake is responsible for the cancer-preventative effect of CR. It was found that increasing energy expenditure via thermoregulation under fixed-food intake conditions, resulted in smaller, leaner mice, which had less cancer incidence and progression. This finding suggests that in terms of cancer prevention, leanness rather calorie intake per se, is more closely associated with the benefits associated with CR. In Experimental Aim 2, results suggest that the longevity protein SIRT1 is elevated in mouse and human prostate cancer, and may be paradoxically implicated in tumor promotion. Experimental Aim 3 found that liver SIRT1 was not significantly affected by acute or chronic exercise. Finally, the results from Aim 4 found that exercised mice were significantly leaner than their weight-matched counterparts which were calorie restricted. However, CR and exercise had disparate effects on longevity biomarkers, including more favorable changes in the insulin/IGF-1 axis with CR, while exercisers had less DNA damage. The results of this experiment imply that the inability of exercise in prior studies to increase lifespan could be due to a failure to fully mimic the physiologic effects of CR. More studies are needed to determine if the inability of exercise to fully mimic CR is inherent to exercise itself, or if the life-prolonging action of CR work independently of changes in energy balance and body composition.

1 online resource (x, 183 p. : ill., digital, PDF file)

Nutrition Sciences;

Health Professions;

exercise cancer aging longevity caloric restriction obesity

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Advisors/Committee Members: Nagy, Tim R., Bamman, Marcas M.<br>, Elgavish, Ada<br>, Grizzle, William E.<br>, Piyathilake, Chandrika J.<br>, Tollefsbol, Trygve O..

▼ Carotenoids are organic pigments that are mainly found in the chloroplasts and chromoplasts of plants and other photosynthetic organisms. Carotenoid molecules containing oxygen, such as lutein, violaxanthin and zeaxanthin are called xanthophylls and the rest containing un-oxygenated carotenoids are known as carotenes. Carotenoids form the integral part of the photosystem II LHC. Xanthophylls mainly aid in light harvesting and dissipation of harmful excess energy from excited chlorophyll molecules, thereby protecting chlorophyll from photo-degradation. The biosynthesis of carotenoids has been widely studied using plant and algae. However, the regulatory mechanisms involved in carotenoid metabolism need better understanding. This thesis identified novel regulatory mechanisms involved in the carotenoid biosynthetic pathway using activation-tagged Arabidopsis mutants. Two screening methods, red seed coat screening and norflurazon resistance screening, were used in this study. Fourteen mutants were screened using red seed coat screening but a successful mutant characterization could not be performed due to the unavailability of mutants with a single copy T-DNA insertion. Norflurazon screening identified eight mutants, out of which two mutants, KN203 and KN231, were characterized. The KN203 mutant had a defective keto-acyl CoA synthase 19 gene. KN203 mutant had lower carotenoid levels in the leaves and increased carotenoid levels in the mature seeds; the mutant was able to revert back to wild type phenotype after complementation of a functional KCS19 gene copy driven by native promoter. The fatty acid analysis indicated that the mutant KN203 had decreased MGDG and increased lysoPG, lysoPC and lysoPE content. Reduced carotenoid content in KN203 leaves was attributed to changes in fatty acid composition of chloroplast envelope membrane. Mutant KN231 had a T-DNA insertion in a gene encoding a RNA binding protein (RBP47C). KN231 leaf carotenoid levels were similar to wild type but their levels were significantly higher in their seeds. Two allelic mutants were selected to characterize the mutants. Overexpression of functional RBP47C in the mutants reverted to wild type phenotype in some overexpression mutants. A tandem repeat homologue of RBP47C, RBP47C’was identified. In-silico analysis predicted RBP47C to be a potential candidate for chloroplast localization. Advisors/Committee Members: Khachatourians, George, Gruber, Margie, Hegedus, Dwayne, Gray, Gordon, Qiu, Xiao.

