UCR

Center for Plant Cell Biology



2005-2011 IGERT Student Descriptions


YIFAN LII

Genetics, Genomics and Bioinformatics Graduate Program
Title of Project: Chemical Genetic Dissection of RNA Silencing in Arabidopsis thaliana 

 

 

Yifan Lii
Background:

I received a B.S. in Biochemistry/Cell Biology and a minor in Psychology from UC San Diego in 2005. While I was an undergraduate I started my research experience in a environmental microbiology lab studying manganese oxidizing bacteria from deep sea hydrothermal vents. Later, I moved to an immunology lab studying rheumatoid arthritis. Before starting graduate school here at UC Riverside, I worked in a lab studying the evolution of cooperation in rhizobia. This was my introduction to working with plants. I am in the Genetics, Genomics and Bioinformatics program and I am interested in plant pathology.

Introduction:

I am currently studying the role of small RNAs in plant immunity. Small RNAs are short (~20-30 nucleotides) non-coding RNA elements that can suppress gene expression in a phenomenon called RNA silencing, which operates in plants and many animals. They regulate gene expression by directing messenger RNA cleavage or protein translation inhibition (posttranscriptional gene silencing) or by mediating chromatin modification (transcriptional gene silencing). In plants, they are involved in a diverse range of pathways and processes including development, genome maintenance, genomic imprinting, and abiotic and biotic stress response. There are many types of small RNAs, one of which is natural antisense transcripts-derived small-interfering RNA (nat-siRNA), so called because it is produced from overlapping sense and antisense transcripts from the same genomic loci. To complement genetic studies, I conducted a screen for compounds that interrupt a nat-siRNA pathway in Arabidopsis thaliana using a luciferase transgenic line exhibiting silencing. Chemicals from the “Library of AcTive Compounds on Arabidopsis” (Sean Cutler) were used to perturb the silencing pathway to identify silencing-inhibition chemicals and new proteins or receptors involved in the biogenesis or function of nat-siRNAs. I used this approach due to its ability to overcome problems found in traditional genetic screens, such as duplicate or essential genes. After several rounds of screening, I found 12 putative hits, currently under further study. One to two molecules effective at perturbing nat-siRNA will be further studied to discover their mode(s) of action. This cross-disciplinary project exemplifies the discovery potential that lies at the interface between chemistry and genetics.


 

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ROBERT KOBLE

Plant Biology Graduate Program
Title of Project: Identifying Boundary Region Processes through Chemical Genomics

Robert Koble

Background:

I received my B.S. in Cell Biology at the University of California, Davis in 2009 alongside a Minor in Music. During my time at UC Davis, I studied putative target genes of the Arabidopsis embryonic transcription factor LEAFY COTYLEDON1 (LEC1) under the tutelage of Dr. John Harada. Through this project, I fell in love with Plant Molecular Biology which led me to join the Plant Biology Graduate Department at the University of California - Riverside in the fall of 2009. Upon completion of my rotations, I decided to join the Springer Laboratory to study the function of the boundary region in Plant Development

Figure 1
Figure 1: A) represents WT seedlings grown in the dark. Note the apical hook. B) LOB overexpressed seedling. Note the lack of apical hook.
Introduction

 

 


The boundary region serves to primarily separate the lateral organs (including leaves) from the Shoot Apical Meristem (SAM) and contains a set of genes that are specifically expressed in this region. One gene that is expressed in the boundary region in Arabidopsis thaliana is LATERAL ORGAN BOUNDARIES (LOB). LOB encodes a DNA-binding transcription factor that differentially regulates target genes to form boundaries.

My Chemical Genomics project will primarily focus on identifying chemicals that perturb the function of LOB. After identifying chemical interactors of LOB, we can apply these chemicals to adult plant to tease apart functions of LOB that have been previously elusive.

To assay for LOB antagonists, I will screen for a gain of apical hook upon application of these chemicals in a LOB over-expression background.  One of the phenotypes of LOB over-expressed etiolated seedlings is a loss of apical hook (see Figure 1), which is due to enhanced LOB activity. I will be taking advantage of this LOB over-expression phenotype by screening for chemicals that suppress the LOB over-expression phenotype by assaying for seedlings that do not have an apical hook.

I have identified two chemicals that show suppression of the LOB over-expression phenotype (apical hook) called LAT24D02 and LAT30D07. To see if these chemicals affect LOB directly, I observed the transcript level of LOB’s target gene, BAS1 (data not shown) and found that BAS1 transcript level is not altered upon chemical application suggesting that these chemicals might work downstream of the LOB pathway

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JESSICA DIAZ

Plant Biology Graduate Program
Title of Project: (TBD)

Jessica Diaz

Background:

I received my Bachelors of Science in biology, biotechnology option, with a minor in chemistry from California State University, Northridge in spring 2009.  I began attending the University of California, Riverside in fall 2009 as a graduate student in the Ph.D. program for the Department of Botany and Plant Sciences.

Introduction:

In rice, Brassinosteroids (BRs), plant steroid hormones that regulate cell division and expansion, have been shown to contribute to shoot architecture, primarily controlling plant height and leaf erectness. BR-deficient rice plants have increased leaf erectness, which is an important agronomic trait, but they also are small in stature and have reduced fertility, which diminishes yield. In Arabidopsis, LATERAL ORGAN BOUNDARIES (LOB), a plant-specific transcription factor, is expressed in the boundary between the SAM and initiating lateral organs. LOB regulates organ separation by modulating accumulation of BRs. LOB expression is positively regulated by BRs and LOB activates expression of BAS1 to inactivate BRs, resulting in a feedback loop to limit BR accumulation in organ boundaries.

Since LOB is expressed in a discrete domain in Arabidopsis and controls BR accumulation, I am investigating the possibility of using LOB orthologs, LOB, and BAS1 in rice to manipulate local BR accumulation in the lamina joint, with a goal of creating plants with more erect architecture than wild type. Modifying rice plants to have reduced BR accumulation only at the lamina joint is predicted to result in increased leaf erectness without the other detrimental effects on shoot architecture that BR deficiency causes. Creating a rice plant with leaf erectness can be extremely beneficial to growers. Plants with erect leaves require less growth space, allowing higher density planting, which contributes to increased grain yields per acre.

To date, transgenic rice plants have been generated expressing the GUS reporter gene under the Arabidopsis LOB promoter. I found that this promoter drives expression in organ boundaries and in the lamina joints in rice. I therefore used the LOB promoter to drive expression of Arabidopsis LOB and BAS1 in rice. We predict that mis-expressing LOB and BAS1 in the organ boundaries in rice should create a rice plant that has reduced amounts of BR at the lamina joint. My goal is to create a rice plant with erect leaves and no other alterations in plant architecture.

The rice genome encodes 35 LOB-domain proteins, which share a conserved DNA binding domain [12]. The complexity of this gene family means that LOB orthologs may not be identified solely on the basis of phylogenetic analyses. I will therefore examine rice genes most closely related to LOB for both possible regulation by BR and for pattern of expression. These criteria should allow us to identify the best candidate LOB ortholog(s) in rice. Rice seedlings will be treated with exogenous BR in a time-course and transcript levels of these genes will be measured using standard approaches. I will also examine the expression pattern of LOB-related genes in rice seedling tissues using in situ hybridization. Observing the pattern of transcript accumulation in rice tissues will determine if any of these genes are expressed in organ boundaries (similar to LOB) or in the lamina joint.

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AARON DEVRIES

Plant Biology Graduate Program
Title of Project: (TBD)

Aaron DeVries

Background:

I originally graduated from Iowa State University in 1999, with a bachelor’s degree in Botany. After graduation, I became employed by the prominent seed company Pioneer Hi-Bred International, where I worked on sunflower seed oil research, and helped validate genetically modified crops for commercial sale. Then in 2006 I went back to school part-time to earn a second degree in Genetics. While there I worked part-time in the lab of Dr. Thomas Baum, who studied the infection process of soybean cyst nematodes. Next, I went back to Pioneer in 2007, and began researching ways to improve the amino acid content of corn seed in the labs of Dr. Rudolf Jung and Dr. Fred Gruis. Finally I moved to UCR in the fall of 2009, in order to begin the PhD program in Plant Biology.

