Cellular differentiation is the cornerstone of metazoan development. Cell-cell signaling mechanisms are responsible for the specification of many cell fates. The response of a particular cell to a given signal is highly context dependent allowing signaling mechanisms to be reused to produce a variety of different outcomes. The EGFR and LIN-12/Notch signaling pathways are well-conserved across metazoan species and govern many fate-specification events. The specification of C. elegans Vulval Precursor Cells (VPCs) offers a powerful system to investigate how these signaling mechanisms specify cell-fates, and previous studies of VPC fate patterning have identified several forms of crosstalk between these two critical signaling mechanisms. In this thesis, I investigate how input from both the EGFR and LIN-12/Notch signaling pathways is integrated by the VPCs. I provide evidence that VPCs respond to the relative levels of LIN-12/Notch and EGFR signaling. I show that LIN-1/Elk1 is critical for VPCs to adopt discrete cell fates. In addition, I show that the Mediator components SUR-2/Med23 and the CDK-8 kinase module (CKM), in cooperation with LIN-1/Elk1, are required for an EGFR-mediated resistance to LIN-12/Notch activity. I also used CRISPR/Cas9 techniques to generate endogenous, fluorescently-tagged LAG-1 proteins. Characterization of tagged LAG-1 accumulation in the VPCs and in the somatic gonad show that LAG-1 is present in all VPCs at low levels in a lin-12/Notch independent manner. Activation of LIN-12/Notch is correlated with higher levels of LAG-1 accumulation compared to cells that do not have activated LIN-12/Notch. These findings suggest a potential autoregulation mechanism for lag-1 in certain contexts. They also suggest that endogenously tagged LAG-1 may be a useful molecular marker of LIN-12/Notch activation.

Notch signaling controls normal and pathological angiogenesis through transcriptional regulation of a wide network of target genes. Despite intensive studies of the endothelial Notch function, a comprehensive list of Notch-regulated genes, especially direct transcriptional targets, has not been assembled in endothelial cells (ECs). Here we uncovered novel EC Notch targets that are rapidly regulated by Notch signaling using several unbiased in vivo and in vitro screening approaches that captured genes regulated within 6 hours or less of Notch signal activation. We used a gamma-secretase inhibitor in neonates to profile Notch targets in the brain endothelium using the RiboTag technique, allowing for isolation of endothelial specific mRNA from a complex tissue without disrupting cell-cell contact. We used two types of primary cultured endothelial cells to define ligand-specific Notch targets by tethered-ligand stimulation. The identified Notch targets were validated by determining their regulation within one to two hours of EGTA-mediated Notch activation. By comparing significantly regulated genes in each of the screens, we assembled a comprehensive database of potential Notch targets in endothelial cells. Of particular interest, we uncovered G protein pathway related genes as potential novel Notch targets. We focused on a novel candidate target passing selection criteria after all screens, a small GTPase RND1. RND1(Rho GTPase1) regulates cytoskeleton arrangement through Rho and Ras signaling. RND1 was validated as an endothelial Notch target in multiple endothelial cell types. In Human Umbilical Vein Endothelial Cells (HUVECs) we established angiogenic activity for RND1 that included regulation of cell migration towards VEGF and function in sprouting angiogenesis. We established that Notch and RND1 suppressed Ras activation but had no effects on Rho activation in HUVECs. These results demonstrate that RND1 expression is regulated by Notch signaling in endothelium and suggest that RND1 functions downstream of Notch in sprouting angiogenesis, revealing an unexplored role of endothelial Notch in regulating G protein pathways.

