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.


Investigation of PERM-1 Localization in Oocytes and Embryos of C. elegans

Anne Berhe ’20; Advisor: Sara Olson; Collaborator: Norani Abilo ’20

The innermost layer of the nematode C. elegans eggshell, the permeability barrier, is one of the most impenetrable materials in the animal kingdom. However, not much is known about the composition or formation of this eggshell layer. C. elegans is a model organism for many parasitic nematodes and further research on this highly impermeable layer of the eggshell could lead to potential drug targets that could halt the viability of worms via the eggshell. Ascarosides, a lipid containing the sugar ascarylose and a fatty acid-like side chain, are predicted to be an essential component of the permeability barrier. Previous studies have revealed that PERM-1 is necessary for the formation of the permeability barrier and this protein’s epimerase and reductase domains are similar to those seen in the final catalyzation steps of ascarylose biosynthesis pathway. A primary goal of this project is to localize PERM-1 before and directly after fertilization. Our experiments this summer focused on analyzing colocalization patterns between PERM-1 and various organelles in the nematode’s oocytes and early embryos. PERM-1 had various levels of colocalization across organelles but did not consistently have high levels of colocalization in any one organelle.
Funding Provided By: Paul K. Richter and Evalyn E. Cook Richter Memorial Fund and The R. Nelson Smith ’38 and Corwin H. Hansch Fund for Summer Chemistry Research

Investigating the Half-site Reactivity of P. Horikoshii CoADR

Yongkang Zhang ’18; Advisor: Matthew Sazinsky; Collaborators: Angela Ling ’19, Maxim Leshchinskiy ’19, Liwam Nerayo ’20

Half-site reactivity is an enzyme characteristic in which the active sites of a multimeric enzyme “fire” alternatively. At any given time, only one-half of the identical enzyme subunits are active. Although many metabolically important enzymes with half-site reactivity have been identified and characterized, the cause of half-site reactivity and its significance on enzymatic catalytic efficiency and mechanisms remain unclear. In this project, we designed, expressed and purified an asymmetrical dimer for P. Horikoshii Coenzyme A disulfide reductase (phCoADR), a member of the pyridine nucleotide disulfide oxidoreductase (PNDORs) family that has shown to display half-site reactivity during in vitro elemental sulfur reduction. Preliminary kinetics data suggest that the asymmetrical dimer loses the ability to fire alternatively. Preliminary data from NADH anaerobic titration of the enzyme reveal a difference in reduction potentials between the active and dead subunit of the enzyme.
Funding Provided By: The R. Nelson Smith ’38 and Corwin H. Hansch Fund for Summer Chemistry Research

Mutating GDI in Drosophila Using CRISPRCas9

Dominique Bruncko ’18; Advisor: Clarissa Cheney

The Rab pathway is extremely important for regulating vesicle transport. Rabs are G-Proteins, meaning that they act as a switch based on whether GTP or GDP is bound. One protein that helps regulate this switch is called a guanine nucleotide dissociation inhibitor (GDI), which holds the Rab in its GDP bound off state and covers its hydrophobic tail to transport it through the cytoplasm. GDI is N-terminally acetelyated post translation by NAT-B based on GDI’s second residue being Asn. Our objective is to mutate the second residue of GDI from Asn to Ser to see how being a substrate of NAT-A instead of NAT-B will change GDI’s interaction with Rabs. We are doing this by creating a Drosophila mutant with the mutated GDI in its genome via the CRISPR-Cas9 system. This system uses a protein to cut in a highly specific location, cleaving out the unwanted gene segment. Cells are then injected with a plasmid containing the Ser mutation as well as regions of homology to the genome to introduce the mutation via homologous recombination. Currently, the CRISPR-Cas9 system and its associated gRNAs have been prepared, and the homology plasmid is in the process of being created by using Gibson Assembly. The first several attempts at assembly were not successful, so several changes were made to optimize assembly in the future. The next step after the creation of the homology plasmid is to send the gRNA and the homology plasmid to be injected into our CRISPR-Cas9 Drosophila strain.
Funding Provided By: The R. Nelson Smith ’38 and Corwin H. Hansch Fund for Summer Chemistry Research

Cause and Effect of Natural Codon Reassignments: eRF1 Structure and Tandem Stop Codons in Ciliates

