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BIO REU Projects

Unraveling the biosynthesis and metabolism of alkenone in marine haptophytes.

Dr. Betsy Read, Professor, Biological Sciences.

The goal of this project is to identify genes and proteins involved in the biosynthesis and metabolism of alkenones. This unusual class of long chain (C35-C40) unsaturated methyl and ethyl ketones are produced by select haptophyte microalgae, are used by geochemists as a paleooceanographic proxy to estimate sea surface temperatures, and recently have attracted attention as a potentially fruitful yet unexplored renewable source of petroleum products. The cellular function and biochemical pathways governing the synthesis and degradation of these lipids, however, are not known. To unravel the metabolism of alkenones this work aims to: 1) compare the genomes of the three alkenone-producing haptophytes, E. huxleyiG. oceanica, and I. glabana (sequenced in our laboratory) with a forth non-alkenone producing species, Chrysochomalina tobin, and 2) generate mutants and characterize genomic lesions responsible for alterations in the accumulation and/or distribution of these very long chain unsaturated ketones.  Students will test the underlying hypotheses that haptophyte genomic diversity co-varies with adaptations to different environments or ecological niches, and the divergent evolution of traits. This will be accomplished by identifying molecular signatures consistent with the ability synthesize alkenones by comparing genome organization and structure, gene composition, metabolic pathways and gene families, with the expression of genes across the four species under conditions known to promote alkenone synthesis. Students will also test the hypothesis that mutants in the genetic circuitry that drives alkenone biosynthesis and degradation will change the lipid profile of these algae without causing lethality. Forward genetic techniques will be employed to generate mutants that will be screened by plating in the presence of alkenone biosynthesis inhibitors and examined using GC/MS. Interesting mutants will be characterized by NGS to establish gene-function relationships with respect to alkenone production.

Student Mentoring

In addition to using a variety of bioinformatics tools and techniques, students will screen mutant libraries, extract and generate lipid profiles using GC/MS, isolate RNA and/or DNA, and prepare libraries for nucleic acid sequencing. Dr. Read led the Emiliania huxleyi (E. huxleyi) genome-sequencing project coordinating the efforts of ~ 75 research scientists. She has extensive experience training undergraduate students (~ 100 students; 17 minorities, 31 who have pursued graduate work; 11 were accepted into major graduate programs and one received an NSF fellowship for graduate studies). Students present their work at local, national, and international meetings. She has published more than 20 peer-reviewed papers, 12 of which include students as co-authors. 5 students have won research awards and/or research fellowships.

Comparative population genomics of three species of coccinellid beetles used in augmentative biological control.

Dr. Arun Sethuraman, Assistant Professor, Biological Sciences.

Three species of predatory coccinellid beetles, Hippodamia convergens (North American Convergent Lady Beetle), Coccinella septempunctata (Eurasian Seven-spotted Lady Beetle), and Harmonia axyridis (Asian Harlequin Lady Beetle) are used extensively in augmentative biological control against common agricultural pests, like aphids and whiteflies, as part of a multi-billion dollar Integrated Pest Management industry. However, little is known about the efficacy of biological control using these three species, and the target and non-target effects of augmentation. Based on our previous work, we will test hypotheses that (1) natural selection due to resource competition, bridgehead, and founder effects of the non-native species of beetles have caused an exponential decline in effective population sizes of the native convergent lady beetle, and (2) augmentation of local populations with industry-reared beetles (of all three species) results in adaptive introgression (from gene flow), and more effective biological control.

NSF REU students will be involved in projects to test these hypotheses that (1) use genome-wide SNP data and coalescent-based modeling to elucidate the effects of human-mediated releases and non-target effects of augmentative biocontrol, (2) identify patterns of adaptive evolution, diversity, and population size trajectories as indicators of the effectiveness of biological control activities, and (3) develop field experiments and predictive computer simulations/models to build a paradigm for using predatory coccinellids in augmentative releases.