Introduction:

When producing commercial crops such as apples, pecans or avocados, the perennial life cycle of the trees often makes it difficult to obtain a consistent yield every year. Instead, the trees display a somewhat cyclical behavior known as alternate bearing, wherein they produce an abundance of fruit one year, and almost nothing at all in the following year. Current evidence suggests that developing fruits inhibit the formation of next year’s flower buds, possibly by altering the distribution of carbohydrates and hormones within the plant. In order to study the former, I am using mass spectrometry to observe the profile of sugars in Avocado meristems, and correlating that with the expression of genes known to be regulated by carbohydrates. When measured over the course of the 2-year cycle, the change in sugar profiles can be used to build up a model of inflorescence inhibition starting from the beginning. In addition, I am also using microarray data obtained from Arabidopsis thaliana inflorescences, in order to identify additional genes correlated with inflorescence inhibition. By treating the plants with different hormones, hormone inhibitors, and sugars, I plan to identify the unique contributions of each molecule (through its associated gene expression pattern), and work backwards towards a common mechanism. Once areas of overlap are identified, I will combine the two data sets and use them to develop a plausible genetic model of how fruits can regulate the number of flowers in the following season.

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RAE EDEN YUMUL

Plant Biology Graduate Program
Title of Project: Exploring Plant Processes with Chemical Biology


Rae Eden Yumul
Background:

I graduated from the University of Pennsylvania in May 2008 and received a Bachelor of Arts in Biology with a concentration in neuroscience. As an undergraduate, I worked in the lab of Dr. Marc Schmidt on a research project aimed at identifying and characterizing neuronal projections underlying behavioral-state dependence in the song control system of zebra finches. In the fall of 2008, I began my graduate studies in the Botany and Plant Sciences program at UCR.

Introduction:

My IGERT project currently remains undefined as I have yet to complete my rotations as a first-year student. I did my first rotation in the lab of Dr. Xuemei Chen, working primarily on mapping several enhancer mutations identified in a sensitized screen in the ag-10 background. These mutants exhibit enhanced defects in floral determinacy and development. My second and current rotation is with Dr. Zhenbiao Yang; I am developing a screen to efficiently assess the effects of chemicals on pavement cell shape. More specifically, the screen would be used to identify small molecules that interfere with or enhance the actions of ROP GTPases, which are critical for growth and polarity in several plant systems (e.g. pollen tubes, root hair cells, pavement cells). I will begin my third rotation with Dr. Patricia Springer before the end of the year.

Publications:

 

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AYESHA BAIG

Plant Biology Graduate Program
Title of Project: Dissection of Conserved Defense Regulated Pathways in Arabidopsis and Tomato by Chemical Genomics

Ayesha Baig

Background:

I received my Bachelor of Science (Honors) in Plant Breeding and Genetics from the N.W.F.P Agricultural University, Peshawar, Pakistan in the year 2000. In 2003 I received my M.Phil degree in Biotechnology from the Institute of Biotechnology and Genetic Engineering (IBGE) at the N.W.F.P Agricultural University, during which I did my research work at the National institute for Biotechnology and Genetic Engineering (NIBGE) Faisalabad on DNA viruses of the family Geminiviridae that have caused major crop losses in Pakistan. Later I worked at the N.W.F.P Agricultural University for three years as a Lecturer and researcher on exploiting the cation co-tolerance phenomenon in potato plants before joining the University of California, Riverside in 2007 as a Fulbright student for my graduate studies.

Introduction:

Currently I am working in Dr. Thomas Eulgem’s lab examining defense mechanisms in Arabidopsis and tomato that involve WRKY70-like transcription factors and members of the LURP cluster of coordinately expressed defense genes. I am performing experiments that examine possible LURP-dependent mechanisms in tomato by silencing the WRKY70 ortholog in tomato and measuring transcript changes of putative orthologs of Arabidopsis LURP genes (which includes CaBP22). This will partially be done by silencing WRKY70 ortholog in -333_CaBP22::GUS tomato lines and examining the effect of WRKY70 orthologs on defense-related expression of -333_CaBP22:: GUS in tomato. These experiments will address if the “WRKY70/CaBP22 regulatory module” is conserved between different plant species.

I am also working on dissecting the biological and molecular roles of the Arabidopsis gene LURP1 and its paralogs LOR1 (LURP-one related 1) and LOR2. Mutations in LURP1 result in reduced disease resistance to the oomycete Hyaloperonospora parasitica. I am in the process of testing if LOR1 and LOR2 T-DNA mutants that show defense-related phenotypes similar to those of LURP1. In addition I will construct LURP1/LOR1 and LURP1/LOR2 double mutants to address if these genes contribute in an additive or synergistic manner to disease resistance.

In addition to this I will carry out screens of Arabidopsis EMS mutants carrying a -333_CaBP22::GUS reporter gene for lines with altered sensitivity for 3,5-Dichloroanthranilic acid (DCA), a potent synthetic elicitor of disease resistance to virulent strains of the oomycete Hyaloperonospora parasitica . These screens may lead to the identification of the biological target of DCA or defense components operating downstream from DCA perception. The overall goal of my work is to get an insight in to key regulatory networks involved in defense mechanisms and dissection of regulatory networks controlling the defense transcriptome across plant species.

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GREGORY BARDING, JR.

Chemistry Graduate Program
Title of Project: Exploring Metabonomics by Chemical Biology

Gregory Barding

Background:

I completed my studies for a Bachelor of Science degree in Chemistry with a Biochemistry option from California State University, San Bernardino in June 2008.  While attending school, I worked on a joint project with the Department of Chemistry, Department of Physics, and NASA concerning hydrogen fuel cells and their efficacy in a high radiation environment.

Currently, I am interested in inter-disciplinary research and the many opportunities it extends to my Biology and Chemistry background.  I am anxious to expand my experience in those areas as well as improve my communication skills with other non-chemistry students.  As the worlds of Chemistry, Biology, and Computer Science collide, the skills and knowledge afforded by such experience will be invaluable.

Introduction:

My initial research project involves the metabolic studies of Oryza sativa (rice).  This is a joint project with Dr. Julia Bailey-Serres and her post-doctoral fellow Takeshi Fukao.  I am working on analytically determining the effects of submergence on different rice cultivars by examining their metabolic profiles through NMR spectroscopy. Future studies would include further analysis by GC-MS.  This study will provide valuable insight into the metabolic response of these plants and help to provide an explanation for the difference in efficacy among the cultivars.

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PATRICK SCHACHT

Genetics, Genomics and Bioinformatics Graduate Program
Title of Project: Studying G Protein Regulation in the Model Organism Neurospora Crassa

Patrick Schacht

Background:
I earned my Bachelor's of Science degree in Biochemistry from Biola University in Spring 2007. Subsequently, I began my work at UCR within the Genetics, Genomics and Bioinformatics graduate program. While rotating in my first year, I joined UCR's IGERT program to extend my research aims into the realm of Chemical Genomics.
Introduction:

Currently I am studying G protein signaling in the Borkovich lab. Using the model organism Neurospora crassa, I am examining proteins that regulate the G-protein signaling cascade through analysis of mutants and protein-protein interaction studies. To start, I will be performing high throughput chemical screens to identify compounds that both promote and inhibit key interactions in G protein signaling. Alongside this screen, I will also use randomly generated suppressors of gene deletion mutants to better understand the roles of uncharacterized interactors within the pathway. Lastly, I will be using site-directed mutagenesis combined with bioinformatics applications to predict and confirm exact points of interaction between proteins essential to G protein signaling.

