Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004. / "February 2004." / Includes bibliographical references. / The experiments presented in this thesis use mutation analysis, and study of the cells of mice with a deletion allele for the Trp53 gene, to explore both the regulation of p53, and its downstream functions mediated by specific activation of target genes. Chapter 2 addresses the regulation of nuclear localization of the p53 protein. Previous reports in the literature had suggested that the p53 negative regulator HDM2 was a nucleocytoplasmic shuttling protein that binds and carries p53 from the nucleus of the cell to the cytoplasm where it is destroyed by the proteasome. We determined that HDM2 with a mutated nuclear export sequence was still able to able to alter p53's cellular localization to a cytoplasmic pattern. The nuclear export sequence in the p53 C-terminus was required for this activity, as was the ability of HDM2 to ubiquitinate p53. Further studies indicated that ubiquitination of the p53 C-terminus was the basis for HDM2's ability to remove it from the nucleus and cause its efficient degradation. C-terminal ubiquitination causes the p53 nuclear export sequence to be activated or made more accessible to the nuclear export machinery of the cell. Chapter 3 summarizes cDNA microarray experiments in which Trp53-/- and Trp53+/+ fibroblasts were treated with a panel of genotoxic agents, and assayed for p53-dependent upregulation or downregulation of the approximately 15,000 gene sequences represented on the microarray. New candidate p53 target genes were revealed, among them the DNA repair gene ErccS, which encodes the xeroderma pigmentosum disease gene homolog Xpg, a participant in nucleotide excision repair and a mediator of base excision repair of oxidative DNA damage. / (cont.) Further analysis of most of the DNA repair genes in the mouse genome using real-time PCR indicated that a second gene, Polk, encoding the translesion DNA polymerase kappa, is also a p53-induced gene. Chapter 4 describes further characterizes the p53-dependent regulation of Ercc5, and shows that it is a directly-regulated p53 target gene with a p53-responsive site in its first intron. TrpS3-null cells show a modest reduction in the ability to repair an oxidatively-damaged DNA construct, and this defect is rescued by exogenous expression of retrovirally transduced XPG, indicating that the lower levels of this gene are likely responsible for the defect. / by Scott D. Boyd. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016. / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / Adhesion-GPCRs, a novel family of G protein-coupled receptors (GPCRs), are characterized by an extended extracellular region linked to a seven-pass transmembrane moiety via GPCR proteolytic site (GPS)-containing stalk region known as GAIN domain. The name adhesion refers to the presence of functional domains in the extracellular region that commonly mediate cell-cell and cell-matrix interactions in various contexts. Recently, many genome-scale analyses of genetic alterations across diverse cancer types have revealed significant alterations (copy number and mutational) in adhesion-GPCRs, yet no comprehensive examination of their roles in cancer biology exists. Through a systematic screening for all adhesion-GPCRs by RT-qPCR in murine mammary carcinoma cell lines with varying metastatic abilities as well as tumor samples of different grades, I have identified several candidate genes with possible roles in breast cancer progression and metastasis. Based on these analyses and cross-referencing with the published gene expression data on human breast cancer cell lines and patient samples, I chose two candidate genes, CELSR2 and GPR126, for more detailed investigation. To elucidate their functions in cancer biology, I investigated the effects of their perturbations using RNAi (loss-of-function) methods both in vitro and in vivo. The results from my work reveal that loss of CELSR2 affects neither tumor growth nor lung metastasis in a xenograft mouse model of breast cancer, despite enhancing invadopodial activity in vitro. I also show that highly metastatic breast cancer and melanoma cells have elevated levels of GPR126, and confirm the significance of this result by revealing (a) reduction in pulmonary metastasis without affecting primary tumor growth in a spontaneous metastasis model of breast cancer, and (b) reduction in lung metastasis in three different experimental metastasis models of breast cancer and melanoma, upon shRNA-mediated knockdown of GPR126. After probing the different steps in the metastatic cascade to investigate how GPR126 promotes metastasis, I demonstrate that GPR126 specifically affects extravasation, most likely through its engagement with type IV collagen in the sub-endothelial basement membrane. Thus, the work described in this thesis contributes to our overall understanding of the perplexing problem of cancer metastasis via identification of novel regulators of distinct steps along the ominous path of malignant cells from primary sites to distant organs. / by Bigyan R. Bista. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1995. / Includes bibliographical references (128-129). / by Juli Dawn Klemm. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011. / Cataloged from PDF version of thesis. [Alpha] and [gamma] in title on title page appear as Greek symbols. Vita. / Includes bibliographical references (p. 148-168). / Cataract, the leading cause of blindness worldwide, is the opacification of the eye lens. In.age-related cataract, as well as roughly one half of congenital cataract cases, aggregation or precipitation of crystallin proteins results in the formation of large structures that scatter light, preventing the pinpoint focusing on the retina normally accomplished by the lens. The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human yD-crystallin (HyD-Crys) is a monomeric, two-domain protein, predominantly localized to the lens nucleus. Both domains of this long-lived protein have double Greek key p-sheet folds with well-packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the y-crystallin buried cores-two in the N-terminal domain (N-td), and one in the C-terminal domain (C-td)-cause earlyonset cataract in mice. It has not been possible to identify the aggregating precursors within lens tissues, and the question persists as to the nature of structure and stability changes in the crystallins leading to their increased propensity for aggregation and cataract formation within the lens environment. To compare in vivo cataract-forming phenotypes with in vitro unfolding and aggregation of y-crystallins, mouse mutant substitutions were introduced into HyD-Crys. WT HyD-Crys unfolds in vitro through a three-state pathway, exhibiting an intermediate with the N-td unfolded, and the C-td native-like. L5S and V75D also displayed three-state unfolding pathways, with the first transition, unfolding of the N-td, shifted to significantly lower denaturant concentrations. 190F was globally destabilized and the overall unfolding transition was shifted to lower denaturant concentrations. During thermal denaturation, the mutant proteins exhibited lowered thermal stability compared with WT. Kinetic unfolding experiments further confirmed this destabilization. The cataract phenotype of increased protein aggregation could be a direct property of the mutation, or could reflect an inability to be recognized by the a-crystallin chaperone. We have therefore carried out experiments comparing the interactions of WT HyD-Crys, V75D and 190F with a major component of the lenticular chaperone system, the small heat shock protein human aB-crystallin. Suppression levels of the aggregation that competes with the refolding pathway after dilution from GdnHCl were indistinguishable between WT and mutant proteins. However, dramatic differences among the mutants were observed under different conditions, including physiological conditions in the absence of denaturant and partially destabilizing acidic conditions such as those that may exist in the vicinity of degrading lysosomes in maturing lens fiber cells. In particular, incubation under physiological conditions highlighted differential interactions between the lens chaperone and the mutant HyD-Crys proteins themselves. Destabilized precursors to the cataractous state may populate various non-native structural conformations that, although aggregation-prone, elude recognition by acrystallin and efficiently continue along the aggregation pathway. Coupled with the observation of different unfolding pathways for the mutant crystallins, these results support the existence of multiple pathways for cataract formation in which partially unfolded species are differentially recognized by the passive lens chaperone system. While these observations were made utilizing destabilized mutant proteins, the same mechanisms may underlie the formation of age-related cataractous aggregates induced in vivo by covalent modification of the lens proteins. / by Kate L. Moreau. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 1998. / Includes bibliographical references (leaves 35-38). / by Brian J. Schneider. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004. / Includes bibliographical references. / The research in this thesis has focused on the analysis of data from two types of microarray technologies with the goal of improving understanding of transcriptional regulatory circuitry in yeast. These microarray technologies, expression analysis and location analysis (ChIP on CHIP), bring unique challenges for data analysis. I have adapted methods described previously and have developing new algorithms for addressing some of these challenges. I generated yeast transcription factor location data as part of a team effort to determine how a large set of transcriptional regulators occupy the yeast genome and to facilitate discovery of regulatory network structures. I also contributed to another team effort to deduce a portion of the yeast transcriptional regulatory code, the set of genomic DNA sequences recognized by transcriptional regulators to effect gene expression programs. Finally, I used these experimental and analytical methods to explore changes in gene expression circuitry that occur after exposure to oxidative stress in yeast. / by Nicola J. Rinaldi. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1997. / Includes bibliographical references (leaves 155-158). / by Nicholas R. Gaiano. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013. / Cataloged from PDF version of thesis. Vita. / Includes bibliographical references. / The retinoblastoma protein (pRB) tumor suppressor is deregulated in the vast majority of human tumors. pRB is a well-established transcriptional co-regulator that influences many fundamental cellular processes. It has been most well characterized in its ability to block cell proliferation by inhibiting the E2F family of transcription factors. Importantly, pRB also plays a pivotal role in apoptosis. This function has been extensively characterized in the context of genotoxic stress. Specifically, these studies have revealed that pRB can act in both an anti-apoptotic manner by inducing cell cycle arrest, and a pro-apoptotic manner by transcriptionally co-activating proapoptotic genes. Here, we show that pRB can also promote TNF[alpha]-induced apoptosis. Moreover, this investigation led us to uncover a novel, non-transcriptional and non-nuclear role of pRB in the induction of apoptosis. Specifically, we found that pRB can enhance TNFainduced apoptosis even in the presence of an inhibitor of translation, and that a fraction of endogenous pRB is localized at the mitochondria both in the absence and presence of treatment with apoptotic stimuli. Further characterization revealed that pRB can directly bind to and activate BAX, resulting in mitochondrial outer membrane permeabilization and apoptosis. Importantly, targeting ectopically expressed pRB specifically to the mitochondria generated a separation-of-function mutant deficient for pRB's classic, nuclear roles. Remarkably, we found that this mito-tagged pRB mutant can promote apoptosis in response to many apoptotic stimuli, arguing that mitochondrial pRB is a general mediator of apoptosis. Moreover, expression of this mito-pRB mutant in vivo was sufficient to suppress tumorigenesis. Taken together, our data uncover a role for pRB in the direct activation of mitochondrial apoptosis. To our knowledge, this is the first characterization of a non-nuclear and transcription-independent function for pRB. Moreover, most human tumors are wild-type for pRB, but contain alterations that result in constitutive phosphorylation of pRB. While this functionally inactivates pRB's cell cycle function, we show that pRB's mitochondrial role is unaffected. This raises the possibility that this novel pro-apoptotic pRB mechanism can be exploited for chemotherapeutic treatment. / by Keren Ita Hilgendorf. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1998. / Includes bibliographical references. / by Andrea Wilder Page. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016. / Cataloged from PDF version of thesis. Vita. / Includes bibliographical references. / The immune system has long been hypothesized to play a role in restraining tumor growth, but compelling evidence for this role evaded scientists for the better part of a century. After many years of skepticism, the field of cancer immunology has recently undergone a major revolution. The success of modern immunotherapeutics has transformed the arenas of oncology and drug development. Large efforts are now focused on understanding the factors that dictate patient responses to immunotherapy, for the identification of possible points of intervention to expand the fraction of patients who benefit from therapy. The majority of approved immunotherapeutics directly target adaptive immune effectors. However, emerging evidence suggests that these treatments preferentially benefit patients with pre-existing immune responses against tumors, and patients who fail therapies often harbor tumors that are poorly infiltrated by adaptive immune cells. I have explored the role of an innate immune effector known for its capacity to kill tumor cells and its importance in stimulating and shaping adaptive immune responses, the natural killer (NK) cell. To this end, I developed a new system for assaying NK cell function in the context of established, autochthonous lung cancer, by engineering vectors for producing tumors with inducible NK cell activating ligands. Using this model, I have shown that NK cells in established tumors exhibit dysfunctional phenotypes, but their responses can be boosted by providing activating stimuli. Strikingly, stimulation of NK cells results in the recruitment of adaptive immune cells to tumors. By developing a next-generation model for inducing activating NK cell ligands in tumors engineered to express T cell antigens, I demonstrated that NK cell activation in immunogenic tumors results in effective immune responses that restrain tumor growth, highlighting the potential for cooperation between innate and adaptive arms of the immune system in anti-tumor immunity. Finally, I developed a novel immunotherapeutic molecule for stimulating NK cell responses against cancer cells. Bifunctional molecules are an emerging class of anti-cancer agents, designed to target immune effectors against tumors. I produced and performed initial functional testing on a bifunctional molecule that stimulates NK cell responses against tumors by 'decorating' the surface of cancer cells with activating NK cell ligands. I demonstrated that this bifunctional molecule induces NK cell cytotoxicity against tumor targets. Based on this work, we hypothesize that strategies for stimulating NK cells in tumors may enhance the efficacy of T cell-targeted therapies in the treatment of cancer. / by Leah Marie Schmidt. / Ph. D.