Specifically, ongoing projects include: (1) Whole genome sequencing, assembly, annotation of all three species, (2) Population genomics using ddRAD-sequencing of 20+ populations of beetles from all three species, (3) Crossing (to simulate the effects of augmentation in the field), and competition assays (between all three species) in controlled green-house experiments. This combination of genomics, and field experiments will help us construct plans for effective biological control using coccinellid beetles.

Student Mentoring

REU students will be involved and mentored in all aspects of these ongoing projects, including organizing and leading field trips for sampling, bench-work in molecular genomics (extraction, quality control, RAD-seq), post-sequencing bioinformatics analyses (quality control, assembly, variant calling, population genomics), and dissemination of findings at conferences, workshops, and in publications. Students will be encouraged to come up with their own testable hypotheses, and experiments, within the scope of our projects. Dr. Sethuraman has previously mentored 24 undergraduate students; 8 have presented at national and international conferences, and 5 are co-authors on publications in high impact journals.

Cellular dynamics of epidermal gd T cells in cytokine signaling

Dr. Julie Jameson, Associate Professor, Biological Sciences.

Cytokine receptor reception is important for cross-talk between gd T cells and keratinocytes in the skin. Stress induces keratinocytes to express an as yet unidentified receptor that activates skin-resident gd T cells to increase transcription of growth factors and cytokines with paracrine and autocrine functions. However, the impact of cytokine reception during activation is little studied. Murine skin-resident gd T cells express both TNFR1 and 2 at varying levels, however it is unknown how TNF-a regulates gd T cell transcription and cellular dynamics. Based on previous studies published by our lab, we will test the hypotheses: (1) short-term TNF-asignaling increases expression of genes and cytoskeletal rearrangement involved in gd T cell activation, while long-term TNF-a signaling reduces the expression of genes and cytoskeletal rearrangement involved in gd T cell activation, and (2) that Akt-mTOR signaling regulates the changes in gene expression and cytoskeletal rearrangement identified with TNF-a signal reception.

Students will be involved in projects that test these hypotheses using (1) RNAseq to identify how short-term versus chronic TNF-a signaling impacts T cell gene expression, 2) western blotting and flow cytometry to determine how signal transduction pathways, such as Akt-mTOR, are impacted by gd T cell stimulation in the presence or absence of TNF-a, and 3) confocal microscopy to determine how TNF-a signaling changes cellular dynamics such as microtubule formation. The use of RNA sequencing data will elucidate signaling pathways and key processes that modulate T cell function in the skin. Students will be involved in the entire experiment from cell culture to data analysis allowing them to expand their knowledge in a variety of techniques, computer programs, and statistical analyses.

Student Mentoring

Students will receive training cellular and molecular biology techniques including: primary cell culture, qPCR, RNAseq, flow cytometry and western blotting. Dr. Jameson has trained 36 undergraduates over the last 12 years, including 17 students from underrepresented minorities and 2 military veterans. Undergraduate and graduate students routinely presented their research at the American Association for Immunologists Conference and the International gd T Cell Conference. She has a long history of mentoring undergraduate students from her lab to join PhD programs.

Defining the protein-protein interactions and gene expression networks that underlie glutaredoxin control of primary root growth

Dr. Matthew Escobar, Professor, Biological Sciences.

