Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2013. / Cataloged from PDF version of thesis. "September 2013." / Includes bibliographical references (pages 54-60). / DnaA is the bacterial replication initiator, which also functions as a transcription factor to regulate gene expression. In B. subtilis, DnaA has previously been shown to repress its own transcription and has also been implicated in directing part of the transcriptional response to replication stress. Because dnaA is essential, most of DnaA's potential effects on gene expression have been determined through indirect methods, which have implemented perturbations in replication and sequence analyses to predict direct effects of DnaA transcriptional regulation. Below, I take a more direct approach to assay DnaA's effect on gene expression and specific transcriptional regulatory networks by deleting dnaA in an oriN+ [delta]oriC strain background, which renders dnaA non-essential. Isogenic dnaA+ cells were constructed similarly and have dnaA constitutively expressed from an ectopic locus. In this background, DNA replication no longer depends on dnaA and is initiated instead by a plasmid replicon, oriN. The native origin of replication, oriC, is also deleted to eliminate differences in replication between [delta]dnaA and dnaA+ cells. Consequently, I can directly compare differences in gene expression due to the presence versus absence of dnaA. Deletion of dnaA results in approximately 463 significant differences in gene expression, most of which I show are due to DnaA direct activation of the gene sda. Many of these genes lie downstream of Sda activity and comprise several regulons, such as the Spo0A, AbrB, and SinR regulons. These regulons are known to become active during the transition from exponential growth to stationary phase. In addition to the many effects on gene expression, I show that deletion of dnaA results in lowered competence development. I also revisit the transcriptional response to replication stress and show that some of the previously predicted targets of DnaA respond to replication stress in a DnaA-dependent manner. Lastly, in collaboration with others, I have studied the relationship between a DnaA regulator, YabA and a nucleoid binding protein Rok. YabA and Rok associate at some of the same chromosomal regions, and at these regions YabA absolutely depends on Rok for its association. We are currently trying to understand the functional relationship between YabA, Rok, and DnaA. / by Tracy Washington. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Metabolic syndrome describes a complex set of obesity-related disorders that enhance diabetes, cardiovascular, and mortality risk. Studies of liver-specific protein-tyrosine phosphatase lb (PTPlb) deletion mice (L-PTPlb-/-) suggests that hepatic PTPlb inhibition would mitigate metabolic syndrome progression through amelioration of hepatic insulin resistance, endoplasmic reticulum stress, and whole-body lipid metabolism. However, the network alterations underlying these phenotypes are poorly understood. Mass spectrometry was used to quantitatively discover protein phosphotyrosine network changes in L-PTP lb-/- mice relative to control mice under both normal and high-fat diet conditions. A phosphosite set enrichment analysis was developed to identify numerous pathways exhibiting PTPlb- and diet-dependent phosphotyrosine regulation. Detection of PTP lb-dependent phosphotyrosine sites on lipid metabolic proteins initiated global lipidomics characterization of corresponding liver samples and revealed altered fatty acid and triglyceride metabolism in L-PTPlb-/- mice. Multivariate modeling techniques were developed to infer molecular dependencies between phosphosites and lipid metabolic changes, resulting in quantitatively predictive phenotypic models. / by Emily R. Miraldi. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / To better understand the tissue-specific regulation of chromatin state in cell-fate determination and development, we defined the tissue-specific expression of all 36 lysine methyltransferase (KMT) genes by endogenous mRNA detection in C. elegans. We found that most KMTs are expressed in only one or two tissues and that the germline is the tissue with the most general KMT expression. We discovered that the germline-expressed C. elegans ortholog of mammalian PRDM9, SET-1 7, promotes fertility through gene regulation in primary spermatocytes. SET-17 drives transcription of spermatocyte-specific genes from four genomic clusters to promote spermatid production. SET-1 7 is concentrated in stable, chromatin-associated nuclear foci at actively transcribed gene clusters, which we term spermatocyte transcription bodies. Our results identify the spatially restricted function of a PRDM9 ortholog in spermatocyte transcription and we propose that the spatial organization of chromatin factors might be a conserved mechanism in tissue-specific control of transcription. / by Christoph G. Engert. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 203-226). / Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. However, the predominant mechanism by which c-Myc regulates global transcription in both healthy and tumor cells is poorly understood. In this thesis, I present evidence that c-Myc is a global regulator of RNA Polymerase II (RNA Pol II) transcriptional pause release. Transcriptional pausing occurs when additional regulatory steps are required to promote elongation of genes after transcription has initiated. Chapter 2 identifies transcriptional pausing as a general feature of transcription by RNA Pol II in mammalian cells. c-Myc is identified as having a major role in promoting release from pause at its target genes. Chapter 3 finds in tumor cells with elevated c-Myc, the transcription factor binds to promoters and enhancers of most actively transcribed genes. The predominant effect of c-Myc binding is to produce higher levels of transcription by promoting RNA Pol II transcriptional pause release. Thus, c-Myc accumulates in the promoter regions of active genes across the cancer cell genome and causes transcriptional amplification, producing increased levels of transcripts within the cells gene expression program. These results imply that transcriptional amplification can reduce rate-limiting constraints for tumor cell growth and proliferation. / by Charles Yang Lin. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2010. / 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. / Excessive collagen degradation (collagenolysis) has been implicated in a series of diseases such as tumor metastasis, atherosclerosis and arthritis. There are still several unresolved questions about the mechanism of collagenolysis. First, the prototypical structure of the collagen triple helix does not fit into the active site of collagenases, the enzymes responsible of cleaving collagen. Moreover, the scissile bond that is degraded during collagenolysis is hidden from solvent. Therefore it is widely agreed that collagen unfolding must occur in order for collagenolysis to proceed. Some proposed mechanisms suggest that collagenases actively unfold collagen in order to expose the cleavage site, but no direct evidence of such mechanisms has been provided. Second, while several potential cleavage sites exist in the sequence of collagen, only one is cleaved in triple helical collagen. The hypothesis of this work is that locally unfolded states exist in collagen in the absence of collagenases. They occur as a result of the natural thermal fluctuations in the structure of collagen. Collagenolysis occurs when collagenases bind and cleave these unfolded states. In this work, a combination of computational and experimental methods is presented in order to test this hypothesis. Initially, computational results suggest that locally unfolded states are ubiquitous along the structure of collagen. However, it is shown that not all unfolded states are created equal, and that the precise sequence in the vicinity of the true collagenase cleavage site in type III collagen allows collagen to sample locally unfolded states that are complimentary to the collagenase active site. Therefore, it is hypothesized that cleavage site specificity is encoded in the nature of the unfolded states. Next, it is shown that types I and III collagen can be bound and cleaved at the actual cleavage site by just the catalytic domain of collagenases, a finding in apparent contradiction with previous work in this field. These results are interpreted in light of a novel conformational selection mechanism in which collagenases only cleave locally unfolded, vulnerable states. Finally, based on the new mechanism of collagenolysis presented here, new strategies to regulate collagenolysis are proposed. / by Ramon Salsas Escat. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2016. / 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 (pages 163-186). / Over the past decade, the advent of next-generation DNA sequencing technologies has ushered in an exciting era of biological research. Through large-scale sequencing projects, scientists have begun to unveil the variability and function of millions of DNA mutations called single nucleotide polymorphisms. Despite this rapid growth in understanding, short tandem repeats (STRs), genomic elements consisting of a repeating pattern of 2-6 bases, have remained poorly understood. Mutating orders of magnitude more rapidly than most of the human genome, STRs have been identified as the causal variants in diseases such as Fragile X syndrome and Huntington's disease. However, in spite of their potentially profound biological consequences, STRs remain systematically understudied due to difficulties associated with obtaining accurate genotypes. To address this issue, we developed a series of bioinformatics approaches and applied them to population-scale whole-genome sequencing data sets. Using data from the 1000 Genomes Project, we performed the first genome-wide characterization of STR variability by analyzing over 700,000 loci in more than 1000 individuals. Next, we integrated these genotypes with expression data to assess the contribution of STRs to gene expression in humans, uncovering their substantial regulatory role. We then developed a state-of-the-art algorithm to genotype STRs, resulting in vastly improved accuracy and uncovering hundreds of replicable de novo mutations in a deeply sequenced trio. Lastly, we developed a novel approach to estimate mutation rates for STRs on the Y-chromosome (Y-STR), resulting in rates for hundreds of previously uncharacterized markers. Collectively, these analyses highlight the extreme variability of STRs and provide a framework for incorporating them into future studies. / by Thomas F. Willems. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2013. / 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 (p. 112-119). / Microbes play a central role in diverse processes ranging from global elemental cycles to human digestion. Understanding these complex processes requires a rm under- standing of the interplay between microbes and their environment. In this thesis, we utilize sequencing data to study how individual species adapt to different niches, and how species assemble to form communities. First, we study the potential temperature and salinity range of 16 marine Vibrio strains. We nd that salinity tolerance is at odds with the strains' natural habitats, and provide evidence that this incongruence may be explained by a molecular coupling between salinity and temperature tolerance. Next, we investigate the genetic basis of bacterial ecological differentiation by analyzing the genomes of two closely related, yet ecologically distinct populations of Vibrio splendidus. We nd that most loci recombine freely across habitats, and that ecological differentiation is likely driven by a small number of habitat-specic alle-les. We further present a model for bacterial sympatric speciation. Our simulations demonstrate that a small number of adaptive loci facilitates speciation, due to the op- posing roles horizontal gene transfer (HGT) plays throughout the speciation process: HGT initially promotes speciation by bringing together multiple adaptive alleles, but later hinders it by mixing alleles across habitats. Finally, we introduce two tools for analyzing genomic survey data: SparCC, which infers correlations between taxa from relative abundance data; and StrainFinder, which extracts strain-level information from metagenomic data. Employing these tools, we infer a rich ecological network connecting hundreds of interacting species across 18 sites on the human body, and show that 16S-defined groups are rarely composed of a single dominant strain. / by Jonathan Friedman. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, February 2013. / 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. "September 2012." / Includes bibliographical references. / All cells sense and respond to their environments using signal transduction pathways. These pathways control a sweeping variety of cellular processes across the domains of life, but the pathways are often built from a small, shared set of protein domains. At the core of tens of thousands of signal transduction networks in bacteria is a pair of proteins, a histidine kinase and a response regulator. Upon receiving an input signal, a histidine kinase autophosphorylates and then catalyzes transfer of its phosphoryl group to a cognate response regulator, which often activates a transcriptional response. Bacteria typically encode dozens of kinases and regulators, and the kinases function as dimers in all known examples. This dimeric state raises two functional questions. Do histidine kinases specifically form dimers? Once a kinase has dimerized, does a chain in the dimer phosphorylate itself (cis) or its partner chain (trans)? Specific kinase dimerization is likely important to avoid detrimental crosstalk between separate signaling pathways, and how autophosphorylation occurs is central to kinase activity. In my thesis, I have taken biochemical and evolutionary approaches to identify molecular determinants for both dimerization specificity and autophosphorylation. To study dimerization specificity, I developed an in vitro binding assay to measure kinase dimerization, and I then showed that a paralogous pair of kinases from E. coli specifically formed homodimers over heterodimers. Residues important for dimerization specificity were predicted by measuring amino acid coevolution within kinases, which leverages the enormous amount of sequence information available for the kinase family. Experimental verification of these predictions showed that a set of residues at the base of the kinase dimerization domain was sufficient to establish homospecificity. This same region of the kinase, in particular the loops at the base of the kinase dimer, was also important for determining autophosphorylation mechanism. Recent work showed that kinases could autophosphorylate either in cis or in trans, and I found that a trans kinase could be made to autophosphorylate in cis by replacing its loop with the loop from a cis kinase. I also found that two sets of orthologs, despite having significantly diverged loop sequences, had conserved their autophosphorylation mechanisms. This raised the possibility that kinase loops may be under selection to maintain the same autophosphorylation mechanism. / by Orr Ashenberg. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 82-85). / Whether at the level of a single protein, or in the cytoplasm as a whole, the diffusive mobility of proteins plays a key role in biological function. To measure protein diffusion in cells, researchers have developed multiple fluorescence microscopy methods, and have tested them rigorously. However, using these methods for precise measurement of diffusion coefficients requires expertise that can be a barrier to broad utilization of these methods. Here, we report on a new method we have developed, which we name Photo-converted Intensity Profile Expansion (PIPE). It is a simple and intuitive technique that works on commercial imaging systems and requires little expertise. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal as diffusion spreads it out. The width of the expanding signal is directly related to the protein ensemble mean-square displacement, from which the diffusion coefficient of the ensemble is calculated. In the main part of the thesis, we demonstrate the success of PIPE in measuring accurate diffusion coefficients in silico, in vitro and in vivo. We then broaden the discussion, and challenge the assumption that the Fickian diffusion equation is the most appropriate model for describing protein motion in the cytoplasm. Since the cytoplasm is crowded with obstacles that trap proteins for a wide range of times, the motion of those proteins may be more accurately described by models of anomalous diffusion. To contribute to the ongoing debate about anomalous diffusion, we show how PIPE can be used to measure the degree of diffusion anomality by examining the temporal scaling of the mean-square displacement. Whether for measuring normal or anomalous diffusion, we suggest that the simplicity and user-friendliness of PIPE could make it a useful tool in molecular and cell biology. / by Rotem Gura Sadovsky. / 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.