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The hypothesis that cell volume and the progression of the cell cycle are interdependent has surfaced off and on in the cell cycle literature for the past 30 years. However, a conclusion as to how cell volume is mechanistically involved in cell division has not been reached in mammalian cells. The aim of this dissertation was to establish how volume changes modulate cell cycle progression. Most of the studies addressing this question have examined mass content yet, more recently, focus has been placed on intracellular water, which is determined by the balance between mechanical and osmotic forces. As a result, ion channels and transporters which regulate intracellular osmotic content are integral to the maintenance of cell volume. In this dissertation, I show that a large, rapid and regulated volume decrease occurs as glioma cells progress through mitosis. I refer to this process as pre-mitotic condensation (PMC). This process is functionally linked to DNA condensation prior to cell division as the two events occur simultaneously, and inhibition of PMC results in a prolongation of DNA condensation. Further, my data demonstrates that glioma cells actively accumulate chloride, which acts as the primary energetic driving for cell volume changes in these cells. During the process of PMC, this gradient drives the efflux of chloride through ClC3 channels, which mediates water loss and the volume decrease. Interestingly, chloride accumulation to similar levels can be observed in immature astrocytes and neurons, suggesting that glioma cells recapitulate the biology of immature proliferating cells in the brain. This also suggests that my findings may have broader applicability to cell division in both neural cells and cancer.

xii, 132 p. : ill., digital, PDF file.

Neurobiology

Joint Health Sciences

Voltage Gated Chloride Channels Pre-miotic Condensation DND Condensation C1C3 Mitosis Volume Regulation

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Advisors/Committee Members: Sontheimer, Harald, Bevensee, Mark<br>Engler, Jeffrey<br>Pozzo-Miller, Lucas<br>Theibert, Anne.

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p63 is a member of the p53 family of transcription factors that is a critical regulator of epithelial development. Studies have shown that p63 does not appear to function as a classical tumor suppressor like p53. Instead, the expression pattern of p63 in cancers suggests that p63 retains the potential to function as either a tumor suppressor or an onco-protein. Here, we provide evidence describing the transcriptional activity of the p63 isoforms and provide mechanisms whereby p63 function is regulated in a context dependent manner. Our data shows that both the [delta]Np63 and TAp63 variants retain the potential to regulate transcription and inhibit proliferation. We suggest that the intrinsic transactivation potential of p63 isoforms is established at the level of transcription. Promoter selection determines which N-terminal activation domain is encoded in the p63 protein. For example, we propose that transactivation by [delta]Np63 is more dependent upon co-activator binding to the C-terminal proline-rich region than the TAp63 variant that possesses the strong, acidic activation domain. In addition, alternative splicing regulates expression of either the [alpha], [beta], or [gamma] C-termini. The [alpha] C-terminus has been previously described as inhibitory. Here we demonstrate that expression of the PPXY motif in the [alpha] and [beta], but not [gamma], termini affords additional transactivation potential to these isoforms. Finally, we show that p63 transactivation potential is dependent upon PXXP and PPXY motifs suggesting that signaling pathways can modulate p63 function through posttranslational modifications or protein-protein interactions occurring at these proline-rich motifs.

vii, 72 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

p63 p53 Family Tumor Suppression Transcription Factor Apoptosis

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Advisors/Committee Members: Chen, Xinbin, Chang, Chenbei <br>, Crawford, David F.<br>, Ruppert, J. Michael <br>, Serra, Rosa A..

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The G protein-coupled receptor, G2A, is expressed by multiple cell-types involved in atherosclerosis and is activated by structurally related lysophospholipids generated during low-density lipoprotein (LDL) oxidation and cholesterol esterification on circulating high-density lipoprotein (HDL) particles. In vitro studies have demonstrated that G2A mediates multiple biological responses relevant to atherosclerosis, including macrophage chemotaxis and apoptosis. To determine the role of these G2Amediated effects in atherosclerosis, we generated hypercholesterolemic G2A deficient (G2A-/-) and G2A sufficient (G2A+/+) LDL receptor knockout (LDLR-/-) mice, a commonly used hypercholesterolemic model of atherogenesis. Although G2A deficiency resulted in slight reductions in lesional macrophage apoptosis at the aortic root without affecting lesion size during early stages of atherogenesis, the major impact of G2A deletion throughout the entire aorta at early and later stages of atherogenesis was a robust reduction in lesion size. Although consistent with the loss of G2A chemotactic function, suppression of atherosclerosis in G2A-/-LDLR-/- mice was associated with increased circulating numbers of apolipoprotein E (apoE)-containing HDL particles. The ability of G2A inactivation to raise HDL in hypercholesterolemic LDLR-/- mice was dependent on apoE expression. In addition, G2A deletion failed to raise HDL levels in apoE knockout (apoE-/-) mice and consequently did not suppress atherosclerosis, irrespective of gender or the type of diet intervention. Hepatocytes, the primary cell-type responsible for HDL biogenesis, were found to express G2A and hepatocytes from hypercholesterolemic G2A-/-LDLR-/- mice secreted significantly more apoE-containing HDL particles compared to those from their G2A+/+LDLR-/- counterparts. This occurred in the absence of any significant alterations in the expression of genes encoding apoE and other key regulators of HDL metabolism. Collectively, these studies demonstrate that G2Amediated chemotaxis does not play a role in vivo during atherogenesis and establish a novel apoE-dependent mechanism mediated by G2A to control HDL biogenesis that is required for the pro-atherogenic action of G2A.

1 online resource (xv, 132 p. : ill., digital, PDF file)

Microbiology

Joint Health Sciences

Atherosclerosis G2A Lysophosphatidylcholine

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Advisors/Committee Members: Kabarowski, Janusz H. S., Barnum, Scott R.<br>, Kearney, John F.<br>, Li, Ling<br>, Murphy-Ullrich, Joanne E..

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Though human papillomavirus infection of the human epidermis is epidemiologically widespread and typically benign, manipulation of the cell cycle within host tissues during infections can predispose the host to malignant neoplastic disease. The frequency of neoplastic progression associated with infection by a particular HPV type is directly related to its risk class; certain virus types are highly prevalent in neoplasia of HPV etiology, whereas other virus types are rarely associated with neoplastic malignancies. Regardless, the gene expression programs of both high-risk and low-risk HPV are sufficient for the production and transmission of viral progeny. In this thesis work we examine the molecular function the E7 protein of both low-risk and the high-risk HPV E7 in the differentiated squamous epithelium. The E7 protein has direct implications in both cell-cycle dependent replication of the viral genome and deregulation of cellular proliferation associated with cancers through inactivation of E2F co-repressor complexes. By comparative and mutational analysis of both oncogenic and non-oncogenic E7 orthologs, we demonstrated the ubiquitous ability of the E7 gene to promote S-phase reentry in the suprabasal, differentiated strata of organotypic raft cultures. Unique molecular properties of the evaluated E7 orthologs provided further insight into the mode of cell-cycle regulation within the stratified epithelium and furthermore, how the function of the E7 protein is regulated. We demonstrated that E7 dependent destabilization of the retinoblastoma family E2F-corepressor p130 in the stratified epithelium is concomitant with S-phase reentry prerequisite for viral DNA amplification. This ability is shared by both oncogenic and non-oncogenic E7 types capable of inducing unscheduled DNA replication. Furthermore, we showed that this function is greatly enhanced by phosphorylation at the casein kinase II motif, which promotes E7 binding and destabilization of pocket protein repressors. In seminal works that both defined the high-risk E7 protein as an oncogene and characterized nonmutagenic, somatic inactivation of the retinoblastoma protein tumor suppressor, the pRB interaction was largely adopted as the biologically pertinent interaction of E7. These results provide a novel perspective on the archetypal role of E7 in cell-cycle regulation and establish precedence for alternative mechanisms of E7 function during the viral life cycle.

1 online resource (xvii, 221 p.) : ill., digital, PDF file.

Biochemistry and Molecular Genetics

Joint Health Sciences

HPV E7 p130 epithelium keratinocyte pRB

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Advisors/Committee Members: Chow, Louise T., Broker, Thomas R.<br>, DeLucas, Lawrence J.<br>, Engler, Jeffrey A.<br>, Lin, Weei-Chin<br>, Wang, Hengbin.

