Browsing by Subject "Signaling"
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Item Identification and characterization of novel ADP-ribosyl cyclase family members(2011-08) Hirte, Renee MarieCD38 and CD157 have been identified as mammalian forms of ADP-ribosyl cyclase (ADPRC). Recently novel membrane bound and cytosolic ADPRCs that are distinct from CD38 and CD157 have been identified in various tissues by our laboratory as well as others. Recently, evidence has indicated the presence of ADPRC in tissues lacking CD38. From this it is clear that there are multiple forms of mammalian ADPRC, many of which have not yet been identified or characterized. The overall goal of the research presented in this thesis was to identify and characterize the novel cytosolic cyclase(s) present in the heart cytosol and determine the mechanism(s) by which cytosolic cyclase(s) are regulated. Previously it has been shown that Aplysia ADPRC, CD38 and CD157 share approximately 30% sequence identity. There are two main features shared by each of these members of the ADPRC family including a conserved region near the center and ten conserved cysteine residues that can be perfectly aligned. We used a molecular approach to identify potential candidates for the cytosolic protein (or other yet unidentified ADPRCs) based on a conserved protein motif search of the mouse genome to identify proteins that shared the feature of the conserved cysteine residues. The existence of novel cyclases has implications for a broad range of cellular processes that are influenced by calcium signaling. Characterization and identification of novel ADPRCs may provide key insight into the function of the novel cyclase(s) as well as interaction and relationships to other members of the ADPRC family, which will further elucidate the complexities of calcium signaling.Item Identification and characterization of three Arabidopsis sugar insensitive genes(2008-10) Huang, YadongCarbohydrates have signaling functions in regulating gene expression, metabolic pathways and developmental processes. Eukaryotic organisms have evolved conserved and novel mechanisms for sensing and responding to sugars. Plant sugar response pathways are complex and exhibit cross-talk with other response pathways. Sugar responses and signaling pathways have been studied via physiological, biochemical and genetic approaches. Genetic screens have identified sugar response mutants with altered seedling growth phenotypes. The Gibson lab has isolated an array of sugar insensitive (sis) mutants by screening mutagenized Arabidopsis seeds on high concentrations of sugars. The identification and characterization of three of the SIS genes, SIS7, SIS3 and SIS8, are presented here. SIS7 is allelic to NCED3/STO1, an abscisic acid (ABA) biosynthetic gene, which is involved in drought and salt stress responses. Lateral root (LR) development of sis7 mutants is resistant to the inhibitory effects of osmotica. Transcriptomic analysis revealed that a set of auxin-related genes are expressed at lower levels in sis7 seeds than in wildtype seeds when incubated with glucose, suggesting that these genes may be involved in controlling LR development by both ABA and auxin. SIS3 encodes a RING finger protein that functions as an E3 ligase in in vitro ubiquitination assays. The sis3 seeds display wild-type germination responses to ABA and GA. However, the root growth of sis3 mutants has slightly reduced sensitivity to ABA. The sis8 mutants have decreased sensitivity to high sugars and hyperosmolarity. Positional cloning of sis8 revealed that the mutation is in a putative mitogen-activated protein kinase kinase kinase gene. Seed germination assays indicate that sis8 mutants have wild-type sensitivity to ABA and GA, whereas overexpression of SIS8 causes slight hypersensitive responses. Potential interaction partners of SIS8 have been identified via yeast two-hybrid screening. A T-DNA insertion in the gene encoding one potential SIS8- interacting protein, UGT72E1, causes a sis phenotype. Further studies of the SIS3 and SIS8 genes will provide more insight into the mechanisms of sugar signaling in plants.Item Single cell analysis of bacterial communication and gene transfer by Enterococcus faecalis(2019-02) Erickson, RebeccaEnterococcus faecalis is a commensal member of the gastrointestinal tract of animals including humans but is also an opportunistic pathogen and a major cause of healthcare-associated infections. Its pathogenicity is thought to arise in immunocompromised people and after infection, treatment is difficult due to antibiotic resistance. E. faecalis is particularly good at transferring antibiotic resistance by mechanisms like conjugation and conjugative transfer of plasmids can occur at a high frequency without antibiotic selection. Conjugative plasmid pCF10 encodes tetracycline resistance and transfer between E. faecalis cells is facilitated by cell-to-cell communication. This signaling triggers expression of genes from pCF10 that encode for transfer machinery. The response to signaling is robust and has been extensively studied at the population level. However, it has recently become apparent that there is response variation. Understanding the mechanisms that underlie variation in response initiation is important to preventing transfer. Studies presented in this dissertation adapt fluorescence in situ Hybridization Chain Reaction (HCR) for single cell analysis of transcripts and explore questions about the pCF10 conjugation system that would not have otherwise been possible. In chapter 3, variation in the signaling response was assessed and the response was shown to be very heterogenous. When the level of signal is low, (like what might occur naturally), a minority of cells respond. Although stochasticity in the system may give rise to such heterogeneity, work in chapter 4 investigates the response impact of a few specific mechanistic players (PrgX, C, and I). Changing the levels of these components was shown to change the outcome. Lastly, single cell analysis was used in chapter 5 to assess the expression of genes required for conjugative transfer. These results show that the few responding cells commit to expression of all the genes encoding for production of the conjugation machinery. Overall, these results suggest that the pCF10 system is evolutionarily tuned for specific levels of each component and poised to have response variation for a population of cells. Thus, a small percent of cells can respond and since the majority of responding cells are able to conjugate, plasmid transfer is highly efficient. These results also exemplify how small differences in two cells can precipitate different responses in otherwise identical cells exposed to very similar conditions. Information about variation in the initiation of the signaling response required for pCF10 transfer is important to understanding the general biology of gene transfer among bacteria. In the future, this information will be important for successful design of effective interventions to the transfer of genes conferring antibiotic resistance.Item Stochastic fluctuations in signaling, gene control and pattern formation.(2011-09) Zheng, LikunStochasticity is inherent in biochemical systems. Noise can come from internal sources such as the random motion and reactions of molecules, and external sources such as environmental fluctuations. The main purpose of this thesis is to study how fluctuations propagate in biological systems. First, we focus on how a signaling molecule called a ligand searches for and binds to its target (receptor). There exist membrane proteins that can bind to the ligand molecule and localize it near receptors, affecting the association between it and the receptor. Our analysis shows that although the membrane protein can concentrate the ligand molecule near the receptor surface, the membrane protein has to pass the localized ligand molecule to the receptor fast enough, in order to enhance signaling. Otherwise, the membrane protein inhibits signaling. Moreover, we also study the effect of localization on signal specificity. In particular, we discuss how the membrane proteins bind to ligand molecules and distribute them to different downstream signaling pathways. Upon ligand binding to receptors, bound receptors can initiate the downstream network, which may finally lead to gene expression. We then study how the noise from the initiation step of transcription propagates in the elongation step. Elongation can be interrupted by the pauses of the transcription complex on the DNA sequence. We give a condition under which the pause of the transcription complex can cause bursts of mRNA production. Finally, we use stochastic simulations to study dorsal-ventral patterning in Drosophila numerically. Our results indicate that a feedback loop can stabilize the determination of the amnioserosa boundary. We then propose a detailed single cell system for the downstream network in nuclei. Our analysis of time scales of reactions and molecular transport shows the phosphorylation of Mad and transport of mRNA across the nuclear membrane are the major limiting steps in the signal transduction pathway. Simulations results show noises are amplified at these limiting steps.