Investigating the Role of the Pentose Phosphate Pathway in Annelid Regeneration

Kathy Gillen, Biology

Some animals can replace body parts lost to injury (restorative regeneration), while others cannot. By comparing elements required for restorative regeneration in a wide variety of species, we may uncover conserved mechanisms that might serve as targets for therapeutic intervention in humans. To this end, our lab investigates the molecular mechanisms underlying the regenerative abilities of the annelid worm Lumbriculus variegatus. When bisected, anterior ends of Lumbriculus re-grow new tails and posterior ends re-grow new heads. Tissue regrowth requires cell proliferation and thus biosynthesis of new cell components. In frogs, successful regeneration of tadpole tails requires a metabolic shift away from aerobic respiration (which completely oxidizes glucose to CO 2 ) and a shunting of glycolytic intermediates into the pentose phosphate pathway (PPP). The PPP helps produce precursors for synthesis of nucleotides, amino acids, and lipids. While the importance of the PPP for regeneration has been demonstrated in frogs, we don’t know whether it is also critical for invertebrate regeneration.

To answer this question, we will take several approaches this summer:

  1. Assessing whether activity of the rate-limiting enzyme in the PPP pathway (glucose-6-phosphate dehydrogenase) is up-regulated in regenerating tissue.
  2. Inhibiting glucose-6-phosphate dehydrogenase (G6PD) and measuring the impacts on regeneration.

If G6PD is up-regulated in regenerating tissue and/or inhibiting it impairs regeneration, then that supports its importance for annelid regeneration and points to the PPP as critical for regeneration across many animal taxa.  

In addition to learning experimental design and lab techniques, the Cascade scholar will maintain a lab notebook, unpack primary literature articles, discuss research ethics, troubleshoot experiments and collaborate with other researchers.

  • Project dates: June 2 - August 2
  • Expected coursework: Bio 115 or equivalent required. Bio 109/110 helpful, but not necessary

Using Genetics to Study Seasonal Responses in the Moss P. Patens

Karen Hicks, Biology

Many organisms use environmental cues, such as day length and temperature, to synchronize reproductive development with favorable climatic conditions, thereby increasing their reproductive success. For example, the moss P. patens, which our group uses as a focal research organism, undergoes sexual reproduction in the fall (short day lengths and cool temperatures), but not in the summer (long day lengths and warm temperature). Although many different flowering plants use the same genetic mechanisms for regulation of seasonal reproduction, our previous research suggests that the moss P. patens uses a distinct and novel downstream pathway to regulate reproductive development in response to seasonal cues. Our current research program seeks to discover how this novel downstream pathway works — what are the genes and proteins involved, how are they regulated, and how do they connect to one another? To ask these questions, we isolated genetic mutants that reproduce in summer conditions, when wild type plants do not reproduce, and identified two closely related genes that are defective in these mutants. We welcome Cascade Scholars to work alongside a Summer Science Scholar to:

  1. create knockout mutations in these and additional closely related genes to confirm their requirement for proper seasonal reproduction;
  2. genetically engineer P. patens to express fluorescently tagged versions of these genes;
  3. use the fluorescent strains and other techniques to study how these genes are regulated.

Through this work, Cascade Scholars will gain experience with genetic cloning, CRISPR-Cas9 targeted mutagenesis, plant transformation, evaluation of gene expression via quantitative reverse transcriptase PCR and fluorescence microscopy, as well as gain expertise in plant development and seasonal responses, all in a collaborative laboratory environment.

  • Recommended coursework: BIOL115-116 and preferably also BIOL109-110

Validating the Indole Lactic Acid Biosynthesis Pathway in Bifidobacteria

Ethan Hillman, Biology

Gut microbes produce a wide array of bioactive metabolites that influence human health, yet the enzymes responsible for many of these transformations remain uncharacterized. Bifidobacterium species, common members of the gut microbiome, have been implicated in the production of Indole-3-lactic acid (ILA), a derivative of tryptophan metabolism with potential immunomodulatory and neuroactive effects. However, the specific genes encoding aromatic lactate dehydrogenases (ALDHs) responsible for this transformation remain to be validated.