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MOSES TATAW

Computer Science Graduate Program
Title of Project: Computational Analysis of Dynamical Processes in Developmental Biology

Moses Tataw

Background:
I graduated from the University of California, Riverside in June 2005, with a bachelor’s degree in computer science.  I transferred to UCR from Mt. San Antonio College, in Walnut, California. As an undergrad, I was involved in research in other areas of physical sciences. For example, I conducted research at NIST-Boulder under the direction of Dr. Elizabeth Donley, working to develop a double pass acousto-optic modulator to be used to develop the next generation atomic clock.  After graduating from UCR, I went on to the University of California, San Diego, where I earned a Master’s of Science degree in computer science in June 2007. My current interests include data mining, bioinformatics, and developmental biology.
Introduction:

I’m currently working with Dr. Reddy Venugopala to study the relationships between cell deformation patterns and cell division patterns. Pattern formation in developmental fields involves precise spatial regulation of gene expression and growth dynamics such as cell division, cell expansion, cell migration and cell death. The information is exchanged between multiple cell layers through cell-cell communication processes to regulate gene expression and growth dynamics. A quantitative and dynamic understanding of the spatial parameters of gene expression dynamics and growth patterns is crucial to model the process of pattern formation. As an example, shoot apical meristem (SAM) stem-cell niche in plants, is a dynamic multilayered structure consisting of about 500 cells and it provides cells for all the above-ground plant structures. The SAM has been divided into distinct functional domains based on differential gene expression patterns and growth patterns. The cells in the outermost L1 layer and the sub-epidermal L2 layer divide in anticlinal orientation (perpendicular to the SAM surface), while the underlying corpus forms a multi-layered structure where cells divide in random orientation.  Within this framework, the SAM is divided into functional zones with distinct gene expression activity.  The central zone (CZ) contains a set of stem-cells and the progeny of stem-cells differentiate within the surrounding peripheral zone (PZ). The CZ also supplies cells to the rib-zone (RZ) located beneath the CZ. Thus, cell fate specification within SAMs is a dynamic process intimately coupled to transient changes in gene activation/repression and changes in growth patterns.

I would like to study cell deformation patterns and how they relate to the cell division patterns. First, by developing computational platforms to identify cells and track their progeny through successive cell divisions from fluorescent 4-D images acquired by using laser scanning confocal microscopy. Second, by introducing experimental manipulations that can alter the cell deformation patterns and analyzing their effects on cell division dynamics. The computationally derived cell growth models will also allow us to understand the causal relationship between cell growth and cell division patterns.

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SHANG WU

Plant Biology Graduate Program
Title of Project: Using Chemical Genetics Approach to Dissect the Function of Protein-Protein Interactions Required for Flower Specification

Shang Wu

Background:
I received my Bachelor’s degree in Molecular Biology and Biochemistry from the China Agricultural University in July 2007. During my undergraduate study, I was accepted into the Honor’s Program In Life Sciences and received basic training in biology for two years.  During my third year of undergraduate in this honors program, I performed my research in the Molecular Biology and Biochemistry Department. My undergraduate research project was focused on utilizing genetic and molecular approaches to dissect the Abscisic Acid signaling transduction pathway in Arabidopsis.  In the fall of 2007, I started pursuing my doctoral degree in the Department of Botany and Plant Sciences at University of California, Riverside.  Currently, my research project is focused on understanding the molecular events that regulate flower formation in Arabidopsis in Harley Smith’s laboratory.
Introduction:

The aerial organs or the plant are originated from a small pool of cells at the growing tip of the shoot called the shoot apical meristem (SAM).  One of the major developmental phase changes in higher plants is the transition from vegetative to reproductive growth, which is controlled by environmental and intrinsic developmental cues that converge at the SAM.  Two redundant functioning BELL1-like homeobox genes, PENNYWISE (PNY) and its paralogy POUNDFOOLISH (PNF), encode DNA-binding proteins that are essential for the specification of flowers during reproductive development. Biochemistry studies show that PNY/PNF associate with specific members of the KNOX class of homeodomain proteins including SHOOTMERISTEMLESS (STM).  Genetic studies indicate that PNY-STM and PNF-STM regulate early internode patterning events and control floral specification. Expressions of PNY, PNF and STM overlap in the vegetative, inflorescence and floral meristems. My research goal is to understand the how PNY/PNF-STM interactions regulate patterning events in the SAM.

Proteins are modular and often contain multiple domains or sequences that facilitate interactions with other proteins.  Transcription factors are highly modular containing multiple protein-protein interaction sequences as well as domains that associate with specific DNA-binding motifs.  Using chemical genetics, we aim to dissect the function of the protein-protein interactions mediated by PNY/PNF and STM.  Genetic approaches to study the function of transcription factors are useful are limited in that the phenotypes produced are the result of immediate and downstream processes.  My research goal is to utilize chemical genetics to dissect the function of PNY/PNF-STM interactions.  This approach will be combined with live imaging to determine the role PNY/PNF-STM regulate early and late patterning events in the SAM.  This research will be essential for understanding the molecular basis of flower specification.

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SEAN BOYLE

Genetics, Genomics and Bioinformatics Graduate Program
Title of Project: Chemical Space Analysis and Virtual Screening in Compound Optimization

Sean Boyle

Background:
I graduated in May 2005 from Indiana University with a Bachelor’s of Science in Informatics and minors in Biology and Chemistry.  During my undergraduate education I experimented on the mes genes of C. elegans determining whether mes-4 functions alone or with other proteins in a macromolecular complex.  Continuing at Indiana University I received a Master’s of Science in Bioinformatics in May of 2007.  During this time my research focused on predicting protein-disease associations through analysis of newly available disease ontology and protein-protein interaction data.  In the fall of 2007 I accepted the IGERT Fellowship at the University of California Riverside and began the doctoral program in Genetics, Genomics, and Bioinformatics with a track in Genomics and Bioinformatics.  During the fall quarter I explored molecular descriptors and chemical space with Dr. Stefano Lonardi.  I am currently rotating in Dr. Michael Pirrung’s laboratory working on molecular descriptors and virtual screening of candidate compounds.
Introduction:

Understanding and identifying ligand enzyme binding is of great importance to the biological community.  This knowledge holds promise of improved drugability as well as providing a fuller understanding of cellular processes.  While High Throughput Screening (HTS) has provided for the identification of many molecular interactions, computational prediction methods classified as Virtual Screening (VS) have been gaining ground in recent years as an alternative or, perhaps more appropriately stated, a complementary approach to compound screening.  VS approaches are designed to computationally bind molecular partners in an attempt to predict biologically true matches.  By applying VS techniques I intend to add to the screening processes in my laboratory.

It was recently estimated that there could be upwards of 1060 small carbon-based compounds with molecular masses around the same range as in living organisms.  This is a truly amazing and in many ways entirely ungraspable number.  While this ability is a wonderful thing, it has recently been noticed that the number of biologically active compounds is strikingly correlated with natural product like scaffolds.  In many ways this makes sense.  Natural products, which are compounds created by cellular machinery, have gone through millions of years of trial and error and contain characteristics that allow them to move and more peacefully exist within the cell.  While we initially thought we should hit chemical genomics with every compound we can create, it is starting to look like we should start that process by looking at natural product like molecules.  By analyzing chemical space I am interested in how to better predict binding patterns.

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MICHELLE BROWN

Genetics, Genomics and Bioinformatics Graduate Program
Title of Project:Identifying Novel Components in Plant Vesicular Trafficking via Chemical Genomics

Michelle Brown

Background:
I obtained my Bachelor of Science degree in Biological Sciences with an emphasis in Microbiology in June of 2007 from the University of California, Riverside. I am currently enrolled in UCR’s Genetics, Genomics and Bioinformatics program and am concentrating in molecular genetics. My main interests are the plant endomembrane system and vesicular trafficking.
Introduction:

Germinating pollen tubes clearly display unidirectional cellular growth. This kind of polarized growth requires calcium gradients, the cytoskeleton, and tip-focused vesicle trafficking, which is organized via Rho-related GTPases. Proteins in somatic cells also travel through a variety of pathways from the ER to the vacuole, and to and from the plasma membrane using endocytic and secretory trafficking pathways (Figure 1). Thus pollen is a good model for other cell types.

