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Tales of three signaling pathways: EGFR, TGFB, and WNT signaling in the GI tractTanksley, Jarred Paul 29 July 2013 (has links)
My study of three signaling pathways in the context of three gastrointestinal diseases is presented here. Firstly, the epidermal growth factor receptor (EGFR) pathway and its role in the pathogenesis of the hypertrophic gastropathy Ménétrier's disease will be introduced. I will discuss the results of a clinical trial demonstrating that cetuximab, an EGFR pathway inhibitor, is the first effective medical therapy against Ménétrier's disease. I will then introduce the transforming growth factor-β (TGFB) pathway, and its role in the disorder juvenile polyposis syndrome (JPS). I will compare the clinico-pathological presentation of JPS to that of Ménétrier's disease, and suggest that there is a role for aberrant EGFR signaling in the pathogenesis of JPS. Finally, I will discuss my studies of the canonical WNT signaling pathway in the context of colorectal cancer (CRC). This part of the dissertation will center on my findings that the E3 ubiquitin ligase, NEDD4L, is a canonical WNT pathway inhibitor that is downregulated early in CRC, and may serve a tumor-suppressive role in CRC.
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INVESTIGATING THE MECHANISM OF GABA NEURON DEGENERATION IN A MODEL OF COENZYME Q DEFICIENCYHacker, Mallory Louise 11 July 2013 (has links)
Neurodegenerative diseases are characterized by inappropriate death of distinct neuronal populations. Although symptoms vary, these diseases share pathogenic features such as age-dependent onset and progressive death of specific neuronal subtypes. Additionally, dysfunction of mitochondria (essential energy-providing organelles) is widely associated with neurodegenerative disorders. Mitochondria are also key regulators of cell death pathways, further underscoring the need to understand how this organelle contributes to disease pathogenesis.
This dissertation characterizes a novel neurodegeneration pathway induced by Coenzyme Q (CoQ) deficiency in C. elegans. CoQ depletion results in an age-related loss in motor coordination and selective degeneration of GABA neurons. My work establishes C. elegans as a model to study CoQ deficiency in the adult nervous system. Age-related onset, selective neuronal vulnerability and mitochondrial dysfunction are features of neurodegenerative diseases that are also prominent in our model of CoQ deficiency.
This study also describes aspects of human CoQ deficiency that are conserved in C. elegans.
My work supports a model of CoQ transport between tissues. This experimental paradigm has translational implications that could provide a genetic system to study regulators of CoQ uptake. Additionally, I show that localization and function of human CoQ biosynthetic enzymes is conserved in C. elegans. Together, this work emphasizes the strength of using C. elegans as a genetic model to study mechanisms that control CoQ uptake and function.
I further investigate the mechanism of GABA neurodegeneration and discover novel roles for canonical apoptotic regulators. In response to CoQ depletion, GABA neurons activate a death pathway that requires CED-4/Apaf-1 but is antagonized by CED-3/caspase. This finding indicates that these components of the core cell death machinery adopt alternative roles in a degeneration pathway arising from CoQ depletion.
I also address the role of necrotic proteases in this neurodegeneration pathway. This work revealed that the CoQ depletion-induced death pathway is controlled by proteases with death-promoting or death-preventing functions. Lastly, ultrastructural analysis identified features of necrosis in CoQ-deficient neurons. Together my work provides a foundation for studying neuronal responses to mitochondrial dysfunction.
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Genetic Analysis of Ninjurin A, A stress-regulated potein that induces nonapoptotic cell deathBroderick, Sarah M 19 April 2013 (has links)
Ninjurins are a conserved family of transmembrane proteins that increase expression in response to injury and stress. There are few in vivo studies of Ninjurin, and little is known about Ninjurin function. In this thesis I investigated the immune function of a recently generated null mutant of NijA, which did not display a detectable phenotype. Expression studies of the NijA protein show redistribution to the cell surface in larval immune tissues after septic injury. The NijA protein is also upregulated by constitutive activation of the Toll pathway. Ectopic expression of NijA induced cell death. These dying cells appeared to die by nonapoptotic cell death because they did not display hallmarks of apoptotic cells including TUNEL staining and inhibition by p35. Preliminary studies investigating UV-irradiation induced cell death in the NijA null mutant suggests that NijA maybe required to induce stress-activated cell death. These results suggest a role for NijA in stress-activated nonapoptoic cell death.
