Thesis (PH. D.)--Michigan State University. Chemistry, 2008. / Title from PDF t.p. (viewed on Sept. 2, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--Carleton University, 1993. / Also available in electronic format on the Internet.

Thesis (Ph. D. in Chemical Engineering)--Vanderbilt University, Aug. 2005. / Title from title screen. Includes bibliographical references.

No description available.

It has recently been established that the activity of crystalline muscle phosphoglucomutase can be greatly stimulated by preincubation of the enzyme with a Mg++-imidazole complex. This observation has aroused interest in the physiological significance of such a system in the possible cellular control of phosphoglucomutase activity. The present study constitutes, in part, an investigation of the properties of phosphoglucomutase from brain tissue. A procedure for the purification of phosphoglucomutase from beef brain is described. The brain enzyme appears to be similar to that from skeletal muscle. Evidence is also presented which indicates that the "activation" produced by Mg++-imidazole is probably of no physiological importance in brain. This observation is consistent with the more recent reports that the phosphoglucomutase reaction is likely not involved in cellular regulatory mechanisms. It is well established that phosphofructokinase is intimately involved in the cellular regulation of glycolysis and the citric acid cycle. Control mechanisms of the phosphofructokinase reaction in mammalian tissues have been postulated on the basis of the complex kinetics of the enzyme. In yeast, however, two enzymatically interconvertible forms of the enzyme have been reported. Preliminary experiments in this study failed to demonstrate a phosphofructokinase system in brain similar to that found in yeast. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

In this dissertation, the emerging stimulated Raman scattering (SRS) microscopy in combination with various vibrational tags was extensively used to explore various aspects of biological systems. New techniques as well as new Raman active materials were also developed to facilitate the applications of SRS in biology. Chapter one introduces and comprehensively reviews vibrational tags that have been developed to date in combination with imaging techniques and their applications in biological sciences to investigate metabolism in living organisms. Chapter two studies lipotoxicity, a phenomenon that is well known but poorly understood. The study found phase separation can form on ER membrane in cells treated with long chain fatty acids due to the high transition temptation of their metabolites. It was also found that the phase separation severely disturbs normal distribution of ER membrane proteins because of hydrophobic mismatching. As the result, ER normal structure is disrupted, luminal space is collapsed, and interconnectivity of ER that ensures normal ER functions is lost. Additionally, ER stress sensor IRE1α was found to be activated directly by the formation of phase separation, which triggers apoptosis and ultimately leads to cell death. Chapter three describes the development of a new method termed as metabolic activity phenotyping (MAP) that acquires quantitative measurements of metabolic activities of individual cells, which is essential to understanding questions in diverse fields in biology. To achieve the goal, an automatic system was designed and built that improves the acquisition speed by more than 100 times compared to commercially available instruments. A set of vibrational probes with deuterium labeling was also carefully selected to enable accurate measurement of metabolic flux. Combining the merits of high throughput measurements and vibrational tags, MAP was applied to investigate the metabolic activity differences among various cancer cells, to study the heterogeneity of drug efficacy, and to facilitate breast cancer subtyping. Chapter four describes the development and application of a new class of Raman active nanoparticles, or Rdots. These Rdots were generated by non-covalently incorporating small molecule Raman probe into polymeric nanoparticles. The resulted Rdots are of compact size (~20 nm) and preserve all Raman spectral features of the small molecule probes used. Rdots were compared to other existing Raman active materials including SERS nanoparticles, and Rdots surpass all the other materials in terms of brightness. In addition, Rdots also possess narrow spectral linewidth (< 3 nm), making them ideal for multiplexed imaging. In the study, Rdots were used as immunostaining reporters to visualize cytoskeleton networks and surface markers in cell and tissue samples.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The clinical efficacy of adoptive T cell therapies including CAR T therapy are limited by poor in vivo persistence and moderate anti-tumor efficacy. According to the literature, metabolism plays a critical role in the phenotypic state and fate of T cells during antigen-driven expansion. During different stages of a T cell life cycle, the predominant pathway used for metabolism changes. Naïve T rely on oxidative phosphorylation, but as the T cells becomes activated, their metabolic profile switches to become more reliant on glycolysis. Most T cells become terminally differentiated and become senescent once they have performed their cytotoxic function. A minority of the activated T cells gradually start to rely on oxidative phosphorylation once again and become memory T cells. Memory T cells can become either effector memory or central memory T cells. These memory T cells, specifically central memory T cells, are the key to T cells persistence during both ex vivo and in vivo expansion and following disappearance of the antigenic stimulus. Since the metabolic profile of the T cells plays a critical role in its differentiation state, we tested the hypothesis that inhibitors of intermediary metabolism could promote a metabolic profile that is more desirable for the optimal phenotype consistent with the memory phenotype that would favor persistence in spite of strong activation signals. The four inhibitors screened were: a PFKFB3 inhibitor, an inhibitor of a key step in glycolysis; ibrutinib, an inhibitor of Bruton’s tyrosine kinase; idelalisib, an inhibitor of PI3K subunit; and duvelisib, an inhibitor of PI3K and PI3K gamma subunits. To test this hypothesis, T cells were cultured with or without each compound and then the analysis included: phenotypic analysis by flow cytometry, quantitative analysis by counting cells with ethidium bromide acridine orange, and metabolic profiling by the Seahorse assay. This study was conducted using T cells from a human healthy volunteer that were collected by apheresis. T cells were cultured in a G-Rex plate for 15 days with complete media supplemented with recombinant human IL-2 (30 U/mL). Cells were activated on day 1 and day 8 by the addition of anti-CD3/CD28 beads and test metabolic inhibitor compounds were added every 4 days. T cells cultured with idelalisib, duvelisib, and ibrutinib had increased expansion (approximately50-fold: idelalisib/ duvelisib and 21-fold ibrutinib) when compared to control (cells with beads alone) with only 6-fold expansion. Phenotypic analysis performed using flow cytometry showed an increased percentage of CD27+ CD28+ in the CD8+ and CD4+ T cell cell populations in the idelalisib treated group and decreased number of senescent T cells that are double negative for CD27 and CD28. Consistent with our hypothesis, metabolic analysis showed that cells treated with idelalisib and duvelisib were more reliant on oxidative phosphorylation, rather than glycolysis as compared to the control cultures. Cells treated with duvelisib also showed an increased spare respiratory capacity (SRC), which is associated with more efficacious memory T cells. The results of these studies show that metabolism plays a critical role in the long-term survival of T cells. We demonstrate that inhibiting intermediary metabolism, specifically inhibiting PI3K, favorably alters the metabolic state of the T cells leading to increased cell numbers and T cells with a phenotype consistent with enhanced ex vivo and in vivo proliferation and persistence.

