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- All Subjects: Microarray
- Creators: Chang, Yung
- Creators: Fu, Jinglin
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
Enzymes which regulate the metabolic reactions for sustaining all living things, are the engines of life. The discovery of molecules that are able to control enzyme activity is of great interest for therapeutics and the biocatalysis industry. Peptides are promising enzyme modulators due to their large chemical diversity and the existence of well-established methods for library synthesis. Microarrays represent a powerful tool for screening thousands of molecules, on a small chip, for candidates that interact with enzymes and modulate their functions. In this work, a method is presented for screening high-density arrays to discover peptides that bind and modulate enzyme activity. A viscous polyvinyl alcohol (PVA) solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide feature. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase (HRP), alkaline phosphatase (APase) and â-galactosidase (â-Gal) and substantially alter their activities by comparing the peptide-enzyme binding levels and bound enzyme activity on microarrays. Several peptides, selected from microarrays, were able to inhibit â-Gal in solution, which demonstrates that behaviors selected from surfaces often transfer to solution. A mechanistic study of inhibition revealed that some of the selected peptides inhibited enzyme activity by binding to enzymes and inducing aggregation. PVA-coated peptide slides can be rapidly analyzed, given an appropriate enzyme assay, and they may also be assayed under various conditions (such as temperature, pH and solvent). I have developed a general method to discover molecules that modulate enzyme activity at desired conditions. As demonstrations, some peptides were able to promote the thermal stability of bound enzyme, which were selected by performing the microarray-based enzyme assay at high temperature. For broad applications, selected peptide ligands were used to immobilize enzymes on solid surfaces. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activities and stabilities. Peptide-modified surfaces may prove useful for immobilizing enzymes on surfaces with optimized orientation, location and performance, which are of great interest to the biocatalysis industry.
ContributorsFu, Jinglin (Author) / Woodbury, Neal W (Thesis advisor) / Johnston, Stephen A. (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010
Description
Breast cancer is the most common cancer and currently the second leading cause of death among women in the United States. Patients’ five-year relative survival rate decreases from 99% to 25% when breast cancer is diagnosed late. Immune checkpoint blockage has shown to be a promising therapy to improve patients’ outcome in many other cancers. However, due to the lack of early diagnosis, the treatment is normally given in the later stages. An early diagnosis system for breast cancer could potentially revolutionize current treatment strategies, improve patients’ outcomes and even eradicate the disease. The current breast cancer diagnostic methods cannot meet this demand. A simple, effective, noninvasive and inexpensive early diagnostic technology is needed. Immunosignature technology leverages the power of the immune system to find cancer early. Antibodies targeting tumor antigens in the blood are probed on a high-throughput random peptide array and generate a specific binding pattern called the immunosignature.
In this dissertation, I propose a scenario for using immunosignature technology to detect breast cancer early and to implement an early treatment strategy by using the PD-L1 immune checkpoint inhibitor. I develop a methodology to describe the early diagnosis and treatment of breast cancer in a FVB/N neuN breast cancer mouse model. By comparing FVB/N neuN transgenic mice and age-matched wild type controls, I have found and validated specific immunosignatures at multiple time points before tumors are palpable. Immunosignatures change along with tumor development. Using a late-stage immunosignature to predict early samples, or vice versa, cannot achieve high prediction performance. By using the immunosignature of early breast cancer, I show that at the time of diagnosis, early treatment with the checkpoint blockade, anti-PD-L1, inhibits tumor growth in FVB/N neuN transgenic mouse model. The mRNA analysis of the PD-L1 level in mice mammary glands suggests that it is more effective to have treatment early.
Novel discoveries are changing understanding of breast cancer and improving strategies in clinical treatment. Researchers and healthcare professionals are actively working in the early diagnosis and early treatment fields. This dissertation provides a step along the road for better diagnosis and treatment of breast cancer.
In this dissertation, I propose a scenario for using immunosignature technology to detect breast cancer early and to implement an early treatment strategy by using the PD-L1 immune checkpoint inhibitor. I develop a methodology to describe the early diagnosis and treatment of breast cancer in a FVB/N neuN breast cancer mouse model. By comparing FVB/N neuN transgenic mice and age-matched wild type controls, I have found and validated specific immunosignatures at multiple time points before tumors are palpable. Immunosignatures change along with tumor development. Using a late-stage immunosignature to predict early samples, or vice versa, cannot achieve high prediction performance. By using the immunosignature of early breast cancer, I show that at the time of diagnosis, early treatment with the checkpoint blockade, anti-PD-L1, inhibits tumor growth in FVB/N neuN transgenic mouse model. The mRNA analysis of the PD-L1 level in mice mammary glands suggests that it is more effective to have treatment early.
Novel discoveries are changing understanding of breast cancer and improving strategies in clinical treatment. Researchers and healthcare professionals are actively working in the early diagnosis and early treatment fields. This dissertation provides a step along the road for better diagnosis and treatment of breast cancer.
ContributorsDuan, Hu (Author) / Johnston, Stephen Albert (Thesis advisor) / Hartwell, Leland Harrison (Committee member) / Dinu, Valentin (Committee member) / Chang, Yung (Committee member) / Arizona State University (Publisher)
Created2015