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2012 Research Showcase
Cellular and Developmental Abstracts
Advisor : DR. WILLIS LI
Abstract Title : Functions of Cathepsins in Drosophila Immunity and Lifespan
Abstract : Aditi Abraham Dr. Willis Li Functions of Cathepsins in Drosophila Immunity and Lifespan Introduction: The JAK/STAT pathway is involved in innate immune response as well as tumor formation and development. Misregulation of this pathway leads to immune diseases and cancer. The JAK/STAT pathway induces its effects by transducing extracellular signals to the nucleus to regulate gene expression. The Li Lab has determined that two proteases, Cathepsin E and Cathepsin L are targets of the JAK/STAT pathway. The simplified yet evolutionarily conserved JAK/STAT pathway in Drosophila presents an ideal model system to study the effect of these target genes on lifespan and immune response. Results: In wild type flies, upregulating cathepsin E decreases lifespan and downregulating it is lethal in the pupa stage. This lethal phenotype is rescued upon administration of antibiotics. On the other hand, in a tumor model, downregulation of cathepsin E extends lifespan. RT-PCR of bacterially infected flies revealed that levels of cathepsin L were increased 6 hours after bacterial infection. Bacterially challenged flies with JAK over activation, cathepsin E deficiency, and both mutations survived longer than wild type flies. Conclusion: These results suggest that optimal levels of cathepsins are important for Drosophila immunity and lifespan, possibly by decreasing inflammation and conferring resistance against microbial infection. Further studies are necessary to determine in what tissues these cathepsins are highly expressed in response to microbial challenge and the mechanism of action through which they affect lifespan. *Preliminary results come from the master?s thesis work of Kimberly Larson M.S. K. Larson M.S., W. Li Ph.D. Evaluating the Role of Cathepsins in the Innate Immune Response through the JAK/STAT Pathway.(2011). Department of Immunology and Microbiology. School of Medicine and Dentristry, University of Rochester.
Abstract Title : Extended Daily Fasting Overrides Harmful Effects of a High-Fat Diet
Abstract : The direct relationship between the consumption of high calorie foods and metabolic diseases such as obesity and type II diabetes is generally accepted. However, there is little evidence regarding the involvement of the timing of such food intake. In our experiment, we chose to restrict the feeding period of standard laboratory mice in order to determine the effects of timing on metabolic diseases caused by high calorie diet consumption. In our experiment, mice were fed a 60% high fat diet but with different temporal patterns of food access: half of the mice had access to the food ad libitum (at all times of the day) whereas the other half were only given food for eight to nine hours a day. As a control, the same experiment was performed with mice that were fed a normal lab diet. Mice weights and food consumption were monitored weekly and glucose and insulin tolerance tests were performed monthly. Our experiment provided shocking results. First, the mice that were fed high fat foods during the restricted time period gained less weight than those that were fed high fat ad libitum, though the total caloric consumption was identical. Second, the insulin and glucose tolerance tests showed that the high fat time restricted mice did not become diabetic like their fat ad lib counterparts. Overall, time restricted feeding protected from diet-induced obesity and metabolic disease. Thus we suggest that a small lifestyle intervention in the temporal spread of food consumption can combat the dramatic increases of diabetes and obesity.
Advisor : SUZI HONG
Abstract Title : Immunosenescence is Associated with Disease Progression but not Depression in Presymptomatic Stage B Heart Failure Patients
Abstract : Immunosenescence is phenotypically characterized by increased cytokine-producing, effector immune cells. Elevated inflammation is associated with the pathophysiology of cardiovascular diseases, including heart failure (HF). Depression rates are greatly elevated in patients with HF, which is prognostic for adverse outcomes and related to disease severity. We hypothesized that individuals at risk for HF, i.e., ACC/AHA presymptomatic stage B HF, particularly with elevated depressive symptoms, would exhibit elevated markers of immunosenescence. We examined relationships of HF severity and depression with combinations of immunosenescence markers (CD45RA-, CCR7-, CD28-, CD57+) using flow cytometry in 29 men with stage B HF (age, 66▒10 years). HF severity was characterized by left ventricular ejection fraction (LVEF), B-type natriuretic peptide levels, and a 6-minute walk test. Depressive mood was assessed by Beck Depression Inventory. The percent and median fluorescence intensity of naive vs. differentiated effector CD8+ T cells were examined. Lower LVEF was associated with lower expression of CD28 on ?na´ve? CD8+ T cells (beta= .412; p= .03) and with higher % partially-differentiated CD28-CD57- CD8+ T cells (beta= -.33; p= .1) after controlling for age and BMI. These initial data suggest that stage B HF severity is associated with immunosenescence, although depressive symptoms are not. Expansion of highly differentiated, cytokine-producing effector immune cells in patients with cardiovascular diseases may lead to disease progression, via increased migration of proinflammatory leukocytes to cardiovascular tissues.