Research Plan:
Plant Endomembrane System
Figure 1: The Plant Endomembrane System. The plant endomembrane system contains compartments and trafficking components that are conserved among all eukaryotes and some that are unique to plants. a) Amino-terminal propeptide (NTPP)pathway. b) Carboxy-terminal propeptides (CTPP). c) ER-to-vacuole pathway. d) ER-to-PAC-to-vacuole pathway. e) Secretion pathway. f) CCV endocytosis. g) Receptor-mediated endocytosis. CCP, clathrin-coated pit; CCV, clathrin-coated vesicle; CV, central vacuole; DV, dense vesicle; ER, endoplasmic reticulum; GA, Golgi apparatus; LV, lytic vacuole, N, nucleus; PAC, precursor-accumulating compartment; PB, protein body; PCR, partially-coated reticulum; PSV, protein-storage vacuole; PVC, pre-vacuolar compartment; SV, secretory vesicle.

A high-throughput strategy employing the Atto Pathway Confocal microscope will be used to carry out a chemical screen to identify small molecules that inhibit tobacco pollen tube germination. Once compounds have been confirmed to inhibit tobacco pollen growth, these compounds will be evaluated for their ability to induce phenotypes in Arabidopsis seedlings. Specifically, I will examine our laboratory’s large library of endomembrane fusion protein marker lines treated with chemicals that inhibit tobacco pollen tube germination to determine whether these compounds inhibit endomembrane trafficking in other tissues. Following the identification and characterization of these compounds, I will focus on target identification studies of one or several informative chemicals in order to determine their effects on dynamic endomembrane trafficking events such as endocytosis as well as to indentify cognate protein-binding partners using forward and reverse genetics.

Surpin and Raikhel (2004). Traffic Jams Affect Plant Development and Signal Transduction. Nature Reviews/Molecular Cell Biology 5:100-109.

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ANNA CHARISI

Genetics, Genomics and Bioinformatics Graduate Program
Title of Project:Compound Selection Strategies for High-throughput Screening (HTS)

Anna Charisi

Background:
I graduated from the National and Kapodistrian University of Athens, Greece in 2000 with a Bachelor’s degree in Informatics and Telecommunications. In 2003 I received a master’s degree in Signal Processing for Communications and Multimedia and afterwards I attended a second MSc in Bioinformatics from the same university. I have worked for 6 years in the University of Athens as a researcher and I have been actively involved in several national and European funded projects in various scientific areas, varying from web-development, database management and virtual environments to satellite image processing and electronic government tools. I have also worked in the National Observatory of Athens and the Hellenic Center of Marine Research developing software tools for the integration of multiple system components. In November 2005 I came to the University of California, Riverside as an exchange visitor in the Database Laboratory under the supervision of Prof. Gunopulos in the Department of Computer Science & Engineering in the framework of the “Health-e-Child” project, working in the area of biomedical data mining. In Fall 2006 I began my graduate studies at the University of California, Riverside in the Genetics, Genomics and Bioinformatics Ph.D. program. Currently I am working in Dr. Thomas Girke’s lab in the area of Chemoinformatics. I am focusing on developing methods and computational tools for the selection of compounds from combinatorial libraries and the prediction of their bioactivity based on structural and physicochemical properties for QSAR analysis.
Introduction:

High-throughput screening (HTS) is a method used in Drug Discovery and Chemical Genomics to assay a large number of compounds in order to identify bioactive compounds with respect to a particular biomolecular pathway. It is desirable to screen a selection of compounds that cover as much of the appropriate chemical space as possible, in order to increase the “hit” rate – the number of the bioactive compounds in the assay. Since the chemical space of all possible chemical compounds is extraordinarily large, most compound libraries available today represent only a part of it. Therefore, there is the need for selection of diverse compounds among all the available compounds that exist in the libraries. Moreover, if we could predict the bioactivity of the compounds in silico, the design of the screening libraries would be more effective and would save a lot of time and money.

The study of my research project is to develop new efficient methods for the selection of most diverse compounds from combinatorial libraries based on their structural and physicochemical properties. At the same time machine learning approaches will be applied in order to build a computational tool for the prediction of the compounds bioactivity using the above properties. These two approaches aim to facilitate the design of compound libraries for screening with higher hit rates.

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ANDREW DEFRIES

Plant Biology Graduate Program
Title of Project:Chemical Genetics and Drug Design

Andrew Defries

Background:

I received my undergraduate degree in Zoology from University of Toronto in the fall of 2006. My area of expertise was endocrinology, embryology, physiology, and the molecular-genetic mechanisms responsible for the orchestration of these complex biological events.

My interest in UCR was initially piqued by one man; Professor Sean Cutler in the Department of Botany and Plant Sciences, previously of University of Toronto. In the summer of 2006, I undertook an undergraduate research project with Dr. Cutler to look for chemical inhibitors of hypocotyl elongation. This screen was motivated by the
estimate that 15% of the Arabidopsis genome is devoted to biogenesis, turnover, and modifications of the cell wall (Carpita et al., 2001). We reasoned that cell wall growth and expansion may be utilized as a reporter for interaction of small molecules with a significant portion of the Arabidopsis proteome. That summer I helped conduct a forward chemical genetic screen of 12,000 small molecules and identified some QTLs involved in drug sensitivity.

To date the Cutler lab has discovered and annotated a 2,000+ bioactive small molecule library called LATCA or Library of AcTive Compounds in Arabidopsis. Subsets of LATCA have been implicated as auxin analogs, cytokinin analogs, brassinosteroid inhibitors, modulators of abscisic acid and ethylene signalling. This is by no means an exhaustive list. Furthermore, compound classes in LATCA closely recapitulate mutants in various pathways including cellulose synthesis. I am currently curating the annotation of LATCA compounds. Involvement with the LATCA project, at the very least, has opened my mind to the power of chemical genetics to systematically perturb biological processes.

Prior to coming to UCR I completed a six-month research project with Dr. James Eubanks, of the Toronto Western Research Institute. The project was designed to test a new technology pioneered by the lab of Steven Dowdy called protein transduction. My project with protein transduction and my early exposure to chemical genetics aroused a lust to develop biological technology. I strongly feel the availability and power of new scientific tools go hand-in-hand with discovery, and intend to devote my Ph.D. to bolster this relationship. In addition, my exposure to the robotic instrumentation at the Institute
for Integrative Genome Biology core instrumentation facility at UCR has endowed me with an awesome power to perform large scale experiments which include but are not limited to high throughput chemical genetic screening.

Chemical genetics is a blooming field, and I aim to help unveil points of intersection between Plant Biology and Chemical Space. Pursuit of a Ph.D. degree in Plant Biology, in conjunction with the ChemGen IGERT program, provides a unique opportunity to perform world-class interdisciplinary research. I am in awe of what the future has in store.

Introduction:

My research project is focused on the following:

  • The modulation of protein-protein interactions involved in plant hormone response using small molecules. Current Y2H project.
  • Investigation into the question, "is glyco-activation of small molecules a ubiquitous or rare event?" Knowledge of this rare chemical genetic phenomenon, glyco-activation, has potential to guide the design of novel and potent glycoside drugs.

Andrew presented a poster [abstract] titled "A LIBRARY OF BIOLOGICALLY ACTIVE SMALL MOLECULES FOR PLANT CHEMICAL GENOMICS" at the International Conference for Arabidopsis Research in Montreal, Canada in August 2008.

