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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
131

The Role of the Transactivation Domain in c-Myc Mediated Cell Cycle Progression and Transformation

West-Osterfield, Kimberly 14 April 2008 (has links)
Dissertation under the direction of Professor Stephen R. Hann<p> c-Myc is a transcription factor whose deregulation has been implicated in numerous cancers. The biological responses resulting from Myc deregulation include hyperproliferation, apoptosis upon serum withdrawal, changes in cellular size, blocking of cellular differentiation, and transformation in conjunction with constitutively activated Ras. c-Myc contains three highly conserved regions within the N-terminal transcriptional transregulatory domain, termed Myc Box I, Myc Box II and Myc Box III. MycS, which is missing the first 100 amino acids, retains several biological functions; however it is unable to transform primary fibroblasts or induce cell cycle entry from quiescence. This suggests that the first 100 amino acids are necessary for some biological functions. It also indicates that specific Myc boxes can each mediate separable and distinct functions through distinct cofactor interactions. I hypothesize that the first 100 amino acids have a role in primary cell transformation and cell cycle progression. Dissection of the transregulatory domain has uncovered a region spanning the first 62 amino acids containing Myc Box I that has the highest transactivation activity. The transactivation activity can be inhibited by interaction with the p19ARF tumor suppressor and be modulated by post-translational modification. When the transactivation domain was fused to the c-Myc C-terminal DNA binding and heterodimerization domain, the resulting protein induced hyperproliferation of immortalized cells, but not Myc-mediated apoptosis. Further assessment of the transactivation activity revealed that a smaller region containing only the first 46 amino acids had substantial transactivation activity. Sequence analysis of this region compared to other Myc proteins uncovered a small sequence of high sequence similarity within the first 46 amino acids, termed MB0. Deletion of this new Myc box demonstrated that this domain is critical for transactivation, cotransformation of primary rat fibroblasts and upregulation of the target genes examined.
132

THE ROLE OF NFATC1 IN PROXIMAL TUBULE INJURY AND REPAIR

Langworthy, Melissa Marie 14 April 2008 (has links)
Recovery from acute kidney injury (AKI) requires renal tubule cell regeneration. The population of cells that repair the damaged proximal tubule epithelial cells (PTC) has been proposed to be derived from an external population circulating in the blood stream, an adjacent less-injured renal cell population, or a resident self-renewing PTC population. The identification of renal progenitor cells has been difficult because such a population remains phenotypically indistinguishable from their terminally differentiated counterparts and would require creation and characterization of transgenic reporters. For my dissertation research, I investigated the role of NFATc1 in PTCs. A single dose of mercuric chloride (HgCl2) was administered to induce AKI. The data presented here show that NFATc1 plays a role in PTC regeneration following AKI and genetic and/or pharmacologic attenuation of NFATc1 results in increased PTC apoptosis, increased serum creatinine, decreased proliferation, and even death. Using novel NFATc1 transgenic lines that reports activation of an NFATc1 enhancer domain important for autoamplification of NFATc1, we identified a subpopulation of proximal tubule progenitor cells that were resistant to apoptosis following HgCl2 injury. Lineage analysis documented that the NFATc1 labeled PTCs proliferate to repair the damaged proximal tubule segment. The expression profile of this labeled cell population and their labeled progeny was compared to unlabeled PTCs and showed that the labeled population were differentiated proximal tubule cells that had increased transcription of pluripotent stem cell and tubule development markers. To our knowledge, the delayed regeneration after AKI is the first example of a phenotype identified in the Nfatc1+/- mouse and proposes a role for NFATc1 in the regeneration of injured proximal tubule cells by as resident population of progenitor proximal tubules accentuated by NFATc1 expression.
133