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Members of the [beta]2-integrin family of adhesion molecules, CD11a, CD11b, and CD11c, have all been shown to play a role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). CD11d had yet to be studied in demyelinating disease and its functions remained unclear. We report here that CD11d is the only member of the [beta]2-integrin family of adhesion molecules that fails to protect against the development of EAE. Surprisingly, the EAE studies suggested that CD11a, CD11b, and CD11c were all contributing to T cell activity during disease development by mechanisms beyond the migration of these cells into the CNS. However, the contributions of individual T cell subsets to the overall phenotypes seen were unclear. Earlier studies show that over the course of EAE a higher proportion of [gamma delta] T cells express the [beta]2-integrins when compared to [alpha beta] T cells. Given this we hypothesized that the [beta]2-integrin family was important to the functions of [gamma delta] T cells that contributed to the development of EAE. However, we show here that even though expression is enriched in this T cell subset the [beta]2-integrins do not seem to be required on [gamma delta] T cells for disease development. The [beta]2-integrin family has also been implicated in regulatory T cell function and homeostasis. Studies using transfer EAE with CD11a-/- mice have suggested these mice may have regulatory defects. Given this we next investigated the role CD11a plays in regulatory T cell biology. We show here that CD11a-/- mice have reduced Treg populations throughout the secondary lymph tissue and that this reduction may be due to a reduced capacity to generate peripheral Tregs. We also found that CD11a is critical to Treg function in vitro, but does not seem to be as important in vivo. Importantly CD11a appears to be mediating its immunosuppressive effects independently of interactions with ICAM-1 on target T cells. Overall, the studies presented here provide further evidence that the ,2-integrin family of adhesion molecules functions in many aspects of T cell biology aside from cellular migration and that these functions differ between T cell subsets.

xi, 100 p. : ill., digital, PDF file

Microbiology

Joint Health Sciences

Integrin EAE Gamma Delta T Cell Regulatory T Cell

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Advisors/Committee Members: Barnum, Scott R., Bullard, Dan C.<br>Raman, Chander<br>Burrows, Peter<br>Justement, Louis.

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Our laboratory reported that a combination of ionizing radiation (IR) or temozolomide (Tmz), a DNA methylating agent clinically approved against glioblastoma multiforme (GBM), a type of human brain cancer, and TRA-8, an anti-DR5 monoclonal antibody produced synergistic cytotoxicity in GBM cells in culture and in mouse xenograft models through an yet unknown mechanism. I hypothesized that understanding of the synergy phenomenon can offer an insight into the nature of GBM vulnerability. The goals of this investigation were two-fold – to elucidate the mechanism of this enhancement in cytotoxicity, and to improve understanding of DR5 mediated apoptosis toward improvement of current anti-GBM therapies. In GBM cells in culture, enhancement of apoptosis induced by TRA-8 was contingent upon pre-treatment with Tmz. Tmz induced cell cycle arrest and senescence, but not apoptosis, at therapeutically relevant doses. Thus, the requirement for pretreatment implicated genotoxic stress response and cell cycle regulation in the mechanism of sensitization. These were investigated along with the key events of apoptosis. As a result, a segment of the apoptotic cascade downstream of mitochondrial depolarization was eliminated as extraneous to the mechanism of enhancement. Additionally, enhancement of apoptosis induced by genotoxic stress appears to be a general phenomenon independent of the mode/source of genotoxicity. Since Tmz pre-treatment is required for enhancement, I investigated global changes in the cellular proteome induced by Tmz using 2-dimensional difference gel electrophoresis (2D DIGE). This approach detected a change in abundance of Apoptosis Inducing Mitochondria Associated Factor (AIF), among other proteins. The necessity of AIF for enhancement was examined with a knockdown experiment. Finally, I developed a mechanistic model of enhancement based on these results. The pro-apoptotic signals generated by DNA damage are transmitted through the Bcl-2 family of proteins. However, they cannot induce apoptosis because genetic alterations in GBM make it highly resistant. This creates a cumulative conditioning effect that “primes” cells for apoptosis. The subsequent treatment with TRA-8 induces apoptosis to a greater extent due to this “priming”. Therefore, I propose that the control of mitochondrial permeability by the Bcl-2 family of proteins is the principal mechanistic event causing the enhancement.

1 online resource (xix, 139 p.) : ill., digital, PDF file.

Biochemistry and Molecular Genetics;

Joint Health Sciences;

glioblastoma TRAIL cell cycle temozolomide antibody therapy apoptosis inducing factor 1

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Advisors/Committee Members: Buchsbaum, Donald J., Barnes, Stephen<br>, Detloff, Peter J.<br>, Elgavish, Gabriel A.<br>, Gillespie, G. Yancey.

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lzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by two major pathophysiological hallmarks, beta-amyloid (A[Beta]) plaques and tau tangles. In AD and other tau associated neurodegenerative disorders, termed tauopathies, a critical role in promoting neuronal degeneration has been demonstrated for hyperphosphorylated forms of tau. Recent findings suggest that cleavage of tau in the carboxyl-terminal region (Asp421) may also promote deleterious effects of tau on neuronal health. In the first half of my study, the relationship between Asp421 cleaved tau and a common AD associated stressor (endoplasmic reticulum [ER] stress) was investigated using an inducible cortical neuronal model. Cells expressing Asp421 tau presented with higher levels of toxicity as compared to cells expressing the full-length protein, even before induction of ER stress. Under ER stress conditions, toxicity was significantly enhanced in Asp421 tau expressing cells. The differences in toxicity were underscored by alterations in caspase activation and cytokine signaling. In the second study, the effect of Asp421 tau expression on mitochondrial function was examined. Mitochondria in cells expressing Asp421 tau showed a shortened and rounded morphology, suggesting fragmentation. Additionally, mitochondrial function was severely inhibited in these cells as demonstrated by mitochondrial membrane potential loss and decreased calcium buffering ability in response to elevated intracellular calcium levels. Further, mitochondria in Asp421 tau cells exhibited increased levels of reactive oxygen species (ROS). Taken together the results suggest that Asp421 tau may negatively impact mitochondrial functioning in affected neurons in AD and other tauopathies, and promote disease progression. Given the importance of proper mitochondrial functioning in maintaining neuronal health, further studies are warranted in order to fully elucidate the mechanisms underlying this toxicity.

1 online resource (x, 105 p. : ill., digital, PDF file)

Cell Biology;

Joint Health Sciences;

tau caspases Alzheimer's disease

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Advisors/Committee Members: Johnson, Gail V. W., Jope, Richard S.<br>, Lesort, Mathieu J.<br>, Sztul, Elizabeth S.<br>, Yoder, Bradley K..

▼  The coordination between cell growth and division is a highly regulated process that is intimately linked to the cell cycle. Efforts to identify an independent mechanism that measures cell size have been unsuccessful. Instead, we propose that size control is an intrinsic function of the basic cell cycle machinery. My work shows that in the fission yeast Schizosaccharomyces pombe Cdc25 accumulates in a size dependent manner. This accumulation of Cdc25 occurs over a large range of cell sizes. Additionally, experiments with short pulses of cycloheximide have shown that Cdc25 is an inherently unstable protein that quickly returns to a size dependent equilibrium in the cell suggesting that Cdc25 concentration is dependent on size and not time. Thus, Cdc25 can act as a sizer for the cell. However, cells are still viable when Cdc25 is constitutively expressed suggesting that there is another sizer in the case that Cdc25 expression is compromised. Cdc13 is a likely candidate due to the similar characteristics to Cdc25 and the ability to activate Cdc2. Cdc13 accumulates during the cell cycle in a manner similar to Cdc25. I show that in the absence of Cdc2 tyrosine phosphorylation, the cell size is sensitive to Cdc13 activity showing that Cdc13 accumulation can determine when cells enter mitosis. These results suggest a two sizer model where Cdc25 is the main sizer with Cdc13 acting as a backup sizer in the event of Cdc25 expression is compromised. Additionally, in the absence of Cdc2 phosphorylation by the kinases Wee1 and Mik1, mitotic entry is regulated by the activity of Cdc2. In the absence of Cdc2 phosphorylation, this activity is regulated by binding of cyclins to Cdc2. Under these circumstances, the activity of Cdc13 can regulate mitotic entry provide further evidence that Cdc13 could be a sizer of the cell in the case where Cdc25 expression is compromised. The results I present in this dissertation provide the groundwork for understanding how cells regulate size and how this size regulation affects cell cycle control in S. pombe . The results show how the intrinsic cell cycle machinery can act as a sizer for the G2/M transition in S. pombe . Interestingly, this mitotic commitment pathway is well conserved suggesting a general solution for size control in eukaryotes at the G2/M transition. Understanding the mechanism of how protein concentration is regulated in a size dependent manner will give much needed insight into how cells control size. Elucidating the mechanism for size control will capitalize on decades of research and deepen our understanding of basic cell biology. Advisors/Committee Members: Nick Rhind, PhD.

