• Biophysical characterization of class II major histocompatibility complex (MHCII) molecules

      Osan, Jaspreet Kaur; Ferrante, Andrea; Kuhn, Thomas; Podlutsky, Andrej; Chen, Jack (2020-05)
      Class II Major Histocompatibility Complex (MHCII) molecules are transmembrane glycoproteins expressed on the surface of antigen-presenting cells (APCs). APCs engulf pathogens and digest pathogenic proteins into peptides, which are loaded onto MHCII in the MHCII compartment (MIIC) to form peptide-MHCII complexes (pMHCII). These pMHCII are then presented to CD4+ T cells on the surface of APCs to trigger an antigen-specific immune response against the pathogens. HLA-DM (DM), a non-classical MHCII molecule, plays an essential role in generating kinetically stable pMHCII complexes which are presented to CD4+ T cells. When a few peptides among the pool of the peptide repertoire can generate the efficient CD4+ T cell response, such peptides are known as immunodominant. The selection of immunodominant epitopes is essential to generate effective vaccines against pathogens. The mechanism behind immunodominant epitope selection is not clearly understood. My work is focused on investigating various factors that help in the selection of immunodominant epitopes. For this purpose, peptides derived from H1N1 influenza hemagglutinin protein with known CD4+ T cell responses have been used. We investigated the role of DM-associated binding affinity in the selection of immunodominant epitopes. Our analysis showed that the presence of DM significantly reduces the binding affinity of the peptides with low CD4+ T cell response and inclusion of DM-associated IC50 in training MHCII algorithms may improve the binding prediction. Previous studies have shown that there is an alternate antigen presentation depending on antigen protein properties. Here, we showed that the immunodominant epitope presentation is dependent on the pH and length of the peptides. To study the MHCII in its native form, we assembled full-length MHCII in a known synthetic membrane model known as nanodiscs. We noted that, based on the lipid composition, assembly of the MHCII differs. Preliminary binding studies with this tool showed that there might be a difference in the binding based on the type of the nanodisc. Collectively, our results showed that the immunodominant epitope selection is a complex process that is driven by various biochemical features.
    • Studies assessing insulin signaling dependent neuronal morphology and novel animal sorting methods in a C. elegans model

      Hunter, Skyler C.; Bult-Ito, Abel; Taylor, Barbara; Podlutsky, Andrej; Vayndorf, Elena (2018-12)
      The purpose of this work is two-part. The primary goal of this thesis is to identify a list of significant target insulin-like peptides (ILPs) that influence the maintenance of neuronal morphology in an aged animal model of Caenorhabditis elegans (C. elegans) and determine whether or not morphological changes have bearing on neuronal function. The second goal is to address and devise a solution for a common laboratory difficulty encountered within the research community, difficulty maintaining large age-synchronous populations of the model organism, C. elegans. Chapter 1 discusses the importance of insulin signaling and how it pertains to the morphology of aging neurons. A reverse genetic screen was conducted to knockdown the expression of individual ILPs in a C. elegans model. The results identify a subfamily of ILPs that play significant roles in maintaining regular morphology of aging mechanosensory neurons. These data corroborate previously published work demonstrating that aberrant morphology of mechanosensory neurons does not directly influence their function and that these two parameters, morphology and function, can be uncoupled and considered mutually exclusive. Chapter 2 describes a main difficulty associated with using C. elegans as a model organism; the problem of maintaining a large age-synchronous population on solid media. To address this difficulty a novel piece of equipment, named the Caenorhabditis Sieve, and an accompanying methodology for its application, were created to mechanically sort and clean C. elegans. The use of this new device facilitates the implementation of assays with animals cultivated on solid media that are normally cost and resource prohibitive. Presented with the protocol for device construction and implementation, are standard experiments that were conducted to verify "proof of concept" of the tool's efficacy. The results demonstrate that the Caenorhabditis Sieve effectively transfers animals from one culture plate to the next in a manner that does not influence common markers of physiological stress; thus validating the sieve's use in future experiments among the research community, as well as highlighting the success of creating a cost-effective, efficient, fast, and simple process to mitigate difficulties and ease progress in research fields using small model organisms.