Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (leaves 92-93). / The two most common forms of inflammatory bowel disease (IBD) are ulcerative colitis (UC) and Crohn's Disease (CD), which affect more than 1 million Americans. Recently the incidence of IBD has been rising in Japan, Europe and North America.' Colorectal cancer is a very serious complication of IBD, and a patient's risk increases with increasing extent and duration of disease.2 There is no cure for CD, and the only cure for UC is removal of the entire colon and rectum. It is thought that cancer risk is based on chronic inflammation of the gastrointestinal mucosa. There have been many studies, which have supported this idea and have made progress toward understanding the link between chronic inflammation and cancer. In both UC and CD, it is known that there are increased levels of EA, cG, and eC, which are potentially miscoding lesions, in the DNA of affected tissues.3 Also, 3-methyladenine DNA glycosylase (Aag in mice), an initiator of the Base Excision Repair pathway, shows adaptively increasing activity in response to increased inflammation in UC colon epithelium.4 This thesis demonstrates the importance of Aag in protecting against the effects of chronic inflammation. / (cont.) It was found that Aag deficient mice, treated with 5 cycles of dextran sulfate sodium (DSS) to induce chronic inflammation, showed significant signs of increased disease including decreased colon length, increased spleen weight, and increase in epithelial defects. Also, when treated with a tumor initiator, azoxymethane, prior to DSS exposure, Aag deficient mice show a 2.95 fold (p<0.0001) increase in tumor multiplicity compared to wild type treated animals, as well as decreased colon length, increased spleen weight, increased dysplasia/neoplasia, and increased area affected by dysplasia/neoplasia. If UC patients had a deficiency in 3-methyladenine-DNA-glycosylase activity, they would likely be more susceptible to mutations and cancer because of their inability to repair DNA damage caused by inflammatory cytokines and reactive oxygen and nitrogen species. In future studies, it would be beneficial to determine if transgenic Aag over-expresser mice show protection against the damage induced by chronic inflammation. This would make intestinal gene therapy a possible approach to finding the first cure for IBD and inflammation associated colorectal cancer. / by Stephanie Lauren Green. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references. / Cells sense mechanical stimuli and respond by changing their phenotype, e.g. shape, gene expression, motility. This process, termed mechanotransduction, was investigated using computational and theoretical approaches, as well as comparisons with experiments. As a first step, a three-dimensional viscoelastic finite element model was developed to simulate cell micromanipulation by magnetocytometry. The model provided a robust tool for analysis of detailed strain/stress fields induced within a single cell or cell monolayer produced by forcing one tethered microbead. On the assumption of structural homogeneity, stress and strain patterns were highly localized, suggesting that the effects of magnetocytometry are confined to a region extending less than 10tm from the bead. Modification of the model to represent experimental focal adhesion attachments supported a non-uniform force transmission to basal surface focal adhesion sites. Proteins in identified zones of high stresses in the cell are candidate mechanosensors and their molecular response to force was hence investigated, A generic model of protein extension under external forcing was created inspired by Kramers theory for reaction rate kinetics in liquids. / (cont.) The protein was hypothesized to have two distinct conformational states: a relaxed state, Ci, preferred in the absence of external force, and an extended state, C2, favored under force application. Appearance and persistence of C2 was assumed to lead to transduction of the mechanical signal into a chemical one. While the level of applied force and the energy difference between states largely determined equilibrium, the dominant influence on the extension time was the height of the transition state. Force-induced distortions in the energy landscape were also shown to have a significant influence on extension time, however, exhibiting a weaker force dependence than exponential. Finally, the link between membrane receptors and the extracellular matrix -- or the bead in magnetocytometry experiments -- was investigated as a primary path for force transduction to the cell interior. To shed light on the role of bonds formed by membrane receptors on measurements of cellular rheology, we modeled the process by which a forced, cell-tethered microbead translates and rotates as influenced by the stochastic formation and. rupture of adhesion bonds. We show that this process is crucial in the inference of cell mechanical properties from microbead experiments. / by Hélène Karcher. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (p. 63-65). / In the wake of the development of technology to sequence the complete genome of an organism, it has become expedient to generate methodologies to elucidate and characterize the function of all genes constituting the complete genetic makeup of an organism, whereby the knowledge of the genetic code may be for scientific and intellectual profit. This work consists of an investigation into two possible methods for determining the role of genes involved in the DNA and cellular damage response, though the methods are generally applicable to investigating a wide variety of biological pathways and responses. A library of approximately 4,800 yeast (Saccharomyces cerevisiae) deletion strains produced by the Saccharomyces Genome Deletion Project and consisting essentially of all possible mutants having one non-essential gene deleted (and replaced with unique identification tags called "bar codes") from the genome are employed in this endeavor. The methods focus on gathering phenotype data in a high-throughput manner and in response to the alkylating agent methyl methanesulfonate (MMS). The first method makes use of a new technology called the Living ChipTM, which can hold libraries of compounds or cell cultures in an array of 50-nl channels and which could ideally accommodate all deletion strains on a single array. The second method involves pooling all strains together in a single culture and allowing them to grow competitively to determine their relative fitness based on a specific treatment. / by Daniel S. Ericksen. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references. / Cholesterol gallstones are exceptionally common and cost nearly 10 billion U.S. dollars annually. Despite a half-century of basic and clinical research questions still remain about cholesterol gallstone pathogenesis. The purpose of the study presented herein is to analyze the roles of infection, and immunity in cholelithogenesis. The first two aims of this work were to analyze the role of enterohepatic Helicobacter spp. and the human gastric pathogen H. pylori in cholesterol gallstone formation. To test this, we prospectively infected C57UJ mice with a variety of Helicobacter spp. and fed infected and uninfected mice a lithogenic diet for eight weeks and analyzed biliary phenotype. Mice infected with H. bilis or coinfected with H. hepaticus and H. rodentium and fed a lithogenic diet developed cholesterol gallstones at 80% prevalence compared with approximately 10% in uninfected controls (P<0.05). Monoinfections with H. hepaticus, H. cinaedi, H. rodentium, and H. pylori gave a cholesterol gallstone prevalence of 40% (P<0.05), 30%, 20% and 20%, respectively; with the exception of H. hepaticus, cholesterol gallstone formation in these groups did not differ significantly from uninfected animals. / (cont.) These findings suggest that some Helicobacter spp. play a role in the cholesterol gallstone formation in mice and perhaps humans. We further hypothesized that inflammation and immunity were important in cholesterol gallstone formation and that cholelithogenic bacteria were promoting gallstones through immune stimulation. To test this we utilized BALB/c and isogenic Rag2-/- mice. When fed a lithogenic diet for eight-weeks, wild-type mice developed cholesterol gallstones (27-80% prevalence) significantly more than Rag2-/- mice (~5%, P<0.05). Transfer of functional splenocytes, or T-lymphocytes to Rag2-/- mice markedly increased cholesterol gallstone formation (26% and 40% respectively, P<0.05) whereas transfer of B-cells did not (13%). The presence of T-cells and solid cholesterol monohydrate crystals induced proinflammatory cytokine expression in the gallbladder. These studies indicate that T-cells are critical in murine cholelithogenesis and function by promoting gallbladder inflammation. In summary, these results illustrate that microbial pathogens can influence cholesterol gallstone formation; this most likely occurs by modulating the immune response with T-cells being a critical component in this immunomodulation. / by Kirk J. Maurer. / Ph.D.

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.