UC San Diego SearchMenu
2012 Research Showcase
Master's Abstracts
Abstract Title : Plasmacytoid Dendritic Cells are Targeted Early During Arenavirus Infection
Abstract : Plasmacytoid dendritic cells (pDCs) are a specialized subset of dendritic cells (DCs) that play a key role in antiviral host defense, capable of rapidly producing potent antiviral mediators Type I Interferons (IFN-I), and, therefore, represent an attractive target for viral immune evasion and immunotherapy. Previous work demonstrates DCs are an immune target of Old World Arenaviruses which can inhibit IFN-I production as an immune evasion strategy. In this study, we investigated both the level of pDC infection as well as the early response of pDCs upon in vivo infection with the DC-tropic arenavirus, lymphocytic choriomeningitis virus clone 13 (LCMV Cl13) in its natural rodent host. Our results demonstrate that LCMV Cl13 targets DCs, especially pDCs, early after in vivo infection. Moreover, pDCs were a major early source of IFN-I, a response that was dissociated from direct infection. These studies help to elucidate processes of DC targeting in viral infections, which may be utilized therapeutically to enhance immune response.
Abstract Title : Assay Development for Novel Drug Discovery Against Whipworm Parasites
Abstract : Intestinal roundworm parasites infect over two billion people in the world. In much of the developing world, children have intestinal worms that cause severe physical and cognitive stunting. Studies have shown that deworming kids is a cost effective way to keep kids in school, yet there is only one drug with adequate efficacy against these parasites to be used for mass drug administration. Of all the soil transmitted helminthes, whipworm proves to be the most difficult problem to solve. Whipworms embed themselves into the walls of the large intestine and proceed to form tunnels composed of enterocyte cells. While buried in this tunnel, the worms are in essence protected by the host cells, thus making whipworm infection (Trichuriasis) incredibly difficult to treat. My proposed research project is to develop an in vitro high-throughput assay system for screening novel compounds that kill or intoxicate intestinal worm parasites. An assay was developed in a 96-well plate format in order to keep the larval stages of Trichuris muris (mouse whipworm) alive and healthy. Health is primarily determined based on a measure of the curvature of the worm body. Novel compounds can then be added to the wells in the hopes of finding a new drug that could kill whipworms. Targeting the larval stages of the worm (before it matures and buries itself in a protective tunnel) is a novel approach to fighting whipworm infections. The hope is to develop an assay that could finally prove effective in finding a cure for Trichuriasis.
Abstract Title : MicroRNAs: The Role of Let-7 in Human Embryonic Stem Cell Derived Neural Precursor Cells
Abstract : Sox2 is a pan-neural transcription factor expressed in neural precursor cells (NPCs) independently of their regional identity. It plays a crucial role in neurogenesis both in development and adult neural stem cells; however the molecular mechanisms underlying its function are poorly understood. We hypothesize that Sox2 may control microRNAs (miRNAs) that hold important functions in neural development through transcriptional or translational regulation. In order to identify miRNAs with this pan-neural Sox2 dependency, I modified a previously established human embryonic stem cell (hESC)-derived dorsal NPC protocol by exploiting the ventral patterning function of the Sonic hedgehog agonist purmorphamine to yield NPCs with a ventral identity. The dorsal and ventral hESC-derived NPC models allowed for the investigation of the Sox2-dependent miRNAs in a human NPC model with different regional identities. Primary screening of Sox2 downregulation in dorsalized and ventralized NPCs highlighted let-7b as a common candidate for Sox2 miRNA regulation. In agreement with this finding, Sox2 knockdown studies revealed a significant increase in the total miRNA levels of let-7b and let-7i. Additional data from our lab suggests that Sox2 regulates let-7 through the well-established Lin28/let-7 pathway which blocks let-7 miRNA maturation. Overexpression studies revealed that let-7b suppresses NPC proliferation with no effect on neuronal differentiation while let-7i does not inhibit NPC proliferation, but abolishes neurogenic potential, phenocopying the defects of Sox2 downregulation. These data suggest that Sox2 regulates NPC self-renewal and neuronal differentiation through the let-7 family.
Abstract Title : GPCR signaling in Cardiac Progenitor Cells
Abstract : G-protein coupled receptors (GPCRs) are abundant, highly expressed and accessible drug targets with documented involvement in myriad physiological and pathological responses. The role of GPCR signaling in regulating responses of cardiac progenitor cells (CPCs) has not been examined, but this ubiquitous class of receptors has documented effects on cell proliferation, migration, and survival. We have determined that c-kit+ CPCs express an impressive complement of GPCRs, quite distinct from that found on cardiomyocytes. We have isolated RNA from CPCs and use TaqMan array to define the population of resident GPCRs at baseline and following differentiation. We found the constellation of GPCRs on the CPCs differ from that of cardiomyocytes and is dynamically regulated. In particular, the 2-AR and AT2 predominate in the CPCs (versus 1-AR and AT1 in the cardiomyocytes). Also, S1P1R predominates in cardiomyocytes while S1P2R is the most highly expressed in CPCs. A variety of GPCR ligands including serum, S1P, LPA, AngII and PE all stimulate proliferation in the CPCs. We tested RhoA involvement in the proliferative response to S1P and serum and demonstrated that these responses are inhibited by treatment with an inhibitor of RhoA. We also demonstrate through siRNA experiments that knockdown of RhoA, S1P2 or S1P3 receptors (the subtypes known to activate RhoA) inhibit S1P induced proliferation while removal of S1P1 receptors (which couple only to Gi) has no effect. Differentiation of CPCs to a cardiac lineage (GATA4, MEF2c) was observed in control cells at 3 days following dexamethasone treatment, as was an increase in the smooth muscle marker (GATA6). In cells treated with S1P, which couples to activation of RhoA, GATA4 was significantly attenuated compared to Dex alone but we observed no significant change in MEF2C or GATA6 transcription. However, LPA induces CPCs towards a cardiac lineage without affecting smooth muscle differentiation. These data implicate S1P mediated Rho signaling may be important in proliferation and survival of cardiac progenitor cells but its role in differentiation needs to be further investigated.