STUDIES ON SRC TYROSINE KINASE IN TUMORIGENIC CELL GROWTH AND INVASION

Lund, Sabata Silva Constancio 27 June 2008 (has links)
This project aims to better understand the role of Src kinase on the development of colon cancer. Enhanced Src expression and activity are commonly elevated in colon cancer, with progressively higher activity observed as tumors progress and metastasize, however, the specific mechanism by which Src promotes cancer progression are still under investigation. First, we examined the potential for biological cooperativity between APC (adenomatous polyposis coli), a tumor suppressor often mutated in early stages of colon cancer, and elevated Src activity, using conditionally immortalized mouse colon epithelial cell lines, IMCE (APC +/min) and YAMC (APC +/+) as a model. The APC genotype did not affect the ability of Src to disrupt cell-cell junctions and promote cell invasiveness. However, IMCE cells exhibited increased capacity for oncogenic Src-mediated anchorage-independent proliferation as compared to YAMC cells. This property was correlated with enhanced â-catenin expression and activity, but not with enhanced ERK phosphorylation. The selective Src inhibitor, AZD0530, was found to be effective in blocking both cell invasion and proliferation. Second, we investigated the possible role for one of the major Src substrates, CAS (Crk associated substrate) in epithelial cell polarization. Stable depletion of CAS did not affect proliferation on the colon epithelial cell lines Caco2 and HCA7, however it increased thee transepithelial cell resistance of Caco2 cells compared to control cells. Furthermore, fluorescently tagged CAS was shown to localize to cell-cell adhesions in polarized Caco2 cells.
134

Receptor-Mediated Activation of Canonical Wnt Signaling

Cselenyi, Christopher Stephen 26 July 2008 (has links)
Wnt/beta-catenin signaling controls various cell fates in metazoan development and is misregulated in several cancers and developmental disorders. Binding of a Wnt ligand to its transmembrane coreceptors, Frizzled (Fz) and LRP5/6, inhibits phosphorylation and degradation of the transcriptional coactivator beta-catenin, which then translocates to the nucleus to regulate target gene expression. To understand how Wnt signaling prevents beta-catenin degradation, I focused on the Wnt coreceptor LRP6, which is required for signal transduction and is sufficient to activate Wnt signaling when overexpressed. LRP6 has been proposed to stabilize beta-catenin by stimulating degradation of Axin, a scaffold protein required for beta-catenin degradation. In certain systems, however, Wnt-mediated Axin turnover is not detected until after beta-catenin has been stabilized. Thus, LRP6 may also signal through a mechanism distinct from Axin degradation. To establish a biochemically tractable system to test this hypothesis, I expressed and purified the LRP6 intracellular domain from bacteria and show that it promotes beta-catenin stabilization and Axin degradation in Xenopus egg extract. Using an Axin mutant that does not degrade in response to LRP6, I demonstrate that LRP6 can stabilize beta-catenin in the absence of Axin turnover. Through experiments in egg extract and reconstitution with purified proteins, I identify a mechanism whereby LRP6 stabilizes beta-catenin independently of Axin degradation by directly inhibiting GSK3's phosphorylation of beta-catenin. In addition to studies of LRP6, I explore the role of the other Wnt coreceptor Fz, which has been suggested to be a G protein coupled receptor. Through biochemical studies in Xenopus egg extract, I demonstrate that Galphao, Galphai, Galphaq, and Gbetagamma promote beta-catenin stabilization by inhibiting GSK3s phosphorylation of beta-catenin. Independently of studies on Wnt signaling, I find that two enzymes involved in glycosylation, NAGK and DPAGT1, regulate anteroposterior patterning in Xenopus embryogenesis. I discover that these enzymes involved in N-glycosylation specifically regulate FGF-mediated events in Xenopus development. Because partial loss-of-function mutations in global regulators of N-glycosylation cause a group of human developmental disorders called Congenital Disorders of Glycosylation (CDGs), I suggest the use of Xenopus as a model organism to study the molecular etiology of CDGs.
135