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NF-κB INTERACTS WITH SP3 TO LIMIT SP1-MEDIATED FGF-10 EXPRESSION IN THE DEVELOPING FETAL LUNGCarver, Billy Joe 18 September 2013 (has links)
Arrested lung development in preterm infants leads to bronchopulmonary dysplasia (BPD). Inflammation and NF-κB activation in the fetal lung inhibit airway morphogenesis and contribute to BPD. The mesenchymal growth factor FGF-10 is crucial for normal airway branching and is decreased in lungs of patients with BPD. I therefore hypothesized that when activated, NF-κB disrupts normal FGF-10 transcription. I discovered that FGF-10 is in fact downregulated by NF-κB activation, but in an indirect manner. Further analysis of the FGF-10 promoter revealed many sites consistent with regulation by Sp proteins. Experiments revealed that Sp1 and Sp3 also regulate FGF-10; Sp1 activates transcription, while Sp3 downregulates Sp1-mediated expression. I further discovered that Sp3 and the NF-κB dimer interact during FGF-10 suppression, suggesting that NF-κB recruits Sp3 to the FGF-10 promoter. This is a novel mechanism of gene regulation in the developing lung.
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BVES FUNCTION IN EPITHELIAL MOVEMENT DURING DEVELOPMENTRipley, Anna Nesset 29 March 2004 (has links)
I will introduce two novel genes isolated by my laboratory, hole and bves. This document will show that the benefits of gene discovery are invaluable. Bves, a novel family of cell adhesion molecules have been identified and my dissertation will be the first illustration of a function of this molecule in vivo. Data derived from this dissertation provide strong in vitro and in vivo evidence that bves plays an adhesive role in epithelial adhesion and morphogenetic movements during gastrulation in Xenopus and eye morphogenesis. Though gastrulation and eye development occur at different times during development and result in two very different structures, they serve as examples of a fundamental role Bves serves throughout embryogenesis. Xbves is expressed in a distinct group of epithelial and migrating cells in the Xenopus embryo. In vivo studies in the Xenopus embryo show that Xbves is required for proper migration of epithelial animal cells. The same migration defect is seen in an in vitro model of corneal epithelial cells. Combined with previous data that show bves participates in epithelial to mesenchymal transition in epicardium and coronary artery development, as well as data that show bves expression across many tissues, the data presented here support the idea that one gene product can participate in a variety of developmental processes. This study is an important contribution to understanding the overall larger concept of how bves functions in embryogenesis and the adult. Taken together, these studies have shown that Bves is important in proper epithelial cell migration and morphogenesis.
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Peroxisome Proliferator-Activated Receptor gamma (PPARg) is a Regulator of Colorectal Cancer Cell Growth and DifferentiationGupta, Rajnish Anand 08 April 2004 (has links)
Peroxisome prolferator-activated receptor g (PPARg) is a member of the nuclear hormone receptor superfamily and is ligand activated by polyunsaturated fatty acids, certain arachadonic acid metabolites, and class of synthetic compounds with insulin sensitizing activity known as thiazoidinediones. PPARg is strongly expressed in the post-mitotic epithelial compartment of the normal human large intestine. Exposure of a panel of human colorectal cancer cell lines to PPARg agonists leads to a decrease in cell growth that is associated with a partial G1 arrest and increased levels of the cyclin dependent kinase inhibitor p21. A subset of these cells lines were resistant to the growth inhibitory effects of PPARg ligands. All four of the resistant lines contained a point mutation at codon 422 of the ligand binding domain of PPARg. Further studies suggested this mutation leads to a loss of functional PPARg due to a defect in the ability of the receptor to repress the transcrption of some target genes in the absence of exogneous ligand. Microrray technology was used to understand the genomic response of intestinal epithelial cells exposed to PPARg agonists. PPARg selective target genes included proteins linked to regulation of cell growth, colon epithelial cell maturation, and immune modulation. One of these genes, Transforming Growth Factor b Clone-22 (TSC-22), is a leucine zipper containing transcription factor that is capable of repressing gene transcription. Inhibition of TSC-22 using a dominant-negative construct partially blocked the ability of PPARg to induce growth arrest. In summary, these studies collective demonstrate that PPARg is a regulator of colorectal cancer cell growth and differentiation.
