Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (p. 43-46). / One erythropoietin molecule binds asymmetrically to two identical receptor monomers via erythropoietin site 1 and site 2, although it is unclear how asymmetry affects receptor activation and signaling. Here we report the computational design and experimental validation of two mutant erythropoietin receptors: one that binds only to erythropoietin site 1 but not site 2, and one that binds only to site 2 but not site 1. Expression of either mutant receptor alone in Ba/F3 cells cannot elicit a signal in response to erythropoietin, but when co-expressed, there is a proliferative response and activation of the JAK2 Stat5 signaling pathway. A truncated erythropoietin receptor with only one cytosolic tyrosine (Y343), on only one receptor monomer is sufficient for signaling in response to erythropoietin, regardless of the monomer on which it is located. The same results apply to having only one conserved juxtamembrane hydrophobic L253 or W258 residue, essential for JAK2 activation, in the full-length receptor dimer. We conclude that despite asymmetry in the ligand-receptor dimer interaction, both sides are competent for signaling, and we suggest that the receptors signal equally. / by Yingxin Zhang. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references. / Deoxyribose oxidation in DNA forms strongly electrophilic terminal blocking groups and abasic sites capable of stopping polymerases, forming DNA-protein crosslinks and nucleobase adducts. These threats to the genetic integrity of cells are caused by exogenous and endogenous oxidizing agents and may contribute to diseases such as cancer. To better understand the role of oxidative DNA damage in these diseases, it is necessary to quantify deoxyribose oxidation both in vitro and in vivo. Current methods lack the sensitivity required to measure low concentrations of deoxyribose oxidation products. The goals of the research findings reported here is to develop a sensitive method that can accurately and reproducibly quantify 3'-phosphoglycolaldehyde (PGA), one example of a deoxyribose oxidation product, and to apply the method to quantify PGA in vitro and in cells. Given the presence of reactive carbonyl groups in many deoxyribose oxidation products, the method should be widely applicable to other studies of oxidative ])NA damage. The analytical method exploits the reactive carbonyl moiety in PGA by derivatization as a stable oxime with pentafluorobenzylhydroxylamine, followed by solvent extraction and gas chromatography/negative chemical ionization/mass spectrometry. A stable isotopically-labeled [13C2]-PGA is synthesized and used as an internal standard. The method is linear in response over the range of 30 fmol to 300 pmol and precision is verified by analysis of a synthetic, PGA-containing oligodeoxynucleotide. The limit of detection in the presence of DNA is 30 fmol of PGA. The analytical method was then applied to the quantification of PGA in purified DNA and cultured cells treated with several oxidants. / (cont.) y-Radiation forms PGA in a linear dose response both in vitro and in cells with a 1000-fold quenching effect in vivo. The total quantity of deoxyribose oxidation, determined by plasmid topoisomer analysis, allows the calculation of PGA formation per deoxyribose oxidation event. PGA is formed in 1% of deoxyribose oxidation events induced by y-radiation versus 7% for a-particles. PGA formation induced by Fe(II), Fe(II)/EDTA/H202, and peroxynitrite follows a nonlinear dose response best fitted to a second-order polynomial, in contrast to a linear response induced by Fe(II)/EDTA, with PGA formation representing 5% of deoxyribose oxidation events. Peroxynitrite-induced DNA damage is studied under conditions of both hydroxyl radical and carbonate radical formation. With increasing carbonate radical formation, PGA induction is suppressed which indicates that the carbonate radical is not capable of inducing PGA. / by Christine Collins. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 81-83). / Although embryonic stem (ES) cells and their pluripotent capability have been elucidated for decades, little study has been done on obtaining the pluripotency profile of ES cells in the incipient stages of differentiation. In this research, an ES cell line with transfected green fluorescent protein (GFP) co-expressed by an Oct-4 promoter was analyzed by fluorescence-activated cell sorter (FACS) to obtain such profile. As Oct-4 is an ES cell differentiation marker whose expression varies with pluripotency, GFP expression could simply be measured in these cells to determine how pluripotent they are as a population. The differentiation characterization of ES cells was also conducted with different culture conditions of reduced serum and glucose concentrations both in the presence and absence of leukemia inhibitory factor (LIF) which prevents spontaneous differentiation, as well as at varied LIF concentrations and seeding densities. In addition, fundamental growth kinetic and metabolic profiles were obtained to get a more complete picture of how ES cells behave under these varied culturing conditions. The doubling time (t[sub]d) of R1 Oct4-GFP cell line was found to be 13 hours in LIF⁺ culture and 8 hours in culture with LIF addition after 7 days of LIF withdrawal, implying that cell proliferation rate is higher for cells receiving a sudden upregulation of genes controlling cell division through LIF addition. Although the upregulation of the genes is rapid, the downregulation of these genes through LIF withdrawal was found to take 6-7 days, while 3-4 days were required to downregulate the pou5f gene (which controls Oct4 expression). Higher