Thesis (Sc. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2007. / Includes bibliographical references (leaves 108-124). / Our understanding of why biological systems are designed in a particular way would benefit from biophysically-realistic models that can make accurate predictions on the time-evolution of molecular events given arbitrary arrangements of genetic components. This thesis is focused on constructing such models for gene expression during bacteriophage T7 infection. T7 gene expression is a particularly well suited model system because knowledge of how the phage functions is thought to be relatively complete. My work focuses on two questions in particular. First, can we address deficiencies in past simulations and measurements of bacteriophage T7 to improve models of gene expression? Second, can we design and build refactored surrogates of T7 that are easier to understand and model? To address deficiencies in past simulations and measurements, I developed a new single-molecule, base-pair-resolved gene expression simulator named Tabasco that can faithfully represent mechanisms thought to govern phage gene expression. I used Tabasco to construct a model of T7 gene expression that encodes our mechanistic understanding. The model displayed significant discrepancies from new system-wide measurements of absolute T7 mRNA levels during infection. / (cont.) I fit transcript-specific degradation rates to match the measured RNA levels and as a result corrected discrepancies in protein synthesis rates that confounded previous models. I also developed and used a fitting procedure to the data that let us evaluate assumptions related to promoter strengths, mRNA degradation, and polymerase interactions. To construct surrogates of T7 that are easier to understand and model, I began the process of refactoring the T7 genome to construct an organism that is a more direct representation of the models that we build. In other words, instead of making our models evermore detailed to explain wild-type T7, we started to construct new phage that are more direct representations of our models. The goal of our original design, T7. 1, was to physically define, separate, and enable unique manipulation of primary genetic elements. To test our initial design, we replaced the left 11,515 bp of the wild-type genome with 12,179 bp of engineered DNA. The resulting chimeric genome encodes a viable bacteriophage that appears to maintain key features of the original while being simpler to model and easier to manipulate. I also present a second generation design, T7.2, that extends the original goals of T7.1 by constructing a more direct physical representation of the T7 model. / by Sriram Kosuri. / Sc.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (leaves 66-75). / Eight distinct nuclear shapes, or morphologies, have been discovered in human proto-organs and tumors, including bell-shaped nuclei with stem-like properties. These bell-shaped, or "metakaryotic," nuclei are abundant in fetal tissues and neoplasias, but rare in normal adult somatic tissues. Metakaryotic nuclei employ an unusual process for division in which DNA synthesis, partial genomic condensation, and separation of the two nuclei in a cup-from-cup fashion occur concurrently, as shown by Feulgen densitometry and single-stranded DNA assays by Dr. Elena Gostjeva. This is clearly different from the sequential steps of S-phase DNA synthesis, chromatin condensation, chromosomal separation, and genomic segregation that occur in mitotic eukaryotic cells. In order to discover how a genome apparently devoid of chromosomes might be organized, this thesis focused on recognizable DNA sequences common to all chromosomes: centromeres and telomeres. Fluorescence In Situ Hybridization (FISH) with pan-centromeric and pan-telomeric probes was applied to samples of human tissue. (A collaborating lab used centromeric and telomeric antibodies to confirm results.) An optimized FISH protocol was developed specifically for metakaryotic nuclei and tested in both human cell lines and eukaryotic cells as experimental controls. Staining of metakaryotic nuclei resulted in approximately 23 centromeric regions in each, unlike the expected number of 46 regions seen in eukaryotic nuclei. Many of these staining regions contained paired centromere signals, or doublets. This suggested a genomic organization of homologous chromosomes paired at their centromere regions. If this were the case, one would expect 46 telomeric signals per nuclei, if telomeres were also homologously paired. / (cont.) Unexpectedly, an average of 23 telomeric regions were found in many, if not all, bell-shaped metakaryotic nuclei. This, along with the observation of a condensed double ring around the mouth of the bell-shaped nuclei, suggested the possibility of a genome organized as paired, continuous genomic circles. Studies of telomere joining in metakaryotic nuclei by Dr. Per Olaf Ekstrom have provided further evidence for the paired genomic circle model. The results in this thesis are an original contribution to the field of stem cell physiology, a starting point for further investigation of DNA organization, synthesis, and repair in these metakaryotic cells, and hopefully will lead to a greater understanding of human development, growth, and cancer. / by Amanda Natalie Gruhl. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references (leaves 40-43). / Adult stem cells are potentially useful in many biomedical applications that can save lives and increase the quality of a patient's life, such as tissue engineering, cell replacement, and gene therapy. However, these applications are limited because of the difficulty in isolating and expanding pure populations of adult stem cells (ASCs). A major barrier to ASC expansion in vitro is their property of asymmetric cell kinetics. Our lab has developed a method, Suppression of Asymmetric Cell Kinetics (SACK), to expand ASCs in vitro by shifting their cell kinetics program from asymmetric to symmetric. We have found that guanine nucleotide precursors can be used to convert the kinetics of adult stem cells from asymmetric to symmetric, which promotes their exponential expansion. Previously, we have used the SACK method to derive hepatic and cholangiocyte stem cell strains from adult rat livers in vitro. These cell strains provide an assay to evaluate whether growth factors and cytokines previously implicated in proliferation of progenitor cells act by converting the kinetics of the stem cells in the population from asymmetric to symmetric, and thus identify new SACK agents. We are evaluating three agents, Wnt, IGF- 1, and Sonic hedgehog (Shh). / (cont.) Wnt has been found to cause self-renewal and proliferation of hematopoietic stem cells (HSCs) in vitro. IGF- 1 also plays a role in hematopoietic progenitor self-renewal in vivo as well as in tissue maturation. Shh has been implicated in the proliferation of primitive neural cells as well as in cellular proliferation during invertebrate development. Thus far, we have found that Wnt peptide shifts the cell kinetics from asymmetric to symmetric and may reduce the generation time, whereas IGF-1 appears only to affect generation time. Studies involving Shh are currently underway. We are also currently investigating whether Wnt acts additively or synergistically with guanine nucleotide precursors to shift cell kinetic symmetry. Discovering new SACK agents will allow us to obtain purer populations of ASCs that can be used to study properties unique to stem cells. Furthermore, the observation that Wnt shifts the kinetics of adult rat hepatic stem cells from asymmetric to symmetric implicates the involvement of similar cell kinetics symmetry mechanisms in the proliferation effect of Wnt on murine and human HSCs. / by Michal Ganz. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (leaves 244-249). / This thesis utilizes modeling approaches rooted in statistical physics and physical chemistry to investigate several aspects of cellular signal transduction at both the modular and global levels. Design principles of biological networks and cell signaling processes pertinent to disease progression emerge from these studies. It is my hope that knowledge of these principles may provide new mechanistic insights and conceptual frameworks for thinking about therapeutic intervention into diseases such as cancer and diabetes that arise from aberrant signaling. Areas of interest have emphasized the role of scaffold proteins in protein kinase cascades, modeling relevant biophysical processes related to T cell activation, design principles of signal transduction focusing on multisite phosphorylation, quantifying the notion of signal duration and the time scale dependence of signal detection, and entropy based models of network architecture inferred from proteomics data. These problems are detailed below. The assembly of multiple signaling proteins into a complex by a scaffold protein guides many cellular decisions. Despite recent advances, the overarching principles that govern scaffold function are not well understood. We carried out a computational study using kinetic Monte Carlo simulations to understand how spatial localization of kinases on a scaffold may regulate signaling under different physiological condition. Our studies identify regulatory properties of scaffold proteins that allow them to both amplify and attenuate incoming signals in different biological contexts. In a further, supplementary study, simulations also indicate that a major effect that scaffolds exert on the dynamics of cell signaling is to control how the activation of protein kinases is distributed over time[2]. / (cont.) Scaffolds can influence the timing of kinase activation by allowing for kinases to become activated over a broad range of times, thus allowing for signaling across a broad spectrum of time scales. T cells orchestrate the adaptive immune response and are central players in maintenance of functioning immune system. Recent studies have reported that T cells can integrate signals between interrupted encounters with Antigen Presenting Cells (APCs) in such a way that the process of signal integration exhibits a form of memory. We carried out a computational