Many students in the Molecular Biology Department undertake focused research during the summer through Pomona's Summer Undergraduate Research Program. Below are recent completed projects by our students.


Computational Analysis of the Taxonomic Content of Bacterial Communities with Metagenomics Software

Liana Piedra (2015); Mentor(s): Andre Cavalcanti

Abstract: A significant amount of microbes have little to no information about them due to the inability to culture them within a controlled environment. Often, this includes microbes that live in places like the deep ocean, the human gut, and hot springs, where conditions are extreme and cannot be replicated in the lab. Metagenomics is the study of these microbes, where the extracted genomic DNA replaces the need to grow cultures. Due to advances in next generation sequencing (NGS), whole genomes can be sequenced faster and cheaper than ever before, meaning that metagenomic studies become viable, but also create increasingly large amounts of data. Genomes from a myriad of species within a sample must be assembled without confusing them with each other, and since these microbes often don’t have genomes already available in databases, they must be built de novo. After the difficult and not yet perfected step of assembly, the genomes eventually have to be annotated for gene calling, putative protein function, and taxonomic organization. Because the field is relatively new, there is no single standardized pipeline to run data from raw sequence to meaningful biological information in just one click. Much of this research has focused on finding the strengths and weaknesses of various open source software, coping with the sheer amount of computational power necessary to run them, and what kind of information can be gleaned by only using the sequences from a heterogeneous microbe sample.
Funding Provided by: Faucett Catalyst Fund

The role of GDI mutations in Drosophila melanogaster vesicle trafficking phenotypes

Lila Hawkinson (2015); Mentor(s): Clarissa Cheney

Abstract: GDP dissociation inhibitor (GDI) is essential to proper vesicle trafficking in its role as a recycler of Rab proteins. Rab proteins direct vesicles to the correct target membrane, after which GDI binds the inactive form, GDP-bound form of the Rab and returns it to its home membrane. Due to the key nature of GDI in cellular functions, individuals with low levels of GDI or non-functional GDI often display severe phenotypes. Better understanding how mutations in GDI can affect vesicle transport will allow further insights into the causes behind diseases tied to GDI. Vesicles in Drosophila melanogaster single-point missense GDI mutants were visualized in hemocytes by using pHrodo Red dextran, a compound that increases in fluorescence with decreasing pH. The number and size of the vesicles in each hemocyte were recorded, and a final statistical analysis showed increased proliferation of vesicles and increased vesicle size in GDI mutant larvae when compared to the wild-type. These results may indicate that the increased vesicle count seen in GDI mutants contributes to homotypic fusion between endocytotic vesicles, and thus leads to increased vesicle size. Future assays will seek to determine the Rabs associated with the enlarged vesicles. Proposed methods for these assays include the use of α-Rab antibodies and imaging of fly stocks with fluorescence-tagged fusion-proteins.
Funding Provided by: Jack Steller Summer Curriculum Enhancement for Molecular Biology Fellowship

Identification of Sulfur Metabolism Products in Microbes using Cyclic Voltammetry

Jerry Lee (2016); Mentor(s): EJ Crane

Abstract: Certain microbes use sulfur compounds as their final electron acceptors in anaerobic metabolism. However, given sulfur’s extreme plasticity (its oxidation states span from +6 to -2, and elemental sulfur can exist in at least 180 different allotropes and polymorphs), the forms of sulfur actually utilized and produced by these microbes remain unclear. We use anodic stripping cyclic voltammetry with micro-solid state electrodes to identify products of microbial and enzymatic sulfur reduction. By comparing voltammograms observed during these experiments to voltammograms of individual sulfur compounds such as sulfide, polysulfide and different polymorphs of sulfur, we can thus identify the sulfur forms that predominate during microbial growth and during enzymatic reduction of sulfur. It was previously assumed that the sole product of enzymatic reduction of sulfur was sulfide. In the studies described here we have shown that the products of the enzymatic reduction of hydrophilic sulfur sols by the NADH and coenzyme A-dependent sulfur/CoA disulfide reductase from Pyrococcus horikoshii (a sulfur-reducing hyperthermophile) are actually a complex mixture of sulfide, polysulfides and nanoparticulate sulfur.
Funding Provided by: Howard Hughes Medical Institute

Preclinical Diagnosis of Parkinson's Disease: α- Synuclein in Saliva Exosomes

Nancy Zhu (2016); Additional Collaborator(s): Jing Zhang (University of Washington), Ane Korff (University of Washington); Mentor(s): Frederick Grieman