▼ Gene expression quantitative trait loci (eQTL) mapping studies can provide mechanistic insights into the functions of disease-associated variants. However, many eQTLs are cell type and context specific. This is particularly relevant for immune cells, whose cellular function and behaviour can be substantially altered by external cues. Furthermore, understanding mechanisms behind eQTLs is hindered by the difficulty of identifying causal variants. We differentiated macrophages from induced pluripotent stem cells from 86 unrelated, healthy individuals derived as part of the Human Induced Pluripotent Stem Cells Initiative. We generated RNA-seq data from these cells in four experimental conditions: naïve, interferon- gamma (IFNɣ) treatment (18h), Salmonella infection (5h), and IFNγ treatment followed by Salmonella infection. We also measured chromatin accessibility with ATAC-seq in 31-42 individuals in the same four conditions. We detected gene expression QTLs (eQTLs) for 4326 genes, over 900 of which were condition-specific. We also detected a similar number of transcript ratio QTLs (trQTLs) that influenced mRNA processing and alternative splicing. Macrophage eQTLs and trQTLs were enriched for variants associated with Alzheimer’s disease, multiple autoimmune disorders and lipid traits. We also detected chromatin accessibility QTLs (caQTLs) for 14,602 accessible regions, including hundreds of long-range interactions. Joint analysis of eQTLs with caQTLs allowed us to greatly reduce the set of credible causal variants, often pinpointing to a single most likely variant. We found that caQTLs were less condition- specific than eQTLs and ~50% of the stimulation-specific eQTLs manifested on the chromatin level already in the naive cells. These observations might help to explain the discrepancy between strong enrichment of diseases associations in regulatory elements but only modest overlap with current eQTL studies, suggesting that many regulatory elements are in a ‘primed’ state waiting for an appropriate environmental signal before regulating gene expression.

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Most patients with advanced breast cancer develop osteolytic bone metastases, which have numerous complications. Because current therapies are not curative, new treatments are needed. Conditionally replicating adenoviruses (CRAds) are anti-cancer agents designed to infect and lyse tumor cells. For increased efficacy, CRAds have been armed with therapeutic transgenes. Osteoprotegerin (OPG), an inhibitor of osteoclastic bone resorption, represents a promising candidate with which to arm a CRAd intended to treat osteolytic bone metastases. We hypothesize that a CRAd armed with OPG will inhibit breast cancer bone metastasis and reduce tumor burden in the bone by directly lysing tumor cells and by inhibiting bone resorption through the action of OPG. We have constructed an armed CRAd (Ad5-[delta]24-sOPG-Fc-RGD) by replacing viral E3B genes with a fusion of the ligand-binding domains of OPG and the Fc portion of human IgG1, for improved secretion. Conditional replication is conferred by a 24 base pair deletion within E1A ([delta]24), which prevents the binding of E1A to cellular Rb and limits replication in normal cells. The incorporation of an RGD peptide sequence within the fiber knob confers transductional selectivity by directing infection to cells expressing [alpha]v integrins. A panel of control viruses has also been constructed. We first confirmed the expression of sOPG-Fc and the remaining E3 genes from our experimental viruses, and showed that expression from E3B inhibits neither the oncolytic potency nor the selectivity of replication of an RGD-modified CRAd. We demonstrated that infection of breast cancer cells by Ad-[delta]24-sOPG-Fc-RGD both kills the infected cells by oncolysis and inhibits the formation of osteoclasts in an in vitro co-culture model. The efficacy of Ad-[delta]24-sOPG-Fc-RGD was evaluated in a model of breast cancer bone metastasis in which athymic mice are injected intratibially with human breast cancer cells stably expressing luciferase, allowing the progression of osteolytic lesions to be monitored in live animals. The results of these studies demonstrated that our armed CRAd reduces tumor burden in the bone and inhibits osteoclast formation more effectively than an unarmed CRAd. We have therefore shown the enhanced therapeutic efficacy of the dual-action, armed CRAd in treating bone metastases of breast cancer.

xiv, 118 p. :$bill., digital, PDF file.

Pathology

Joint Health Sciences

conditionally replicating adenovirus bone metastasis breast cancer osteoprotegerin virotherapy gene therapy

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Advisors/Committee Members: Douglas, Joanne T., Curiel, David T. <br>, Selander, Katri <br>, Siegal, Gene P. <br>, Welch, Danny R..

