| Advisor : | ADAM ENGLER | ||
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| Abstract Title : | Novel Mechanisms of Non-Coding Genomic Regulation Identified in Cardiac Disease-in-a-dish Models | ||
| Abstract : | Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the non-coding 9p21 gene locus associated with increased risk of sudden and arrhythmic cardiac death, even after accounting for patient and family history for CAD and MI, suggesting an altered cardiac remodeling response. However, little is known about a possible cardiac phenotype as studies have largely focused on its effect on CAD and have trouble describing regulation with non-coding loci. Using induced pluripotent stem cell-derived CMs from patients that are homozygous risk/risk (R/R) and non-risk/non-risk (N/N) for 9p21 SNPs, we assessed cardiomyocyte (CM) function when cultured on hydrogels capable of mimicking the fibrotic stiffening associated with disease post-heart attack, i.e. stiffening from 10 kiloPascals (kPa) to 50 kPa. While all CMs independent of genotype beat synchronously on soft matrices, R/R CMs cultured on dynamically stiffened hydrogels exhibited asynchronous contractions versus N/N CMs in the same conditions. Dynamic stiffening reduced connexin 43 expression and gap junction assembly in R/R CMs but not N/N CMs. To eliminate patient-to-patient variability, we created an isogenic line by deleting the 9p21 locus from a R/R patient, i.e. R/R KO. R/R KO CMs maintained synchronous contractions and organized connexin 43 junctions after stiffening. The 9p21 locus suppresses the activity of the cell cycle regulator CDKN2A. p16, a protein produced by CDKN2A, prevents JNK phosphorylation (p-JNK), which in turn reduces gap junction expression in CMs and contributes to the development of arrhythmias in rabbit myocardium in response to stress. We observed increased p-JNK expression in R/R CMs after stiffening compared to N/N and R/R KO CMs. Furthermore, treatment with the p-JNK antagonist SP600125 after stiffening restored synchronous contractions and organized gap junction assembly to R/R CM, indicating that JNK phosphorylation is sufficient to induce the phenotype observed in the R/R CMs. As a non-coding locus, 9p21 appears to repress connexin transcription, but only when the niche is stiffened as in disease. These data are the first to demonstrate that disease-specific niche remodeling can differentially affect CM function depending on SNPs within a non-coding locus. | ||
| Advisor : | MEI HUA GAO | ||
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| Abstract Title : | Urocortin 2 Effects on Liver Metabolic Gene Expression in Mice with Insulin Resistance | ||
| Abstract : | Urocortin 2 (Ucn2) is an endogenous hormone belonging to the corticortropin-releasing-factor family. Adeno-associated virus mediated Ucn2 gene transfer (GT) increases insulin sensitivity and glucose disposal, resulting in normalization of glucose homeostasis in insulin-resistant (IR) mice fed with a high fat diet (HFD). To determine the mechanism for the Ucn2 effects, alterations in mice-liver gene expression were examined using focused gene arrays and RT-PCR. Mice that received Ucn2 GT prior to HFD prevented IR development, with five genes (Abca1, Akt1, CEBPB, Lepr, and G6PC) being up-regulated and eight genes (Foxa2, Slc27a5, Slc2a2, Frs3, Map2k1, Pik3r1, Raf1, and Sorbs1) demonstrating down-regulation compared against saline injected HFD-fed control mice. In HFD mice that already developed IR, Ucn2 GT was able to up-regulate the expression of two genes (Abca1 and Acox1) and down-regulate four genes (Fabp1, Ras2, Adrb3, and Dok2.) Our findings demonstrate that Ucn2 improves glucose homeostasis in HFD-fed mice by modulating metabolic gene expression in liver. | ||
| Advisor : | KARL WILLERT | ||
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| Abstract Title : | The Role of Wnt-Fzd Specificity in Hematopoietic Stem Cell Development | ||
| Abstract : | Hematopoietic Stem Cells (HSCs) can differentiate into any type of mature blood cell. One candidate pathway for HSC regulation is the Wnt/β-Catenin (Wnt hereafter) pathway. Our laboratory has already shown that the Wnt pathway is necessary for proper HSC development through the ligand Wnt9a and the Fzd receptor Fzd9b. My research focuses on the specificity of the interaction between Fzd9b and Wnt9a. The overall aim of my project is to characterize how features of Fzd9b and Wnt9a affect Wnt ligand signaling through Fzd receptors. Our laboratory previously utilized a Wnt signaling assay to determine that Wnt9a signals through Fzd9b but not Fzd8a. I made different Fzd9b constructs that have one or more domain that are replaced by the corresponding domain in Fzd8a, which Wnt9a does not signal through, and then assessed their ability to signal in our Wnt reporter assay. From the Wnt signaling assay results and western blot protein visualizations, we determined that the ICL-3 and CTT confer Wnt-Fzd signaling specificity. We hope to validate these Wnt-Fzd specificity characterizations in a zebrafish model and relate them to Hematopoietic stem cell development in zebrafish. | ||
| Advisor : | DR. ALEXANDRA C. NEWTON | ||
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| Abstract Title : | Rare Protein Kinase C Variants in Alzheimer's Disease | ||
| Abstract : | Protein kinase C (PKC) is involved in the signal transduction of cellular processes such as proliferation and apoptosis. Its signaling output is controlled through precise mechanisms to maintain homeostasis, and the deregulation of PKC activity is associated with pathological states ranging from cancer to neurodegeneration. We have previously shown that PKC mutations found in cancer were loss-of-function, while rare PKCα variants that co-segregate with late-onset Alzheimer’s disease (LOAD) exhibit enhanced cellular signaling output relative to wild type PKCα. Here, we use a variety of approaches to characterize the inhibitor sensitivity of the AD-associated M489V variant in PKCα in order to understand the mechanisms by which this mutation confers increased agonist-evoked activity. Our studies show that compared to wild type PKCα, the M489V variant is more sensitive to active site inhibitor Gö6976 but equally sensitive to allosteric inhibitor BISIV. Furthermore, we show that another recently identified AD-associated PKC variant, K65R in PKCη, also displays enhanced signaling output relative to wild type PKCη. The altered pharmacological profiles of these AD-associate PKC variants provide insight into the role of PKC activity in disease and may be of potential therapeutic use. | ||