Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (leaves 56-59). / Gene Fabrication technology involves the development and optimization of methods relevant to the in vitro synthesis of any given target gene sequence(s) in the absence of template. The driving purpose of this field of research is to bring about the capability for on-demand fabrication of a DNA construct of arbitrary length and sequence quickly, efficiently, and cost-effectively. The first part of this document describes many of the important considerations in performing successful de novo gene synthesis from a survey of the literature as well as from our own work. Recommendations are made for a universally effective, robust, and simple protocol for potential users of gene synthesis, discussing important factors such as choice of protocol, source of commercial oligonucleotides, and polymerase choice. The second part of this document focuses on error correction. Reducing error rates is one of the main challenges in gene fabrication because high error rates preclude the possibility of fabricating long gene targets in a practical and economical manner. Improvements in error rates are essential for continued progress in the development of gene fabrication technology. I discuss the importance of error rate in gene synthesis from a practical standpoint and show results in the development of novel methods for the removal of errors from a pool of synthesized DNA. / by Jason Sun-hyung Park. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (leaves 149-151). / Linkage disequilibrium studies have discovered few gene-disease associations for common diseases. The explanation has been offered that complex modes of inheritance govern risk for cancers, cardiovascular and cerebrovascular diseases, and diabetes. Such studies, however, depended on the untested assumption of monoallelic risk. My research advisor and I set out to investigate whether simple forms of inherited risk, monoallelic or multiallelic, could be excluded by analysis of familial risk for a common disease, such as colorectal cancer (CRC). First, we derived formulae that describe the risk for monogenic, multigenic, and polygenic possibilities of Mendelian inheritance. Next, we obtained an estimate of minimum lifetime risk for CRC of >0.26. Then, we examined the case of late-onset CRC, using the Swedish Family Cancer Database (1958-2002) to estimate the familial relative risk for CRC diagnosis at age 50 or older, and obtained an estimated range of 1.5 to 3.0. We compared this range of actual values to the ranges of expected values for monogenic, multigenic, and polygenic modes of inheritance. / (cont.) We delimited bounds that can be placed on the conditions for various modes of inheritance. The key observation is that monogenic risk for CRC is included among various possibilities, and cannot be eliminated by existing observations. The arguments herein indicate that further efforts can and should be made to obtain more precise estimates of familial risk for CRC and other common forms of cancer. / by Helen Banava. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references (p. 119-134). / How do cells sense their environment and decide whether to live or to die? This question has drawn considerable interest since 1972, when it was first discovered that cells have an intrinsic ability to self-destruct through a process called apoptosis. Since then, apoptosis has been shown to play a critical role in both normal physiology and disease. In addition, many of the basic molecular mechanisms that control apoptosis have been revealed. Yet despite the known list of interactions and regulators, it remains difficult to inspect the network of apoptosis-related proteins and predict how cells will behave. The challenge is even greater when one considers interactions with other networks that are anti-apoptotic, such as growth-factor networks. In this thesis, we develop an approach to measure, analyze, and predict how complex intracellular signaling networks transduce extracellular stimuli into cellular fates. This approach entails three interrelated aims: 1) to develop high-throughput, quantitative techniques that measure key nodes in the intracellular network; 2) to characterize the quantitative changes in network state and cell behavior by exposing cells to diverse fate-changing stimuli; and 3) to use data-driven modeling approaches that analyze large signaling-response datasets to suggest new biological hypotheses. / (cont.) These aims were focused on an apoptosis-survival cell-fate decision process controlled by one prodeath cytokine, tumor necrosis factor (TNF), and two prosurvival stimuli, epidermal growth factor (EGF) and insulin. We first developed radioactive- and fluorescence-based high-throughput assays for quantifying activity changes in the kinases that catalyze key phosphorylation events downstream of TNF, EGF, and insulin. By combining these assays with techniques measuring other important posttranslational modifications, we then compiled over 7000 individual protein measurements of the cytokine-induced network. The signaling measurements were combined with over 1400 measurements of apoptotic responses by using partial least squares (PLS) regression approaches. These signaling-apoptosis regression models predicted apoptotic responses from cytokine-induced signaling patterns alone. Furthermore, the models helped to reveal the importance of previously unrecognized autocrine cytokines in controlling cell fate. This thesis has therefore shown how cell decisions, like apoptosis-versus-survival, can be understood and predicted from the quantitative information contained in the upstream signaling network. / by Kevin A. Janes. / Ph.D.