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MELINDA RODGRIGUEZ-SALUS

Plant Biology Graduate Program
Title of Project: Identification of SA-Independent Defense Elicitors by Chemical Genomics

Melinda Salus

Background:
I received my Bachelors of Science with a major in Botany from the University of Wisconsin-Madison in 2006.  At UW-Madison I worked for Dr. Douglas P. Maxwell, Emeritus Professor in the Plant Pathology Department, there I did research on disease resistance in tomato.  After graduation I came to the University of California-Riverside to begin a PhD program in the Department of Botany and Plant Sciences.  I work in the lab of Dr. Thomas Eulgem and am currently interested in dissecting plant pathogen interactions using chemical genomics and the model plant system Arabidopsis and oomycete pathogen, Hyaloperonospora parasitica.
Introduction:

Using chemical genomics, I will perform high-throughput screens to identify compounds able to activate PDF1.2a-promoter/reporter fusions in transgenic Arabidopsis seedlings.  PDF1.2a was one of a group of five genes that were shown to be up-regulated, in a JA-dependent manner in response, to infection of Hyaloperonospora parasitica (Peronospora).  This group of genes was termed the “JEDIs” which stands for Jasmonic Acid and Ethylene Dependent Induced genes.  My long term goal is the identification of a suite of elicitors capable of targeting defined points and thus specific branches of the defense network. These synthetic elicitors will be powerful tools for a fine dissection of defense mechanisms, because they will trigger strong, uniform and synchronous defense responses in plants and cell cultures.  Previously identified synthetic elicitors specific to the SA-pathway of the plant immune response allow us to stimulate that branch of the web and the identification of additional elicitors specific to the JA branch will allow for experiments that mimic the complex state of the plant after infection.  The protein targets of any identified synthetic elicitors will be identified using mutant screens with altered sensitivity to the compound as well as a variety of biochemical approaches, such as phage display.  Compounds discovered that manipulate the immune systems in Arabidopsis show potential for application on agriculturally relevant plants.  These identified synthetic defense elicitors may facilitate the development of new pesticides that are able to stimulate the plant’s inherent defense capabilities and reduce the quantities of older more harmful pesticides that agricultural growers use.

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MELISSA SCRANTON

Plant Biology Graduate Program
Title of Project: Use of Chemical Genomics to Understand Aminopeptidase Regulation in Plant Defense Signaling

Melissa Smith
Background:
I graduated in spring of 2007 from Harvey Mudd College with a Bachelor's degree in biology with a concentration in molecular biology. In the fall of 2007 I began my graduate studies at the University of California, Riverside in the Botany and Plant Sciences program. I am currently starting research in Dr. Linda’s Walling lab where I will focus my studies on plant defense signaling in tomato.
Introduction:

Aminopeptidases are a small subset of the peptidases that are ubiquitous in all living organisms. More than simply housekeeping proteins, aminopeptidases have been shown to have roles in the regulation of plant cell growth, development, homeostasis, and stress response. In particular, leucine aminopeptidases (LAP) are highly conserved proteins that have multiple functions in both eukaryotes and prokaryotes. The solanaceous-specific LAP-A has been shown to be upregulated in floral and fruit development as well as in response to biotic and abiotic stress. The focus of my research will be to understand LAP-A’s role specifically in the JA-dependent plant defense response in Solanum lycopersicum (tomato). I intend to screen chemical libraries for small molecules which either stimulate or inhibit LAP-A’s activity in this pathway. Identified chemicals will hopefully be useful tools to identify LAP-A’s regulation and mechanism of action which will in turn help to gain understanding in the regulation of an important plant signaling pathway.

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EDDIE CAO

Computer Science Graduate Program
Title of Project: Local Structure Similarity Searches to Identify and Predict Bioactive Substructures in Drug Databases

Eddie Cao
Background:
I graduated from Tsinghua University, Beijing in 2003 with a Bachelor's degree in electronic engineering. After that, I studied computer engineering and received a master's degree in 2005 from National University of Singapore. I began my Ph.D. study in computer science here in University of California Riverside in fall of 2005. My research focuses on design and analysis of algorithms. Since joining ChemGen IGERT program, I have been working on applying combinatorial algorithms and statistical learning methods to identification and prediction of bioactive chemical (sub)structures, using efficient and effective computational method to measure structure similarity among compounds in drug databases.
Introduction:

Structure similarity searching is one of the major techniques for retrieving chemical and bioactivity information from databases, as well as predicting drug properties of small molecules. The two most commonly used structure search approaches, substructure and structure similarity searches, both have major limitations in identifying local similarities between compounds. New computational methods for identifying these local similarities will have important applications in drug discovery and chemical genomics research, such as advanced QSAR studies, large-scale docking and diversity predictions of entire compound libraries.

The goal of my research project is to build a cheminformatics framework for efficient local similarity searching. This will enable chemical genomics researchers to search for bioactive compounds with higher confidence. In the initial stage, my project will evaluate the usefulness of 'Maximum Common Subgraphs' for local stucture similarity searching. The approach will include the following steps: (1) The computational complexity of the problem will be reduced by using heuristics that are specific to chemical structures and that can result in more effective local similarity measures. (2) Imperfect matching will be allowed by using an error tolerant method. (3) Further performance improvements will be achieved by designing an efficient approximation algorithms.

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JOLENE DIEDRICH

Analytical Chemistry Graduate Program
Title of Project: Capillary Electrophoresis Separation of RNA for Enzyme Analysis; Investigation of Protein Folding Using ESI-MS

Jolene Diedrich

Background:

I received my Bachelors of Science in Chemistry in the spring of 2006 from the University of Denver. I am currently in my first year at UCR in the Analytical Chemistry PhD. Degree program. I have conducted research in two analytical chemistry labs this fall under the guidance of Dr. Wenwan Zhong and Dr. Ryan Julian.

Introduction:

Capillary Electrophoresis Separation of RNA for Enzyme Analysis: Zhong Lab

During my rotation in Wenwan Zhong’s lab I worked on developing a capillary electrophoresis (CE) method to separate methylated and nonmethylated RNA strands. This research was conducted in collaboration with Dr. Xuemei Chen’s lab which is investigating HEN1. HEN1 is an enzyme which methylates RNA causing it to be resistant to degradation. In the absence of methylated RNA the cell dies. This CE separation would provide a screening method to identify chemical inhibitors of HEN1. Identification of a chemical inhibitor of HEN1 can then be used to select for HEN1 mutants in Arabidopsis

Investigation of Protein Folding using ESI-MS: Julian Lab

Julian’s Lab has developed an electrospray ionization-mass spectrometry (ESI-MS) method for analyzing protein folding. In this particular application 18-crown-6 ether is used to probe the availability of lysine side chains. The non-covalent attachment of the crown ether to the lysine will be effected by the intramolecular interactions within the protein. Changes in the proteins structure will result in changes in the intramolecular interactions. Reduced intramolcular interactions will increase number of lysine side chains available for interactions with the crown ether. By changing the composition of the solution a comparison of the number of attached crown ethers can be done to analyze changes in the protein structure. I am currently using this method to analyze differences in the folding of hemoglobin and sickle cell hemoglobin. Future plans include using this same method to analysis other proteins of interest, along with plans to study protein folding kinetics.

Identifying Post Translational Modifications of Proteins by Mass Spectrometry

Diedrich JK, Julian RR (2008) Site-Specific Radical Directed Dissociation of Peptides at Phosphorylated Residues.Journal of the American Chemical Society 130: 12212-12213. *Featured in C&E News!

Li N, Nguyen A; Diedrich J, Zhong W (2008)  Separation of miRNA and its methylation products by capillary electrophoresis. Journal of Chromatography A 1202: 220-223.

Many signaling pathways in organisms, including plant systems, are controlled in part by post-translational modifications of the proteins involved. While standard protocols can be used to determine if a protein is modified, it is more challenging to determine the specific site of modification. However the specific location of the modification is important to function and improper sites of modification are often found in diseased states. The focus of this work is to develop alternative methods for identifying sites of post translational modification using mass spectrometry.