ANALYSIS OF EPH RECEPTOR SIGNALING DURING OOCYTE MEIOTIC MATURATION IN CAENORHABDITIS ELEGANS

cheng, hua 06 December 2008 (has links)
A conserved biological feature of sexual reproduction in animals is that oocytes arrest in meiotic prophase and resume meiosis in response to extra-ovarian signals. In Caenorhabditis. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation. Sperm release the major sperm protein (MSP) signal to promotes meiotic maturation by antagonizing Eph receptor signaling and counteracting inhibitory inputs from the gonadal sheath cells. I show that MSP promotes oocyte meiotic maturation in part through direct interaction with the VAB-1/Eph receptor. Four conserved proteins, including a disabled protein (DAB-1), a vav family GEF (VAV-1), a protein kinase C (PKC-1), and a STAM homolog (PQN-19), function with VAB-1 in oocytes. We also show that antagonistic Gáo/i and Gás signaling pathways function in the soma to regulate meiotic maturation in parallel to the VAB-1 pathway. Furthermore, I show that in the absence of MSP VAB-1 inhibits meiotic maturation while either in or in transit to the endocytic recycling compartment (ERC). VAB-1::GFP localization to the RAB-11-positive ERC is antagonized by MSP signaling. Two negative regulators of oocyte meiotic maturation, DAB-1/Disabled and RAN-1, interact with the VAB-1 receptor and are required for its accumulation in the ERC in the absence of MSP/sperm. Inactivation of the endosomal recycling regulators rme-1 or rab-11.1 causes a vab-1-dependent reduction in the meiotic maturation rate in the presence of MSP/sperm. In addition, I show that Gás signaling in the gonadal sheath cells, affects VAB-1::GFP trafficking in oocytes. Taken together, our findings show that oocyte Eph receptor and somatic cell G protein signaling pathways control meiotic diapause in C. elegans, highlighting contrasts and parallels between MSP signaling in C. elegans and luteinizing hormone signaling in mammals. Moreover, my finding suggests that regulated endocytic trafficking of the VAB-1/Eph receptor contributes to the control of oocyte meiotic maturation in C. elegans. Eph receptor trafficking in other systems may be influenced by the conserved proteins DAB-1/Disabled and RAN-1 and by cross-talk with G-protein signaling in neighboring cells.
136

The role of alpha-endosulfine in the female meiotic cell cycle in Drosophila

Von Stetina, Jessica Rivera 29 December 2008 (has links)
CELL AND DEVELOPMENTAL BIOLOGY THE ROLE OF α-ENDOSULFINE IN THE FEMALE MEIOTIC CELL CYCLE IN DROSOPHILA JESSICA R. VON STETINA Dissertation under the direction of Professor Daniela Drummond-Barbosa Meiosis is coupled to gamete development and must be well regulated to prevent chromosomal abnormalities such as aneuploidy. During meiotic maturation, Drosophila oocytes progress from prophase I to metaphase I. The molecular factors controlling meiotic maturation timing, however, are poorly understood. My work in this thesis shows that Drosophila α-endosulfine (endos) plays a key role in this process. endos mutant oocytes have a prolonged prophase I arrest and fail to progress to metaphase I. This phenotype is similar to that of mutants of cdk1 and of twine, the meiotic homolog of cdc25, which is required for Cdk1 activation. I found that Twine and Polo kinase levels are reduced in endos mutants and identified Early girl (Elgi), a predicted E3 ubiquitin ligase, as a strong Endos-binding protein. In elgi mutant oocytes the transition into metaphase I occurs prematurely, but Polo and Twine levels are unaffected. These results suggest that Endos controls meiotic maturation by regulating Twine and Polo levels and, independently, by antagonizing Elgi. Interestingly, I also found that endos genetically interacts with Matrimony (Mtrm), which is a negative regulator of polo required to set the timing of meiotic maturation and the proper orientation of chromosomes. endos dominantly rescues the premature nuclear envelope breakdown defects observed in mtrm heterozygous females but enhances their chromosome misorientation defects, demonstrating that these processes are independent from each other. It is possible that the genetic interactions between endos and Mtrm occur via their effects on Polo, but other mechanisms are also conceivable. Finally, germline-specific expression of the human α-endosulfine ENSA rescues the endos meiotic defects and infertility, and α-endosulfine is expressed in mouse oocytes, suggesting potential conservation of its meiotic functions. Approved: Daniela Drummond-Barbosa
137

