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The Howard Hughes Undergraduate Research Scholars Program

2006-2007

Ms. Rebecca Boyle
Mr. Thomas Clarke
Mr. Bryan Harada
Ms. Ashley Koegel
Ms. Yara Mikhaeil
Ms. Alissa Minkovsky
Ms. Katherine Ng
Ms. Sacha Prashad
Mr. Kevin Roy
Mr. Grant Sanders
Mr. Aswin Sekar
Mr. Kevin Yackle


Ms. Rebecca Boyle
Mentor: Cue-Wei Xie
Title:
Opioid-Adrenergic Interactions in Sensory Neurons

Rebecca is a third year neuroscience major investigating the functional properties of G-Protein Coupled Receptor (GPCR) interactions in neurons. Under the mentorship of Dr. Cui Wei Xie, Rebecca examines the relationship between the μ-opioid and α 2a adrenergic receptors. Simultaneous activation of these two GPCRS has been shown to synergistically enhance the analgesic properties of opioid treatment. In heterologous cell systems it has been demonstrated that the μ -opioid and α 2a adrenergic receptors are capable of forming heterodimers. However, the mechanisms behind opioid analgesia, tolerance, and dependence have yet to be elucidated in vivo. Previous work in Dr. Xie’s lab has demonstrated that μ-opioid and α 2a adrenergic receptors can undergo cross-desensitization and co-internalization in mouse dorsal root ganglia (DRG) neurons. Using the whole-cell voltage clamp technique to measure Ca 2+ current inhibition upon μ-opioid or α 2a agonist activation, Rebecca is examining whether both receptors must be able participate in ligand binding in order for either homologous or cross desensitization to occur. The results from these recordings in DRG neurons will help elucidate the requirements for μ-opioid and α 2a interaction and whether or not this interaction occurs at the receptor level or intracellularly at a point downstream of ligand binding.

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Mr. Thomas Clarke
Mentor: Dr. Rachelle Crosbie
Title: The Role of Phosphorylation in the Binding of Gas11 to Microtubules

(Left to right: Dr. Janine Bekker, Tom Clarke, Dr. Rachelle Crosbie)

Tom Clarke, a fourth year majoring in Molecular, Cell, and Development Biology, is currently conducting research under the guidance of Dr. Rachelle Crosbie. His research focuses on the microtubule binding protein Gas11. Dr. Crosbie’s lab identified a microtubule association domain within Gas11 called the “Gas11 Microtubule Association Domain” (GMAD). Tom is working to delineate the key regulatory features within the GMAD region. His preliminary results demonstrate that the GMAD is phosphorylated and that mutagenesis of predicted phosphorylation sites within the GMAD reduces Gas11 binding to microtubules. Tom is currently working to characterize the binding properties of these phosphorylation mutants in vivo and in vitro. He then plans to test whether these mutants affect cell motility. Additionally, Gas11 functions in the context of a larger protein complex, called the “Dynein Regulatory Complex” (DRC). Dr. Crosbie’s lab has recently designed two methods to biochemically purify the DRC from mouse tissue. It is hypothesized that this complex contains kinases and phosphatases that modify Gas11. Tom proposes to isolate and characterize these Gas11-regulatory proteins. After graduation, Tom plans to continue his education in graduate school.

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Mr. Byran Harada
Mentor: Dr. James Bowie
Title: Structure and Function of the SAM Domain of Diacylglycerol Kinase δ

Bryan is a third-year biochemistry major, mathematics minor researching under the direction of Dr. James Bowie in the Department of Chemistry and Biochemistry. For his project, Bryan is studying the structure and function of diacylglycerol kinase δ (DGKδ). DGKδ is a member of a family of kinases which convert diacylglycerol to phosphatidic acid. Since both diacylglycerol and phosphatidic acid are important lipid second messengers, the DGK isozymes may play an important role in regulating the signaling pathways which involve these two molecules. DGKδ possesses a sterile alpha motif (SAM) domain, which is a conserved structural motif found in a variety of proteins. Most SAM domains mediate protein-protein interactions, and the SAM domain of DGKδ has been shown to mediate the homo-oligomerization of DGKδ. This oligomerization is thought to be involved in determining the intracellular localization of DGKδ. In order to study the role of this oligomerization, Bryan has developed a novel in vivo fusion-reporter screen to identify monomeric mutants of the SAM domain of DGKδ. Identification of these monomeric mutants will reveal which amino acid residues are critical for oligomer formation as well as provide mutant proteins for further biochemical, biophysical, and structural characterization. Ultimately, Bryan aims to map the oligomeric interface of the DGKδ and solve the structure of the SAM domain of DGKδ, so that he can use this structural information to study the function of DGKδ oligomerization in vivo. This information may give insight into the regulation of DGKδ’s activity and its role in intercellular signaling. After graduating from UCLA, Bryan plans to attend graduate school and pursue a Ph.D. in biochemistry or biophysics.

