The role of calcium in the destruction of target cells by cytotoxic T cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Harvard-MIT Divison of Health Sciences and Technology Program in Medical Engineering and Medical Physics, 1987.Title as it appears in M.I.T. Graduate List, June 1987: The role of calcium in the destruction of target cells by cytotoxic T lymphocytes.Bibliography: leaves 224-239.by Nancy L. Allbritton.Ph.D

In situ Roughening of Polymeric Microstructures

A method to perform in-situ roughening of arrays of microstructures weakly adherent to an underlying substrate was presented. SU8, 1002F, and polydimethylsiloxane (PDMS) microstructures were roughened by polishing with a particle slurry. The roughness and the percentage of dislodged or damaged microstructures was evaluated as a function of the roughening time for both SU8 and 1002F structures. A maximal RMS roughness of 7-18 nm for the surfaces was obtained within 15 to 30 s of polishing with the slurry. This represented a 4-9 fold increase in surface roughness relative to that of the native surface. Less than 0.8% of the microstructures on the array were removed or damage after 5 min of polishing. Native and roughened arrays were assessed for their ability to support fibronectin adhesion and cell attachment and growth. The quantity of adherent fibronectin was increased on roughened arrays by two-fold over that on native arrays. Cell adhesion to the roughened surfaces was also increased compared to native surfaces. Surface roughening with the particle slurry also improved the ability to stamp molecules onto the substrate during microcontact printing. Roughening both the PDMS stamp and substrate resulted in up to a 20-fold improvement in the transfer of BSA-Alexa Fluor 647 from the stamp to the substrate. Thus roughening of micron-scale surfaces with a particle slurry increased the adhesion of biomolecules as well as cells to microstructures with little to no damage to large scale arrays of the structures

Effect of the DEF motif on phosphorylation of peptide substrates by ERK

MAP kinase ERK maintains specificity by binding to docking sites such as the DEF domain or D domain. It was previously shown that appending peptides derived from D domains to a substrate peptide increased apparent efficiency of peptide phosphorylation while preserving its apparent specificity for ERK. Here we determine the effect of the DEF motif on efficiency and specificity of peptide phosphorylation by ERK. The DEF motif modulated the apparent affinity of the peptide for ERK while the substrate motif dominated the apparent catalytic rate. Attachment of the DEF sequence improved apparent phosphorylation efficiency by 60-fold. Addition of peptides possessing both the DEF and D motif to a substrate sequence did not yield additive effects on the KM of the substrate for ERK. Further, the DEF motif diminished the apparent specificity for ERK and increased the apparent efficiencies of phosphorylation of the substrate peptide by p38α kinase and JNK1

An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells

Tools to evaluate oncogenic kinase activity in small clinical samples have the power to guide precision medicine in oncology. Existing platforms have demonstrated impressive insights into the activity of protein kinases, but these technologies are unsuitable for the study of kinase behavior in large numbers of primary human cells. To address these limitations, we developed an integrated analysis system which utilizes a light-programmable, cell-permeable reporter deliverable en masse to many cells. The reporter's ability to act as a substrate for Akt, a key oncogenic kinase, was masked by a 2-4,5-dimethoxy 2-nitrobenzyl (DMNB) moiety. Upon exposure to ultraviolet light and release of the masking moiety, the substrate sequence enabled programmable reaction times within the cell cytoplasm. When coupled to automated single-cell capillary electrophoresis, statistically significant numbers of primary human cells were readily evaluated for Akt activity

Efficient division and sampling of cell colonies using microcup arrays

A microengineered array to sample clonal colonies is described. The cells were cultured on an array of individually releasable elements until the colonies expanded to cover multiple elements. Single elements were released using a laser-based system and collected to sample cells from individual colonies. A greater than an 85% rate in splitting and collecting colonies was achieved using a 3-dimensional cup-like design or “microcup”. Surface modification using patterned titanium deposition of the glass substrate improved the stability of microcup adhesion to the glass while enabling minimization of the laser energy for splitting the colonies. Smaller microcup dimensions and slotting the microcup walls reduced the time needed for colonies to expand into multiple microcups. The stem cell colony retained on the array and the collected fraction within released microcups remained undifferentiated and viable. The colony samples were characterized by both reporter gene expression and a destructive assay (PCR) to identify target colonies. The platform is envisioned as a means to rapidly establish cell lines using a destructive assay to identify desired clones