Tag: The Substance of Us

The role of the Chemoreceptor Zinc-Binding Domain in bacterial signal transduction

Presenter(s): Dan Tudorica—Biochemistry

Faculty Mentor(s): Arden Perkins

Session 3: The Substance of Us

Previous work presented at the undergraduate research symposium hypothesized that the chemoreceptor zinc-binding (CZB) domain acted to sense bleach in certain bacteria’s environment and correspondingly direct bacterial swimming patterns . This project presents an expanded view of the CZB domain as being responsible not just for informing bacterial swimming patterns in the presence of bleach, but also for regulating the formation and dispersal of bacterial biofilms . Through the use of genetically-modified bacteria and biofilm-quantification assays, we determined that bleach in the bacteria’s environment encourages the formation of biofilms . In addition, we find that modifying the active site of the CZB domain in such a way as to make the domain “always on” increases the amount of biofilm produced by the bacteria in a fashion largely insensitive to subsequent addition of bleach . This evidence suggests that the CZB domain, known to regulate bacterial swimming patterns, is also used by bacteria to modulate the amount of biofilm that they form . This work helps us understand the biochemistry of how bacteria, particularly gut-colonizing human pathogens, behave in order to survive and thrive in their environment, possibly setting the groundwork for future therapeutic interventions .

Defining the roles of conserved DNA repair complexes in maintenance of C. elegans meiotic genome integrity

Presenter(s): Alina Salagean—Biology

Faculty Mentor(s): Erik Toraason, Diana Libuda

Session 3: The Substance of Us

Most organisms utilize meiosis, a specialized form of cell division, to produce reproductive cells such as sperm and eggs . Failure to maintain genomic integrity during meiosis can result in serious diseases, including infertility and cancer . The Structural Maintenance of Chromosomes 5/6 complex (SMC-5/6), its E3 SUMO ligase subunit NSE-2, and the BRCA1/BARD1 heterodimer are conserved protein complexes implicated in ensuring accurate meiotic DNA repair and are known to genetically interact . However, the specific mechanisms by which these proteins interact to preserve genome integrity is unknown . To determine the NSE-2 specific and NSE-2 independent meiotic functions of the SMC-5/6 complex in meiotic DSB repair, we utilized immunofluorescence imaging and a mortal germline phenotype assay to assess smc-5 and nse-2 C . elegans mutants . Our findings suggest a separation of function within the SMC-5/6 complex, which performs NSE-2 dependent functions promoting efficient meiotic DSB repair and NSE-2 independent functions in preservation of germline immortality . Finally, to define epistatic relationships between BRC-1/BRD-1, SMC-5/6, and NSE-2 in DNA repair, we assessed the germline sensitivity to exogenous DNA damage by scoring the brood viability of pairwise brc-1, smc-5, and nse-2 double mutants . These data reveal that exogenous DNA damage repair is differentially regulated within meiotic prophase I and implicate SMC-5/6 as a central regulator of both NSE-2 and BRC-1 dependent DSB repair . Taken together, our research defines fundamental genetic mechanisms and interactions preserving genomic integrity .

Glomerular Signals Underlying Olfactory Navigation

Presenter(s): Nelly Nouboussi—Biology

Faculty Mentor(s): Matt Smear, Amanda Welch

Session 3: The Substance of Us

The olfactory system is the least studied sense although it is very important for our existence . Our lab has examined the behavioral structure of olfactory navigation . Our next goal is to compare sampling movements directly against sensory input in order to establish a correlation between neural activity and behavior . The first step in this goal, which is the topic of my thesis, is to successfully express fluorescence indicators in the olfactory bulb and to detect this expression using our imaging apparatus . We are focusing specifically in the glomeruli, which contains the neurons responsible for converting odor information into action potentials . To achieve the expression of our fluorescence sensor GCaMP, we either injected a virus encoding the fluorescence protein into mice brains or engineered mice to encode the sensor gene in their genome . We worked with three mice strains:
B6 mice which can express GCaMP anywhere, Tbet-Cre mice which can express the virus only in the mitral layer and Tbet-Cre-Ai148D mice which contain the GCaMP gene in their genome . Histology revealed that we successfully expressed GCaMP in B6 mice, but we could only observe background fluorescence in Tbet-Cre and Tbet-Cre-Ai148D mice . This could result from the frying of the bulb due to continuous expression of the protein or the degradation of the virus . Despite the difficulty of the surgeries, we could visualize activity in the glomeruli of live mice with the two-photon microscope, although our success rate remains low . We are continuously adjusting our protocol to improve our techniques, so we can move on to the next stage of our project .

Differences in Old and Young Patient-Derived Myotubes Response to Amino Acid Stimulation

Presenter(s): Jeanette Helgerson—Human Physiology

Faculty Mentor(s): Hans Dreyer, Doug Foote

Session 3: The Substance of Us

Human muscle cell growth is regulated through the protein complex mTOR, the mammalian target of rapamycin, which is activated by nutrients such as amino acids and growth factors such as insulin . We wanted to know if there were differences in how old and young patient derived muscle cells responded to amino acids, particularly how the mTOR cascade was affected . The mTOR response to amino acids has been studied in many types of cells, but this study’s approach using patient derived human skeletal muscle cells to analyze mTOR response has yet to be explored . Using purified samples of these patient derived cells, a mixture of amino acids (LRK) that are known to activate mTOR were given, and the cellular signaling of certain proteins were quantified to measure mTOR activation . Both young and old patient derived cells were given these conditions and the averages of the groups were compared . This research is currently ongoing, so not all of the data is available yet . Knowing the differences in how young and old patient derived cells respond to amino acids is important because it could help alleviate muscle loss . Muscle loss shortens lifespans and diminishes the quality of life for many people, so understanding the underlying mechanisms of how to gain muscle back after the aging process, injury, or surgery, is imperative .

Development of a new live imaging technique to uncover the mechanisms of heat- induced male infertility

Presenter(s): Cailan Feingold—Biology

Faculty Mentor(s): Diana Libuda, Cori Cahoon

Session 3: The Substance of Us

Male infertility affects approximately one-third of couples who are unable to conceive . Exposing mammalian spermatogenesis to elevated temperatures causes 40% of primary male infertility cases; however, the mechanisms behind this heat-induced male-specific infertility are largely unknown . Similar to mammals, Caenorhabditis elegans also display heat-induced sperm-specific infertility . Following heat-stress, C . elegans spermatocytes have increases in DNA damage that correlate with a premature loss of chromosome structures essential for meiotic chromosome segregation . Using live imaging, I will examine the dynamic relationship between this heat-induced DNA-damage and disassembly of meiotic chromosome structures in spermatocytes . To circumvent immobilization issues with existing current live imaging techniques, I am developing and implementing a new conditional, immobilization method for live imaging fluorescently tagged proteins in both sexes of intact worms . This novel method utilizes the auxin-inducible degron system, which targets degradation of degron tagged proteins in the presence of auxin, and thus can be used to specifically degrade genes that cause severe paralysis . Based on the gene location and predicted function, I selected three genes to degron tag (unc-104, unc-52, unc-18) . Using fertility assays, I confirmed that loss of these three gene products does not interfere with meiosis or fertility . Overall, this novel live imaging system will allow for conditional paralysis of living worms during live imaging experiments, enabling us to examine the dynamics of the heat-induced defects during spermatogenesis .