Ira Fleming ’18; Advisor: Andre Cavalcanti

Tandem stop codons, used as a safety-net in the event of codon readthrough, are found to be highly over-represented at the +2 position alone or the +3 and +4 positions together after the annotated stop codon in several ciliate species as well as in drosophila melanogaster, the latter known for its high rates of stop codon readthrough. The reassignment of stop codons in many of these ciliate species and the speculated reduction in termination efficiency, hinted at by higher levels of tandem stops, may be related to several identified point mutations within and adjacent highly conserved codon recognition sites and the GGQ’ peptide release site within eukaryotic release factor one.
Funding Provided By: The Professors Corwin Hansch and Bruce Telzer Undergraduate Research Fund

Protein Complexes that are Scaffolded by a Common Protein and Regulate C. elegans Eggshell Formation Localize Independently

Julian Prieto ’20; Advisor: Sara Olson

In the nematode C. elegans, fertilization of an oocyte leads to the hierarchical development of different eggshell layers and a reorganization of the embryo that serves to protect the embryo from polyspermy and assure its development. The vitelline layer, the outermost layer of the eggshell, is present on oocytes and separates from the plasma membrane immediately after sperm entry. Very little is known about the mechanics and composition of this layer, but CBD-1 (chitin-binding domain protein) has been identified as essential for this point of embryonic development. Through both RNAi and CRISPR experiments, PERM-2 and PERM-4 were identified as proteins having a co-dependent function essential in the formation of the vitelline layer. CBD-1 scaffolds both the PERM complex and the EGG complex (EGG-1-5, which is required for fertilization and egg activation).  By using CRISPR null mutants and domain mutants, I showed that the localization of each of these protein complexes are independent of each other. Interestingly, some of the perm-2(null) mutants still form some viable eggshells.
Funding Provided By: The Professors Corwin Hansch and Bruce Telzer Undergraduate Research Fund

The Roles of Rad50 and TFIIH in Double Strand Break Repair in saccharomyces cerevisiae

Robert Buxton ’18; Advisor: Tina Negritto

The transcription factor TFIIH has long been theorized to play an important role in the repair of double strand breaks (DSBs) in DNA. In this study, DSBs were induced in various Saccharomyces cerevisiae strains displaying both wild types (WT) alleles and mutations in either the Rad50 or Rad3 gene, and samples were taken of each strain at various time points. Either the Rad50 protein or the TFB1 component of the TFIIH transcription factor was tagged with FLAG peptide tags. The DNA associated with the tagged protein was then isolated via chromatin immunoprecipitation (CHIP) and quantified via QPCR to analyze the degree of occupancy of the target protein at the DSB at various time points in both WT and mutant yeast. In both TFB1 and Rad50 pull down, primer choice was found to play a large role in determining the magnitude of the degree of occupancy, but the shape of the curve was preserved. Rad50 pull down at the first of two recruitment events was substantially decreased in the Rad3-G595R mutant but was not completely knocked down, hinting at a potential role of TFIIH in facilitating recruitment of Rad50 to the DSB. The Rad3 mutant does not display the second of two increases in occupancy shown by the WT strain, showing that TFIIH may be required for the second recruitment of Rad50 to the DSB but not the first. An assay must be designed to determine whether increases in occupancy are due specifically to the behavior of the protein of interest at the experimental region or the primer region.
Funding Provided By: The Professors Corwin Hansch and Bruce Telzer Undergraduate Research Fund

The Characterization of rad3 Mutant Phenotypes in Nucleotide Excision Repair

Zoe Zhou ’18; Advisor: Tina Negritto

UV radiation can cause covalent linkages to form between adjacent DNA base pairs. Normally, this type of damage is repaired by a process called Nucleotide Excision Repair (NER). However, in a number of human diseases, including trichothiodystrophy (TTD), mutations in the genes encoding NER proteins prevent DNA repair from occurring. In some cases of TTD, this confers a photosensitive phenotype. For this study, we want to characterize the phenotypes of four different NER mutants, rad4, rad3-G595R, rad3-R660C, and rad3-A596P in S. cerevisiae. The rad3 mutants were created to mimic known mutations in human TTD patients. During NER, Rad4 plays the role of recognizing UV-induced DNA damage, and Rad3 acts as a DNA helicase that unwinds the DNA and recruits subsequent repair proteins. To characterize the mutants, a pinning assay and pulsed-field gel electrophoresis (PFGE) assay were used. For the pinning assay, cells were exposed to four different levels of UV radiation: 0, 50, 75, or 100 J/m2 and then grown over two days. For the PFGE assay, time points were collected after treatment with UV. Samples were subsequently treated with either mung bean nuclease or TUNEL staining to detect formation of single-stranded breaks after thymine dimer excision. The results from these assays suggest the mutant strains behave differently from the wild-type and also behave differently from one another. These differences may explain the underlying molecular defects in DNA repair that lead to TTD.
Funding Provided By: The Doudna and Cate Chemistry, Biology and Molecular Biology Fund, The Doudna and Cate Chemistry Fund