The Major Histocompatibility Complex class II B (MHC II B) gene encodes a protein that is part of the adaptive immune system and critical for the non-self recognition ability of immune cells. This gene has been characterized in the Bald Eagle, ten unique alleles were found in two subpopulations at the geographic extremes of the range margins. Geographic genetic variation is suggested by the presence of population specific alleles. The results showed considerable divergence of groups of Bald Eagle alleles when compared to alleles from other birds of prey. Particular codons within the exon II show signs of balancing selection driving the evolution of the MHC II B. Transcription data showed statistically significant differential expression of alleles. This can be interpreted as meaning a particular locus is being preferentially expressed in blood. The analysis of the polymorphism of this adaptive marker may aid managers of wildlife during this age of global climate change and the biodiversity crisis. / by Andrew Smith. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

In this thesis, we propose to discover co-regulated genes using microarray expression data, as well as providing visualization functionalities for domain experts to study relationships among discovered co-regulated genes. To discover co-regulated genes, we first use existing gene selection methods to select a small portion of genes which are relevant to the target diseases, on which we build an ordered similarity matrix by using nearest neighbor based similarity assessment criteria. We then apply a threshold based clustering algorithm named Spectral Clustering to the matrix to obtain a number of clusters. The genes which are clustered together in one cluster represent a group of co-regulated genes and to visualize them, we use Java Swings as the tool and develop a visualization platform which provides functionalities for domain experts to study relationships between different groups of co-regulated genes; study internal structures within each group of genes, and investigate details of each individual gene and of course for gene function prediction. Results are analyzed based on microarray expression datasets collected from brain tumor, lung cancers and leukemia samples. / by Vaibhan Lad. / Thesis (M.S.C.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Biotic stress is one of the most serious constraints on rice productivity. Strategy adopting regulators in signal transduction of systemic acquired resistance for conferring long-lasting disease resistance against broad spectrum of pathogens become highly favorable. To achieve this, signal transduction of disease resistance should be well characterized. / OsGPBP1 is a putative G-protein binding protein and interacts with a member of the YchF G-protein subfamily that has not been thoroughly studied in plants, while OsRHC1 is a novel RING zinc finger protein harboring multiple transmembrane domains at the N-half and a unique RING-HC domain at the C terminus. Both of them were induced in the bacterial blight resistant near isogenic rice line upon wounding. Gain-of-function tests in transgenic Arabidopsis thaliana showed that their ectopic expressions are able to trigger the expression of both defense marker genes mediated either by SA- or JA/ET-pathways and led to increased resistance toward the pathogen Pseudomonas syringae pv. tomato DC3000 and both of the two clones seemed to rely on NPR1 (disease resistance key regulator) for function. Furthermore, over-expressions of the two clones in its native system are also able to activate rice defense marker genes. / Suppression subtractive hybridization experiment, using RNA samples from a pair of near-isogenic rice lines either containing the R gene Xa14 (CBB14) or its susceptible recurrent parent (SN1033), were previously performed in our laboratory. Two gene candidates ( OsGPBP1 and OsRHC1) probably encoding two novel types of signal transduction components related to disease resistance are chosen for further analysis. / Cheung, Ming Yan. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4555. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 148-168). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The Hippo pathway is a master regulator of cell proliferation and organ size, namely through regulation of transcriptional co-activators YAP and TAZ which bind TEAD1-4 transcription factors. The Hippo effector YAP is dysregulated in many human solid tumours including rhabdomyosarcoma and oesophageal cancer. Additionally, persistent hyperactivity of YAP in activated but not quiescent satellite cells can give rise to embryonal rhabdomyosarcoma. However, the question of exactly how YAP acts as an oncogene and actively gives rise to tumour progression in these cancers remains unknown. In this thesis I characterised the mechanisms which determine the functional role of YAP in driving instability in the genome. Secondly, lentiviral mediated knockdown of YAP is performed to determine and investigate its effect on tumorigenesis. Thirdly, gene sets from constitutive YAP S127A induced mouse ERMS tumours subjected to array-CGH were further analysed. Finally, I cloned chicken Yap1, Tead1 and Fstl5 to identify its role during chick embryonic development, by the retroviral mediated loss of function approach. The results demonstrated that constitutive YAP S127A expression in-vitro as well as in-vivo induces chromosomal instability by increasing the rate of mitotic chromosome segregation errors and copy number alterations of oncogenes and other cancer related genes. Recurrent copy number gains of the p53 inhibitor Mdm2 were observed in YAP S127A-driven ERMS tumours. Moreover, lentiviral mediated YAP knockdown showed significant reduction in proliferation, migration and invasion as well as transformation potential in human cultured cancer cells. Moreover, retroviral YAP S127A expression during early stages of chick embryo development did not lead to an overt phenotype and showed poor survival. Additionally, I have cloned RCAS-RNAi vectors to study the loss of function effect on Hippo targets and Fstl5 during chicken embryo development. Collectively, my data provides insight into the mechanisms with which YAP could drive tumorigenesis and that YAP knockdown can be considered a potential therapeutic target to reduce cancer progression.