Abstract Title : The Role and Therapeutic Potential of gp130 Receptor in Chronic Viral Infection
Abstract : Chronic infections directly affect hundreds of millions of people worldwide. The transmembrane receptor, gp130, is a common signaling protein utilized by the interleukin-6 family of cytokines, potent immune signaling molecules. Using lymphocytic choriomeningitis virus clone 13 (LCMV Cl13), a mouse model of chronic viral infection, we examined the role of gp130 receptor signaling in immune responses. Infection of mice with a genetic ablation of gp130 signaling on T cells led to a complete failure in viral control, with both antibody mediated and cellular immunity being compromised compared to wild type mice. Interestingly, gp130 signaling was not found to be important in the control of an acute viral infection. We next determined the therapeutic potential of this pathway by treating wild type LCMV Cl13 infected mice with HYPER-IL6, a man-made cytokine capable of universally activated of the gp130 pathway. Mice receiving HYPER-IL6 showed slightly enhanced viral control compared to PBS treated control mice, however there was no observable difference in anti-viral immune responses. This study identifies gp130 signaling as a vital component of the immune response during chronic infection, and suggests that therapeutically targeting this pathway on CD4 T cells may have more profound effects.
Abstract Title : Novel role of MBL in HIV-1 related Neuroinflammation and CNS Impairment in Methamphetamine Users
Abstract : Methamphetamine (METH) is a highly addictive neurotoxic psychostimulant abused commonly by individuals infected by human immunodeficiency virus (HIV). METH enhances HIV-1 replication in vitro. HIV-1 infection and METH use cause synergistic neuronal death and neurotoxicity via oxidative stress and inflammatory immune responses. Mannose binding lectin (MBL), an innate immune protein, binds HIV-1 envelope protein gp120 and activates the complement pathway for HIV-1 opsonization and phagocytosis. Dr. Singh?s lab has observed increased MBL expression in HIV encephalitis (HIVE) suggesting MBL-mediated neuroinflammation. Our goal is to evaluate the role of the lectin complement pathway in METH induced neuroinflammation in HIV-1 infected individuals.
Abstract Title : Performance of native and exotic species following fire in San Diegan coastal sage scrub communities
Abstract : Coastal sage scrub communities in Southern California are increasingly impacted by environmental changes including habitat loss, invasive species, and accelerating fire regimes. Our experiment sought evidence of whether these disturbances, specifically fire and invasion, could result in alternate community states, as defined by Multiple Stable Equilibrium (MSE) theory, where herbaceous invaders would have long term dominance of the plant community structure. We surveyed sites where pre-fire data was available for up to 25 years and also collected seeds in order to look at germination and growth rates of natives and exotics under different soil conditions. The data we found suggest that dynamics of invaded post-fire communities do not strictly fit within MSE theory, but rather there is a variable (site specific) time period following burning in which conditions favor invasion by exotic species over native shrub re-establishment.
Abstract Title : Mating System and Evolutionary Genetics of an Invasive African Drosophilid: Zaprionus indianus
Abstract : Zaprionus indianus is an invasive Drosophilid that poses major threats to a host of fruits including figs, barbados cherry, and longan. They arrived in the new world from Africa where they were first recorded in Brazil in 1999, and has expanded their range reaching California, Arizona, Texas, South Carolina and Florida in the U.S. Very little is known, however, about those aspects of their reproductive behavior that make them a successful invasive. In order to characterize their reproductive biology, I asked the following questions: How does the mating system of Zaprionus indianus compare to that of other fruit flies? What reproductive characteristics does it share with other invasive species? And what might be some ways used by this fly to adapt to new environments?
Abstract Title : RON Overexpression Induces Acincar-Ductal Metaplasia and Accelerates Tumorigenesis in a KRAS Mutant Mouse Model of Pancreatic Cancer
Abstract : Introduction: The RON receptor is increasingly over-expressed during pancreatic cancer progression and has been implicated as an important mediator of KRAS oncogene addiction. It is unclear whether RON plays a causal role in tumor formation, or if its aberrant expression represents an epiphenomenon. We hypothesized that RON overexpression would accelerate pancreatic tumorigenesis in the setting of oncogenic KRAS. Methods: To test this hypothesis, we generated a transgenic mouse model which over-expressed wt-RON in a pancreas-specific manner using Pdx-1, the identical promoter used to drive cre expression in the Kras-LSLGD12/Pdx-1-cre (KC) strain that develops pancreatic duct neoplasia. RON overexpression was verified via Western Blot Analysis and immunohistochemistry (IHC). Pdx-1-RON mice were crossed with LSL-KRASG12D mice to yield Pdx-1-RON/ LSL-KRASG12D mice (RK). RK mice were then bred to Pdx-1-Cre mice in order to generate mice over-expressing RON in the presence of oncogenic KRAS (RCK). RCK mice were aged and sacrificed at various time points (6 weeks-12 months) and histology compared to KC controls. Immunofluorescent staining was used to analyze cellular expression of amylase and keratin 19. Results: Pdx-1-RON mice developed no pancreatic phenotype prior to 12 months. At 18 months, one of four animals developed primary pancreatic adenocarcinoma with lung metastasis. RON overexpression in KRAS mutant mice (RCK) led to accelerated PanIN progression compared to KC mice (p<0.05). RCK mice displayed visible high-grade PanIN lesions at 6 weeks, with invasive carcinoma and metastasis detectable as early as 3 months. Acinar-ductal metaplasia (ADM) lesions were present beginning at 6 weeks in RCK mice, which were increased compared to KC controls (p<0.05). RCK mice had decreased survival compared to KC mice, with a median survival of 47 weeks (95% CI 43-51 weeks). Conclusions: RON overexpression alone results in pancreatic cancer at long latency. In the presence of oncogenic KRAS, RON overexpression markedly accelerates PanIN progression to primary and metastatic pancreatic ductal cancer. This data implicates a novel role for RON signaling in acinar-ductal metaplasia, validates RON as a potential therapeutic target in pancreatic cancer, and provides a model that will be useful for studying the role of RON signaling in pancreatic tumor biology.
