Biotechnologies for cancer diagnostics : cell sorting, protein analysis and imaging of cellular metabolism

This thesis presents the development of chip-based technology for informative in vitro cancer diagnostics. In the first part of this thesis, I will present my contribution in the development of a technology called “Nucleic Acid Cell Sorting (NACS)”, based on microarrays composed of nucleic acid encoded peptide major histocompatibility complexes (p/MHC), and the experimental and theoretical methods to detect and analyze secreted proteins from single or few cells.

Secondly, a novel portable platform for imaging of cellular metabolism with radio probes is presented. A microfluidic chip, so called “Radiopharmaceutical Imaging Chip” (RIMChip), combined with a beta-particle imaging camera, is developed to visualize the uptake of radio probes in a small number of cells. Due to its sophisticated design, RIMChip allows robust and user-friendly execution of sensitive and quantitative radio assays. The performance of this platform is validated with adherent and suspension cancer cell lines. This platform is then applied to study the metabolic response of cancer cells under the treatment of drugs. Both cases of mouse lymphoma and human glioblastoma cell lines, the metabolic responses to the drug exposures are observed within a short time (~ 1 hour), and are correlated with the arrest of cell-cycle, or with changes in receptor tyrosine kinase signaling.

The last parts of this thesis present summaries of ongoing projects: development of a new agent as an in vivo imaging probe for c-MET, and quantitative monitoring of glycolytic metabolism of primary glioblastoma cells. To develop a new agent for c-MET imaging, the one-bead-one-compound combinatorial library method is used, coupled with iterative screening. The performance of the agent is quantitatively validated with cell-based fluorescent assays. In the case of monitoring the metabolism of primary glioblastoma cell, by RIMChip, cells were sorting according to their expression levels of oncoprotein, or were treated with different kinds of drugs to study the metabolic heterogeneity of cancer cells or metabolic response of glioblastoma cells to drug treatments, respectively.

An in vivo approach to tRNA identity

A leucine-inserting tRNA has been transformed into a serine-inserting tRNA by changing 12 nucleotides. Only 8 of the 12 changes are required to effect the conversion of the leucine tRNA to serine tRNA identity. The 8 essential changes reside in basepair 11-24 in the D stem, basepairs 3-70, 2-71 and nucleotides 72 and 73, all of the acceptor stem.

Functional amber suppressor tRNA genes were generated for 14 species of tRNA in E. coli, and their amino acid specificities determined. The suppressors can be classified into three groups, based upon their specificities. Class I suppressors, tRNA^(Ala2)_(CUA), tRNA^(GlyU)_(CUA), tRNA^(HisA)_(CUA), tRNA^(Lys)_(CUA), and tRNA^(ProH)_(CUA), inserted the predicted amino acid. The Class II suppressors, tRNA^(GluA)_(CUA) , tRNA^(GlyT)_(CUA), and tRNA^(Ile1)_(CUA) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase (AAS). The Class III suppressors, tRNA^(Arg)_(CUA), tRNA^(AspM)_(CUA), tRNA^(Ile2)_(CUA), tRNA^(Thr2)_(CUA), tRNA^(Met(m))_(CUA) and tRNA^(Val)_(CUA) inserted predominantly lysine.

On an embedding property of generalized Carter subgroups

If <i>Ei> and <i>Fi> are saturated formations, we say that <i>Ei> is strongly contained in <i>Fi> if for any solvable group G with <i>Ei>-subgroup, E, and <i>Fi>-subgroup, F, some conjugate of E is contained in F. In this paper, we investigate the problem of finding the formations which strongly contain a fixed saturated formation <i>Ei>.

Our main results are restricted to formations, <i>Ei>, such that <i>Ei> = {G|G/F(G) ϵ<i>Ti>}, where <i>Ti> is a non-empty formation of solvable groups, and F(G) is the Fitting subgroup of G. If <i>Ti> consists only of the identity, then <i>Ei>=<i>Ni>, the class of nilpotent groups, and for any solvable group, G, the <i>Ni>-subgroups of G are the Carter subgroups of G.

We give a characterization of strong containment which depends only on the formations <i>Ei>, and <i>Fi>. From this characterization, we prove:

If <i>Ti> is a non-empty formation of solvable groups, <i>Ei> = {G|G/F(G) ϵ<i>Ti>}, and <i>Ei> is strongly contained in <i>Fi>, then

(1) there is a formation <i>Vi> such that <i>Fi> = {G|G/F(G) ϵ<i>Vi>}.

(2) If for each prime p, we assume that <i>Ti> does not contain the class, <i>Si>_p’, of all solvable p’-groups, then either <i>Ei> = <i>Fi>, or <i>Fi> contains all solvable groups.

This solves the problem for the Carter subgroups.

We prove the following result to show that the hypothesis of (2) is not redundant:

If <i>Ri> = {G|G/F(G) ϵ<i>Si>_r’}, then there are infinitely many formations which strongly contain <i>Ri>.