Abstract: Parkinson’s Disease (PD) is currently diagnosed based on the presence of clinical motor symptoms that appear when a substantial number of neurons in certain parts of the brain are already lost. Neuropathologically, PD is characterized by aggregates of proteins within neurons called Lewy bodies. Alpha-synuclein (α-syn) is a major component of Lewy bodies and mutations and variations in the α-syn gene (SNCA) have been linked to familial PD and increased risk of sporadic PD, respectively. α-Syn is secreted in the extracellular space and has been studied extensively as a potential biomarker for PD with varying success. CSF α-syn levels show promise in this regard; CSF collection, however, is an invasive procedure and therefore not ideally suited for routine screening. α-Syn has also been identified in the submandibular gland as well as in blood and saliva. However, its biomarker performance in these fluids have been less promising, possibly due to the influence of peripheral α-syn. It has been shown that a fraction of extracellularly secreted α-syn is contained in exosomes and we have developed an immunoprecipitation technique to isolate exosomes in plasma that are likely CNS derived. In the current study, we adapt this technique to isolate exosomes from saliva, followed by measurement of exosomal saliva α-syn via xMAP Luminex assay.
Funding Provided by: Evelyn B. Craddock-McVicar Memorial Fund

Requirements for Maximal Knockdown of Mannitol Transporter

Naomi Vather (2015); Student Collaborator(s): Maya Tsao-Wu (2017); Mentor(s): Jane Liu

Abstract: Vibrio cholerae is the causative agent of cholera, which predominantly affects the developing world, killing approximately 3-5 million people worldwide each year. In its life cycle, it inhabits aquatic environments and later, upon ingestion of contaminated food or water, it can inhabit the human small intestines. How V. cholerae is able to transition and persist in these aquatic environments before infecting a host is largely unknown. It has been posited that small non- coding regulatory RNAs (sRNAs) may contribute to its adaptability. In particular, the MtlS sRNA regulates mtlA expression, a gene that encodes for the EII-mannitol specific phosphotransferase system that transports mannitol into the cell. We previously observed that MtlS is expressed only in the absence of mannitol. The sRNA is transcribed antisense to the 5’ untranslated region (UTR) of mtlA and consists of 70 nucleotides of complementarity that can bind to the 5’UTR of mtlA mRNA and occlude its ribosome binding site. Consequently, translation of mtlA mRNA is inhibited. Here, we questioned whether the binding of MtlS to the 5’UTR of mtlA mRNA is sufficient to repress MtlA synthesis in the absence of mannitol. By conducting fluorescence assays, using a gfp reporter, we investigated the requirements for knockdown of mtlA expression by MtlS. Our results suggest that maximal knockdown of MtlA protein occurs when more of the coding region of mtlA is present and when MtlS is overexpressed.
Funding Provided by: Rose Hills Foundation (NV); National Science Foundation #CBET-1258307: PI J. Liu (MTW)

Impact of the Antimicrobial Peptide Temporin-SHf on Membrane Structure and Integrity: A QCM-D Study

Andrea Diaz (2015); Mentor(s): Malkiat Johal, Daniel O'Leary

Abstract: The need for alternatives to traditional antibiotics has been highlighted in recent years by an increase in multidrug resistant bacterial strains. Antimicrobial peptides (AMPs) are promising candidates for the next generation of antibiotic therapeutic agents. AMPs constitute a part of the innate immune system in mammals and plants. They fight against a broad range of bacteria, yeast, fungi, and protozoa mainly through the disruption of pathogen cell membrane integrity. Temporin-SHf (FFFLSRIFa) is the shortest (8-residues) naturally occurring antimicrobial peptide, isolated from the amphibian Pelophylax saharicus. It has broad-range activity against gram-positive and gram-negative bacteria, with no hemolytic activity. Using the quartz-crystal microbalance, this study monitored the interaction of Temporin-SHf with model lipid bilayers composed of 1, 2- dioleoylphosphatidylcholine (POPC) for eukaryotic systems, and a mixture of POPC and 1, 2- dioleoylphosphatidylglycerol (POPG) for bacterial systems. Other stereoisomers of Temportin-SHf, for example the D-form of Temporin-SHf, were also used in order to investigate the potential influence of stereochemical effects on membrane disintegration. The next stage of this project will involve monitoring the system using the dual polarization interferometer and modeling the interaction using molecular dynamics simulations.
Funding Provided by: Linares Family SURP for Chemistry

Lipid droplets may play a role in the formation of the C. elegant eggshell permeability barrier

Min Joo Kim (2016); Mentor(s): Sara Olson

Funding Provided by: Sherman Fairchild Foundation

Expression of a Putative Ascarylose Biosynthetic Enzyme from C. elegans

Sabari Kumar (2017); Mentor(s): Sara Olson, Matthew Sazinsky

Funding Provided by: Howard Hughes Medical Institute

Characterization of Vitelline Layer Proteins and Endocytic Machinery in the Nematode Eggshell

Zachary Wilson (2016); Mentor(s): Sara Olson

Funding Provided by: Jack Steller Summer Curriculum Enhancement for Molecular Biology Fellowship

Quantifying differential gene expression in the MAIDS model

Donghyung Lee (2015); Student Collaborator(s): Samuel Du (2016), Graham Barlow (2016); Mentor(s): Sharon Stranford