This project will use E. coli to express and purify candidate ALDH genes from Bifidobacterium species to assess their role in ILA biosynthesis. Students will clone target genes into expression vectors, induce protein production in E. coli, and purify the recombinant enzymes. Enzyme function will be confirmed through biochemical assays measuring ILA production. This work builds on previous efforts in our lab to functionally characterize microbial enzymes involved in biosynthesis of host-interacting metabolites, expanding our understanding of gut microbial contributions to host health.

Cascade scholars will gain hands-on experience with molecular cloning, recombinant protein expression and purification, and enzymatic assays. Students interested in microbiology, genetics, and microbial metabolism will gain valuable research skills while contributing to the broader understanding of beneficial microbial metabolites. Students will also learn how to engage with scientific literature and craft both oral and written presentations. 

  • Expected start date: Week of May 27
  • Students should have completed BIOL 115/116 and BIOL 109/110

Identifying Tissue-Specific Causes of Chemosensory Dysfunction in Caenorhabditis Elegans

Peter Kropp, Biology

From bacteria up to the largest animals, it is important to be able to detect and respond to stimuli that lead us toward good things (e.g. food) or away from bad things (e.g. hazardous chemicals). This function is called chemotaxis. In the Kropp lab, we use the microscopic round worm Caenorhabditis elegans to model rare mitochondrial diseases. In our model of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1), we know that there are neuromuscular impairments, but we’ve also noticed that the mutant worms tend to avoid food: a chemotaxis defect. While it may seem obvious that this defect is due to neuronal dysfunction, we know that other tissues in the body (e.g. the intestine) signal to neurons to modulate their activity. This intercellular signaling is especially important to change food-seeking behavior in response to nutritional status in a paradigm known as the gut-brain signaling axis.

The goal of this project is to use neuron-specific and intestine-specific knockouts of the gene nfu-1, the causative factor of MMDS1, to identify which tissue is responsible for the food avoidance phenotype of global nfu-1 mutants. The Cascade Scholar working on this project will learn about genetic engineering, C. elegans. husbandry, neuronal function, behavior assays, and statistical analysis. Having taken BIOL 115/116 and 109/110 will be very helpful.

Pathway of Sugar Transport in Non-Vascular Land Plants

Kamesh Regmi, Biology

My research tries to contextualize, within the anatomical context of cells, tissues, and organs, how photosynthesized sugar is translocated, stored, and utilized in phylogenetically diverse lineages of land plants including mosses, liverworts, lycopods, and flowering plants. One of the major innovations that facilitated the explosive radiation of land plants was the evolution of vascular tissues – namely, xylem and phloem tissues. Analogous to our own circulatory system, the sugar-conducting phloem cells and the water-conducting xylem cells in plants form an extensive vascular network in the plant body, with the phloem cells translocating photosynthesized sucrose from the source of synthesis (i.e., leaves) to the heterotrophic sink tissues (i.e., flowers, roots). How sugars are transported in vascular plants has been fully molecularly dissected in the model flowering plant Arabidopsis thaliana.

Interestingly, mosses are non-vascular plants and do not harbor true xylem and phloem tissues. Therefore, it is natural to ask how non-vascular plants like mosses transport sugars. In trying to answer this question, my lab uses a model moss Physcomitrium  patens. Based on basic bioinformatic research, it is immediately apparent that the molecular toolkit required for sugar transport in Arabidopsis has homologs in the Physcomitrium genome. Whether these molecular homologs perform the same functions in Physcomitrium is unknown. 