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The connecting cilium serves as the major route for protein transport from the inner to outer segment of photoreceptor cells. The hypothesis that all Congenital Stationary Night Blindness (CSNB) rhodopsin mutants travel properly to the rod outer segment in Xenopus laevis will be tested by preparing transgenic X. laevis expressing a rhodopsin mutation found in human CSNB, A295V. In addition, probing for the existence of potential proteins involved in regulating rhodopsin trafficking and monitoring their localization will contribute to a better understanding of the underlying pathway of intracellular photoreceptor trafficking.

M.S.

1 online resource (viii, 36 p.) : ill., digital, PDF file.

Vision Science

Optometry

Rhodopsin Mutation Congenital Stationary Night Blindness TULP1 Septin-2 Localization in Retina Protein Trafficking

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Advisors/Committee Members: Gross, Alecia K., Kraft, Timothy<br>, Semes, Leo.

▼ The polysaccharide capsules of Streptococcus pneumoniae represent the most important virulence determinant produced by this organism. Ninety-one different serotypes have been identified, but only a small number of these serotypes are responsible for most of the infections caused by S. pneumoniae. The reasons why certain serotypes predominate in infections over others are not well understood. One common serotype found in invasive diseases is type 2, however, it is unclear what characteristics of a type 2 capsule makes strains producing it virulent. The type 2 capsule consists of repeat units made of a backbone of Glc-Rha-Rha-Rha, with a side chain Glc-GlcUA linked to the terminal Rha. In type 2 S. pneumoniae, neither the role the capsular side chain Glc-UA has in capsule assembly nor the role it has in virulence is known. UDP-GlcUA is synthesized from the oxidation of UDP-Glc by the activity of cps2K, a type-specific gene encoded within the type 2 capsule locus containing homology to known UDP-Glc dehydrogenses. Deletion of cps2K should eliminate the production of UDP-GlcUA, allowing us to assess the effects of alteration of the side chain on both capsule synthesis and virulence. Deletion of cps2K resulted in the production of only low levels of high molecular weight polymer that were retained on the cell membrane but not transferred to the cell wall, demonstrating a requirement for GlcUA in this process. Repair of this mutation only partially restored capsule production, suggestive of secondary mutations selected for during isolation of [Delta]cps2K mutants. Linkage analyses were used to localize the suppressor mutations to a region containing cps2E, the glycosytransferase responsible for the addition of Glc-1-P to a C55 polyprenol acceptor (Und-PP) to initiate repeat unit synthesis. Sequence analyses revealed that the original [Delta]cps2K mutants contained different point mutations in cps2E, all reducing Cps2E activity. Construction of multiple independent [Delta]cps2K mutants resulted in the same phenotype as the original and a selection for suppressor mutations, the majority of which were located in cps2E. Mutants that were reduced in capsule production due to either mutations in cps2E or the capsule promoter region, resulting in 0.1% and 60% of the parental levels of capsule, respectively, were unable to efficiently colonize or cause disease in murine models of pnuemococcal infection, demonstrating a requirement for full capsule production by type 2 for these processes. Deletion of cps2H, the repeat-unit polymerase, and cps2J, the repeat-unit flippase, also selected for suppressor mutations in cps2E. Collectively, the data presented here suggests a selection for suppressor mutations may have resulted from the sequestration of undecaprenyl-pyrophosphate due to retention of a repeat-unit on the cytoplasmic side of the cell membrane or an inability to transfer a single repeat-unit or altered polymer to the cell wall. Suppressor mutations which reduced capsule synthesis, such as those in cps2E, would help alleviate… Advisors/Committee Members: Yother, Janet L., Benjamin, William H.<br>, Niederweis, Michaerl E.<br>, Pritchard, David G.<br>, Steyn, Andries J..

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Many different tumors have been documented to have elevated levels of the enzyme ST6Gal I, a Golgi glycosyltransferase that adds [alpha]2-6 sialic acids to glycoproteins. Concurrently, upregulated ST6Gal I is associated with metastasis and poor patient prognosis. We initially showed that HD3 colonocytes made to express oncogenic-ras, a common mutation in colon carcinoma, have increased expression of ST6Gal I and elevated [alpha]2-6 sialylated integrins. Having established that ras regulates ST6Gal I expression in colon epithelial cells, we found that colonocytes carrying hyper [alpha]2-6 sialylated [beta]1 integrins exhibit increased adhesion to and migration toward collagen I. Further, we found that [beta]1 integrins in human colon adenocarcinoma samples consistently carry higher levels of [alpha]2-6 sialic acids. These results led us to hypothesize that hypersialylated [beta]1 integrins may have a role in augmenting tumor progression by affecting cell adhesion and motility. To determine whether the effects of oncogenic ras on cell behavior were mediated via upregulated ST6Gal I and consequent hypersialylation of [beta]1 integrins, we individually manipulated ras and ST6Gal I levels in HD3 colonocytes. These cells were engineered to express oncogenic ras with high and low ST6Gal I levels. Initially we confirmed that shRNA mediated dowregulation of ST6Gal I in HD3 cells having oncogenic-ras intact leads to decreased [alpha]2-6 sialylation of [beta]1 integrins. We found that cells with diminished integrin sialylation show decreased in vitro invasiveness and impaired binding to and migration toward collagen I, as compared with oncogenic-ras expressing HD3 cells that have high ST6Gal I. We also found that ST6Gal I can protect against apoptosis. Specifically, while downregulating ST6Gal I in HD3 cells restored the sensitivity to apoptotic stimuli, forced ST6Gal I expression in SW48 cells actually protected cells from entering the apoptotic cascade. This finding, to our knowledge, represents a novel function attributable to ST6Gal I. Taken together, results presented in this dissertation establish that altered sialylation of surface glycoprotein receptors in general, and [beta]1 integrins in particular, plays a direct role in regulating tumor cell behavior.

x, 103 p. : ill., digital, PDF file

Physiology and Biophysics

Joint Health Sciences

Metastasis Integrin Sialylation ST6Gal-I colon Apoptosis

UNRESTRICTED

Advisors/Committee Members: Welch, Dan.

