Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (p. 157-169). / Cells use a complex web of protein signaling pathways to interpret extracellular cues and decide and execute cell fates such as survival, apoptosis, differentiation, and proliferation. Cell decisions can be triggered by subtle, transient signals that are context specific, making them hard to study by conventional experimental methods. In this thesis, we use a systems approach combining quantitative experiments with computational modeling and analysis to understand the regulation of the survival-vs-death decision. A second goal of this thesis was to develop modeling and analysis methods that enable study of signals that are transient or at intermediate activation levels. We addressed the challenge of balancing mechanistic detail and ease of interpretation in modeling by adapting fuzzy logic to analyze a previously published experimental dataset characterizing the dynamic behavior of kinase pathways governing apoptosis in human colon carcinoma cells. Simulations of our fuzzy logic model recapitulated most features of the data and generated several predictions involving pathway crosstalk and regulation. Fuzzy logic models are flexible, able to incorporate qualitative and noisy data, and powerful enough to generate not only quantitative predictions but also biological insights concerning operation of signaling networks. To study transient signals in differential-equation based models, we employed direct Lyapunov exponents (DLEs) to identify phase-space domains of high sensitivity to initial conditions. These domains delineate regions exhibiting qualitatively different transient activities that would be indistinguishable using steady-state analysis but which correspond to different outcomes. / (cont.) We combine DLE analysis of a physicochemical model of receptor-mediated apoptosis with single cell data obtained by flow cytometry and FRET-based reporters in live-cell microscopy to classify conditions that alter the usage of two apoptosis pathways (Type I/II apoptosis). While it is generally thought that the control point for Type I/II occurs at the level of initiator caspase activation, we find that Type II cells can be converted to Type I by removal of XIAP, a regulator of effector caspases. Our study suggests that the classification of cells as Type I or II obscures a third variable category of cells that are highly sensitive to changes in the concentrations of key apoptotic network proteins. / by Bree Beardsley Aldridge. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Vita. / Includes bibliographical references (p. 108-111). / The objective of this work is to functionalize an existing polymer such that it better mimics natural tissue for tissue growth and regeneration. Numerous other processes have tried and accomplished this by non-specific protein adsorption, covalent attachment, biomolecule entanglement, and synthesis of new polymers with the desired functionality. The focus of this work is to modify the polymer's binding capability to cells while not altering the bulk properties. Through the use of both phage display of peptide libraries and yeast surface display of scFv libraries the surface of chlorine-doped polypyrrole (PPyCl) has been modified to facilitate binding of neuronal phenotype cells. The selection of peptides using phage display found a surface specific recognition peptide (T59) that was made bivalent by altering the C-terminus with an integrin binding epitope. The bivalency of the modified T59 peptide was exploited to tether phenochromocytoma (PC12) cells to the surface of PPyCl. Furthermore the tethering of the cells to PPyCl through the peptide does not decrease the cells neuronal function and maintains the bulk conductive polymers characteristics. Using the peptide as a bivalent linker, the addition of other types of cells, drugs, growth factors, and enzymes could be incorporated for various biomedical applications. / (cont.) An antibody (Y2) specific to PPyC1 was found using yeast surface display. This antibody was utilized to mediate cellular binding to PPyCl by expression of the antibody on the surface of PC12 cells. Complimenting the peptide studies of having an exterior bivalent linker the antibody recognition provides the means for any cell type to adhere to PPyCl, through expression of the antibody on the surface of the cell. This type of system could be used for various types of tissue growth supports. / by Kiley Preston-Halfmann Miller. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references (p. 163-168). / Biological engineers have constructed a number of multi-part synthetic biological systems that conduct logical operations on input signals, produce oscillatory output signals, store memory, or produce desired products. However, very few of these genetically-encoded systems worked as originally designed. The typical process of constructing a functional system involves a period of tuning the system properties to find a functional variant. This tuning process has been optimized and applied with great success to the engineering of individual biological parts by directed evolution. For instance, researchers developing improved enzymes, transcriptional promoters, and fluorescent proteins have generated large libraries of variants and screened these libraries to find individual mutants that met desired performance specifications. In this thesis, I address some of the bottlenecks preventing the application of directed evolution to more complex devices and systems. First, I describe an input / output screening plasmid that was designed to enable screening of higher-order genetic devices based on the equilibrium response of the device. This plasmid includes two fluorescent reporters and an inducible promoter to enable screening of device libraries across a range of inputs. Second, I describe measurement kits and reference standards designed to improve the characterization of promoter and RBS parts that are used as input substrates for device evolution. By using the kits, researchers are able to report promoter and RBS activities in standard units (Standard Promoter Units, SPUs, and Standard RBS Units, SRUs) enabling the growth of a collection of well-characterized parts to draw on for assembling device variants. Finally, I describe a new microfluidic device, the Sortostat, that integrates a cell sorting chamber with a previously published microscope-mounted microfluidic chemostat. / (cont.) Researchers can use the Sortostat to apply morphological, time-varying, or other complex selective pressures to cells in continuous culture. / by Jason R. Kelly. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references. / The current gold standard for bone graft material is autologous bone, which provides mechanical support, possesses factors that promote bone formation, and contains connective tissue progenitors (CTPs), a heterogeneous population of connective tissue stem and progenitor cells that contribute to neotissue formation. A major limitation to autologous bone grafts is the risk of surgical complications associated with graft harvesting as well as significant donor-site morbidity. Available bone graft substitutes are not as efficacious as autologous bone, resulting in a prescient need for improved bone grafting materials. A promising tissue engineering approach involves the use of bioactive biomaterials that can promote the selective retention of CTPs from pre-seeded autologous bone marrow. When presented in a tethered form, EGF has been shown to promote the survival and enhance the adhesion of culture expanded CTPs. Therefore, the hypothesis of this work was that tethered EGF could be used to enhance the retention of osteogenic CTPs from freshly aspirated bone marrow. Numerous adhesion ligands and growth factors have been investigated for use as candidates for the functionalization of bioactive materials. In this work, we showed that synergy-RGD peptides, which incorporate the putative synergy site on fibronectin, can promote cell adhesion through both a5pl and av33 integrins. We then investigated the effects of tethered EGF on CTP colony formation in the context of defined adhesion environments using a functionalizable comb copolymer. We found that tethered EGF increased the colony forming efficiency of CTPs from fresh human marrow when cell attachment was promoted by either non-specific protein adsorption, fibronectin pre adsorption, or through the synergy-RGD ligand. In contrast, soluble EGF did not increase colony formation, demonstrating the importance of the modality of ligand presentation. / (cont.) Quantitative image analysis also suggested that while tethered EGF did not promote increased osteogenesis at early times after cell seeding tethered EGF may induce the proliferation and migration of cells within osteogenic colonies. These results provide important insight into both the study of the effect of EGF on CTP behavior, as well as the use of tethered EGF as a potential ligand for use in biomaterials that promote the selective retention of CTPs. / by Nicholas A. Marcantonio. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (leaves 28-34). / CD8+ T cells are the main adaptive immune system cell type responding to intracellular pathogens, particularly viruses, and tumor antigens. In the case of influenza, activated T cells migrate from the mediastinal (draining) lymph nodes to the lung where they perform their cytolytic function. After pathogen clearance, memory CD8+ T cells are generated, giving rise to long-term protection from reinfection. However, these cells are no longer detectable in the lung parenchyma six months post-infection, and cell-mediated immunity, and protection is lost. Knock-out studies in mice show that interleukin 15 (IL-15) is essential for memory CD8+ T cell proliferation. Fibroblasts, macrophages, dendritic cells and epithelial cells express IL-15 and its receptor isoform [alpha] (IL-15R[alpha]). Histological studies suggest that memory CD8+ T cells preferentially reside in peribronchiolar and perivascular areas, the stroma, of the lung. We hypothesize that memory CD8+ T cells preferentially reside in regions where molecules necessary for their maintenance, for example, IL-15/R secreting cells, are located. In this study, we have shown that antigen-specific 2C GFP effector memory CD8+ T cells are generated in B6 recipient mice 30-32 days after influenza virus infection, preferentially reside in peribronchiolar areas. Both 2C and 2C GFP recipient mice have severe vasculitis and widely distributed inflammatory infiltrates 7 days post-infection. Lower lung lobes appear to be more affected than upper lobes at this time point. On day 30, most of the airways have been cleared and restored. Although lymphoid-appearing nodules were detected in the lungs 31 dpi, no clusters of B cells and T cells suggesting induced BALT were identified by immunofluorescence. / (cont.) Interestingly, antigen-specific GFP cells preferentially remained in the lung tissue and were almost undetectable in spleens, lymph nodes, and livers. This preference was not observed in 2C (non-GFP) recipient mice. Immunofluorescence studies showed no colocalization between 2C GFP T cells and dendritic cells that might suggest stable dendritic cell interactions contribute to antigen-specific cells preferentially residing in the lung stroma. Further studies are necessary to determine what other cell types might contribute to this phenomenon. These results provide some insight into how structural elements in non-lymphoid tissue influence cell-mediated immunity. / by Mobolaji O. Olurinde. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 113-123). / Hematopoietic stem cells (HSCs) hold great promise for the treatment of disease. The rare frequency at which HSCs occur in the bone marrow under homeostatic conditions is a limiting factor in both their study and clinical use. ex vivo expansion of these cells is therefore a necessary step to maximizing their potential. In this thesis I explore the concept that signals from the extracellular matrix can direct differentiation, survival and self-renewal decisions in hematopoietic cells, and thus can provide a foundation for the design of ex vivo expansion strategies. This work is focused on the role integrins, the major class of cell-extracellular matrix adhesion molecules, play in mediating these signals to hematopoietic cells at two developmental stages. In the erythroid lineage, I show that expansion of committed erythroid progenitors is regulated by growth factor and integrin-mediated signals in temporally distinct regimes. I establish a biologically relevant role for [alpha]401 but not [alpha]501 integrins in erythropoiesis and provide evidence that erythroid differentiation and expansion are regulated by separate processes. / (cont.) In the study of uncommitted HSCs, I identify several integrin subunits that are differentially expressed on highly purified HSC populations that correlate with long term repopulating ability. One of these subunits, [alpha]2 integrin, specifically mediates adhesion of HSCs to bone marrow extracellular matrix proteins, thereby providing a potential mechanism for stem cell self-renewal. This work establishes that integrin-mediated interactions between hematopoietic cells and the extracellular matrix are dynamic and provide important developmental cues. / by Shawdee Eshghi. / Ph.D.

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.