The goal of this dissertation research is to determine novel regulatory mechanisms of neurotrophin signaling mediated by protein phosphatase 2A (PP2A). PP2A is a ubiquitous Ser/Thr phosphatase that removes phosphates from proteins to switch their activity on or off. The substrate specificity and subcellular localization of PP2A is determined by almost 20 regulatory subunits that associate with a core dimer built of catalytic and scaffold subunits. Since there are more than 48 possible heterotrimers, studying the function of PP2A poses many challenges. Therefore we have devised a strategy, using scaffold subunit knockdown and mutant replacement, to discern the function of specific families of regulatory subunits. With this approach, I have identified specific PP2A holoenzymes that modulate nerve growth factor (NGF) signaling pathways by positively regulating TrkA receptor tyrosine kinase activity. Many studies have shown that NGF is required for the survival and differentiation of sensory and sympathetic neurons. Additionally, NGF is implicated in many neurodegenerative diseases including Alzheimer's disease, Parkinson's disease as well as neuropathic pain. NGF elicits its biological effect through sustained activity of the TrkA receptor and stimulated signaling cascades, including the MAP kinase pathway. Although PP2A has been shown to modulate the mitogen-activated protein (MAP) kinase pathway both positively and negatively at multiple levels, work described herein introduces yet another level of regulation. Specifically, I have shown that PP2A/B' holoenzymes complex with the TrkA neurotrophin receptor to potentiate receptor tyrosine kinase activity, downstream effector kinase activation, neurite outgrowth, and neuronal differentiation. On the other hand, extracellular signal regulated kinase (ERK), a terminal effector in the MAP kinase pathway was shown to phosphorylate a residue in the juxtamembrane region of TrkA and impose feedback inhibition of receptor activity. Collectively, these data suggest a model in which PP2A and ERK oppose each other in the regulation of TrkA receptor activity and downstream signaling cascades that govern neuronal differentiation and maintenance.

Erythropietin (Epo), acting through its receptor (EpoR), is an essential hemotopietic growth factor that regulates the proliferation, differentiation, and survival of erythroid progenitor cells. Perturbations of Epo/EpoR function cause myeloproliferative disease, such as erythrocytosis, or myelodeficient disease, such as anemia. Therefore, defining the mechanisms by which Epo/EpoR control proliferation and differentiation of erythroid cell lineages attracts interest. Ubiquitin-dependent internalization and degradation is a common regulatory mechanisms affecting signaling from a variety of receptors. Although EpoR has been found to be ubiquitinated, the function of EpoR ubiquitination in the regulation of Epo signaling remains unclear. Therefore, the primary goal of this study was to define the role of EpoR ubiquitination in regulating Epo signaling activities and erythroid cell growth. We showed that EpoR was ubiquitinated in response to ligand stimulation, and that loss of EpoR ubiquitination reduced signaling activity and biological responses to low dosages of Epo. We also identified two EpoR lysines that were the primary targets for ubiquitination, and showed that either ubiquitination site supported the enhanced activities of wild-type-EpoR. Ubiquitination of EpoR was also associated with a change in the endocytic pathway mediating internalization of EpoR. Specifically, constitutive internalization of non-ubiquitinated EpoR was found to depend on dynamin activity, while internalization of ubiquitinated EpoR was dynamin-independent but could be inhibited by disrupting lipid raft microdomains in the plasma membrane. Interestingly, inhibiting internalization of ubiquitinated EpoR (by disrupting lipid rafts) specifically reduced signaling from ubiquitinated receptors without affecting signaling from non-ubiquitinated receptors. Conversely, reducing internalization of non-ubiquitinated EpoR (by inhibiting dynamin) reduced its signaling activity without affecting signaling from ubiquitinated receptors. This strong correlation between EpoR internalization and signaling activity suggests a novel regulatory mechanism in which internalization of EpoR facilitates its signaling activity. In this regard, Epo-induced ubiquitination of EpoR promotes more efficient internalization of ligand-activated receptor and may contribute to enhanced responsiveness to low concentrations of Epo.