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Neural cell death plays a critical role in normal nervous system development and dysregulated neural stem cell death contributes to brain malformation, tumorgenesis, and possibly, neurodegenerative disease. The two major forms of cell death in the nervous system are apoptotic and autophagic. Altered clearance of proteins may lead to neuronal dysfunction and neuron loss via apoptotic and/or autophagic pathways. The research presented here examined the molecular regulation of the autophagic and apoptotic pathways in vivo and in vitro by using models of lysosome dysfunction and hypoxia. In vivo investigation of lysosome dysfunction-induced death utilized the cathepsin D (CD)-deficient mouse model. CD is a major lysosomal aspartate protease and mice deficient for this gene show increased accumulation of autophagic vacuoles (AVs) and neurodegeneration (Koike et al. 2003;Koike et al. 2000). This study showed CD deficiency resulted in AV accumulation followed by an age-dependent decrease in Akt signaling. To test the hypothesis that altered lysosome function would lead to neural precursor cell (NPC) death via an interaction between autophagy- and apoptosisassociated proteins, we treated NPCs with potent inducers of autophagy, such as the lysosomotropic agent chloroquine (CQ), and the vacuolar ATPase inhibitor bafilomycin A1 (Baf A1), or starvation. NPCs deficient for bax or p53 exhibited decreased caspase activation and NPC death in response to these agents. Lysosome dysfunction triggered an Atg7-dependent up-regulation of phospho-p53, caspase-3, and LC3-II, indicating that Atg7 lies upstream in this cell death pathway. To study hypoxia-induced NPC death in vitro, NPCs were treated with hypoxia mimetics desferroxamine (DFO) or cobalt chloride (CoCl2), or oxygen glucose deprivation (OGD). Hypoxia exposure resulted in increased hypoxia inducible factor alpha (HIF1α) and bcl-2/adenovirus E1B 19 kDa interacting protein-3 (BNIP3) expression. BNIP3 shRNA knockdown failed to affect hypoxia -induced caspase-3 activation but was able to attenuate cell death and nuclear translocation of apoptosis inducing factor (AIF). These findings indicate that multiple Bcl-2 family members critically regulate hypoxia through a caspase-independent and -dependent NPC death pathway. In total, these studies provide new insights into the molecular pathways regulating neural stem cell death and suggest possible molecular targets for the development of neuroprotective agents.

xv, 178 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

Apoptosis Neural Stem Cells Autophagy Neurodegeneration Bc1-2 p53

UNRESTRICTED

Advisors/Committee Members: Roth, Kevin A., Marques, Guillermo<br>, Theibert, Anne<br>, Zhang, Jianhua<br>, Wilson, Scott.

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The LIM domain-containing Thyroid Receptor-Interacting Protein 6 (TRIP6) is a zyxin family member that has been implicated in actin dynamics and cell motility. In this study, we have demonstrated that LPA stimulation promotes the recruitment of TRIP6 to the activated LPA2 receptor, and induces the association of TRIP6 with the components of focal complexes, including paxillin, p130cas, FAK and c-Src. Overexpression of TRIP6 enhances LPA-induced SKOV-3 ovarian cancer cell migration; in contrast, suppression of the endogenous TRIP6 expression by its specific small interfering RNA inhibits it, suggesting a physiological role for TRIP6 in LPA-induced cell migration. The function of TRIP6 in cell motility is regulated by c-Src-mediated phosphorylation at Tyr- 55. This phosphorylation is required for TRIP6 coupling to Crk SH2 domain and ERK activation, thereby enhancing LPA-induced morphological changes and chemotaxis. We further demonstrate that TRIP6 phosphorylation and function are negatively modulated by a PTP-L1/FAP-1 (Fas-associated phosphatase-1)-dependent mechanism. PTP-L1 dephosphorylates phosphotyrosine-55 residue of TRIP6 in vitro, and inhibits LPAinduced tyrosine phosphorylation of TRIP6 in cells. In contrast, deletion of the carboxylterminal LIM3 and PDZ-binding domains of TRIP6 disrupts the interaction with PTP-L1, and abolishes PTP-L1-mediated negative regulation of TRIP6 phosphorylation. As a result, LPA-induced association of TRIP6 with Crk, and the function of TRIP6 in promoting LPA-induced morphological changes and cell migration are inhibited by PTP L1. Taken together, a switch from c-Src-mediated phosphorylation to PTP-L1-dependent dephosphorylation serves as a negative regulatory mechanism to control TRIP6 function in LPA-induced cell migration.

xvi, 130 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

TRIP6 Lysophosphatidic Acid Cell Migration C-Src PTPL1 Focal Adhesions

UNRESTRICTED

Advisors/Committee Members: Lin, Fang-Tsyr<br>, Benveniste, Etty (Tika)<br>Frank, Stuart J.<br>Lin, Weei-Chin<br>Woods, Anne C.

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ErbB receptor tyrosine kinases have long been implicated in cancer formation and progression by regulating cell division, migration, and survival. ErbBs are also essential in multiple processes during invertebrate development; however, their activities during vertebrate embryogenesis are not well understood. For functional characterization of ErbB signaling during vertebrate development, frog model Xenopus laevis was used in our studies. The expression pattern and the general activities of ErbB receptors during early frog development were first analyzed and results shown that ErbBs regulate gastrulation, somite organization and head patterning. As gastrulation is the first major morphogenetic event in vertebrate development, I focused the following studies on ErbB signaling in frog gastrulation. During Xenopus gastrulation, mesendodermal cells are internalized and display different movements. Head mesoderm migrates along the blastocoel roof, while trunk mesoderm undergoes convergent extension (C&E). Inhibition of ErbB pathway blocks both C&E of trunk mesoderm and migration of head mesoderm. Cell polarization and intercalation, cell-cell and cell-matrix interaction, as well as formation of membrane protrusions are also impaired, demonstrating that modulation of cell adhesive properties and cell morphology may underlie the functions of ErbBs in gastrulation. These results reveal for the first time that vertebrate ErbB signaling modulates gastrulation movements, thus providing a novel pathway, in addition to noncanonical Wnt and FGF signals, that controls gastrulation. To further elucidate mechanisms of ErbB signaling in Xenopus gastrulation, I next examined downstream signals employed by ErbBs during this process. From rescue experiments, PI3K and Erk MAPK were shown to mediate ErbB signaling to regulate gastrulation morphogenesis. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways. While activation of either pathway rescues gastrulation defects induced by translational inhibitory ErbB4 morpholino oligonucleotides, the two signals preferentially regulate different aspects of cell behaviors. PI3K is more efficient in rescuing cell adhesion and cell spreading while MAPK is more effective in stimulating the formation of filopodia. The data reveal that PI3K and Erk MAPK, which previously considered as mesodermal inducing signals, also act downstream of ErbB signaling to regulate gastrulation morphogenesis.

xi, 190 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

Xenopus gastrulation ErbB Erk MAPK PI3K

UNRESTRICTED

Advisors/Committee Members: Chang, Chenbei, Carroll, Steven L.<br>, Mayne, Richard <br>, Wyss, J. Michael <br>, Yoder, Bradley K..

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Proper regulation of survival signaling is critical for all organisms. One important signaling cascade involved in the coordinated effort to control signals influencing cell fate is the phosphatidylinositol 3-kinase (PI3K)-Akt-glycogen synthase kinase 3[beta] (GSK3[beta]) signaling cascade. Following activation by growth factors the PI3K-Akt pathway promotes cell survival and, cell death is facilitated following inhibition of Akt. Many factors influence the function of the Akt-GSK3[beta] signaling dyad, including phosphorylation, protein complex formation and subcellular localization. Previous work had demonstrated that the 90kDa heat shock protein, HSP90, directly binds and stabilizes Akt. Furthermore, several heat shock proteins including HSP90 and HSP105 can modulate cell survival following various insults. Therefore, the goals of this research were to test if heat shock proteins, either directly or indirectly, could regulate signaling to GSK3[beta], modulate GSK3[beta]-mediated apoptosis, or direct nuclear localization of GSK3[beta]. The HSP90 inhibitor geldanamycin was used to block HSP90 function followed by examination of insulin like growth factor-1 (IGF-1) or insulin-induced signaling. HSP90 inhibition enhanced growth factor-induced phosphorylation and activation of Akt and subsequent phosphorylation and inactivation of GSK3[beta], indicating that HSP90 normally acts to buffer the magnitude of growth factor signaling. Hsp90 dampened Akt signaling by facilitating phosphatase-mediated dephosphorylation of Akt. Endoplasmic reticulum (ER) stress, in opposition to growth factors, promotes apoptotic signaling. GSK3 and HSP105 were found to interact and both modulate ER stress-induced apoptotic signaling. Knockdown of HSP105 attenuated ER stress-induced caspase-3 activation down stream of the proapoptotic molecule C/EBP homologous protein (CHOP), indicating that HSP105 facilitates ER stress-induced caspase-3 activation. GSK3 also facilitates ER stress-induced apoptotic signaling, however GSK3 is acting upstream of CHOP expression. Thus HSP105 and GSK3 interact and regulate distinct points in the ER stress-induced apoptotic signaling pathway. Many of the actions of GSK3[beta] are influenced by tightly controlled nuclear localization. Nuclear localization of GSK3[beta] is controlled by a cohort of factors including a bipartite nuclear localization sequence (NLS), intramolecular interactions and stimulus-induced release from protein complexes. Overall, this work reveals novel roles for heat shock proteins in the regulation of survival signaling. Moreover, the molecular mechanism regulating the nuclear localization of GSK3[beta] were discovered.

vii, 143 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

Apoptosis Akt ER stress Glycogen synthase kinase Nuclear localization Heat shock protein

UNRESTRICTED

Advisors/Committee Members: Jope, Richard S., Johnson, Gail V. W.<br>, Lesort, Mathieu <br>, Wilson, Scott <br>, Wyss, J. Michael.

