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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2013., Cataloged from PDF version of thesis., Includes bibliographical references (p. 103-111)., Using models to simulate and analyze biological networks requires principled approaches to parameter estimation and model discrimination. We use Bayesian and Monte Carlo methods to recover the full probability distributions of free parameters (initial protein concentrations and rate constants) for mass action models of receptor-mediated cell death. The width of the individual parameter distributions is largely determined by non-identifiability but co-variation among parameters, even those that are poorly determined, encodes essential information. Knowledge of joint parameter distributions makes it possible to compute the uncertainty of model-based predictions whereas ignoring it (e.g. by treating parameters as a simple list of values and variances) yields nonsensical predictions. Computing the Bayes factor from joint distributions yields the odds ratio (~20-fold) for competing "direct" and "indirect" apoptosis models having different numbers of parameters. The methods presented in this thesis were then extended to make predictions in eight apoptosis mini-models. Despite topological uncertainty, the simulated predictions can be used to drive experimental design. Our results illustrate how Bayesian approaches to model calibration and discrimination combined with single-cell data represent a generally useful and rigorous approach to discriminating between competing hypotheses in the face of parametric and topological uncertainty., by Hoda Eydgahi., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011., This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections., Cataloged from student submitted PDF version of thesis., Includes bibliographical references., The nuclear factor-kappa B (NF-kappaB) family of transcription factors play a pivotal role in the inflammatory and immune responses, as well as cell growth and survival. Since its discovery 25 years ago, NF-kappaB has emerged as one of the most intensely studied eukaryotic transcription factors, due to its pleiotropic functions and its inducible pattern of expression that is subject to multi-level regulation. Many modulatory events affect NF-kappaB function and thus to have a better understanding of the NF-kappaB pathway and its gene regulation will require additional studies of NF-kappaB's modulators. The goal of this thesis was to investigate the role of intra- and extra-cellular modulators of NF-kappaB. To study how intracellular modulators affect NF-kappaB function, various genetic and biochemical approaches were used to identify potentially new NF-kappaB's interacting proteins. Two AGC family kinases, STK38 and STK38L, were chosen for further study. These two proteins were found to not only interact with NF-kappaB's p65 subunit but also act as its kinases in vitro. shRNA mediated knockdown combined with gene expression analysis revealed that STK38 and STK38L via their kinase function regulated p65 activity on some, but not all NF-kappaB-responsive inflammatory genes in response to TNFa stimulation. To study the role of extracellular modulators on the apoptotic behaviors of cells, a panel of human immortalized and cancer liver cell lines were pre-treated with pro-inflammatory cytokines TNFa and IL-1a followed by TRAIL, a potent inducer of cell death. This context-dependent approach revealed that TNFa and IL-1a had diverse effects on cell death behaviors and inhibiting the NF-kappaB pathway was enough to block pro-inflammatory cytokines-mediated pro-death or pro-survival outcomes. Chromatin immunoprecipitation followed by high throughput sequencing, realtime PCR and Western blotting were used to identify NF-kappaB's target genes involved in the cell death pathway, by Hung Nhat Nguyen., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011., This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections., Cataloged from student submitted PDF version of thesis., Includes bibliographical references., The nuclear factor-kappa B (NF-kappaB) family of transcription factors play a pivotal role in the inflammatory and immune responses, as well as cell growth and survival. Since its discovery 25 years ago, NF-kappaB has emerged as one of the most intensely studied eukaryotic transcription factors, due to its pleiotropic functions and its inducible pattern of expression that is subject to multi-level regulation. Many modulatory events affect NF-kappaB function and thus to have a better understanding of the NF-kappaB pathway and its gene regulation will require additional studies of NF-kappaB's modulators. The goal of this thesis was to investigate the role of intra- and extra-cellular modulators of NF-kappaB. To study how intracellular modulators affect NF-kappaB function, various genetic and biochemical approaches were used to identify potentially new NF-kappaB's interacting proteins. Two AGC family kinases, STK38 and STK38L, were chosen for further study. These two proteins were found to not only interact with NF-kappaB's p65 subunit but also act as its kinases in vitro. shRNA mediated knockdown combined with gene expression analysis revealed that STK38 and STK38L via their kinase function regulated p65 activity on some, but not all NF-kappaB-responsive inflammatory genes in response to TNFa stimulation. To study the role of extracellular modulators on the apoptotic behaviors of cells, a panel of human immortalized and cancer liver cell lines were pre-treated with pro-inflammatory cytokines TNFa and IL-1a followed by TRAIL, a potent inducer of cell death. This context-dependent approach revealed that TNFa and IL-1a had diverse effects on cell death behaviors and inhibiting the NF-kappaB pathway was enough to block pro-inflammatory cytokines-mediated pro-death or pro-survival outcomes. Chromatin immunoprecipitation followed by high throughput sequencing, realtime PCR and Western blotting were used to identify NF-kappaB's target genes involved in the cell death pathway, by Hung Nhat Nguyen., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2010., Cataloged from PDF version of thesis., Includes bibliographical references (p. 157-168)., The epidermal growth factor receptor (EGFR, also known as ErbB 1) is a prototypical receptor tyrosine kinase (RTK) that activates multi-kinase phosphorylation cascades to regulate diverse cellular processes, including proliferation, migration and differentiation. ErbB 1 heterooligomerizes with three close homologues: ErbB2, ErbB3 and ErbB4. ErbB1-3 receptors are frequently mutated, overexpressed or activated by autocrine or paracrine ligand production in solid tumors and have been the target of extensive drug discovery efforts. Multiple small molecule kinase inhibitors and therapeutic antibodies against ErbB receptors are in clinical use or development. Despite their importance as RTKs, oncogenes and drug targets, regulation of ErbB receptors by the interplay of conformational change, phosphorylation, phosphatases and receptor trafficking remains poorly understood, and the impact of these dynamics on physiological activity and cellular responses to anti-ErbB drugs is largely unknown. This thesis investigates the dynamic opposition of kinases and phosphatases within the ErbB pathway. By standard biochemical analysis, ErbB receptors and downstream proteins appear to become phosphorylated and then dephosphorylated in approximately 30 minutes. However, pulse chase experiments where cells are exposed to ligand and then to small molecule kinase inhibitors reveal that individual proteins must in fact cycle rapidly between being phosphorylated and dephosphorylated in seconds. We construct a succession of differential equation-based models of varying biochemical resolution, each model appropriate for analyzing a different aspect of ErbB regulation, to help interpret the data and gain quantitative insight into receptor and drug biology. Rapid phosphorylation and dephosphorylation of receptors has important implications for the assembly dynamics of signalosomes. We find that signals are rapidly propagated through some downstream pathways but slowly through others, resulting in, By Laura B. Kleiman., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2009., Cataloged from PDF version of thesis., Includes bibliographical references (p. 127-142)., Diversity within a population of organisms is typically ascribed to genetic