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Failure to reduce the number of cancer deaths over the last 50 years is due to the inability to selectively target metastatic disease. Recently, the KISS1 metastasis suppressor has emerged as a promising molecular agent for the management of metastatic disease. Although KISS1 has been implicated in the regulation of the metastatic phenotype in human cancers and in in vivo mouse models, little is known about its mechanism. Recent evidence suggests that KISS1 is a neuropeptide that is processed and secreted to interact with its cognate receptor GPR54 in the hypothalamus to trigger puberty and maintain the reproductive state. However, whether KISS1 secretion is required for metastasis suppression is not clear. The goal of this study was to test the hypothesis that KISS1 secretion is required for multiple organ metastasis suppression using the highly metastatic human melanoma cell line C8161.9. To detect secretion, KISS1 was engineered with an internal FLAG epitope in order to identify processed forms in the absence of a suitable antibody. Green fluorescent C8161.9 cells were stably transfected with pcDNA3-FLAG KISS1 (KFM) or pcDNA3-FLAG KISS1 lacking the putative secretion signal (KFM)SS). Secretion and processing of KISS1 proteins could only be detected in C8161.9KFM cells despite equal KISS1 protein expression in whole cell lysate for both constructs. Only C8161.9KFM cells suppressed the incidence and frequency of metastases in the lung, kidney, eye and bone by 56%-100% when compared to C8161.9vector or C8161.9KFM)SS cells. In the lung, C8161.9KFM cells remained dormant while C8161.9KFM)SS and C8161.9vector cells formed overt metastatic lesions. These results suggest that KISS1 secretion is necessary to prevent colonization at the secondary site. More importantly, the dormancy of C8161.9KFM cells was sustained providing a significant survival advantage in these mice when compared to mice receiving C8161.9vector controls. These data indicate that KISS1 secretion is required for multiple organ metastasis suppression and for the maintenance of metastatic cells in a dormant state. Soluble Kisspeptin (mimetics) could potentially be used to maintain tumor dormancy, rendering disseminated micrometastasis a legitimate treatment target.

xi, 221 p. : ill., digital, PDF file

Pathology

Joint Health Sciences

UNRESTRICTED

Advisors/Committee Members: Welch, Danny R., Chow, Louise T. <br>, Hardy, Robert W. <br>, Justement, Louis B. <br>, Marchase, Richard B. <br>, Murphy-Ullrich, Joanne <br>, Ruppert, J. Michael.

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Osteogenesis and angiogenesis are tightly coupled during bone formation and repair. Blood vessels not only carry oxygen and nutrients to the developing bone, but also play an active role in bone formation and remodeling by mediating the interaction between osteoblasts, osteocytes, osteoclasts and vascular cells at a variety of levels. Tissue hypoxia is believed to be a major stimulus for angiogenesis. Hypoxia activates the hypoxia- inducible factor (HIF) pathway and triggers hypoxia-responsive gene expression such as vascular endothelial growth factor (VEGF), which plays a critical role in angiogenesis, endochondral bone formation and bone repair following fracture. In this dissertation, I investigated the mechanisms through which osteoblasts use the HIF pathway to sense reduced oxygen tension and transmit signals that impinge on angiogenic and osteogenic gene programs during bone formation. Using a genetic approach, I have demonstrated that overexpression of HIF in mouse osteoblasts through disruption of the von Hippel-Hindau (pVHL)-mediated degradation pathway results in profound increases in angiogenesis and osteogenesis which appear to be mediated by cell nonautonomous mechanisms involving VEGF. Based on these results, the role of the HIF pathway during bone repair was also investigated by using a murine model of distraction osteogenesis in which repair is accomplished through osteoblast-mediated new bone formation. Mice with overexpressed HIF, through either deletion of Vhl gene or treatment of prolyl hydroxylase inhibitors, showed a markedly increased vascularity and accelerated bone regeneration in response to distraction osteogenesis. The increased bone regeneration in Vhl mutant mice was eliminated by concomitant infusion of VEGF receptor antibodies, suggesting that VEGF is required for bone regeneration. These findings suggest that the HIF/VEGF pathway also plays an important role in coupling angiogenesis and bone formation during repair. The precise mechanism for angiogenicosteogenic coupling during skeletal development, as to what extent VEGF functions independent of endothelial cells, is currently under investigation.

