Several published functions associated with the CHK1 histidine kinase of Candida albicans resemble those of the MAPK Cek1p and its cognate receptor Sho1p (SSU81). To explore this further, we have compared mutants lacking the proteins mentioned above and have constructed a double sho1/chk1Δ null mutant to determine relationships among these proteins. We observed that the sensitivity to Congo red (CR), calcofluor white (CW), as well as clumping of cells, was slightly increased in the double mutant compared to the single chk1Δ or sho1Δ mutants. However, Cek1p phosphorylation via Sho1p, which occurs during log phase growth in the presence or absence of CR in Wt cells, does not require Chk1p. These data suggest that Chk1p and Sho1p are components of parallel but independent signal pathways. In addition, bulk mannan of strains was analyzed by GLC/MS and GPC MALLS and NMR. Compared to Wt and a CHK1 gene-reconstituted strain (CHK23) that contained high, intermediate and low Mw mannan species, we found that the mannan of strains CHK21 (chk1Δ null), the cek1Δ null, and the double mutant consisted only of low Mw mannan. The sho1Δ null mutant only demonstrated a reduced intermediate type of mannan. Alcian blue binding was lower in cek1Δ, chk1Δ, and the double sho1/chk1Δ null mutant lacking high and intermediate Mw mannan than in the sho1Δ null which had a partial loss of intermediate Mw mannan only. We conclude that the Chk1p HK is part of a functionally similar but parallel pathway to the Sho1p-Cek1p pathway that confers resistance to the cell wall inhibitors CR and CW. However, a functional relationship in mannan biosynthesis of Chk1p and Cek1p exists that only partially requires Sho1p.

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The LIN-12/Notch signaling pathway is highly conserved in all animals, and is crucial for proper development. It is a key pathway in specifying cell fate in many cellular contexts, and dysregulation of the pathway can have deleterious consequences. Therefore, understanding how LIN-12/Notch signaling is regulated in different contexts has been a main area of interest in the field. Previous studies in different model organisms have identified many modes of regulation of the signaling pathway, one of which is endocytosis of the ligand and receptor. Here, I further investigated the role of endocytosis in LIN-12/Notch signaling in multiple developmental contexts in Caenorhabditis elegans. Work in Drosophila and vertebrates had previously established that ligand-mediated activation of Notch requires ubiquitination of the intracellular domain of the transmembrane ligand and the activity of the endocytic adaptor Epsin in the signaling cell. The consensus in the field is that Epsin-mediated endocytosis of mono-ubiquitinated ligand generates a pulling force that exposes a cleavage site in Notch for an ADAM protease, a critical step in signal transduction. In contrast, in this thesis, I examined two different transmembrane ligands in several different cell contexts and found that activation of LIN-12/Notch and the paralogous GLP-1/Notch in C. elegans does not require either Epsin-mediated endocytosis or ubiquitination of the intracellular domain of the ligand. Results obtained by a collaborator indicate that C. elegans ligand and receptor interactions are tuned to a lower force threshold than are Drosophila ligand and receptor interactions, potentially accounting for these differences. I also looked at the role of endocytosis in regulating LIN-12 signaling in the context of vulval development. The cell fate pattern of six vulval precursor cells (VPCs) is mediated by EGFR and LIN-12/Notch signaling. Previous work using multicopy transgenes in fixed specimens indicated that LIN-12 is post-translationally downregulated via endocytosis in response to EGFR activation in the VPC named P6.p, an event that appeared essential for ligands to activate LIN-12/Notch in neighboring VPCs. In this thesis, I manipulate the endogenous lin-12 gene and examine live specimens to show that LIN-12 appears to be regulated transcriptionally in P6.p and evidence that there may be additional potential endocytic motifs that may regulate LIN-12 in this context.

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Helicobacter pylori infection is a leading cause of gastric cancer, which is the second-most common cancer-related death in the world. The chronic inflammatory environment in the gastric mucosal epithelia during H. pylori infection stimulates intracellular signaling pathways, namely inflammatory signals, which may lead to the promotion and progression of cancer cells. We herein report two important signal transduction pathways, the LPS-TLR4 and CagA-MET pathways. Upon H. pylori stimulation, lipopolysaccharide (LPS) binds to toll-like receptor 4 (TLR4) mainly on macrophages and gastric epithelial cells. This induces an inflammatory response in the gastric epithelia to upregulate transcription factors, such as NF-κB, AP-1, and IRFs, all of which contribute to the initiation and progression of gastric cancer cells. Compared with other bacterial LPSs, H. pylori LPS has a unique function of inhibiting the mononuclear cell (MNC)-based production of IL-12 and IFN-γ. While this mechanism reduces the degree of inflammatory reaction of immune cells, it also promotes the survival of gastric cancer cells. The HGF/SF-MET signaling plays a major role in promoting cellular proliferation, motility, migration, survival, and angiogenesis, all of which are essential factors for cancer progression. H. pylori infection may facilitate MET downstream signaling in gastric cancer cells through its CagA protein via phosphorylation-dependent and/or phosphorylation-independent pathways. Other signaling pathways involved in H. pylori infection include EGFR, FAK, and Wnt/β-Catenin. These pathways function in the inflammatory process of gastric epithelial mucosa, as well as the progression of gastric cancer cells. Thus, H. pylori infection-mediated chronic inflammation plays an important role in the development and progression of gastric cancer.

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DAWDLE (DDL) is one of the eighteen genes in Arabidopsis thaliana that encodes a protein with a Fork-Head Associated domain, a phospho-threonine binding domain providing a role in DNA repair and cell cycle regulation. DDL also contains an arginine-rich N terminal domain with putative Nuclear Localization Signals and a region for RNA binding. Two ddl knockout alleles in the WS-2 ecotype exhibit a strong pleiotropic phenotype with developmental defects such as short root and hypocotyl, reduced fertility, and distorted organs. This developmentally delayed phenotype is due to reduced accumulation of microRNAs and the phenotype is similar to those displayed by mutants involved in microRNA biogenesis pathway, suggesting a function for DDL in the microRNA biogenesis. One of the goals of my research was to characterize DDL protein through a structureunction study. Twelve point mutants were isolated in a mutagenesis screen and a comparative phenotypic and molecular characterization of each mutant with wildtype (WT) plants was performed. This revealed a few functionally significant amino acid residues of DDL. Traits for comparison included hypocotyl and root length, plant height, fertility, and microRNA accumulation. While all the mutants displayed reduced fertility, some of them had significantly varying stem height, hypocotyl and root length, and microRNA accumulation compared to the WT. Another objective of my research was to identify components involved in the DDL pathway, which in-turn would contribute to discovering additional components in microRNA biogenesis pathway. One such component, ddl suppressor1 (dds1), was identified through a second site mutagenesis screen of ddl-2. Phenotypic and molecular characterization revealed that dds1 is a strong suppressor of ddl that was mapped on chromosome 3 of Arabidopsis. Another component identified was MODIFIER OF DDL (MDL), a natural variation between Col and WS-2 ecotype of Arabidopsis. The variation has been mapped to an interval consisting of 37 genes on chromosome 5. MDLCol is the dominant allele and imparts a weak phenotype to ddl knockouts, whereas the recessive MDLWS-2 does not modify a strong ddl knockout phenotype. ddl MDLCol does not display abnormality in microRNA accumulation unlike ddl MDLWS-2 indicating that MDL has a function related to microRNA biogenesis.