Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 57-60). / Cells respond to the application of force with a variety of biochemical responses modulating their shape, structure, function, and proliferation. Two force-responsive links between the inside and outside of a cell are integrin proteins, which link a cell to the extracellular matrix (ECM), and cadherin proteins, which link neighboring cells to each other. The strength of integrin-ECM bonds has been noted to increase in response to the application of force. However, the strengthening of cadherin-cadherin bonds in response to force has not been studied. Here, we use magnetic trapping to probe adhesion strengthening at cadherin adherens junctions, using cadherin-coated magnetic beads to simulate neighboring cells and apply force at adherens junctions. 43% of beads exposed to a high force (2.1 nN) detached, compared to 31% of those exposed to a low-to-high force ramp followed by high force. This indicates that adherens junctions are strengthened by force application. The actin cytoskeleton and vasodilator-stimulated phosphoprotein (VASP) both associate with adherens junctions, so their role in adhesion strengthening at adherens junctions was also studied. Cells treated with actin-inhibitor cytochalasin D showed no difference in bead detachment from constant high force and from ramped followed by high force, indicating that the actin cytoskeleton is crucial in the adhesion strengthening response. Beads attached to cells expressing GFP-VASP, which behave like VASP-overexpressing cells, detached in 24% of trials when exposed to constant high force, compared to 39% of trials in response to ramped force. Cells expressing GFP-MITO-FPPPP, which behave like VASP-downregulated cells, showed no difference in bead detachment between application of high force and ramped force followed by high force. / (cont.) These experiments indicate that VASP is necessary for the adhesion strengthening response, but high levels of VASP may slow actin restructuring and diminish the ability of the cytoskeletal linkages to respond to increasing force. The importance of VASP in cells' responses to forces from other cells suggest that modulation of VASP activity may play a role in tissue development, where cell-cell force responses are important, and the pathogenesis of certain diseases, where cell-cell adhesion is affected. / by Anita S. Kris. / M.Eng.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (p. 70-72). / Self-assembling peptides are a promising new area of research with usage in numerous areas, from tissue engineering to membrane protein biology. This work is to further study the characteristics of the peptides KLD12 and RID12 and to generate new ways to control the properties of them. Peptide structures in solution were studied with circular dichroism and dynamic light scattering, and material properties of the peptide solutions were studied with rheology. Nanofiber structure was studied through atomic force microscopy. Sonication was found to have a minimal effect on KLD12, while pH could alter the length of the fibers formed. The presence of a second type of peptide in solution interacted with nanofibers of another peptide and resulted in a decrease of average fiber length. Peptide solutions transitioned from a viscous solution to a gel as the concentration of peptide increased, both alone and in mixture with another peptide. Understanding the properties of these peptides will help researchers design new biomaterials and improve applications of self-assembling peptides. / by Jessica Dai. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 141-153). / Creating a physiologically relevant in vitro liver model requires reproducing the cellular heterogeneity of in vivo liver in a functional state. However differentiated sinusoidal endothelial cells (SECs), marked by SE-1 expression are difficult to maintain in culture while stellate cells easily activate and over-proliferate. We hypothesized that recreating a liver tissue system that captured in vivo like paracrine influences would foster survival of these cells, and predicted that stimuli resulting from flow and oxygen gradients close to physiological conditions would preserve the delicate balance between the cell types. Spheroids containing hepatoctyes with incorporated non-parenchymal cells (NPCs) were seeded into capillary bed sized channels in polycarbonate scaffolds, housed in a three-dimensional perfused system, and maintained for two weeks. Micro-flow rates of different media through the formed tissue units in scaffolds were controlled using pneumatic pumps and microfluidics. Staining and confocal imaging of endpoint tissue showed that lower flow rates closer to physiological regimes allowed the survival of SE-1+ SECs, regardless of exogenously added growth factors in the medium. Higher flow rates, exogenous growth factors, and scaffold contact were associated with activation of stellate cells (alpha-smooth muscle actin staining). / Since oxygen measurements in the system coupled low flow rates with hypoxic tissue outlet concentrations, we parsed out these variables by repeating flow experiments