FG NUCLEOPORINS COORDINATE MULTIPLE TRANSPORT PATHWAYS THROUGH THE NUCLEAR PORE COMPLEX

Terry, Laura Jennings 30 December 2008 (has links)
Transport of nucleic acids and proteins between the nucleus and cytoplasm occurs exclusively through nuclear pore complexes (NPCs), large transport channels embedded in the nuclear envelope. Molecules larger than ~40kDa are largely excluded from moving through the NPC unless bound by a specialized transport receptor. Cargo macromolecules bind a transport receptor, which in turn interacts with a subset of NPC proteins containing phenylalanine-glycine (FG) repeat domains. Trafficking of a transport receptor-cargo complex through the NPC requires stochastic, low-affinity interactions between the transport receptor and FG repeat domains. More than a third of the ~30 NPC proteins harbor FG repeat domains, and each FG repeat domain potentially serves as a binding site for transport receptors at intermediate points in nucleocytoplasmic transport. Whether each of the ~15 transport receptors in yeast preferentially binds a subset of these FG domains is unresolved. In this study, we used genetic strategies in Saccharomyces cerevisiae to systematically delete (∆) combinations of FG domains. We assayed these FG∆ mutants for defects in trafficking of several different transport receptors, including a specific focus on messenger RNA (mRNA) export. We found that mRNA export and specific protein import receptors require different subsets of FG domains. This result indicates that there are multiple transport pathways through the NPC, each of which is defined by preferential binding of a transport receptor to a subset of FG domains. Additionally, we found that FG domains located on the nuclear side of the NPC contribute to recruiting the mRNA export receptor to the NPC. Further, FG domains positioned on the cytoplasmic side of the NPC might regulate terminal events in mRNA export. As a whole, these results suggest that each FG domain might play a regulatory role in mediating efficient movement of specific transport receptors through the NPC.
138

Endocardial cells are a distinct endothelial lineage derived from multipotent cardiovascular progenitors

Misfeldt, Andrew Michael 30 December 2008 (has links)
Identification of multipotent cardiac progenitors has provided fresh insight into the mechanisms of myocardial lineage specification, yet has done little to clarify the origin of the endocardium. Despite its essential role in heart development, the lineage classification of endocardium has remained undefined due to a lack of specific markers of this early vascular subpopulation. To distinguish endocardium from other vasculature, we generated an NFATc1-nuc-LacZ BAC transgenic mouse line that faithfully recapitulated endogenous NFATc1 expression in the heart and other cell populations during development. Utilizing this novel specific marker, the endocardium can be distinguished from other endothelial subpopulations, and tracked as it emerges from the cardiac mesoderm and participates in early cardiac morphogenesis. To further characterize endocardiogenesis, embryonic stem cells (ESCs) derived from NFATc1-nuc-LacZ blastocysts were utilized to demonstrate that endocardial differentiation occurs in a pattern consistent with its mesodermal origin and its close association with myocardium. Endocardium is specified as a cardiac cell lineage, independent from other vascular populations, responding to BMP and Wnt signals that enhance cardiomyocyte differentiation. Furthermore, a population of Flk1+ cardiovascular progenitors, distinct from hemangioblast precursors, represents a mesodermal precursor of the endocardial endothelium, as well as other cardiovascular lineages. Taken together, this work emphasizes the endocardium as a unique cardiac lineage and provides further evidence that endocardium and myocardium are derived from a common precursor. Furthermore, we have established a method to identify the endocardium at the onset of differentiation and throughout subsequent stages of development, providing the framework for future work to delineate mechanisms important in the ontogeny of this unique population.
139