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Parkinson Disease (PD) is the second most common age-related neurodegenerative disorder and is characterized pathologically by the loss of dopaminergic (DA) neurons in the stubstantia nigra pars compacta (SNpc). Mitochondrial dysfunction, increased oxidative stress, and accumulation of aggregated α-synuclein (α-syn), an intracellular protein involved in synaptic function, are all pathological hallmarks of PD have been implicated in PD pathogenesis. However, it is debated whether α-syn aggregates themselves are responsible for neurodegeneration in PD, cellular pathways involved in degradation of α-syn aggregates are believed to promote neuron survival. The autophagy lysosomal pathway (ALP), a physiological mechanism for recycling of intracellular components, has been shown to clear α-syn, its aggregates, and regulate neuron survival in PD. iv The first part of this dissertation reviews the developments regarding the contribution of the ALP to neuron survival and death regulation. It discusses the effects of oxidative stress on ALP function and vice versa. The role of the lysosome, a cellular organelle responsible for digestion of intracellular constituents delivered via ALP, in regulation of neuron survival is highlighted. Increased oxidative stress and α-syn aggregate accumulation has been reported in PD models of mitochondrial dysfunction such as that induced by the insecticide rotenone. Another part of this dissertation focused on rotenone’s effects on ALP function. Rotenone-induced inhibition of lysosomal function has been observed. Our findings suggested that lysosomal dysfunction may be responsible for α-syn accumulation and neuron death observed in PD, implying that mechanisms improving lysosomal function may be cytoprotective. This hypothesis was tested in the second chapter of this dissertation using a chloroquine (CQ) model of lysosome dysfunction. CQ, a known antimalarial agent, induced α-syn accumulation and neuron death by inhibiting ALP function. Bafilomycin A1, a plecomacrolide antibiotic, attenuated CQ-induced neuron death, an effect accompanied by restoration of lysosomal function. These studies expand our knowledge of the potential role of the ALP in PD pathogenesis and suggest that further studies are needed to both decipher the neuroprotective role of the ALP in preventing neuron death in response to PD-associated stimuli, such as rotenone, and to identify novel ALP-related molecular targets for PD therapy.

1 online resource (xi, 170 p. : ill., digital, PDF file)

Pathology

Joint Health Sciences;

autophagy lysosome cell death rotenone chloroquine bafilomycin A1

UNRESTRICTED

Advisors/Committee Members: Roth, Kevin A., Shacka, John J.<br>, Carroll, Steven L.<br>, Sztul, Elizabeth<br>, Burton Theibert, W. Anne<br>, Yacoubian, Talene Alene.

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According to the Central Brain Tumor Registry of the United States, the most common primary brain tumors are gliomas, tumors composed of cells of glial origin, most commonly astrocytes and oligodendrocytes. The most aggressive of these tumors are characterized by hyperproliferation, marked cellular and nuclear atypia, extensive infiltration into surrounding normal brain tissue, and large areas of cell and tissue death. Previous data published by our lab and others suggest that these biological processes may involve regulated cell volume changes. Using cell volume regulation in the presence of an anisosmotic challenge as a model for cell swelling and shrinkage, cell volume changes have been shown to involve the movement of molecules, or osmolytes, across the plasma membrane through channels and transporters. Water is osmotically obliged to follow the net movement of molecules, resulting in a net flux of water across the plasma membrane and an overall change in cell volume. As the most abundant anion in biological systems, chloride has been shown to be involved in the volume regulation of several cell types. However, chloride-mediated volume changes in human glioma cells have not been extensively studied. It is the primary goal of this dissertation to examine the mechanisms employed by human glioma cells to dynamically regulate their cell volume at rest, in the presence of anisosmotic conditions, and importantly during the biological processes of apoptosis and migration. We confirm the expression of the voltage gated chloride channels ClC-2, 3, and 5 in human glioma cell lines and patient biopsies. In addition, we demonstrate the expression of the cation chloride cotransporters, KCC1, KCC3, and NKCC1. Using a variety of techniques, including electrophysiology, Coulter Counter, and chloride-sensitive fluorescent dyes, we establish that the resting intracellular chloride concentration and cell volume are maintained by the basal activity of chloride cotransporters. While pharmacological inhibition of these cotransporters suggests that they are also involved in cell volume regulation during an aniosomotic challenge, chloride efflux through channels plays a more significant role in post-hyposmotic volume decrease. Similarly, inhibition of chloride channels, but not chloride cation cotransporters, inhibits the cell condensation that occurs in the presence of apoptotic stimuli. Cells treated with chloride channel inhibitors also demonstrated limited caspase 3 activity and DNA fragmentation, suggesting that the volume decrease is necessary for apoptosis. Finally, transwell migration of human glioma cells was blocked in the presence of chloride channel and transport inhibitors, suggesting that the mechanisms involved in cell shrinkage are necessary in glioma cell migration.

xi, 176 p. : ill., digital, PDF file

Neurobiology

Joint Health Sciences

glioma astrocytoma chloride channels migration apoptosis volume regulation

UNRESTRICTED

Advisors/Committee Members: Sontheimer, Harald, Benos, Dale <br>, Brenner, Michael <br>, Guay-Woodford, Lisa <br>, Hablitz, John.

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The t(8;21)(q22;q22) AML1-ETO translocation is one of the most frequent translocations in acute myeloid leukemia (AML), occurring in approximately 12% of cases. Our laboratory has generated a mouse model in which AML1-ETO is expressed in hematopoietic progenitor cells using a retroviral vector and assayed using bone marrow transplantation. Animals transplanted with AML1-ETO-expressing cells failed to develop leukemia. However, AML1-ETO promoted significant expansion of immature myeloid cells suggesting a “pre-leukemic” state that requires secondary mutations for disease progression. In addition, analysis of cell cycle kinetics in AML1-ETOexpressing myeloid progenitor cells showed that AML1- ETO slowed G1 to S phase progression compared to control animals. Further analysis using quantitative RT-PCR showed that AML1-ETO increased expression of p15Ink4b and p16Ink4a, which may account for the inhibition of the G1 to S phase transition. Interestingly, p15INK4b is a tumor suppressor that is inactivated by hypermethylation in over 70% of human AML patient samples. In addition, p16INK4a is frequently inactivated in the FAB-M2 subset of AML that expresses the t(8;21). These observations suggest that loss of p15Ink4b or p16Ink4a may cooperate with AML1-ETO in disease progression. To test this hypothesis, p15Ink4b-/-, p16Ink4a-/-, or wild-type bone marrow cells were transduced with AML1- ETO or control retroviruses and then transplanted into lethally irradiated recipient animals. Analysis of reconstituted animals at two months post-transplant showed that p15Ink4b or p16Ink4a deficiency did not accelerate disease progression in the presence of AML1-ETO. However, loss of p15Ink4b alone did cause a modest expansion of hematopoietic stem cells and a 2-fold increase in myeloid progenitor cells based in FACS analysis and differential bone marrow cell counts. These data suggest that the loss of either p15Ink4b or p16Ink4a alone is not a major contributing factor to pathogenesis associated with the AML1-ETO translocation.

vii, 66 p. : ill., digital, PDF file

Biochemistry and Molecular Genetics

Joint Health Sciences

AML1-ETD Leukemia Myeloid Development Hematopoietic Stem Cell

UNRESTRICTED

Advisors/Committee Members: Klug, Christopher A., Chen, Xinbin <br>, Emanuel, Peter D. <br>, Ruppert, J. Michael <br>, Townes, Tim M..