Abstract Title : Isolation of potential inner membrane proteins targeting the Yersinia pseudotuberculosis YopE effector to the Type III Secretion System in preparation for secretion and translocation
Abstract : Numerous pathogenic bacteria infect and regulate characteristics of their host through the transfer of virulence factors. One method occurs via the type III secretion (T3S) system for movement of proteins, known as effectors, into the host's extracellular environment and translocation directly into the cell's interior. The T3S system is found solely in gram negative bacteria due to their elevated likelihood for high penetration and pathogenic ability from an additional outer membrane of potent lipopolysaccharides (LPS) in the cell envelope. Previous studies have shown that the Y. pseudotuberculosis 15kDa SycE chaperone binds to the 23kDa YopE effector in a 2:1 stoichiometric fashion. Common to most chaperone-effector complexes, YopE requires both an N-terminal region termed the Signal Sequence (SS) and the Chaperone Binding (Cb) region for correct secretion and translocation. Although its location is certain, previous research has indicated the SS region may be composed of the first 15 amino acids of YopE, or the first 15 mRNA codons, indicating uncertainty regarding its molecular composition. The studies reported here present the ability for use of Formaldeyde as a cross-linking reagent to isolate potential inner membrane proteins that interact with the first 15 amino acid SS region of the Y. pseudotuberculosis YopE effector prior to secretion and translocation. Formaldehyde, both highly reactive and cell permeable, is one of the shortest available chemical cross-linkers at 2.3-2.7 . Its ability to covalently link primary amines on lysine residues in close proximity ensures high specificity and isolation of the most transient interactions. The focus of this project is to obtain all experimental parameters for future identification of the proteins involved in signaling the Yersinia pseudotuberculosis YopE effector in preparation for secretion and translocation through the T3S system.
Abstract Title : The role of Src kinase proteins in pDC cytokine response to TLR ligands
Abstract : Plasmacytoid dendritic cells (pDCs) are a dendritic cell subset specialized to rapidly secrete copious amounts of Type I Interferon (IFN-I), a group of innate mediators that play key roles in antiviral immune defense and autoimmune diseases. Loss of pDC-derived IFN-I during chronic viral infection enhances susceptibility to secondary infection while excessive pDC IFN-I production contributes to autoimmune pathology, demonstrating the need for further investigation into the mechanisms of IFN-I regulation in pDCs. We recently identified greater expression of Src family kinase proteins in pDCs compared to other dendritic cell subsets and sought to determine whether these proteins contributed to the regulation of IFN-I as well as pro-inflammatory cytokine production in pDCs. To address this question, we performed in vitro studies in murine bone marrow derived dendritic cells (BM-derived DCs). Using chemical inhibitors of the src family kinases, we found that Src kinases contribute to both IFN-I and pro-inflammatory cytokine production in BM-derived wild type pDCs in response to TLR9 ligand stimulation. We further analyzed BM-derived pDCs from mice deficient in Lyn and Fyn, two members of the src kinase family, which also demonstrated a role in pDC cytokine response but no significant difference in dendritic cell differentiation or survival. Our data suggests the Src family of proteins may play an important role in pDC cytokine response during pathogenic challenge. Future studies will determine the in vivo role of this family of kinases as well as the mechanisms mediating cytokine regulation.
Abstract Title : An MICROscopic analysis of fruit patterning in Arabidopsis thaliana
Abstract : The model organism Arabidopsis thaliana has long been used to dissect different developmental processes in plants, including fruit morphogenesis. Anatomically, the Arabidopsis fruit can be divided into three main territories: the replum, which retains meristematic characteristics, and the valves and valve margin. Through our research we have been able to identify a handful of transcriptional regulators controlling the formation of these tissues, which helped us to elaborate a working model for fruit patterning. Interestingly our recent findings uncovered the role of several regulatory small RNAs controlling different aspects of fruit development. We have identified a family of microRNA (miR) genes that control the final size of the replum domain as well as its inner/outer polarity. We are currently generating an expression map for these miR genes and their corresponding targets. We are also developing several molecular and genetic experiments to include these new genes into the current regulatory network controlling fruit patterning.
Advisor : BENJAMIN D. YU
Abstract Title : A scalable system for identifying changing patterns of DNA-binding proteins during stem cell differentiation
Abstract : Transcription factors are recognized for their ability to specify and even reprogram distinct cell lineages during differentiation. A variety of approaches allow for the detection of the changing landscape of transcription factors during differentiation. Transcription factors are regulated by multiple mechanisms, many of which control their stability and their ability to bind DNA. The majority of screening approaches, e.g. microarray, RNA sequencing, qPCR, do not address whether detected transcription factors are stable or even capable of binding DNA. Moreover, antibodies necessary to characterize thousands of different transcription factors are not available. My proposal examines the use of a non-radioactive DNA binding assay to facilitate the detection of one or perhaps hundreds of transcription factors in a short time frame. With the new advance in nucleotide detection method using end-labeled fluorescent dye, we propose an alternative electrophoretic mobility shift assay (EMSA) protocol with a LiCOR imaging system that provides solution to these disadvantages. By using EMSA probes tagged with a fluorophore that can be visualized at a wavelength of 700nm with the LiCOR imaging system, the result can be observed within three hours. This protocol will provide a scalable method to create signature TF binding profile for ES and varying cell lineages.
Abstract Title : A Study of Death Associated Protein Kinase Function in C. elegans
Abstract : The innate immune response is an important component of wound healing, but must be negatively regulated to prevent continuous reaction to a one-time injury. Death Associated Protein Kinase (DAP Kinase) has been shown to negatively regulate this response in C. elegans by acting upstream of the TIR-1/p38 MAP Kinase cascade controlling the neuropeptidelike antimicrobial peptides. We show in our experiments that DAP Kinase acts upstream of the Protein Kinase C TPA-1, and downstream of the Gα subunit GPA-12 to regulate not only this pathway but a second TGF-β pathway controlling the caenacin antimicrobial peptides as well. Our results also suggest that the worm can compensate for the loss of components in its innate immune response pathways, both with the activity of parallel pathways and with redundant components within a pathway. dapk-1(ju4) morphological defects were previously found to be suppressed by the sydn-1(ju541) mutation. We show that DAP Kinase and SYDN-1 interact autonomously in the epidermis, and that DAPK possibly regulates SYDN-1's role in mRNA 3' end processing. Finally, we mapped two novel suppressors of dapk-1(ju4) defects, ju697 and ju698, to chromosome I and chromosome X respectively. We predict that these mutations are most likely in the genes dhc-1, which encodes a dynein heavy chain homolog, and pqn-34, which encodes a microtubule minus-end binding protein, known to promote microtubule stability and anchoring in other species. Our results hint at previously unknown roles for DAP Kinase, including involvement in mRNA 3' end processing and microtubule dynamics in the worm.