study using a simple mathematical model of T cell activation to investigate the ramifications of interrupted T cell-APC contacts on signal integration. We considered several mechanisms of how signal integration at these time scales may be achieved. In another study, we investigated the role of spatially localizing signaling components of the T cell signaling pathway into a structure known as the immunological synapse. We constructed a minimal mathematical model that offers a mechanism for how antigen quality can regulate signaling dynamics in the immunological synapse These studies involving the analysis of signaling dynamics led us to investigate how differences in signal duration might be detected. Signal duration (e.g. the time scales over which an active signaling intermediate persists) is a key regulator of biological decisions in myriad contexts such as cell growth, proliferation, and developmental lineage commitments. Accompanying differences in signal duration are numerous downstream biological processes that require multiple steps of biochemical regulation. We present an analysis that investigates how simple biochemical motifs that involve multiple stages of regulation can be constructed to differentially process signals that persist at different time scales[3]. / (cont.) Topological features of these networks that allow for different frequency dependent signal processing properties are identified. One role of multisite phosphorylation in cell signaling is also investigated. The utilization of multiple phosphorylation sites in regulating a biological response is ubiquitous in cell signaling. If each site contributes an additional, equivalent binding site, then one consequence of an increase in the number of phosphorylations may be to increase the probability that, upon disassociation, a ligand immediately rebinds to its receptor. How such effects may influence cell signaling systems is not well understood. A self-consistent integral equation formalism for ligand rebinding, in conjunction with Monte Carlo simulations, was employed to further investigate the effects of multiple, equivalent binding sites on shaping biological responses. Finally, this thesis also seeks to investigate cell signaling at a global scale. Advances in Mass Spectrometry based phosphoproteomics have allowed for the real-time quantitative monitoring of entire proteomes as signals propagate through complex networks in response to external signals. The trajectories of as many as 222 phosphorylated tyrosine sites can be simultaneously and reproducibly monitored at multiple time points. We develop and apply a method using the principle of maximum entropy to infer a model of network connectivity of these phosphorylation sites. The model predicts a core structure of signaling nodes, affinity dependent topological features of the network, and connectivity of signaling nodes that were hitherto unassociated with the canonical growth factor signaling network. Our combined results illustrate many complexities in the broad array of control properties that emerge from the physical effects that constrain signal propagation on complex biological networks. / (cont.) It is the hope of this work that these studies bring coherence to seemingly paradoxical observations and suggest that cells have evolved design rules that enable biochemical motifs to regulate widely disparate cellular functions. / by Jason W. Locasale. / Ph.D.

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 (p. 195-208). / Citrobacter rodentium is a naturally occurring murine bacterial pathogen which is used to model human diarrheagenic E. coli (EPEC and EHEC) infections in mice. C. rodentium causes colonic hyperplasia and a variable degree of colitis and mortality in the majority of inbred and outbred lines of mice. Differences in C. rodentium-induced disease are determined by the genetic background of the host. Here, C. rodentium infection in resistant outbred Swiss Webster (SW) mice was compared with infection in the cognate inbred FVB strain for the first time. In contrast to subclinical infection in SW mice, adult FVB mice developed overt disease with significant weight loss, severe colitis, and over 75% mortality. Fluid therapy intervention completely prevented mortality in FVB mice, and expression of pro-inflammatory and immunomodulatory genes in the colon was similar in both lines of mice, suggesting that mortality in C. rodentiuminfected FVB mice is due to hypovolemia resulting from severe dehydration. To identify host factors responsible for the development of mortality, gene expression in the distal colon of FVB and SW mice was investigated using a whole mouse genome Affymetrix array. / (cont.) Transcripts represented by 1,547 probe sets (3.4%) were differentially expressed between FVB and SW mice prior to infection and at 4 and 9 days post-inoculation. Data analysis suggested that intestinal ion disturbances rather than immune-related processes are responsible for susceptibility in C. rodentium-infected FVB mice. Marked impairment in intestinal ion homeostasis predicted by microarray analysis was confirmed