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The p53 tumor suppressor is the most commonly mutated gene in human cancers. As a transcription factor, p53 exerts its tumor suppressor function through the activation of downstream target genes involved in cell cycle arrest, apoptosis, or cellular senescence. Cellular senescence plays an important role in tumor suppression. However, the p53 target genes that mediate senescence remain unknown. Here, we characterized the role of DEC1, a novel target gene of the p53 family, in p53-dependent cellular senescence. Specifically, we found that DEC1, a bHLH transcription factor, is induced by the p53 family and DNA damage in a p53-dependent manner. DEC1 has been found to be up-regulated in K-ras-induced premature senescence. However, it is not clear whether DEC1 is capable of inducing premature senescence. Here, we found that over-expression of DEC1 induces G1 arrest and promotes premature senescence. Moreover, we found that knockdown of endogenous DEC1 attenuates p53-mediated premature senescence in response to DNA damage. Furthermore, to uncover potential DEC1 targets regulating senescence, an Affymetrix GeneChip assay was performed with RNAs purified from an MCF7 cell line which was uninduced or induced to express DEC1. We identified ID1, inhibitor of differentiation/DNA binding, to be a novel gene down-regulated upon over-expression of DEC1. ID1 is known to be down-regulated in senescent cells. Interestingly, we found that ID1 is down-regulated by DNA damage in both p53- and DEC1-dependent manners. We also provided multiple lines of evidence that ID1 is a direct target of DEC1 and p53 repression of ID1 is mediated by DEC1. Finally, we showed that over-expression of ID1 or ID1’, an alternative spliced isoform of ID1, promotes cell proliferation. In sum, we concluded that DEC1 is one of the p53 effectors involved in promoting premature senescence and that the inhibition of ID1 expression is an important mechanism by which p53 and DEC1 inhibit cell proliferation and promote premature senescence.

ix, 75 p. : ill., digital, PDF file.

Cell Biology

Joint Health Sciences

P53 Tumor Suppressor DEC1 Transcriptional Regulation Cellular Senescence DNA Damage Response ID1

UNRESTRICTED

Advisors/Committee Members: Chen, Xinbin, Chang, Chenbei<br>Crawford, David F.<br>Lin, Fang-Tsyr<br>Miller, Michael.

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Aquaporins (AQP) constitute the primary pathway for water movement across cellular membrances. As a result, their expression and function are important for regulating cell volume. Both gliomas and astrocytes have highly effective volume regulatory mechanisms capable of rapidly moving ions and water into and out of cells following edemic episodes. In this dissertation, we examined the expression of AQPs in both gliomas and astrocytes in order to understand their function role as well as their regulation. We have shown that primary gliomas highly express AQP1 and AQP4 whereas cell lines express no AQP4 and variable AQP1. We reintroduced AQP1 and AQP4 into D54MG cell line lacking AQPs and examined their function. AQP1 enhanced tumor swelling and migration while AQP4 enhanced cell swelling and adhesion. PKC activation is known to regulate AQP function. Using PKC modulators, we found that while PKC did not regulate AQP1, AQP4 function was highly dependent on PKC activity. Specifically, phorbol 12-myristate 13-acetate, PKC activator, reduced cell swelling in AQP4-expressing tumors as well as migration. Chelerythrine, PKC inhibitor, enhanced both cell swelling and migration. Additionally, knocking out the PKC phosphorylation site at S180 eliminated the regulation of AQP4 water permeability and tumor migration. We verified this data by implanting AQP1, AQP4 and S180-AQP4 intracranially into mice and examined glioma invasion. We found that AQP1 and S180-AQP4 invaded on average significantly further than either AQP4 or control tumor cells lacking AQPs. AQP1 and S180-AQP4 invaded much further distances (>1500 μm) suggesting that AQP1 expressing tumors invade into tissue to set up satellite colonies characteristic of gliomas whereas AQP4 mediated invasion is regulated by PKC activity. Finally, we wanted to understand the regulation of AQP1 expression in astrocytes following injury. AQP1 is upregulated following cortical stab injury, and we could mimic this upregulation using an in vitro wound assay. We found that pMEK1/2 and pERK1/2 was increased up to 60 min post-injury but then subsequently decreased. AQP1 expression was increased by 16hrs. MEK1/2 and ERK1/2 activity as well as AQP1 expression was completely inhibited by U0126, a MAPK inhibitor. Astrocyte upregulation following injury is regulated by MAPK signaling.

1 online resource (x, 127 p. : ill., digital, PDF file)

Neurobiology

Joint Health Sciences

volume regulation tumor edema migration invasion cell signaling

UNRESTRICTED

Advisors/Committee Members: Sontheimer, Harald, Benos, Dale<br>, Brenner, Michael<br>, Wilson, Scott<br>, Yoder, Bradley.

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Cystic fibrosis (CF) is caused by mutation of one protein, the cystic fibrosis transmembrane conductance regulator (CFTR), which normally functions as a chloride channel at the apical surface of epithelial cells. The most common CFTR mutation results in the deletion of a single amino acid (phenylalanine) at position 508, which causes the protein to fold improperly. The [Delta]F508 CFTR is a temperature-sensitive (TS) processing mutant: At the restrictive temperature, 37°C, [Delta]F508 CFTR misfolds in the endoplasmic reticulum (ER) and is degraded, but at the permissive temperature, 27°C, it is “rescued” from degradation (r[Delta]F508). In particular, r[Delta]F508 CFTR folds correctly enough to exit the ER and produces a chloride channel at the cell surface, similar to the wild-type (WT) protein. Unfortunately, r[Delta]F508 is rapidly degraded after returning to the restrictive temperature, suggesting a surface stability defect. Therefore, it is clear that the [Delta]F508 CFTR defect can be corrected under certain conditions, but first we must understand how the WT and [Delta]F508 CFTR proteins are handled differentially by the ER and at the cell surface, which is the aim of the work presented herein. We first characterized an interaction between CFTR and a protein associated with ER degradation, valosin-containing protein (VCP), and found that it interacts with [Delta]F508 CFTR, but not with the WT protein. This study provides further evidence that WT CFTR is processed efficiently in the ER. Second, with regard to [Delta]F508 CFTR, we found that culture at the permissive temperature corrects not only the folding defect in the ER, but also the impaired surface stability of [Delta]F508 CFTR. This result suggests that, like the folding defect, the surface stability defect of [Delta]F508 CFTR is also TS. Additionally, we showed that two small molecular compounds known to mimic permissive temperature culture of [Delta]F508 by correcting its ER processing defect also enhance its surface stability at 37°C. These studies add significantly to current CF knowledge and may aid in the design of future therapeutics for CF and other diseases resulting from protein folding mutations. Specifically, we identify specific differences between ER processing and surface trafficking of WT and [Delta]F508 CFTR, and we reveal a novel mode of action for two known pharmacological interventions.

xiv, 118 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

Cystic fibrosis VCP/p97/cdc48 Trafficking CFTR Biogenesis Temperature-sensitive

UNRESTRICTED

Advisors/Committee Members: Collawn, James F., Morrow, Casey D.<br>, Parker, Scott D.<br>, Timares, Laura <br>, Weaver, Casey T..

