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- All Subjects: proteomics
Description
The goal of this thesis is to test whether Alzheimer's disease (AD) is associated with distinctive humoral immune changes that can be detected in plasma and tracked across time. This is relevant because AD is the principal cause of dementia, and yet, no specific diagnostic tests are universally employed in clinical practice to predict, diagnose or monitor disease progression. In particular, I describe herein a proteomic platform developed at the Center for Innovations in Medicine (CIM) consisting of a slide with 10.000 random-sequence peptides printed on its surface, which is used as the solid phase of an immunoassay where antibodies of interest are allowed to react and subsequently detected with a labeled secondary antibody. The pattern of antibody binding to the microarray is unique for each individual animal or person. This thesis will evaluate the versatility of the microarray platform and how it can be used to detect and characterize the binding patterns of antibodies relevant to the pathophysiology of AD as well as the plasma samples of animal models of AD and elderly humans with or without dementia. My specific aims were to evaluate the emergence and stability of immunosignature in mice with cerebral amyloidosis, and characterize the immunosignature of humans with AD. Plasma samples from APPswe/PSEN1-dE9 transgenic mice were evaluated longitudinally from 2 to 15 months of age to compare the evolving immunosignature with non-transgenic control mice. Immunological variation across different time-points was assessed, with particular emphasis on time of emergence of a characteristic pattern. In addition, plasma samples from AD patients and age-matched individuals without dementia were assayed on the peptide microarray and binding patterns were compared. It is hoped that these experiments will be the basis for a larger study of the diagnostic merits of the microarray-based immunoassay in dementia clinics.
ContributorsRestrepo Jimenez, Lucas (Author) / Johnston, Stephen A. (Thesis advisor) / Chang, Yung (Committee member) / Reiman, Eric (Committee member) / Sierks, Michael (Committee member) / Arizona State University (Publisher)
Created2011
Description
Our goal was to design a method to express soluble folded major histocompatibility complex (MHC) proteins using human cell line HeLa lysate with the novel 1-Step Human In Vitro Protein Expression by Thermo Scientific in the presence of β2 microglobulin (β2m) and antigenic peptide.
We confirmed that the soluble protein MHC-A2.1 could be successfully attached to the Luminex magnetic beads and detected using the primary antibody anti-GST and the detection antibody goat mAb mouse PE. The average net MFI of the attached pA2.1-bead complex was 8182. Biotinylated A2.1 MHC complexes pre-folded with β2m and FLU M1 peptide (A2.1 monomers) were also successfully attached to Luminex magnetic beads and detected with BB7.2. The average net MFI of the detected A2.1 monmer-bead complexes was 318. The protein MHC complexes were multimerized on magnetic beads to create MHC tetramers and detected with BB7.2, PE labeled monoclonal antibody, via median fluorescent intensity with the Luminex platform. Varying protein, β2 microglobulin (β2m), and peptide concentrations were tested in a number of MHC-A2.1 protein refolding trials. Different antigenic peptides and attachment methods were also tested. However, none of the MHC-A2.1 protein folding and capture trials were successful. Although MHC-A2.1 complexes and recombinant MHC molecules could be attached to Luminex magnetic beads and be detected by Luminex arrays, soluble protein A2.1 could not be successfully expressed, refolded, captured onto Luminex beads, and detected. All refolding trials resulted in a net MFI of <25. The failed refolding and capture trials of A2.1 lead to the conclusion that human cell line HeLa lysate cannot be used to properly fold MHC molecules. However, efforts to refold the complexes onto Luminex magnetic beads are ongoing. We are also using the baculovirus expression system to refold soluble A2.1 lysate onto peptide-bead complexes.
We confirmed that the soluble protein MHC-A2.1 could be successfully attached to the Luminex magnetic beads and detected using the primary antibody anti-GST and the detection antibody goat mAb mouse PE. The average net MFI of the attached pA2.1-bead complex was 8182. Biotinylated A2.1 MHC complexes pre-folded with β2m and FLU M1 peptide (A2.1 monomers) were also successfully attached to Luminex magnetic beads and detected with BB7.2. The average net MFI of the detected A2.1 monmer-bead complexes was 318. The protein MHC complexes were multimerized on magnetic beads to create MHC tetramers and detected with BB7.2, PE labeled monoclonal antibody, via median fluorescent intensity with the Luminex platform. Varying protein, β2 microglobulin (β2m), and peptide concentrations were tested in a number of MHC-A2.1 protein refolding trials. Different antigenic peptides and attachment methods were also tested. However, none of the MHC-A2.1 protein folding and capture trials were successful. Although MHC-A2.1 complexes and recombinant MHC molecules could be attached to Luminex magnetic beads and be detected by Luminex arrays, soluble protein A2.1 could not be successfully expressed, refolded, captured onto Luminex beads, and detected. All refolding trials resulted in a net MFI of <25. The failed refolding and capture trials of A2.1 lead to the conclusion that human cell line HeLa lysate cannot be used to properly fold MHC molecules. However, efforts to refold the complexes onto Luminex magnetic beads are ongoing. We are also using the baculovirus expression system to refold soluble A2.1 lysate onto peptide-bead complexes.
ContributorsChang, Peter S (Author) / Anderson, Karen (Thesis director) / Chang, Yung (Committee member) / Sundaresan, Krishna (Committee member) / Barrett, The Honors College (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
Description
While the entire human genome has been sequenced, the understanding of its functional elements remains unclear. The Encyclopedia of DNA Elements (ENCODE) project analyzed 1% of the human genome and found that the majority of the human genome is transcribed, including non-protein coding regions. The hypothesis is that some of the "non-coding" sequences are translated into peptides and small proteins. Using mass spectrometry numerous peptides derived from the ENCODE transcriptome were identified. Peptides and small proteins were also found from non-coding regions of the 1% of the human genome that the ENCODE did not find transcripts for. A large portion of these peptides mapped to the intronic regions of known genes, thus it is suspected that they may be undiscovered exons present in alternative spliceoforms of certain genes. Further studies proved the existence of polyadenylated RNAs coding for these peptides. Although their functional significance has not been determined, I anticipate the findings will lead to the discovery of new splice variants of known genes and possibly new transcriptional and translational mechanisms.
ContributorsWang, Lulu (Author) / Lake, Douglas (Thesis advisor) / Chang, Yung (Committee member) / Touchman, Jeffery (Committee member) / Arizona State University (Publisher)
Created2010