Abstract: The murine leukemia virus (MuLV) induces a disease in some mouse strains that mimics human acquired immunodeficiency syndrome (AIDS) called murine AIDS (MAIDS). A specific isolate of MuLV known as LP-BM5 is comprised of three components: a mink cell focus-forming (MCF) virus and an ecotropic virus that are replication-competent, and a replication- incompetent defective virus that, while being the etiologic agent, requires the two helper viruses to infect cells. The C57BL/6 mouse strain is highly susceptible to MAIDS while the BALB/c strain is resistant to the disease following MuLV exposure. Our lab uses quantitative real-time PCR (qPCR) to quantify differential gene expression observed in DNA microarray experiments (eg., carboxypeptidase B1), and to measure individual LP-BM5 components (Defective, Ecotropic, MCF) in infected tissue.
Funding Provided by: Sherman Fairchild Foundation (DL); Howard Hughes Medical Institute (GB); Pomona College SURP (SD)

Chromosomal Translocations due to Short- Sequence Recombination in Saccharomyces cerevisiae RAD3 and SSL1 Mutants

Maria Arciniega (2016); Additional Collaborator(s): Johan Martinez; Mentor(s): Tina Negritto

Funding Provided by: Corwin Hansch and Bruce Telzer Fund

Ssl1 Recruitment to a Double-stranded Break suggests TFIIH Involvement in Double-stranded DNA Break Repair

Owen Chapman (2017); Student Collaborator(s): Jorge Mejia (2015); Mentor(s): Tina Negritto

Funding Provided by: Pomona Alumni SURP Fund (OC)

A novel approach to study the recruitment of TFIIH to DSBs in S. cerevisiae

Luis Díaz-Ruiz (2015); Mentor(s): Tina Negritto

Funding Provided by: Rose Hills Foundation

Effect of the Phenols BHA and BHT on DNA Breakage and Apoptosis in Saccharomyces cerevisiae

Bradley King (2017); Mentor(s): Tina Negritto

Funding Provided by: Howard Hughes Medical Institute

TFIIH recruitment to double-stranded breaks in noncoding DNA in S. cerevisiae

Jorge Mejia V. (2015); Mentor(s): Tina Negritto

Funding Provided by: Jack Steller Summer Curriculum Enhancement Fellowship

The genetic control of large-scale CAG repeat expansions

Tanner Byer (2017); Additional Collaborator(s): Jane Kim (Tufts University); Mentor(s): Sergei Mirkin (Tufts University)

Abstract: Peppered throughout the human genome are areas of repetitive DNA called microsatellites. Structurally, microsatellites are tandem reiterations of a simple sequence of DNA, between 2-5 base pairs long. These DNA repeats are dynamic genomic features with the capacity to expand to longer repeat lengths, which can alter gene expression. Consequently, the expansion of microsatellite DNA has already been implicated in the development of 30 different hereditary, degenerative diseases. Across the globe, in an array of different experimental systems, is directly aimed at providing a mechanism by which to explain microsatellite repeat expansion. To facilitate this process, I worked closely with large-scale manifestations of the trinucleotide repeat CAG in order to better characterize this specific type of microsatellite expansion. Through a candidate gene assay and an unbiased genetic screen, I looked to investigate and uncover genes involved in these large-scale CAG repeat expansions. The work done this summer strongly suggests that the Srs2 gene, a gene previously implicated in large-scale GAA repeat expansions, has not affect on the CAG type. Furthermore, a series of eight novel genes were found with the potential to affect these large-scale CAG expansions, some coding for ubiquitination components and other involved in the phosphorylation critical to the cellular signaling pathways.
Funding Provided by: National Science Foundation (Tufts University)

Hydrogen Production by a Bioassisted Nanophotocatalyst

Aleksandra Karapetrova (2015); Mentor(s): Elena Rozhkova (Argonne National Laboratory), Matthew Sazinsky

Abstract: Nanophotocatalysis is a potentially efficient way of capturing and storing solar energy in the form hydrogen. Semiconductor photocatalysts such as TiO2 can generate hydrogen via water splitting under ultraviolet light irradiation. There is great interest in extending the visible light reactivity of the TiO2 photocatalyst with synthetic or natural dyes, or light-harvesting proteins. These systems that employ dyes or biomolecules often have limited stability but the light harvesting proton pump bacteriorhodopsin (bR) has shown more robustness. In a nanoscale system consisting of bR assembled onto Pt/TiO2 photocatalysts in the presence of methanol, a significant amount of visible-light generated hydrogen was detected. In order to obtain bR for this energy system it was harvested from the archaea Halobacterium salinarum. This project involved the culturing, atomic force microscopy (afm), and assemblage of H. salinarum membrane/protein complexes onto the surface of TiO2 nanoparticle arrays. This research which was done at Argonne National Laboratory began with large scale culturing of H. salinarum. Imaging of the halophiles, that tend to be approximately between one and two microns, was successful only with AFM and important for further hypothesis about variations in the nanobiophotocatalyst system. In addition, the assembly of H. salinarum membrane with TiO2/Pt nanoparticles was attempted and subsequent hydrogen production measured with gas chromatography.
Funding Provided by: Department of Energy