Abstract Title : Role of the Cytoplasmic Domain in Alpha-4 Signaling Mechanisms
Abstract : The alpha-4 integrin, a receptor on the cell surface, has been shown to play a vital role in the cell migration of both neuroblastoma and normal cells. Past research has also proved that the cytoplasmic domain of alpha-4 is required for these effects. This study used an ovarian cancer cell model to establish which portion of the cytoplasmic domain caused enhanced migration. It was determined that the alpha-4 sequence is not essential for alpha-4 mediated effects, including adhesion, downstream signaling, and migration. A random amino acid sequence (gfp) attached to the truncated alpha-4 can restore alpha-4 function to near wild-type levels.
Abstract Title : The inductive effects of mercury (II) chloride on S. purpuratus ATP-binding cassette transporter genes
Abstract : Mercury is a pollutant that is nearly universally present in water sources worldwide. Studies have been done demonstrating that ATP-binding cassette(ABC) transport proteins help to limit accumulation of inorganic mercury in sea urchin embryos,1 but it is unknown whether the mercury itself has any effect on the expression of these genes and the amount of ABC transporter protein present in organisms. This experiment attempted to address the question of whether there is a correlation between exposure to mercuric chloride (HgCl2) and the level of gene expression of certain ABC transporters that are thought to possibly play a role in mercury efflux. Additionally, it attempted to address the morphological changes associated with non-lethal levels of mercury in sea urchin embryos. Even at concentrations as low as 1nM, embryos exhibited changes such as abnormal gastrulation, misshapen borders, and malformed blastocoels. These abnormalities increased in severity and amount of embryos affected with an increase in mercury concentration, and persisted through development until the end of the study period. Additionally, mercuric chloride exposure induced the increased expression of certain ABC transporter genes, particularly ABCB1a, ABCC1, and ABCC4b. Although little is known about the specific function of these proteins with regards to how (or if) they confer protective effects to sea urchin embryos, the results of this assay are a good starting point for further exploration of the role of ABC transporters and mercury toxicity.
Abstract Title : Protective activities of aldolase on Lactate Dehydrogenase against various effects
Abstract : It was shown that the enzyme, aldolase, from rabbit muscle protected muscle phosphofructokinase-1, muscle adenylate (AK), and lactate dehydrogenase (LDH) from inhibition by ascorbate (AA) and some AA-fatty acid derivatives. Preliminary studies showed that a plant enzyme, spinach aldolase, had similar properties. This study extends the protective activities of spinach aldolase with respect to muscle LDH and introduces an interesting topic of the relationships between plant and animal proteins in terms of evolution. Rabbit and spinach belong to different Kingdoms and it would not be expected that their proteins would interact. Experiments comparing protective effects of rabbit muscle aldolase and spinach aldolase on inhibitions of LDH by AA and some AA-fatty acid derivatives were conducted. In addition to the inhibitor AA, the following AA-fatty acid derivatives were also studied: ascorbyl phosphate (AAPO4), ascorbyl stearate (AAS), and ascorbyl dipalmitate (AADP). It has been discovered that they have very similar protective behavior for LDH against inhibitions by dilution, AA, AAP, AADP, and AAS; however, spinach aldolase seems to protect and recover the activity of LDH better than rabbit muscle aldolase does. It is believed that this and related basic science research has some relation to cancer and diabetes studies.