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Gliomas, primary brain tumors derived from glial cells, constitute the majority of malignant tumors within the central nervous system. The most malignant of these tumors, grade IV Glioblastoma multiforme, are characterized by extensive proliferation, cellular and nuclear atypia, angiogenesis, areas of necrosis, and widespread invasion into the brain parenchyma. Data from our lab and others have implicated ion channels in the invasion and proliferation of glioma cells. Moreover, calcium signaling in gliomas and other cells has been implicated in both migration and proliferation. The aim of this dissertation was to investigate Transient Receptor Potential Canonical (TRPC) channels role in glioma cell biology. TRPC channels are non-selective cation channels whose activation is downstream of the phospholipase C cascade and their role in glioma biology was previously unknown. In this dissertation, I show expression of TRPC channel subunits within glioma cell lines and glioblastoma multiforme patient biopsy lysates. Further, I demonstrate that these subunits form functional channels on the plasma membrane. Whole-cell patch clamp electrophysiology shows that TRPC channel inhibitors block small, linear currents and affect glioma calcium signaling by decreasing store-operated calcium entry. These pharmacological inhibitors additionally chronically impair glioma proliferation. To assess whether TRPC channels specifically impact glioma calcium signaling and proliferation, shRNA plasmids directed against TRPC1 were utilized. These plasmids specifically decrease TRPC1 subunit expression as shown by western blot analysis and I show through whole-cell recordings and calcium imaging that TRPC1 channels are functionally impaired. Furthermore, TRPC1 shRNA plasmids decrease glioma proliferation. With TRPC1 channel inhibition, glioma cells become larger and, often, multinucleated indicating a role for TRPC1 channels in cytokinesis. In this dissertation, I also show that TRPC1 channels localize to lipid raft fractions of the plasma membrane and propose that the localization and timing of TRPC channel activation plays a crucial role in glioma cell biology.

Neurobiology;

Joint Health Sciences;

TRPC proliferation multinucleation calcium

UNRESTRICTED

Advisors/Committee Members: Sontheimer, Harald, Kirk, Kevin<br>, Lester, Robin<br>, Nabors, L. Burt<br>, Pozzo-Miller, Lucas.

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During monocyte activation and differentiation along the macrophage lineage, the activity of [alpha]4[beta]1 integrins is upregulated, which promotes extravasation from the vasculature and migration through inflamed tissues. In this dissertation research, we investigated the mechanisms regulating [alpha]4[beta]1 activation, utilizing U937 promonocytic cells as a model system for studying the monocyte/macrophage transition. When treated with PMA, these cells acquire functions characteristic of mature phagocytes. We demonstrate that PMA induces a rapid, yet transient, activation of [alpha]4[beta]1 receptors, which is followed by a more pronounced and sustained increase in [alpha]4[beta]1 binding activity. We hypothesize that this second increase in binding allows sustained recruitment of monocytes, which are known to traffic to sites of inflammation much later than neutrophils. Our results suggest that this latter adhesive phase occurs as a consequence of a structural change in [alpha]4[beta]1 receptors, namely, the expression of a [beta]1 integrin subunit that lacks [alpha]2,6 linked sialic acids. During in vitro differentiation of both U937 cells and primary CD14+ human monocytes, the ST6Gal-I sialyltransferase is markedly downregulated, which leads to hyposialylation of the [beta]1 subunit. The expression of hyposialylated integrins is temporally correlated with increased cell adhesion to VCAM-1. In addition, pharmacological intervention, as well as ectopic expression of activated MEK, in U937 cells reveal that ST6Gal-I downregulation, integrin hyposialylation, and VCAM-1 binding are all directed by a PKC/ras/ERK signaling cascade, a pathway known to be in involved in monocyte differentiation. Interestingly, the activity of BACE1, a protease not previously identified in monocytes, appears to be responsible for decreased ST6Gal-I levels. We find that in U937 cells and primary monocytes, differentiation along the macrophage lineage dramatically increases the expression of BACE1 (via a PKC/ras/ERK signaling cascade), which in turn mediates ST6Gal-I cleavage and shedding from the cell. Importantly, preventing ST6Gal-I downregulation, through both BACE1 inhibition and constitutive overexpression of ST6Gal-I, eliminates [alpha]4[beta]1 dependent VCAM-1 binding. In addition, ST6Gal-I overexpression also prevents a PMA-induced conformational change of the [beta]1 integrin into a more activated state. Taken together, these results describe a novel mechanism for regulation of [alpha]4[beta]1 integrins, and further suggest a previously unanticipated role for BACE1 in immune cell function.

xv, 119 p. : ill., digital, PDF file

Joint Health Sciences

Alpha4beta1 Integrin Sialylation STGal-I VCAM-1 BACE1

UNRESTRICTED

Advisors/Committee Members: Bellis, Susan L., Bullard, Daniel<br>Frank, Stuart<br>Kucik, Dennis<br>Murphy-Ullrich, Joanne<br>Weigent, Douglas.

▼  The fission yeast Schizosaccharomyces pombe has become a powerful model system for studying cytokinesis, a process of cytoplasmic division by which one cell divides into two identical daughter cells. Like mammalian cells, S. pombe divides through the use of an actomyosin contractile ring, which is composed of a set of highly conserved cytoskeletal proteins. Cytokinesis in S. pombe is primarily regulated by the SIN pathway, which is activated in late mitosis and is required for actomyosin contractile ring and septum assembly, and also plays a role in spindle checkpoint inactivation, and telophase nuclear positioning. The various functions of the SIN are carried out by the terminal kinase in the pathway called Sid2. The lack of information in the downstream targets of Sid2 has limited our understanding of the different functions of the SIN. We recently showed that, in addition to its other functions, the SIN promotes cytokinesis through inhibition the MOR signaling pathway, which normally drives cell separation and initiation of polarized growth following completion of cytokinesis (Ray et al, 2010). The molecular details of this inhibition and the physiological significance of inhibiting MOR during cytokinesis was unclear. The results presented in Chapter II describe our approach to identify Sid2 substrates, particularly focusing on Nak1 and Sog2 that function in the MOR signaling cascade. We identified and characterized Sid2 phosphorylation sites on the Nak1 and Sog2 proteins. Chapter III explores how post translational modification of MOR proteins by Sid2 regulates polarized growth during cytokinesis. This includes delineating the effect of Sid2 mediated phosphorylation of Nak1 and Sog2 on protein-protein interactions in the MOR pathway as well as on the regulation of their localization during late mitosis. Finally, results in Chapter IV demonstrate that failure to inhibit MOR signaling is lethal because cells initiate septum degradation/cell separation before completing cytokinesis thereby emphasizing the importance of cross-regulation between the two pathways to prevent initiation of the interphase polarity program during cytokinesis. Advisors/Committee Members: Dannel McCollum, PhD.

▼  The midbody (MB) is a proteinaceous complex formed between the two daughter cells during cell division and is required for the final cell separation event in late cytokinesis. After cell division, the post-mitotic midbody, or midbody derivative (MB^d), can be retained and accumulated in a subpopulation of cancer cells and stem cells, but not in normal diploid differentiated cells. However, the mechanisms by which MB^ds accumulate and function are unclear. Based on this, I hypothesize that the MB^d is degraded by autophagy after cell division in normal diploid differentiated cells, whereas non-differentiated cells have low autophagic activity and would accumulate MB^ds. Indeed, I found this to be the case. MB^d degradation occurred soon after cytokinesis in differentiated cells that possess high autophagic activity. Specifically, I found MB^d degradation to be mediated by binding of the autophagy receptor, NBR1, to the MB protein Cep55. Moreover, by performing proteomic analysis of NBR1 interactions I found additional MB-localized proteins that are potential substrates for NBR1. In contrast to differentiated cells, stem and cancer cells have low autophagic activity thus MB^ds evade autophagosome encapsulation and accumulate. To examine whether MB^ds can define the differentiation status of a cell, we depleted NBR1 from differentiated fibroblasts causing an increase in MB^d number. Strikingly, under these conditions, reprogramming of fibroblasts to pluripotent stem cells is increased. Equally interestingly, cancer cells with increased MB^ds have increased in vitro tumorigenicity. In conclusion, this study gives an insight into the fates of post-mitotic midbodies and also suggests a non-cytokinetic role of midbodies in enhancing pluripotency in stem cells and cancer stem cells. Advisors/Committee Members: Stephen Doxsey, PhD.

