Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Protein interaction networks and protein compartmentalization underlie all signaling and regulatory processes in cells. Traditional approaches to proteomics employ mass spectrometry (MS) coupled to biochemical fractionation or affinity purification but require cell lysis prior to analysis which often results in false-negatives from missed interactions or incomplete purification and false-positives from contaminants. Enzyme-catalyzed proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in which a PL enzyme can be genetically targeted to a subcellular region and used to tag surrounding endogenous proteins with a chemical handle that allows their identification by MS. Tagging is carried out in living cells in a distance-dependent manner, allowing data collection from a physiologically relevant environment with preservation of spatial information. Current PL methods are limited by poor catalytic efficiency or toxic substrates that limit their application in vivo. Therefore, we have developed a new proximity labeling method, called TurboID, that uses non-toxic labeling conditions and has high catalytic efficiency that allows its use in a wide variety of biological contexts. Here, we describe our use of yeast display-based directed evolution to engineer two promiscuous mutants of biotin ligase, TurbolD and miniTurbo. We describe our characterization of the evolved PL enzymes in microbes, cultured cells, in vitro, and in vivo in flies and worms, and show that TurbolD and miniTurbo have much greater catalytic efficiency than any other biotin ligase-based PL method currently available. Lastly, we demonstrate that TurbolD and miniTurbo can be used to obtain proteomes with the same size, specificity, and depth-of-coverage as existing biotin-ligase based PL techniques with over 100- fold shorter labeling times. In the Appendix, we discuss two separate projects. In Part I, we describe how fusion of the PL enzyme APEX2 to various mitochondrial proteins could be used to map the proteomes of mitochondrial subdomains and be used to visualize the localization of mitochondrial proteins in mitochondrial subdomains using APEX2 to generate contrast for electron microscopy imaging. In Part II, we discuss the development of two platforms that could be used to temporally control genome editing using light. / by Tess C. Branon. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1994. / Includes bibliographical references (leaves 229-252). / by Kevin J. Yarema. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1997. / Includes bibliographical references (leaves 134-140). / by Florian Johannes Schattenmann. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1996. / Includes bibliographical references (leaves 192-199). / by Sherri L. Oslick. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1998. / Includes bibliographical references. / by Thomas J. Tolbert. / Ph.D.

Thesis (S.M. in Inorganic chemistry)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012. / Cataloged from PDF version of thesis. Vita. / Includes bibliographical references (p. 38-40). / Nucleotide excision repair, a DNA repair mechanism, is the major repair pathway responsible for removal of platinum-based anticancer drugs. In this study, 146 bp duplexes were prepared containing either a site-specific cisdiammineplatinum( Il)-DNA intrastrand d(GpG) cross-link or a cisdiamminephenanthridinechloroplatinum( Il)-DNA dG adduct. Comparison of the repair efficiencies of the two adducts reveals that the diamminephenanthridinechloroplatinum(lI)-DNA dG lesion eludes the nucleotide excision repair pathway better than diammineplatinum(lI)-DNA intrastrand d(GpG) cross-link. A factor that may be relevant to the difference is the influence of platination on DNA-mediated charge transfer. Atomic force microscopy is a method by which we can explore the possibility that phenanthriplatin influences charge transfer properties of DNA. Long DNA duplexes site-specifically modified with cisplatin or phenthanriplatin were prepared for AFM studies. / by Meiyi Li. / S.M.in Inorganic chemistry