TMEM16A Promoter Methylation and Protein Expression in HPV Unrelated Squamous Cell Carcinoma of the Head and Neck

Alicia Mizes (2016); Student Collaborator(s): Ronak Dixit (University of Pittsburgh Medical Center); Additional Collaborator(s): Uma Duvvuri (University of Pittsburgh); Mentor(s): Scott Kulich (University of Pittsburgh), Karl Johnson

Abstract: Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide. Evidence shows that the overexpression of TMEM16A, a transmembrane calcium dependent chloride channel, in SCCHN is associated with decreased patient survival. We hypothesized that the CpG hypomethylation of the TMEM16A promoter may be a novel mechanism through which TMEM16A expression is increased in human papillomavirus unrelated (HPV negative) SCCHN. Data mining of SCCHN cases in the cancer genome atlas (TCGA) database were performed with a focus on TMEM16A mRNA expression, TMEM16A promoter methylation, and HPV status. Immunohistochemistry (IHC) for p16, a surrogate marker for HPV in SCCHN, and DOG1, a polyclonal antibody for TMEM16A, were performed on oropharyngeal SCCHN resection specimens using the Benchmark Ultra. Methylation levels were quantified on SCCHN resection specimens and cell lines by bisulfite conversion of purified DNA, followed by real-time quantitative methylation- specific PCR (RT qMSP) analysis of the TMEM16A promoter region and beta actin gene (ACTB) for normalization. TCGA data mining revealed a relationship between HPV negativity, higher TMEM16A mRNA levels, and decreased promoter methylation. Preliminary comparison of DOG1 IHC staining quantification and RT qMSP does not demonstrate a clear-cut association between TMEM16A protein expression and promoter methylation in SCCHN resection specimens. Further analysis according to site of resection and TMEM16A gene copy number from FISH studies has begun to offer more insight.
Funding Provided by: University of Pittsburgh Medical Center


Ciliate Dynamics: Selection and Senescence

David Morgens (2014); Mentor(s): Andre Cavalcanti

Abstract: Ciliates are single cellular eukaryotes whose unusual life cycle and nuclear dimorphism can give rise to novel dynamics on both an evolutionary and population level. We use simulations and modeling to address the effects on chromosomal imbalances as well as population genetics. This gives us insight into a variety of processes such as protein evolution, effective population size, senescence, regulation, and evolutionary patterns.
Funding Provided by: Howard Hughes Medical Institute

Studies on Vesicle Transport in Drosophila

Alan Chen (2014); Student Collaborator(s): Karen Leung (2014 CMC); Michelle Ozaki (2016 SCR); Additional Collaborator(s): Zhaohua Tang (W.M. Keck Science Center of the Claremont Colleges); Ruye Wang (HMC); Mentor(s): Clarissa Cheney

Abstract withheld upon request.
Funding Provided by: Howard Hughes Medical Institute

Studies on Vesicle Transport in Drosophila

Mary (Molly) Horgan (2014); Mentor(s): Clarissa Cheney

Abstract withheld upon request.
Funding Provided by: Pomona College SURP

Studies on Vesicle Transport in Drosophila

Howard Lee (2014); Mentor(s): Clarissa Cheney

Abstract withheld upon request.
Funding Provided by: Corwin Hansch and Bruce Telzer Fund

An Epigenetic Analysis of Apis mellifera

Jingyuan Liao (2015); Student Collaborator(s): Tobin Ivy (2013 HMC); Sherry Zhang (2015 HMC); Mentor(s): Robert Drewell

Abstract: Apis mellifera, the European honey bee, has a caste system consisting of one fertile female queen, fertile males (drones) and infertile female (workers). Differentiation of the three is suspected to be through epigenetic modification of DNA methylation performed by the queen when she lays eggs. Previous work has shown differential DNA methylation between the egg and sperm of A.mellifera. Further DNA methylation analysis of A.mellifera genomic DNA is performed in this project to identify the differential methylated genes (DMGs) involved in worker sterility. However, future work is needed to ensure the proper amplification of our targeted DMGs.
Funding Provided by: Howard Hughes Medical Institute

The Role of PDGF Signaling in Epicardial Cell Activation/Migration and Blood Vessel Formation in Regenerating Zebrafish Hearts after Cryoinjury

Luis Francisco Díaz (2015); Additional Collaborator(s): Jieun Kim (Saban Research Institute, Children’s Hospital Los Angeles); Gayatri Nair (Saban Research Institute, Children’s Hospital Los Angeles); Mentor(s): Ellen Ching-Ling; M. Cristina Negritto