1 online resource (x, 110 p. : ill., digital, PDF file)

Pharmacology and Toxicology;

Joint Health Sciences;

bone angiogenesis VEGF HIF repair

UNRESTRICTED

Advisors/Committee Members: Barnes, Stephen, Cao, Xu<br>, Clemens, Thomas L.<br>, Frank, Stuart J.<br>, Serra, Rosa.

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Translation of the majority of eukaryotic mRNAs is initiated upon recognition of its 5′ cap structure by translation initiation factors in so-called cap-dependent translation. Capdependent translation is repressed during cell stress and translation initiation by internal ribosome entry sites (IRESs) predominates. IRESs, located in the 5′ untranslated region of specific mRNAs, recruit the ribosome independent of the 5′ cap and are essential regulatory elements in many RNA viruses and cellular messages. The dysregulation of cellular IRESs has been linked to cancer because they regulate the expression of genes involved in growth, development and stress response. In comparison to cap-dependent translation, the mechanism of IRES-mediated translation is poorly understood. The intergenic region (IGR) IRES from the cricket paralysis virus serves as a model IRES because it is the most simplified one known, however little is known about other IGR IRESs. We compared the activity of nine IGR IRESs in yeast and HeLa cells. Our analysis demonstrated that all IGR IRES are functional and that the strength of the IRES is influenced by the ribosome binding domain and the sequence downstream of the IRES. We also discovered that many viral and cellular IRESs use a common strategy for translation initiation which requires ribosomal protein S25 (Rps25). Deletion of Rps25 in yeast, or depletion in human cells, modestly affects global translation but causes a specific defect in IRES-mediated translation which leads to inefficient replication of IRES-containing viruses and lower protein expression from IRES-containing cellular mRNAs. Rps25 is also vital for prostate cancer (PCa) survival in the bone. Rps25 depletion in the highly aggressive ARCaPM PCa cell line resulted in a failure of these cells to thrive in vivo, most likely because these cells were less invasive and more susceptible to apoptosis in vitro. Because Rps25 depletion specifically affects IRES-mediated translation, these data strongly suggest that translation by cellular IRESs is essential for establishing prostate cancer in the bone. Taken together, this work revealed the widespread, conserved and vital role of Rps25 in IRES-mediated translation and suggests that Rps25 would be a good target for drug development for anti-viral and anti-cancer therapeutics.

1 online resource (x, 108 p. : ǂb ill., digital, PDF file)

Microbiology;

Joint Health Sciences;

ribosome IRES translation ribosomal protein S25 prostate cancer Hepatitis C virus

UNRESTRICTED

Advisors/Committee Members: Thompson, Sunnie, Bedwell, David<br>, Chen, Ching-Yi<br>, Lefkowitz, Elliot<br>, Morrow, Casey.

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The protein alpha-synuclein (α-SYN), which is found in the Lewy bodies of dopaminergic (DA) neurons in the substantia nigra (SN), has an important role in the pathogenesis of Parkinson's disease (PD). Fcγ receptors (FcγR) are proteins present on the surface of microglia, which bind immunoglobulin G (IgG) and other ligands. Our studies in an AAV-synuclein mouse model of PD showed that over-abundance of α-SYN triggered the expression of NF-κB p65, and led to microglial activation and DA neurodegeneration; however, in mice deficient of gamma chain subunit of the Fc receptors (FcγR^-/- mice), α-SYN-induced NF-κB signaling was blocked, while microglial activation and DA neurodegeneration were attenuated. In order to study whether α-SYN interacts directly with microglia, we treated the primary microglia of wild-type (WT) and FcγR^-/- mice with aggregated human α-SYN in vitro. We found that α-SYN was taken up by both WT and FcγR^-/- microglia though with different internalization patterns. α-SYN-induced nuclear accumulation of NF-κB p65 and downstream chemokine expression in WT microglia were blocked in FcγR^-/- ones. Moreover, we hypothesize that excess α-SYN causes further microgliosis through major histocompatibility complex-II (MHC-II) antigen presentation to T-lymphocytes, and this process is FcγR-dependent. To test this hypothesis, in vivo we selectively over-expressed human α-SYN in the SN of WT and FcγR^-/- mice, and in vitro, we performed T-cell co-culture with WT and FcγR^-/- microglia. Over-abundance of α-SYN in mouse SN triggered IgG deposition and vigorous MHC-II expression in the brain, and these immune responses were markedly attenuated in FcγR^-/- mice. α-SYN-treated microglia-T cell co-culture produced increased pro-inflammatory molecules and this elevated cytokine expression was blocked with FcγR^-/- microglia. Our results suggest that the humoral adaptive immune response triggered by excess α-SYN plays a causative role in microglial activation through IgG-FcγR interaction. This involves NF-κB signaling, and leads to DA neurodegeneration. To further activate microglia, α-SYN-induced microglial MHC-II antigen presentation to CD4+ T-lymphocytes is a crucial step, and FcγRs are indispensable in this process. Therefore, blocking either FcγR signaling or specific intracellular events downstream of FcγR-IgG interaction, such as NF-κB activation or MHC-II antigen presentation may be viable therapeutic strategies in PD.

