Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002. / Includes bibliographical references. / All cells must compact their chromosomes in order for the DNA to fit inside the cell or nucleus. In Bacillus subtilis, and other bacteria, replication occurs simultaneously with the organization, compaction and segregation of newly duplicated chromosomal regions. My work indicates that the B. subtilis Structural Maintenance of Chromosomes (BsSMC) protein is involved in compacting and organizing the chromosome. Increasing the amount of supercoiling of DNA is a means to compact the chromosome. This thesis describes a role for BsSMC in supercoiling. I determined that BsSMC can alter the DNA topology of plasmids in vivo. There is also genetic evidence that BsSMC is involved in supercoiling. An smc null mutant is hypersensitive to inhibitors of DNA gyrase, which reduce the level of negative supercoiling in the cell. Conversely, depletion of Topoisomerase I, which increases the amount of negative supercoiling of the chromosome, partially suppresses the phenotype of an smc null mutant. These data are consistent with the model that BsSMC affects chromosome compaction by constraining positive supercoils. Interestingly, SMC-containing complexes in eukaryotes are able to constrain positive supercoils in vitro and affect chromosome architecture suggesting that there is a conserved function for SMC proteins in chromosome structure. I also determined the subcellular localization of BsSMC. I found that BsSMC is a moderately abundant protein that can bind to many regions of the chromosome. A portion of BsSMC localizes in a pattern similar to the replication machinery. / (cont.) Simultaneous localization of BsSMC and a component of the replisome revealed that they are usually in the same region of the cell but are not always colocalized. Finally, the formation of BsSMC foci is dependent on the presence of the nucleoid but not ongoing replication. I propose that BsSMC is acting to compact newly replicated DNA by affecting DNA topology and is thereby facilitating the partitioning of sister chromosomes to opposite halves of the cell. / by Janet C. Lindow. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005. / Includes bibliographical references. / Variants of the canonical cell cycle are frequently used in nature to accomplish specific developmental goals. In one such variant, the endocycle, synthesis phase alternates with a gap phase without an intervening mitosis, producing cells that have multiple copies of the genome. These cells show diversity in their chromosome structure; at one extreme, the sister chromatids are separate (polyploid) and at the other extreme, the sisters are held together (polytene). The endocycle itself can be modified and these variations are speculated to correlate with the observed differences in chromosome structure. In this thesis, we have analyzed the contribution of mitotic regulators to the endocycle and polytene chromosome structure in Drosophila. We show that morula, a gene required for the transition from polytene to polyploid chromosome structure in Drosophila nurse cells, is a subunit of the anaphase-promoting complex/cyclosome. Increasing levels of cyclin B, a known mitotic target of the APC/C, does not alter the timing of the transition, indicating that CYCLIN B is not the only APC/C target at the polyteny-polyploidy transition. In mitosis, activity of APC/C and POLO lead to the loss of sister-chromatid cohesion and we find that mutants in polo are unable to progress through the polyteny-polyploidy transition. Finally, we find that the cohesin complex, a complex required for the physical attachment of sister chromatids in mitosis, is required for proper polytene chromosome structure in the salivary gland. These results describe a requirement for the cohesin complex in a variant of the cell cycle lacking mitosis and indicate that sister-chromatid cohesion differentiates polytene and polyploid chromosome structures. / by Julie Ann Wallace. / Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002. / Includes bibliographical references. / Although enormous progress has occurred in the field of human genetics, the cloning of complex trait mutations remains a challenging and unresolved process. This continuing difficulty is responsible for an ever-increasing awareness of the phenomenon of linkage disequilibrium (LD). The principle behind LD is relatively simple. Over the lifetime of a population, the genetic markers that are adjacent to an ancestral mutation will recombine less often than more distant markers. Therefore, the ancestral alleles of the markers closest to the mutation should be most frequent in a collection of disease chromosomes. The allelic association should decrease as the distance from the ancestral disease mutation increases. This thesis is a collection of ideas and experiments aimed at dissecting the behavior of LD in the human genome. Specific studies examine LD in a variety of populations including isolated founder populations, as well as globally diverse population samples. A large number of regions throughout the genome are investigated using both pairwise comparisons of markers, as well as multimarker haplotypes. The X chromosome is more closely scrutinized because of its unique population history, as well as the advantages afforded to haplotyping due to hemizygosity of the X