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EXPRESSION OF DUB-1 AND DUB-2 AND ANALYSIS OF A ROLE FOR DEUBIQUITINATION IN THE REGULATION OF NUCLEAR FACTOR-KAPPA BStrayhorn, William David 08 April 2004 (has links)
The ubiquitin-proteasome pathway (UPP) is the principal mechanism for the selective degradation of short-lived proteins. The proximal signal for UPP-mediated proteolysis is the covalent modification of target proteins with multiple ubiquitin polypeptides. Protein-ubiquitin conjugation is catalyzed by ubiquitinating enzymes, which assemble the polyubiquitin degradation signal on a target protein. It is postulated that removal of ubiquitin by deubiquitinating enzymes may also regulate protein targeting to the UPP. However, in comparison to ubiquitinating enzymes, relatively little is known about the functions or regulation of deubiquitinating enzymes. Dub-1 and Dub-2 are closely related deubiquitinating enzymes that were initially identified in hematopoietic cell lines as cytokine-inducible proteins. To gain insights into the substrate(s) and function(s) of the Dub enzymes, I examined the expression of Dub-1 and Dub-2 mRNA and protein, and investigated a potential role for these enzymes in regulation of signal transduction through the nuclear factor-kappa B (NF-kB) family of transcription factors. Expression analyses indicated that Dub-1 is expressed in the developing murine limb bud and in interleukin-3-stimulated FL5.12 pro-B cells. To investigate whether the principal inhibitory protein of NF-kB, IkBa, is a substrate for Dub-1 and Dub-2, a novel in vitro deubiquitination assay was established using polyubiquitinated IkBa as the substrate. In addition, I provide evidence for an IkBa-directed deubiquitinating activity in cytoplasmic lysates from a panel of cell lines. Using this and other complementary assays, I show that Dub-1 and Dub-2 do not deubiquitinate IkBa, do not stabilize IkBa, and do not modulate NF-kB activity. In addition, I show that Dub-1, but not the closely homologous Dub-2, is degraded via the UPP in HEK-293T cells. The UPP-mediated degradation of Dub-1 does not require an intact Dub-1 catalytic domain, thus indicating that this process does not proceed via Dub-1-catalyzed transfer of ubiquitin from a substrate to itself. Overall, these studies provide valuable insights as to the regulation of Dub-1 and Dub-2 that may help elucidate the substrate(s) and biological role(s) of these enzymes. Furthermore, the reagents generated for this dissertation will be useful for the study of Dub biochemistry and IkBa deubiquitination.
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THE PCH FAMILY PROTEIN, CDC15P, INTERACTS DIRECTLY WITH TWO ACTIN NUCLEATION PATHWAYS TO CONTRIBUTE TO CYTOKINETIC ACTIN RING FORMATION IN SCHIZOSACCHAROMYCES POMBECarnahan Jr., Robert Herschel 10 November 2003 (has links)
Cytokinetic actomyosin ring formation (CAR) in S. pombe requires two independent actin nucleation pathways, one dependent on the Arp2/3 complex and another involving the formin Cdc12p. Here we investigate the role of the PCH family protein, Cdc15p, in CAR assembly and find that it interacts with proteins from both of these nucleation pathways. Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants. Cdc15p also binds directly to Cdc12p. Cdc15p and Cdc12p not only display mutual dependence for CAR localization, but also exist together in a ring nucleating structure prior to CAR formation. The disruption of these interactions in cdc15 null cells is likely to be the reason for their complete lack of CARs. We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.
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FORKHEAD TRANSCRIPTION FACTOR REGULATES PROSTATE-SPECIFIC GENE EXPRESSION AND PROSTATIC MORPHOGENESIS: A FUNCTIONAL INTERACTION WITH ANDROGEN SIGNALINGGao, Nan 15 October 2004 (has links)
Comparative genome analysis implied that a progressively more elaborate regulation of gene expression rather than invention of new genes is responsible for the organismal complexity, and that a constrained organization of metazoan enhancers is essential for the precise patterns of gene expression during development. This project was focused on studying of the transcriptional regulatory complex that directs tissue-specific gene expression and organ development in the prostate.