differences. However, even members of a genetically identical group of cells or organisms in identical environments can exhibit variability in state and phenotype. One striking example of such heterogeneity is revealed when a genetically identical population of human cells is exposed to saturating doses of a death-inducing drug called TRAIL - many cells in the population will undergo apoptosis, a form of controlled cell death, but a fraction of cells always survives the treatment. The goal of this thesis was to understand the origins of variability in both the timing and the probability of death in TRAIL-induced apoptosis. To this end, both experimental and computational methods were implemented. Experiments examining the response of sister cells to TRAIL provided strong evidence that variability in initial conditions played a key role, and ruled out genetic, stochastic, and cell cycle effects as possible causes of heterogeneity in response. A detailed analysis of the relative contributions of three segments of the TRAIL pathway revealed that the majority of the variability in time-to-death arose upstream of mitochondrial outer membrane permeabilization (MOMP), with little contribution from downstream reactions. More specifically, the rate of cleavage of initiator caspase substrates was highly predictive of a cell's death time. However, to determine whether (as opposed to when) a cell will die, variation in the MOMP threshold became critical., (cont.) This dependency was indicated by observation of the height of the MOMP threshold in surviving and dying cells and by modulation of this threshold via overexpression of anti-apoptotic regulators of MOMP. Simulations of cell-to-cell variability in TRAIL-induced apoptosis confirmed that the endogenous variability in apoptotic regulators was sufficient to produce the observed variability in death time. However, knowledge of the concentration of individual proteins did not allow prediction of death time because variation in other proteins masked the underlying trends. The ability to simulate heterogeneity in cellular response also led to the development of novel, biologically intuitive methods of sensitivity analysis, which revealed that sensitivities shift depending on whether knowledge of covariance in initial conditions is included. The ability to predict sensitivity and resistance of tumors to TRAIL would be clinically valuable, as TRAIL is currently in clinical trials as an anti-cancer therapy. The results described here represent progress toward understanding the "fractional killing" of tumor cells following exposure to chemotherapy, and for understanding variability in mammalian signaling pathways in general., by Sabrina Leigh Spencer., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2009., Cataloged from PDF version of thesis., Includes bibliographical references (p. 127-142)., Diversity within a population of organisms is typically ascribed to genetic differences. However, even members of a genetically identical group of cells or organisms in identical environments can exhibit variability in state and phenotype. One striking example of such heterogeneity is revealed when a genetically identical population of human cells is exposed to saturating doses of a death-inducing drug called TRAIL - many cells in the population will undergo apoptosis, a form of controlled cell death, but a fraction of cells always survives the treatment. The goal of this thesis was to understand the origins of variability in both the timing and the probability of death in TRAIL-induced apoptosis. To this end, both experimental and computational methods were implemented. Experiments examining the response of sister cells to TRAIL provided strong evidence that variability in initial conditions played a key role, and ruled out genetic, stochastic, and cell cycle effects as possible causes of heterogeneity in response. A detailed analysis of the relative contributions of three segments of the TRAIL pathway revealed that the majority of the variability in time-to-death arose upstream of mitochondrial outer membrane permeabilization (MOMP), with little contribution from downstream reactions. More specifically, the rate of cleavage of initiator caspase substrates was highly predictive of a cell's death time. However, to determine whether (as opposed to when) a cell will die, variation in the MOMP threshold became critical., (cont.) This dependency was indicated by observation of the height of the MOMP threshold in surviving and dying cells and by modulation of this threshold via overexpression of anti-apoptotic regulators of MOMP. Simulations of cell-to-cell variability in TRAIL-induced apoptosis confirmed that the endogenous variability in apoptotic regulators was sufficient to produce the observed variability in death time. However, knowledge of the concentration of individual proteins did not allow prediction of death time because variation in other proteins masked the underlying trends. The ability to simulate heterogeneity in cellular response also led to the development of novel, biologically intuitive methods of sensitivity analysis, which revealed that sensitivities shift depending on whether knowledge of covariance in initial conditions is included. The ability to predict sensitivity and resistance of tumors to TRAIL would be clinically valuable, as TRAIL is currently in clinical trials as an anti-cancer therapy. The results described here represent progress toward understanding the "fractional killing" of tumor cells following exposure to chemotherapy, and for understanding variability in mammalian signaling pathways in general., by Sabrina Leigh Spencer., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2009., Cataloged from PDF version of thesis., Includes bibliographical references (p. 127-142)., Diversity within a population of organisms is typically ascribed to genetic differences. However, even members of a genetically identical group of cells or organisms in identical environments can exhibit variability in state and phenotype. One striking example of such heterogeneity is revealed when a genetically identical population of human cells is exposed to saturating doses of a death-inducing drug called TRAIL - many cells in the population will undergo apoptosis, a form of controlled cell death, but a fraction of cells always survives the treatment. The goal of this thesis was to understand the origins of variability in both the timing and the probability of death in TRAIL-induced apoptosis. To this end, both experimental and computational methods were implemented. Experiments examining the response of sister cells to TRAIL provided strong evidence that variability in initial conditions played a key role, and ruled out genetic, stochastic, and cell cycle effects as possible causes of heterogeneity in response. A detailed analysis of the relative contributions of three segments of the TRAIL pathway revealed that the majority of the variability in time-to-death arose upstream of mitochondrial outer membrane permeabilization (MOMP), with little contribution from downstream reactions. More specifically, the rate of cleavage of initiator caspase substrates was highly predictive of a cell's death time. However, to determine whether (as opposed to when) a cell will die, variation in the MOMP threshold became critical., (cont.) This dependency was indicated by observation of the height of the MOMP threshold in surviving and dying cells and by modulation of this threshold via overexpression of anti-apoptotic regulators of MOMP. Simulations of cell-to-cell variability in TRAIL-induced apoptosis confirmed that the endogenous variability in apoptotic regulators was sufficient to produce the observed variability in death time. However, knowledge of the concentration of individual proteins did not allow prediction of death time because variation in other proteins masked the underlying trends. The ability to simulate heterogeneity in cellular response also led to the development of novel, biologically intuitive methods of sensitivity analysis, which revealed that sensitivities shift depending on whether knowledge of covariance in initial conditions is included. The ability to predict sensitivity and resistance of tumors to TRAIL would be clinically valuable, as TRAIL is currently in clinical trials as an anti-cancer therapy. The results described here represent progress toward understanding the "fractional killing" of tumor cells following exposure to chemotherapy, and for