Of the mineral nutrients necessary for plant growth, nitrogen is required in the largest amounts and is often limiting. Beyond its fundamental role in biosynthesis of cellular macromolecules, the availability and distribution of soil nitrogen is an important signal that regulates plant growth and metabolism. We recently showed that nitrate activates the expression of a group of six genes encoding glutaredoxin enzymes in Arabidopsis thaliana. Silencing these glutaredoxins in transgenic plants caused increased primary root growth and a decreased sensitivity to nitrate-mediated inhibition of root growth. These findings demonstrated that nitrate-induced glutaredoxins act as negative regulators of primary root growth. However, the downstream signaling mechanisms connecting glutaredoxins to root growth remain unknown. The primary objectives of the current project are to: 1) define the protein interaction partners of nitrate-regulated glutaredoxins, and 2) characterize glutaredoxin-regulated gene expression networks via comparative transcriptomics. In objective one, we will use yeast two hybrid analyses to identify potential glutaredoxin interaction partners, and selected interactions will be verified in planta by bimolecular fluorescence complementation. Based on previous studies of other plant glutaredoxins, we hypothesize that the nitrate-induced glutaredoxins interact with and regulate the activity of TGA transcription factors. In objective two, we will use RNAseq to compare the transcriptomes of wild-type plants and glutaredoxin mutants (created by CRISPR-Cas9 genome editing) in the presence and absence of nitrate. We hypothesize that glutaredoxin mutants will exhitib a defective transcriptional response to nitrate, resulting in altered synthesis and distribution of the hormones auxin and ethylene in the root.

Student Mentoring

Students will receive training in modern molecular biology and genomics techniques, including CRISPR-Cas9 genome editing, RNAseq, and bimolecular fluorescence complementation. Over the past 12 years Dr. Escobar has supervised 26 undergraduate researchers and 5 graduate (Master’s) students. Almost all of these students have presented research posters at scientific conferences and/or were authors on peer-reviewed publications. Nine of Dr. Escobar’s former students went on to pursue doctoral degrees in science (five of these were underrepresented minority students). 

Complex genome rearrangements initiated by microsatellite DNA repeats

Dr. Jane C. Kim, Assistant Professor, Biological Sciences

Approximately 30 neurological, neurodegenerative, and developmental diseases are caused by expanded microsatellite repeats. Microsatellites are naturally occurring repetitive DNA sequences within the genome that tend to be one to ten base pairs in length. Friedreich’s Ataxia is a neurodegenerative disorder caused by expanded GAA trinucleotide repeats. Using a yeast experimental system, it was previously shown that long GAA repeat tracts cause chromosomal breakage and gross chromosomal rearrangements (GCRs). Myotonic Dystrophy 2 is another repeat expansion disorder caused by long CCTG tetranucleotide repeats. CCTG repeats are not well-characterized, and it is not known whether long CCTG repeats also cause GCRs. The objective of this research is to construct yeast strains with CCTG and reverse complementary CAGG repeats to investigate their effect on GCR rates and determine the patterns of complex genome rearrangements these DNA repeats initiate. GCRs and their genomic breakpoints will be assessed using Oxford Nanopore sequencing, which has the capability of sequencing ultra-long read lengths (>30 kb). Such events will be characterized by comparing single reads to the reference genome of the background strain. This research will contribute new information about genome instability instigated by disease-associated CCTG repeats.

 Student Mentoring: Students will receive laboratory training in molecular biology and genetics, gaining specific experience in molecular cloning, yeast genetics, nucleic acid biology, and bioinformatics analyses. In her previous role as a postdoctoral fellow, Dr. Kim mentored two undergraduates who are co-authors on her publications. In her first four years at CSUSM, Dr. Kim has mentored 16 undergraduate students, including five from a local community college participating in the BRIDGES to the Future program and two who are currently enrolled in biology PhD programs.

 

Metagenomic analyses of microbial abundance and diversity in semi-arid soils exposed to long-term experimental nitrogen deposition

Dr. George L Vourlitis, Professor, Biological Sciences.

Chronic N inputs affect soil carbon (C) storage through changes in microbial activity, abundance, and species diversity; however, N effects on diversity are less well-known because the molecular tools required for quantifying microbial diversity have only recently been developed. The objective of this research is to utilize metagenomics tools to assess how chronic N exposure alters soil microbial species abundance and diversity of chaparral shrublands. Based on previous studies, the work will test the hypotheses that long-term exposure to experimental N deposition will significantly increase microbial groups that utilize labile soil organic carbon (SOC) and significantly decrease microbial groups that utilize recalcitrant SOC. This hypothesis will be tested utilizing a long-term field N addition experiment in chaparral where four-10 x 10 m plots have received 50 kgN ha-1 annually since 2003 and an additional four-10 x 10 m plots have served as controls. Soil samples will be collected from each plot in the spring (May) and fall (October). DNA will be extracted, purified, and subjected to targeted NGS sequencing for soil bacteria (16S rRNA) and fungi (ITS1-4 rRNA). Sequence reads will be subjected to BLAST analysis against the NCBI nucleotide database to determine how chronic N exposure alters microbial species abundance and diversity. This research will generate novel data to assess how human inputs of atmospheric N affect soil microbial community structure and function.

