Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references (leaves 38-44). / Nitric oxide is postulated to protect cancer cells from the death-inducing effects of tumour necrosis factor alpha by S-nitrosating the active site cysteines, inhibiting cleavage of caspase-9. We aimed to test this hypothesis and to determine its validity across cancer cell types. In addition, we hoped to explain the involvement of certain kinases in nitric oxide-induced apoptosis. The experimental setup involved stimulating human colorectal cancer cells, HT-29 and HCT- 116, and human prostate cancer cells, LNCaP, with cytokines in order to induce cell death. Then, we observed the effects of NO inhibitors, kinase inhibitors, and activation of Akt, a kinase up-stream of the caspase cascade, following transfection of a DNA sequence that was proven to protect cells against apoptosis induction. In our series of experiments, inhibition of the nitric oxide synthases removes nitric oxide protection from apoptosis, but inhibition of only the inducible synthase has opposite effects with prostate and colon cancer cells that are considered insignificant, and its effects on the two types of colon cancer cells are in discord. Transformation and transfection of ARK5 into the colorectal cancer cell line, HT-29 did not prove beneficial. Similarly, glucosamine showed no clear pattern of reducing apoptosis in the cells. Therefore, we propose further exploration of the inhibition of constitutive nitric oxide synthases as a potential therapy. / by S. Julie-Ann Lloyd. / S.M.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Vita. / Includes bibliographical references (p. 82-89). / TLR-4 is essential in host defense against bacterial infection. By recognition of specific pathogen-associated molecular patterns such as lipopolysaccharide (LPS), TLR-4 can in tandem initiate a pair of downstream signaling pathways to regulate cytokine/chemokine release, endotoxin tolerance and apoptosis, which have been suggested to directly or indirectly participate in the regulation of innate and adaptive immune responses. However, little is known about their detailed signal-response relationships. In this thesis, we sought to identify these potential signal-response relationships in RAW264.7 cells through systematic analysis. We first measured LPS stimulated dynamic signaling profiles over a range of an inhibitor of p38 MAPK, SB202190 concentrations for a distribution of kinases centrally involved in TLR-4 signaling network. We then applied quantitative analytical approaches to determine the most important signals or signal combinations contributing to induction of either IL-6 and TNF-ao secretion or apoptosis and construct their corresponding predictive mathematical models. Particularly, we found that the partial least squares regression (PLSR) models built using the ratio of phosphorylated Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) predicted LPS plus SB202190-induced apoptosis accurately even following perturbation with pharmacological inhibitors of JNK and ERK. Thus, by combining experimental and computational approaches, this thesis has proposed two new potential targets, JNK and ERK, for development of drug therapies against bacterial infection. / by Rongcong Wu. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / MIT Institute Archives copy: DVD inserted in pocket on p. [3] of cover on v. 1. / "c2007"--p. ii. / Includes bibliographical references (v. 2, leaves 292-302). / A grand challenge of systems biology is to model the cell. The cell is an integrated network of cellular functions. Each cellular function, such as immune response, cell division, metabolism or apoptosis, is defined by an interconnected ensemble of biological pathways. Modeling the cell or even one cellular function requires a computational architecture that integrates multiple biological pathway models in a scalable manner while ensuring minimal effort to maintain the resulting integrated model. Scalable is defined as the ease in which more and more biological pathway models can be integrated. Current architectures for integrating biological pathway models are primarily monolithic and involve combining each biological pathway model's software source code to build one large monolithic model that executes on a single computer. Such architectures are not scalable for modeling complex cellular functions or the whole cell. We present Cytosolve, a new computational architecture that integrates a distributed ensemble of biological pathway models and computes solutions in a parallel manner while offering ease of maintenance of the integrated model. The individual biological pathway models can be represented in SBML, CellML or in any number of formats. The EGFR model of Kholodenko with known solutions is used to compare the Cytosolve solution and computational times with a known monolithic approach. A new integrative model of the interferon (IFN) response to virus infection is developed using Cytosolve. Each model within the integrated model, spans different time scales, is created by different authors from four countries and three continents across different disciplines, is written in different software codes, and is built on different hardware platforms. / (cont.) A new quantitative methodology and formalism is then derived for evaluating different types of monolithic and distributed architectures for integrating biological pathway models. As more biological pathway models develop in a disparate and decentralized manner, the Cytosolve architecture offers a unique platform to build and test complex models of cellular function, and eventually the whole cell. / by V.A. Shiva Ayyadurai. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / "June 2005." / Includes bibliographical references. / Micro- and nanofabrication approaches have dramatically changed our society through their use in microelectronics and telecommunication industries. These engineering tools are also useful for many biological applications ranging from drug delivery to DNA sequencing, since they can be used to fabricate small features at a low cost and in a reproducible manner. The goal of this thesis was to develop techniques based on the merger of novel materials and nano and microfabrication approaches to manipulate cell microenvironment in culture. To control cell migration and to restrict cell or colony size, cells and proteins were patterned by using molding or printing methods. Poly(ethylene glycol)-based molecules and polysaccharides were used to control cell-substrate interactions and to prevent cell adhesion on specific regions of a substrate. To control cell-cell contact, layer-by-layer deposition of ionic biopolymers (i.e. negatively charged hyaluronic acid and positively charged poly-L-lysine) was used to generate patterned co-cultures. In addition, to control cell-soluble factor interactions, microfluidic-based approaches were developed. To pattern cells and proteins within microchannels, a soft lithographic method was developed to pattern microchannel substrates using printing and molding approaches. / (cont.) To easily immobilize cells within channels, poly(ethylene glycol) microstructures were used to capture cells within low shear stress regions. These techniques also allowed for the fabrication of multiphenotype cell arrays. In addition, techniques were developed to control the interaction of cells within hydrogels by controlling the spatial properties of hydrogels. / by Ali Khademhosseini. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 51-53). / Neurodegenerative diseases typically affect a limited number of specific neuronal subtypes, and the death of these neurons causes permanent loss of a specific motor function. Efforts to restore function would require regenerating the affected cells, but progress is limited by a narrow understanding of the mechanisms that underlie the generation of these neurons from their progenitor cells. In order to prevent neuronal degeneration and potentially repair or regenerate the damaged motor output circuitry, it will be necessary to understand the molecular and genetic factors that control, direct, and enhance differentiation, axonal projection and connectivity. While techniques are available to separate specific populations of neurons once they are fully-differentiated, current methods make it nearly impossible to monitor or control the development of a neural precursor in standard open culture. To carry out directed differentiation experiments effectively, it will be critical to control how signals are introduced to the cells. In this study, we present a microfluidic system to address the limitations of previous research. / (cont.) The device is capable of generating a controlled gradient of chemoattractant or growth factor of interest and directing axonal growth through an extra-cellular matrix material. Once the cells have grown into the device, signals and gradients can be applied directly to either the cell bodies or the axons. This device will serve as a platform technology for future experimentation with biomaterial scaffolds for neural tissue engineering, drug design or testing, and eventually directed differentiation of neural precursor cells. / by Johanna Varner. / M.Eng.

Thesis (S.M. in Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2002. / Includes bibliographical references (leaf 23, 1st group). / A formal series of conditions of lifetime genetic risk of cancer is explored, and algebra is provided for applications in human population genetics. Risks are considered in terms of alleles necessary and/or sufficient for carcinogenesis. Alleles are first classified with respect to their effects on reproductive fitness, and then in terms of their potential effects on carcinogenic pathways. The algebraic formulations for a series of genetic possibilities: monogenic, multigenic, and polygenic, are provided. It is expected that technology will be developed to identify and enumerate rare inherited alleles in large general and cancer proband populations. / by Helen Banava. / S.M.in Toxicology

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references (leaves 112-127). / The design and development of new proteins and small molecules has considerable practical application in medicine, industry, and basic science. Frequently, progress in this area is made by altering an existing small molecule or protein for new function. This thesis presents methods for the analysis and design of rationally and evolutionarily designed molecules and focuses on applying these methods to make protein and small molecule changes more strategically. First, electrostatic analysis of a series of small molecule neuraminidase inhibitors was used to demonstrate that charge optimization improves the electrostatic component of the binding free energy, despite changes in binding mode and discrete chemical constraints. Additionally, chemical changes suggested by charge optimization frequently corresponded to tighter-binding inhibitors, indicating that this technique would be useful for the design of future inhibitors. Second, computational sequence and structure analysis were used to study the PDZ3-CRIPT binding interaction and a method for sequence analysis was developed to locate residues important for binding specificity. Third, computational analysis of the horseradish peroxidase active site suggested five positions as candidates for mutation, and further studies of new mutant enzymes let to ideas for the improvement of computational enzyme design procedures. Finally, both computational protein design techniques and a model of the evolutionary process were used to study the efficiency of evolution as a tool for creating new proteins in the laboratory. We identified sequences that serve as better evolutionary starting points that others and provide a general framework for considering the impact of protein structure on the allowed sequence space and therefore on the challenges that each protein presents to evolutionary protein engineering procedures. / by Kathryn Anne Armstrong. / Ph.D.