Abstract: Approximately 600,000 people die of heart disease in the United States alone every year according to the Centers for Disease Control and Prevention (CDC). Myocardial infarction (MI) in humans causes severe heart damage leading to scarring of the heart, which leads to impaired cardiac function and eventual heart failure. Unlike mammals, zebrafish have to ability to regenerate their hearts after severe injury, however, the mechanism for this is not well understood. During zebrafish heart regeneration epicardial cells proliferate in a platelet-derived growth factor (PDGF) manner and can undergo an epithelial-mesenchymal transition (EMT) contributing to the formation of blood vessels. In this study we investigated the role PDGF signaling plays in epicardial migration/activation and blood vessel formation during zebrafish heart regeneration after cryoinjury. We used the cryoinjury model to injure the zebrafish hearts (n=19) as doing so causes their hearts to scar making it more relevant to the manner in which mammalian hearts respond to MI. In our study, we blocked PDGF signaling using the platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor V (0.25µM). To monitor epicardial migration/activation in regenerating zebrafish hearts we used transgenic fish expressing fluorescent reporters under the control of two epicardial markers, tcf21 and wt1. To monitor blood vessel formation we used the endothelial cell marker fli1a. We used confocal microscopy to observe blood vessel formation and epicardial migration/activation using these transgenic fish.
Funding Provided by: Office of Minority Health Research Coordination (OMHRC) in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH)

Studying the role of the mtlA 5’-UTR in the regulation of the mannitol transporter by the small RNA MtlS in Vibrio cholera

Howard Chang (2014); Mentor(s): Jane Liu

Abstract: Small, non-coding RNAs (sRNAs) may contribute to the ability of V. cholerae, the causative agent of cholera, to adapt quickly to different environments. The MtlS sRNA, expressed in non-mannitol carbon sources, is hypothesized to repress translation of the mannitol transporter MtlA by binding to the 5’-untranslated region (UTR) of the mtlA mRNA. We investigated whether the mtlA 5’-UTR is sufficient for knockdown of MtlA expression by MtlS in Escherichia coli and V. cholerae, or if the full-length mtlA sequence is required. The following plasmids were used: pXGmtlA5’UTR-gfp (reporter, with a fusion between the mtlA 5’-UTR and gfp), pXGmtlAfull-gfp (reporter, with a fusion between the entire mtlA gene and gfp), pMtlS (allows for inducible expression of MtlS), and pVector (control). We observed by fluorescence assays that E. coli cells with pXGmtlA5’UTR-gfp and pMtlS had a ≥3-fold reduction in fluorescence compared to the control. The same experiment in V. cholerae resulted in a modest reduction in fluorescence. When only the reporter plasmid (pXGmtlAfull-gfp) was used, V. cholerae cells in glucose had reduced fluorescence compared to cells grown in mannitol; prior research showed no reduction in fluorescence under the same conditions using cells with pXGmtlA5’UTR-gfp. Our results suggest that the mtlA 5’-UTR is sufficient for repression of MtlA synthesis in V. cholerae when MtlS is expressed in cis to the mtlA 5’-UTR or when MtlS is over-expressed in trans to the mtlA 5’-UTR.
Funding Provided by: Rose Hills Foundation

Post-translational Regulation of the Mannitol Transporter by the small RNA MtlS

Maxwell Coyle (2014); Additional Collaborator(s): Daniel O'Leary; Sonja Hess (Caltech); Mentor(s): Jane Liu

Abstract: Vibrio cholerae regulates the influx of mannitol through the small RNA MtlS – if mannitol is removed from the Vibrio environment, MtlS is transcribed and decreased concentrations of the mannitol-specific transporter MtlA are observed. The same reduction in MtlA levels is seen in V. cholerae grown in mannitol media where MtlS is overexpressed. Since MtlA is a stable protein (half-life > 100 minutes), I hypothesized that reduction of MtlA levels proceeds through active proteolysis of MtlA involving increased translation of a protease or adaptor protein and directed by the expression of MtlS. To test this hypothesis, I treated V. cholerae cells grown in mannitol with chloramphenicol (Cm), a translational inhibitor, and subsequently induced MtlS transcription. In many trials, Cm-treatment rescued degradation of MtlA, although the induced transcription of MtlS in Cm¬treated samples was impaired compared to control samples. These results suggest that Cm-treatment paired with induced transcription of MtlS is an ineffective way to study the MtlA/MtlS system. I now plan to use mass spectrometry-based quantitative proteomics to identify changes in protein expression following MtlS induction. I have demonstrated that “heavy” Lys and Arg can be incorporated into V. cholerae proteins, indicating that I will be able to use stable incorporation of labeled amino acids in cell culture (SILAC) to study changes to the V. cholerae proteome in relation to changing levels of MtlS.
Funding Provided by: Dale N. Robertson Fund