Thesis (Ph. D. in Molecular Systems Toxicology and Pharmacology)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Vita. / Includes bibliographical references. / High affinity antibody binding interactions are important for both pharmaceutical and biotechnological uses. However, designing higher affinity interactions has remained difficult. Both high affinity interactions from nature and the results from directed evolution affinity maturation processes may yield clues about the important structural and energetic contributions to attain these tight associations. In this Thesis, we investigate affinity maturation of antibodies for very high affinity binding. Two single chain antigen-binding fragment (scFv) antibody systems that were engineered to obtain higher affinity interactions through directed evolution were probed using biophysical techniques to illuminate affinity modulation in proteins. First, anti-c-erbB-2 antibodies and their binding partner, the extracellular domain of the glycoprotein tumor antigen c-erbB-2, were examined. Thermodynamic studies were carried out on the originally identified human scFv and three higher affinity mutants. Although the first two steps included either entropic or enthalpic gains to affinity, the third improvement came from both types. This study demonstrates that a single energetic component is not generally responsible for the increased affinity within a given protein-protein affinity maturation pathway. Second, a family of anti-fluorescein antibodies and their binding to the small molecule fluorescein-biotin were explored. The femtomolar affinity matured anti-fluorescein antibody, 4M5.3, was compared to its wildtype high affinity precursor, 4-4-20. Affinity, thermodynamic, kinetic, and structural characterization of the binding identified 4M5.3 as one of the highest engineered affinity protein binding interactions known and / (cont.) illuminated how subtle structural changes can lead to large consequences for the kinetics and free energy of binding. The affinity mechanisms were further studied by the creation of a series of partial mutants. Context dependent and independent mutational effects on binding affinity indicated the extent of complexity in higher affinity mechanisms attained through directed evolution affinity maturation processes. These studies emphasize the importance of a large number of residues working in concert to create a very high affinity binding molecule. Based on these results, both rational design and directed evolution studies will need to allow for mutations in a spatially broad range around the binding site and involve many biophysical contributions to the binding free energy to reach very high antigen binding affinities. / by Katarina S. Midelfort. / Ph.D.in Molecular Systems Toxicology and Pharmacology

Thesis (Ph. D. in Molecular and Systems Toxicology and Pharmacology)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references. / Tetracyclines, a broad-spectrum class of antibiotics, were discovered in the late 1940s, and became widely used because of their important advantages: they are inexpensive, safe, demonstrate good oral absorption, and are active against a broad range of bacterial pathogens. Unfortunately, as with most antibiotics, the emergence of microbial resistance to tetracyclines has become a serious problem. Today, most genera examined have tetracycline-resistant isolates, although the percentage varies according to species and geographic location. Due to the emergence of resistance, tetracyclines are no longer the antibiotics of choice in treatment of many conditions, although they are still extensively used to treat a variety of bacterial infections. Substantial research efforts have been directed towards reversing tetracycline resistance in bacteria. This work describes the development of a novel anti-bacterial treatment for diseases caused by bacteria resistant to tetracycline. It was found that tetracycline-resistant bacteria expressing the TnlO gene of tetracycline resistance, upon induction with tetracycline, became extremely susceptible to destruction by the DNA-damaging anti-cancer drug cisplatin. Tetracycline-resistant bacteria grown in tetracycline and subsequently treated with cisplatin in the presence of tetracycline were killed about 10⁵-fold more effectively than wild-type bacteria and tetracycline-resistant bacteria not exposed to tetracycline. This phenomenon was observed in different strains of tetracycline-resistant E. coli. Other antibiotics tested with respective antibiotic-resistant bacteria did not produce the same effect of sensitization to cisplatin, suggesting a unique relationship among cisplatin, tetracycline and the tetracycline resistance / (cont.) gene. It was determined that levels of platinum DNA damage were higher in sensitized tetracycline-resistant cells than in wild-type cells, although total cellular platinum levels in sensitized tetracycline-resistant cells were not increased. At this time, the mechanism of increased DNA damage formation and the mechanism underlying sensitization to cisplatin are still matters of speculation. The experiments reported here, however, demonstrate that cells expressing the genes of tetracycline resistance actually became primary targets for destruction by cisplatin. Based on this study, it is suggested that the therapeutic power of the tetracyclines could be restored and enhanced by using a complementary drug that, in combination with tetracycline, would induce selective destruction of tetracycline-resistant bacteria. / by Doriana Froim. / Ph.D.in Molecular and Systems Toxicology and Pharmacology