Thesis (Ph. D. in Bioengineering)--Massachusetts Institute of Technology, Biological Engineering Division, 2003. / Includes bibliographical references. / Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix accumulation in a three-dimensional geometry. In these studies, a self-assembling peptide hydrogel is evaluated as a potential scaffold for cartilage repair using a model bovine cell source. A seeding technique is developed for 3-D encapsulation of chondrocytes in a peptide hydrogel. The chondrocyte-seeded peptide hydrogel was then evaluated cellular activities in vitro under standard culture conditions and also when subjected to dynamic compression. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically-functional neo-tissue. Culture of chondrocyte-seeded peptide hydrogels in ITS-supplemented medium was investigated as an alternative to high serum culture. Low serum (0.2%), ITS-supplemented medium was found to maintain high levels of cell division and extracellular matrix synthesis and accumulation, as seen in high serum culture. Furthermore, low serum, ITS medium induced minimal chondrocyte de-differentiation on the surface of the hydrogel. This is in contrast to high serum culture, where surface de-differentiation and subsequent proliferation led to a 5-10 cell thick layer that stained positive for type I collagen. / (cont.) The effects of dynamic compression of chondrocyte-seeded peptide hydrogels were evaluated over long-term culture. A non-continuous loading protocol was identified in which proteoglycan, but not protein, synthesis increased over static, free-swelling culture. Increases in GAG matrix accumulation were observed after at least 8 days of loading, while hydroxyproline accumulation was unaffected by dynamic compression. These data demonstrated dynamic compression differentially regulated the synthesis of proteoglycans. Analysis of GAG loss to the medium indicated peak proteoglycan catabolism occurred immediately after the initiation of loading. This phenomenon was further explored using a modified loading protocol that increased GAG loss to the medium. Peak GAG loss to the medium was 2-fold higher than previously observed, resulting in GAG accumulation values significantly less than controls. Hydroxyproline accumulation was minimally affected by loading, demonstrating that dynamic compression also differentially regulated the catabolism of proteoglycans. Proteoglycan catabolism was not predominantly due to physical disruption accumulated extracellular matrix or loss of newly-synthesized molecules. Instead, the presence of MMPs in the medium that coincided with GAG loss suggest a potential enzymatic mechanism. These results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like extracellular matrix ... / John D. Kisiday. / Ph.D.in Bioengineering

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (leaves 57-62). / Implantable, controlled release drug delivery devices offer several advantages over systemic oral administration routes and immediate drug release treatments including direct therapy to target organ, more continuous maintenance of plasma and tissue drug levels and the potential for reduced side effects or toxicity. Urology has emerged as a unique field in which minimally invasive implantation techniques are available and such devices could provide improved beneficial therapies over conventional treatments. Urological indications for which localized drug therapy is already being advocated and investigated are highly suitable for treatment with implantable controlled release devices. This thesis describes the in vitro performance evaluation of an implantable, bio-resorbable device that can provide localized drug therapy of ciprofloxacin (CIP) to the seminal vesicle and nearby prostate gland for treatment of chronic prostatitis (CP). The device functions as an elementary osmotic pump (EOP) to release CIP for a period of 2-3 weeks after implantation in the seminal vesicle (SV) through transrectal needle injection or cystoscopic methods. The device is composed of an elastomeric, resorbable polymer cast in a tubular geometry with solid drug powder packed into its core and a micromachined release orifice drilled through its wall. Drug release experiments were performed to determine the effective release rate from a single orifice and the range of orifice size in which osmotic-controlled zero-order release was the dominant mechanism of drug delivery from the device. Device stability and function in an alkaline environment of similar pH to that of the SVs and infected prostate gland was also assessed in vitro. The device was found to function well in both de-ionized water and NaOH pH-8 solution with a sustained zero-order release rate of 2.47 ± 0.29 jtg/hr when fabricated with an orifice of diameter 100-150pm. / by Irene S. Tobias. / M.Eng.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 91-93). / This work describes the integration of novel microfabrication techniques for vascular tissue engineering applications in the context of a novel biodegradable elastomer. The field of tissue engineering and organ regeneration has been born out of the high demand for organ transplants. However, one of the critical limitations in regeneration of vital organs is the lack of an intrinsic blood supply. This work expands on the development of microfluidic scaffolds for vascular tissue engineering applications by employing microfabrication techniques. Unlike previous efforts, this work focuses on fabricating this scaffolds from poly(glycerol-sebacate) (PGS), a novel biodegradable elastomer with superior mechanical properties. The transport properties of oxygen and carbon dioxide in PGS were measured through a series of time-lag diffusion experiments. The results of these measurements were used to calculate a characteristic length scale for oxygen diffusion limits in PGS scaffolds. Microfluidic scaffolds were then produced using fabrication techniques specific for PGS. Initial efforts have resulted in solid PGS-based scaffolds with biomimetic fluid flow and capillary channels on the order of 10 microns in width. These scaffolds have also been seeded with endothelial cells and perfused continuously in culture for up to 14 days resulting in partially confluent channels. More complex fabrication techniques were also demonstrated. A novel electrodeposition technique was used in the fabrication of biomimetic microfluidic masters. Thin-walled devices were also synthesized to accommodate the relatively low gas permeability of PGS. / by Christopher John Bettinger. / M.Eng.