understanding variability in mammalian signaling pathways in general., by Sabrina Leigh Spencer., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2010., Cataloged from PDF version of thesis., Includes bibliographical references (p. 157-168)., The epidermal growth factor receptor (EGFR, also known as ErbB 1) is a prototypical receptor tyrosine kinase (RTK) that activates multi-kinase phosphorylation cascades to regulate diverse cellular processes, including proliferation, migration and differentiation. ErbB 1 heterooligomerizes with three close homologues: ErbB2, ErbB3 and ErbB4. ErbB1-3 receptors are frequently mutated, overexpressed or activated by autocrine or paracrine ligand production in solid tumors and have been the target of extensive drug discovery efforts. Multiple small molecule kinase inhibitors and therapeutic antibodies against ErbB receptors are in clinical use or development. Despite their importance as RTKs, oncogenes and drug targets, regulation of ErbB receptors by the interplay of conformational change, phosphorylation, phosphatases and receptor trafficking remains poorly understood, and the impact of these dynamics on physiological activity and cellular responses to anti-ErbB drugs is largely unknown. This thesis investigates the dynamic opposition of kinases and phosphatases within the ErbB pathway. By standard biochemical analysis, ErbB receptors and downstream proteins appear to become phosphorylated and then dephosphorylated in approximately 30 minutes. However, pulse chase experiments where cells are exposed to ligand and then to small molecule kinase inhibitors reveal that individual proteins must in fact cycle rapidly between being phosphorylated and dephosphorylated in seconds. We construct a succession of differential equation-based models of varying biochemical resolution, each model appropriate for analyzing a different aspect of ErbB regulation, to help interpret the data and gain quantitative insight into receptor and drug biology. Rapid phosphorylation and dephosphorylation of receptors has important implications for the assembly dynamics of signalosomes. We find that signals are rapidly propagated through some downstream pathways but slowly through others, resulting in, By Laura B. Kleiman., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2010., Cataloged from PDF version of thesis., Includes bibliographical references (p. 157-168)., The epidermal growth factor receptor (EGFR, also known as ErbB 1) is a prototypical receptor tyrosine kinase (RTK) that activates multi-kinase phosphorylation cascades to regulate diverse cellular processes, including proliferation, migration and differentiation. ErbB 1 heterooligomerizes with three close homologues: ErbB2, ErbB3 and ErbB4. ErbB1-3 receptors are frequently mutated, overexpressed or activated by autocrine or paracrine ligand production in solid tumors and have been the target of extensive drug discovery efforts. Multiple small molecule kinase inhibitors and therapeutic antibodies against ErbB receptors are in clinical use or development. Despite their importance as RTKs, oncogenes and drug targets, regulation of ErbB receptors by the interplay of conformational change, phosphorylation, phosphatases and receptor trafficking remains poorly understood, and the impact of these dynamics on physiological activity and cellular responses to anti-ErbB drugs is largely unknown. This thesis investigates the dynamic opposition of kinases and phosphatases within the ErbB pathway. By standard biochemical analysis, ErbB receptors and downstream proteins appear to become phosphorylated and then dephosphorylated in approximately 30 minutes. However, pulse chase experiments where cells are exposed to ligand and then to small molecule kinase inhibitors reveal that individual proteins must in fact cycle rapidly between being phosphorylated and dephosphorylated in seconds. We construct a succession of differential equation-based models of varying biochemical resolution, each model appropriate for analyzing a different aspect of ErbB regulation, to help interpret the data and gain quantitative insight into receptor and drug biology. Rapid phosphorylation and dephosphorylation of receptors has important implications for the assembly dynamics of signalosomes. We find that signals are rapidly propagated through some downstream pathways but slowly through others, resulting in, By Laura B. Kleiman., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2010., Cataloged from PDF version of thesis., Includes bibliographical references (p. 157-168)., The epidermal growth factor receptor (EGFR, also known as ErbB 1) is a prototypical receptor tyrosine kinase (RTK) that activates multi-kinase phosphorylation cascades to regulate diverse cellular processes, including proliferation, migration and differentiation. ErbB 1 heterooligomerizes with three close homologues: ErbB2, ErbB3 and ErbB4. ErbB1-3 receptors are frequently mutated, overexpressed or activated by autocrine or paracrine ligand production in solid tumors and have been the target of extensive drug discovery efforts. Multiple small molecule kinase inhibitors and therapeutic antibodies against ErbB receptors are in clinical use or development. Despite their importance as RTKs, oncogenes and drug targets, regulation of ErbB receptors by the interplay of conformational change, phosphorylation, phosphatases and receptor trafficking remains poorly understood, and the impact of these dynamics on physiological activity and cellular responses to anti-ErbB drugs is largely unknown. This thesis investigates the dynamic opposition of kinases and phosphatases within the ErbB pathway. By standard biochemical analysis, ErbB receptors and downstream proteins appear to become phosphorylated and then dephosphorylated in approximately 30 minutes. However, pulse chase experiments where cells are exposed to ligand and then to small molecule kinase inhibitors reveal that individual proteins must in fact cycle rapidly between being phosphorylated and dephosphorylated in seconds. We construct a succession of differential equation-based models of varying biochemical resolution, each model appropriate for analyzing a different aspect of ErbB regulation, to help interpret the data and gain quantitative insight into receptor and drug biology. Rapid phosphorylation and dephosphorylation of receptors has important implications for the assembly dynamics of signalosomes. We find that signals are rapidly propagated through some downstream pathways but slowly through others, resulting in, By Laura B. Kleiman., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2009., Cataloged from PDF version of thesis., Includes bibliographical references (p. 127-142)., Diversity within a population of organisms is typically ascribed to genetic differences. However, even members of a genetically identical group of cells or organisms in identical environments can exhibit variability in state and phenotype. One striking example of such heterogeneity is revealed when a genetically identical population of human cells is exposed to saturating doses of a death-inducing drug called TRAIL - many cells in the population will undergo apoptosis, a form of controlled cell death, but a fraction of cells always survives the treatment. The goal of this thesis was to understand the origins of variability in both the timing and the probability of death in TRAIL-induced apoptosis. To this end, both experimental and computational methods were implemented. Experiments examining the response of sister cells to TRAIL provided strong evidence that variability in initial conditions played a key role, and ruled out genetic, stochastic, and cell cycle effects as possible causes of heterogeneity in response. A detailed analysis of the relative contributions of three segments of the TRAIL pathway revealed that the majority of the variability in time-to-death arose upstream of mitochondrial outer membrane permeabilization (MOMP), with little contribution from downstream reactions. More specifically, the rate of cleavage of initiator caspase substrates was highly predictive of a cell's death time. However, to determine whether (as opposed to when) a cell will die, variation in the MOMP threshold became critical., (cont.) This dependency was indicated by observation of the height of the MOMP threshold in surviving and dying cells and by modulation of this threshold via overexpression of anti-apoptotic regulators of MOMP. Simulations of cell-to-cell variability