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Chapter 1 is an introduction to the work detailed in this thesis and details the background of the main biological molecules that are studied. The recent discovery of novel long-non coding RNAs (lncRNAs) reveals a new class of molecules with immense biological relevance and promising candidates for therapeutic targeting. Classified as RNA molecules longer than 200 nucleotides in length, lncRNAs have been found to be dysregulated in various disease conditions, and their return to steady-state levels through transcript level manipulation has been observed to induce cell and tissue survival. Of great interest is the lncRNA MALAT1 which is known to be dysregulated in various cancer tissues and neurodegenerative diseases. Localized within the nucleus, MALAT1 is involved in a variety of important biological processes including mRNA transcriptional regulation, alternative splicing, and stabilizing nascent nuclear speckles. A detailed analysis of direct protein binders to MALAT1 reveals TDP-43 as a strong binder with common functionalities present. Both MALAT1 and TDP-43 are dysregulated in neurodegenerative diseases, and their common functionalities overlap in alternative splicing and transcript regulation. An increase in MALAT1/TDP-43 binding is also observed in neurodegenerative diseased tissues, but the reason for this interaction is yet to be explored fully in literature. Thus, it is important to study the common functions of MALAT1 and TDP-43 in the context of neurodegeneration and understand if the MALAT1/TDP-43 complex plays a role in regulating biological mechanisms for cell survivability, which can then be used for therapeutic advancements in this field. Chapter 2 is focused on the regulation of TDP-43 binding to mRNA targets by MALAT1. The increase in MALAT1/TDP-43 interaction in neurodegenerative disease tissues indicates a potential function for this interaction. Given TDP-43’s known function of regulating gene expression level, we hypothesized that MALAT1 may

Non-coding RNA transcripts regulate diverse functions including gene expression, splicing, and cell growth in human cells through their interactions with RNA binding proteins. The full biological relevance of these RNA/protein interactions and the role of RNA in regulating protein function is still unclear in many cases. This work investigates two specific cases of lncRNA regulating gene expression to better understand the cellular functions and molecular details of lncRNA/protein interactions. Specifically, the ability of the lncRNAs, MALAT1 and XIST, to regulate gene expression through their interactions with the RNA binding proteins TDP-43 and SHARP, respectively, is examined in this thesis. Chapter 2 is focused on the interaction of TDP-43 protein and MALAT1 non-coding RNA. TDP-43 protein is associated with neurodegenerative disease and has been shown to bind directly to MALAT1 non-coding RNA. We investigated the dependence of TDP-43 localization to the 3’ untranslated regions (3’ UTRs) of other mRNAs upon the level of MALAT1 expression in human cells. Target RNAs selected for analysis were representative of high, moderate, and low expression mRNAs bound by TDP-43 in multiple previous CLIP-seq studies. To measure the affinity of TDP-43 binding to MALAT1 long noncoding RNA and 3’ UTR mRNA targets, we used a newly designed fluorescent multiplexed version of the electrophoretic mobility shift assay (mEMSA), described in Chapter 3. Binding affinities of TDP-43 for mRNA 3’ UTRs correlated positively with the number of UG repeats in the identified CLIP-seq peak binding region. In addition, CLIP-seq peak signal height directly correlated with TDP-43 in vitro binding affinity for mRNA 3’ UTR targets. The binding of TDP-43 protein to messenger RNA 3’ UTR targets was evaluated by immunoprecipitation and quantitative real-time PCR (RT-qPCR). Decreased MALAT1 expression resulted in re-localization of TDP-43 protein to mRNA 3’ UTRs. TDP-43 binding stabilized the mRNA express

RNA molecules interact with proteins to perform a variety of functions in living cells. The binding partners of many RNAs, in particular the newly discovered class of long noncoding RNAs (lncRNAs), remain largely unknown. RNA antisense purification coupled with mass spectrometry (RAP-MS) is a method that enables the identification of direct and specific protein interaction partners of a specific RNA molecule. Because RAP-MS uses direct RNA–protein cross-linking methods coupled along with highly denaturing purification conditions, RAP-MS provides a short list of high confidence protein interactors.

