Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references (p. 252-285). / Glycosaminoglycans (GAGs) are complex polysaccharides that exist both on the cell surface and free within the extracellular matrix. The intrinsic sequence variety stemming from the large number of building blocks that compose this biopolymer leads to substantial information density as well as to the ability to regulate a wide variety of important biological processes. With the recent and progressive emergence of biochemical and analytical tools to probe GAG structure and function, efforts can be taken to understand the role of GAGs in cell biology and in disease in the various physiological locations where GAGs can exist. As a first step to probe the functions of GAGs, the heparin/heparan sulfate-GAG (HSGAG)-fibroblast growth factor (FGF) system was examined. Understanding the role of HSGAGs in inducing FGF2 dimerization led to the development of a novel engineered protein that was found to be effective at promoting functional recovery in stroke. Subsequently, methods to isolate HSGAGs from the cell surface were optimized and the ability of HSGAGs to support FGF signaling was investigated. Cell surface HSGAGs can define the responsiveness of a given cell to FGF1 and FGF2 through multiple receptor isoforms. Stromal cell derived HSGAGs were also identified as critical regulators of tumor cell growth and metastasis, effecting not only FGF2., but also 1-integrin signaling. / (cont.) Other GAGs, including dermatan sulfates, were characterized as modulators of FGFs and vascular endothelial growth factors. Finally, FGFs and HSGAGs were found to have important roles in maintaining epithelial monolayer integrity, with syndecan-l serving as a critical factor in inflammatory bowel disease. In addition to understanding HSGAGs in their normal physiological settings, techniques to internalize them were developed. Poly(3-amino ester)s were found to condense heparin and enable its endocytosis into cells. Internalized heparin is preferentially taken up by cancer cells, which often have a faster endocytic rate than non-transformed cells, and promotes apoptotic cell death. Internalized heparin can also be used as a tool to probe cell function. In Burkitt's lymphoma, poly(3-amino ester)-heparin conjugates served to identify cell surface HSGAGs as an important modulator of cell growth that can be harnessed to inhibit growth. Finally, studies that sought to broaden the scope of GAG biology were undertaken. Cell surface HSGA(:is were identified as mediators of vascular permeability. Furthermore a novel technique to immobilize GAGs was employed. The interactions between GAG and substrate were via hydrogen bonding. Immobilization of GAGs alters their properties, such that they can affect cells in ways distinct from GAGs free in the ECM. / (cont.) Furthermore, immobilized GAGs can regulate cancer cell adhesion, growth and progression, and may offer a new way to regulate the activity of cancer cells. In addition to directly providing new potential therapeutics and drug targets, these studies represent a foundation to enable additional studies of GAG function. Future work harnessing the techniques presented may open new avenues of research and facilitate the development of novel GAG-based therapeutics. / by David Berry. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (leaves 44-45). / There is a great need in the fields of biology, medicine, and pharmaceuticals to create high-throughput devices for the detection of specific cell states in a heterogeneous mixture of cells. The desire is to differentiate among diseased and healthy cells, cell age, and cell type with the minimum amount of sample pretreatment. This project addresses this need by developing microfluidic devices that exploit the adhesion differences between cell states and cell types to rapidly count cells of different types without the need for labels. There are two avenues in which to explore cell adhesion differences with these devices, the first is a net electrostatic change at the surface of the cell wall and the second is the presence of specific cell-membrane adhesion proteins. It is hypothesized that the forced interaction of the cell wall with the microfabricated microcapillary walls would result in a differential velocity based on cell type that could be detected simply using a microscope and video camera or an interferometer. The eventual integration of cell velocity detection would result in a portable all-inclusive lab-on-a-chip system that could be used in the field for detecting the presence of diseases, such as malaria and cancer as well as in a lab setting for drug discovery. / by Kristen M. Naegle. / S.M.