PERM-1 ligand screening through Differential Scanning Fluorimetry

Inga Van Buren ’18; Advisor: Matthew Sazinsky

Parasitic nematode infections pose a major worldwide health problem affecting more than 2 billion individuals each year. Current treatments, however, are specific for worms in the larval stage as embryos are protected by a permeability barrier. Previous studies have identified PERM-1 as a transmembrane protein implicated in the synthesis of the permeability barrier in C. elegans, a model organism for nematode infections. The goal of this project was to utilize differential scanning fluorimetry (DSF) to better characterize the binding partners of PERM-1, serving as a starting point for future drug screening. A truncated recombinant construct was expressed in a Shuffle cell overexpression system and purified using a detergent-based protocol. DSF was then used to tentatively assess the extent to which PERM-1 was folded, as well as its ability to bind NAD+, CDP, and UDP. Preliminary results suggest that PERM-1 has a melting temperature of ~53C and exhibits a thermal shift upon substrate exposure. However, observed thermal shifts are not consistent and could stem from the inherent instability of PERM-1 after purification. As a result, buffer, dye, and protein concentrations used in DSF must be further optimized to enhance protein stability and its behavior.
Funding Provided By: HHMI

Experimental Examination of C. elegans Ovulation Differences in Wild Type and trp-3 Mutants Through Live Imaging Microscopy

Katiannah Moise ’18; Advisor: Sara Olson

C. elegans has been used as model organisms to better understand fertilization and egg development. Easy view of meiotic division of oocytes without dissection has been helpful in developing the field around in vivo egg development. Egg activation is prompted by biochemical reactions. Calcium signals in the early stages of oocyte fertilization facilitates the transition from egg to embryo. This egg activation includes the formation of the eggshell that prevents polyspermy and allows for reactivation of the cell cycle. Calcium signaling has been modeled as a two-step process. The first step is an initial spike when the egg and sperm fuse, and the second is the propagation of a slow wave of calcium throughout the length of the egg. Takayama & Onami (2016) observed that the initial Ca2+ spike is produced by a sperm-specific Ca2+-permeable transient receptor potential (TRP)-3 channel that is delivered to the egg upon fusion. Without it, the initial spike is missing, but there is still a delayed calcium wave propagation. Not much is known about different sources of calcium channels that participate in the event, so I am examining the role of the egg’s ER in providing calcium for the slow wave. In addition, I am using live imaging microscopy to visualize ovulation and calcium wave differences between trp-3 mutant and wild-type embryos. The data will be used by collaborators in the math department to develop better 1D/2D mathematical models to describe the calcium wave propagation.
Funding Provided By: J. Steller Summer Curriculum Enrichment Fund

Engineering riboswitch-based whole cell biosensors through dual genetic selections and fluorescence activated cell sorting

Maryann Zhao ’18; Advisor: Jane Liu

Whole-cell biosensors are tools used to assess the bioavailability and bioaccessibility of small molecules. Compared to traditional instruments, biosensors offer the advantage of not only being easy and cheap to replicate but also requiring minimal specialized expertise. Riboswitches, nature’s own versions of biosensors, are well suited for this purpose because of their molecular recognition capabilities. Generally found in the 5’-untranslated region of prokaryotic mRNA, riboswitches are genetic regulatory elements that switch between conformational states in the presence of a small molecule ligand. The binding of the small molecule to the mRNA aptamer domain induces structural changes in the expression platform that result in transcriptional and/or translational modulation of gene expression. My project harnessed riboswitch’s natural mechanism to answer the question, is it possible to engineer an artificial riboswitch that can sense a selected small molecule and control gene expression in a characteristic manner? Using dopamine as the small molecule, our platform used libraries of Escherichia coli, each harboring a mutagenized variant of a natural riboswitch, and performed dual genetic selection and fluorescence activated cell sorting to isolate functional riboswitches. Developing an effective platform to engineer riboswitch-based biosensors will provide researchers with a powerful detection tool that can be applied in diverse situations.
Funding Provided By: Department Funding

Investigating a Functional Role for N-terminal Acetylation of Rab GDI in Drosophila