Abstract Title : Axonal growth and myelination of E-14 transplanted spinal cord stem cells
Abstract : In order for grafted cells to become normal functioning spinal cord neurons they must become mylenated by oligodendrocytes. During neurogenesis, oligodendrocytes that are not stimulated by neurons will simply die off or have a morphological change, which may make them inactive. One current debate is whether this same apoptosis or necrosis of oligodentrocytes occurs post spinal cord injury (Yang 2006). This project involves quantifying the amount of E-14 growing axons that have become myelinated based on their diameter and G-ratio, as well as determining the origin of the myelinating Oligodendrocytes. Finally this project is looking at a possible mechanism for the promotion of growth by the degeneration of myelin.
Abstract Title : The role of Foxo3 in neutrophil homeostasis
Abstract : Foxo transcription factors are critical mediators and have a highly conserved role in regulating hematopoietic homeostasis by limiting physiologic oxidative stress. A loss in Foxo3 causes a striking myeloproliferation, induced in part by increased accumulation of reactive oxygen species (ROS), that amplifies that Akt/mTOR signaling pathway. Here we found that a Foxo3-deficient mouse strain, Foxo3Lex, exhibited increased neutrophil production from an expansion of myeloid progenitor cells: CMPs, Pre GMs, and GMPs. Such neutrophilia and increase in progenitor cells were more apparent and exaggerated in the spleen, in comparison to the BM of Foxo3Lex mice. Interestingly, although neutrophils are known to exaggerate conditions of neutrophilic inflammation, Foxo3Lex mice, exhibited normal conditions of peritonitis and reduced severity of rheumatoid arthritis despite their increased neutrophil numbers. The increase in splenic neutrophils also compelled us to examine their possible novel role as B cell-helper neutrophils, which have been recently identified as neutrophils in the marginal zone of the spleen that activate B cells to generate an antimicrobial immunoglobulin defense. In conclusion, Foxo3 appears to regulate and promote the proper balance of neutrophil populations to avoid overproduction possibly from exaggerated granulopoiesis, especially in the spleen.
Advisor : DR. PAUL INSEL
Abstract Title : Disease-stage specific G-protein coupled receptor expression in clinical disorders: Chronic lymphocytic leukemia as a model.
Abstract : G protein-coupled receptors (GPCRs) are attractive targets in disease since they are expressed on the plasma membrane and show tissue-specific expression. A clinical problem for many heterogeneous diseases, such as chronic lymphocytic leukemia (CLL), is the lack of markers that can predict prognosis. CLL, which is characterized by the accumulation of B-cells, is classified as aggressive, which requires immediate treatment, or indolent, which does not require treatment. We hypothesized that patterns of GPCR expression are disease stage-specific and used CLL as a model to test if such patterns can identify biomarkers and therapeutic targets. Using a TaqMan GPCR array we found that normal B-cells (n=10) express greater than 200 GPCRs (74 orphan receptors), indolent CLL cells (n=10) express greater than 170 GPCRs (74 orphan GPCRs) and aggressive CLL cells (n=10) express greater than 117 GPCRs (51 orphan GPCRs). Numerous GPCRs were uniquely expressed or altered in CLL and expression differed in the 2 stages of CLL. Expression of vasoactive intestinal polypeptide receptor 1 (VIPR1), a Gs-coupled GPCR, increased >700-fold in aggressive CLL compared to indolent and VIP (1 μM, 48h) induced apoptosis of CLL cells (P<0.05, n=3). Thus, expression of particular GPCRs can provide stage-specific markers and identify novel targets for the treatment of CLL or other heterogeneous diseases. Funded by NIH.
Abstract Title : Dentate Gyrus Network Reorganization During Medial Temporal Lobe Epilepsy Leads to Dysfunctional Dentate Neural Network in Rats
Abstract : Theoretical models have suggested that the DG plays a significant role in pattern separation, which is thought to be important for memory formation. Patients with medial temporal lobe epilepsy (MTLE), defined by having chronic unprovoked seizures and is usually caused by head trauma, typically complain of memory impairments. There are many distinct anatomical changes that occur in many MTLE patients, including mossy fiber (mf) sprouting from hippocampal region the dentate gyrus (DG) granule cells. It is unknown as to whether the observed anatomical reorganization that occurs in the development of MTLE contributes to changes in memory processing and consequently memory impairments. I therefore proposed to test network functions of the DG in a MTLE rat kainate model. In this study we looked at the permanent anatomical changes (mf sprouting) of the DG in relation to the functional changes (pattern separation) of the DG and its downstream target, cornu ammonis region 3 (CA3), in awake behaving animals. Experiments included rats foraging in a series of square and circular environments, while electroencephalograph and extracellular action potentials were recorded. The results showed that the increased amount of pathological reorganization found in the DG (mf sprouting) correlated with the DG cell populations? impaired ability to pattern separate. However, there was no effect on the CA3 region in epileptic rats when compared to control rats.
Abstract Title : Co-culturing nitrogen-fixing cyanobacteria and diatoms in nitrogen-deplete media for biofuels production
Abstract : Culture of microalgae on a large scale, such as biofuels production, requires vast nutrient inputs. Some species of cyanobacteria, like the heterocyst forming Nodularia harveyana, can fix diatomic nitrogen (N2) into ammonia (NH4+) and thus do not require added nitrogen as a nutrient. Growth in co-cultures of this species and three species of diatoms in nitrogen-deplete media was characterized. Growth rates were measured using accessory pigment concentrations as proxies for biomass. The diatoms in the three co-cultures grew in nitrogen-deplete media, but at different rates. The diatoms exhibited no growth in the absence of the nitrogen fixer. Lipid production of the co-cultures was qualitatively evaluated using the fluorescent lipid dyes Nile Red and BODIPY 493 and appears to be equal to or greater than conventionally nitrogen starved cultures. 100L bag cultures of Nodularia harveyana and Phaeodactylum tricornutum have been successfully grown at UCSD?s biology field station in nitrogen-replete media and both species have remained in culture over multiple transfers. These results demonstrate that it may be possible to grow algal biofuel strains using a nitrogen fixer as the sole source of nitrogen once further strain selection is carried out and growth conditions are optimized.