chromosome in males. Major conclusions include the observation that LD between pairs of markers is highly variable even at extremely close distances and multimarker haplotypes better serve to resolve the underlying haplotype structure of the genome. / (cont.) The genome appears to be structured as blocks of limited haplotype diversity that do not exhibit much internal recombination but which are separated by segments that show little or no LD. The lack of LD between haplotype blocks appears to be due to clustering of recombination events into specific hotspots. The size of the blocks and haplotype diversity varies slightly by population. In addition, the identity of the haplotypes varies between populations. The existence of 3-4 major haplotypes for specific regions in a diverse human population sample is a surprising finding that was originally believed to have only existed in very special isolated and young populations. / by Shau Neen Liu-Cordero. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Protein-protein interactions are central to all biological processes. Designer reagents that selectively bind to proteins and inhibit their interactions can be used to probe protein interaction networks, discover druggable targets, and generate potential therapeutic leads. Current technology makes it possible to engineer proteins and peptides with desirable interaction profiles using carefully selected sets of experiments that are customized for each design objective. There is great interest in improving the protein design pipeline to create protein binders more efficiently and against a wider array of targets. In this thesis, I describe the design and development of selective peptide inhibitors of anti-apoptotic BcI-2 family proteins, with an emphasis on targeting Bfl-1. Anti-apoptotic Bcl-2 family proteins bind to short, pro-apoptotic BH3 motifs to support cellular survival. Overexpression of BfI-1 has been shown to promote cancer cell survival and the development of chemoresistance. Prior work suggests that selective inhibition of Bfl-1 can induce cell death in Bfl-1 overexpressing cancer cells without compromising healthy cells that also rely on anti-apoptotic BcI-2 proteins for survival. Thus, Bfl-1-selective BH3 mimetic peptides are potentially valuable for diagnosing Bfl-1 dependence and can serve as leads for therapeutic development. In this thesis, I describe three distinct approaches to designing potent and selective Bfl-1 inhibitors. First, I describe the design and screening of libraries of variants of BH3 peptides. I show that peptides from this screen bind in a previously unobserved BH3 binding mode and have large margins of specificity for Bfl-1 when tested in vitro and in cultured cells. Second, I describe a computational model of the specificity landscape of three anti-apoptotic Bcl-2 proteins including Bfl-1. This model was derived from high-throughput affinity measurement of thousands of peptides from BH3 libraries. I show that this model is useful for designing peptides with desirable interaction profiles within a family of related proteins. Third, I describe the use of a scoring potential built on the amino acid frequencies from well-defined structural motifs complied from the Protein Data Bank to design novel BH3 peptides targeting Bfl-1. / by Justin Michael Jenson. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006. / Includes bibliographical references. / Mammalian germ cells arise during early embryogenesis and migrate to the developing gonad where, under the direction of the somatic environment, they initiate distinct sex-specific developmental programs resulting in the production of egg or sperm. Our understanding of the molecular mechanisms governing many stages of germ cell development has advanced greatly in recent years. However, many aspects of germ cell development remain entirely uncharacterized at the molecular level. In this thesis I will present projects utilizing forward and reverse genetics that generate new points of entry into poorly understood transitions during germ cell development. The X and Y chromosome do not have pairing partners during male meiosis. As a result they become silenced during this time. One mechanism that has been proposed to compensate for inactive X-linked housekeeping genes during male meiosis is X-to-autosome retropositions. We have identified a mutation within an X-to-autosome retrogene in the mouse spermatogenic mutant jsd/jsd that provides the first supporting evidence for this model. / (cont.) Evolutionary analysis indicates that since the X and Y chromosome evolved from a pair of autosomes, retroposition of this gene occurred and was maintained independently in several different mammalian lineages, demonstrating a positive selective pressure for this event. Through targeted disruption of the vertebrate-specific Stra8 gene, we have generated a point of entry into the study of meiotic initiation in mammals. Stra8, which is expressed exclusively in premeiotic germ cells, is required for the initiation of meiosis in mice. In female mice Stra8 is required after the last mitotic division, but prior to meiotic DNA replication. In Stra8-deficient male mice, germ cells arrest at the onset of meiosis, but in a less stringent manner than observed in female germ cells. Additionally, Stra8 appears to be required for proper regulation of spermatogonial stem cells, as Stra8-deficient male mice undergo gradual germ cell depletion, followed by a high frequency of testicular germ cell tumor formation. Gaining a better understanding of these events in the Stra8-deficient mice will provide insight into the regulation of spermatogonial stem cell activity. / by Andrew Edmund Baltus. