Tag: Biochemistry

Determining the role of the pulvinar in visual attentional control

Presenter(s): Emmalyn Leonard—Biochemistry

Faculty Mentor(s): Cristopher Niell, Philip Parker

Session 5: The Wonders of the Brain

Visual attentional control is a behavior that is critical for survival; despite its importance, the specific neural mechanisms underlying the process remain unclear . Upon perception, visual information is routed from the retina through the thalamus, which relays signals to the cortex for further processing . The pulvinar, a nucleus of the thalamus, has strong connections to both visual cortex (V1) and areas involved with attentional control, such as the superior colliculus and prefrontal cortex . The pulvinar has been implicated in attentional control from studies of human patients, as pulvinar lesions are correlated with an inability to ignore distracting visual information during performance of a behavioral task . Studies have also shown that mice are capable of learning similar tasks; given that their visual system is highly analogous to that of humans, mice serve as an optimal model for important behaviors such as visual attentional control . We targeted mouse pulvinar neurons with a GCaMP-expressing virus to allow measurement of brain activity through a cranial window . Utilizing both widefield and two-photon microscopic imaging, we found that axons projecting from the pulvinar to V1 are visually responsive and appear to be organized in a retinotopic manner . Future work on this project will include introduction of a visually guided behavioral task alongside silencing of pulvinar neurons using a DREADDs-expressing virus . We expect to find that, when mouse pulvinar neurons are silenced, important signals for visual attention sent from the thalamus to V1 will be interrupted, resulting in poor performance of a task requiring visual attentional control .

Characterizing the Conformational Fluctuations of DNA Under Physiological and Salt-Stabilized Conditions

Presenter(s): Anabel Chang—Biochemistry

Co-Presenter(s): Maya Pande

Faculty Mentor(s): Andrew Marcus

Session: Prerecorded Poster Presentation

The Marcus Group conducts studies on the dynamics of macromolecules in biological environments . In our experiments, we used a variety of techniques to analyze the structure of DNA with the
overall goal of better understanding the conformations it can take . Our studies were focused in two areas: (1) understanding the mechanisms of DNA breathing, and (2) conducting experiments on the stabilizing and destabilizing properties of salt solutions on DNA . Techniques included circular and linear dichroism, UV-Vis spectroscopy, and Förster Resonance Energy Transfer (FRET) . Determining the structure of DNA is crucial to understanding biochemical and molecular events essential for gene expression and DNA replication . For these processes to occur, various proteins must access single- stranded DNA coding templates which are otherwise inaccessible due to complementary base pairing in double-stranded DNA . Proteins rely on thermal fluctuations in the DNA double-stranded region at physiological temperatures known as DNA ‘breathing .’ Studies are ongoing, but thus far have led us to a better understanding of the energetic favorability of various conformations of DNA .

 

Determining detergent dependence of Cytolysin A oligomeric state through native mass spectrometry

Presenter(s): Lejla Biberic—Biochemistry

Faculty Mentor(s): Amber Rolland, James Prell

Session: Prerecorded Poster Presentation

Membrane proteins, including pore-forming toxins (PFTs), play important roles in human health . PFTs are promising for nanopore sequencing and drug delivery, but to maximize success in these applications, it is important to know the size of the pore and thus the oligomeric state (number of identical subunits) . The flexibility of alpha-PFT transmembrane helices may allow their oligomeric state to vary in different environments . Elucidating the relationship between oligomeric state and detergent environment is thus important for PFT bionanotechnology applications . Here, we studied how native oligomeric states of Cytolysin A (ClyA), an alpha-PFT found in pathogenic strains of Escherichia coli, varied in different detergent environments using native mass spectrometry (MS) . Native MS enables preservation of noncovalent complexes and accurate measurement of the complex mass . Together with a known monomer mass, this allows unambiguous determination of oligomeric state . ClyA was incubated with various detergents and screened for complex formation using Blue Native PAGE . Preliminary native MS results show that ClyA forms various oligomeric complexes ranging from octameric to dodecameric in n-dodecylÎ2-D-maltoside and octaethylene glycol monododecyl ether. ClyA forms no identifiable pore complexes in n-octyl-Î2-D-glucoside, in contrast to previous reports, while n-tetradecylphosphocholine heavily adducts to and stabilizes ClyA monomers only . Combining these experimental results with computational modeling enables further investigation into the relationship between detergent properties and oligomeric state . These findings will not only advance the fields of MS and structural biology but also provide new insight for PFT applications in bionanotechnology through manipulation of desired oligomeric state and pore size .

Influence of a prion protein on the TOR pathway in Saccharomyces Cerevisiae

Presenter(s): Zack Basham—Biochemistry

Faculty Mentor(s): David Garcia

Session: Prerecorded Poster Presentation

Pseudouridine synthases are critical RNA modifiers in eukaryotes . One member of this family of enzymes, encoded by the Pus4 gene in the budding yeast Saccharomyces cerevisiae, forms a prion protein, named [BIG+] . Rather than resulting in cell death, as for known mammalian prion diseases, [BIG+] promotes increased cell proliferation and cell size . These observations raise the question of how the prion promotes cell growth . The increased cell size and growth rate suggests an alteration to a fundamental eukaryotic growth control pathway, mediated by the TOR complex (“target of rapamycin”) . One target of TOR, a protein kinase, is Sch9, an AGC kinase, which is activated via phosphorylation by the TOR complex . Sch9 is involved in multiple processes essential for growth such as ribosome biogenesis, translation control, and cAPK activity . To better understand the relationship between [BIG+] and TOR, we have introduced hyperactive mutants of TOR or Sch9 into [BIG+] and naive cells . By monitoring growth rate in media with varying levels of arginine, we can monitor [BIG+] response to different nutrient conditions . We have also made progress in monitoring the expression of Sch9 in [BIG+] cells compared to cells without the prion . This contributes to our understanding of how the prion and TOR complex are interacting to affect cell growth .