<p> Sphingolipids (SL) are essential components of eukaryotic cells involved maintaining membrane integrity. They are important for membrane trafficking and function in signaling as messenger molecules. SLs are synthesized <i> de novo</i> from very long chain fatty acids (VLCFA) and sphingoid long-chain bases (LCB), which are amide-linked to form ceramide and further processed by addition of various head-groups. Little is known concerning the regulation of VLCFA levels and how cells coordinate their synthesis with the availability of LCBs for SL synthesis. Here I show that Elo2, a key enzyme of VLCFA synthesis, is controlled by signaling of the guanine nucleotide exchange factor Rom2, initiating at the plasma membrane. This pathway controls Elo2 phosphorylation state and VLCFA synthesis. My data identify a regulatory mechanism for coordinating VLCFA synthesis with SL metabolism and link signal transduction pathways from the plasma membrane to the regulation of lipids for membrane homeostasj.</p>

<p> Progression through the mammalian cell cycle is a tightly regulated process that allows cells to replicate their genomes and divide properly. In growth factor-deprived conditions or in response to stress, the cell will exit the cell cycle either reversibly through quiescence, or permanently via senescence. Studies have shown that the SIN3 family of proteins plays a crucial role in these cell cycle exit processes. SIN3 proteins are highly conserved, and exist in mammals as two family members: SIN3A and SIN3B, which function as flexible scaffolding proteins to assemble co-repressor complexes. Our laboratory has recently implicated SIN3B as a critical mediator of each of these cell cycle exit processes. However, its mechanism of action and the consequences of its disruption pertaining to cancer progression have not been comprehensively elucidated. Here we demonstrate that SIN3B is required for the induction of senescence in a mouse model of prostate cancer, and thus prevents the progression to aggressive and invasive carcinoma. In addition, through interaction analysis, we uncovered a novel and robust association between SIN3B and the DREAM complex. The DREAM complex, comprised of p107/p130, E2F4/5, DP1 and the MuvB core complex, is responsible for the repression of hundreds of cell cycle-related transcripts during quiescence. We determined that the deletion of <i>SIN3B</i> resulted in the derepression of DREAM target genes during quiescence, but was not sufficient to allow quiescent cells to resume proliferation. However, the ectopic expression of APC/C^CDH1 inhibitor EMI1 was sufficient for <i>SIN3B</i> deleted cells, but not wild-type cells, to reenter the cell cycle. These studies demonstrate a critical role for SIN3B in the senescence and quiescence programs, and provide important mechanistic insight into the molecular pathways that exquisitely regulate cell cycle exit.</p><p>

<p> Ovarian cancer (OC) takes the lives of nearly 14,000 US women every year. Although platinum is one of the most effective drugs in treating ovarian cancer, the development of platinum resistance is one of the biggest challenges facing patients. I have shown that the long non-coding RNA HOTAIR contributes to platinum-resistant OC and determined the regulators and targets of HOTAIR during the platinum-induced DNA damage response. My published data supports the role of HOTAIR in contributing to DNA damage induced cellular senescence and secretion of pro-inflammatory cytokines leading to cisplatin resistance. My unpublished work (under review) analyzed the interaction of HOTAIR with the PRC2, its known interacting partner. In this study, I developed a novel strategy blocking HOTAIR-PRC2 interaction and resensitized ovarian tumors to platinum in mouse studies. The results offer a pre-clinical proof of concept for targeting long non-coding RNAs as a therapeutic approach and may represent a strategy to overcome chemotherapy resistance in tumors exhibiting high expression of HOTAIR, a frequent observation in high grade serous OC.</p>