Thesis (Ph. D. in Molecular and Systems Bacterial Pathogenesis)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references. / Helicobacter hepaticus infection of A/JCr mice is a model of liver cancer resulting from chronic active inflammation. We monitored hepatic global gene expression profiles and correlated them to histological liver lesions in H. hepaticus infected and control male A/JCr mice at 3 months, 6 months, and 1 year of age. We used an Affymetrix-based oligonucleotide microarray platform on the premise that a specific genetic expression signature at isolated time points would be indicative of disease status. Model based expression index comparisons generated by dChip yielded consistent profiles of differential gene expression for H. hepaticus infected male mice with progressive liver disease versus uninfected control mice within each age group. Linear discriminant analysis and principal component analysis allowed segregation of mice based on combined age and lesion status, or age alone. Up-regulated genes present throughout the 12 month study involved inflammation, tissue repair, and host immune function. Upregulation of putative tumor and proliferation markers correlated with advancing hepatocellular dysplasia. Transcriptionally down-regulated genes in mice with liver lesions included those related to peroxisome proliferator, cholesterol, and steroid metabolism pathways. Transcriptional profiling of hepatic genes documented gene expression signatures in the livers of H. hepaticus infected male A/JCr mice with chronic progressive hepatitis and preneoplastic liver lesions, complemented the histopathological diagnosis, and suggested molecular targets for the monitoring and intervention of disease progression prior to the onset of hepatocellular neoplasia. Our laboratory, in collaboration with Professors Suerbaum and Schauer, recently identified a / (cont.) 70kb genomic island in Helicobacter hepaticus strain ATCC 51488 as a putative pathogenicity island (HhPAI) (Suerbaum et al, PNAS, 2003). This region within H. hepaticus contains genes HH0233-HH0302, a differential GC content, several long tandem repeats but no flanking repeats, and three components of a type IV secretion system (T4SS). A/JCr mice were experimentally infected with three naturally occurring strains of H. hepaticus including the type strain H. hepaticus ATCC 51488 strain (Hh 3B1) isolated from A/JCr mice, MIT 96-1809 (Hh NET) isolated from mice shipped from the Netherlands, and MIT-96-284 (HhG) isolated from mice acquired from Germany.4 HhNET (missing most of the HhPAI) infected male A/JCR mice exhibited a significantly lower prevalence (p<.05) of hepatic lesions at 6 months post infection than Hh 3B1 with an intact HhPAI. Hh G also has a large segment of the genomic island deleted, but not as many genes are deleted as compared to Hh NET. Hh G also demonstrated a lower prevalence of hepatic lesions. This variable pathological effect was evident in male mice only. The severity of chronic active inflammation in the liver of the H. hepaticus infected A/JCr mice depended on H. hepaticus liver colonization levels. The in vivo results support the presence of the HhPAI as a legitimate virulence determinant and predictor of severity of liver lesions in H. hepaticus infected A/JCr male mice. To further determine the differences in virulence of the H. hepaticus strains Hh 3B1, Hh NET, Hh G and an isogenic mutant H. ... / by Samuel R. Boutin. / Ph.D.in Molecular and Systems Bacterial Pathogenesis

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 84-93). / Non-parenchymal cells (NPC's) are integral to recreate the native hepatic microenvironment and necessary to maintain in vivo liver function. A variety of in vitro culture systems have been developed to address different aspects of liver physiology and architecture in order to recreate the microenvironment. These in vitro co-culture strategies have been limited by their inability to systematically characterize the addition of non-parenchymal cells. In this dissertation, I use gene expression levels quantified by real-time RT-PCR to determine tissue composition. The identified genes demonstrate significant cell-type specificity, magnitude, and stability of expression in vivo and throughout each step of cell isolation process. In the course of this development, we establish protocols to accurately isolate and count an enriched fraction of primary NPC's. Experiments on the perfusion and isolation process prove that there exists an inverse correlation between perfusion flow rate and NPC yield and viability. Further, we have characterized the tissue composition of each step in the cell isolation process and the resulting NPC population to confirm that a significant number of each NPC type is delivered to in vitro co-culture. System output analysis of spheroids co-cultured at physiological ratios and seeded into the milliF bioreactor shows the presence of stellate cells, but the absence of endothelial (EC) and kupffer cells (KC). The same analysis of 2D collagen gel sandwiches shows the presence of all NPC cell types. This indicates that our process is currently limited by the ability of EC's and KC's to incorporate into spheroid aggregates. Future work that validates the temporal expression stability of the identified genes in different in vitro culture systems / (cont.) and environments will enable determination of relative levels of NPC incorporation and will allow correlations to be made between operational features of in vitro systems, the resulting culture microenvironment, and observed tissue function. / by Brent M. Schreiber. / M.Eng.

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.