Abstract Title : Redox Regulation by Thioredoxins in Plant Immunity
Abstract : Plants are regularly exposed to stresses that negatively affect their development and nutritional content. One of the most severe stresses that plants are exposed to is pathogen infection, caused by plant pathogens such as Pseudomonas syringae. Plants have two main types of defense against pathogens, the PAMP triggered immunity (PTI) and effector triggered immunity (ETI), which recognizes and defends the plants against pathogen associated molecular patterns (PAMPs) and effector molecules, respectively. When a plant is infected by pathogens there is an increase in salicylic acid (SA) which causes an important reduction in Nonexpresser of Pathogenesis Related genes-1 (NPR1) by the oxidoreductase Thioredoxin 5 (TRX5). This reduction in NPR1 allows its monomerized form to become nuclear localized and this leads to transcription of defense related genes. In order to determine what the role of redox signaling is in plant defense against pathogen infection, we are studying plant TRX3, TRX4, and TRX5 and their role in reducing the disulfide bonds of various proteins that are involved in plant defense against pathogen, such as NPR1. Our research involves creating double and triple mutants of trx3, trx4 and trx5 and subjecting these mutants to pathogen infection to determine their defense phenotype. We are also interested in using in vivo TRX pull down to determine the interacting partners of these oxidoreductases and how these interactions change when the plant is treated with different defense elicitors. This study will help us better understand the plant immunity.
Abstract Title : Interleukin 33 Recruits Lineage Negative Myeloid Cells Into The Lung And Replicates The Allergic Asthma-like Phenotype In Mice In The Absence Of Antigen Or Lymphocytes
Abstract : RATIONALE: Interleukin 33 (IL-33) is a cytokine associated with allergic inflammation and host defense against helminths. Although IL-33 plays an important role in the Th2 adaptive immune response, we now recognize an important role in the innate immune response. Here we address the role of IL-33 in the asthma-like phenotype independent of antigen. METHODS: C57BL/6 mice (20-25 gm) were either immunized and challenged with ovalbumin (OVA) over 21 d or had intratracheal administration of 500 ng of IL-33/d for 4 d. The next day animals were intubated and ventilated. Baseline airway resistance was measured and methacholine dose-response curves were performed, followed by post-mortem bronchoalveolar lavage, cytokine measurements, lung histology, and immunohistochemistry. Similar experiments were conducted in RAG1-/- and ST2 -/-. Also, lungs from IL33- and OVA- treated C57BL6 mice were digested and stained for flow cytometry. We gated on the lineage negative cells and identified those positive for ICOS and CD45. This population, known as nuocytes, was then adoptively transferred into ST2 -/-mice and these mice challenged with IL-33. RESULTS: IL-33 induced airway hyperresponsiveness (6.9 1.2 vs. 6.1 1.2 cm H2O*s/m; IL-33 vs. OVA) and eosinophilic airway inflammation (1.3 0.14 vs. 0.9 0.13 x 106/lavage) similar to OVA. GM-CSF, IL-5, and IL-13 were greater in the IL-33 treated animals when compared to OVA. Expression of both IL-33 and its receptor, ST2, were increased in the lungs of both models. RAG1-/- (lacking mature T and B cells) had no response to OVA, but with IL-33 treatment, responses were not different from wildtype. Since the receptor for IL33 is ST2, we challenged ST2-/- with IL-33 and there was no response. When nuocytes (non-T, non-B, non-granulocyte cells) were administered intratracheally and the mice challenged with IL-33, airway hyperresponsiveness to IL33 was restored. Additionally, IL33 increased nuocytes in lung to 1.11% compared to 0.22% in OVA and 0.004% in control. CONCLUSIONS: IL-33 can replicate an allergic asthma-like phenotype in a mouse model. The response is independent of an adaptive immune response, but is ST2 dependent, due to the stimulation and recruitment of nuocytes to the lung.
Abstract Title : Endothelial-Specific FGF Signaling Is Required for HSC Formation
Abstract : Exciting advances in stem cell biology have clinical applications in the field of regenerative medicine which aims to replenish damaged tissues via cell replacement therapy. In the vital process of blood formation, or hematopoiesis, self-renewing hematopoietic stem cells (HSCs) give rise to all blood cell lineages throughout the life of an organism. Zebrafish have evolutionarily conserved hematopoietic genes and regulatory networks, and possess all mature blood cell lineages found in mammals. Moreover, the zebrafish offers numerous experimental advantages, such as visual accessibility during embryonic stages and diverse genetic experimental approaches including both large-scale forward screening and feasible transgenesis. These advantages make the zebrafish a powerful model organism for studying vertebrate hematopoiesis. Previously, we studied the role of fibroblast growth factor (Fgf) signaling on HSC development using transgenic zebrafish embryos. To analyze HSC specification in the absence or presence of Fgf signaling, we utilized transgenic zebrafish and examined the expression of definitive HSC marker genes, runx1 and cmyb in the hemogenic endothelium at the dorsal aorta, using whole-mount in situ hybridization (WISH). Interestingly, blockade of Fgf signaling reduces runx1 and cmyb expression (hsp70:dn-fgfr1), while enhancement of constitutively active Fgf receptor 1 (Fgfr1) increases runx1 expression (hsp70:ca-fgfr1). Both blockade and ectopic activation of Fgf signaling have global effects on the developing embryo, however. To isolate the role of Fgf signaling on HSC formation, we have designed endothelium-specific transgenic animals. We found that endothelium-specific knockdown of Fgf signaling (lmo2:dn-fgfr1) blocks HSC formation. We have now generated a construct to study the effects of endothelium-specific overexpression of Fgf signaling (lmo2:ca-fgfr1). Together, these studies will allow us to further characterize the role of Fgf signaling on HSC development.