PhD

1 online resource (x, 140 pages) :illustrations

Ph.D.University of Alabama at Birmingham2012.

Genetics

Nursing

Fc-gamma;R MHC-II microglia neurodegeneration neuroinflammation NF-κB

UNRESTRICTED

Advisors/Committee Members: David G. Standaert, Barnum, Scott R. Benveniste,Etty Kimberly, Robert P. West, Andrew B..

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Mycoplasma pulmonis is a murine respiratory pathogen and is used as a model for studying chronic mycoplasmal respiratory disease. This organism has been used by many in vivo and in vitro studies to gain a better understanding of host-pathogen interactions in chronic respiratory infection. Studies have shown that alveolar macrophages play an im-portant role in the removal of this pathogen from the host. Given that M. pulmonis is able to produce a chronic disease state, this organism should produce antiphagocytic factors that aid in avoidance of killing by alveolar macrophages. The Vsa proteins are involved in the avoidance of killing by complement and in modulating the ability of the mycoplasma to form a biofilm. These proteins contain an extensive tandem repeat region, a pattern seen in several other microbial proteins. The length of the tandem repeat unit varies from as few as 11 amino acids to as many as 19. The number of tandem repeats can be as high as 60 and varies at a high frequency due to slipped-strand mispairing events that occur during DNA replication. Capsular polysac-charides are involved in host immune avoidance in several other bacterial species. The EPS-I polysaccharide is produced on the surface of M. pulmonis and is made up of the monosaccharides glucose and galactose in a 1:1 ratio. Few mycoplasma polysaccharides have been described and little is known about their role in pathogenesis. We hypothesize that the Vsa proteins and the surface polysaccharide EPS-I would function as antiphago-cytic factors. We show that cells producing a Vsa protein with many tandem repeats are rela-tively resistant to killing by alveolar macrophages. We also show that mutants lacking the EPS-I polysaccharide are more susceptible to binding and subsequent killing by macro-phages. These results may be pertinent to understanding the function of proteins similar to Vsa in other microbes and suggest that polysaccharides produced by other species of mycoplasma are involved in immune avoidance. The results give further insight into how mycoplasmas are able to avoid the host immune system and sustain a chronic infection.

1 online resource (xii, 81 p.) :ill., digital, PDF file.

Genetics

Joint Health Sciences

Vsa, capsule, alveolar macrophages, mycoplasma, M. pulmonis, antiphagocytic

UNRESTRICTED

Advisors/Committee Members: Kevin Dybvig, Additional advisors: David E. Briles, Daniel Bullard, John F. Kearney, Warren Simmons..