Student(s) will use Real-time Polymerase Chain Reaction (qPCR) to quantitatively ascertain whether the candidate homologous genes in Physcomitrium are in fact transcribed. Specifically, student(s) will (i) use basic bioinformatic tools to find the homologs of an Arabidopsis Sucrose Symporter, SUC2, encoded in the Physcomitrium genome, (ii) extract total RNA from Physcomitrium plants, (iii) synthesize complementary DNA from RNA using Reverse Transcriptase, (iv) design primers to amplify candidate gene transcripts, (v) perform qPCR, (vi) perform empirical analyses to quantify the levels of transcripts from the candidate genes.

Why Do Male and Female Birds (Sometimes) Differ in Their Flight Anatomy?

Natalie Wright, Biology

My lab is studying why in some species of birds, males have larger flight muscles than females, but in other species, the pattern is reversed, and in still others, the sexes exhibit no difference in flight muscle size. We take two approaches to these questions: First, we are compiling a dataset of skeletal measurements of a thousand species of birds (measurements of bones are excellent proxies for the sizes of the muscles that attach to those bones). Second, we test the functional effects of sexual dimorphism in flight muscle size by studying flight biomechanics. This summer, we will have active projects using both approaches. The Cascade Scholar may choose which project they wish to focus on, but there will be opportunities to help with all projects in the lab and therefore learn many skills.

For the first project, we will travel to a museum research collection to take digital photographs of bird skeletons. Back in the lab, we will measure bones from the digital photographs we took in the museums. We will also compile data on life history and ecological traits to test which of these traits predicts sexual dimorphism in flight muscle size. We will spend most of the summer in Gambier, but will make a one-week trip to natural history museums to collect data (TBD; e.g., the Smithsonian National Museum of Natural History in Washington, DC, the Field Museum in Chicago, or the Burke Museum in Seattle). All travel expenses will be paid for by Professor Wright’s grant, including hotel and food while away from Gambier, and all members of the lab are welcome on this trip, even if this is not their primary research project.

For the second project, we will examine whether male and female House Sparrows, which differ in their flight muscle size, have different takeoff flight biomechanics. We will capture House Sparrows around Gambier and use high-speed video cameras to film standardized takeoff flights in the lab. We then digitize these videos to calculate velocity, acceleration, wingbeat frequency and other measures of flight performance. After experimentation, we will euthanize the birds (they are an invasive species), dissect them to measure flight muscle and heart sizes, and test whether these or other morphological characters (e.g., wing size, shape) predict flight performance within this species.

The Cascade Scholar will learn basic bird biology and ecology, physics and aerodynamics of flight, how to identify bird bones, how use ImageJ to take measurements from photographs, how to safely handle wild birds, how to collect data from videos, museum-quality dissection techniques, and data analysis and visualization in R. No previous knowledge is necessary, but some prior experience using R is helpful (e.g., having taken BIOL 109/110, STAT 106, STAT 206, or STAT 226).

  • Summer research in the Wright Lab will take place from June 16 - August 16.

Bespoke Polymer Degradation

Yutan Getzler, Chemistry

If you love working with both your hands and your mind, and will have completed two semesters of chemistry lab by this summer, this may be the project for you.
 
We seek to build polymeric materials whose functional lifetimes can be rationally tailored. Polymers, sometimes called plastics, are large molecules synthesized by the repeated linking together of many small molecules (monomers). The properties of a polymeric material stem from its size, shape, and repeat unit. Properties we value at one point in a compound’s life, such as durability, may become harmful. Controlling how material properties change over time may mitigate these harms.

Cascade Scholars who join the group this summer will help complete the synthesis of a small library of monomers for use in this project. Most of these monomers have not previously been reported in the scientific literature, so you may bring a new molecule into being. The monomer synthesis is three steps long, two of which have been completed by your predecessors. To honor the value of their work and these materials, you will start the summer learning to perform the relevant synthetic transformation on a closely related, commercially available, starting material. When you feel ready, you will tackle the new work.

You will master the standard techniques and tools of organic synthesis, including aqueous workups, thin layer chromatography, rotary evaporation, flash column chromatography, recrystallization and NMR spectroscopy.