The Gram positive soil dwelling bacteria Bacillus subtilis produces spores when encountered with a low nutrient environment. However, B. subtilis can delay spore production by a mechanism known as cannibalism. Cannibalism is a process by which B. subtilis delays commitment to sporulation by killing a subpopulation of its cells. This process involves production of two toxins, SDP and SKF. SDP is a 42 amino acid peptide with a disulfide bond derived from the internal cleavage of its precursor protein pro-SdpC. pro-SdpC is part of the sdpABC operon. Production of extracellular SDP induces expression of the sdpRI operon. Encoded in this operon is the negative regulator SdpR and SdpI. SdpI is a dual function protein which acts both as a signal transduction protein and the immunity factor against SDP. The current model states that production of SDP is sensed via SdpI. SdpI will sequester SdpR to the membrane in response and allow for sdpRI expression. The aims of this dissertation are to establish the requirements for SDP production and its response via SdpI/SdpR during cannibalism. Studies in Chapter II were carried out to determine the factors required for production of the antimicrobial peptide SDP. Site directed mutagenesis of the leader signal peptide sequence in pro-SdpC demonstrated that proper signal peptide cleavage was required for SDP production. Additional site directed mutants of the cysteine residues in pro-SdpC revealed that these are not required for SDP toxic activity. These studies also included deletions within the sdpABC operon and revealed that the two proteins of unknown function, SdpA and SdpB are required for SDP production. Using mass spectrometry analysis, we found that SdpA and SdpB together are required to produce the active 42 amino acid peptide SDP. Taken together we concluded that SDP production was a multi step process which required proteins encoded within the operon and additional processing supplemented in the cell. In Chapter III we investigated the role of SdpI, specifically what residues were required for the signaling and immunity functions observed. Our initial screen, included site directed mutagenesis of highly conserved residues between the 4th and 5th transmembrane domains of SdpI. These resulted in over 20 SdpI mutants generated. From these, only two SdpI mutants had defects in either signal transduction or SDP immunity. Additional localized mutagenesis was used to isolate two other mutants in SdpI which only affected signal transduction or SDP immunity. SdpI signaling-immunity+ mutants presented a defect in SdpR membrane sequestration and sdpRIinduction. Our findings suggest these types of SdpI mutants may be important for the downstream effect of SdpR membrane sequestration. SdpI signaling+ immunity- mutants revealed defects in SDP protection. Some of the residues mutated were conserved in other SdpI homologs. Site directed mutagenesis of these conserved residues in the SdpI ortholog YfhL showed these are also required for SDP resistance. For the first time, we were able to identify mutations which affected only SDP immunity and gained further insight into how SdpI signaling-immunity+ mutants play a role during signal transduction. In Chapter IV we initiated studies to define what regions of the negative regulator SdpR are important for its function during cannibalism. We employed localized mutagenesis to identify SdpR mutants which decreased sdpRIexpression even in the presence of inducing signal. We isolated three such SdpR mutants, referred to as super repressors. We expect these SdpR super repressors are unable to be sequestered to the membrane in the presence of SDP.

No description available.