in TRAIL-induced apoptosis confirmed that the endogenous variability in apoptotic regulators was sufficient to produce the observed variability in death time. However, knowledge of the concentration of individual proteins did not allow prediction of death time because variation in other proteins masked the underlying trends. The ability to simulate heterogeneity in cellular response also led to the development of novel, biologically intuitive methods of sensitivity analysis, which revealed that sensitivities shift depending on whether knowledge of covariance in initial conditions is included. The ability to predict sensitivity and resistance of tumors to TRAIL would be clinically valuable, as TRAIL is currently in clinical trials as an anti-cancer therapy. The results described here represent progress toward understanding the "fractional killing" of tumor cells following exposure to chemotherapy, and for understanding variability in mammalian signaling pathways in general., by Sabrina Leigh Spencer., Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2008., Includes bibliographical references (leaves 39-43)., Protein phosphatases regulate the phosphorylation state of intracellular signaling molecules in conjunction with protein kinases. In order to better understand the role of phosphatases in the ErbB signaling network, experimental studies were carried out to validate assays and phosphatase inhibitors for gathering dynamic, system-wide data of phosphatase activity. We verified the use of In-Cell Westerns and phospho-ErbB ELISAs for these studies, as well as the use of different cell lines and both general and specific phosphatase inhibitors in system-wide experiments. The phosphatase inhibitors we used perturbed cellular systems in complex ways that can help elucidate the regulation and activity of specific phosphatases in the ErbB signaling pathway in the future., by Kuojung Gordon Lu., S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008., Includes bibliographical references (p. 157-169)., Cells use a complex web of protein signaling pathways to interpret extracellular cues and decide and execute cell fates such as survival, apoptosis, differentiation, and proliferation. Cell decisions can be triggered by subtle, transient signals that are context specific, making them hard to study by conventional experimental methods. In this thesis, we use a systems approach combining quantitative experiments with computational modeling and analysis to understand the regulation of the survival-vs-death decision. A second goal of this thesis was to develop modeling and analysis methods that enable study of signals that are transient or at intermediate activation levels. We addressed the challenge of balancing mechanistic detail and ease of interpretation in modeling by adapting fuzzy logic to analyze a previously published experimental dataset characterizing the dynamic behavior of kinase pathways governing apoptosis in human colon carcinoma cells. Simulations of our fuzzy logic model recapitulated most features of the data and generated several predictions involving pathway crosstalk and regulation. Fuzzy logic models are flexible, able to incorporate qualitative and noisy data, and powerful enough to generate not only quantitative predictions but also biological insights concerning operation of signaling networks. To study transient signals in differential-equation based models, we employed direct Lyapunov exponents (DLEs) to identify phase-space domains of high sensitivity to initial conditions. These domains delineate regions exhibiting qualitatively different transient activities that would be indistinguishable using steady-state analysis but which correspond to different outcomes., (cont.) We combine DLE analysis of a physicochemical model of receptor-mediated apoptosis with single cell data obtained by flow cytometry and FRET-based reporters in live-cell microscopy to classify conditions that alter the usage of two apoptosis pathways (Type I/II apoptosis). While it is generally thought that the control point for Type I/II occurs at the level of initiator caspase activation, we find that Type II cells can be converted to Type I by removal of XIAP, a regulator of effector caspases. Our study suggests that the classification of cells as Type I or II obscures a third variable category of cells that are highly sensitive to changes in the concentrations of key apoptotic network proteins., by Bree Beardsley Aldridge., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005., Includes bibliographical references., The spindle checkpoint ensures the fidelity of chromosome segregation by delaying anaphase until all sister chromatids form proper bipolar attachments to the mitotic spindle. Spindle checkpoint proteins localize to unattached or maloriented kinetochores in mitosis and generate a signal that prevents dissolution of sister chromatid cohesion. Checkpoint signaling requires binding of Mad2 to the checkpoint protein Madl and Cdc20, a subunit of the Anaphase Promoting Complex. We have characterized the interactions of human Mad2 with Madl, Cdc20, and CMT2, a checkpoint inhibitor. Cdc20 and Madl form competitive high affinity complexes through contacts in the peptide binding cleft of Mad2, while CMT2 binds noncompetitively to the closed conformation of the Mad2 C-terminus. I propose a model by which conformation- specific binding of CMT2 silences Mad2 signal generation. The requirement for active checkpoint inhibition in mitosis is not known. We examined the role of CMT2 in mitosis by fixed- and live-cell microscopy. CMT2 localizes to kinetochores in a Mad2-dependent manner and forms ternary complexes with Madl-Mad2 and Cdc20-Mad2 in vivo. Surprisingly, CMT2 is required for completion of mitosis even in the absence of spindle damage., (cont.) I show that CMT2 opposes Mad2 function at kinetochores and in the cytosol and propose that active silencing of the Mad2- dependent checkpoint is required for completion of mammalian mitosis., by Robert S. Hagan., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007., Includes bibliographical references., Kinetochores are proteinacious structures that assemble on centromeric DNA and fulfill several important functions during chromosome segregation. They attach chromosomes to microtubules of the mitotic spindle in a bipolar fashion and monitor the state of the kinetochore-microtubule attachment. In case errors are present, a monitoring mechanism, the spindle assembly checkpoint, inhibits metaphase to anaphase transition until proper bipolar attachments are formed. The mechanics of the attachment process is poorly understood and functions of many players involved are largely unknown. The relationship between the formation of kinetochore-microtubule attachment and its monitoring by the checkpoint remains unclear. I combined an RNAi technology with the high resolution microscopy to analyze the function of six microtubule-binding proteins and a checkpoint protein complex ROD/ZW 10/Zwilch (RZZ) in chromosome segregation and spindle checkpoint signaling. I discovered a non-redundant role for CLIP- 170, dynein/dynactin, LIS 1 and TOG I in chromosome congression to the metaphase plate. Selective depletion of dynein/dynactin and CLIP- 170 from mitotic kinetochores by RNAi of ZW 10, uncovered a novel role for these proteins in the initial kinetochore-microtubule encounter. Surprisingly, my results also demonstrate that ZW10 functions in the spindle checkpoint signaling independently of previously proposed downstream player, Mad2. In addition, I identified a ZW10's interaction with BubRI that may suggest a possible role for ZW 10 in the checkpoint signaling pathway. Thus RZZ complex acts at the interphase of attachment and signaling at kinetochores suggesting a close link between structural establishment of kinetochore-microtubule binding and its monitoring by the spindle checkpoint, in contrary to classical thinking., by Irina Michailovna Shapiro., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007., Includes bibliographical references., Kinetochores are proteinacious structures that assemble on centromeric DNA and fulfill several important functions during chromosome segregation. They attach chromosomes to microtubules of the mitotic spindle in a bipolar fashion and monitor the state of the kinetochore-microtubule attachment. In case errors are present, a monitoring mechanism, the spindle assembly checkpoint, inhibits metaphase to anaphase transition until