Creation LRB (Light-Response BTB) /PIF (Phytochrome-Interacting Factor) Mutant Lines in Arabidopsis thaliana

Color poster with text, charts, and graphs.Light is vital to plant survival and thus plants have developed sophisticated pathways to respond properly to their light environments. Plants sense specific wavelengths of light via photoreceptors, one family of which are the red (R)/far-red (FR)-absorbing phytochromes (phys). Absorption of red light activates the phys, which causes their translocation from the cytosol to the nucleus where they modulate gene expression. They do so by regulating the activity and levels of a family of transcription factors called Phytochrome-Interacting Factors (PIFs). In response to red light the active phys cause PIFs to be ubiquitylated and degraded, which activates expression of PIF-repressed genes. There is feedback regulation of this pathway as, in response to red light, the PIFs also induce ubiquitylation and degradation of the phys. The genes Light-Response BTB 1 and 2 [LRB1 and LRB2]) are critical regulators of the phy/PIF light-response pathway. LRB1 and LRB2 encode BTB (Bric-a-Brac, Tramtrack, Broad Complex) domain-containing proteins that act as target adapters in E3 ubiquitin-ligase complexes. Plants with disruptions of the LRB genes have reduced light-dependent degradation of phys and, like plants with disruptions of PIF genes, exhibit hypersensitivity to red light. The mechanism by which the LRBs modulate phy levels is not entirely clear, however it has been shown that the LRBs can bind to a complex of a PIF protein (PIF3) and a phy (phyB), leading to ubiquitylation and degradation of both. In order to better understand how the LRB and PIF genes interact we are taking a genetic approach, creating plants with T (transfer)-DNA disruptions of both LRB and PIF genes. Study of the phenotypes of these plants may shed light on how these two families of genes work together to regulate light responses or plant growth and development in general.National Science Foundation-Research in Undergraduate Institutions (RUI) grants (#1354438); University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Polycystic Kidney Disease and Protein Localization : Analysis of the Role of gar-3 in Protein Localization to Cilia in Caenorhabditis Elegans

Color poster with text, images, and diagrams.Gar-3, a gene that encodes a G protein-coupled acetylcholine receptor, has been shown to play roles in both neurotransmission and protein localization to synapses. We asked whether gar-3 also affects the localization of PKD-2 protein to primary cilia in C. elegans, a free-living nematode. Dysregulated PKD-2 localization has been linked to polycystic kidney disease. Thus, understanding the factors that mediate PKD-2 protein localization to cilia might help us better understand the molecular basis of polycystic kidney disease. We chose to utilize C. elegans as our model organism because it is transparent and has a cilia structure similar to cilia found in mammalian kidneys. In order to address our question, we used both genetic and reverse genetic techniques to reduce gar-3 gene expression in C. elegans transgenic for PKD2::GFP. This allowed us to use fluorescent microscopy to assess PKD-2 localization to primary cilia in a living animal and draw conclusions about how this might affect the progression of polycystic kidney disease. If gar-3 is associated with PKD-2 localization, this would provide new insight into polycystic kidney disease.University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Involving Students in Curriculum Development : Design of a New Genetics Laboratory

Color poster with text, images, charts, and graphs.In Fall 2017, the Biology Department introduced a new laboratory-based genetics course. To develop the course, we modified a team-based approach successfully used in the past. During the summer, I tested and revised protocols, researched background literature, and gave feedback from a student perspective. We also figured out how long it took do experiments and other activities for the students to make a course schedule. During the first offering of the course, I served as a student academic apprentice. Although we found some drawbacks to our modification of the team-based approach, the involvement of a student in course development helped the course run more smoothly in its initial offering and be more interesting for the enrolled students.University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Elucidating the Role of Spinner in Cystic Kidneys : Using a Zebrafish Model to Better Understand Polycystic Kidney Disease

Color poster with text, images, charts, photographs, and graphs.Patients with autosomal domain polycystic kidney disease (ADPKD) suffer from disrupted kidney function due to large, fluid-filled cysts that form in the collecting ducts and kidney tubules. There is no known cure and current treatments for the disease only manage cyst growth but do not prevent cyst formation. In collaboration with the Sussman and Ekker labs at Mayo Clinic (Rochester), we have begun to characterize the changes that take place during kidney cyst formation using spinner, a zebrafish mutant that develops kidney cysts. Previous work in our lab identified a region of chromosome 17 that appears to be deleted in spinner mutant zebrafish. This region contains at least 6 protein coding genes. We hypothesize that deletion of one or more of these genes causes the spinner phenotypes. None of these genes has previously been identified to play a role in cystic kidney disease. Thus, if we can identify the causative gene(s), we can potentially understand more about the disease. Utilizing DNA extraction and PCR techniques we are working to redefine the deletion boundaries.University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Creation and Characterization of LRB (Light-Response BTB) IPIF (Phytochrome-Interacting Factor) Mutant Lines in Arabidopsis thaliana