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 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. Vita. / Includes bibliographical references. / MicroRNAs (miRNAs) are post-transcriptional regulators that tune gene expression. Despite the modest 2-fold repression that miRNA activity generally confers on a target, miRNAs are critical for many biological processes including development and differentiation. Due to this mild repression directly conferred by miRNA activity, miRNAs coordinate with other regulators such as transcription factors to shape the gene expression landscape and phenotypes of a cell. To understand the function of global miRNA activity in regulating the specification of the somatic state, we deleted Dicer in a murine mesenchymal stem cell model. Upon exploring the consequences of Dicer deletion, we identify a specific let-7 regulated mid-embryonic program within the global de-repression of miRNA targets accompanying Dicer loss. We further observe within the landscape of let-7 regulated targets, an activation of greater than 50-fold of known oncofetal (Igf2bp1/2/3) genes, an effect much greater than that typically reported for miRNA-mRNA interactions. This suggests a requirement of let-7 for the continual suppression of mid-embryonic programs in adult cells. To investigate the regulation of these oncofetal genes, we restored miRNAs through re-expression of Dicer. Despite complete reconstitution of the posttranscriptional activity of miRNAs, the activated oncofetal genes are incompletely suppressed. Igf2bp1-3 are components of a larger set of irreversible oncogenes whose chromatin signature indicate that they are transcriptionally activated upon Dicer deletion. This transcriptional activation is maintained, despite miRNA restoration in Dicer rescued cells. Consistent with this expression pattern, Dicer rescued cells are able to form tumors in mice, a phenotype absent in the parental wild-type and Dicer knockout cells. Moreover, the irreversible gene set is amplified in human cancers and is predictive of patient survival indicating that our observations are relevant to human disease. Finally, we develop a computational method to decipher the indirect, transcription factor mediated effects of miRNAs on gene expression. Through comprehensive analysis of ChIP-Seq, CLIP-Seq and RNA-Seq datasets, we quantitatively assess the relative contributions of direct posttranscriptional miRNA activity and transcriptional activity on gene expression changes resulting from Dicer deletion. We find that transcriptional changes contribute significantly to perturbations in gene expression resulting from global miRNA loss upon Dicer deletion. In summary, our work expands the current knowledge of fundamental roles for miRNAs in differentiated mammalian cells. As further work builds on our observations, the increased understanding of miRNA-mediated regulation will inform therapeutic strategies for human disease. / by Courtney K. JnBaptiste. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004. / Includes bibliographical references. / (cont.) the RTT phenotype. / Deletion of the Xist gene results in skewed X-inactivation. To distinguish primary non-random choice from post-choice selection, we analyzed X-inactivation in early embryonic development in the presence of two different Xist deletions. We found that Xist is an important choice element, and that in the absence of an intact Xist gene, the X chromosome will never be chosen as the active X. To understand the molecular mechanisms that affect choice we analyzed the role of replication timing prior to X-inactivation. The X chromosomes replicated asynchronously before X-inactivation but analysis of cell-lines with skewed X-inactivation showed no preference for one of the two Xist alleles to replicate early, indicating that asynchronous replication timing prior to X-inactivation does not play a role in skewing of X-inactivation. Expression of the Xist is negatively regulated by its antisense gene, Tsix. In order to determine the role of transcription in Tsix function, we modulated Tsix transcription with minimal disturbance of the genomic sequence. Loss of Tsix transcription lead to non-random inactivation of the targeted chromosome, whereas induction of Tsix expression caused the targeted chromosome always to be chosen as the active X. These results for the first time establish a function for antisense transcription in the regulation of Xist expression. The X-linked disease Rett syndrome (RTT), a neurodevelopmental disorder, is caused by mutations in the MECP2 gene. We used a mouse model to test the hypothesis that RTT is exclusively caused by neuronal MeCP2 deficiency. Expression of an Mecp2 transgene in postmitotic neurons resulted in symptoms of severe motor dysfunction. Transgene expression in Mecp2 mutant mice, however, rescued / by Sandra Luikenhuis. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008. / Includes bibliographical references. / E2F transcription factors were originally identified as regulators of cell cycle and cellular proliferation. In vivo mouse models have uncovered novel roles for these proteins in different developmental processes. This dissertation examines the biological role of E2F6 in mammalian development. E2F6 functions as a repressor of transcription in concert with the polycomb group (PcG) proteins and chromatin modifiers. PcG proteins regulate processes required for proper embryonic development and differentiation. E2F6 interacts with core components of Polycomb Repressive Complex 1 (PRC1) and participates in PcG-mediated repression of Hox genes. Hox genes are required for correct patterning of the mammalian skeleton. Loss of E2f6 results in posterior axial skeletal transformations. Mice deficient for both E2f6 and Bmil, a component of PRC1, exhibit increased penetrance of axial skeletal transformations. Thus, E2F6 and Bmil cooperate in the regulation of Hox genes and axial skeletal development. Bmi 1 also represses transcription of the Ink4a-Arf locus, and it is consequently required to maintain the proliferative and self-renewal properties of hematopoietic and neural stem cells. However, E2F6 does not participate in the repression of the Ink4a-Arflocus. These findings underscore the significance of the E2F6-Bmil interaction in vivo and suggest that the Hox and Ink4a-Arfloci are regulated by somewhat different mechanisms. In addition to axial skeletal transformations, E2f6' mice exhibit a suppressed gliotic response after neural injury. Gliosis occurs in response to neurodegeneration, ischemia, and neuronal cell death. This process provides neuronal protection by restricting inflammation and regulating the concentration of molecules in the extracellular environment. / (cont.) However, gliosis has potentially detrimental effects such as the inhibition of axonal regeneration or the release of cytotoxic agents that trigger degeneration of neighboring neurons. The molecular mechanisms required to initiate and sustain a gliotic response are poorly understood. Gliosis is the focus of therapy for neurodegenerative diseases and ischemia, and complete understanding of the mechanisms underlying this process will lead to more effective therapies for neurodegenerative disease and ischemia. / by Laurie Beth Friesenhahn. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, June 2004. / Includes bibliographical references (p. 175-185). / Postsynaptic density protein-95 (PSD-95) is the second most abundantly expressed synaptic protein in the postnatal forebrain. It is an integral part of the postsynaptic scaffolding complex and helps recruit receptors, channels and associated factors involved with synaptic transmission. A mouse whose wildtype gene was replaced with truncation mutant of PSD-95 preserving two PDZ binding domains causes a spatial learning and memory deficit and a dramatic enhancement of synaptic strengthening. Long Term Potentiation is enhanced at all frequencies of stimulation (1-100Hz), while Long Term Depression is absent in the mutants. This study explores CA1 pyramidal cell spatial representations in the PSD-95 mutant mice. Mutants are not significantly different than controls in running velocity. Nor are its pyramidal cells or interneurons different than controls in: place cell firing rates, sparsity of run active cells, bursting behavior, or theta modulated activity. However, mutants do exhibit significantly larger place fields and wider spike waveforms. Mutants also expressed enhanced directionality of place fields and increased post-run sleep correlation of firing for overlapping place fields. Mutants also exhibited disruption of asymmetrical place fields and phase precession, the first such observation reported in mice. In conclusion, LTP alone is not enough for the active process of encoding experience. Instead, bi-directional synaptic plasticity is necessary for proper place field formation, correlation, directionality, asymmetry, phase precession, and the formation of spatial memories. / by Linus Da-Shih Sun. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005. / Includes bibliographical references. / The regulation of eukaryotic cell division, which involves the faithful segregation of a complete DNA complement to each daughter cell, is a fundamental area of research in biology. Entry into mitosis is initiated by the action of mitotic cyclins complexed with the cyclin dependent kinase (CDK). Once the chromosomes have been successfully segregated, the exit from mitosis ensues. In order for cells to exit from mitosis, mitotic CDKs must be inactivated. The inactivation of mitotic CDKs, in turn, promotes cytokinesis. In S. cerevisiae, mitotic exit is controlled by the Mitotic Exit Network (MEN). In this simple eukaryote, the tight coupling of nuclear migration and mitotic exit is achieved in part by the spatial segregation of Lte1, a positive activator of the MEN, and Teml, a GTPase that acts at the top of the MEN signaling cascade. The spatial segregation of Lte1 and Teml is particularly important in cells with mispositioned anaphase spindles, and plays a role in the prevention of aneuploidy. A model for the regulation of Lte1 localization across the cell cycle is proposed. Additionally, the role of Lte1 localization in mediating its ability to promote mitotic exit is examined. This work identifies novel connections between polarity determinants, Ras signaling, and mitotic exit. / Anupama Seshan. / Ph.D.