<p> Chronic alcohol induced liver disease (ALD) comprises of a spectrum of disease stages progressing from fatty liver, steatohepatitis, fibrosis, to cirrhosis that may eventually lead to death. Although, the early stages of ALD are reversible, 40% of the patients develop advanced stage liver disease characterized by significant hepatic fibrosis and cirrhosis, for which, currently, liver transplantation is the only curative approach. However, the number patients waiting for liver transplantation far exceeds the meager number of available donors resulting in premature mortality of such patients. Hence, there is an urgent need for therapies for not only prevention and early intervention to stop the disease progression, but also to effectively regenerate the remaining healthy liver so that the patient can be reasonably functional before they can fully recover with a liver transplantation. Thus, any biologically natural modulator that can effectively prevent the progression of ALD until the donor liver is available for transplantation would be desirable even if it cannot completely cure the disease. </p><p> Thymosin &beta;4 (T&beta;4) is an immune modulating natural peptide secreted by thymus gland that is known to prevent inflammation and fibrosis, and promote wound healing and regeneration in the eye, skin and heart. Previous work from our laboratory has also shown that T&beta;4 protects against carbon tetrachloride induced acute liver injury in rat. However, not much is known of the role of T&beta;4 in alcoholic liver injury. Therefore, in this dissertation research, the role of T&beta;4 was investigated in acute on chronic ethanol and lipopolysaccharide (LPS) induced hepatic oxidative stress, inflammation, and fibrosis in an in vivo mouse model, as well as its regenerative potential was studied in chronic ethanol fed mice after partial hepatectomy. Furthermore, the underlying molecular mechanism by which T&beta;4 exerts its action, particularly on fibrosis was examined using human hepatic stellate cells (HSC), the main fibrogenic cells of the liver. </p><p> Based on the well accepted two-hit model for ALD, in the hepatocytes, ethanol acts as the first hit and is oxidized to acetaldehyde, the highly toxic first metabolite of ethanol oxidation by alcohol dehydrogenase (ADH) and ethanol-inducible cytochrome P450 2E1 (CYP2E1) leading to the generation of reactive oxygen species (ROS), resulting in oxidative stress. On the other hand, ethanol-induced leaky gut results in the release of endotoxin (LPS) that acts as the second hit and activates nuclear factor Kappa B (NF?B) in the Kupffer cells and the subsequent production of the pro-inflammatory cytokines that propagates liver inflammation. ROS and the pro-inflammatory cytokines act as fibrogenic stimuli for the activation of HSC and their trans-differentiation from quiescent lipid storing phenotype to activated myofibroblasts that express fibrogenic genes and proliferate and migrate to the site of injury and form a fibrous scar, resulting in fibrosis. This is essentially due to the fact that the quiescent HSC exhibit up-regulated adipogenic gene, peroxisome proliferator-activated receptor gamma (PPAR&gamma;), and down-regulated fibrogenic gene, methyl CpG binding protein (MeCP2), whereas the reverse is true upon their activation to myofibroblasts. </p><p> The experimental results showed that T&beta;4 reduced the ethanol and LPS induced levels of ROS by increasing the levels of the antioxidants, glutathione and superoxide dismutase. It also inhibited the nuclear translocation of NF&kappa;B by blocking the phosphorylation of the inhibitory protein I&kappa;B and thereby prevented the up regulation of pro-inflammatory genes, TNF-&alpha;, IL-1&beta;, and IL-6. T&beta;4 also prevented the activation of HSC by up-regulating miRNA 132, thus suppressing MeCP2, that coordinately reversed the down-regulated adipogenic gene, PPAR&gamma;, and the up-regulated fibrogenic genes (&alpha;-smooth muscle actin, PDGF-&beta; receptor, collagen 1, and fibronectin), and fibrosis. Moreover, T&beta;4 also promoted liver regeneration after partial hepatectomy in chronic ethanol fed mice by increasing hepatocyte growth factor and its receptor, c-Met; &alpha;-fetoprotein; proliferation markers, proliferating cell nuclear antigen and Ki-67 as well as the liver progenitor cell marker, cytokeratin 19.</p><p> Furthermore, it was discovered that in human HSC cultures, T&beta;4 prevented PDGF-BB induced fibrogenesis and also abolished PDGF-BB induced HSC proliferation and migration by blocking the phosphorylation of Akt by preventing the binding of Akt to actin. Moreover, experiments with two bioactive peptides of T&beta;4, the amino terminal peptide (1-15 aa) and the actin binding peptide (17-23 aa) revealed that T&beta;4 exerts most of its anti-fibrotic actions <i> via</i> its actin binding domain.</p><p> In conclusion, these data suggest that T&beta;4 has antioxidant, anti-inflammatory, anti-fibrotic and hepatic regenerative potential against alcoholic liver injury. </p>