Advisor : PAUL A. INSEL
Abstract Title : G protein-coupled receptor (GPCR) arrays identify physiologically relevant targets in Pulmonary Artery Smooth Muscle Cells (PASMC): mRNA to Function
Abstract : Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance, in part due to pulmonary artery smooth muscle cells (PASMC). Since cyclic AMP (cAMP) decreases the contraction and proliferation of PASMC, G protein-coupled receptors (GPCRs) that couple G-alpha s and increase the intercellular accumulation of this second messenger, are potential targets for PAH. Using a TaqMan GPCR array and human PASMC we identified >130 GPCRs, at least 50 of which regulate cAMP formation. We then used real-time PCR and GPCR agonists to investigate the relationship between GPCR expression and function by comparing the concentrations of cAMP (determined by radioimmunoassay) that are generated by the highest, intermediate and lower expressed G-alpha s-coupled GPCRs (determined by cycle threshold [ΔCt]) in PASMC. As examples of the highest, intermediate, and lower expressed G-alpha s-coupled GPCR, we compared the adenosine 2B receptor (A2BR, ΔCt = 18), the vasoactive intestinal peptide receptor (VIPR1, ΔCt = 19), the prostaglandin I receptor 2 receptor (PGI2R, ΔCt =20), the prostaglandin E receptor 2 receptor (EP2R, ΔCT = 22) and the gastric inhibitory polypeptide receptor (GIPR, ΔCt = 24), respectively, cAMP accumulation (fmol / cell / 10 min treatment with agonist) in response to receptor agonists, CV1808 (A2BR, 1M, 0.6 fmol / cell / 10 min), VIP (VIPR1, 1M, 0.2 fmol / cell / 10 min), epoprostenol (PGI2R, 10M, 0.4 fmol / cell / 10 min), butaprost (EP2R, 1M, 0.4 fmol / cell / 10 min) and GIP (GIPR, 1M, 0.2 fmol / cell / 10 min) correlated, in part, with receptor mRNA expression (r2=0.31). Moreover, cAMP dose-dependently decreased PASMC proliferation. Overall these data show that the mRNA expression of G-alpha s-coupled GPCRs predicts functional response in PASMC, implying that this approach can identify GPCRs that may be therapeutic targets for PAH. Funded by NIH and the Doris A. Howell Foundation for Women?s Health Research.
Advisor : AMY KIGER
Abstract Title : The role of Atg18 and Atg18-like proteins in D.melanogaster autophagy
Abstract : Autophagy describes a conserved process that utilizes a double membrane in isolating cytoplasmic cargo for lysosomal degradation. Nutrient-regulation can be used to control the ?on/off? state of the process. In yeast and mammals, the PI(3)P binding protein Atg18, has been determined to localize to the omegasome, isolation membranes and autophagosomes, while positively regulating autophagy. Drosophila melanogaster encodes for three Atg18 family homologues: Atg18, CG11975 and CG8678. Here, I explored their role in autophagy, by analyzing their protein sequences and phylogenetic relationships, autophagy associated phenotypes, and regulated cellular localization. I also investigated the possibility of Atg18, CG11975 or CG8678 being autophagy or PI(3)P markers. Here, I show that the D. melanogaster Atg18 family share conserved sequences and motifs. Phylogenetic analysis revealed close ancestry between the D. melanogaster, yeast and mammal Atg18 family. Overexpression and loss of function studies indicated that Atg18 positively regulates autophagy, and partially localizes to both acidified compartments and autophagosomes. In addition, overexpression of Atg18 induced lysosome acidification in larval fat body. In contrast, overexpression of CG11975 inhibited formation of acidified compartments and autophagosomes. CG11975 also showed low localization to acidified compartments and autophagosomes. Furthermore, coexpression of either Atg18 or CG11975 with the class II PI3-kinase Pi3K68D, mutually altered localizations, suggesting that both Atg18 and CG11975 can detect a Pi3K68D-specific PI(3)P pool. I also show that overexpression of CG8678 does not affect starvation-induced autophagy. These studies showing D. melanogaster Atg18s autophagy phenotypes and PI(3)P localization establish inroads for new markers and functional insights into phosphoinositide-dependent functions in autophagy.
Abstract Title : Long-Distance Outgrowth from Neural Stem Cell Grafts in Chronic Spinal Cord Injury
Abstract : Neural Stem Cells (NSCs) grafts have the ability to fill spinal cord lesion sites and act as neuronal relays, which re-establish connectivity between rostral and caudal segments of the injured spinal cord. In the current study, we examine the effects of NSC transplantation into chronic spinal cord injury models. Rats underwent C4 dorsal column lesions and 6 months later receive NSCs transplantation derived from GFP transgenic fetal rat spinal cords. Transplanted neurons extend large numbers of axons from the lesion site, both rostrally and caudally. Number and length of NSC-derived axons in a chronic lesion was qualitatively lower compared to NSC-derived axons in an acute lesion. Our findings indicate NSCs retain the ability to extend axons over long distances in a chronic spinal cord injury environment, although there are intrinsic mechanisms which appear to hinder the overall length and distance of outgrowth. Identification and manipulation of these intrinsic mechanisms of the chronically injured state may enhance growth.
Abstract Title : Peroxisomal Degradation and Hepatitis C Virus Proliferation
Abstract : Cellular peroxisomes function in detoxification of reactive oxygen species (ROS), beta-oxidation of lipids, and in innate immune defense, cooperatively with mitochondria. Hepatitis C virus influences these functions to promote a lipid and ROS rich micro-environment to facilitate its proliferation, and to inhibit the innate immunity defense mechanisms. Several studies have shown that HCV induces bulk autophagy, via endoplasmic reticulum stress response. HCV promotes autophagy to utilize the autophagy vesicles for its replication and to inhibit the innate immune response as well. In this study we look into the possibility of Hepatitis C virus induced selective degradation of peroxisomes (?pexophagy?), in order to observe their affect on these processes. We observe that HCV does not promote pexophagy and the HCV infected cells display normal physiological levels of peroxisomal degradation.