Michael Poeschla ’18; Advisor: Clarissa Cheney

Rab GTPases comprise a large family of proteins that regulate a variety of membrane trafficking processes related to vesicular transport. 1 GDI is an essential part of the Rab pathway, responsible for recycling inactivated Rabs from membranes back into the cytosol. 2 Previous data from the Cheney laboratory has shown that GDI is N-terminally acetylated in Drosophila, and that mutations in the acetyltransferase that catalyzes this modification are lethal in the late larval (3rd instar) stage. 3 Though over 80% of eukaryotic proteins have been found to be N-terminally acetylated, the functional consequences of this modification are poorly understood. 4 The aims of this study are to construct an in-vitro system by which we can assess the effects of N-terminal acetylation of Rab GDI on its ability to bind and extract Rabs from membranes.
Funding Provided By: HHMI

Using FUCCI to investigate Mitotic Activity in Rab5 knockdown Hemocytes and Imaginal Wing Discs in Drosophila

Priyanka Ramanan ’19; Advisor: Clarissa Cheney

Rabs are G-proteins that are important directors of vesicle traffic in the cell. Previous studies demonstrate that knockdown of Rab5 in the hemocytes of Drosophila results in an overabundance of circulating hemocytes. Knockdown of Rab5 in the imaginal wing discs also results in malformed wings. The goal of this project was to use the FUCCI system to explore cell over proliferation as a possible explanation. FUCCI transgenes were introduced to specifically be expressed in the imaginal wing disc tissue and in hemocytes in order to visualize the cell cycle. No significant difference was found in mitotic activity between wild-type and Rab5 knockdown imaginal wing disc and circulating hemocytes.
Funding Provided By: J. Steller Summer Curriculum Enrichment Fund

Identifying MtlS Targets in Vibrio cholerae

Sabrina Mendez-Contreras ’18; Advisor: Jane Liu

MtlS is a small RNA transcribed antisense to the 5’ untranslated region of mtlA, which codes for the sugar transporter for mannitol. MtlS works in cis as a translational repressor of mtlA. Because bacterial sRNAs can target multiple genes, we hypothesized that MtlS may regulate other genes in addition to mtlA. We used RNA target-prediction programs and mass spectrometry-based proteomics to identify additional MtlS targets. The results identified 6 potential targets, including mtlA. To confirm that MtlS is regulating the predicted targets, we created translational fusions of each target with gfp and introduced these constructs into V. cholerae strains with and without mtlS. This allowed us to monitor the effects of mtlS expression on fluorescence levels, and thus the amount of target-gfp being expressed. The results were also confirmed by western blot analysis. As a positive control, an mtlA-GFP fusion was constructed and I observed the expected down-regulation. I observed stationary phase down-regulation of VC1899 in the presence of MtlS, contrary to the up-regulation indicated by the proteomics results. Moving forward, I will determine where MtlS is binding VC1899 transcript to affect down-regulation of the gene. Additional putative targets of MtlS are also in the process of being tested. Ultimately, this work will expand our knowledge on MtlS’s role in V. cholerae and sRNAs in general.
Funding Provided By: Department Funding

Evolution of RNA-based biosensors in E. coli yields riboswitches with over three-fold activation

Samuel Lin ’20; Advisor: Jane Liu

Small molecules play important roles in cell metabolism and function; thus, developing efficient small-molecule sensors is integral to our further understanding of cell functions with many potential applications. In order to create a small-molecule sensor, we created a RNA-based biosensor constituted of two main components: in the 5’ untranslated region, an aptamer domain that binds the ligand of interest, and between the aptamer domain and the Shine-Dalgarno (SD) Sequence, an expression platform that mediates conformational changes of the mRNA, which upon ligand binding, causes a change in downstream gene expression. For use as a biosensor, gfp was placed downstream of the SD sequence, and thus cell fluorescence would be turned “on” and “off” following ligand binding. Also placed downstream of the SD sequence is the gene tetA, which allows for genetic selection of riboswitches from a randomized RNA library. In order to evolve the Liu Lab’s previously found theophylline riboswitch, the aptamer domain of the initial “hit” was mutagenized, thus creating a 107 library of E. coli, each harboring a unique RNA library member on which to perform genetic selections and fluorescence-activated cell sorting (FACS). Three rounds of selections on the mutagenized library yielded riboswitches exhibiting over three-fold activation upon ligand binding. Refinement of our selection and screening protocol may lead to discovery of riboswitches with even higher activation and overall fluorescence.
Funding Provided By: Department Funding

Clinically Tested NDV-3A Vaccine May Protect Against Highly Virulent Multi-drug Resistant Fungal Infection