MtlS, an sRNA in Vibrio cholerae, Regulates Translation of mtlA in vitro

John Replogle (2014); Mentor(s): Jane Liu

Abstract: As the causative agent of cholera, Vibrio cholerae moves between human small intestines and aquatic environments throughout its life cycle. In order to thrive, this bacterium must use a variety of carbon sources for energy. We previously showed that a small RNA (sRNA) expressed by V. cholerae, MtlS, affects the pathogen’s gene expression and metabolism. MtlA is the mannitol transporter protein necessary for V. cholerae to metabolize mannitol and is produced exclusively in the presence of mannitol. On the other hand, MtlS sRNA is only produced in the absence of mannitol and appears to post-transcriptionally downregulate expression of mtlA. We hypothesized that since it is complementary to the 5’ untranslated region (UTR) of mtlA mRNA, MtlS may affect expression of MtlA by forming an mtlA-MtlS complex that blocks translation of this mRNA. We used in vitro methods in order to examine the role MtlS plays in mtlA regulation. Using a gel shift assay, we showed that the sRNA is able to bind specifically to the mtlA mRNA. Next, we used an in vitro translation assay with varying amounts of MtlS to demonstrate that MtlA production decreases with increasing levels of MtlS. Thus, these results are consistent with our hypothesis that MtlS is sufficient to block ribosomes from translating mtlA mRNA. These findings will allow us to further understand how mannitol metabolism is regulated in V. cholerae and how these bacteria quickly adapt to new environments.
Funding Provided by: Sherman Fairchild Foundation

Determining the Role of Tfb4 in DSB Repair via TAP-tag Immunopreciptation in Yeast

Colin Belanger (2014); Mentor(s): M. Cristina Negritto

Abstract: The repair of DNA damage is an extraordinarily complex process vital to overall cell function and survival, actively studied in the model organism Saccharomyces cerevisiae. One of the primary mediators suggested for this process include the TFIIH complex, a previously characterized transcription factor that may act through its Rad3 subunit to promote DNA end degradation at double-strand breaks. To further determine the extent of TFIIH involvement at DNA breaks, three subunits of the larger complex (Tfb4, SSL1, and Rad3) were labeled with the previously-characterized TAP tag and assayed for recruitment to a known DSB using a combination of both chromatin immunoprecipitation and quantitative real-time PCR. Preliminary results indicate a significant fold-increase in recruitment of the Tfb4::TAP subunit to a known DSB relative to intact control regions of DNA, as expected. Reproducing this trend for TAP-tagged SSL1 and Rad3 subunits will further confirm the notion that TFIIH is recruited to and actively involved with the DSB repair in vivo.
Funding Provided by: Pomona College Department of Molecular Biology; Jack Steller Curricular Development Fund

Toxicity Analysis of Novel Fluorescent Nucleic Acid Probe DANPY-1 and Traditional Intercalator Dyes

Emma Carroll (2014); Mentor(s): M. Cristina Negritto; Lewis Johnson

Abstract: Novel fluorescent nucleic acid probe DANPY-1 has a high affinity for nucleic acids, is soluble in biologically relevant solvents, and exhibits superior photostability. The DEL assay is a well-established and robust technique for assessing intrachromosomal recombination in S. cerevisiae caused by DNA damage in the presence of potential mutagens. Here, we use the DEL assay to assess mutagenicity of DANPY-1 and comparable nucleic acid probes ethidium bromide, SYBR® Green I, and SYBR® Safe and an additional control dye, DAST. Due to its relatively small size, DANPY-1 was hypothesized to exhibit lower mutagenicity and toxicity than traditional intercalator dyes. We find in our preliminary results that rates of intrachromosomal recombination caused by DNA damage in the presence of DANPY-1 are reduced when compared to recombination rates of classic DNA intercalators ethidium bromide and SYBR® Green, indicating that DANPY-1 may be a potentially less toxic alternative to traditional fluorescent nucleic acid probes. Furthermore, SYBR® Green I, marketed as a desirable alternative to ethidium bromide, exhibits the highest rates of intrachromosomal recombination and DNA damage in the study. These results highlight the need for additional toxicity testing of DANPY-1 and traditional fluorescent probes using assays for different types of DNA damage, such as frameshifts and gross chromosomal rearrangement, and reveal the potential to use QSAR techniques based on dye structure/toxicity relationships to design less toxic fluorescent nucleic acid probes.
Funding Provided by: Howard Hughes Medical Institute