proper bipolar attachments are formed. The mechanics of the attachment process is poorly understood and functions of many players involved are largely unknown. The relationship between the formation of kinetochore-microtubule attachment and its monitoring by the checkpoint remains unclear. I combined an RNAi technology with the high resolution microscopy to analyze the function of six microtubule-binding proteins and a checkpoint protein complex ROD/ZW 10/Zwilch (RZZ) in chromosome segregation and spindle checkpoint signaling. I discovered a non-redundant role for CLIP- 170, dynein/dynactin, LIS 1 and TOG I in chromosome congression to the metaphase plate. Selective depletion of dynein/dynactin and CLIP- 170 from mitotic kinetochores by RNAi of ZW 10, uncovered a novel role for these proteins in the initial kinetochore-microtubule encounter. Surprisingly, my results also demonstrate that ZW10 functions in the spindle checkpoint signaling independently of previously proposed downstream player, Mad2. In addition, I identified a ZW10's interaction with BubRI that may suggest a possible role for ZW 10 in the checkpoint signaling pathway. Thus RZZ complex acts at the interphase of attachment and signaling at kinetochores suggesting a close link between structural establishment of kinetochore-microtubule binding and its monitoring by the spindle checkpoint, in contrary to classical thinking., by Irina Michailovna Shapiro., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006., "February 2006.", Includes bibliographical references., Kinetochores are large, multi-protein complexes that bind centromeric DNA to the microtubules of the mitotic spindle and mediate chromosome movement throughout the cell cycle. The proteins that regulate both force generation at kinetochores as well as and the cell-cycle-dependent changes in kinetochore architecture are largely unknown. The relative simplicity of centromere specification and kinetochore-microtubule attachment make Saccharomyces cerevisiae an attractive model organism for investigations into kinetochore-microtubule attachment and regulation. We used a combination of cell biology and biochemistry to study the roles of the four nuclear kinesin motor proteins at budding yeast kinetochores. We discovered that each of the four nuclear kinesins localizes to kinetochores. Three of these, Cin8p, Kip1p, and Kip3p are present at mature chromosome-microtubule attachments in metaphase. Cin8p and Kip1p align metaphase chromosomes into the characteristic bilobed configuration that is analogous to the metaphase plate in higher eukaryotes. Kip3p regulates microtubule dynamics throughout the cell cycle and regulates poleward movement during anaphase. Kar3p, the final nuclear kinesin, is recruited specifically to detached kinetochores., (cont.) In addition, we have discovered that kinetochore-microtubule attachments alter during the cell cycle indicating that kinetochore function is temporally regulated., by Jessica Dawn Tytell., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007., Includes bibliographical references., Kinetochores are proteinacious structures that assemble on centromeric DNA and fulfill several important functions during chromosome segregation. They attach chromosomes to microtubules of the mitotic spindle in a bipolar fashion and monitor the state of the kinetochore-microtubule attachment. In case errors are present, a monitoring mechanism, the spindle assembly checkpoint, inhibits metaphase to anaphase transition until proper bipolar attachments are formed. The mechanics of the attachment process is poorly understood and functions of many players involved are largely unknown. The relationship between the formation of kinetochore-microtubule attachment and its monitoring by the checkpoint remains unclear. I combined an RNAi technology with the high resolution microscopy to analyze the function of six microtubule-binding proteins and a checkpoint protein complex ROD/ZW 10/Zwilch (RZZ) in chromosome segregation and spindle checkpoint signaling. I discovered a non-redundant role for CLIP- 170, dynein/dynactin, LIS 1 and TOG I in chromosome congression to the metaphase plate. Selective depletion of dynein/dynactin and CLIP- 170 from mitotic kinetochores by RNAi of ZW 10, uncovered a novel role for these proteins in the initial kinetochore-microtubule encounter. Surprisingly, my results also demonstrate that ZW10 functions in the spindle checkpoint signaling independently of previously proposed downstream player, Mad2. In addition, I identified a ZW10's interaction with BubRI that may suggest a possible role for ZW 10 in the checkpoint signaling pathway. Thus RZZ complex acts at the interphase of attachment and signaling at kinetochores suggesting a close link between structural establishment of kinetochore-microtubule binding and its monitoring by the spindle checkpoint, in contrary to classical thinking., by Irina Michailovna Shapiro., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006., "February 2006.", Includes bibliographical references., Kinetochores are large, multi-protein complexes that bind centromeric DNA to the microtubules of the mitotic spindle and mediate chromosome movement throughout the cell cycle. The proteins that regulate both force generation at kinetochores as well as and the cell-cycle-dependent changes in kinetochore architecture are largely unknown. The relative simplicity of centromere specification and kinetochore-microtubule attachment make Saccharomyces cerevisiae an attractive model organism for investigations into kinetochore-microtubule attachment and regulation. We used a combination of cell biology and biochemistry to study the roles of the four nuclear kinesin motor proteins at budding yeast kinetochores. We discovered that each of the four nuclear kinesins localizes to kinetochores. Three of these, Cin8p, Kip1p, and Kip3p are present at mature chromosome-microtubule attachments in metaphase. Cin8p and Kip1p align metaphase chromosomes into the characteristic bilobed configuration that is analogous to the metaphase plate in higher eukaryotes. Kip3p regulates microtubule dynamics throughout the cell cycle and regulates poleward movement during anaphase. Kar3p, the final nuclear kinesin, is recruited specifically to detached kinetochores., (cont.) In addition, we have discovered that kinetochore-microtubule attachments alter during the cell cycle indicating that kinetochore function is temporally regulated., by Jessica Dawn Tytell., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007., Includes bibliographical references., The ubiquity of aneuploidy in human cancers, particularly solid tumors, suggests a fundamental link between errors in chromosome segregation and tumorigenesis. The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase onset until all kinetochores achieve bipolar attachment to the mitotic spindle. Abrogation of the mammalian spindle checkpoint can cause chromosomal instability (CIN), the frequent gain or loss of entire chromosomes during cell divisions. While CIN has been proposed to be sufficient to initiate tumorigenesis, no direct evidence has shown that CIN itself is a causal agent for human cancer. To determine if CIN can promote tumor formation, I generated mice containing a conditional mutant allele of the spindle checkpoint kinase MPS1 (MpslA) and induced its expression in thymocytes. I show that MpslA is a partial loss of function allele for spindle checkpoint activation which results in chromosome missegregation in MEFs and embryonic lethality in mice. Expression of MpslA in thymocytes causes sporadic lymphomas and a decrease in thymus size. Strikingly, when MpslA was introduced into a p53 heterozygous background, all mice developed thymomas due to a loss of heterozygosity of the wildtype p53 allele., (cont.) In contrast, no p53 heterozygous micedeveloped thymomas in the same time frame when MPS1 is wildtype. Moreover, MpslA also accelerated tumor formation in p 19AF heterozygous thymocytes. I propose that MpslA-induced CIN activates p53, and results in apoptosis of the majority of SpsI A- expressirn cells. Therefore, there- ;is strong selecptiove pressure- for ;nactivation of the p53 pathway, and in those cells where p53 loss does occur, MpslA-induced CIN drives tumor development. In summary, I show that CIN caused by a weakened checkpoint is sufficient to facilitate tumorigenesis when the p53 pathway is also impaired., by Stephanie Zhi-Juan Xie., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007., Includes