Abstract Title : Physical and genetic interactions of proteins required for asymmetric cell division in maize
Abstract : Asymmetric cell division is associated with the development of new cell lineages and is important in an organism?s embryogenesis and development. In asymmetric cell division, it is essential to generate correct cues for polarity in order to correctly specify two different daughter cells. Since plant cells are held in place by rigid cell walls, it is crucial the asymmetric division is oriented correctly as it cannot later be corrected by cell migration. In the crop plant Zea mays (maize), stomatal formation by division of subsidiary mother cells (SMCs) is an excellent model for asymmetric cell division. We previously identified two classes of mutants defective in this asymmetric cell division: the pangloss1 (pan1) and pan2 mutants; and the brick1 (brk1), brk2, and brk3 mutants. . PAN1 and PAN2 are inactive leucine-rich repeat kinases (LRRKs), a family of proteins predominately known for their roles in signaling. BRK1, 2, and 3 are proteins involved in stimulating actin branching. I initiated a series of experiments to determine the physical and genetic interactions of the PAN and BRK proteins. Genetic analyses suggest that pan and brk work synergistically, indicating that pan and brk are part of the same pathway. Similar to previous results with PAN1-YFP and PAN2-YFP, BRK 1-CFP polarly localizes to patches in SMCs prior to division. Since PAN1-YFP was previously found to have reduced polar patch intensity in brk mutants, I measured PAN protein levels and found that they are unchanged compared to wild type in brk mutants. This implies a reduction in PAN1 recruitment, rather than expression, and that brk is important for this recruitment. To identify physical interactions I performed yeast 2-hybrid experiments PAN1, PAN2 and six other candidate signaling proteins, which were identified from a separate proteomic study. PAN2 was found to interact with itself, however no other interactions were identified, . Together, these data suggest that the PAN receptor kinases act together with actin branching to specify polarity in SMCs.
Abstract Title : Spermidine as a Stimulus of Protein Tyrosine Phosphatase N2 Mediated Protection of Intestinal Epithelial Barrier Function
Abstract : Genome-wide association-studies (GWAS) have revealed that single nucleotide polymorphisms (SNP) in the gene locus encoding a particular phosphatase, protein tyrosine phosphatase non-receptor type 2 (PTPN2), are associated with the chronic intestinal inflammatory condition, Crohn?s disease. PTPN2 is ubiquitously expressed. However, its expression in intestinal epithelial cells (IEC) has been shown to play an important role in the protection of epithelial barrier function during periods of inflammation by acting as a negative regulator of the pro-inflammatory cytokine, IFN-γ. IFN-γ is known to play a major role in the pathophysiology of Crohn?s disease and part of this effect is thought to be linked to its capacity to decrease intestinal epithelial barrier function. Therefore, agents that increase the activity of PTPN2 are of general interest as modifiers of inflammatory signaling events, and chronic inflammatory disease states. A recent study demonstrated that the small molecule, spermidine, is a selective activator of PTPN2 in vitro. Spermidine belongs to a class of molecules called polyamines, which are involved in many physiological processes including cell growth and immunity. Here, I describe the effects of spermidine on PTPN2 expression and activity, as well as its effect on IFN-γ signaling and barrier function in the human colonic epithelial cell line, T84. My studies revealed that while spermidine had no effect on PTPN2 mRNA transcription, PTPN2 protein levels were increased in a dose-dependent manner following 24-hour incubation with the polyamine. Spermidine also increased PTPN2 enzymatic activity. This correlated with a decrease in phosphorylation of the signal transducers and activators of transcription (STAT)1 and 3, downstream mediators of IFN-γ signaling, upon co-administration of spermidine to IFN-γ treated cells. Additionally, spermidine protected barrier function under the setting of inflammation, restricting the decrease in transepithelial electrical resistance (TER) measurements induced by IFN-γ in co-incubation experiments. Spermidine?s ability to increase PTPN2 levels and activity, as well as reduce IFN-γ signaling in IECs indicate spermidine as a potential therapeutic agent for treating conditions associated with dysregulated IFN-γ signaling and a faulty mucosal barrier.
Advisor : YIMIN ZOU
Abstract Title : Inhibiting Wnt-Ryk signaling to promote functional recovery after spinal cord injury
Abstract : Wnt proteins pattern the developing nervous system and are re-expressed after spinal cord injury (SCI). I am studying the effects of blocking Wnt signaling through the repulsive receptor Ryk after SCI. Previous work from our laboratory showed that Wnt-Ryk signaling after SCI results in axonal die-back of the motor axons that comprise the corticospinal tract. My project looks into the effects of blocking Wnt-Ryk signaling on functional recovery after injury. We have used intrathecal infusion of monoclonal antibody to block Wnt-Ryk signaling after SCI in rats. Forelimb function was tested on both skilled and unskilled behavioral tasks. In preliminary experiments, animals infused with function blocking Ryk antibody have shown marked improvement on skilled forelimb testing when compared to IgG control infused animals. Functional testing was performed over a 16 week time period followed by anterograde labeling of the corticospinal tract. Histological methods will be used to determine if increases in functional recovery correlate with anatomical evidence of axonal plasticity.
Abstract Title : The Mechanism of Fibroblast Activation in Cancer
Abstract : The past decade of cancer research has shown that in addition to the intrinsic properties of the cells themselves, cancer development relies largely on the properties of the tumor stroma, especially the extracellular matrix. A key regulator of extracellular matrix homeostasis is the fibroblast, which has been found to become activated in the presence of cancer and reorganize the tumor stroma. In addition many carcinomas have been shown to express the transcription factor Snail. Although the mechanism by which fibroblast activation in cancer occurs is largely unknown, it has been shown that Snail-transgenic mice, in which Snail is expressed throughout the skin, fibroblasts acquire an activated state similar to that of cancer-associated fibroblasts. As such, the expression of Snail in cancer cells may be a contributing factor to how cancer cells hijack fibroblasts and cause them to remodel the tumor stroma in such a way that favors tumor growth. In a screen of cell lines, three cancer cells lines and one normal keratinocyte line were shown to express Snail. All four of these lines were also able to activate cAMP activity in dermal fibroblasts, while primary keratinocytes were not. This suggests that Snail expression may be a contributing factor to fibroblast activation in cancer.
Abstract Title : The Search for Novel Anthelmintics: Assay Development as Critical for New Drug Discovery Against Human Intestinal Parasites
Abstract : The most recent estimates from the World Health Organization indicate that around 2 billion people worldwide harbor worm infections. With such a staggering prevalence and the only partially effective mass-administered pharmaceutical treatment facing resistance and loss of efficacy, it is difficult to comprehend why more is not being done to find a solution. The effective goal of this study was to develop an assay for high throughput in-vitro drug screening against human intestinal parasites. After testing a number of nematode systems, it was determined that the hamster/ human hookworm Ancylostoma ceylanicum was most accurate in demonstrating toxicity to existing anti-parasitic (anthelmintic) compounds in the order of their clinical relevance to humans. An assay was then optimized to keep larval stages of the parasite alive and healthy in a 96-well format for up to two weeks. The screening method was initially tested with a 1,000 compound library and proved effective by identifying four novel compounds with anthelmintic activity. It is our aim that this system be used to transform the status quo and find a superior alternative to current anthelmintics. An effective approach to deworming the populations of the developing world may be a significant step in helping these individuals fight and overcome poverty, we hope that this assay is a leap in that direction.