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Ms. Ashley Koegel
Mentor: Dr. Dennis Slamon, Dr. Juliana Oh
Title: Characterization of H37/RBM5 lung cancer tumor suppressor gene

(Ashley Koegel and Dr. Juliana Oh)

Ashley Koegel is a fourth year biochemistry major. She has been conducting research under the guidance of Drs. Dennis Slamon and Juliana Oh since the start of her second year at UCLA. She is currently working in the Slamon Lab in MRL on a project characterizing the H37/RBM5 tumor suppressor gene in lung and breast cancers. H37 is located at chromosome 3p21.3. Deletion at 3p21.3 is one of the most prevalent (occurs in >80% of lung cancers) and one of the earliest genetic alterations in lung cancer. Therefore uncovering the molecular pathways of this gene holds particular promise for the development of new cancer diagnostics and therapeutics. Compared to adjacent normal tissue, the H37 transcript and/or protein is underexpressed in ~75% of primary non-small cell lung carcinomas. In addition, when transfected into A549 lung cancer cells, H37 significantly inhibits cell growth both in vitro and in vivo. Using A549/H37 cells, Ashley has participated in research to show that the molecular mechanism of H37 tumor suppression involves both G1 cell cycle arrest and apoptosis.

Based on data from earlier yeast-two hybrid screenings, Ashley is currently investigating the H37-MTA1 (Metastasis Associated Protein 1) protein-protein interaction in vivo. MTA1 protein is an Estrogen Receptor (ER) co-regulator which binds to ER and is also known to be involved in the HER2 oncogene pathway in breast cancer. Given that H37 was initially discovered to be differentially expressed in HER2 overexpressing breast cancers, and that the 3p21.3 region is also frequently deleted early in breast cancer, the H37-MTA1 interaction may explain the potential role that H37 has in the HER-2/ Estrogen Receptor (ER) pathway in human breast and lung cancers. In addition, Ashley is using siRNA techniques to knock down the expression of H37 mRNA in immortalized breast and lung epithelial cells in order to investigate the role of H37 in preventing tumor initiation. This research could potentially lead to earlier detection of lung (and breast) cancer and the development of novel therapeutics. Upon graduation, Ashley plans to pursue a joint M.D./Ph.D program studying translational oncology research. Ashley would like to thank all of the members of the Slamon Lab for their support and invaluable guidance.

 

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Ms. Yara Mikhaeil
Mentor: Dr. V. Reggie Edgerton and Dr. Niranjala Tillakaratne
Title: Spinal AMPA receptors in spinally transected mice after step training .

(Yara Mikhaeil and Dr. Niranjala Tillakaratne)