bibliographical references., Apoptosis is a form of programmed cell death that is essential for the elimination of damaged or unneeded cells in multicellular organisms. Inactivation of apoptotic cell death is a necessary step in the development of cancer, while hypersensitivity to apoptosis is a factor in degenerative diseases. Many of the molecular components controlling apoptosis have been identified, including the central effectors of apoptosis, a family of proteases known as caspases that efficiently dismantle the cell when active. While many of the molecular details of apoptotic regulators are now understood, a major challenge is to integrate this information to understand quantitatively how sensitivity to apoptosis and the kinetics of death are determined, in both single cells and populations of cells. We have approached this problem with a combined experimental and computational approach. Using single-cell observations, genetic and pharmacological perturbations, and mechanistic mathematical modeling, we have dissected the mechanism by which cells make a binary decision between survival and apoptosis. We identified conditions under which the apoptotic decision system fails, allowing cells to survive with caspase-induced damage that may result in damage to the genome and oncogenesis., (cont.) We further used live-cell imaging to identify and characterize a kinetic threshold at which slow and variable upstream signals are converted into rapid and discrete downstream caspase activation. Lastly, we examined the integration of multiple pro-and apoptotic signal transduction pathways by constructing a principal component-based model that linked apoptotic phenotypes to a compendium of signaling measurements. This approach enabled the identification of the molecular signals most important in determining the level of apoptosis across a population of cells. Together, our findings provide insight into the molecular and kinetic mechanisms by which cells integrate diverse molecular signals to make a discrete cell fate decision., by John G. Albeck., Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2006., Includes bibliographical references., In this project, we have proposed a decorrelator-based single antenna interference cancellation algorithm for the asynchronous GSM/GPRS network. The algorithm is tested according to the current SAIC/DARP performance requirement in the computer simulation, and is shown to give various gains in different test scenarios., by Sheldon Y. Chan., M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007., Includes bibliographical references., The ubiquity of aneuploidy in human cancers, particularly solid tumors, suggests a fundamental link between errors in chromosome segregation and tumorigenesis. The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase onset until all kinetochores achieve bipolar attachment to the mitotic spindle. Abrogation of the mammalian spindle checkpoint can cause chromosomal instability (CIN), the frequent gain or loss of entire chromosomes during cell divisions. While CIN has been proposed to be sufficient to initiate tumorigenesis, no direct evidence has shown that CIN itself is a causal agent for human cancer. To determine if CIN can promote tumor formation, I generated mice containing a conditional mutant allele of the spindle checkpoint kinase MPS1 (MpslA) and induced its expression in thymocytes. I show that MpslA is a partial loss of function allele for spindle checkpoint activation which results in chromosome missegregation in MEFs and embryonic lethality in mice. Expression of MpslA in thymocytes causes sporadic lymphomas and a decrease in thymus size. Strikingly, when MpslA was introduced into a p53 heterozygous background, all mice developed thymomas due to a loss of heterozygosity of the wildtype p53 allele., (cont.) In contrast, no p53 heterozygous micedeveloped thymomas in the same time frame when MPS1 is wildtype. Moreover, MpslA also accelerated tumor formation in p 19AF heterozygous thymocytes. I propose that MpslA-induced CIN activates p53, and results in apoptosis of the majority of SpsI A- expressirn cells. Therefore, there- ;is strong selecptiove pressure- for ;nactivation of the p53 pathway, and in those cells where p53 loss does occur, MpslA-induced CIN drives tumor development. In summary, I show that CIN caused by a weakened checkpoint is sufficient to facilitate tumorigenesis when the p53 pathway is also impaired., by Stephanie Zhi-Juan Xie., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007., Includes bibliographical references., Apoptosis is a form of programmed cell death that is essential for the elimination of damaged or unneeded cells in multicellular organisms. Inactivation of apoptotic cell death is a necessary step in the development of cancer, while hypersensitivity to apoptosis is a factor in degenerative diseases. Many of the molecular components controlling apoptosis have been identified, including the central effectors of apoptosis, a family of proteases known as caspases that efficiently dismantle the cell when active. While many of the molecular details of apoptotic regulators are now understood, a major challenge is to integrate this information to understand quantitatively how sensitivity to apoptosis and the kinetics of death are determined, in both single cells and populations of cells. We have approached this problem with a combined experimental and computational approach. Using single-cell observations, genetic and pharmacological perturbations, and mechanistic mathematical modeling, we have dissected the mechanism by which cells make a binary decision between survival and apoptosis. We identified conditions under which the apoptotic decision system fails, allowing cells to survive with caspase-induced damage that may result in damage to the genome and oncogenesis., (cont.) We further used live-cell imaging to identify and characterize a kinetic threshold at which slow and variable upstream signals are converted into rapid and discrete downstream caspase activation. Lastly, we examined the integration of multiple pro-and apoptotic signal transduction pathways by constructing a principal component-based model that linked apoptotic phenotypes to a compendium of signaling measurements. This approach enabled the identification of the molecular signals most important in determining the level of apoptosis across a population of cells. Together, our findings provide insight into the molecular and kinetic mechanisms by which cells integrate diverse molecular signals to make a discrete cell fate decision., by John G. Albeck., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007., Includes bibliographical references., The ubiquity of aneuploidy in human cancers, particularly solid tumors, suggests a fundamental link between errors in chromosome segregation and tumorigenesis. The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase onset until all kinetochores achieve bipolar attachment to the mitotic spindle. Abrogation of the mammalian spindle checkpoint can cause chromosomal instability (CIN), the frequent gain or loss of entire chromosomes during cell divisions. While CIN has been proposed to be sufficient to initiate tumorigenesis, no direct evidence has shown that CIN itself is a causal agent for human cancer. To determine if CIN can promote tumor formation, I generated mice containing a conditional mutant allele of the spindle checkpoint kinase MPS1 (MpslA) and induced its expression in thymocytes. I show that MpslA is a partial loss of function allele for spindle checkpoint activation which results in chromosome missegregation in MEFs and embryonic lethality in mice. Expression of MpslA in thymocytes causes sporadic lymphomas and a decrease in thymus size. Strikingly, when MpslA was introduced into a p53 heterozygous background, all mice developed thymomas due to a loss of heterozygosity of the wildtype p53 allele., (cont.) In contrast, no p53 heterozygous micedeveloped thymomas in the same time frame when MPS1 is wildtype. Moreover, MpslA also accelerated tumor formation in p 19AF heterozygous thymocytes. I propose that MpslA-induced CIN activates p53, and results in apoptosis of the majority of SpsI A- expressirn cells. Therefore, there- ;is strong selecptiove pressure- for ;nactivation of the p53 pathway, and in those cells where p53 loss does occur, MpslA-induced CIN drives tumor development. In summary, I show that CIN caused by a weakened checkpoint