concentration of LIF resulted in higher ES cell proliferation rate, but GFP⁺ expression was unaffected by / (cont.) concentration. Higher seeding density resulted in greater improvement in GFP⁺ expression for LIF⁺ culture but lower non significant reduction in GFP⁺ expression in LIF⁻ culture. Low level of glucose in medium led to reduction in the rate of ES cellular mechanisms and lower Y[sub]lac/gluc (8-49 % versus 40-60 % in high glucose), but metabolic rates were consistent with cells grown in high glucose medium, implying more efficient glucose metabolism through oxidative phosphorylation. The level of serum in medium had no effect on GFP⁺ expression or cell proliferation rate in LIF⁺ cultures, but reduction in GFP⁺ expression level was higher and t[sub]d was longer in low-serum culture (71 [plus-minus] 33 hours versus 35 [plus-minus] 9 hours) in the absence of LIF. / by Yasunori Hashimura. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references. / The procession of helix-tracking enzymes along a DNA molecule results in the formation of supercoils in the DNA, with positive supercoiling (overwinding) generated ahead of the enzyme, and negative supercoiling (underwinding) in its wake. While the structural and physiological consequences of negative supercoiling have been well studied, technical challenges have prevented extensive examination of positively supercoiled DNA. Studies suggest that at sufficiently high levels of overwinding, DNA relieves strain by adopting an elongated structure, where the bases are positioned extrahelically and the backbones occupy the center of the helix. This transition has only been identified, however, at a degree of supercoiling substantially higher than is generated physiologically. To examine the structural changes resulting from physiological levels of positive DNA supercoiling, I have developed a method for preparing highly purified positively supercoiled plasmid substrates. Based on a method previously developed in this laboratory, this allows for preparation of large quantities of very pure, highly positively supercoiled plasmid. It also expands on earlier methods by exploiting ionic strength to modulate the direction of supercoiling introduced, allowing preparation of either positively or negatively supercoiled substrates. / (cont.) A combination of approaches has been used to elucidate changes to DNA structure that result from physiological levels of positive supercoiling. Enzymatic probes for regions of single-stranded character are not reactive with positively supercoiled plasmid, indicating that stably unpaired regions are not present. Additionally, the effect of supercoiling on the activity of restriction enzymes has been examined. With the enzymes tested, no substantial differences in cleavage rates were observed with either positively or negatively supercoiled substrates. To examine structural changes at a wider range of superhelical densities, design and preparation was undertaken on 2-aminopurine-containing DNA substrates for use in fluorescence studies with a magnetic micromanipulator. Technical limitations rendered these experiments infeasible with current instrumentation, but important insights were gained for future fluorescence-based A destabilizing effect on the base pairs, however, can be seen using Raman difference spectroscopy, suggesting a subtle shift toward the more extreme extrahelical state. / (cont.) The Raman data suggest that structural adjustments due to positive supercoiling are small but significant, and in addition to the base-pairing effects, alterations are observed in phosphodiester torsion and the minor groove environment, as well as a slight shift in sugar pucker conformation to accommodate lengthening of the DNA backbone. These results point to subtle changes in DNA structure caused by biologically relevant levels of positive superhelical tension and positive supercoiling. All of the changes are consistent with the mechanical effects of helical overwinding and suggest a model in which base pair destabilization in overwound DNA could affect the search mechanisms used by DNA repair enzymes and the binding of other proteins to DNA. / by Marita Christine Barth. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2007. / Includes bibliographical references. / Ligand binding to cell surface receptors initiates a cascade of signaling events regulated by dynamic phosphorylation on a multitude of pathway proteins. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues play a role in determining cellular response. Mass spectrometry has been previously used to identify and catalog phosphorylation sites or quantify the phosphorylation dynamics of proteins in cell signaling networks. However, identification of phosphorylation sites presents little insight on cellular processes and quantification of phosphorylation dynamics of whole proteins masks the different roles that several phosphorylation sites within one protein have in the network. We have designed a mass spectrometry technique allowing site-specific quantification of dynamic phosphorylation in the cell. We have applied this technique to study signaling events triggered by different members of the epidermal growth factor receptor (EGFR) family. Self organizing maps (SOMs) analysis of our data has highlighted potential biological functions for phosphorylation sites previously unrelated to EGFR signaling and identified network modules regulated by different combinations of EGFR family members. Partial least square regression (PLSR) analysis of our data identified combination of signals strongly correlating with cellular proliferation and migration. / (cont.) Because our method was based on information dependent acquisition (IDA) the reproducibility of peptides identified across multiple analyses was low. To improve our methodology to permit both discovery of new phosphorylation sites and robust quantification of hundreds of nodes within a signaling network we combined IDA-analysis with multiple reaction monitoring (MRM) of selected precursor ions. MRM quantification of high resolution temporal profiles of the EGFR network provided 88% reproducibility across four different samples, as compared to 34% reproducibility by IDA only. In summary, we have developed a new robust mass spectrometry technique allowing site specific identification, quantification and monitoring of dynamic phosphorylation in the cell with high temporal resolution and under any number of biological conditions. Because the data obtained with this method is not sparse it is especially well suited to mathematical and computational analyses. The methodology is also broadly applicable to multiple signaling networks and to a variety of samples, including quantitative analysis of signaling networks in clinical samples. / by Alejandro Wolf Yadlin. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references. / Epidermal growth factor (EGF) receptor autocrine and/or paracrine signaling plays an important role in normal epithelial cell proliferation, survival, adhesion and migration. Aberrant expression of the EGF receptor and its cognate ligands have been implicated in various types of cancers, hence EGF receptor autocrine activation is thought to also be involved in tumorigenesis. EGF family ligands are synthesized as membrane-anchored proteins requiring proteolytic release to form the mature soluble, receptor-binding factor. Despite the pathophysiological importance of autocrine systems, how protease-mediated ligand release quantitatively influences receptor-mediated signaling and consequent cell behavior is poorly understood. Therefore, we explored the relationship between autocrine EGF release rate and receptor-mediated ERK activation and migration in human mammary epithelial cells. A quantitative spectrum of EGF release rates was achieved using chimeric EGF ligand precursors modulated by the addition of the metalloprotease inhibitor batimastat. We found that ERK activation increased with increasing ligand release rates despite concomitant EGF receptor downregulation. / (cont.) Cell migration speed depended linearly on the steady-state phospho-ERK level, but was much greater for autocrine compared to exogenous stimulation. In contrast, cell proliferation rates were constant across the various treatment conditions. In addition, we investigated an EGFR-mediated positive feedback through ERK that stimulated a 4-fold increase in release rate of our TGFa based construct. Thus, in these cells, ERK-mediated migration stimulated by EGF receptor signaling is most sensitively regulated by autocrine ligand control mechanisms. / by Elizabeth Jane Joslin. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (leaves 56-59). / Gene Fabrication technology involves the development and optimization of methods relevant to the in vitro synthesis of any given target gene sequence(s) in the absence of template. The driving purpose of this field of research is to bring about the capability for on-demand fabrication of a DNA construct of arbitrary length and sequence quickly, efficiently, and cost-effectively. The first part of this document describes many of the important considerations in performing successful de novo gene synthesis from a survey of the literature as well as from our own work. Recommendations are made for a universally effective, robust, and simple protocol for potential users of gene synthesis, discussing important factors such as choice of protocol, source of commercial oligonucleotides, and polymerase choice. The second part of this document focuses on error correction. Reducing error rates is one of the main challenges in gene fabrication because high error rates preclude the possibility of fabricating long gene targets in a practical and economical manner. Improvements in error rates are essential for continued progress in the development of gene fabrication technology. I discuss the importance of error rate in gene synthesis from a practical standpoint and show results in the development of novel methods for the removal of errors from a pool of synthesized DNA. / by Jason Sun-hyung Park. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (leaves 149-151). / Linkage disequilibrium studies have discovered few gene-disease associations for common diseases. The explanation has been offered that complex modes of inheritance govern risk for cancers, cardiovascular and cerebrovascular diseases, and diabetes. Such studies, however, depended on the untested assumption of monoallelic risk. My research advisor and I set out to investigate whether simple forms of inherited risk, monoallelic or multiallelic, could be excluded by analysis of familial risk for a common disease, such as colorectal cancer (CRC). First, we derived formulae that describe the risk for monogenic, multigenic, and polygenic possibilities of Mendelian inheritance. Next, we obtained an estimate of minimum lifetime risk for CRC of >0.26. Then, we examined the case of late-onset CRC, using the Swedish Family Cancer Database (1958-2002) to estimate the familial relative risk for CRC diagnosis at age 50 or older, and obtained an estimated range of 1.5 to 3.0. We compared this range of actual values to the ranges of expected values for monogenic, multigenic, and polygenic modes of inheritance. / (cont.) We delimited bounds that can be placed on the conditions for various modes of inheritance. The key observation is that monogenic risk for CRC is included among various possibilities, and cannot be eliminated by existing observations. The arguments herein indicate that further efforts can and should be made to obtain more precise estimates of familial risk for CRC and other common forms of cancer. / by Helen Banava. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references (p. 119-134). / How do cells sense their environment and decide whether to live or to die? This question has drawn considerable interest since 1972, when it was first discovered that cells have an intrinsic ability to self-destruct through a process called apoptosis. Since then, apoptosis has been shown to play a critical role in both normal physiology and disease. In addition, many of the basic molecular mechanisms that control apoptosis have been revealed. Yet despite the known list of interactions and regulators, it remains difficult to inspect the network of apoptosis-related proteins and predict how cells will behave. The challenge is even greater when one considers interactions with other networks that are anti-apoptotic, such as growth-factor networks. In this thesis, we develop an approach to measure, analyze, and predict how complex intracellular signaling networks transduce extracellular stimuli into cellular fates. This approach entails three interrelated aims: 1) to develop high-throughput, quantitative techniques that measure key nodes in the intracellular network; 2) to characterize the quantitative changes in network state and cell behavior by exposing cells to diverse fate-changing stimuli; and 3) to use data-driven modeling approaches that analyze large signaling-response datasets to suggest new biological hypotheses. / (cont.) These aims were focused on an apoptosis-survival cell-fate decision process controlled by one prodeath cytokine, tumor necrosis factor (TNF), and two prosurvival stimuli, epidermal growth factor (EGF) and insulin. We first developed radioactive- and fluorescence-based high-throughput assays for quantifying activity changes in the kinases that catalyze key phosphorylation events downstream of TNF, EGF, and insulin. By combining these assays with techniques measuring other important posttranslational modifications, we then compiled over 7000 individual protein measurements of the cytokine-induced network. The signaling measurements were combined with over 1400 measurements of apoptotic responses by using partial least squares (PLS) regression approaches. These signaling-apoptosis regression models predicted apoptotic responses from cytokine-induced signaling patterns alone. Furthermore, the models helped to reveal the importance of previously unrecognized autocrine cytokines in controlling cell fate. This thesis has therefore shown how cell decisions, like apoptosis-versus-survival, can be understood and predicted from the quantitative information contained in the upstream signaling network. / by Kevin A. Janes. / Ph.D.

Thesis (Ph. D. in Molecular Systems Toxicology and Pharmacology)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Vita. / Includes bibliographical references. / High affinity antibody binding interactions are important for both pharmaceutical and biotechnological uses. However, designing higher affinity interactions has remained difficult. Both high affinity interactions from nature and the results from directed evolution affinity maturation processes may yield clues about the important structural and energetic contributions to attain these tight associations. In this Thesis, we investigate affinity maturation of antibodies for very high affinity binding. Two single chain antigen-binding fragment (scFv) antibody systems that were engineered to obtain higher affinity interactions through directed evolution were probed using biophysical techniques to illuminate affinity modulation in proteins. First, anti-c-erbB-2 antibodies and their binding partner, the extracellular domain of the glycoprotein tumor antigen c-erbB-2, were examined. Thermodynamic studies were carried out on the originally identified human scFv and three higher affinity mutants. Although the first two steps included either entropic or enthalpic gains to affinity, the third improvement came from both types. This study demonstrates that a single energetic component is not generally responsible for the increased affinity within a given protein-protein affinity maturation pathway. Second, a family of anti-fluorescein antibodies and their binding to the small molecule fluorescein-biotin were explored. The femtomolar affinity matured anti-fluorescein antibody, 4M5.3, was compared to its wildtype high affinity precursor, 4-4-20. Affinity, thermodynamic, kinetic, and structural characterization of the binding identified 4M5.3 as one of the highest engineered affinity protein binding interactions known and / (cont.) illuminated how subtle structural changes can lead to large consequences for the kinetics and free energy of binding. The affinity mechanisms were further studied by the creation of a series of partial mutants. Context dependent and independent mutational effects on binding affinity indicated the extent of complexity in higher affinity mechanisms attained through directed evolution affinity maturation processes. These studies emphasize the importance of a large number of residues working in concert to create a very high affinity binding molecule. Based on these results, both rational design and directed evolution studies will need to allow for mutations in a spatially broad range around the binding site and involve many biophysical contributions to the binding free energy to reach very high antigen binding affinities. / by Katarina S. Midelfort. / Ph.D.in Molecular Systems Toxicology and Pharmacology