<p> Autophagy is a cellular recycling process in which cytoplasmic proteins and organelles are sequestered in a double membrane vesicle, delivered to the lysosome, and degraded following fusion of the two vesicles. A key part of the initiation signaling for autophagy is the generation of phosphoinositol 3-phosphate (P13P) by class III phosphoinositol 3-kinase also knows as Vps 34. In humans there are eight P13K isoforms divided into three classes, four class I enzymes, three class II enzymes, and a single class III enzyme. Of these eight enzymes, only the class III isoform is thought to participate directly in autophagic signaling. A quantitative microscopy based, loss-of-function survey of all eight P13K isoforms was used to determine their relative contribution to autophagic signaling, as measured by LC3 positive autophagic vesicles. As predicted, knockdown of P13K-class III reduced the number of autophagic vesicles in cells. Interestingly, knockdown of the P13K-class II&alpha; isoform had an even more potent effect on reducing the number of autophagic vesicles than knockdown of P13K-class III. In follow up studies, knockdown of P13K-class II&alpha; reduced endogenous LC3 conversion, caused the accumulation of p62 and lipid droplets, and colocalized with endosomal markers. These results suggest P13K-class II&alpha; may act to promote autophagy through the shuttling of endosomal vesicles into the autophagic pathway and approaches to test this hypothesis will be discussed. The requirement of P13K-class II&alpha; for autophagy is an important finding as it indicates a role for class II P13Ks in autophagy.</p>

<p> Chemoresistance is a highly significant problem affecting a diverse array of cancers at all clinical stages. In an attempt to identify molecular mechanisms leading to chemoresistance, we performed a RNAi screen against all known and putative kinases and phosphatases in the human genome. The knockdown of one of these genes, MK-STYX, resulted in potent chemoresistance in response to a diverse array of chemotherapeutic agents. As many of these drugs function through the induction of the apoptotic program, we hypothesized that the RNAi-mediated knockdown of MK-STYX blocks the cellular response to chemotherapeutic-induced apoptosis. </p><p> To investigate this hypothesis, we determined the ability of both control and MK-STYX knockdown cells to undergo apoptosis after exposure to an array of cell death inducing agents with different mechanisms of action. The results of these experiments demonstrated that MK-STYX knockdown protects against intrinsic, but not extrinsic apoptotic stimuli. These data were recapitulated with knockdown of the pro-apoptotic genes caspase-9 and Bax/Bak, suggesting that MK-STYX may modulate the regulation of one of these key apoptotic regulatory nodes. We demonstrated that the loss of MK-STYX blocks cytochrome c release, placing the apoptotic deficiency at the level of Bax/Bak-mediated mitochondrial outer membrane permeabilization, or MOMP. MK-STYX was found to localize to the mitochondria, but is neither released from the mitochondria upon apoptotic stress nor localized proximal to the machinery currently known to control MOMP. These results are summarized in Chapter 2. </p><p> In an effort to more fully define molecular mechanism of MK-STYX, we performed an unbiased TAP-tagging experiment to identify its interaction partners. The most significant and unique protein identified was the mitochondrial phosphatase PTPMT1. Interestingly, MK-STYX is a catalytically inactive dual specificity phosphatase, and catalytically inactive phosphatases have a precedent for regulating the activity and/or localization of active phosphatases. Because of this potential phosphatase regulatory mechanism, as well as similar localization patterns of both genes, we chose to further explore the interaction between PTPMT1 and MK-STYX. </p><p> Due to the robust survival phenotype seen in MK-STYX knockdown cells when treated with chemotherapeutic, we predicted that the knockdown of PTPMT1 may have a similar phenotype. Surprisingly, we found that PTPMT1 knockdown causes a Bax/Bak dependent cell death, suggesting that MK-STYX and PTPMT1 may functionally oppose one another in the mitochondria. Experiments in which both enzymes are downregulated show that PTPMT1 is epistatic to MK-STYX, as cells are resensitized to chemotherapeutic agents and cytochrome c release under these conditions. Interestingly, PTPMT1 was recently shown to be an important enzyme in the cardiolipin biosynthetic pathway, positively regulating the synthesis of this mitochondrial lipid. The genetic interaction provided by the robust changes in viability seen when these enzymes are downregulated suggests that MK-STYX may function to dampen PTPMT1 enzymatic activity. This allows us to hypothesize that the loss of MK-STYX results in increased cardiolipin biosynthesis, leading to altered mitochondrial membrane composition and subsequently, an altered apoptotic response. These results are summarized in Chapters 3 and 4. </p><p> We further hypothesize that the upregulation of cardiolipin levels directly inhibits the ability of Bax/Bak to permeabilize the outer mitochondrial membrane, effectively blocking the induction of mitochondrial apoptosis. These data suggest a novel mechanism by which dysregulated cardiolipin can facilitate chemoresistance, and suggest that this pathway could be exploited by recurrent cancers to evade therapies.</p><p>