Recruitment of TFIIH to DSBs in Saccharomyces cerevisiae by Rad4

Tyler Oe (2014); Mentor(s): M. Cristina Negritto

Abstract: Our lab focuses on TFIIH and its recruitment to double strand breaks (DSB) in Saccharomyces cerevisiae. We have shown that a subunit of TFIIH is recruited to DSBs and propose that the complex in its entirety is recruited as well. We also propose that TFIIH is responsible for the resection of DNA ends at DSB sites. In this study, we focus on what may be recruiting TFIIH to the DSB itself. RAD4 is known to recruit TFIIH during NER and we propose that it is also associated with TFIIH’s recruitment at DSB sites. We analyzed the recruitment of TFIIH by means of ChIP and qPCR in a rad4 null mutant. If RAD4 is imperative for TFIIH recruitment, we expect to see decreased TFIIH recruitment to a DSB in the null mutant compared to the wildtype.
Funding Provided by: Sherman Fairchild Foundation; Pomona College Department of Biology

Patched family member PTR-2 implicates cortical granules in formation of the C. elegans eggshell permeability barrier

Alexander Bell (2014); Mentor(s): Sara Olson

Abstract withheld upon request
Funding Provided by: Jack Steller Summer Curriculum Enhancement Fellowship

Investigating the Role of Sugar Modification Enzymes in Formation of the C. elegans Eggshell Permeability Barrier

Karen Hou (2014); Mentor(s): Sara Olson

Abstract withheld upon request
Funding Provided by: Kenneth T. and Eileen L. Norris Foundation

Recreating the Corneal Extracellular Matrix: The Effects of Material and Alignment on Corneal Fibroblasts

Molly Kupfer (2014); Student Collaborator(s): Jennifer Zheng (2015 HMC); Mentor(s): Elizabeth Orwin; Laura Hoopes

Abstract: Corneal blindness affects more than 10 million people worldwide. An artificially grown corneal tissue construct would help satisfy the worldwide need for corneal transplants and serve as a model for further corneal research. Corneal transparency is a function of the arrangement of type I collagen fibers in the extracellular matrix and the presence of the corneal crystalline proteins transketolase (TKT) and aldehyde dehydrogenase class 1A1 (ALDH1A1) in the fibroblasts of the corneal stroma. Fibroblasts in a healthy cornea exhibit the quiescent keratocyte phenotype and express these corneal crystallins. When the cornea is wounded, cells can differentiate into myofibroblasts that express high levels of alpha-smooth muscle actin (α-SMA) stress fibers, decreasing corneal transparency. In this study we used electrospinning to create aligned and unaligned fibrous scaffolds on which to seed rabbit corneal fibroblasts. Using three materials—collagen, gelatin, and laminin—we sought to compare the relative importance of scaffold material and fiber alignment in controlling cell phenotype, with the goal of reverting cells from the wound-healing phenotype to the quiescent phenotype. SEM imaging and analysis revealed that fiber density and diameter, both of which can affect cell phenotype, were not uniform among the electrospun samples. In some cases, cells cultured for a week on aligned scaffolds could be seen aligning in the direction of the fibers. Future work will focus on obtaining more uniform samples and comparing expression of TKT, ALDH1A1, and α-SMA among the six conditions.
Funding Provided by: Kenneth T. and Eileen L. Norris Foundation; Engman Foundation

Understanding the Cytotoxicity of Permalloy Disks

Aleksandra Karapetrova (2015); Student Collaborator(s): Sam Ciocys (2015 Drexel University); Brian Flores (2013 California State University, Long Beach); Philip Gach (Postdoctoral Student, Argonne National Laboratory); Additional Collaborator(s): Dr. Elena Rozhkova (Argonne National Laboratory-Center for Nanoscale Materials); Dr. Valentin Novosad (Argonne National Laboratory-Materials Science Division); Mentor(s): Matthew Sazinsky

Abstract: Nanomagnetic materials offer exciting opportunities to remotely control biological processes. For example, ferromagnetic microdisks induce apoptosis in cancer cells via external magnetomechanical stimulus. Iron-nickel permalloy disks, which have these capabilities, are fabricated using optical lithography and metal deposition. They are removed from an array using organic solvents and are transferred to aqueous mediums for further applications. The permalloy disks can be made with a layer of gold on the top and bottom sides for the purpose of surface functionalization. The disks can bond with fluorescent dyes and biological compounds to be used as probes or drug deliverers in biological systems. Since the magnetic cores of the disks consist of a transition metal alloy, there is a possibility of reactive oxidative species (ROS) forming in aqueous solution. This occurs through a Fenton reaction and ROS are considered harmful. The chemical stability of permalloy disks not coated with a gold layer were studied. ROS formation was detected using fluorescent probe hydroxyphenyl fluorescein, X-ray fluorescence microscopy, and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). No significant levels of hydroxyl radicals were detected at neutral pH. However, X-Ray fluorescence did detect leaching of nickel and iron from the disks on a model cancer tissue.
Funding Provided by: Argonne National Laboratory

Engineering and Purifying ToMO Mutants to Enhance Terminal Hydroxylation of Alkanes