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There is a growing body of evidence linking preeclampsia to future development of cardiovascular disease (CVD). Although CVD is well-known as the leading cause of death in women, a lack of evidence exists demonstrating that women with preeclampsia are routinely informed of their risks for future CVD. The specific aims of this study were to: (1) examine the levels of CVD knowledge and perception of CVD risk pre-CVD education; (2) explore relationships among age, race, parity, marital status, previous preeclampsia, income, education, CVD knowledge, and perception of CVD risk; and (3) examine the effect of a postpartum CVD educational intervention on personal perception of CVD risk in women with new-onset preeclampsia.Using telephone-based interviews, baseline levels of CVD knowledge and CVD risk perception were examined among 64 women with recent, new-onset preeclampsia. Relationships among CVD knowledge, CVD risk perception, and several covariates (age, race, parity, marital status, previous preeclampsia, income, and education) were also examined. The intervention of CVD education was provided after baseline data were collected; CVD risk perception was reexamined post-CVD education. At baseline, CVD knowledge was found to be a significant predictor of CVD risk perception, accounting for 8.4% of explained variance (p = 0.011). Although none of the covariates significantly influenced CVD risk perception, the factors of age and income significantly influenced CVD knowledge (R2 = 0.226; p =.001). After CVD education, levels of CVD risk perception were significantly higher than at baseline (paired t = - 2.3; p = 0.003).Accurate perceptions of CVD risk have been associated with demonstrable behaviors suggestive of risk reduction. As an intervention, CVD education, provided by telephone, served as a practical and effective approach to reaching women with recent, new-onset preeclampsia. Results of the CVD risk perception scale from baseline to post-CVD education demonstrated that CVD education significantly increased perceptions of personal CVD risk among women with recent, new-onset preeclampsia.

PhD

1 online resource (x, 116 p.) :ill., digital, PDF file.

Nursing

Nursing

Cardiovascular Disease in Women Preeclampsia

UNRESTRICTED

Advisors/Committee Members: Erica R. Pryor, Hodges, Ashley L. Martin, Jr., James N. Moneyham, Linda D. White-Williams,Connie.

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We have examined effects of hypoxia signaling on epithelial gene expression, focusing on the cystic fibrosis transmembrane conductance regulator (CFTR), the loss of which leads to destruction of exocrine tissues, including those of the GI tract and lung. With the use of environmental hypoxia and pharmacologic induction of hypoxic signaling, we conducted microarray studies to evaluate global changes in gene expression and quantitative real-time reverse transcription-PCR to specifically measure the repression of CFTR. We found CFTR mRNA under these conditions to be downregulated 10 to 100 fold (p<0.01). Immunoprecipitation, subcellular localization, and transepithelial chloride transport studies indicated that repression of CFTR mRNA is accompanied by substantial decreases in protein and activity. Studies in mice exposed to chronic hypoxia in vivo, as well as in lung tissue from humans with chronic lung disease, supported the in vitro results. These findings are relevant to progression of cystic fibrosis (CF) caused by partial function mutant CFTR alleles and also to pathogenesis of common non-CF lung diseases in man. To further elucidate the mechanisms by which CFTR mRNA is affected by hypoxia, we hypothesized that CFTR transcription may be affected indirectly by hypoxic signaling and that many cellular pathways may contribute to this process. We chose to perform global analyses of microRNA (miRNA) expression in human epithelia and correlated those findings with mRNA expression array studies under identical conditions. We found that although the specific candidate miRNAs predicted did not appear to participate in CFTR repression, there are a substantial number of miRNAs that are differentially expressed in hypoxia. The hypoxic miRNA profile was evaluated for strength of correlation with levels of potential mRNA targets on the basis of the number of miRNA binding sites. Combinatorial regulation was evaluated by examining the coregulation of groups of miRNAs. We found that although a small number of the miRNAs had an inverse correlation with their targets, the majority did not have such a correlation; this finding lends credence to the hypothesis that translational repression (as opposed to effects on mRNA transcription or stability) may be a key mechanism of action of miRNAs in mammalian cells. These findings have important implications for the study of miRNA regulation of gene expression.

xi, 110 p. : ill., digital, PDF file

Genetics

Joint Health Sciences

UNRESTRICTED

Advisors/Committee Members: Sorscher, Eric J., Clancy, John P.<br>Engler, Jeffrey A.<br>Schoeb, Trenton R.<br>Schroeder, Harry W..