Zhaoqi Huang ’19; Advisor: Jonathan Wright

C. auris is an emerging opportunistic fungus that has recently presented itself as an enormous threat in hospital settings around the world. Of the few infectious species of candida, C. auris has gained attention because of its growing resistance to all three major classes of antifungal drugs. In immunocompromised patients, the fungus can enter the bloodstream and cause invasive candidiasis, affecting the central nervous system, organs, and may lead to death. The current mortality rate for patients infected with C. auris is estimated to be 30-60%. Candida albican’s Als3 protein based NDV-3A vaccine has shown protection against C. albican infections in preclinical and clinical trials (phase I/IIb). Through bioinformatics, we found that C. auris contains a cell surface protein that shows amino acid homology to the Als3 protein. Using various molecular biology and immunodetection tools, we identified and isolated the Als3 protein homologue in various C. auris strains. This finding has serious implications in Candida vaccine development as previously tested NDV-3 vaccine might show protection against highly virulent and drug resistant strains of Candida auris.
Funding Provided By: Department Funding


Characterization of a Halophilic Archaeon Isolated from Saturated Salt Pools in the Salton Sea

Jerry Lee ’16; Mentor: EJ Crane

A halophilic species was isolated from saturated salt pools in the Salton Sea. The species exhibited a distinct pink color in both solid and liquid media and grew optimally in 3-4 M NaCl. The species was a facultative anaerobe capable of using elemental sulfur and iron (III) oxide as terminal electron acceptors. 16S rRNA gene analysis places this species within the Haloferax genus with Haloferax sulfurifontis as its most closely related species.
Funding Provided By: Fletcher Jones

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

Maria Arciniega ’16; Mentor: Tina Negritto

DNA double-strand breaks (DSBs) can result from endogenous or exogenous hazards such as enzymatic errors during replication and ionizing radiation respectively. In S. cerevisiae, homologous recombination is a relevant mechanism used to repair DSBs. TFIIH is a multisubunit protein complex that is known to be involved in the repair of DSBs, and Rad3 and Ssl1 are two core subunits shown to play a key role in inhibiting homologous recombination between short-homologous sequences. Our goal in this study was to analyze the role of RAD3 and SSL1 in translocation formation by assaying two mutant strains, rad3-G595R and SSL1-T242I, via a haploid translocation assay. Mutant strains for the translocation assay were built, consisting of HO endonuclease cut sites engineered on different chromosomes next to substrates for homologous recombination. Previously it was hypothesized that rad3-G595R and SSL1-T242I mutants would have increased recombination between short homologous sequences, as compared to wild-type, but preliminary results contradicted our prediction. A molecular approach was implemented to corroborate the recombination frequencies observed. Pulsed-Field Gel Electrophoresis (PFGE) and Southern blot were utilized to check for the induction of the DSB at HIS3 locus. Induction of DSB is absent on chromosome III of the rad3-G595R strain. The absence of DSB at chromosome III may explain the deviation of recombination frequencies from the hypothesized trend.
Funding Provided By: Rose Hills

Cytotoxicity of Butylparaben, Phenoxyphenol, and Trifluoromethyl Phenol on Saccharomyces Cerevisiae

Prisca Diala ’18; Mentors: Tina Negritto and Cynthia Selassie; Collaborators: Katiannah Moise ‘18

Phenolic compounds are incorporated as antioxidants in the development of cosmetics, plastics and processed food. While previous research supports the notion that the antioxidant nature of the phenols may function to suppress the genotoxic effects of free radicals that may evolve as a result of oxidative reactions, recent studies suggests that many of these same phenolic compounds can become free radicals, causing oxidative stress themselves. This oxidative stress can induce double strand breaks or the formation of DNA adducts, leading to mutations that suggests the carcinogenicity of these compounds. In the present study, we focused on evaluating the cytotoxicity of butylparaben, 4-phenoxyphenol, and 4-trifluoromethyl phenol, three phenols that are commonly used in cosmetics. We first determined the inhibitory concentration (IC) values (20, 50, 80) of these phenol compounds in killing yeast cells. We then screened different null mutant strains of yeast cells for growth defects in the presence of these phenols using a Pinning Assay. Further experiments were done to determine apoptosis and double strand breaks in response to the phenols. However the results were inconclusive and future experiments will focus on determining if in fact there are incidences of apoptosis invoked by the phenols.
Funding Provided By: Pomona Unrestricted (Diala), Telzer/Hansch (Moise)