Color poster with text, graphs, charts, and images.In order to better understand how the LRB and PIF genes interact we are taking a genetic approach, creating plants with T (transfer)-DNA disruptions of both LRB and PIF genes. Study of the phenotypes of these plants may shed light on how these two families of genes work together to regulate light responses or plant growth and development in general.National Science Foundation-Research in Undergraduate Institutions (RUI Grants #1354438); Unversity of Wisconsin--Eau Claire Differential Tuition; University of Wisconsin--Eau Claire Office of Research and Sponsored Programs

Zebrafish, C. elegans and Human Polycystic Kidney Disease

Color poster with text, graphs, charts, and images.The focus of this project is to identify the downstream effects of mutations in the causative genes (PKD1 and PKD2) of polycystic kidney disease (ADPKD). ADPKD is responsible for 5% of all endstage renal disease and is characterized by large, fluid-filled cysts forming in kidney tubules and collecting ducts that disrupt the normal functioning of the kidneys. It is currently thought that primary cilia (1) and different signaling pathways may play a major role in the progression of ADPKD.University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Identification and characterization of a novel allele of Caenorhabditis elegans bbs-7.

Primary cilia play a role in the sensation of and response to the surrounding environment. Caenorhabditis elegans (C. elegans) have primary cilia only on the distal tips of some dendrites. In order to better understand the relationship between receptor localization to cilia, cilia structure and cilia function, we have characterized a mutation originally identified in a forward genetic screen for mutants with defective PKD-2 ciliary localization. Through behavioral assays and examination of the structure of cilia in the cil-5 (my13) mutant animals, we have found that my13 disrupts not only receptor localization, but also some cilia-mediated sensory behaviors and cilia structural integrity. We have identified the my13 lesion and found that it is a missense mutation in bbs-7, an ortholog of human BBS-7, a gene known to affect human cilia and to be involved in Bardet-Biedl syndrome. Finally, we show that bbs-7(my13) also affects the glia cells which support the cilia

Studying the Effects of Extra Sets of Chromosomes on C. elegans Neurons : Using Tetraploid C. Elegans to Investigate Genetic Abnormalities

Color poster with text, images, photographs, and graphs.Polyploidy is a condition in which organisms possess multiple sets of chromosomes as a result of nondisjunction events during meiosis. In most organisms, polyploidy results in sterility or lethality, making it difficult to study These characteristics make C. elegans a good model to investigate the effects of changes in ploidy on living multicellular organisms. Primary cilia are organelles that extend from the surface of some sensory neurons in C. elegans. In animals, cilia are thought to function as regulators of cell growth, division and proliferation, and defects in cilia function have been linked to several human diseases and health concerns. Polyploidy has been associated with cyst growth in patients with Polycystic Kidney Disorder (PKD). We hypothesized that analysis of primary cilia structure and function in C. elegans might yield insights into connections between ploidy and pathology.University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Course Development : Building an Advanced Cell/Molecular Lab as Undergraduate Research

Color poster with text, charts, and tables.The biology department seeks to provide authentic experiences for students both within and outside of the classroom. Teaching labs are a venue for development of technical and critical thinking skills, and upper level labs foster research skill development initiated in the introductory science labs. BIOL 405 is a new laboratory-based course designed to immerse students in current molecular biology techniques.CETL Summer SoTL Grant; University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Can These Mutations Lead to Blindness? : Analyzing the Effects of Genetic Variants of Uncertain Significance

Color poster with text, charts, and images.Genetic testing involves examining a patient's DNA sequence to look for changes (mutations) in the DNA that can potentially cause disease or illness. While some of the changes are benign, many have not yet been characterized and are thus classified as variants of uncertain significance (VUS). In collaboration with Prevention Genetics, our lab has begun to analyze VUS that are predicted to affect the splicing of genes related to ocular diseases. Disruption or alteration of splicing can affect gene function and lead to disease. We are using a minigene system in which the gene segment under investigation is cloned into a plasmid vector and then transfected into eukaryotic cell culture. The mRNA transcripts are then collected and analyzed to determine the effects of the variant on the transcript. Analysis of VUS could lead to a change in variants’ interpretation and directly have an impact on patients and their families by supporting diagnosis and access to treatment.University of Wisconsin--Eau Claire Office of Research and Sponsored Program