Identification of phosphorylation sites by mass spectrometry is often difficult and results in ambiguous assignment. A selective method for assignment of phosphorylation sites was developed. (JACS, 2008, 130: 12212-12213) This method incorporates two levels of selectivity, first phosphorylation sites are chemically modified with naphthalenethiol which is reactive only to phosphorylation sites. Secondly the sites of modification are determined by photodissociation mass spectrometry. The chromophore which was incorporated in the first step causes specific fragmentation of the peptide backbone only at the sites of original phosphorylation. Fragmentation is detected by mass spectrometry allowing easy and unambiguous identification of the phosphorylation sites.

Phosphorylation Sites

Future work will apply this method to identifying unknown sites of phosphorylation in Rop signaling pathways through collaboration with the Yang lab. Further work could be envisioned wherein this technique is used to profile the proteome in the presence of a drug which affects phosphorylation.

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THERESA DINH

Plant Biology Graduate Program
Title of Project: Determine Function of a Putative PH Domain

Theresa Dinh

Background:

I obtained my B.S. at UC Davis with a minor in Communications in 2003.  Upon graduation, I worked at Protein Research and Phoenix Pharmaceuticals for one year.  I then participated in a post-bac program at the University of Missouri-Columbia.  I am currently a first year graduate student in the Department of Botany and Plant Sciences.  I research interest in studying plant development and stem cells.

Introduction:

A functionally important protein in plant developmental regulation has been found to have a homology domain to a protein that contains a pleckstrin homology (PH) domain.  This domain in our protein of interest has been found to bind lipids. The specific goal of my project is to determine which specific lipids bind to this domain and its biological relevance.

I will lipid binding and chemical assays such as NMR and capillary electrophoresis to deduce what specific lipids bind the PH domain.  In addition, I will utilize a battery of genetic as well as molecular biology assays to determine the function of the PH domain. 

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KAYLA KAISER

Analytical Chemistry Graduate Program
Title of Project: Probing the Metabolome of Arabidopsis thaliana upon Anaerobic Stress Using 1H-NMR

Kayla KaiserPublications:
  • "Metabolic Profiling Unravels Complex Biological Phenomena in the Model Plant A. thaliana." Presented at the NSF 2008 IGERT Project Meeting held May 18-20, 2008 at the Westin Arlington Gateway. [poster]
  • Kaiser KA , Merrywell CE, Fang F, Larive CK "Metabolic Profiling" in Quantitative NMR Spectroscopy in Drug Analysis U. Holzgrabe, Ed. In press.Expected Release Date: Oct 2008  Available online: Aug 2008.
    [Book Description; Chapter Abstract]
  • Branco-Price , Kaiser KA, Jang CJH, Larive CK, Bailey-Serres J "Selective mRNA translation coordinates energetic and metabolic adjustments to hypoxia and reoxygenation in Arabidopsis thaliana" Plant J. Early View Date: August 2008. [Full Text pdf]
  • Kaiser KA, Barding GA, Larive CK "Metabolic profiling of plants by 1H-NMR: A comparison of metabolite extraction strategies using rosette leaves of the model plant Arabidopsis thaliana" Magnetic Resonance in Chemistry. Accepted May 2009. [Full Text PDF]
  • Kaiser, K., Plant Responses to Abiotic Stress. In Consilience:Building Bridges Across Disciplines. Presentation at the Graduate Student Association Conference, University of California, Riverside held May 23,2009. [Powerpoint]
Background:

I received my Bachelor of Science degree in chemistry from the University of Nebraska at Kearney in 2002. While at Kearney, I carried out collaborative research with the Department of Environmental Quality, United States Geological Survey, and the Environmental Protection Agency on problems such as volatile organic compound contamination in soil, acetanilide herbicide contamination in groundwater, and the accumulation of antibiotics in groundwater. While attending school, I worked for an agricultural testing company called Ward Laboratories, where I analyzed soils, feeds, waters, plants, fertilizers and manures.

After graduating from UNK, I attended Arizona State University, earning a Master of Science degree in 2005. My research at ASU involved the development of implantable surface-plasmon resonance biosensors for the characterization of non-healing cutaneous wounds in human diabetic patients. The complex nature of the data generated necessitated the use of multivariate statistics such as principal components analysis for signal processing.

I am excited to be an IGERT fellow because it brings scholars from different areas of study into the same arena, which I believe will better overcome the scientific challenges we will face in the 21st century. In addition to valuing integration in science, I feel it is important to be a well-rounded person. Some of my extracurricular interests include teaching science at the community college, enjoying all kinds of dancing, cooking international cuisine at my home, and taking outdoor adventures to the mountains, deserts, forests, and beaches.

Introduction:

The aim of my project is to dissect the metabolic responses of Arabidopsis thaliana to environmental and chemical stress. One environmental stress we are investigating is low oxygen, which occurs naturally through soil compaction and flooding, but is practically carried out by replacing laboratory air with argon gas in a small chamber or by complete submergence in water. One method of applying chemical stress is by spraying seedlings with a small molecule known to have herbicidal activity. This project relies on chemistry, multivariate statistics, computer science, bioinformatics, plant biology and biochemistry to examine and interpret the plant's complex metabolic profile and how it changes upon these stresses.

Experiments in progress will probe the plant's response using wildtype and mutant plants. The tissue samples have kindly been prepared by Dr. Cristina Branco-Price, Dr. Takeshi Fukao, Dr. Angelika Mustroph, and Seung Cho Lee in the Bailey-Serres lab, although through my lab rotation I have learned how to raise and stress my own plants. In the work that has been carried out, A. thaliana was grown for seven days, harvested, and freeze dried. Small molecule metabolites were extracted from dry tissue and subjected to analysis by nuclear magnetic resonance spectroscopy (NMR). NMR is capable of providing information about which metabolites are present in the plant extract and their relative amounts.

Using a non-targeted analysis known as metabolic fingerprinting, we gained insight into unexpected metabolic adjustments that occurred in the plants in response to a hypoxic environment. Metabolite data were compared to transcript profiling data. These experiments generate large datasets with hundreds of highly correlated variables, therefore a battery of statistical tests were employed to extract biologically meaningful patterns from the data. Metabolic profiling and flux balancing experiments will be used to quantify the direction and magnitude of these adjustments in future experiments. The data will be used to construct predictive models of primary metabolism, which put the results into context at the molecular, cellular and organismal levels. This work allows us to build an arsenal of knowledge about the plant metabolome and its dynamic nature within a model system.

Kaiser Research

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AUGUSTA JAMIN

Genetics, Genomics and Bioinformatics Graduate Program
Title of Project: Chemical Genetics Approach in Studying ROP Signaling Pathway that Regulates Pollen Tube Growth in Arabidopsis thaliana

Augusta Jamin

Background:

I graduated in June 2003 with a Bachelor degree from California State University, San Bernardino in Chemistry – Biochemistry Option and a minor in Psychology. After graduation, I worked at Robert Mondavi winery and Charles Krug winery in Napa Valley. My work involved various aspects of winemaking such as performing various chemical tests on wine, preparing yeast culture for inoculation, handling tasting set-up, and performing quality control tests during bottling. During this time, I developed interest in understanding simple questions such as how temperature affects maturity of grapes or how nitrogen level changes during maturation of grapevine. I became very interested in studying molecular genetics and decided to pursue graduate study in this field. In fall 2006, I was accepted to the Genetics, Genomics, and Bioinformatics program at University of California, Riverside. I joined Dr. Zhenbiao Yang’s lab and am currently working on understanding the ROP signaling network in regulating pollen tube growth in Arabidopsis.