Development of bacterial encapsulation using polymeric microparticles in a W/O emulsion for the targeted delivery of P. Distasonis

Erik Guillen ’18; Mentor: Quiaobing Xu (Tufts University); Collaborator: Feng Jia (Tufts University)

A recent study on colorectal cancer suggests that P. Distasonis may play an anti-inflammatory role in the gut, which may reduce the incidence of colorectal cancer. In order to study the role this bacteria plays, it is necessary to target its delivery to the colon. Microencapsulation is known to be an effective method for targeted and controlled drug delivery, however, conventional techniques require complicated processes and are geared towards small and rigid molecules (i.e. RNA and enzymes). The purpose of this project is to develop a delivery method specific for the encapsulation of live bacteria by an oil-in-water emulsion using FS30D, a methacrylic co-polymer that is able to target the colon. The hydrophilic-hydrophobic interactions within the emulsion and the polymerization of the wall material are expected to result in an enteric matrix that can protect the bacteria in gastric conditions and deliver to the colon. Encapsulation trials were performed by using E. coli as a model for P. Distasonis and the quantification of bacteria release were calculated by performing colony counts on release samples. We found that using the proposed technique FS30D may successfully encapsulated 1.58*103 cells per gram of polymer, which suggests that a new polymer and/or a changes to the current technique must be implemented for future experimentation.
Funding Provided By: Tufts University Building Diversity in Biomedical Sciences Program

DNA Damage Induced by Phenolic Compounds in Saccharomyces cerevisiae

Sarai Santos ’16; Mentors: Tina Negritto and Cynthia Selassie

A variety of phenolic compounds serve as antioxidants in everyday products such as cosmetics and processed foods. While antioxidants function to inhibit the genotoxic effects of free radicals that may arise from oxidation, recent studies have also implicated the potential of antioxidants to form hydroxyl radicals and cause oxidative stress themselves. This oxidative stress can lead to DNA damage which may signal the potential carcinogenicity of these compounds. In the present study, we focus on a series of phenolic compounds previously studied in our lab that have shown to cause DNA degradation in vitro. Our in vivo study evaluates the relative amount of breaks that these phenolic compounds induce in Saccharomyces cerevisiae chromosomal DNA using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and pulsed-field gel electrophoresis (PFGE) assay. PFGE of chromosomal DNA from cells treated with the phenolic compounds should reveal chromosomal band smearing as a result of induced DNA breaks or degradation. In order to quantify the intensity of the smearing, we coupled PFGE with the TUNEL assay. In essence, this would allow for the quantitation of break induction since the TUNEL assay serves to catalyze the addition of flourescein-labeled dUTPs to 3’OH ends that result from DNA strand breakage.
Funding Provided By: Howard Hughes Medical Institute

Effect of Transcription Factor II H on Recruitment of Rad50 and Rad52 to Double Strand Breaks in Yeast

Nancy Zhu ’16; Mentor: Tina Negritto

Funding Provided By: Elgin

Exploring the Role of MtlR in Vibrio cholera

Naomi Vather ’15; Mentor: Jane Liu

Funding provided by: Rose Hills

Investigating genetic control of the interaction between Rad59 and Rad52 using the two-hybrid system

Kathie Tang ’17; Mentors: Tina Negritto, Adam Bailis (Beckman Research Institute at the City of Hope), and Glenn Manthey (Beckman Research Institute at the City of Hope)

Radiation causes loss of genome stability that can stimulate tumorigenesis. Homologous recombination (HR) promotes genome stability by repairing breaks in DNA. RAD52 is an HR gene that works with breast cancer susceptibility genes BRCA1 and BRCA2 to maintain genome stability. Rad59 is an HR protein that works with Rad52 by facilitating association of Rad52 with broken chromosome ends. Studies have shown that loss of Rad59 has an effect on Rad52’s ability to anneal single stranded DNA in non-conservative HR. We have found three mutations in RAD59 that disable this association: rad59-K166A, -K174A, and -F180A. We investigated the effects of these mutations on association between Rad59 and human and yeast Rad52 using the two-hybrid system. The two-hybrid system allows for quantitative study of protein-protein interactions by utilizing the yeast GAL4 transcription factor that can be separated into two functionally essential domains, DNA-binding domain and activation domain, which are required for transcription of the Escherichia coli lacZ reporter gene. Plasmids are engineered to produce protein products in which each domain is fused onto a protein. If the proteins interact, then the DNA-binding domain and activation domain are indirectly connected, allowing for reporter gene transcription. In this experiment, the proteins were human and yeast Rad52 and Rad59. Preliminary results have shown that rad59-K166A, -K174A, and -F180A have little interaction with human or yeast Rad52.
Funding Provided By: Rose Hills