Abstract Title : Comparison of Glaucoma Progression in Series of Heidelberg Retina Tomograph Images with the Visual Field
Abstract : It is well established that the structural changes at the optic nerve head are an early and important feature of the glaucomatous process. Optic disc photographs provide qualitative assessment, but it provides subjective interpretations and only limited quantitative information. To overcome these limitations, methods for objective and quantitative analysis of the optic nerve have been developed. Among them, Heidelberg Retina Tomograph (HRT) is the widely used confocal scanning laser ophthalmoscopy, which provides objective and reproducible images of the optic disc and peripapillary retina. It enables not only objective quantification of the optic disc and diagnosis of glaucoma, but also the detection of changes of optic disc changes over time. The primary method for assessing glaucomatous change using HRT is Topographic Change Analysis (TCA), a technique that compares the variability within a baseline examination to that between baseline and follow-up examinations. TCA describes a significant, repeatable change on the superpixel (4 x 4 pixels) level with a statistical method to evaluate the presence of change in a series of images acquired over time with HRT. Its sensitivity and specificity for detection of glaucoma progression was good. However, there are few reports that compared serial HRT findings with the visual field.
Abstract Title : The Interplay of AMPK and GIV in Linking Energetic Stress to Loss of Epithelial polarity and Oncogenesis
Abstract : Cell polarity, the asymmetry in cell shape, functions, and protein distributions, is a fundamental mechanism that regulates the apical-basal organization of epithelial cells. Maintenance of cell polarity is necessary to prevent neoplastic transformation, a multistep process in which a cell accumulates a series of mutations in genes that control crucial cellular processes, such as tight junction (TJ) stability in cancer forming cells. Here, I investigated how AMPK (a kinase involved in cell metabolism) phosphorylates a novel protein effector, GIV, to regulate epithelial integrity and cell polarity in mitosis. I obtained evidence that GIV and AMPK form a complex in cells, with AMPK phosphorylating GIV?s serine 245. Upon challenge with calcium deprivation (which disrupts TJ structure), I found that compared to the MDCK GIV-WT cells, the TJs were highly sensitive and rapidly disassembled in MDCK GIV-S245A cells, while MDCK GIV-SD cells were highly resistant and maintained integrity for prolonged periods of time. These findings indicate that phosphorylation of GIV at S245 plays a significant role in maintenance of TJ integrity. Furthermore, TJ in GIV WT were more sensitive than in GIV SD to an AMPK inhibition, indicating that phosphorylation at S245 by AMPK works in sustaining the TJs. These results help us gain a deeper molecular insight on how cell polarity is regulated and what mechanisms trigger its loss during oncogenesis.
Abstract Title : Identification and characterization of a regulatory mutation that causes hypervariable phenotypes in Drosophila embryos
Abstract : The overall goal of my project is to study the molecular basis of variable gene expression, with a gene in the Toll signaling pathway serving as the experimental system. The Toll pathway is highly conserved between both mammals and flies. In Drosophila melanogaster, one of the functions of this pathway is to specify ventral fate in embryos. When this pathway is interrupted, embryos display a dorsalized phenotype. The severity of such dorsalization can range from severe to mild, depending on the degree to which Toll signaling is decreased. One gene in this pathway is Tube, which encodes an adaptor protein. Most mutations in the Tube gene lead to a set level of dorsalization, whether it be strong or weak. I am studying the tub8 allele, which causes single female flies to produce embryos that range across the entire spectrum of dorsal phenotypes (from D0, the strongest degree of dorsalization, to D3, the mildest phenotype). Previous research show that tub8 mutants contain no sequence change in the mature protein. However, quantitative PCR results reveal a decreased amount of transcript, suggesting that a regulatory mutation might be the cause of decreased signaling. My experimental focus is to map and identify this regulatory mutation using site-specific recombination in combination with sequencing. To date, I have mapped the regulatory mutation to a 16 kilobase region containing the Tube gene. Using sequencing, I will identify the regulatory mutation and subsequently determine the molecular mechanisms of variable transcriptional repression of the Tube gene. These results may help the understanding of transcriptional regulation and stochastic processes in the cell, as well as the molecular mechanisms of variable phenotypes that occur in numerous organisms including humans.