Student Mentoring

Students will receive valuable training in field research and laboratory techniques. They will be involved in sample collection; optimizing soil DNA extraction, purification, and quantification; bioinformatics analyses including species identification, and computational methods to quantify species abundance.  Over the last 3 years Dr. Vourlitis has mentored 18 undergraduate students, 7 were underrepresented minority students. Six of these students are authors on conference proceedings, 2 are co-authors on recent peer-reviewed publications, and 4 are currently preparing manuscripts.

Comparative genomics of ranaviruses in the western United States bait trade.

Dr. James K. Jancovich, Associate Professor, Biological Sciences.

Ranaviruses (genus Ranavirus, family Iridoviridae) are large double stranded DNA viruses that infect a wide variety of cold-blooded vertebrates. Numerous worldwide outbreaks of ranavirus disease over the last 25 years in reptiles, diverse amphibian species and commercially valuable fish suggest that these viruses are ecologically and economically important pathogens. Ranaviruses are thought to spread with cold-blooded vertebrates that are transported globally for bait, food, and pets and it has been suggested that the anthropogenic movement of ranavirus host species has influenced the host range of this group of viruses, with naïve species becoming susceptible to infection because of pathogen introduction and subsequent speciation by the virus. We hypothesize that ranavirus host shifts have taken place because of the anthropogenic movement of ranavirus hosts. To test this hypothesis, we are proposing to sequence ranavirus genomic DNA isolated from our unique collection of salamander bait trade samples. We have a one-of-a-kind collection of bait salamander samples that differ temporally and spatially from around the western United States and our goal is to sequence the ~105 kbp ranavirus genomes we will isolate from our collection using next-generation sequencing technology. Using assembled genomic sequence information, comparative analyses will generate a molecular clock to describe the evolution of the ranavirus and provide insight into the proliferation of these viruses.

Student Mentoring

Students will receive training in cell culture, virology, molecular biology and bioinformatics.  Dr. Jancovich has mentored over 30 undergraduate students over the last 6 years. Twelve of these students are authors on conference proceedings, two are first authors and five are co-authors on recent peer-reviewed publications, and one is currently preparing a manuscript.

Understanding Biomineralization in Coccolithophores by Integrative Multi-omics Analyses

Dr. Xiaoyu Zhang, Professor, Computer Science and Information Systems.

Coccolithophores are one of the most spectacular calcifying microalgae and the third most prominent group of phytoplankton. Because of their ability to fix inorganic carbon photosynthetically and via biomineralization, they play an important role in global carbon cycling and are of great interest to biogeochemists.  The nanoscale architecture, and light reflecting capabilities of select coccoliths, have also captured the attention of material scientists. Information relating to the genetic and biochemical underpinnings of the biomineralization, however, is lacking. The primary goal of this work is to improve our understanding of genes and proteins involved in coccolithophore biomineralization by performing integrated comparative analyses of genomic, transcriptomic and epigenetic data generated from next-generation sequencing (NGS). In this NSF-REU project, students will be involved in developing and applying computational tools and algorithms to discover patterns and biologically significant information in large multi-omics data from different sources. Students will also practice using different visualization tools to present and interpret results. Specifically, potential REU projects include: (1) Applying Bayesian learning models to effectively integrate the gene expression and methylation data from calcifying vs. non-calcifying strains to prioritize genes of interest; (2) Improving the current assembly and annotation of coccolithophore genomes, and constructing their gene regulatory networks from expression data of multiple coccolithophore species under different growth conditions; (3) Performing cross-species co-expression network analysis to identify conserved gene modules that are potentially related to biomineralization; (4)  Developing machine learning models that integrate multi-omics data and other important features of biomineralization genes and proteins.