Investigating the half-site reactivity of an oxidoreductase family

Wuyi Li ’16; Mentors: EJ Crane and Matthew Sazinsky

Enzymes are described to exhibit the half-site reactivity if reactions they catalyze show stoichiometry equal to one-half the number of identical subunits [1]. Early literature proposed that the half-site reactivity is caused when one substrate molecule induces a change in an adjacent subunit, preventing a subsequent substrate molecule from reacting [2]. Although the half-site reactivity is well documented, its impacts on enzyme catalytic efficiency and mechanism remain poorly understood. In this project, we genetically designed and screened enzyme ‘heterodimers', which are dimers with a mutated (functionally dead) subunit and a wild-type one. Through characterizing these heterodimers kinetically and structurally, we hope to further our understanding of the half-site reactivity. Preliminary kinetics data suggests that one ‘ heterodimer', coenzyme A disulfide reductase from Pycococcus horikossi, exhibits enhanced catalytic efficiency than wild-type one for a reaction it catalyzed. This result contradicts with early literature, suggesting communication between subunits either doesn’t exist or is prevented by construction of heterodimers. In addition, it implies half-site reactivity slows down enzymatic catalysis.
Funding Provided By: Howard Hughes Medical Institute

Jurassic Park in Miniature: Resurrecting the Function of Inactive Homing Endonucleases

Ellen Green ’16; Mentor: Lenny Seligman

In the film Jurassic Park, scientists recreate long-extinct dinosaurs by using DNA from their living relatives to fill in the gaps of their genome. In this research, we attempted to restore function to an inactive Homing Endonuclease by using information from the genome of its active family member. Homing Endonucleases, or HEs, are enzymes that cleave highly specific DNA sequences. This specificity makes them useful tools for genome editing. We focus on the LAGLIDADG family of HEs, named for its distinctive amino acid motif. In this study, we sought to restore function to an inactive LAGLIDADG HE encoded in the Lon gene from the archaea Pyrococcus furiosus (Pfu). We were able to compare the inactive HE with an active HE in the same gene from a close relative, Pyrococcus horikoshii (Pho). Pfu Lon lacks a key amino acid in its LAGLIDADG motif, and has other deletions relative to Pho Lon, including one large N-terminus deletion, as well as other smaller deletions. We initially attempted to restore function to Pfu Lon by restoring the LAGLIDADG motif through PCR mutagenesis. This mutation did not restore function, suggesting that at least one other mutation is responsible for the loss of function. For future research, we are attempting to restore the large N terminus deletion found in Pfu Lon relative to Pho Lon. We are using PCR to combine a portion of Pho Lon containing the deleted region with the remainder of Pfu Lon. Currently we are working to isolate this hybrid intein.
Funding Provided By: Fletcher Jones

Mechanism of Pathogenesis in Mucormycosis

Graham Barlow ’16; Mentor: Jonathan Wright; Collaborators: Sameh Soliman, Jack Edwards, Jr., Ashraf S. Ibrahim

Mucormycosis is a life-threatening disease caused by Mucoralean fungi which afflict immunocompromised patients (e.g. diabetic ketoacidotic, deferroxamine-treated, organ transplantation, or those with trauma). A striking increase in the number of mucormycosis cases for the last two decades has been reported. Recently we were able to identify two novel toxins from Mucorales, H and S-toxin. Both toxins showed significant damaging effect to mammalian cells. The combination of the two toxins caused death in neutropenic mouse model. The expression of both toxins depends greatly on the fungal culture conditions. H-toxin is expressed in the fungal hyphae at sufficient aeration condition, while S-toxin is expressed when oxygen is limited and secreted into the culture media. Both toxins were tested for their apoptotic, oligomerization and phospholipase-like activities. Both toxins increased the release of FAS apoptotic factor, indicating apoptosis-like effect. In addition to DNAse and caspase3 induction activities, S-toxin showed apparent oligomerization, indicating a perforation at the mammalian cell membrane. The S-toxin inflicts significant damage within 15 minutes, and complete damage in 4 hours. These toxins are likely to be critical factors in the pathogenesis of mucormycosis.
Funding Provided By: Gift of Joan Hanley and Donald Hanley

Metabolic Engineering of Methanothermobacter For Producing Fuels From CO2

Christian Gonzalez ’18; Mentor: Laurens Mets ’68 (University of Chicago)