Abstract Title : The role of the homeodomain protein Six6 in pituitary gonadotrope gene expression
Abstract : The hypothalamus orchestrates the release of FSH and LH from the pituitary through its pulsatile release of GnRH. FSH and LH in turn act on the gonads to stimulate hormonal secretions and gametogenesis. This research aims at better understanding regulation of gonadotrope gene expression through studying the mechanism by which Six6 represses the expression of LHβ, FSHβ, and GnRH receptor (GnRH-R). Six6, a member of the SIX/Sine oculis family of homeobox genes, stimulates progenitor cell development in the growing pituitary, eye, and brain. In mice, absence of Six6 causes a hypo-pituitary phenotype and a striking decrease in fertility. I have shown repression of LHβ, FSHβ, and GnRH-R by Six6 and a relief of repression of the genes when transfecting with Six6 carrying a mutation in the eh1 domain. The region of repression by Six6 is in the 800 to 600 region for GnRH-R, in the -300 to -87 region for LHβ and inside of -95 in FSHβ. I will address two non-exclusive hypotheses: displacement of activators from binding sites and recruitment of TLE co-repressors. To build on these findings and to test the hypothesis that Six6 represses by recruiting co-repressors to the chromatin of gonadotrope genes, I will investigate the interaction with TLE/Grg proteins. The murine TLE (Grg, Groucho-related gene) proteins are co-repressors regulating development. Research conducted in Dr. Mellon?s laboratory has shown that both long-form (Grg4) and short form (Grg5) proteins regulate the GnRH gene by co-localizing with through Oct1 and Msx1 in vivo. Elaborating on these findings, I will identify the possible role of TLE/Grg proteins in the repression of LHβ, FSHβ or GnRH-R. I will perform co-transfections using Grg4 and Six6 expression vectors and test the effects on LHβ, FSHβ or GnRH-R Luciferase reporter genes. Overall, the aim of the project is to further understand the role played by TLE/Grg co-repressing proteins in the effect of Six6 on gonadotrope genes. Such findings would help increase our knowledge of the mechanisms regulating GnRH release?mechanisms that play a key role in physiology and pathophysiology dealing with development and reproduction.
Abstract Title : Role of Mindin in Fibroblast Activation
Abstract : My research aims to understand the mechanism behind fibrosis, the over accumulation of fibrous connective tissue, using the skin as a model. The skin is primarily composed of two layers, the epidermis and dermis. The epidermis serves as the protective, outermost layer, and the dermis which consists of connective tissue and provides elasticity and strength to the skin. The main function of dermal fibroblasts, the main cell type in the dermis of the skin is to secrete collagen and fibronectin to maintain homeostasis of the extracellular matrix, the structural support of the tissue. In normal wound healing, fibroblasts become activated to make up for the loss of connective tissues and to aid in wound closure. However, prolonged activation of the fibroblasts leads to the disease pathology of fibrosis. Dr. Colin Jamora's laboratory in the Division of Biological Sciences previously identified potential secreted factors from keratinocytes, the main cell type of the epidermis that cause fibroblasts activation in the dermal layer. This is an unknown central process leading to fibrotic development in multiple organs and causes death of the tissue. The proteins secreted by keratinocytes expressing the gene Snail are potentially causing the activation of fibroblasts, which over secrete collagen. In order to investigate how fibroblasts are activated, I will analyze a transgenic mouse model that is engineered to overexpress Snail. The effect of over expressing Snail in the skin of mice results in similar characteristics to the fibrotic skin disease known as scleroderma, an increased hardening and thickening of the skin. My project included testing the role of Mindin, one of the identified proteins that may be contributing to the fibrosis pathology through its activation of the NFκB, a transcription factor involved in inflammation.
Abstract Title : Investigating the cytokine activity of the Drosophila Toll Pathway
Abstract : Comparisons between Drosophila species based on genome-wide transcription profiling have identified a core set of Toll-induced genes of unknown function that potentially play a role in the Drosophila innate immune response. We are currently developing assays to elucidate the function of these immune effectors and, for those that are likely to act as cytokines, identify their receptors. Ectopic expression of one candidate cytokine is associated with the proliferation of lamellocytes and melanotic tumor formation in larvae and adult flies. Bioinformatics studies suggest that this immune effector may act through a G protein-coupled receptor (GPCR) pathway. In order to study this, I have generated a stable cell line expressing a genetically encoded calcium indicator, GCaMP. Using a synthetic peptide and assaying for calcium influx in GCaMP expressing cells, we hope to identify the mechanism by which this immune effector acts. The results of these studies hold promise for providing a better understanding of these novel immune effectors and of their roles in the innate immune response.