Yara Mikhaeil is a third year Neuroscience major working with Drs. Reggie Edgerton and Niranjala Tillakaratne examining the biochemical pathways underlying spinal learning. Edgerton lab has previously demonstrated that even in the absence of supraspinal input, mice are able to learn and retain specific motor tasks. Yara is focusing on comparing and contrasting the biochemical pathways that have been established in the hippocampus, to those that may be occurring in the spinal cord. More specifically, Yara examines the changes of AMPA receptors in adult mice whose spinal cord had been completely transected at a mid-thoracic level and had learned to step through a robotic assisted step training regimen. AMPA receptors are fast-acting ionotropic glutamate receptors that consist of a combination of four subunits: GluR1, GluR2, GluR3 and GluR4, and are critically involved in excitatory transmission. GluR1 is the subunit of interest in most research projects due to the fact that it is phosphorylated in two specific sites: ser-831 by CaMKII and PKC and ser-845 by PKA. Yara performs immunohistochemistry on the spinal cord tissue to determine the levels of phosphorylated and non-phosphorylated AMPAR in the muscle specific motoneurons using specific antibodies to these receptors. These changes will reflect both the phosphorylation of pre-existing AMPA receptors as well as the insertion of silent synapses, which is the addition of new AMPA receptors. The findings of this project will provide more insight in the molecular pathways involved in spinal learning. For example, the phosphorylation of serine 845 will tell us that the following pathway is involved in spinal cord learning: Adenyl Cyclase ® cAMP ® Protien Kinase A ® serine 845. The project relates the behavioral learning of motor tasks by spinally transected mice to the learning that occurs in the brain, especially in the hippocampus. In addition, it compares the pathways involved in cumulating long term memories in both the brain and the spinal cord. Eventually, these findings will be beneficial in development of therapeutic treatments to improve stepping in spinal injured patients. In the future, Yara plans to pursue a joint MD/PhD degree and continue her research in the field of neuroscience.

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Ms. Alissa Minkovsky
Mentor: Dr. Christopher Denny
Title: Establishing stable expression of EWS/FLI1 in primary cell lines in the search to find a Ewing's Sarcoma cell of origin

(Left to right: Christopher Denny, Alissa Minkovsky, and Gary Potikyan)

Ewing’s sarcoma is a poorly lethal pediatric cancer characterized by a chromosomal translocation that results in the juxtaposition of the EWS gene on chromosome 22 with one of five different ETS family transcription factors of chromosome 11, the most common of which is the Fli1 gene. Alissa Minkovsky, a Junior Microbiology, Immunology, and Molecular Genetics Major, is studying this EWS/Fli1 chimeric gene in the lab of Dr. Christopher Denny her first year here at UCLA. She is working, with the guidance of Gary Potikyan, towards attaining stable expression of the EWS/Fli1 protein in murine embryonic stem cells by altering tumor suppression pathways in order to come closer to finding the cell of origin in EFTs. Stable expression of EWS/Fli1 has been achieved in NIH3T3 murine cell lines which already possess numerous mutations in tumor suppression pathway genes but expression has been toxic to most primary cell lines. Strong evidence exists that alteration of the INK4a/ARF network, is necessary for oncogenesis in Ewing’s. The knockdown of p16, a protein that is upstream in the p53 and RB tumor suppressor pathways, will hopefully make expression of EWS/Fli1 stable in mouse embryonic cells so that the cell types that do tolerate expression of EWS/Fli1 when the ES cells are differentiated in an tetracycline-inducible system can be identified. Alissa is studying towards a MD/PhD and a career in biomedical research.

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Ms. Katherine Ng
Mentor: Dr. Steven Smale
Title: Chromatin Remodeling During an Inflammatory Immune Response

Katharine Ng is a third year Microbiology, Immunology and Molecular Genetics major conducting research under the guidance of Dr. Stephen Smale. The Smale laboratory is interested in understanding the transcriptional regulation of pro-inflammatory genes.

Previous work done in the Smale lab has grouped bacterial lipopolysaccharide (LPS)-inducible genes into three major regulatory classes based on kinetics of induction, requirement for de novo protein synthesis and requirement for nucleosome remodeling complexes. Efforts to decipher the differential transcriptional regulation of these classes of pro-inflammatory genes have increasingly focused on the regulatory role of chromatin structure and nucleosome remodeling. Assaying for inducible DNase I hypersensitivity can provide information about changes in accessibility of local chromatin structure that take place upon stimulation, and DNAse I hypersensitivity has been found to be associated with regulatory regions of many genes. Katharine's project involves the utilization of DNase I hypersensitivity assays to monitor nucleosome remodeling at the promoters of LPS-inducible genes in each of the three classes and locate novel regulatory elements of these genes. It is hoped that understanding the pattern of DNase I hypersensitivity will aid in a more comprehensive understanding of the complex differential regulation of these genes.