in low oxygen environments. Retention of SE-i staining cells even in higher flow rates demonstrated that hypoxic conditions in the tissue could play a role in aiding their survival by overcoming negative effects brought about by high flow. The relationship of stellate cells with flow rate was unaffected by oxygen concentrations. To explore if the negative effects of high flow on SE-i expression were mediated by transforming growth factor-beta (TGF-[beta]), we added a TGF-[beta] inhibitor SB-431542 in our cultures, and found that it greatly enhanced the presence of SE-1 staining SECs at high flow rates. In conclusion we successfully created a three-dimensional flow controlled hepatic culture system that allows balanced survival of hepatocytes and non-parenchymal cells, making it useful as a potential model for studies such as cancer metastasis that require interactions between tumor cells and heterotypic host tissue. Key Words: Liver, In vitro, co-culture, sinusoidal, endothelial, stellate, oxygen, flow, shear. / by Ajit Dash. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references (p. 131-142). / The mechanical properties of articular cartilage are associated with the extracellular matrix network of type II collagen and the proteoglycan, aggrecan, which in combination provide the tensile, shear, and compressive stiffness of the tissue. While the collagen network mainly provides resistance to tensile and shear deformation, aggrecan enmeshed within this network contributes significantly to the tissue's compressive and shear properties under equilibrium as well as dynamic loading conditions. Aggrecan has a "bottle-brush" structure that includes -100 negatively charged chondroitin sulfate glycosaminoglycan (CS-GAG) chains attached covalently to a core protein. Electrostatic interactions between these GAGs contribute to the compressive and shear stiffness of the tissue. Variations in the structure of aggrecan and its GAG constituents are known to exist as a function of tissue age, disease, and species. Using atomic force microscopy (AFM), we directly visualized the nanometer scale structure of aggrecan deposited on a 2-D substrate, including the first high resolution imaging of individual GAG chains along the core protein. We also visualized and quantified the differences in structure between aggrecan obtained from fetal epiphyseal and mature nasal bovine cartilages. / (cont.) A combination of AFM, biochemical, and polymer statistical methodologies was used to better understand the dependence of aggrecan structure and stiffness on the properties of its constituent GAG chains. The fetal epiphyseal aggrecan had a denser GAG brush region and longer GAG chains, which correlated with a higher effective persistence length of fetal core protein compared to that of mature nasal aggrecan. The effect of increasing the concentration of aggrecan on the substrate resulted in a decrease in molecular extension, suggesting a flexible protein core backbone, which allowed aggrecan to entangle and interact with neighboring molecules. AFM imaging of the conformation of aggrecan that had been deposited on substrates from solutions of varying ionic strength (IS), from DI water to the hysiological IS of 0.1 M NaCl, allowed for direct visualization of the collapse of the molecule on the substrate at the highest IS, due to charge shielding of the CS-GAGs by by Na+ counter-ions. Lastly, the nanomechanical properties of cartilage cells (chondrocytes) and their aggrecan-collagen-rich pericellular matrix (PCM) were probed via AFM nanoindentation using both a sharp nano tip and a larger micro-colloidal tip to better understand the deformation of cells in cartilage. / (cont.) The properties of cells freshly isolated from cartilage tissue, devoid of PCM, were compared to that of cells isolated and then cultured for selected times in 3-D alginate gel to obtain cells surrounded by their newly developed PCM. Using Hertzian contact mechanics as well as finite element analyses, material properties were estimated from the AFM force-indentation curves measured with these cell preparations. We also studied the effects of culture conditions on the resulting PCM properties, comparing 10% fetal bovine serum vs. medium containing a combination of insulin growth factor-i (IGF-1) + osteogenic protein-i (OP-1). While both systems showed increases in matrix stiffness with time in culture between days 7 to 28, the IGF-1 + OP-1 combination resulted in a higher effective modulus for the cell-PCM composite. These AFM cell indentation studies were enabled by the use of microfabricated chips containing wells designed to immobilize the spherical chondrocytes during testing. Due to the nonconventional but known geometry of the microfabricated wells, finite element analysis was used to include the effects of the cell-well boundary conditions and tip geometries on the calculated cell-PCM material properties. / (cont.) Taken together, these studies examining cartilage mechanics at the molecular and cellular levels give insight into the intricate roles that proteoglycans and collagen play in governing tissue-level mechanical properties. / by Laurel Jean Ng. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 161-172). / Gene therapy has the potential to cure thousands of diseases caused by genetic abnormalities, provide novel combination therapies for cancers and viral infections, and offer a new and effective platform for next generation vaccines. However, after more than three decades of research and development efforts, clinical success has yet to be realized. Successful delivery of DNA is a crucial first step in attaining safe and effective gene therapeutics. While vectors based upon recombinant viruses have shown high delivery and transfection efficiencies, they may also pose certain health risks to patients, can be difficult to target to cell or tissue types of interest, and present difficulties for large-scale manufacturing. Non-viral vectors look to offer a safer alternative and can be engineered to more effectively treat a specific cell type, tissue, or pathology, but these vectors are still plagued with low transfection levels and cannot provide adequate and sustained levels of gene expression. Continued efforts focus on producing next generation non-viral vectors that safely deliver therapeutic transgenes with the efficiency of their viral counterparts. Many barriers exist in the successful trafficking of these non-viral complexes to the nucleus. / (cont.) Current evaluations of non-viral gene delivery treatments in more clinical settings often focus on a single barrier at a time, and as a result, may not lead to an overall improvement in gene delivery. Concurrently, more quantitative or systematic in vitro experiments may not correlate well with in vivo data. Our combined approach of quantitative vector trafficking and expression experiments coupled with computational simulation of vector specific mathematical models that describe every step of the gene delivery process has shown that a systems level approach can glean insight into the most rate-limiting steps for a given vector and generate hypotheses for future vector development and improvement. These studies have been extended to primary liver cultures, coupled with device development to attain a more clinically relevant model system and more spatial resolution to study intracellular vector trafficking and localization. A larger perfused 3-D liver bioreactor has been built that allows for long-term culture of primary hepatocytes that more closely mimic hepatic phenotype than in conventional 2-D cultures and for multiplexed quantitative measurement that is not possible in animal models. / (cont.) A newly constructed density gradient electrophoresis device can separate vesicular organelles and track vector dynamics throughout the cell. These systems have provided more comprehensive data sets which show that vectors behave differently in different culture systems and that different vectors show unique cell trafficking dynamics. These results lend insight for future vector screening methodologies and provide vector specific mathematical models for primary cell transfection that can lead to further optimization of the polymer vectors studied in this work, which can contribute to the development of more efficient next generation in vivo delivery agents. / by Nathan C. Tedford. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (leaves 60-61). / Idiosyncratic drug reactions are a subset of adverse reactions frequently targeting the liver, which become obvious only in large sample populations. Drug-induced hepatotoxicity, occurring in a very small fraction of patients, poses a major challenge to pharmaceutical companies due to its unknown mechanism(s) of action and deficient models for study. In vitro model systems may have the potential to predict this liver injury by generating conditions possibly representing key processes involved, both directly and indirectly, in drug effects on cellular physiology. Our ultimate goal is to develop an in vitro model effectively mimicking certain relevant aspects of the in vivo response of the human liver. In our initial effort described herein, we have designed a novel cell-based system using alternatively in both a human hepatoma cell line and primary rat hepatocytes to study toxic effects in a background reflecting in vivo inflammatory conditions. This background incorporates bacterial lipopolysaccharide (LPS) administration along with inflammatory cytokines (tumor necrosis factor, interferon y, interleukin-1 a, interleukin-113, and interleukin-6) previously shown to increase in LPS-administrated rats. Our study began with an investigation of toxicities that are induced by combinations of five cytokines and LPS in HepG2 and C3A human hepatoma cell lines and in primary rat hepatocytes. Informed by the results of these experiments, we selected representative cytokine/LPS treatments and cell systems to examine drug-cytokine synergies in vitro and were able to identify multiple idiosyncratic hepatotoxins that induced synergistic toxicity in either the HepG2 cell line or primary rat hepatocytes. / (cont.) Finally, we measured the sensitization of these cell systems to a panel of these drugs, given an inflammatory background induced by an abbreviated set of cytokine treatments including four cytokines and LPS. Analysis of this multivariate drug-cytokine toxicity data set yielded a subset of representative cytokine treatments for future drug-cytokine synergy investigations. This subset will be used to characterize the differences between cell systems, including cultured human hepatocytes, and to hopefully develop a data-driven partial least squares regression model that predicts idiosyncratic liver injury. The implications are two-fold. First, this model could provide direction to pharmaceutical companies in focusing their drug discovery and development. Second, it could help physicians design better treatment plans for their patients. / by Maya Hasan. / M.Eng.