is sufficient to facilitate tumorigenesis when the p53 pathway is also impaired., by Stephanie Zhi-Juan Xie., Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2006., Includes bibliographical references., In this project, we have proposed a decorrelator-based single antenna interference cancellation algorithm for the asynchronous GSM/GPRS network. The algorithm is tested according to the current SAIC/DARP performance requirement in the computer simulation, and is shown to give various gains in different test scenarios., by Sheldon Y. Chan., M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2006., Includes bibliographical references (leaves 181-191)., Surface-based sensors take advantage of the natural high surface-to-volume ratios in microfluidic devices, low reagent consumption and high potential for integration in more complex micro total analysis systems (microTAS or pTAS). This thesis studies the fundamental limits of on-chip integrated microfluidic sensors. More specifically, it focuses on detection methods involving surface interaction in channels with thicknesses on the order of a few microns or less. Through mass transfer analysis, we demonstrate that, for thin enough channels, sample detection is limited by the convective transport of analytes, and neither by diffusion nor reaction. The results provided extend the validity of transport models to include transport in the absence of mass transfer boundary layer. All existing analytic solutions to the Graetz problem are described and compiled. The analysis, complemented by finite element simulations, successfully predicts experimental observations made for on-chip immunoassays in micron-thick fluidic channels. Subsequently, our study of on chip detection systems is carried on with emphasis on resonating cantilever sensors. In order to interpret the output signal from these devices, we develop a dynamic cantilever model to link spatially and temporally dependent mass adsorption with resonance frequency change., (cont.) The mass adsorption is then directly related to the sensors' operating conditions via the mass transfer models previously developed. We then develop a 2D finite-element model capable of predicting the devices response and of extracting bimolecular rate constants. Finally, since hydraulic resistance severely increases as channels get shallower, we provide a structural analysis of polymer-based microsystems. Through scaling and numerical simulations we demonstrate the effect of channel deformation on the flow conditions inside the device and vice versa. Finally, channel deformation is experimentally quantified using optical methods and compared with the models developed. Throughout this thesis, the approach to physical modeling has been to use mathematical and numerical analysis as predictive tools in the design of integrated lab-on-a-chip systems. Whenever possible, scaling and analytic solutions are developed, since they provide a direct relationship between experimental observations, geometry and the multiple dependent variables in the system, and can be readily used as design criteria by the experimentalist., by Thomas Gervais., Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2006., Includes bibliographical references., In this project, we have proposed a decorrelator-based single antenna interference cancellation algorithm for the asynchronous GSM/GPRS network. The algorithm is tested according to the current SAIC/DARP performance requirement in the computer simulation, and is shown to give various gains in different test scenarios., by Sheldon Y. Chan., M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2006., Includes bibliographical references (leaves 181-191)., Surface-based sensors take advantage of the natural high surface-to-volume ratios in microfluidic devices, low reagent consumption and high potential for integration in more complex micro total analysis systems (microTAS or pTAS). This thesis studies the fundamental limits of on-chip integrated microfluidic sensors. More specifically, it focuses on detection methods involving surface interaction in channels with thicknesses on the order of a few microns or less. Through mass transfer analysis, we demonstrate that, for thin enough channels, sample detection is limited by the convective transport of analytes, and neither by diffusion nor reaction. The results provided extend the validity of transport models to include transport in the absence of mass transfer boundary layer. All existing analytic solutions to the Graetz problem are described and compiled. The analysis, complemented by finite element simulations, successfully predicts experimental observations made for on-chip immunoassays in micron-thick fluidic channels. Subsequently, our study of on chip detection systems is carried on with emphasis on resonating cantilever sensors. In order to interpret the output signal from these devices, we develop a dynamic cantilever model to link spatially and temporally dependent mass adsorption with resonance frequency change., (cont.) The mass adsorption is then directly related to the sensors' operating conditions via the mass transfer models previously developed. We then develop a 2D finite-element model capable of predicting the devices response and of extracting bimolecular rate constants. Finally, since hydraulic resistance severely increases as channels get shallower, we provide a structural analysis of polymer-based microsystems. Through scaling and numerical simulations we demonstrate the effect of channel deformation on the flow conditions inside the device and vice versa. Finally, channel deformation is experimentally quantified using optical methods and compared with the models developed. Throughout this thesis, the approach to physical modeling has been to use mathematical and numerical analysis as predictive tools in the design of integrated lab-on-a-chip systems. Whenever possible, scaling and analytic solutions are developed, since they provide a direct relationship between experimental observations, geometry and the multiple dependent variables in the system, and can be readily used as design criteria by the experimentalist., by Thomas Gervais., Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2004., Includes bibliographical references (p. 83)., The Open Microscopy Environment (OME) image browser enables biologists to quickly analyze, manipulate and modify large imaging datasets. The browser includes a variety of features that facilitate image classification, annotation, visualization, and organization. The browser displays image metadata using a variety of techniques, including visual cues, context-sensitive overlays, and color-coding. The application explicitly supports visualization of screening datasets, but also supports multidimensional images, as well as standalone images. When integrated with the rest of the applications of the OME client software, the browser allows users to view images in greater resolution, analyze multiple dimensions, and in future releases will support analysis routines., by Jeffrey C. Mellen., M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2003., Includes bibliographical references., Kinetochores bind to microtubules and are responsible for chromosome segregation and the accurate transmission of genetic information during cell division. Kinetochores are DNA-protein complexes that assemble on centromeric DNA sequences. In budding yeast, the kinetochores consist of approximately fifty proteins that are organized into a large multi-subunit complex. Multiple kinetochore proteins are thought to work together in establishing, sensing and maintaining the microtubule attachment. Following attachment, kinetochores act to couple microtubule force generation to chromosome movements. Microtubule associated proteins are also thought to play a key role in this process by regulating plus-end microtubule dynamics and tensile force generation. However, the actual molecular mechanism of force generation is unclear. We have used a combination of live-cell imaging, biochemical and genetic techniques in the budding yeast, S. cerevisiae, to identify ten kinetochore subunits and elucidate their roles in microtubule attachment. Among these proteins are microtubule binding proteins and a molecular motor with homologues in animal cells. By analyzing the changes in chromosome positioning and dynamics in various kinetochore mutants, we show that different kinetochore proteins are required for the imposition of tension on paired