Many long non-coding RNAs (lncRNAs) affect gene expression, but the mechanisms by which they act are still largely unknown. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell. Here we develop a method to purify a lncRNA from cells and identify proteins interacting with it directly using quantitative mass spectrometry. We identify ten proteins that specifically associate with Xist, three of these proteins—SHARP, SAF-A and LBR—are required for Xist-mediated transcriptional silencing. We show that SHARP, which interacts with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of RNA polymerase II (Pol II) from the inactive X. Both SMRT and HDAC3 are also required for silencing and Pol II exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for Xist-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X chromosome. Our results suggest that Xist silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude Pol II across the X chromosome., Gordon and Betty Moore Foundation (Grant GBMF775), Beckman Institute, National Institutes of Health (U.S.) (Director’s Early Independence Award DP5OD012190), Rose Hills Foundation, Edward Mallinckrodt, Jr. Foundation, Sontag Foundation, Searle Scholars Program, California Institute of Technology

Thesis (M.C.P.)--Massachusetts Institute of Technology, Dept. of Urban Studies and Planning, 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. [184]-187)., There were approximately 17,000 vacant lots in New Orleans in 2012, amounting to over 11 percent of total parcels in the city. Many of these lots have become vacant since Hurricane Katrina hit the Gulf Coast in 2005, but many were already empty. The population in parts of the older core of the city significantly declined from World War II until 2000. The migration of people into the recently drained low-lying subdivisions both within and outside of the city limits led to disinvestment and high vacancy rates in central neighborhoods of the city. This thesis seeks to define the current physical landscape of vacancy in New Orleans, within the context of these two historic narratives, Katrina and suburbanization before the storm, in order to appropriately target policy strategies for the reuse of vacant lots. This thesis uses images collected by the author of vacant lots throughout the city to define spatial types and conditions common to vacant land in New Orleans. A rigorous, data-driven mapping exercise explores patterns of vacancy in relation to physical and socioeconomic measures. This analysis supports the definition of three neighborhood types in which vacant land should be treated differently. These three types are based on pre-Katrina vacancy and post-Katrina flood depths, and consist of: 1) areas with significant pre-Katrina vacant land and little flooding, 2) areas with little pre-Katrina vacant land and high flood levels, and 3) areas with both significant pre-Katrina vacant land and high flood levels. The findings of this research indicate the need to revisit the physical footprint of New Orleans, with an emphasis on how the city should target its limited resources in the future to maximize both social justice and environmental justice imperatives, as well as mitigate the negative impacts of future disasters., by Colleen M. McHugh., M.C.P.

Ricin is a potent toxin presenting a threat as a biological weapon. The holotoxin consists of two disulfide-linked polypeptides: an enzymatically active A chain (RTA) and a galactose/N-acetylgalactosamine-binding B chain. Efforts to develop an inactivated version of the A chain as a vaccine have been hampered by limitations of stability and solubility. Previously, recombinant truncated versions of the 267-amino-acid A chain consisting of residues 1-33/44-198 or 1-198 were designed by protein engineering to overcome these limits and were shown to be effective and nontoxic as vaccines in mice. Herein we used CD, dynamic light scattering, fluorescence, and Fourier-transform infrared spectroscopy to examine the biophysical properties of these proteins. Although others have found that recombinant RTA (rRTA) adopts a partially unfolded, molten globule-like state at 45 degrees C, rRTA 1-33/44-198 and 1-198 are significantly more thermostable, remaining completely folded at temperatures up to 53 degrees C and 51 degrees C, respectively. Deleting both an exposed loop region (amino acids 34-43) and the C-terminal domain (199-267) contributed to increased thermostability. We found that chemically induced denaturation of rRTA, but not the truncated variants, proceeds through at least a three-state mechanism. The intermediate state in rRTA unfolding has a hydrophobic core accessible to ANS and an unfolded C-terminal domain. Removing the C-terminal domain changed the mechanism of rRTA unfolding, eliminating a tendency to adopt a partially unfolded state. Our results support the conclusion that these derivatives are superior candidates for development as vaccines against ricin and suggest an approach of reduction to minimum essential domains for design of more thermostable recombinant antigens., The original document contains color images.