▼  Key regulatory events take place at very early stages of human embryonic stem cell (hESC) differentiation to accommodate their ability to differentiate into different lineages; this work examines two separate regulatory events. To investigate precise mechanisms that link alterations in the cell cycle and early differentiation, we examined the initial stages of mesendodermal lineage commitment and observed a cell cycle pause that occurred concurrently with an increase in genes that regulate the G2/M transition, including WEE1. Inhibition of WEE1 prevented the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. These findings reveal a novel G2 cell cycle pause required for endodermal differentiation. A role for phenotypic transcription factors in very early differentiation is unknown. From a screen of candidate factors during early mesendodermal differentiation, we found that RUNX1 is selectively and transiently up-regulated. Transcriptome and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that RUNX1 knockdown impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion compromised TGFβ2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFβ2, but not TGFβ1. These findings identify novel roles for RUNX1-TGFβ2 signaling in mesendodermal lineage commitment. Advisors/Committee Members: Gary S. Stein, PhD.

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A microarray study was performed which identified G2E3 as a novel, putative ubiquitin ligase that was both G2/M-specific in expression and down regulated at the transcriptional level following DNA damage by [gamma]-irradiation. The initial characterization of this protein is described herein. G2E3 is composed of three PHD/RING (plant homoedomain/really interesting new gene) domains within its amino-terminal half and a carboxy-terminal HECT (homologous to E6AP carboxy-terminus) domain. G2E3 is regulated in part by its subcellular localization. The protein normally resides within the nucleoplasm of cultured cells and distinctly within the nucleolus of HeLa cells. G2E3 is a nucleo-cytoplasmic shuttling protein with its nuclear export mediated by a novel, CRM1-independent nuclear export domain located within the HECT domain. Furthermore, in response to DNA damage, G2E3 rapidly delocalizes from the nucleolus of HeLa cells. In vitro ubiquitination experiments demonstrate that G2E3 is able to mediate auto-ubiquitination and that this activity is dependent upon the third PHD/RING domain. To examine the function of G2E3 in vivo, a knockout mouse model has been generated. G2E3 heterozygous animals develop normally and display no overt defects. However, G2E3 knockout animals die prior to embryonic day 8.5. Northern blotting demonstrates that G2E3 is expressed throughout development, and [beta]-galactosidase staining indicates those tissues where the protein is most predominantly expressed. These experiments demonstrate that G2E3 is a highly regulated ubiquitin ligase that is required for mammalian embryonic development.

vi, 94 p. : ill., digital, PDF file.

Cell Biology

Joint Health Sciences

G2E3 Subcellular Localization Ubiquitin Ligase Embryonic Lethal

UNRESTRICTED

Advisors/Committee Members: Crawford, David F., Bullard, Daniel C.<br>, Chen, Xinbin <br>, Lin, Weei-Chin <br>, Prince, Lawrence S..

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Infections by the high-risk human papillomavirus type 18 (HPV-18) or type 16 (HPV-16) can lead to cancers of the anogenital tract. Because the propagation of HPVs is restricted to differentiated human squamous epithelia, experimental studies of the productive program are conducted in organotypic cultures. The current methods are of low efficiency and time consuming and have relied on immortalized epithelial cell lines that do not support efficient viral DNA amplification or virion production, compromising HPV genetic analyses and obscuring normal virus-host cell interactions. I used Cre-loxP site-specific recombination to develop a relatively simple, fast and highly efficient system to produce HPV-18 genomic plasmids in primary human keratinocytes (PHKs). The transfected primary human keratinocytes can be quickly developed into organotypic raft cultures, obviating the need for immortalized cells or the immortalization function of the high-risk HPV types. Upon keratinocyte differentiation and early viral gene expression, the HPV-18 replicons amplified to high copy numbers in raft cultures. The major capsid protein (L1) expression was widely detected in superficial cells and in cornified layers on and after day 14 or 16. Electron microscopy examination revealed HPV-18 particles were packed in paracryatalling arrays in shrunken nuclei with condensed chromatins. These virions infected PHKs efficiently and were passaged in fresh PHK raft cultures, a feat not achieved until now. Thus, this system should be a useful tool to study HPV mutants or low-risk HPV genotypes. I further verified the utility of this system in HPV genetic analyses. Indeed, I was able to analyze the phenotypes of the HPV-18 E6*I and E6-null mutant replicons which had not been studied previously in raft cultures due to their inability to immortalize PHKs or failure to be maintained in transfected PHKs as plasmids. The mutant genomes were highly deficient in L1 production but were complemented in trans by a retrovirus expressing wild type HPV-18 E6. In support of translating basic scientific capabilities into clinical outcomes, this system should also be highly informative for testing agents that can prevent or treat human papillomavirus infections.

1 online resource (xiv, 128 p. : ill., digital, PDF file)

Biochemistry and Molecular Genetics

Joint Health Sciences;

organotypic raft cultures of primary human keratinocytes Cre-loxP site specific recombination HPV-18 infection cycle viral DNA amplification HPV-18 E6*I and E6-null mutants genetic complementation in trans

UNRESTRICTED

Advisors/Committee Members: Chow, Louise T., Britt, William J.<br>, Broker, Thomas R.<br>, Chen, Ching-Yi<br>, Kappes, John C.<br>, Ruppert, J. Michael.

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Members of the Fc receptor-like (FCRL) family are cell-surface proteins with ancient conservation, distinct lymphocyte expression patterns, and tyrosine-based signaling capabilities that imply a fundamental role for them in modulating immune responses. Though they share many features with the Fc receptors (FCR) for IgG and IgE, FCRLs have not been found to bind immunoglobulin. In these studies, we sought to characterize the cellular expression pattern, binding partner, and function of a recently identified FCRL family member, human FCRL6. By developing specific monoclonal antibodies (mAbs) we determined that FCRL6 is distinctly expressed by cytotoxic lymphocytes, namely NK cells, CD8+ T cells, γδ T cells, and rare cytotoxic CD4+ T cells. To elucidate its extracellular ligand(s), a cell-based GFP reporter system was developed to assay FCRL6 surface receptor engagement. These studies revealed that FCRL6 binds to HLA-DR, an MHC class II molecule. This interaction was confirmed by generating a panel of HLA-DR-reactive mAbs that blocked the FCRL6/HLA-DR association and by demonstrating that soluble recombinant FCRL6-Fc molecules specifically bind to HLA-DR transductants. The functional relevance of this discovery was also examined. First, by blocking the FCRL6/HLA-DR interaction, CD8+ T cell functions could be enhanced in response to antigenic peptide stimulation. Second, forced expression of FCRL6 in a human NK cell line inhibited its killing of HLA-DR expressing cells. Collectively, these studies demonstrate that FCRL6, a molecule distinctly expressed by cytotoxic lymphocytes, inhibits their effector function upon engagement of its ligand, MHC class II. These findings reveal an intriguing evolutionary relationship between receptors for immunoglobulin and MHC and demonstrate that NK cells and CD8+ T cells, whose activities are traditionally considered to be governed by MHC class I interactions, are also functionally regulated by MHC class II. This newfound interface may have important implications for better understanding HLA class II disease association and its manipulation could be of therapeutic benefit to patients with disorders of cell-mediated immunity.

1 online resource (v, 86 p.) : ill., digital, PDF file.

Microbiology;

Joint Health Sciences;

FCR FCRL FCRL6 MHC class II NK cells CD8+ T cells

UNRESTRICTED

Advisors/Committee Members: Davis, Randall S., Burrows, Peter D.<br>, Justement, Louis B.<br>, Lorenz, Robinna G.<br>, Zajac, Allan J..

▼ The utilization of Electrical Pulse Stimulation (EPS) has been predominantly used to study the physiological, cellular, and molecular responses of excitable cells such as nerve and muscle. Based on previous work on myotubes, non-excitable myoblasts, and non-excitable cancerous rhabdomyosarcoma cells, this study looks into the effects of EPS on breast cancer cells (MCF7s). My aim is to characterize the response of MCF7 cells to EPS and add to a working model of cell cycle arrest, autophagy, and cell death mediated by calcium signaling through cell cycle signaling proteins. This is based on the body of literature detailing the effects of EPS on excitable cell types. Ultimately, by stressing cancer cells with EPS, we can learn more about potential novel modes to induce mechanisms of proliferative failure. Advisors/Committee Members: Connor, Michael K. (advisor).