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (leaves 61-67). / The distribution and generation of force within the myocardium during normal contractility is dictated by the tissue's underlying 3D myoarchitecture. The presence of disordered myoarchitecture may in turn constitute the pathological basis of impaired cardiac mechanics in numerous clinical conditions, such as the remodeling heart following myocardial infarction and cardiomyopathies. To investigate the multi-scale nature of architectural disarray in the setting of myocardial disease, a dual imaging approach consisting of diffusion spectrum magnetic resonance imaging (DSI) and high-speed multislice two-photon microscopy (TPM) was used. DSI is a technique that derives fiber orientation from directionality of proton diffusion, whereas TPM derives cellular alignment from an autocorrelation of 3D resolved images of cells and subcellular structure. Mesoscale tract representations of myofiber orientation are generated from similarly aligned diffusion or autocorrelation vectors. These methods were applied to study induced myocardial infarction in the rat and hypertrophic cardiomyopathy associated with deletion of the gene for myosin binding protein C (cMyBP-C) in the mouse. Normal cardiac muscle fiber alignment within the ventricular wall was characterized by a series of helical tracts transitioning from a lefthanded orientation in the subepicardium to circumferential in the mid-myocardium to righthanded in the subendocardium. Infarcted hearts displayed a fiber void in the infarct zone and an extension of both subepicardial and subendocardial fibers beyond the border zone. It's hypothesized that the growth of fibers contributes to the remodeling process and provides tensile strength to the myocardium during contraction. / (cont.) The hearts obtained from the cMyBP-C knockouts displayed significant myoarchitectural disarray characterized by a loss of voxel to voxel orientational coherence for fibers located from the mid-myocardium to subendocardium, resulting in a change in the transmural progression of remaining helical fibers. These observations suggest an association between cMyBP-C expression and cardiac fiber alignment, where variations in torsional rotation may constitute a mechanism for pump failure in hypertrophic cardiomyopathy. These results substantiate the use of multi-scale imaging methods to enhance understanding of molecular and cellular contributions to tissue mechanical function. / by Teresa T. Wang. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references. / Saccharomyces cerevisiae stimulates dendritic cells and represents a promising candidate for cancer immunotherapy development. Effective cross-presentation of antigen delivered to dendritic cells is necessary for successful induction of cellular immunity. Using a yeast vaccine model, we investigated the phagosome-to-cytosol pathway of cross-presentation. We demonstrate that the rate of antigen release from phagocytosed yeast directly affects cross-presentation efficiency, with an apparent time limit of about 25 min post-phagocytosis for antigen release to be productive. Antigen expressed on the yeast surface is cross-presented much more efficiently than antigen trapped in the yeast cytosol by the cell wall. The cross-presentation efficiency of yeast surface-displayed antigen can be increased by the insertion of linkers susceptible to cleavage in the early phagosome. Antigens indirectly attached to yeast through antibody fragments are less efficiently cross-presented when the antibody dissociation rate is extremely slow. Next, we present a yeast-based cancer vaccine approach that is independent of yeast's ability to express the chosen antigen, which is instead produced separately and conjugated to the yeast cell wall. The conjugation method is site-specific (based on the SNAP-tag) and designed to facilitate antigen release in the dendritic cell phagosome and subsequent translocation for cross-presentation. / (cont.) Phagosomal antigen release was further expedited through the insertion of the invariant chain ectodomain as a linker, which is rapidly cleaved by Cathepsin S. The dose of delivered antigen was increased in several ways: by using yeast strains with higher surface amine densities, by using yeast cell wall fragments instead of whole cells, and by conjugating multiple layers of antigen. The novel multi-layer conjugation scheme is site-specific and takes advantage of Sfp phosphopantetheinyl transferase, enabling the antigen dose to grow linearly. We show that whole yeast cells coated with one layer of the cancer-testis antigen NY-ESO-1 and yeast hulls bearing three layers were able to cross-prime naive CD8+ T cells in vitro, with the latter resulting in higher frequencies of antigen-specific cells after ten days. This cross-presentation-efficient antigen conjugation scheme is not limited to yeast and can readily be applied towards the development of other particulate vaccines. / by Shanshan W. Howland. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2003. / Includes bibliographical references (p. 211-223). / Glycosaminoglycans (GAGs) are a family of complex carbohydrates whose known biological roles have dramatically increased over the recent years. It is now becoming increasingly evident that sequence specific GAG-protein interactions play critical roles in cell growth, development, angiogenesis, cancer, anticoagulation and microbial pathogenesis. Therefore it is important to understand the specificity of glycan-protein interactions and how these specific interactions influence their structure-function relationships. This thesis addresses many challenges in this emerging area of glycomics, by taking an integrated approach that couples biophysical and biochemical methods with a bioinformatics framework to represent and process sequence information content in GAGs. With this motivation the thesis is divided into 4 components 1. Using heparin/heparan sulfate GAGs (HSGAGs) - fibroblast growth factor (FGF) as a model GAG-protein system, the first part focuses on determining the structural basis of FGF oligomerization, sequence specific FGF-HSGAG interactions and FGF-receptor (FGFR) interactions which collectively influence the specificity of HSGAG mediated FGF signaling. 