by quantitative RT-PCR and serum electrolyte measurements that showed hypochloremia and hyponatremia in susceptible FVB mice. C. rodentium infection was next characterized in additional inbred strains of Swiss origin. SWR and SJL mice developed minimal morbidity and no mortality in response to the pathogen, demonstrating resistance to disease. Furthermore, C3H mice developed severe diarrhea and gene expression changes comparable to those in infected FVB mice, suggesting common pathogenic mechanisms in susceptible strains. In conclusion, C. rodentium infection in FVB mice is a useful model for fatal infectious diarrhea. These studies contribute to our understanding of C. rodentium pathogenesis and identify possible candidates for susceptibility to fatal enteric bacterial infection. / by Diana Borenshtein. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (p. 139-153). / Many cellular processes are governed by large and highly-complex networks of chemical interactions and are therefore difficult to intuit. Computational modeling provides a means of encapsulating information about these interactions and can serve as a platform for gaining understanding of the biology and making predictions about cellular response to perturbation. In particular, there has been considerable interest in ordinary differential equation (ODE) models, which have several attractive features: ODEs can describe molecular interactions with mechanistic detail, it is relatively straightforward to implement perturbations, and, in theory, they can predict the concentration and activity of every species as a function of time. However, both the topology and parameters in such models are subject to considerable uncertainty. We explore the ramifications of these sources of uncertainty for making accurate predictions and develop methods of selecting high confidence predictions from uncertain models. In particular, we promote a shift in emphasis from model selection to prediction selection, and use consensus among model ensembles to identify the predictions most likely to be accurate. By constructing decision trees, this consensus can also be used to partition the space of potential perturbations into regions of high and low confidence. We apply our methods to the Fas signaling pathway in apoptosis to satisfy two goals: first, to design a therapeutic cocktail to reduce cell death in the presence of high levels of stimulus, and second, to design experiments that may lead to a better understanding of the biological network. / by Caitlin Anne Bever. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (p. 309-336). / Molecular signaling systems allow cells to sense and respond to environmental stimuli. Quantitative modeling can be a valuable tool for evaluating and extending our understanding of signaling systems. In particular, studies of the mating pheromone response system in yeast (Saccharomyces cerevisiae) have revealed many protein families and regulatory motifs also found in higher eukaryotes. This thesis develops several computational and experimental approaches that facilitate characterization of cellular signaling systems, and tests these approaches using yeast mating response as a model. Limitations in the current approach to building models of molecular systems were addressed first. For example, published computational models are often difficult to evaluate and extend because researchers rarely make available the information and assumptions generated throughout model building. I developed tools that facilitate model construction, evaluation, and extension. I used these tools to develop the YeastPheromoneModel (YPM) information repository, in which construction of an exhaustive model of the yeast mating system is documented (http://www.YeastPheromoneModel.org). Next, motivated by an ability to rapidly make many derivative models from the YPM repository and by carefully measured abundances of mating system proteins, I analyzed a model of the mating system mitogen activated protein kinase cascade. I found that varying the abundance of the scaffold protein Ste5, but not the abundances of other proteins, is expected to result in a quantitative tradeoff between total system output and dynamic range. Thus, the abundance of scaffold proteins in signaling systems may generally be under selective pressure to support specific quantitative system behavior. / (cont.) Finally, because traditional methods for characterizing signaling systems can be slow and tedious, I postulated that time-dependent stimulation of signaling systems might increase the richness and value of data derived from individual experiments. To do this, I devised a custom microfluidic device to expose yeast cells to pheromone in a time-dependent manner. I also developed computational approaches to investigate the use of time-dependent stimulation to characterize receptor and G protein response dynamics. I found that, at least for the receptor/G protein portion of the mating system, time-dependent stimulation does not appear to offer significant gains for constraining kinetic parameters relative to traditional step-response experiments. / by Ty M. Thomson. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (v. 2, leaves 195-258). / The anticancer drug cisplatin is in widespread use but its mechanism of action is only poorly understood. Moreover, human cancers acquire resistance to the drug, which limits its clinical utility. A paradox in the field is how loss of mismatch DNA repair leads to clinical resistance to this widely used drug. The phenomenon of cisplatin tolerance in mismatch repair deficient cells was initially discovered in E. coli, where methylation deficient dam mutants show high sensitivity to cisplatin and dam mutants with an additional mutation in either of the mismatch repair genes mutS or mutL show near wildtype levels of resistance. A prevalent explanation for this observation is the abortive repair model, which proposes that in dam mutants, where the strand discrimination signal is lost, mismatch repair attempts futile cycles of repair opposite cisplatin-DNA adducts. Previous findings have supported this model to the extent that MutS, the E. coli mismatch recognition protein, specifically recognizes DNA modified with cisplatin. However it has recently been shown that MutS binding to cisplatin adducts may contribute to toxicity by instead preventing the recombinational repair of a cisplatin-modified substrate, and we have previously shown that recombination is an essential mechanism for tolerating cisplatin damage. / (cont.) In the present study, we examined the global transcriptional responses of wildtype, dam, dam mutS, and mutS mutant E. coli after treatment with a toxic dose of cisplatin. We also determined any dose-response at the transcriptional level of several SOS response genes and other genes involved in DNA repair by real time RT-PCR. Furthermore, we performed single-cell electrophoresis in order to determine the effect of mismatch repair on the level of double-strand break formation in cisplatin-treated cells. Our results show that Dam-deficient strains exhibit unique gene regulation that may be due to mismatch-repair induced DNA damage in the absence of adenine methylation. In addition, cisplatin treatment induces double-strand break formation and the SOS response in a dose-dependent manner, and both break formation and the SOS response are greatest in the hypersensitive dam mutant strain. The higher level of cisplatin-induced double-strand breaks in the dam mutant may be dependent on functional mismatch repair. / by Jennifer L. Robbins. / Ph.D.

Thesis (S.M. in Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 45-47). / Design and fabrication of a microfluidics system capable of generating reproducible and controlled micro-biochemical environments that can be used as a diagnostic assay and microreactor is important. Here, a simple technique was developed to create a robust microfluidics system capable of generating precise gradients of biochemical properties within its channels. Through this approach, it is possible to create a gradient generator with mammalian cells patterned and seeded under its poly(dimethylsiloxane) (PDMS) channels. Cells that were seeded and patterned under the PDMS channels remained viable and capable of performing intracellular reactions. Using the gradient generator within the PDMS microfluidic device, a gradient of specific and controlled biochemicals can be flowed on seeded cells allowing for high-throughput molecular interaction analysis. The microfluidics system provides a way to study and analyze cell response in the presence of a combination of biochemical signals. / by Guan-Jong Chen. / S.M.in Toxicology

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (p. 139-145). / Developments in light microscopy over the past three centuries have opened new windows into cell structure and function, yet many questions remain unanswered by current imaging approaches. Deep ultraviolet microscopy received attention in the 1950s as a way to generate image contrast from the strong absorbance of proteins and nucleic acids at wavelengths shorter than 300 nm. However, the lethal effects of these wavelengths limited their usefulness in studies of cell function, separating the contributions of protein and nucleic acid proved difficult, and scattering artifacts were a significant concern. We have used short exposures of deep-ultraviolet light synchronized with an ultraviolet-sensitive camera to observe mitosis and motility in living cells without causing necrosis, and quantified absorbance at 280 nm and 260 nm together with tryptophan native fluorescence in order to calculate maps of nucleic acid mass, protein mass, and quantum yield in unlabeled cells. We have also developed a method using images acquired at 320nm and 340nm, and an equation for Mie scattering, to determine a scattering correction factor for each pixel at 260nm and 280nm. These developments overcome the three main obstacles to previous deep UV microscopy efforts, creating a new approach to imaging unlabeled living cells that acquires quantitative information about protein and nucleic acid as a function of position and time. / by Benjamin J. Zeskind. / Ph.D.