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 66-75). / (cont.) of transcription factors and subsequently, induction of RNA processing (35% of genes incrementally induced) and kinases involved in protein phosphorylation. In the WT, the response was restricted to a transient repression of fundamental biochemical processes. Interestingly, a gene whose repression is known to mimic apoptosis was found to be repressed in the WT. The overwhelming induction of ribosomal protein synthesis genes in both WT and mgtl in response to MNNG is an unexpected result that could signify a successful recovery following wide-spread cellular damage. / Damage to DNA can occur by means of endogenous biochemical processes or exogenous chemicals such as alkylating agents. If left unrepaired, alkylated bases, most notably, O⁶ Methylguanine (O⁶MeG) can be mutagenic and cytotoxic to the cell. Luckily, DNA methyltransferase (encoded by the gene MGT1 in yeast), repairs this damage. By using transcriptional profiling as a tool, an attempt to elucidate the role of MGT1 has been made. First, the basal expression profile of the mgtl was established. Then, the response of wild-type (WI) yeast and yeast lacking MGT1 (mgt1) to the alkylating agent, MNNG was studied using exponentially growing WT and mgti cultures which were exposed to 30[mu]g/ml of MNNG for 10 to 60 minutes. Basal expression profile of yeast lacking MGT1 showed up-regulation of RETV7, a gene implicated in spontaneous mutagenesis. Response to MNNG was invoked immediately and was dramatic and widespread involving 30% of the genome in both WT and mgt1. Cell-cycle checkpoints, damage signal amplifiers, DNA repair genes (nucleotide excision repair, photoreactive repair, mismatch repair) and chromatin remodeling genes were induced. Genes involved in maintaining mitochondrial structure and mitochondrial genome were also induced. Intriguingly, RPN4, a key regulator of proteasomal system was found to be repressed. Environmental stress response genes were culled out to examine the effects of MNNG on WT and mgtl, more carefully. Temporal gene expression profiles in WT and mgtl were informative in delineating differences in the distinct responses mounted by WT and mgtl. The magnitude of response in mgt1 is more profound than in WT. The differences in the dynamic trends between the two suggest that mgt1 initiates a coordinated response involving repression / by Anoop Rao. / S.M.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (leaves 139-141). / The purpose of this thesis was to provide a broad survey of bioprocess options for typical drug production vehicles in the biotechnology industry. This goal was accomplished by comparing the growth and monoclonal antibody production by HPCHO Chinese hamster ovary cells and IB4 hybridoma cells in batch, fed-batch, and three perfusion systems: the stirred ceramic membrane reactor (SCMR), the alternating tangential flow (ATF) hollow fiber membrane system, and the external spin filter (ESF) system. The batch experiments for each cell line were used as base case scenarios for the comparisons of cell growth and monoclonal antibody production. The fed-batch experiments for each cell line did not vastly improve the cell growth and monoclonal antibody production over the batch cases. The SCMR perfusion system greatly improved the cell growth and monoclonal antibody production for each cell line. The maximum viable cell concentration of the IB4 hybridoma cells in the SCMR experiment was over seven times that found in the batch experiment and the monoclonal antibody production in the SCMR experiment was over ten times that found in the batch experiment. / (cont.) The ATF perfusion system greatly increased the cell growth for each cell line over the batch cases, but the shear forces created by the system adversely affected the viability of the cells and the monoclonal antibody production was less than that of the SCMR experiments. The ESF perfusion system damaged the HPCHO Chinese hamster ovary cells. For the production of the IB4 hybridoma cells, the ESF system, as well as a modified ESF system, did not vastly improve the cell growth and monoclonal antibody over the base case due to cell aggregation and filter fouling. The cell growth and productivity data, along with economic considerations, were evaluated for the purpose of recommending the best feeding strategy for each of the two cell lines studied. It was concluded that the most successful operation mode for the two cell lines studied was the SCMR perfusion system. More research is needed regarding the scale-up ramifications of the SCMR perfusion system as well as in the quantification of monoclonal antibodies, the optimization of fed-batch systems, and the use of alternative perfusion systems. / by Kathy Hufford. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (p. 117-127). / One goal underlying synthetic biology is to develop standard biological parts that can be reliably assembled into devices encoding higher-order functions. Here, I developed a framework for engineering post-translational devices, which are devices whose inner workings are modulated by non-covalent protein interactions and covalent protein modifications. To test the framework, I designed a scaffold for engineering post-translational devices in yeast, the Phospholocator, that can be used to assemble peptide parts in order to produce devices that couple upstream kinase activity to regulated nuclear translocation. I used the Phospholocator to design, build, and characterize a Phospholocator device, the Cdc28-Phospholocator, whose location is regulated by the activity of cyclin-dependent kinase Cdc28. I next engineered and tested a Fus3-Phospholocator device, whose location is regulated by the activity of the mitogen-activated protein kinase Fus3, in order to demonstrate that the Phospholocator scaffold supports the engineering of many post-translational devices. I used the Cdc28-Phospholocator to follow Cdc28 activity levels throughout the yeast cell cycle, thereby illustrating the utility of the Cdc28-Phospholocator as a tool for biological inquiry. To implement more complex functions, device engineers will want to connect post-translational devices to build multi-component systems. I thus developed a model for device composition that features a universal signal carrier that is both input into and output from post-translational devices. The universal signal could enable engineers to easily combine devices in any desired order, and thus build many new post-translational systems. / (cont.) I next developed a set of specifications and guidelines for designing prototypical protein parts for engineering post-translational devices that communicate via the universal signal carrier. I used the universal signal model and the corresponding set of device specifications to design and model a proof-of-principle. multi-device post-translational system, a post-translational latch, that functions as designed. Taken together, my initial experiences in engineering post-translational devices, defining universal device signals that enable device interconnectivity, and designing, modeling, and analyzing the model of a functional multi-device system, along with the work of many other groups, are sufficiently encouraging to motivate continued work on post-translational devices. / by Samantha C. Sutton. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (p. 143-160). / Live or attenuated pathogens are the basis of many successful vaccines due in part to the orchestrated response of dendritic cells (DCs) triggered by these immunizations, which includes (1) DC and DC precursor attraction to the immunization site, (2) efficient antigen delivery to class I and class II MHC loading pathways coincident with maturation of DCs, and (3) emigration to draining lymph nodes for T cell activation. We have developed a model immunization system designed to allow these steps in the DC life cycle to be controlled in the context of a subunit vaccine. The system is comprised of microspheres encapsulating chemokines and hydrogel nanoparticles; each nanoparticle contains antigen and DC maturation signals (e.g., TLR ligands). The nanoparticles remain sequestered within the carrier microspheres but the chemokine is released at a controllable rate, creating a local chemoattractant gradient centered on each microsphere. DCs are attracted to individual microspheres where nanoparticles are concentrated; attracted DCs extract nanoparticles from the carrier microspheres, and receive maturation signals coincident with the delivery of antigen into both class I and class II MHC processing pathways. / (cont.) In addition, the nanoparticles may be labeled to allow subsequent tracking of particle-carrying DCs in vivo. These components allow the attraction (or if desired, emigration) of dendritic cells and their precursors to be selectively modulated at an immunization site, and the activation signals received by these cells when they encounter antigen to be tailored. In vitro experiments indicate that chemokine-releasing microspheres effectively attract DCs and monocytes over significant distances, and that the gel nanoparticles efficiently trigger DC maturation and lead to both CD4+ and CD8+ T cell activation in vitro and in vivo. This system provides both a platform for rational immunotherapy as well as a powerful set of tools by which the function of dendritic cells can be manipulated and dissected to improve our understanding of how DC trafficking and functional state impacts immune responses. / by Siddhartha Jain. / Ph.D.