Introduction:

ROPs (Rho GTPases from plants), subfamilies of Rho GTPases, are involved in various cellular signaling processes including pollen tube growth. ROP signaling pathway is turned on when ROPs bind GTP (active form) and turned off when ROPs bind GDP (inactive form). Regulatory proteins, RopGAPs (GTPase-activating proteins) help increase GTPase activity of ROPs. Another class of regulatory proteins, RopGEFs (GDP exchange factors) help promote GDP-for-GTP exchange. Out of 11 members of ROPs, three of them ROP 1, 3, and 5 are expressed in pollen and are functionally redundant. Genetic analysis of ROP 1 has shown that overexpressing the constitutively active (CA) mutants of ROP1 leads to swelling of pollen tubes. In contrast, overexpressing the dominant negative of dominant negative (DN) mutants of ROP1 leads to inhibition of pollen tube elongation. The observation of terminal phenotypes of these mutants provided evidence that ROP signaling is involved in pollen tube growth. However, events following ROP activation in regulation pollen tube growth are difficult to determine solely by genetic analysis. Thus, another technique such as chemical genetics would be particularly useful for this study.

Research Project:

Chemical genetics is a relatively recent approach that has been used to study and understand molecular mechanisms and complex interactions in living systems. Identification of chemicals that can specifically modify gene/proteins of interest is key to the success of this chemical genetics approach. In addition, the rapid effect of chemicals allows one to perform a time course study following chemical treatment. Therefore, the initial goal of my work is to identify chemicals that inhibit ROP and RopGAP interaction which lead to accumulation of active form of ROP. For the chemical screen, I use yeast-two-hybrid system which is a tool to study protein-protein interaction. It assumes that when two proteins interact, transcription machinery can be recruited to drive the expression of reporter genes which can be easily quantified. Thus, it provides an excellent system for my screen. Using this system, potential chemicals will be identified as those that cause low expression of reporter gene activities which include histidine and beta-galactosidase. Once chemicals have been identified, other in vitro as well as in vivo assays will be utilized to confirm its effects on ROP and RopGAP interaction. In addition, the specificity of chemicals will also be tested against other type of protein-protein interaction. And the effect of chemicals on pollen growth will also be observed.

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CHARLES JANG

Genetics, Genomics and Bioinformatics Graduate Program
Title of Project: Chemical Genetics of Hypoxia Signaling in Arabidopsis thaliana

Charles Jang

Background:

I graduated in 2001 from California State University in Fullerton with my Bachelors in Biology and minors in Chemistry and Biotechnology. Following graduation, I worked in the private sector for Isis Pharmaceuticals manufacturing antisense therapeutics. I left Isis to pursue my graduate studies in the fall of 2004 in the Genomics and Bioinformatics track of the Genetics, Genomics, and Bioinformatics Department here at UCR. Here I began work in the Julia Bailey-Serres lab working on the understanding the response of Arabidopsis to hypoxia and the genes of unknown function in Arabidopsis using publicly available microarray data. Upon acceptance into the IGERT fellowship, I have focused my work on microarrays to apply to chemical genetics. I am also planning a reverse chemical genetics screen for inhibitors of MPK3/6 (MAP kinase 3 and 6) in Arabidopsis..

Introduction:

The field of chemical genetics is an adaptation of the field of drug discovery to the understanding of mechanisms in organisms. The field mirrors classical genetics in that the organism is perturbed using small molecules rather then mutations to dissect processes. With advances in diversity oriented chemical synthesis, the universe of available small molecules have exploded, allowing biologists a large set of tools with which to perturb organisms to study their function. In forward chemical genetics, a library of chemicals are screened against an organism to illicit a desired phenotype. Upon determining a chemical that produced the proper phenotype, the molecular target of that chemical must be determined.

Kinase Inhibitor Assay:

MAP kinase cascades are important signal transduction pathways for many different processes including hormonal responses, cell cycle regulation, stress signaling, and defense mechanisms. I am interested in the role of MPK3/6 in the Arabidopsis response to hypoxia. MPK3, 4, and 6 are the only three of the 20 putative MPK genes in Arabidopsis that has been experimentally shown to have MAP kinase activity. I plan to screen activated MPK3 and 6 extracted from hypoxia stressed Arabidopsis plants with small chemicals looking for inhibitors of kinase activity.

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SAMER ELKASHEF

Genetics, Genomics and Bioinformatics Graduate Program
Title of Project: Chemical Genetics Approach to Understanding Virus-Host Interactions in Arabidopsis thaliana

Samer ElkashefBackground:

I received my bachelor’s degree in Genetics from the University of California, Davis in the spring of 2004 and began my graduate studies at the University of California Riverside in the fall of 2004 in the Genetics, Genomics and Bioinformatics program (GGB). Before joining the ChemGen IGERT program, I was conducting research in a collaborative effort between the lab’s of Shou-wei Ding and Morris Maduro to understand the antiviral silencing pathway in the nematode C. elegans. Since joining the ChemGen IGERT program, I have started work in Arabidopsis thaliana to apply chemical genetics to understanding viral immunity in plants.

Introduction:

Chemical genomics is an approach in which synthetically created small molecules are used to block the activities of proteins of interest, a method often used as a means of drug discovery. My research involves generating transgenic plants that carry a modified viral genome that is compromised in its ability to successfully replicate in a wild-type Arabidopsis plant. I intend to screen for small molecules that can inhibit host proteins that will allow the virus to replicate unhindered. It is my hope that this screening strategy will yield to the discovery of novel host defense factors that will shed light on how a plant responds to an invading viral pathogen.

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JAMES KIM

Cell, Molecular & Developmental Biology Graduate Program
Title of Project: Using a Chemical Genomics Approach to Elucidate the Mechanism of G-protein Coupled Receptor Regulated Pathways in Neorospora crassa

James Kim
Background:

My educational journey can only be described as a long, circuitous one. After getting my bachelor’s degree of botany from University of California at Davis , I worked in the vegetable seeds industry for a company called PetoSeed (they are now called Seminis). My job involved working with fungal plant pathogens, and it really peaked my interest in these organisms. After 8 years of working in the industry, I decided to go back to school and get more formal education in science and accomplished this by getting a Master’s degree from California Polytechnic University of Pomona. My research involved genetically analyzing a genus of medicinal mushrooms called Ganoderma using amplified fragment length polymorphisms technology (AFLP). Finally, my journey has brought me to UCRiverside where I am now established in Dr. Katherine Borkovich’s lab investigating the mechanism of signal transduction in a model organism called Neurospora crassa.  

Introduction:

I am currently screening the ChemBridge chemical library for compounds that will either cause a wild-type strain of Neurospora crassa to conidiate under submerged conditions or correct the submerged conidiation of a G-protein mutant (Δgna-3). By finding “hits” that will make the wildtype behave like the mutant or vice-versa, I will be able to use them as tools to better dissect the GPCR-involved process of conidiation. Also, an additional goal of screening the chemical library is to look for anti-fungal compounds that will prevent germination of conidia or suppress growth of the fungus, a potential aid in fighting pathogens of plants and animals.

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COLLEEN KNOTH

Plant Biology (Genetics) Graduate Program
Title of Project: Function and Regulation of Genes Showing Late/Sustained Up-Regulation in Response to Peronospora parasitica

Colleen Knoth
Background:
I graduated Magna Cum Laude in the spring 2003 from California State Polytechnic University, Pomona California with a Bachelors of Science in Biology. I began my graduate work at UCR in the Fall of 2003, where I work in the lab of Dr. Thomas Eulgem.
Introduction:
Figure 1. Peronospora parasitica infected Arabidopsis plant
Plants are constantly under attack from a wide range of pathogens. How plants are able to recognize and activate specific defense responses is an active area of research. Interactions of Arabidopsis thaliana (Arabidopsis) and the fungus-like pathogen Peronospora parasitica (Peronospora) are a useful model system to study host gene regulation during plant-pathogen interactions (Figure 1). In this system specific disease resistance (R) genes recognize distinct Peronospora isolates and trigger signaling cascades leading to resistance. Using microarrays, a large group of Arabidopsis genes were identified that exhibit elevated mRNA levels during defense reactions. I focus on a subset of these genes that show a coordinated sustained Late Up-regulation in Response to Peronospora parasitica genes (LURP) (Figure 2A).Using T-DNA insertion mutants I have shown that several genes from this set are important for defense.
Research Plan:

Figure 2. LURP1 shows strong and localized expression in response to Peronospora parasitica. A . Normalized mRNA levels at 0, 12, and 48 hours post infection with Peronospora. LURP genes show a late/sustained up-regulation during resistant interactions that is disrupted in susceptible interactions. B. -114 LURP1::GUS lines show localized GUS expression at infection sites 48 hours after infection with Peronospora. GUS expression is absent in the water control. These photos represent preliminary data of the average behavior from 10 independent transformation events. 