Abstract Title : Chytrid pathogenesis programs the algal host proteotype
Abstract : Some species of single-celled green algae in the genus Scenedesmus are highly susceptible to infection by single-celled fungi in the phyla Chytridiomycota (i.e., chytrids). Infection in outdoor ponds can be so severe that cultivation becomes impossible. While chemical control measures may be feasible, it is less costly and more convenient to deploy resistant strains. As a step toward development of resistant strains we begin with a proteomic interrogation of a compatible interaction (where the algal strain is susceptible to chytrid infection) to establish the baseline molecular reaction of na´ve algae cells to chytrid attack. There is no direct evidence for innate immunity of algae to chytrids but we have observed varying levels of susceptibility associated with growth conditions or strain variation. We study signaling in algae by observing infection-induced changes in the levels and post-translational modifications of proteins. Mass spectrometry is used to quantify changes in levels, splice isoforms, sites of phosphorylation, and sites of acylation (methylation and acetylation) of proteins at the proteome-wide level. We will search for pattern recognition receptors and signaling triggered by infection and then use genetics to evaluate candidate receptors and signal transduction proteins. Whole-cell protein extracts and protein complexes from pulldowns are analyzed using Thermo LTQ linear ion trap tandem mass spectrometers. We use iTRAQ mass tagging to measure precise differences in protein levels between samples. Quantitative differences are confirmed and converted to absolute values using MRM assays with synthetic peptides on our chip-cube equipped Agilent 6410 triple quadrupole mass spectrometer. Mass spectra are interpreted using Agilent Spectrum Mill software running on our custom-built servers.
Abstract Title : Protective effects of spinach aldolase on muscle adenylate kinase and phosphofructokinase-1 activities
Abstract : From previous studies, a hypothesis was developed that ascorbic acid (AA) inhibits muscle glycolytic enzymes during periods of rest to facilitate the storage of glucose as glycogen. Phosphofructokinase-1 (PFK-1) is considered the enzyme that controls the rate of glycolysis and along with lactate dehydrogenase (LDH) and adenylate kinase (AK), PFK-1 is inhibited by AA. During contraction, AA does not inhibit glycolysis due to the formation of a complex of glycolytic enzymes with contractile muscle proteins that protects them. Muscle aldolase prevents AA inhibitions and its protective properties are considered a microcosm of the complex formed with contractile muscle proteins. It was observed that spinach aldolase appeared to protect muscle PFK-1 activity from AA inhibition. The current study focuses on this ability of a plant enzyme, spinach aldolase, to interact and protect animal enzymes. Specifically the studies showed that spinach aldolase protected muscle AK activity loss due to dilution and protected muscle PFK-1 from inhibition by each AA and ascorbyl dipalmitate, an AA derivative shown to be more inhibitory than AA alone.
Abstract Title : Intestinal Intraepithelial Lymphocyte (IEL) Decline in the Obese Mouse Model
Abstract : The intestine is a key organ that digests food and absorbs nutrients. In the small intestine villi are found in the mucosal layer increasing the overall surface area for nutrient absorption. The epithelial layer of the villi contains intraepithelial intestinal lymphocytes (IEL) that are important in maintaining epithelial homeostasis and repairing damaged tissue. Obesity is caused by nutrient excess leading to a state of chronic inflammation that impacts epithelial tissues such as the intestine. Our goal is to study how obesity causes epithelial barrier disruption that impacts the severity of colitis. To mimic obesity, mice are placed on a high fat diet (HFD) and IEL numbers are monitored in the epithelial layer. HFD mice show dramatically reduced numbers of IEL in the epithelium of the intestine. In addition, obesity results in increased intestinal villi length. Furthermore, imunofluorscence imaging of lamina propria macrophages indicates that there are increased numbers of M1 CD11b+ CD11c+ proinflammatory type macrophages that may be exacerbating the severity of chronic inflammation in obesity on the IELs. Partial IEL numbers and function can be rescued after the mice are taken off the HFD. These data show the detrimental effects of obesity on the small intestine and in future studies we specifically hope to find treatments to fully restore IEL numbers and function.