Lily Zhuang (2014); Mentor(s): Matthew Sazinsky

Abstract: Toluene/o-xylene monooxygenase (ToMO) belongs to a family of proteins called Bacterial Multicomponent Monooxygenase (BMM). Proteins of this family are capable of hydroxylating many different organic compounds despite having similar and sometimes even identical active sites. However, ToMO demonstrates poor terminal alkane hydroxylation capability as it hydroxylates toluene to form mainly cresol (99%), and a small amount of benzyl alcohol (1%) (Pikus, et. al., 1997). Mutations can be made on ToMO, however, to improve its ability in terminal hydroxylation. In order to accomplish these mutations, site directed mutagenesis is used to create a combination of single, double, and triple ToMO mutants Q141C, E214G, T201S. Additional single mutants Q141S, Q141A, Q141G, H96A, and H111D were also made. These mutant strains were then inserted into cell line Top 10, expressed, and screened for terminal hydroxylation activity using Whole Cell Assay and a Purpald Screen. Mutants that showed promising hydroxylation capabilities went through a series of protein purification steps. Hanging drop crystallization was subsequently performed on the isolated protein in an attempt to crystallize the structure of the respective ToMO mutant.
Funding Provided by: Kenneth T. and Eileen L. Norris Foundation

Purification and crystallization of intein-encoded homing endonucleases in P. horikoshii and M. jannaschii

William Reilly (2014); Mentor(s): Len Seligman; Matthew Sazinsky

Abstract: This study aims to employ X-ray crystallography to discern the structure of archaeal homing endonucleases (HEs) encoded by inteins within the Lon protease of P. horikoshii and replication factor C of M. jannaschii. To purify the proteins, we engineered a construct to add an N-terminal Strep-tag and a C-terminal 6xHis tag to both proteins. Following successful tagging of the P. horikoshii HE, large-scale expression and sequential affinity chromatography steps using Nickel-NTA and Strep-Tactin columns were completed. The eluate from the final column was estimated via protein gel to be ~95% pure (i.e. sufficiently pure for crystallization), so following size-exclusion concentration to 5 mg/mL, crystallization was attempted with 384 ‘core’ conditions outlined by the Joint Center for Structural Genomics. 16 conditions were found to promote protein microcrystal growth, and variations of these conditions are currently being tested to grow single crystals suitable for X-ray crystallography. Furthermore, in an attempt to co¬crystallize our HE with its DNA binding site, a 22 b.p. DNA strand derived from the protein binding site was prepared and added to the purified protein. Crystallization using this sample was attempted, and promising early results suggest that microcrystals have grown under a number of conditions.
Funding Provided by: Sherman Fairchild Foundation

Novel Surgical Aid: Fluorescent Nerve Peptide Labeling of Degenerate Neural Tissue

Melina Mastrodimos (2016); Additional Collaborator(s): Roger Tsien (UCSD School of Medicine -Department of Chemistry & Pharmacology); Mentor(s): Quyen Nguyen (UCSD School of Medicine)

Abstract: One of the most significant obstacles surgeons face in modern times is visual ambiguity within the human body. Various pathologies can obscure normal anatomy, making identification and preservation of normal structures challenging for the surgeon. With the use of a series of newly engineered, fluorescent peptides designed to bind to and illuminate specific anatomic structures, however, easy identification of obscured or damaged tissues is possible. In this study a comprehensive look was taken at the interaction between these fluorescent peptides and chronically denervated peripheral nerves. Using a series of these probes, nerves were highlighted at different locations, and stages of function. Degenerate distal branches were imaged 1-9 months post proximal surgical transection. At just 72 hours post transection, nerve electrical function shuts down and nerve stumps cannot be stimulated with electromyography (EMG) monitoring. Without the aid of stimulation feedback, most surgeons abandon any attempt at surgical repair, leaving patients with little to no options for recovery from paralysis. In our study a fluorescence microscope was utilized to obtain images of the degenerate nerves otherwise invisible to the eye with white light alone. With this specific targeting and fluorescence illumination capability, this study indicates the treatment window for paralysis could be extended up to 9 months post injury. To test this claim, he next phase of this study will explore fluorescence guided repair via surgical neurorrhaphy.
Funding Provided by: Howard Hughes Medical Institute; National Institute of Health (UCSD) SUR2695 -Award Title: Testing Fluorescently Labeled Probes for Nerve Imaging During Surgery

Eukaryotic evolutionary relationships in the methionine salvage pathway

Bianca Villavicencio (2016); Mentor(s): Andre Cavalcanti

Abstract: The methionine salvage pathway (MSP) is a nuclear encoded pathway present in all eukaryotic supergroups, responsible for recycling methionine from 5’¬methylthioadenosine. Some eukaryotic species have gene fusions resulting in multi-domain proteins, such as mtnKA and mtnBD in Tetrahymena thermophila, and mtnBC in Arabidopsis thaliana. In this project we used similarity searches to characterize the MSP proteins in 270 eukaryotes with fully sequenced genomes. MSP and its many fusion genes might give insight on eukaryotic evolution and phylogenetic relationships.
Funding Provided by: Pomona College SURP