Student Mentoring

Working at the intersection of computer science, mathematics, statistics, and biology REU students will develop skills and knowledge in bioinformatics and machine learning; developing, testing and running programs to process and mine large datasets on the parallel servers housed in the computer science department. Dr. Zhang has advised more than 30 graduate and undergraduate students in the last 13 years; several have co-authored manuscripts and/or presented research at national conferences, and 6 have been admitted to PhD programs. He and an undergraduate student were awarded the faculty-student research collaboration award by the college of science and mathematics in 2015.

Employing MetaHiC to map the movement potential of antibiotic resistance in microbial populations

Dr. Elinne Becket, Assistant Professor, Biological Sciences.

The spread of microbial antibiotic resistance, particularly pathogenic microbes with multidrug resistance, is a global healthcare and ecological problem driven by the extensive medical and agricultural use of antibiotics1. Commensal human or environmental microbial populations harbor reservoirs of antibiotic resistance known as the resistome. Exposure to antibiotics, environmental/diet changes, or other factors can drastically change the composition of the microbiome and resistome2. Specific microbes increase or decrease in abundance due to selective advantage in a given environment3, and resistance markers can be transmitted to other bacterial species through horizontal gene transfer. In fact, many pathogenic microbes obtain genetic resistance determinants from resistant commensal microbes within a niche2. Additionally, antibiotics in the environment can influence transfer within the resistome, by inducing transfer of resistance elements4 or increasing the competency of recipient cells5. Therefore, we seek to elucidate changes in the resistome of marine microbiome samples in response to common antibiotics seen in agricultural and urban waste (notably fluoroquinolones, beta-lactams, and tetracyclines). This will be accomplished by treating sampled populations with/without antibiotics and overlaying three NGS techniques: MetaHiC6 (to map the resistance elements to specific host organisms), plasmid-prep coupled with shotgun sequencing (to measure global excision/insertion of resistance elements), and metatranscriptome sequencing (to assess competency and expression of resistance elements). Soil and water samples near and far from ocean sewage outlets will be compared to assess if priming with antibiotics affects the movement of the resistome.

The overarching hypotheses are: 1) exposure to antibiotics will increase cell competency and resistance element transfer in these sampled populations, 2) different response patterns will occur depending on the antibiotic used and cells within the population, and 3) populations primed with antibiotics (near sewage outlets) will demonstrate greater resistome mobility compared to other populations. 

Student Mentoring

REU students will be involved in all aspects of the project, including procuring environmental samples, isolating and culturing bacterial strains, and performing the MetaHiC pipeline, NGS library preparation, and established NGS/HiC bioinformatics analyses. Students will be trained in molecular biology and NGS techniques, coupled with extensive data analysis with open-ended applications in ecological studies. They will also acquire BSL2 laboratory skills and training. As a post-doc, Dr. Becket mentored 27 undergraduates, 20 of whom have co-authored papers publications.

Next generation sequencing on Major Histocompatibility Complex (MHC) genes reveals founder effect in non-human primates.

Dr. Bianca R. Mothé, Professor, Biological Sciences. 

Non-human primates, including Chinese-origin, Indian-origin and Cynomolgus macaques, amongst others, have been bred in captivity for a wide range of reasons. Many of the captive-bred colonies have been in place for over 50 years and no information on their origin is available.  The objective of this research is to utilize next generation sequencing, based on the PacBio and Ion Torrent platforms, to analyze the MHC diversity in several colonies, including those at Tulane National Primate Research Center (TNPRC) and FIOCRUZ in Rio de Janeiro to examine founder effects.  Based on previous studies, we will test the hypotheses that specific non-human primate colonies that have been in captivity demonstrate founder effect based on limited MHC diversity.  Blood samples will be collected from non-human primates and RNA will be extracted. cDNA will be generated from these samples and NGS sequencing will be performed to identify MHC class I and II genes. This research will identify novel MHC genes as well as provide information into non-human primate colony formation.