In the lab we worked with Methanothermobacter and producing isobute through their metabolic pathway of Isoprene. Our work for the summer was to identify a kinase enzyme that could fulfill one of the last two missing steps in the Archaeal isoprenoid synthesis pathway and postulated the existence of a novel decarboxylase enzyme that would be necessary to complete the pathway. Until now, efforts to identify this novel decarboxylase by direct experimentation have not been fruitful. Thus, the majority of my project’s efforts was to characterize the last missing enzyme in the isoprenoid biosynthesis pathway of Archaea in order to give off the production of isobute. Nonetheless, we attempted to do this through measuring the activity of the enzyme and then pursuing with column chromatography to diverge anything other then our decarboxylase. However, our project ran in to obstacles when the 3-hydroxymethylbuturate (substrate) was spontaneously reacting at a similar rate with or without the presence of the enzyme. This led to making it close to impossible to measure the activity of the enzyme and thus not identify its presence, which led to incompetent results. All reactions have a spontaneous rate, therefore the next step is to figuring out where that threshold of minimal spontaneous rate and maximal enzyme activity intersects.
Funding provided by: Pomona 1415

Endosome trafficking and regulation of cellular signals in Drosophila

Min Joo Kim; Mentor: Clarissa Cheney

Funding Provided By: Norris

Role of the Transcription Initiation Factor II H (TFIIH) in double strand break end processing in yeast

Luis F. Diaz-Ruiz; Mentor: Tina Negritto; Collaborators: Greg Elison ’13, Tyler Oe ‘14

DNA double-strand breaks (DSBs) are a particularly dangerous type of DNA damage, and homologous recombination is an important mechanism for their repair. We propose that transcription factor IIH (TFIIH), a multi-subunit complex with known functions in nucleotide excision repair (NER) and transcription initiation, also plays a role in the repair of DSBs in S. cerevisiae. Rad3, one of the TFIIH subunits, when mutated (rad3-G595R) causes increased homologous recombination between short sequences (SSR) and an increase in the stability of broken DNA ends. Using chromatin immunoprecipitation (ChIP) studies we have shown that TFIIH associates with DSBs and that in a rad3-G595R mutant, this association is lost. These results help support the hypothesis that TFIIH is necessary for DNA end degradation. What remains unknown is what factor is responsible for its recruitment. Since Rad4 is one of the factors known to recruit TFIIH during NER in yeast, we propose that this factor may also be involved in the recruitment of TFIIH to DSBs. To test this possibility, we have determined the effect of a rad4 null mutant strain on SSR and on the recruitment of TFIIH to a DSB via ChIP. Our results indicate that Rad4 may be the recruiting factor since the association of TFIIH with an induced double strand break is decreased in the absence of Rad4 and SSR is increased. In conjunction, our results ultimately help postulate new ideas on the exact role TFIIH may be playing in DSB repair.
Funding Provided By: Rose Hills (Diaz-Ruiz)

The Repair of Thymine Dimers in Saccharomyces cerevisiae rad3 Mutants

Zoe Zhou; Mentor: Tina Negritto

Thymine dimers are a form of DNA damage incurred when a cell is exposed to ultraviolet (UV) radiation. Normally, cells have a method of repairing damage caused by UV radiation called nucleotide excision repair (NER). The proteins involved in NER excise the damaged bases and then a polymerase arrives at the site of the excision to insert the correct bases. DNA ligase then seals the nicks. However, individuals who suffer from disorders such as xeroderma pigmentosum (XP) have mutations in the genes that code for proteins in the NER pathway. These individuals are unable to repair UV-induced damage to their DNA and therefore are highly sensitive to light and susceptible to skin cancer. Genes similar to those involved in NER in humans are also found in yeast. One such gene, RAD3, encodes for a protein that is a 5’ to 3’ helicase that is also a subunit of the TFIIH initiation factor. We are interested to see how three different rad3 mutants (rad3-G595R, rad3-R660C, and rad3-A596P) respond to UV radiation by quantifying thymine dimer repair. To do so, we had to develop a method of detecting thymine dimers similar to a Western blot. Additionally, we used pulsed field gel electrophoresis (PFGE) to quantify the repair of thymine dimers in these mutants. Currently, the results from these two assays are inconclusive; however, we were able to fine tune the thymine dimer detection method and determine an effective concentration of the anti-thymine dimer antibody to use. We were also able to find an effective concentration of T4 PDG to use for the PFGE.
Funding Provided By: Steller


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