Abstract Title : Proteins that Interact with Arabidopsis TANGLED
Abstract : TANGLED is a plant-specific protein that plays an important role in cell division plane orientation. The mechanisms by which TANGLED functions in cell division plane orientation are unknown. There are most likely proteins that interact with TANGLED to help it function. Using yeast-two-hybrid assay, I screened through a cDNA library of Arabidopsis thaliana and identified eight proteins that interact with TANGLED. For two of the eight proteins, localization experiments revealed that they localize to the cell plate in dividing Arabidopsis thaliana cells.
Abstract Title : EhCP1 and EhCP5: key released cysteine proteinases of Entamoeba histolytica as drug targets
Abstract : Protozoan Entamoeba histolytica is the causative agent of amebiasis, which affects more than 50 million people worldwide resulting in 70,000 deaths annually. Current treatment relies on metronidazole; however, metronidazole-resistant E. histolytica have been induced in the laboratory. Therefore, new drugs based on defined targets are needed to treat amebiasis. Recently, new drugs designed to inhibit E. histolytica cysteine proteinases, which are major virulence factors in amebic invasion and evasion of the host immune system, have been developed. To gain a better understanding of how these inhibitors irreversibly bind their target proteinases, recombinant EhCP1 has been resynthesized and expressed in bacteria to cocrystallize with cysteine proteinase inhibitors. Although soluble rEhCP1 has been expressed, obtaining the necessary large quantity for crystallization is still in progress. Biologically active rEhCP5, the other cysteine proteinase unique to invasive E. histolytica was assayed against vinyl sulfone derivatives of K11777, a cysteine proteinase inhibitor of cruzain that is currently in Phase I clinical trials for treatment of Chagas disease. Through these inhibition assays, both rEhCP5 and rEhCP1 have a substrate preference for positively charged amino acids at the P2 position, more specifically an arginine, despite possessing a different amino acid residue at the base of the active pockets. While the readily cell-permeable inhibitors WRR-666 and WRR-668 were effective against rEhCP1 and rEhCP5 in vitro, minimal inhibition of infection by E. histolytica trophozoites was observed in mouse models, possibly due to drug instability. rEhCP1 and rEhCP5 expression and corresponding inhibition assays provide insight into the development of new therapeutics to treat amebiasis.
Abstract Title : Co-expression Analysis of the Maize Seed
Abstract : Biological processes are a complex mesh of interactions of which high throughput methodologies are attempting to unravel. Using proteomic data from ~99 million tandem mass spectra, we have quantified protein abundance from 13,000 genes and localized 8,889 sites of phosphorylation in the developing maize seed. Weighted gene co-expression analysis has been completed on 8 independent time points in the seed development and a scale-free biological network has been created. Using network theory to investigate these interactions, we have determined clusters of proteins that share similar expression profiles and have found enriched associated ontology terms describing these proteins. Additionally, biologically interesting network features such as nodes with high degree centrality and cliques have been identified. Co-expression analysis of seed data, as well as the eventual larger scale maize protein atlas data, will assist in illuminating important regulatory interactions which will extend the understanding of developing maize and plants.
Abstract Title : Effects of In Vivo Expression of Gap Junction Mutant Hve-innexin1PL on Neural Coupling & Coexpression of Other Innexins
Abstract : Gap junctions (GJs) are membrane channels that form between apposed cells and mediate electrical communication as well as the transfer of small molecules. GJ hemichannels are hexamers comprised of subunits called innexins (Inxs) in invertebrates and pannexins (Pnxs) or connexins (Cxs) in vertebrates. In addition to some of their shared structural motifs, Inxs, Pnxs, and Cxs also possess a highly conserved Proline residue in their second transmembrane domain (TM2). Studies with Cxs describe this Proline as introducing a kink into the protein?s three-dimensional structure, which has been implicated in mediating conformational changes in the hemichannel in response to voltage. To study the role of the Proline residue in TM2 of Inxs in the developing CNS of the leech Hirudo verbana, it was mutated to a Leucine in pan- neuronally expressed Hve-inx1 (Hve-inx1PL). Through a series of experiments involving the expression of this mutant inx1PL construct in developing neurons of the intact embryo, it became evident that mutant expression conferred a dominant negative effect on INX1 GJs as witnessed by the loss of GJ plaques in neuronal arbors and the decoupling of expressing neurons from their normal cellular networks. Furthermore, coexpression studies using this mutant inx1PL along with wild-type (WT) inx1 and other leech Inxs has revealed that normal punctal expression of WT Inx transgenes could also be abolished for some pairs of transgenes (inx1PL with WT inx1 or inx14), but not for others (inx1PL with inx6). Such results suggest that the dominant negative effects of INX1PL expression can be useful for not only studying INX1 function in the CNS, but also for revealing heteromeric composition of hemichannels in individual neurons.
Abstract Title : Treating Atherosclerosis by Targeting Heparan Sulfate and RAGE Interactions
Abstract : RAGE, the Receptor for Advanced Glycation End products, is highly expressed and associated with many inflammation-related pathological conditions, especially in atherosclerosis. Past studies have shown RAGE is a heparan sulfate binding protein and receptor signaling is dependent upon this proteoglycan. Where heparan sulfate interacts and binds to RAGE is still unknown. Site directed mutagenesis of RAGE was performed to identify four sets of double mutations (K43A-K44A, R216A-R218A, V78A-L79A, and F85A-L86A) critical for heparan sulfate binding. RAGE containing these sets of double mutations expressed a significant reduction in heparan sulfate binding shown by a filter binding assay. Lastly, gel filtration chromatography suggested RAGE oligomer tetramerization was affected by these mutant heparan sulfate binding and hydrophobic patch residues. Developing a specific monoclonal antibody targeting these significant heparan sulfate binding residues in RAGE could possibly control atherosclerosis progression.