2. The second part focuses on developing enzymatic tools for analysis of GAGs using chondroitinase B, HSGAG 2-0 sulfatase and 3-0 sulfotransferase as model enzymes. For each of these enzymes a theoretical model for the enzyme-substrate structural complex is developed and it is coupled with the site directed mutagenesis and biochemical studies to determine its catalytic mechanism and substrate specificity. 3. To deal with the heterogeneity and high information density of GAG sequences, an informatics based approach to decode GAG sequence information has been developed. / (cont.) A new property encoded nomenclature (PEN) computational framework has been formulated to encode and process information content in GAG sequences. The numerical nature of the PEN code facilitated the incorporation of diverse data sets from different analytical methods including mass spectrometry, electrophoresis and NMR as constraints to accurately determine the sequence of GAGs. Two practical methodologies for sequencing GAGs have been developed based on this approach. 4. The last part outlines the development of a powerful relational database that is capable of bridging sequence, structure and function information in glycomics. Thus this database and its associated computational tools to search and mine the data is an important resource for advancing glycomics. / by Rahul Raman. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references. / Cancer is a leading cause of death in the world. Much work has been done to study the role of inflammation in carcinogenesis. One hypothesis suggests that inflammation causes oxidative stress that induces damage to cellular targets, including DNA. The multistep model of cancer proposes that cancer is a genetic disease in which mutations are required in carcinogenesis. When this theory was championed, research focused on somatic mutations. The focus has broadened to include epigenetic mechanisms in changing gene expression. The association between chronic infection, chronic inflammation and increased cancer risk has been supported by epidemiologic studies. Data link chronic inflammation associated with infectious disease to increased cancer risk. Some examples of such infectious agents include hepatitis B virus, Schistosoma haemotobium, and Helicobacter pylori. One objective of this thesis was to investigate the role of inflammation in self-limiting infection. Additional objectives focus on evaluating a novel model of intestinal and extraintestinal cancer, and using immune regulating cells as treatment for intestinal cancer. For the first objective, a murine mutational analysis model was used to study infection with Citrobacter rodentium, an enteric bacterium that causes self-limiting hyperplasia and inflammation. Increased mutant frequency was observed in association with elevated levels of iNOS 13 days post infection. The second aim, to characterize a novel model of neoplasia, led to the discovery of basosquamous cancer in mice with intestinal tumors. Finally, Apc-[Min] mice, a model of intestinal neoplasia, were treated with T regulatory cells to investigate the role of these cells on tumor development. These / (cont.) cells were previously observed to have an anti-inflammatory and therapeutic effect on an infection-driven model of colon cancer. It was shown that T regulatory cells led to a decrease in the number of adenomas. In conclusion, it has been shown that self-limiting infection can increase mutant frequency. In addition, a novel model of intestinal and basosquamous cancer has been characterized, and a promising therapy for intestinal cancer has been validated. / by Jane Joo-hee Sohn. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2006. / Includes bibliographical references (p. 40-44). / The MilliF bioreactor offers great potential for the formation of i vivo-like liver tissue outside the body, making it a valuable tool for applications such as drug toxicity models and biosensors. Cell adhesion is an important factor in the maintenance of differentiated hepatocyte functions. Hepatocyte adhesion environments were examined in two settings: spheroid culture prior to seeding in the bioreactor and 2D surface culture methods that could be applied to the bioreactor scaffold. Spheroids were formed either by culturing in spinning suspension or on a static, non-adherent surface. In spheroid culture, the addition of extracellular matrix (ECM) signaling through the use of soluble Matrigel or adhesion protein-coated microspheres did not improve hepatocyte viability or function as assessed by liver-specific gene expression. These results suggest the importance of cell-cell rather than cell-surface interactions in maintaining hepatocytes. Optimal culturing of spheroids in spinning suspension without the ECM addition was found to be 3 days without media changes. 2D surfaces were treated with an adhesion peptide-conjugated comb polymer, preventing nonspecific cell adhesion and allowing attachment through the [alpha]₅[beta]₁ integrin. / (cont.) Varying the proportion of adhesion peptide presented to cells was found to regulate hepatocyte morphology and function; a surface with decreased hepatocyte spreading and liver-specific gene expression closer to in vivo was characterized. Immunoblotting for activated focal adhesion kinase (FAK) revealed that FAK signaling was not induced by attachment to the comb polymer surfaces. Immunostaining for other liver cell types demonstrated that the surface allowed hepatic stellate cell and Kupffer cell adhesion. / by Michael B. Wongchaowart. / M.Eng.