Organic Semiconductors in the Solid State

Katie Mauck, Chemistry

The Mauck Lab uses physical chemistry to study the properties of organic semiconductors in the solid state. Students work across many different areas of chemistry, ranging from instrumental techniques such as UV-Vis absorption and FTIR spectroscopy and cyclic voltammetry, to organic synthesis, crystal growth, and thin film preparation. Students often also pursue some computational work, either through spectroscopic data analysis or in quantum mechanical calculations by using density functional theory to model electronic structure, molecular geometry, and vibrational modes. The Cascade scholar will work collaboratively with other Mauck Lab student researchers and receive training in both synthetic and spectroscopic techniques. 

  • Dates: June2 - July 18
  • Preferred coursework: At least CHEM 121 or CHEM 122

From Sweet to "Salty": Identifying and Characterizing Enzymes Involved in Plant Defense Mechanisms

Kerry Rouhier, Chemistry

Tomatoes synthesize a class of molecules called acyl-sugars, which are excreted from hair-like tissues on the plant’s leaves. Acyl-sugars come in a variety of shapes and sizes and their structural diversity is specific to the plant and its ecological pressures, such as pests and disease. This project will look at the process (pathways) tomatoes use to synthesize these molecules. The project will focus on the synthesis of the acyl chains that ultimately get attached to the acyl-sugar molecule by primarily looking to express and purify functional 3-ketoacyl ACP synthase I (KAS I). Previous work in the lab focused on a related enzyme, KAS III.  The Cascade scholar will learn many common molecular biology and biochemistry lab techniques such as cloning and protein expression and purification. Additionally, the scholar will learn how to read and analyze primary literature and improve their written and oral scientific communication skills. No previous knowledge or experience is necessary, but having taken courses such as CHEM 123/126 or BIOL 109/110 is preferred.

  • Research for this project will be conducted from May 27 - July 18.

How Mosquito Kidneys Sort Friend From Foe: Using Dyes to Understand Xenobiotic Transport

Matthew Rouhier, Chemistry

My research group is interested in how mosquitoes remove unwanted or toxic molecules.  This summer my research group is using microinjection to determine if urine excretion can be halted by the introduction of dye molecules. The project will introduce the scholar to microscopy (to preparing and injecting mosquitoes with dyes) and molecular biology (extracting RNA, amplifying DNA, and potentially sequencing of DNA). In addition to lab work, the scholar will practice electronic notebook keeping, discuss their research project with other scientists and non-scientists, and practice applying the scientific method within the context of fighting mosquito-borne disease.

Insect-Mediated Ecosystem Services on Farms

Lauren Schmitt, Environmental Studies

Insects interact with plants throughout their lifecycle. Some of these interactions, like herbivory, can be problematic for farmers. Other interactions, like pollination and pest control, can be beneficial in improving yield or reducing crop damage. Farmers and land managers are incentivized to manage farms and land to encourage beneficial insect ecosystem services, while minimizing antagonistic interactions. Research in the Schmitt lab investigates how management choices, such as intercropping flowering plants, and landscape patterns, including the land-use of adjacent properties, shape insect communities and plant-insect interactions in agroecosystems. Projects this summer will investigate how the abundance and diversity of flowers in and around Brassica crop fields and apple orchards alters the interactions between insect herbivores and their natural enemies. We will also ask how the landscape around the farm changes the insect community and how insect interactions change between crops with different physical and chemical characteristics.

A Cascade student will be involved in all aspects of the research project, from troubleshooting methods and collecting insects to lab-based insect feeding trials and science communication. Students will work on field-based research projects at the Kenyon Farm, Glen Hill Orchards and other gardens, orchards, and farms around Knox County. Lab work may include insect identification, feeding trials and greenhouse projects. An ideal candidate is enthusiastic about working outdoors and willing to learn basic insect identification skills.

  • No prior knowledge is necessary, though ENVS 112 or the Introductory Biology series would be helpful background.