sister kinetochores and for correct chromosome motion throughout the cell cycle. Utilizing 3D comparative motion analysis of the chromosomes has revealed that kinetochore proteins essential for microtubule attachment in early S-phase are not required at other points in the cell cycle. Our results suggest that different subsets of kinetochore proteins regulate the dynamic nature of microtubule growth and shrinkage for the generation of mechanical force and proper chromosome segregation., by Daniel R. Rines., Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001., "June 2001.", Includes bibliographical references., by Max C. Dobles., Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002., Vita., Includes bibliographical references., The separation and partitioning of sister chromatids prior to cytokinesis must be tightly regulated to insure that progeny cells receive the correct number of chromosomes. The spindle assembly checkpoint prevents chromosome missegregation by arresting the cell before sister chromatid separation if any one chromatid is not properly attached to the spindle microtubules. Defects in chromosome segregation lead to the creation of aneuploid cells that contain too much or too little genetic material. Some aneuploid cells have gained oncogenes or lost tumor suppressor genes, and these genetic alterations allow the cells to ignore signals that regulate growth and proliferation. A majority of tumors are aneuploid, but it is not yet known whether chromosome instability is a driving force or merely a secondary effect of tumor development. In Chapter 2, I will present work showing that the protein most often altered in colon cancers, Adenomatous Polyposis Coli (AdPC) (Kinzler and Vogelstein 1996), is modified by members of the spindle assembly checkpoint and that a truncating mutation of the AdPC protein leads directly to aneuploidy in mouse cells. Although genetic disruption of any member of the spindle assembly checkpoint signaling cascade has proven lethal for metazoans on both the cellular and organismal level (Basu et al. 1999; Kitagawa and Rose 1999; Dobles et al. 2000; Kalitsis et al. 2000), Chapter 3 will detail the creation and analysis of a Mad2-/- mouse embryonic fibroblast cell line that is viable due to a simultaneous disruption in p53., (cont.) Together these two bodies of research support the possibility that a chromosome missegregation event can be a defining, early step in tumorigenesis while providing unique tools to investigate this possibility., by Aurura A. Burds., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2004., Includes bibliographical references (p. 65-66)., Gene expression arrays are a technology used to measure quantities of messenger ribonucleic acid (mRNA). Application of the technology involves a variety of physical processes beginning with the acquisition of mRNA samples and ending with the fluorescence imaging of a gene expression array. This thesis examines these physical processes, develops a mechanistic model, and derives the analysis procedure based on the model. Chief advantages of this approach are that it accounts for certain previously unexplained array phenomena and is based in a clear way on physical knowledge allowing non-arbitrary determination of both the probability that any given gene has altered expression ratio relative to a control as well as the magnitude of this induction or repression. We demonstrate its use on simulated and real array data, and show that a considerable amount of previously unrecognized information concerning gene expression differences is inherent in the array measurements., by Keith Howard Duggar., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2003., Includes bibliographical references., Kinetochores bind to microtubules and are responsible for chromosome segregation and the accurate transmission of genetic information during cell division. Kinetochores are DNA-protein complexes that assemble on centromeric DNA sequences. In budding yeast, the kinetochores consist of approximately fifty proteins that are organized into a large multi-subunit complex. Multiple kinetochore proteins are thought to work together in establishing, sensing and maintaining the microtubule attachment. Following attachment, kinetochores act to couple microtubule force generation to chromosome movements. Microtubule associated proteins are also thought to play a key role in this process by regulating plus-end microtubule dynamics and tensile force generation. However, the actual molecular mechanism of force generation is unclear. We have used a combination of live-cell imaging, biochemical and genetic techniques in the budding yeast, S. cerevisiae, to identify ten kinetochore subunits and elucidate their roles in microtubule attachment. Among these proteins are microtubule binding proteins and a molecular motor with homologues in animal cells. By analyzing the changes in chromosome positioning and dynamics in various kinetochore mutants, we show that different kinetochore proteins are required for the imposition of tension on paired sister kinetochores and for correct chromosome motion throughout the cell cycle. Utilizing 3D comparative motion analysis of the chromosomes has revealed that kinetochore proteins essential for microtubule attachment in early S-phase are not required at other points in the cell cycle. Our results suggest that different subsets of kinetochore proteins regulate the dynamic nature of microtubule growth and shrinkage for the generation of mechanical force and proper chromosome segregation., by Daniel R. Rines., Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001., "June 2001.", Includes bibliographical references., by Max C. Dobles., Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002., Vita., Includes bibliographical references., The separation and partitioning of sister chromatids prior to cytokinesis must be tightly regulated to insure that progeny cells receive the correct number of chromosomes. The spindle assembly checkpoint prevents chromosome missegregation by arresting the cell before sister chromatid separation if any one chromatid is not properly attached to the spindle microtubules. Defects in chromosome segregation lead to the creation of aneuploid cells that contain too much or too little genetic material. Some aneuploid cells have gained oncogenes or lost tumor suppressor genes, and these genetic alterations allow the cells to ignore signals that regulate growth and proliferation. A majority of tumors are aneuploid, but it is not yet known whether chromosome instability is a driving force or merely a secondary effect of tumor development. In Chapter 2, I will present work showing that the protein most often altered in colon cancers, Adenomatous Polyposis Coli (AdPC) (Kinzler and Vogelstein 1996), is modified by members of the spindle assembly checkpoint and that a truncating mutation of the AdPC protein leads directly to aneuploidy in mouse cells. Although genetic disruption of any member of the spindle assembly checkpoint signaling cascade has proven lethal for metazoans on both the cellular and organismal level (Basu et al. 1999; Kitagawa and Rose 1999; Dobles et al. 2000; Kalitsis et al. 2000), Chapter 3 will detail the creation and analysis of a Mad2-/- mouse embryonic fibroblast cell line that is viable due to a simultaneous disruption in p53., (cont.) Together these two bodies of research support the possibility that a chromosome missegregation event can be a defining, early step in tumorigenesis while providing unique tools to investigate this possibility., by Aurura A. Burds., Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2004., Includes bibliographical references (p. 83)., The Open Microscopy Environment (OME) image browser enables biologists to quickly analyze, manipulate and modify large imaging datasets. The browser includes a variety of features that facilitate image classification, annotation, visualization, and organization. The browser displays image metadata using a variety of techniques, including visual cues, context-sensitive overlays, and color-coding. The application explicitly supports visualization of screening datasets, but also supports multidimensional images, as well as standalone images. When integrated with the rest of the applications of the OME client software, the browser allows users to view images in greater resolution, analyze multiple dimensions, and in future releases will support analysis routines., by Jeffrey C. Mellen., M.Eng.