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Neuroinflammation and Fragile X syndrome (FXS) are two particularly devastating neurologic conditions for which no adequate treatment exists and much is still unknown about the underlying cellular and molecular processes. Neuroinflammation contributes to the pathogenesis of many neurologic and psychiatric disorders, thus treatment of the underlying inflammation has broad implications. FXS is the most common cause of inherited intellectual and developmental delay and one of the few known genetic causes of autism. Neuroinflammation and FXS have potential links with glycogen synthase kinase-3 (GSK3) and its inhibitor, lithium. Therefore, the overall goal of this research was to examine the role of GSK3 in neuroinflammation and FXS, as well as the potential therapeutic value of GSK3 inhibitors in these neurologic conditions. Most neuroinflammatory processes result in activation of microglia, the resident immune cells of the brain. Microglial responses include migration to an injury site and the release of pro- and anti-inflammatory factors, which ultimately can affect neuronal survival. Treatment with GSK3 inhibitors attenuated several microglial responses in vitro and in situ after lipopolysaccharide (LPS)-induced activation of microglia, including migration, activation, and production of pro-inflammatory molecules. GSK3 inhibitors also provided protection from inflammation-induced neuronal toxicity. Thus, the utilization of GSK3 inhibitors provides a means to limit the inflammatory actions of microglia and provides neuroprotection after an inflammatory insult. FXS results from an expansion-mutation upstream of the fragile X mental retardation 1 (Fmr1) gene causing loss of the gene product. Together with previous results, it was discovered that impaired inhibitory serine-phosphorylation of GSK3 is a robust phenotype in the brains of Fmr1 knockout mice and two potential therapeutics for FXS, a metabotropic glutamate receptor antagonist and lithium, converge to inhibit GSK3. Chronic lithium treatment reversed the hyperactive GSK3 in the brains of Fmr1 knockout mice and improved many of the tested FXS-associated behaviors. Therefore, impaired inhibitory regulation of GSK3 plays a prominent role in the pathogenesis of FXS and supports GSK3 as a potential therapeutic target. Overall, this work provides novel insight regarding the function of GSK3 in two pathologic processes in the central nervous system, and further supports GSK3 inhibitors as viable therapeutics for both neuroinflammation and FXS.

vii, 141 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

Neuroinflammation Microglia Glycogen Synthase Kinase 3 Lithium Neurodevelopmental Disorders Fragile X Syndrome

UNRESTRICTED

Advisors/Committee Members: Jope, Richard S., Barnum, Scott R.<br>, Cowell, Rita M.<br>, Michalek, Suzanne M.<br>, Sontheimer, Harald W..

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During nervous system development, neurons proliferate, differentiate, project their axons to their targets and make synapses with them. At the neuromuscular junction of Drosophila, these functional synapses enlarge as the body grows to maintain physiologically effective synaptic transmission with the muscles. Drosophila Bone Morphogenetic Protein (BMP) type II receptor, Wishful thinking (Wit), is important in this proper synaptic growth and function. To identify transcriptional targets of Wit signaling, we performed microarray experiments comparing normal controls to wit mutants. Through this study, we found that some of the verified target genes showed isoform-specific regulation in wit mutants, and that several neuropeptide hormones were down-regulated in wit mutants. One of the most promising targets is the novel gene CG9335 that shows 2.35 fold decrease in microarrays and about 3 fold decrease by Quantitative Real time PCR (QRT-PCR) in mutants. In situ hybridization results show that CG9335 is expressed in the optic lobe proliferating center, ventral ganglion of larval CNS and eye imaginal discs. CG9335 expression is specifically eliminated in the ventral ganglion of wit mutants. Molecular characterization of CG9335 shows that it is a Ly-6 domain-containing, GPI-anchored cell surface protein. Co-expression studies by in situ hybridization strongly suggest that CG9335 is a motoneuron expressed gene. Functional characterization using a null allele revealed that CG9335 mutants have defects in spontaneous synaptic vesicle release. The significantly decreased frequency of spontaneous vesicle release of CG9335 mutants can be rescued by neuronal expression of a CG9335 transgene. Wit mutants also show a decreased frequency of spontaneous synaptic vesicle release, and the phenoptype can be partially rescued by presynaptic expression of CG9335. Through our study, we proved our tested hypothesis; the canonical BMP signaling pathway regulates downstream target genes that are important for synaptic development and function. Further validation and study of other targets identified in our microarray study will provide much more valuable information about how BMP signaling orchestrates synapse formation and function.

1 online resource (x, 137 p. : ill., digital, PDF file)

Cell Biology;

Joint Health Sciences;

BMP Motoneuron Drosophilia Synapse Neuromuscular Junction mEJP

UNRESTRICTED

Advisors/Committee Members: Marques, Guillermo, Woods, Anne C.<br>, Chang, Chenbei<br>, Miller, Michael A.<br>, Wilson, Scott M..