 

 

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Ms. Sacha Prashad
Mentor: Dr. Hanna Mikkola
Title:

Sacha Prashad is a third year Molecular, Cell and Developmental Biology major conducting research under the mentorship of Dr. Hanna Mikkola. Dr. Mikkola has previously shown that in mice, the placenta is one of the first organs to harbor self-renewing, multipotential hematopoietic stem cells (HSCs). Sacha is investigating whether the human placenta possesses a similar ability to support HSC development, and possibly even generate HSCs de novo. Recent work in the Mikkola lab has shown that placental hematopoiesis in the human occurs as early as 4 weeks developmental age, as evidenced by the presence of multilineage progenitors/candidate HSCs in 4 week placental tissue explant cultures. Sacha’s project involves localizing these putative HSCs in their placental niche using candidate stem cell surface markers. In the future, these putative HSCs will be isolated, and their ability to engraft and function in vivo will be examined by using immundeficient animal models. The characterization of the placental niche of the HSC will illustrate how HSCs are generated and protected during fetal development, and ultimately allow for the recreation of this microenvironment ex vivo so that HSCs may be expanded from cord blood, or generated from embryonic stem cells for therapeutic purposes.

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Mr. Kevin Roy
Mentor: Professor Guillaume Chanfreau
Title: The role of double-stranded RNA endonucleases in eukaryotic gene regulation

Kevin, a fourth-year major in Biochemistry, performs research under the direction of Dr. Guillaume Chanfreau in the Department of Chemistry and Biochemistry. The Chanfreau Laboratory investigates the regulation of gene expression primarily at the post-transcriptional level, using the model eukaryote Saccharomyces cerevisiae, commonly known as baker’s yeast. In particular, the laboratory is interested in the regulatory role of class III riboendonucleases, which cleave double-stranded RNA (dsRNA), focusing on the yeast nuclear RNase III homolog Rnt1p. Rnt1p is responsible for the regulation of various mRNAs, some of which control the import of iron, as well as the processing of a number of non-coding RNAs, including precursors to ribosomal RNA and small nuclear and nucleolar RNAs. Kevin is investigating the structure of Rnt1p by mapping its independently folded domains through limited proteolysis. While the structure of the dsRNA binding domain of Rnt1p is well characterized, the role of other domains in assisting substrate recognition and catalyzing cleavage is unknown. Identifying these stable domains may facilitate further structural studies with the goal of understanding the mechanism of RNase III binding and cleavage. Kevin is also investigating the function of two nuclear-encoded proteins found to associate with the large subunit of the mitochondrial ribosome. These two proteins have been found by sequence homology to contain the class III riboendonuclease catalytic domain, but there is no known functional role for either protein. Kevin is analyzing the processing of mitochondrial RNA in yeast strains deficient in each of these proteins. Accumulation of unprocessed mitochondrial RNA may reveal substrates for these putative RNases III and identify a role for dsRNA processing in mitochondrial gene regulation. Greater understanding of the regulation of mitochondrial genes in yeast may elucidate common mechanisms for mitochondrial gene expression in higher eukaryotes. Furthermore, a comparison of the mode of regulation in mitochondria with that in the prokaryotes may reveal evidence for the endosymbiosis theory of the origin of mitochondria. More complete characterization of all yeast RNase III activity will bring insight into the mechanisms by which eukaryotic RNases III recognize their dsRNA substrates and catalyze cleavage while preventing cleavage of non-substrate dsRNA in vivo. Kevin will continue his research while pursuing his Masters degree as a Departmental Scholar from Fall 2007 to Spring 2008. Afterwards, he intends to pursue an M.D./Ph.D. degree to combine his research and clinical aspirations.