Student Mentoring: Students will be involved in sample preparation including RNA extractions, cDNA synthesis, library preparation, and NGS; bioinformatics analyses; and computational methods.  Since Dr. Mothé’s arrival at CSUSM in 2003, she has produced 34 publications; 12 with student co-authors.

Understanding the toxicity of stream benthic cyanobacteria exposed to variable culturing conditions using genomic and transcriptomic analyses

Dr. Rosalina Hirstova, Adjunct Professor, Biological Sciences.

Benthic cyanobacteria has recently been recognized as a source of multiple cyanotoxins recorded ~30% of the streams studied in central and southern California. Toxigenic cyanobacteria blooms in freshwater ecosystems are an escalating threat to environmental and human health worldwide. The benthic cyanobacteria species responsible for producing the various toxins are virtually unknown, as are the environmental cues that trigger the harmful blooms. Following recent toxic events in California rivers, several toxin-producing strains of filamentous cyanobacteria (i.e. Phormidium, Anabaena and Geitlerinema) were isolated and have been maintained in culture. The objective of this research is to: 1) characterize the toxin produced by each strain by GC/MS analysis, and 2) utilize whole genome and transcriptome sequencing to confirm the presence and expression of toxin related genes. NGS genomic and transcriptomic data will be used to identify key genes and proteins essential to toxin synthesis under variable growth conditions, such as different levels of nitrogen, phosphorus, chlorides, and sulfates. The work will test the hypotheses that increased nutrient and ion content stimulate growth and toxin-production in studied cyanobacteria and will determine threshold levels of water constituents relevant to preventing or controlling harmful algal blooms.

Student Mentoring

Students will isolate and culture algae, characterize harmful algal communities in fresh water streams, measure toxins levels in streams and cultures, isolate RNA and DNA from toxin producing species, prepare libraries for genomic and transcriptomic sequencing, and participate in sequence and bioinformatic analyses. In the past 5 years Dr. Histova has mentored over 20 students and published 16 peer-reviewed journal articles or book chapters which include five student co-authors.

Transcriptomic survey of animal epithelia for novel mechanisms of rapid regulation of epithelial ion transport

Dr. Dennis Kolosov, Assistant Professor, Biological Sciences.

Epithelia are tissues of multicellular organisms that specialize in directional transport of ions and water in and out of the animal. Faced with rapid changes in environmental and systemic variables, animals rely on epithelia to mount a rapid response in a timely manner aimed at maintaining homeostasis. Recent work in my research group has identified several novel molecular mechanisms used by epithelia of the insect ‘kidney’ to rapidly adjust their ion transport. These novel molecular mechanisms include the use of voltage-gated, ligand-gated and mechanosensitive ion channels, as well as the gap junctional coupling between epithelial cells. Despite the fact that these molecular components appear to be very important for the function of insect epithelia, there is no consensus on whether these ion channels are expressed in the epithelia of other animals. Projects use transcriptomics (RNAseq) to (i) identify the above-described ion channels expressed in animal epithelia, and (ii) determine which environmental (e.g., salinity) and systemic (e.g., hormone treatment) factors rely on these ion channels to adjust epithelial function. A mix of lab-generated and publicly available transcriptomic data will be used to carry out these projects.

Student Mentoring

Students will be involved in sample preparation (e.g., RNA extraction) for NGS workflow, NGS data access, manipulation, and bioinformatics and computational analyses (quality control, trimming, mapping to genome/transcriptome, differential expression and gene ontology enrichment analyses). Dr. Kolosov arrived to CSUSM in 2021 and is excited to welcome the first generation of students into his lab.