Abstract Title : Signaling Pathways Governing Cardiac Inflow Tract Formation in Zebrafish
Abstract : The inflow tract is a structure that serves as a passageway for blood to enter the heart. However, very little is known about how the inflow tract is initially formed and the signaling pathways that are required for its proper specification. Through the use of molecular markers and different pathway inhibitors, I aim to gain a better understanding of the formation of the zebrafish inflow tract. Recent studies have utilized bone morphogenetic protein 4 (bmp4) as a marker for the inflow tract, but it is unclear when bmp4 expression is initially activated in the inflow tract. To test this, I have performed in situ hybridization on zebrafish embryos at different stages in development to determine when bmp4 is expressed in the cells that go on to form the inflow tract. I have found that bmp4 expression transitions from a broad region to a defined region over time. With this knowledge in hand, I will examine zebrafish embryos at key time points using molecular markers to determine the pathways that regulate this transition. The inflow tract forms adjacent to the atrium, but it is not known whether pathways that regulate atrial development will affect the inflow tract in the same way. Based on previous experiments, we know that there are two signal transduction pathways that are important for atrial cardiomyocyte development: the BMP and retinoic acid pathways. A decrease in BMP signaling reduces the number of atrial cardiomyocytes, while inhibition of retinoic acid synthesis increases the number of atrial cardiomyocytes. I am currently testing the roles of these pathways on inflow tract development by treating embryos with chemical inhibitors and examining their effects on inflow tract markers, and I will present my latest findings.
Abstract Title : Characterizing divergent bacteriophage-encoded tubulin homolog "PhuZ"
Abstract : We have identified and characterized a protein encoded by a bacteriophage that shares high sequence homology with divergent prokaryotic tubulins FtsZ and TubZ. Named PhuZ, short for Phage TubZ, this divergent tubulin is synthesized by 201ϕ2-1, a large dsDNA myoviridae phage that infects bacterial host Pseudomonas chlororaphis. While the crystal structure of PhuZ, determined by the Agard Lab at UCSF, greatly resembles that of FtsZ, its C-terminal hook-like tail is unique. From structure-based interaction predictions, we hypothesize that this tail plays a crucial role in filament assembly. Through fluorescence microscopy and growth curve experiments comparing the wild type protein with proteins mutated at amino acids expected abolish tail function, we have begun to characterize this filament-forming tubulin-like protein encoded in the genome of a bacteriophage. The results suggest that PhuZ, like TubZ, polymerizes into filaments and exhibits treadmilling in vivo. However, it is singular in that its C-terminal hook-like tail plays a crucial role in its assembly. Mutations at this site increase the critical concentration for assembly, but assembled polymers show the same dynamics as wild type. The study of PhuZ has uncovered new possibilities in our understanding of tubulin polymer assembly and in our conception of how large phage successfully replicate and lyse their hosts.
Abstract Title : Effects of kisspeptin signaling on body weight in mice
Abstract : GPR54, also known as kisspeptin receptor, is known to function in the onset of puberty and the regulation of the endocrine reproductive system. However, the effects of GPR54 on energy balance and metabolism in mice is not yet known. We aim to track the body weights of GPR54 knock-out, heterozygote, and wild-type mice in order to study the relationship between GPR54 function and energy balance. We also look at glucose tolerance in mice with and without the kisspeptin receptor. This poster presents preliminary data that has been completed thus far.
Abstract Title : Using Rodent Parasite Systems to Study Anthelmintics (anti-worm drugs) for Human Intestinal Parasites
Abstract : Soil-transmitted helminths, commonly known as intestinal roundworms, are the most common parasitic infections worldwide, affecting upwards of two billion of the world?s poorest people. Infections most seriously impact children, leading to significant cognitive impairment and stunted growth. Sadly, research efforts dedicated to studying and developing anthelmintics are limited. Our lab focuses on studying soil-transmitted helminths through the use of rodent-infected parasites that closely mimic parasite infections in humans. We assessed the efficacy of different anthelmintics and the advantages and limitations of each rodent parasite system by conducting in vitro assays. Our research could indicate an ideal system for studying anthelmintics.