Genetic manipulation of the rat probasin (Pbsn) gene promoter in transgenic mice uncovered a ~150 base pair core DNA fragment, which confers prostate-selectivity to the expression of this gene. Among a cluster of DNA sequence-specific transcription factors directly binding to this region, there are two cell-type limited proteins: the androgen receptor, a nuclear receptor, and Foxa1, a forkhead protein. Reporter assays indicated the forkhead response elements are crucial for AR-mediated transcriptional regulation. In vitro and in vivo binding assays established that the two proteins can bind adjacent DNA sequence concomitantly while the binding is not interdependent. A physical interaction occurs between Foxa1 and AR when the ligand for AR is present. This interaction is mediated through the forkhead domain and the AR DNA binding domain. Alignment of the forkhead and the androgen response elements resulted in the identification of a shared cis-regulatory code that is present in a variety of prostate-specific enhancers across species.
The impact of Foxa1 loss-of-function and haploinsufficiency on mouse prostatic organogenesis was analyzed. Foxa1-/- prostates showed drastic morphologic alteration including a disorganized epithelial pattern resembling primitive epithelial cords, and an expansion in the mesenchymal smooth muscle layer. Cell type and ultrastructural studies demonstrated that Foxa1-/- epithelium is predominantly arrested as immature basal cells, consistent with a failure of luminal determination. Foxa1-/- basal cells actively express Sonic hedgehog (Shh), Ptc1 and Foxa2, proteins that are normally elevated during embryonic ductal budding in the UGS. Alteration of these signals correlates with the expansion of precursor cells and the modified epithelial-stromal pattern in Foxa1-/- prostates. In contrast, expression of the Nkx3.1 homeobox protein is absent in these Foxa1-/- cells. In addition a haploinsufficient phenotype was observed in heterozygous dorsal prostates showing a similar ductal morphologic defect. A novel Foxa1 target gene was identified. Upon examination of its promoter, critical forkhead response elements were identified to be immediately flanked by androgen response elements. We propose that Foxa1 regulates genes involved in prostatic ductal morphogenesis, and promotes epithelial cell maturation through balancing the effects of Shh, Foxa2, and Nkx3.1.
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The role of Nuclear Factor-kappa B (NF-kB) in the regulation of lung inflammation.Everhart, Michael Brett 01 December 2004 (has links)
The NF-kappaB pathway has been shown to play a critical role in both adaptive and innate immunity and has been implicated as a focal point for induction of lung inflammation by a variety of inflammatory stimuli; however, the role of NF-kappaB in specific lung cell types remains unclear. We hypothesized that individual cell types in the lungs make important and unique contributions to the NF-kappaB dependent innate immune response. To determine the temporal and cell specific activation of NF-kappaB in vivo, an NF-kappaB reporter mouse in which expression of an enhanced green fluorescent protein (eGFP)/luciferase fusion protein cDNA driven by an NF-kappaB inducible promoter (NGL mouse) was generated. NF-kappaB activity was detected in intact, anesthetized animals by bioluminescence imaging and at the cellular level by detection of GFP on lung tissue sections. Using Eschericia coli lipopolysaccharide (LPS) and Pseudomonas aeruginosa models of lung inflammation, the timing and duration of NF-kappaB activation in alveolar macrophages, neutrophils, airway epithelium, and endothelium was determined to be dependent on the dose, duration, and route of delivery. To determine the role NF-kappaB in alveolar macrophages, bone marrow chimeras were generated by fetal liver transplantation (FLT) using the HLL transgenic NF-kappaB reporter mouse (HIV-LTR driving expression of luciferase) as the recipient and WT, IkappaB-alpha heterozygous, IkappaB-alpha knockout, and p50 knockout mice as donors. After LPS administration, lung NF-kappaB activation (bioluminescence imaging) and inflammation (neutrophil influx) were detected. IkappaB-alpha heterozygous FLT chimeras displayed enhanced and prolonged NF-kappaB activation and inflammation compared to WT FLT chimeras, while IkappaB-alpha knockout FLT and p50 knockout FLT chimeras displayed severe NF-kappaB activation and inflammation accompanied by mortality. To study macrophage/epithelium communication, co-culture studies using bone marrow derived macrophages from the above donors and epithelial cells containing an NF-kappaB reporter were performed. After LPS treatment, IkappaB-alpha knockout and p50 knockout macrophages demonstrated prolonged NF-kappaB activation, compared to WT, resulting in sustained NF-kappaB activation in epithelial cells. Taken together, these studies provide important insight into the importance of the NF-kappaB pathway in regulating lung inflammation and suggest IkappaB-alpha and p50 play a critical role in the active turn-off of NF-kappaB.
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