No description available.

Src Homology 3 (SH3) domains are small protein- protein interaction domains that bind to proline-rich peptides, mediating a range of important biological processes. Because the deregulation of events involving SH3 domains forms the basis of many human diseases, the SH3 domains are appealing targets for the development of potential therapeutics. Previously in the field, no examples of entirely small-molecule ligands for the SH3 domains have been identified. However, in our research group, we have discovered a class of heterocyclic compounds that bind to the Tec SH3 domain at conserved residues in the proline-rich peptide binding site, with weak to moderate affinity. The highest affinity of these was 2- aminoquinoline (Kd = 125 mM). In this thesis, a range of approaches are described, that were intended to contribute towards development of higher affinity small-molecule ligands for the Tec SH3 domain. Preliminary experiments, involving testing a variety of compounds structurally related to 2- aminoquinoline, provided new structure activity information, and led to a better understanding of the 2-aminoquinoline/SH3 domain binding event. The major component of this thesis is a thorough investigation into the synthesis of a range of 2- aminoquinoline derivatives. N-Substituted- 2-aminoquinolines were synthesised, however these compounds bound the SH3 domain with slightly lower affinity than 2-aminoquinoline. 6- Substituted-2-aminoquinolines were subsequently prepared, and ligands were identified with up to six-fold improved affinity relative to 2-aminoquinoline, and enhanced selectivity for the Tec SH3 domain. The techniques used for the ligand binding studies were Nuclear Magnetic Resonance (NMR) chemical shift perturbation and Fluorescence Polarisation (FP) peptide displacement assays. As part of the ligand binding studies, it was intended that the 3D tructure of a 2- aminoquinoline ligand/SH3 complex would be obtained using NMR methods, provided that a ligand was identified that bound the SH3 domain in slow exchange on the NMR timescale. However, this goal was not fulfilled. Despite this, the work presented in this thesis provides a solid foundation for the development of potent 2-aminoquinoline ligands for SH3 domains, with engineered specificity. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.