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Mr. Grant Sanders
Mentor: Dr. Michael Teitell
Title: Bacterial Production of GST-labeled Polynucleotide Phosphorylase (PNPase) for Use in Identifying Novel Interacting Proteins of PNPase by Far Western Analysis

Grant Sanders is a third year student majoring in Microbiology, Immunology, and Molecular Genetics who is conducting research under the guidance of Dr. Michael Teitell in the department of Pathology and Laboratory Medicine. His research focuses on the protein, Polynucleotide Phosphorylase (PNPase), which is a 3’-5’ exoribonuclease localized to the inner mitochondrial membrane. Immunoprecipitation experiments have shown a tight binding between PNPase and the oncoprotein, T Cell Leukemia-1 (TCL1). TCL1 is a 14-kDa protein that has been studied extensively by the Teitell Lab and its overexpression in TCL1 transgenic mice has been shown to result in B cell lymphomas of the germinal center. Mammalian PNPase, however, has only been recently identified and both its cellular function and the role it plays in conjunction with TCL1 remain unclear. Grant’s project will include elucidating the role PNPase plays is cancer formation by identifying any additional binding partners in the cell, something that has been troublesome in the past due to artifacts produced with immunoprecipitation experiments. Grant will perform a Far Western Analysis to identify any binding partners of PNPase in both the mitochondria and cytosol. He is currently optimizing a protocol to purify large quantities of Glutathione S-Transferase (GST) tagged PNPase to be used as a probe in his experiment. Being extremely insoluble, the GST-PNPase probe is difficult to purify yet Grant has successfully altered the protocol to efficiently create substantial amounts of the intact protein. The Far Western Analysis will hopefully shed light onto PNPase’s function in the cell and will help Grant further characterize the protein’s role in B cell lymphoma formation. After graduating from UCLA, Grant plans on attending medical school and continuing biomedical research in an academic setting. Grant would like to sincerely thank Dr. Michael Teitell and Cynthia Balatoni, as well as all of the other members of the Teitell Lab, for their guidance and unwavering support. He would also like to thank the Howard Hughes Undergraduate Research Program for the opportunity they have provided him.

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Mr. Aswin Sekar
Mentor: Dr. Desmond Smith
Title: Identifying genetic circuits in cells

Aswin Sekar is a third year Molecular, Cell and Developmental Biology student conducting research in Dr. Desmond Smith's laboratory in the Molecular and Medical Pharmacology Department. Aswin is working on a project that is part of a recent trend in systems biology that involves the investigation of a methodical perturbation of biological systems. His project seeks to identify and map regulatory gene circuits in a mammalian system using microarrays. Identifying regulatory genes has vast implications because it allows for the detection of putative control genes in humans by homology. After completion of his bachelor's degree, Aswin plans to attend medical school and become a physician-scientist. He would like to thank Dr. Smith and Chris Park for their mentorship, as well as the Howard Hughes Undergraduate Research Program for fostering his research efforts.

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Mr. Kevin Yackle
Mentor: Dr. Utpal Banerjee and Dr. Gerald Call
Title: Characterization of Two Nuclear-Encoded Genes Putatively Involved in Mitochondrial Protein Translation

(Dr. Gerald Call and Kevin Yackle)

Currently Kevin is conducting research in genetics to study developmental biology in Drosophila melanogaster under the mentorship of Dr. Utpal Banerjee and Dr. Gerald Call. He is working to characterize two nuclear-encoded genes that are putatively involved in mitochondrial protein translation. These genes were identified through a genetic screen on the 3R chromosome which utilized mitotic recombination technology in order to reveal interesting cell cycle phenotypes caused by lethal mutations. These mutations were mapped and homology searches classified as nuclear encoded genes whose proteins function within the mitochondrial protein synthesis. This mechanism of function within the mitochondria of higher organisms like Drosophila has not been shown thus far. Experiments will be conducted in an in vivo (adult fly) and in vitro system (cell culture). The in vivo research will involve the re-introduction of wild type genes in order to rescue the mutant phenotypes of mitochondrial staining which show respiration and protein phenotypic effects in mutant organisms. The in vitro experiments will use dsRNA to knockout these genes in cell culture. To study mitochondrial function ATP levels will be measured to show respiration and Western analysis will reveal effects on protein translation. Lastly, fluorescent fusion proteins of these genes show localization patterns within the cell. Through these assays these nuclear genes can be characterized as functioning within the mitochondria where they play important roles in protein translation. After graduation Kevin plans on joining an MD/Ph.D program with the goal of becoming a medical scientist. Kevin would like to thank and acknowledge Dr. Utpal Banerjee, Dr. Gerald Call, everyone in his lab, and the Howard Hughes Undergraduate Research Program.

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Profiles of Students