BMP SIGNALING IN THE MORPHOGENESIS OF THE ESOPHAGUS AND TRACHEA

Li, Yina 28 February 2007 (has links)
The trachea and esophagus are respectively respiratory and digestive organs that originate from a common foregut endoderm during development. Perturbed patterning in this endodermal tissue can result in a variety of foregut anomalies, such as tracheal agenesis (TA), esophageal atresia (EA) and tracheoesophageal fistula (TEF); however, their etiologies remain unknown. We found that mice lacking Noggin displayed Type C EA/TEF, the most common form in humans, and notochordal defects strikingly similar to the adriamycin-induced rat EA/TEF model. In accord with esophageal atresia, Noggin-/- embryos displayed reduction in the dorsal foregut endoderm which was associated with reduced adhesion and disrupted basement membrane. However, no significant apoptosis in the Noggin-/- dorsal foregut was observed. Instead, non-notochordal, likely endodermal, cells were found in Noggin-/- notochord suggesting that Noggin function is required in the notochordal plate for its proper delamination from the dorsal foregut. Notably, ablating Bmp7 function in Noggin-/- embryos rescued EA/TEF and notochord branching defects, establishing a critical role of Noggin-mediated Bmp7 antagonism in EA/TEF pathogenesis. In addition to the critical role of Bmp signaling in EA/TEF pathogenesis, it also plays a vital role in tracheal development. We found that conditional ablation of Bmp4 in the primitive foregut by Foxg1Cre resulted in complete loss of trachea. While tracheal specification was not affected in Bmp4-deficient foreguts, tracheal outgrowth was severely impaired. The anterior foregut domain also displayed significantly reduced epithelial and mesenchymal proliferation without apparent alteration in apoptotic cell death. While we did not observe alteration in Wnt/â-catenin signaling in the Bmp4-deficient foregut, we detected consistent reduction in the expression level of Shh, a signaling molecule known to promote cell proliferation. In line with this observation, expression of Cyclin D1, a known downstream target of Shh signaling, was also found to be reduced in Bmp4- deficient foreguts. Taken together, these findings elucidate a critical role of Bmp signaling and cell proliferation in tracheal morphogenesis and implicate potential Bmp-Shh crosstalk in anterior foregut morphogenesis.
140

The Role of Rab11-FIP2 in Epithelial Cells

Ducharme, Nicole Annette 03 April 2007 (has links)
The small GTPase Rab11 family proteins have been implicated in the plasma membrane recycling system in such diverse model systems as H/K-ATPase trafficking in parietal cells to GLUT4 trafficking in heart and skeletal muscle. Rab11 family interacting protein 2 (Rab11-FIP2) was previously identified as binding to both Rab11a and the motor protein, myosin Vb. Therefore, we hypothesized that Rab11-FIP2 is a critical regulator of the plasma membrane recycling system. In these studies, we sought to characterize the role of Rab11-FIP2 function in polarized epithelial cells. To address the role of Rab11-FIP2, we uncovered a new role for Rab11-FIP2 in the establishment of polarity. We found that Rab11-FIP2 is phosphorylated by MARK2, and that this phosphorylation is necessary for the proper formation of the adherens junction. Next, we characterized a new dominant mutant Rab11-FIP2 mutant, which has unique effects on the recycling system than all previously characterized mutants. Analysis of the data suggests that FIP2 is involved in multiple stages in passage through the Rab11a associated recycling system. Multiple points of entry into the Rab11a/ FIP2 recycling system may be exploited depending upon the origin of the protein and possibly, its destination. This model supports a dynamic vision of the recycling system trafficking. We also analyzed new Rab11-FIP2 interacting proteins utilizing a novel approach of immunoprecipitation from stable cell lines overexpressing either wild type Rab11-FIP2 or its mutants followed by identification of the associated proteins using mass spectrometry. This proteomics approach revealed novel interactions with proteins known to be involved in trafficking (dynein and Rab10) in early endosomal membrane regulation (Rab5b and EpsinR), and vesicle coat proteins (AP-1 and clathrin heavy chain). Validation of these interactions proves that this methodology reveals robust interactions that are readily confirmed. All together, this body of work significantly advances our understanding of the diverse roles of Rab11-FIP2 in the regulation of epithelial cell function.

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