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Development of the vertebrate eye is controlled by a network of regulatory genes including those in the basic loop-helix-loop (bHLH) family. In this study, we explored the function of a proneural bHLH gene, achaete-scute homologue 1 (ash1) in the chick eye. The Replication Competent Avian Splice (RCAS) retrovirus was used to overexpress ash1 in the developing chick eye to study its role in ocular morphogenesis. Overexpres-sion of ash1 was embryonically lethal and led to gross abnormalities in the embryo. In the retina, overexpressing ash1 resulted in overproduced amacrine cells at the expense of bipolar and Müller glial cells. A reduction of photoreceptors, probably secondary to Müller glial cell deficiency, was also observed. These alterations were accompanied with impaired cell proliferation and enhanced apoptosis in the retina. By studying truncated ASH1 proteins, we found the N-terminus may contain a functionally important domain. Ash1 was also misexpressed in cultured chick embryonic retinal pigment epithelial (RPE) cells. It reprogrammed RPE cells into cells morphologically resembling neurons that form clusters. These cells expressed retinal neural markers de novo. Calcium imaging demonstrated that the reprogrammed RPE had physiological responses to glutamate or N-methyl-D-aspartic acid (NMDA) similar to that of dissociated retinal neurons. Unexpect-edly, ash1 misexpression also resulted in multiple developmental defects similar to ante-rior segment dysgenesis (ASD). Transmission electron microscopy (TEM) showed disor-ganized collagen fibrils associated with excessive, broad, and heavily stained sulfated proteoglycans (PGs) in the corneal stroma. The epithelial and endothelial layers remained intact, and corneal transparency related genes were not significantly decreased. Ash1 misexpression did not upregulate six3. Our data implied important functions of ash1 in the developing chick eye.

xiii, 131 p. : ill., digital, PDF file

Vision Science

Optometry

BHLH Gene Ash1 Development Retina RPE Anterior Segment Dysgenesis

UNRESTRICTED

Advisors/Committee Members: Wang, Shu-Zhen, Guidry, Clyde<br>, Keyser, Kent T.<br>, Mayne, Richard<br>, Pittler, Steven J..

▼ The <i>C. elegans</i> genome has been completely sequenced, and the developmental anatomy of this model organism is described at single-cell resolution. Here we utilize strategies that exploit this precisely defined architecture to link gene expression to cell type. We obtained RNAs from specific cells and from each developmental stage using tissue-specific promoters to mark cells for isolation by FACS or for mRNA extraction by the mRNA-tagging method. We then generated gene expression profiles of more than 30 different cells and developmental stages using tiling arrays. Machine-learning–based analysis detected transcripts corresponding to established gene models and revealed novel transcriptionally active regions (TARs) in noncoding domains that comprise at least 10% of the total <i>C. elegans</i> genome. Our results show that about 75% of transcripts with detectable expression are differentially expressed among developmental stages and across cell types. Additionally, we used self-organizing maps to define groups of co-regulated transcripts and applied regulatory element analysis to identify known transcription factor– and miRNA-binding sites, as well as novel motifs that likely function to control subsets of these genes. By using cell-specific, whole-genome profiling strategies, we have detected a large number of novel transcripts and produced high-resolution gene expression maps that provide a basis for establishing the roles of individual genes in cellular differentiation. In a second project, I have identified an immunoglobulin-domain containing cell adhesion molecule that promotes synaptic-connectivity between the AVA command interneuron and A-class motor neurons in the <i>C. elegans</i> motor circuit. Animals carrying a mutation in <i>rig-3</i> show moderate backward locomotion defects. Additionally, <i>rig-3</i> mutants show minor AVA axon guidance defects and most synapses between AVA and A-class motor neurons are lost. These data suggest <i>rig-3</i> plays a critical role in synapse formation. It will be interesting to determine the involvement of <i>rig-3</i> function in connectivity between other neurons in the motor circuit and the entire nervous system. Advisors/Committee Members: James Goldenring (committee member), Randy Blakely (committee member), Ethan Lee (committee member), David Miller (committee member), Guoqiang Gu (Committee Chair).