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 (leaves 180-203). / The aim of this thesis is to consider how fundamental engineering principles might best be applied to the design and construction of engineered biological systems. I begin by applying these principles to a key application area of synthetic biology: metabolic engineering. Abstraction is used to compile a desired system function, reprogramming bacterial odor, to devices with human-defined function, then to biological parts, and finally to genetic sequences. Standardization is used to make the process of engineering a multi-component system easier. I then focus on devices that implement digital information processing through transcriptional regulation in Escherichia coli. For simplicity, I limit the discussion to a particular type of device, a transcriptional inverter, although much of the work applies to other devices as well. First, I discuss basic issues in transcriptional inverter design. Identification of key metrics for evaluating the quality of a static device behavior allows informed device design that optimizes digital performance. Second, I address the issue of ensuring that transcriptional devices work in combination by presenting a framework for developing standards for functional composition. The framework relies on additional measures of device performance, such as error rate and the operational demand the device places on the cellular chassis, in order to proscribe standard device signal thresholds. Third, I develop an experimental, proof-of-principle implementation of a transcriptional inverter based on a synthetic transcription factor derived from a zinc finger DNA binding domain and a leucine zipper dimerization domain. Zinc fingers and leucine zippers offer a potential scalable solution to the challenge of building libraries of transcription-based logic devices for arbitrary information processing in cells. / (cont.) Finally, I extend the principle of physical composition standards from parts and devices to the vectors that propagate those parts and devices. The new vectors support the assembly of biological systems. Taken together, the work helps to advance the transformation of biological system design from an ad hoc, artisanal craft to a more predictable, engineering discipline. / by Reshma P. Shetty. / 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. 165-176). / The aim of my thesis is to help enable the engineering of biological systems that behave in a predictable manner. Well-established techniques exist to engineer systems that behave as expected. Here, I apply such techniques to two aspects of the engineering of biological systems. First, I address the design and construction of standard biological devices in a manner that facilitates reuse in higher-order systems. I describe the design and construction of an exemplar device, an engineered cell-cell communication receiver using standard biological parts (refined genetic objects designed to support physical and functional composition). I adopt a conventional framework for describing the behavior of engineered devices and use the adopted framework to design and interpret experiments that describe the behavior of the receiver. The output of the device is the activity of a promoter reported in units of Polymerases Per Second (PoPS), a common signal carrier. Second, I begin to address the coupling that exists between engineered biological systems and the host cell, or chassis. I propose that the coupling between engineered biological systems and the cellular chassis might be reduced if fewer resources were shared between the system and the chassis. I describe the construction of cellular chassis expressing both T7 RNA polymerases (RNAP) and orthogonal ribosomes that are unused by the chassis but are available for use by an engineered system. I implement a network in which the orthogonal ribosomal RNA and the gene encoding T7 RNAP are transcribed by T7 RNAP. In turn, the orthogonal ribosomes translate the T7 RNAP message. In addition, the T7 RNAP and orthogonal ribosomes express a repressor that inhibits transcription of both the T7 RNAP and orthogonal ribosomes. / (cont.) As a result, the orthogonal RNAP and ribosomes are auto-generating and self-regulating. The provision of resources unused by the cellular chassis and dedicated to an engineered biological system forms the beginnings of a biological virtual machine. / by Bartholomew Canton. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (leaves 100-112). / Cell survival, growth, differentiation, migration, and communication all depend on the appropriate combination of specific interactions between proteins and biomolecules. Therefore, understanding the molecular mechanisms influencing protein-biomolecule binding interactions is important both for fundamental knowledge and as a foundation for therapeutic applications and biotechnology. This thesis presents two applications of computational modeling to study protein-biomolecule binding in different contexts. First, we sought to characterize effects of applied mechanical force on protein structural and biochemical properties. Despite growing experimental evidence of force-regulated cell behavior, the molecular mechanisms involved in force sensing and transmission are still largely unknown. We adapted a free energy method to directly compute the change in binding affinity upon force application. Our simulations demonstrated that differential responses in the bound and unbound state of a protein-ligand complex can lead to graded force-modulation of binding affinity. Application to a prototypical protein system - the helical bundle complex of a paxillin fragment bound to the FAT domain of focal adhesion kinase (FAK) revealed several structural mechanisms responsible. Second, we used computational methods to design individual mutations computed to improve binding affinity of an antibody-small molecule complex with relevance to cancer treatment. Our calculations suggested several beneficial mutations for experimental characterization. The work illustrates the value of computational modeling for understanding protein-biomolecule interactions with application to therapeutic development and advances in biotechnology. / by Aurore Zyto. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / "September 2006." / Includes bibliographical references (p. 141-147). / Nonviral vector research and development has been stunted by a lack of knowledge and understanding of how vectors are trafficked within the cell. Research currently involves mass screenings of different combinations of vector components without a true understanding of how each component interacts with the target cell. Few tools are currently available for scientists to quantitatively examine these vector-to-cell interactions or determine the rate limiting steps within the gene delivery pathway. Thus, researchers cannot fully optimize the vector design to reach maximal delivery efficiency. This project seeks to address this issue by modifying a density gradient electrophoresis (DGE) device originally developed on Mel Juso cells to segregate primary rat hepatocyte lysate into nuclear, early endosomal, late endosomal/lysosomal, and cytoplasmic fractions. We found that according to the Horseradish Peroxidase assay, late endosomes and lysosomes consistently localize to fractions 11-13 and early endosomes in fractions 18 to 21. There was minimal labeling in fractions 14 through 17 demonstrating that separation of the organelles was achieved. With this higher resolution fractionation, movement through the endosomal pathway can be studied in greater detail. / (cont.) The rates with which each vector moves from outside of the cell into the early endosome, to the late endosome, to the cytoplasm and into the nucleus can be quantified. The steps affected by specific modifications to the vector design and the vector properties most important for delivery efficiency can be identified. As vectors are sorted differently in different cell types, this DGE device will allow researchers to gain insight of the cell-specific sorting mechanisms. Ultimately, DGE can aid design of vectors that reach delivery efficiencies comparable to viruses and tailor the vectors to the tissue of interest. / by Jennifer Fang. / M.Eng.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 35-40). / The study of stem cells is one of the most fascinating topics in biology. Adult stem cells (ASC), which play the prime role in the maintenance and restoration of tissues, are thought to hold great potential for the advancement of medicine. It has been postulated that adult stem cells are able to retain "immortal" DNA template strands over successive generations by non-random chromosome co-segregation, and in so doing, to protect the long-term genomic fidelity of whole tissue compartments. The investigation of this theory may yield insights into areas such as the development of cancer and the process of aging. In addition, it may lead to the discovery of an effective method for the unique identification of adult stem cells, the study of which has thus far suffered from the lack of unique identifiers. Thus, the goal of this research was to develop an assay for the detection of immortal DNA strand co-segregation that could be applied to the detection and analysis of adult stem cells. It is proposed that such an assay may in itself serve as a unique identification method for adult stem cells. In this thesis, the development of such an assay is described. This assay, referred to as the label release assay, has provided further evidence for the existence of immortal strand co-segregation in model cell lines, and will potentially be useful in the study of adult stem cells in tissues. / by Jennifer J. Cheng. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 111-127). / To shed light on the cell's response to its mechanical environment, we examined cell rheology at the single cell level and quantified it with nanometer spatial and microsecond temporal resolutions over a five-decade frequency range (- 0.5 Hz to 50 kHz). To this end, we developed and optimized an instrument for fast fluorescence laser tracking microrheology (FLTM). This novel method aims at experimentally deriving cellular viscoelastic properties from the passive monitoring of fluorescent microspheres undergoing Brownian motion inside the tested sample. Further instrument enhancement even broadens the FLTM frequency span up to seven decades by modulating data acquisition speed or complementing FLTM with a two-particle microrheology modality. In living cells, FLTM accurately characterizes the solid-like vs. liquid-like cytoskeletal behavior from measurements based on endocytosed micron-sized beads, independently of probe size or surface chemistry. FLTM also demonstrates the existence of two distinct rheological regimes on the cell surface and in the cell interior: While the former surface investigations show power-law frequency variations of the complex shear modulus G*(co), the latter intracellular experiments identify multiple time and length scales affecting cell rheological features. Finally, FLTM evaluates frequency-specific stretch-induced cell mechanics and thus promises to broaden and diversify the scientific knowledge on mechanotransduction, from a molecular and cellular standpoint. / (cont.) FLTM also demonstrates the existence of two distinct rheological regimes on the cell surface and in the cell interior: While the former surface investigations show power-law frequency variations of the complex shear modulus G*(co), the latter intracellular experiments identify multiple time and length scales affecting cell rheological features. Finally, FLTM evaluates frequency-specific stretch-induced cell mechanics and thus promises to broaden and diversify the scientific knowledge on mechanotransduction, from a molecular and cellular standpoint. / by Maxine Jonas. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, June 2006. / "February 2006." / Includes bibliographical references (leaves 56-59). / Following exposure to DNA damage, cells initiate a stress response involving multiple protein kinase signaling cascades. The DNA damage response results in one of several possible cell-fate decisions, or cellular responses: induction of cell-cycle arrest, initiation of DNA repair, activation of transcriptional programs, and either apoptosis, necrosis or cell senescence. The mechanisms by which cells make these decisions, and how cell fate depends upon variables such as DNA damage type and dose, and other environmental factors, is unknown. The process by which cells select among alternate fates following such stimuli, or "cues" is likely to involve a dynamic, multi-variate integration of signals from each of the kinase signaling components. A major goal of signal transduction research is to understand how information flows through signal transduction pathways downstream of a given cue, such as DNA damage, and how signals are integrated, in order to mediate cellular responses. Mathematical modeling approaches are necessary to advance our understanding of these processes. / (cont.) Indeed, statistical mining and modeling of large datasets, consisting of quantitative, dynamic signaling and response measurements, is capable of yielding models that identify key signaling components in a given cue-response relationship, as well as models that are highly predictive of cellular response following novel cues that perturb the same network. We have validated a novel assay system that allows for the high throughput collection of quantitative and dynamic signaling data for 7 protein kinases or phospho-proteins known to be "hubs" in the DNA damage response and/or general stress response networks, including ATM, Chk2, H2AX, JNK, p38, ERK and p53. This novel high-throughput In-cell Western assay is based on immuno-fluorescent staining and detection of target proteins in a "whole cell" environment, performed and visualized in a 96-well plate format. This assay allows for the detection and measurement of up to 7 target proteins in triplicate, over up to 3 treatment regimes, or up to 21 signals for a single treatment, simultaneously. Pre-processing steps, and steps involved in the protocol itself are significantly fewer (and require smaller amount of most reagents and biological material), / (cont.) as compared to traditional signal measurement methods, such as quantitative Western analysis and kinase assays. We have used this novel high-throughput In-cell Western assay to investigate the DNA damage response after the specific induction of DNA double strand breaks (DSB). We have measured the dynamics of seven "hub" proteins modified with activating phosphorylations (as a surrogate measure of protein activity) that span major branches of the DNA damage, stress, and death signaling networks, following the specific induction of DNA double strand breaks. Signaling proteins measured include ATM, Chk2, H2AX, JNK, p38, ERK and p53. In parallel with these signaling measurements, we have quantitatively measured corresponding phenotypic responses, such as cell cycle profile and apoptosis. In future work, we will use a Partial Least Squares (PLS) regression analysis approach to construct a statistical model using this data, which is predictive of the cellular responses included in our measurements, following perturbation of this branch of the DNA damage response network. This analysis should reveal key signaling components involved in the decision-making process (possible molecular targets for the improvement of cancer therapy regimens that rely upon the induction of DSB, e.g. the topoisomerase inhibitor, cisplatin), and provide a basis for constructing new, and improving existing, physics-chemical models of this branch of the DNA damage response network. / by Andrea R. Tentner. / S.M.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaf 39). / Dynamic material properties of the isolated mouse tectorial membrane (TM) were quantified by applying sinusoidal shearing forces to the TM with a microfabricated probe (contact area 30 x 30 [micro]m²). Forces in the range 30-300 nN with frequency in the range 10-9000 Hz were applied tangentially to the surfaces of four TM specimens. We found that TM displacement was linear with respect to force. The TM exhibited both elastic and viscous characteristics, with the elastic behavior being more prominent. The elastic and viscous components of TM impedance remained proportional through two decades of frequency. The radial impedance was approximately three times larger than the longitudinal impedance. The point stiffness of the TM increased with frequency when radial forces were applied but showed no trend with frequency for longitudinal forces. Displacement of surrounding tissue decreased as distance from the probe increased. Space constants were on the order of tens of micrometers. These results represent the most detailed shear measurements to date of the isolated TM and are consistent with those obtained using the magnetic bead method [AF00]. / by Jianwen Wendy Gu. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references. / A multi-faceted approach was applied to the molecular design of substrates for the selective adhesion, proliferation and differentiation of connective tissue progenitors (CTPs) from human bone marrow aspirates. The basic premise of the thesis is that integrin-specific adhesion peptides, when presented in a biophysically appropriate spatial arrangement against an inert background, allow enrichment of CTPs in vitro. Comb copolymer comprising a methyl methacrylate backbone with 10-mer poly(oxyethylene) sidechains was selected as the vehicle to present small adhesion peptides at the surface. This polymer shows excellent performance in cell resistance studies and offers sufficient functionalizable sites to create high local densities of ligands. Methods for preparing comb copolymer substrates with peptides in [approx.] 300 nm² clusters with inter-ligand spacings closer than integrins were developed. This nanometer-scale clustered presentation was favorable to integrin binding. Cells were more spread on RGD peptide substrates with a higher degree of nanoscale clustering but of the same overall peptide surface density as comparable substrates with lower degree of peptide clustering. We evaluated adhesion peptides for their ability to support CFU formation of marrow-derived CTPs using colony forming unit (CFU) assay. / (cont.) The results, analyzed with a statistical model implemented to capture characteristics of CFU assay, showed that while RGD substrates supported a moderate amount of alkaline phosphatase -positive CFU (CFU-AP) formation, the bone sialoprotein peptide FHRRII(A, and two [alpha]4 [beta]1 peptides demonstrated the best performance in promoting CFU-AP formation. Patient variability in CFU data could be partially explained by the variations in marrow aspirate cell integrin expression, particularly [alpha]5 and [alpha]v [beta]3. The high level of ECM protein association seen with aspirate cells, as revealed by immunoblotting, may inhibit cell adhesion and account for the fairly low CFU counts observed. Treatments of marrow aspirate with phosphate saline buffer (PBS) and RGD solution reduced a significant amount of protein association. A comprehensive study showed that patients' marrow aspirates were naturally partitioned into two groups of very different colony formation behavior and integrin and AP expressions but consistency was observed within each group. Upon treatment of marrow cells with divalent ion-free PBS, CFU-AP formation on RGD substrates drastically increased in one group of patients. The designs and assays developed in this thesis could be applied for the further understanding of marrow aspirates, such as their interaction with a high-affinity [alpha]5 [beta]1 peptide, and with that knowledge, further optimize the surface design of bone marrow grafts. / by Ada Au. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, June 2008. / "May 2008." / Includes bibliographical references. / Despite increased awareness of the obesity epidemic and a higher incidence of the metabolic syndrome in humans, the incidence of obesity and its comorbidities-most notably, type II diabetes (T2D) and cardiovascular disease (CVD)-continues to increase. Although numerous animal models are available to study the molecular pathways, genetics and therapeutic/prevention strategies for T2D, no model completely recapitulates T2D or it's comorbidities in humans. Therefore, novel animal models represent valuable research tools in understanding T2D. To develop a novel mouse model of T2D, I characterized an outbred mouse discovered at MIT that displayed clinical signs of diabetes. Prevalence of glucosuria in the Swiss Webster colony reached 60% (n=70) in males 8 weeks to 6 months of age. Despite severe obesity in some females, no females were diabetic. Pathologic findings in affected males included cachexia, dilated gastrointestinal tracts with poor muscular tone, pancreatic islet degeneration and atrophy with compensatory metaplasia and/or neogenesis, bacterial pyelonephritis, membranous glomerulopathy, and late-onset hepatic tumors with macrosteatosis, microsteatosis and hydropic change in aged males. Serum insulin correlated with blood glucose in a nonlinear pattern suggestive of islet exhaustion. Circulating leptin levels showed a weak inverse correlation with glucose. Diabetic males were bred with obese colony females to produce 20 male and 20 female offspring. Prevalence of diabetes in male offspring was 80% (16/20) with a median age of onset of 18 weeks. In contrast, no diabetic females were identified, despite being significantly more obese than males. Male predominance is likewise a feature of T2D in humans. / (cont.) To our knowledge, this is the first documentation of hepatocellular carcinoma and islet metaplasia and/or neogenesis in a spontaneous outbred mouse model of T2D. The SW availability and histolopathologic features represent a promising new model for the study of T2D. Further studies are required for complete molecular and genetic characterization of the diabetic SW mouse. These studies are outlined in this thesis. / by Laura B. Lemke. / S.M.