Tunable Microchips for Imaging Protein Structures formed in Breast Cancer Cells

dc.contributor.authorAlden, Nicholas Andrewen
dc.contributor.committeechairKelly, Deborah F.en
dc.contributor.committeememberBehkam, Baharehen
dc.contributor.committeememberNain, Amrinderen
dc.contributor.departmentMechanical Engineeringen
dc.description.abstractThe breast cancer susceptibility protein, BRCA1, is a tumor suppressor that helps maintain genomic integrity. Changes in BRCA1 that effect DNA repair processes can fuel cancer induction. The Kelly lab, at the Virginia Tech Carilion Research Institute, has recently developed a new methodology that employs silicon nitride (SiN) microchips to isolate BRCA1 assemblies from the nuclear material of breast cancer cells. These microchips are coated with adaptor proteins that include antibodies against target proteins of interest. The adaptor proteins are added in sequential steps to the coated microchips, followed by an aliquot of sample containing the protein of interest, such as BRCA1. The Kelly lab, partnered with Protochips Inc., developed these devices as a robust, tunable platform to monitor molecular processes, and refer to them as 'Cryo-SiN' in cryo-Electron Microscopy (EM) imaging. We are currently using Cryo-SiN to recruit BRCA1 protein assemblies to the microchip surface under mild conditions, while simultaneously preparing them for cryogenic preservation and EM imaging. This strategy presents a viable alternative to antibody affinity columns that require stringent elution steps to obtain protein complexes from the column. Another advantage of the microchip strategy is that it requires only a 30-minute nuclear extraction, a 60-minute enrichment procedure, and a 5-minute microchip capture step--a total of 95 minutes from initially lysing the cells to plunge-freezing the EM specimens. Therefore, these novel approaches represent a major departure from classical separation procedures that often require days to complete, during which time active protein assemblies can readily dissociate or become inactive. Overall, our use of BRCA1-specific microchips may reveal changes in the BRCA1 architecture during various stages of cancer progression--a major gap in knowledge that persists in cancer research.en
dc.description.degreeM. S.en
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.subjectCryo-Electron Microscopyen
dc.subjectTransmission Electron Microscopyen
dc.subjectSilicon Nitrideen
dc.subjectSilicon Nitride Microchipsen
dc.subjectMolecular Imaging Strategyen
dc.subjectProtein Enrichmenten
dc.titleTunable Microchips for Imaging Protein Structures formed in Breast Cancer Cellsen
thesis.degree.disciplineMechanical Engineeringen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.nameM. S.en
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