<p> The mitochondrion is a complex, double membrane organelle that serves several important cellular functions including ATP synthesis, Ca ²⁺ buffering, and ROS homeostasis. Although classic mitochondrial diagrams depict the mitochondrion as a simple oval or &ldquo;bean&rdquo; shaped organelle, the mitochondria can form extensive tubular networks or numerous small spheres in response to various cellular environments through two opposing processes, mitochondrial fission and fusion. Deregulation of mitochondrial dynamics has been implicated in a wide range of diseases, including Parkinson&rsquo;s disease, heart disease and cancer. While significant emphasis for the last 15 years has been placed on the identification of the protein machinery responsible regulating mitochondrial morphology, it remains less clear how mitochondrial morphology affects various cellular functions and cellular fate outcomes. This thesis summarizes our findings on how mitochondrial morphology regulates cellular fate in the context of mitotic cell division and apoptosis. Using live cell microscopy and image analysis software we characterized mitochondrial dynamics with single cell resolution. We found that loss of key components of the mitochondrial fission machinery promotes a defect in cell cycle progression, characterized by an inability for cells to exit G2/M. Prolonged periods of mitochondrial fusion induced potent cell death, suggesting a novel mechanism to target the replicative potential of cancer cells. We also found that mitochondrial fission and fusion can alter the kinetics of cell death following apoptotic stimuli by inducing mitochondrial fusion prior to the commitment step in apoptosis, mitochondrial membrane permeabilization. This thesis summarizes our work in trying to elucidate how the structure of the mitochondria influences both mitochondrial and cellular fate.</p><p>

RAC/ROPs are a unique group of RAS-related monomeric G proteins (small G proteins) that constitute the sole family of Rho GTPases in plants. RAC/ROPs, like their counterpart Rho GTPases from mammalian and fungal systems, can interconvert between an active GTP and an inactive GDP bound state. These powerful signaling molecules lie upstream in many diverse signal transduction pathways. Their controlled regulation is critical to overall plant fitness, growth, development, and responses to abiotic and biotic stress. RAC/ROP activation is regulated by guanine nucleotide exchange factors (GEFs). The work in this dissertation initiated from the characterization of the expression and functions of RopGEF1, a broadly expressed GEF that localizes to the sites of root hair formation and regulates polarized cell growth. The focus of this dissertation is on FERONIA (FER), an upstream regulator of RopGEF1 that was initially identified as a RopGEF1 interacting protein in a yeast two-hybrid screen. FERONIA (FER) was found to regulate RAC/ROP-mediated signal transduction for auxin-regulated root hair growth and a number of other auxin-dependent responses, consistent with RAC/ROPs playing an important role in auxin signaling. ^ In flowering plants, pollen tubes deliver sperm to fertilize the female gametophytes located inside the ovules. Once a pollen tube enters it subsequently bursts releasing its sperm, which enables fertilization. Mechanisms are in place to coordinate tube rupture as well as repel late arriving pollen tubes from an already visited ovule. In this way not yet visited ovules have higher chances of fertilization and fertilized ovules avoid polyspermy. In this work, FER is demonstrated to mediate NADPH oxidase-dependent reactive oxygen species production required for pollen tube rupture. In addition, FER is also required for de-esterified pectin deposition outside the female gametophyte, which correlates with the ability of ovules to divert late arriving pollen tubes. Furthermore, the extracellular domain of FER, which contains predicted carbohydrate-binding malectin-like domains, interacts directly with pectin. This finding establishes FER as a cell wall-binding receptor kinase in plants and illuminates unprecedented mechanisms of pollen tube reception. ^ The presence of carbohydrate binding motifs in the extracellular domain of FER and its direct interaction with pectin prompted my investigation of its role in sugar sensing and signaling pathways. My work shows that feronia (fer) mutants are hypersensitive to sucrose, while over-expressing FER suppresses sugar signaling. Moreover, fer mutants accumulate elevated levels of starch, which demonstrates defects in their distribution of carbohydrate resources in source (sugar producing) and sink (sugar consuming) tissues. The fer mutants also display sucrose-induced cell wall defects, alterations to cellular morphology, and enhanced production of the stress-associated pigment, anthocyanin. These results suggest that FER functions as a negative regulator of sugar sensing and signaling pathways. Additional support for this model stems from the finding that FER is highly expressed in tissues involved in sucrose transport and its expression is stimulated by sucrose. Furthermore, and consistent with the hormone abscisic acid being part of the overall sugar sensing network, fer mutant seedlings are hypersensitive to ABA and display enhanced ABA response gene expression. Taken together, the data presented in this dissertation reveal the regulation of widespread and essential plant functions including RAC/ROP signaling, pollen tube reception, cell wall integrity, and sugar signaling by a single cell surface receptor kinase.^