Identification of Functional Promoter Elements of LURP Genes by Delegation Analysis and Cloning of Cognate Transcription Factors

I intend to identify cis-elements responsible for the co-regulation of the LURP gene set by deletion analysis with promoter::reporter (GUS) fusion constructs stably transformed into Arabidopsis plants. Any cis-elements identified will then be used by yeast one-hybrid screen or DNA-affinity chromatography coupled with mass-spectroscopy to identify their cognate transcription factors. Once identified the biological roles of these transcription factors can be studied through T-DNA insertion mutants.

Chemical Genomics

A fast and efficient way to identify novel regulatory cis-elements is to test promoter-reporter fusions in transient expression assays for their response to a defined physical or chemical stimulus in cell cultures. However, chemical substances (elicitors) to trigger expression of genes targeted by defined R-pathways are not available. I will conduct a screen to find chemicals that activate a GUS reporter fused to a LURP gene promoter in the absence of Peronospora. A good candidate reporter will show low background expression and be strongly expressed in response to the recognition of Peronospora (Figure 2B).

These screens are likely to identify several classes of chemicals. I anticipate identification of chemicals (elicitors) that activate parts of the Peronospora defense pathway by interference with R proteins or other pathway components. This screen will likely produce a large amount of data that will require the use of bioinformatics tools for interpretation. For example, informatics tools will be vital for the structural analysis and the retrieval of the any information currently known about the biological activity of the compounds identified. The discovery of substances specifically interacting with R proteins could be checked by their inability to induce LURP gene expression in mutants for these R genes. Chemicals will also be tested against other known defense mutants to determine their placement in the signaling hierarchy. Microarray experiments will also be conducted to compare pathogen-induced and chemical elicitor-induced gene expression profiles. This will show if the elicitor and Peronospora recognition activate the same pathway. Also, if the transcriptome responds in the same manner it will provide information as to what hierarchical level the elicitor is acting on. Screens for mutants that are insensitive or hypersensitive to the respective chemical will be used to identify protein targets of promising compounds. Substances that effectively stimulate LURP expression may lead to the development of agrochemicals with the ability to utilize the gene-for-gene resistance program inherent to plants. These elicitors will be invaluable tools for the dissection of mechanisms controlling the plant defense transcriptome.

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CHRISTIANA MERRYWELL

Chemistry Graduate Program
Title of Project: Metabolic Analysis of Vacuolar Protein Sortin Inhibitors in Arabidopsis thaliana

Christiana Merrywell

Background:

I received my bachelors in Chemistry from the University of Southern California in the spring of 2004 and began my graduate studies at the University of California, Riverside in the fall of 2004. I have been carrying out my research under the guidance of Professor Cynthia K. Larive and recently began working in Natasha V. Raikhel's group. Prior to starting the ChemGen IGERT program, I used metabonomics to study oxidative stress in microdialysis samples taken from rats subjected to ischemia/reperfusion. Since starting the ChemGen IGERT program, I have redirected my attention to the plant Arabidopsis thaliana. I am currently studying the effects of two drugs, Sortin1 and Sortin2, in A. thaliana using 1H-NMR and LC-MS.

Christiana mentored an REU student named Thao Nguyen from Ohio Wesleyan University in the summer of 2006.  Her project title was METABONOMICS STUDIES OF ARABIDOPSIS THALIANA USING LC-MS AND LC-NMR. Photos and more information can be found at: http://research.chem.ucr.edu/groups/larive/reu/2006/participants_abstracts.html   or http://research.chem.ucr.edu/groups/larive/reu/2006/metabonomics.html

Introduction:

Chemical genomics is a systematic approach in biological research and drug discovery. Synthetic organic chemists create combinatorial libraries of compounds to be used in screening. My research uses the plant Arabidopsis thaliana as a model organism for screening compounds from combinatorial libraries. The complete genome of A. thaliana has been sequenced, making it an ideal organism for chemical genomics research. In a screen of 4,800 compounds from the ChemBridge DIVERSetE library performed by previous researchers, fourteen compounds caused excretion of carboxypeptidase Y, a protein normally confined to vacuoles, in yeast. These compounds were then analyzed for their effects on A. thaliana. The two compounds causing the most severe phenotypic effects in A. thaliana were selected for further analysis. The major effects are disruption of vacuole biogenesis and protein sorting as well as root growth inhibition. Characterization of the Sortins and their effects was performed by Lorena Norambuena and Glenn Hicks. These sorting inhibitors are now referred to as Sortin1 and Sortin2. This project uses metabonomics to investigate the metabolic profiles of both Sortin molecules as well as endogenous metabolites in A. thaliana in an attempt to identify the metabolic pathways targeted by Sortin1 and Sortin2. Metabonomics is the study of relative changes of endogenous small molecules in response to a chemical, environmental, or genetic stimulus. Metabonomic analysis of A. thaliana cell and plant extracts is carried out using proton nuclear magnetic resonance spectroscopy (H-NMR) and liquid chromatography coupled with mass spectrometry (LC-MS).

H-NMR and MS data for seedlings grown 7 days in the absence of Sortin1
Metabolic Analysis:

Extracts of seedlings grown in the presence of Sortin1 have been analyzed by 1H-NMR and LC-MS. These methods complement each other very well. While NMR is not as sensitive as MS, it is a good non-destructive method for the identification and quantification of small molecule metabolites. MS is a much more sensitive method, but is destructive to the sample and is mainly used for identification of larger molecular weight metabolites. To capitalize on the complementary nature of NMR and MS, sample extracts are first analyzed by NMR before being transferred to the autosampler for LC-MS analysis. Our preliminary NMR and MS data provide detailed information about the biochemistry of Arabidopsis. To date, many amino acids, sugars, hormones, and other metabolites have been identified. One metabolite of interest is glutamine, showing a dramatic increase in concentration when A. thaliana is grown in the presence of Sortin1. Further analysis is currently underway to characterize the action of Sortin1 and the metabolic pathways that it perturbs. It is known that the Sortins affect vacuolar biogenesis. For this reason, it may be of interest to investigate the effects of the Sortins on lipid biosynthesis. Because of the difficulty in getting both hydrophilic and hydrophobic metabolites into the same solution, extractions to separate lipids, waxes, and chlorophyll from more hydrophilic metabolites will be performed and NMR data acquired for both phases.

Waters QTOF Micromass Mass Spectrometer
Waters Acquity UPLC
 
Bruker Avance 600MHz Spectrometer
Agilent 1100 HPLC

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ChemGen IGERT Students: 2005-07
From left to right: Kayla Hamersky, Jolene Diedrich, Samer Elkashef, Charles Jang, Theresa Dinh, Colleen Knoth, Augusta Jamin, Christiana Merrywell, Eddie Cao

 

grp07
ChemGen IGERT Student Participants at Third Annual Retreat:
November 2-4, 2007

 

Joshua Tree

Students with Guest Speaker at Joshua Tree National Park: February 10, 2009 


L to R: Andrew Defries (IGERT student), Dr. Renier van der Hoorn (Guest Speaker, Max Planck Institution for Plant Breeding Research), Kayla Hamersky (IGERT student), and Shang Wu (IGERT student)

"We had an awesome time... Dr. van der Hoorn was very special and open with us about conducting science at the interface between two disciplines ... It was a bonding experience among each of us trainees and the speaker." --Kayla Hamersky (Student)


More Information

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E-mail: genomics@ucr.edu

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