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2015. / Cataloged from PDF version of thesis. Vita. Page 216 blank. / Includes bibliographical references. / Complexes (THF)₀-₂E[P₃Nb(ODipp)₃]₂ (E = Sn, Pb; Dipp = 2,6-iPr₂C₆H₃) were prepared and characterized as potential precursors to new tetrel phosphides. Treatment of (THF)Sn[P₃Nb(ODipp)₃]₂ with pyridine-N-oxide effected the formation of ONb(ODipp)₂(py)₂ and a new tin phosphide, [(THF)SnP₆]x, characterized using a multitude of techniques. Additionally, P₃Nb(ODipp)₂(py)₂, prepared from [Na(THF)₃][[P₃Nb(ODipp)₃] in the presence of pyridine and salts of coordinating cations, was found to successfully produce AsP₃ upon treatment with AsCl₃ Dimer [P₂Nb(ODipp)₃]₂ has been obtained via a novel "2(3-1)" synthetic strategy. The mononuclear diphosphorus complex P₂Nb(ODipp)₃ proposed to be generated transiently by formal P- abstraction from previously reported [Na(THF)₃][P₃Nb(ODipp)₃] undergoes irreversible dimerization to form the [P₂Nb(ODipp)₃]₂ complex, and is alternatively trapped reversibly by a 1,3-cyclohexadiene (CHD) to form in situ C₆H₈P₂Nb(ODipp)₃ Unprotected dibenzo-7[lambda]³A3-phosphanorbornadiene derivatives RPA (A= C₁₄H₁₀ or anthracene; R = tBu, dbabh, HMDS, iPr₂N, cis-Me₂ Pip, Cy₂N, Me₂N) were synthesized by treatment of the corresponding phosphorus dichloride RPCl₂ with MgA-3THF. Thermolysis of RPA benzene-d₆ solutions leads to anthracene extrusion. Experimental and computational thermodynamic activation parameters suggest this process occurs through the intermediacy of a singlet phosphinidene for the iPr₂N derivative. The [RP] unit could be transferred to CHD, a carbene and an ortho-benzoquinone, with formation of the corresponding anti-7-phosphanorbornene, phosphaalkene and phospholane. The anthracene diphosphorus adduct P₂A₂ was synthesized from Me₂NPA, through isolated intermediates CIPA and [P₂A₂Cl][AlCl₄]. P₂A₂ was found to transfer P₂ efficiently to N-₃ CHD, 1,3-butadiene and (C₂H₄)Pt(PPh₃)₂ to form 1,2-cyclo-[P₂N₃]-, P₂(CHD)₂, P₂(BD)₂ and (P₂)[Pt(PPh₃)₂]₂ . A complete kinetic and computational study of the P₂(CHD)₂ formation from P₂A₂ suggests P₂ is a productive intermediate in solution. Additionally, a molecular beam mass spectrometry study on the thermolysis of solid P₂A₂ reveals the direct detection of molecular fragments of only P₂ and anthracene thus establishing a link between solution-phase P₂-transfer chemistry and production of gas-phase P₂ by mild thermal activation of a molecular precursor. The HCP precursor Ph₃PC(H)PA was synthesized directly from C1PA. The activation of P₄ with [Na][SnPh₃ ] and sodium naphthalenide led to the synthesis of phosphide compounds [Na(benzo-15-crown-5)][P(SnPh₃)₂], P(SnPh₃)₃, (XL)₂InP(SnPh₃)₂, (Ph₃P)AuP(SnPh₃)₂, P₇(SnPh₃)₃ and P7(SiMe₃)₃, characterized using a variety of techniques. / by Alexandra Velian. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 123-134). / Humans are exposed to a variety of infectious pathogens, such as virus, bacteria, etc. But we survive thanks to our immune systems. One of the important immune systems is the adaptive immune system, which mounts pathogen specific immune responses. The orchestrators are lymphocytes, including B cells and T cells. When lymphocytes are stimulated strongly enough by infected or antigen-presenting cells, the signal will be transmitted through a complex network of biochemical reactions underlying the cellular functions. Such a complex network is fundamentally regulated by stochastic principles with intriguing behaviors. This thesis aims to understand the signaling network in lymphocytes and the stochastic regulation behind. In lymphocytes, mitogen-activated protein (MAP) kinases can be triggered by various stimuli (growth factors, cytokines, etc.) with significant cellular responses (proliferation, inflammation, etc.). We first investigate the roles of upstream proteins, Son of Sevenless (SOS) and Ras guanyl-releasing protein (RasGRP), in MAP kinases, Erk and P38, activation. The signaling pathways of P38 activation are still elusive. In Chapter 3, we study two signaling pathways for P38 activation, the classical pathway through the linker for activation of T cells (LAT) complex and the alternative pathway directly via ZAP-70 molecules. In both of studies, we bring together computational tools, stochastic theories and cell biology by collaborating with biologists, Professor Jeroen Roose and Dr. Jesse Jun, in University of California, San Francisco. In biochemical systems, like lymphocytes, fluctuations are ubiquitous. Such noise may drive biochemical systems from one stable state to the other with biological significance. In this part of the work, we investigate fluctuation-driven transitions in two biochemical models: genetic toggle switches (GTS) and self/non-self peptide-major histocompatibility complex (MHC) discrimination models. In the GTS model, we introduce dynamical disorder in rate coefficients and study its influences in optimal transition paths, transition rates, and the stationary probability distribution of the system. In peptide-MHC discrimination part, we investigate the effects of a conformational change step of ZAP-70 molecules on the sensitivity and robustness of the discrimination. / by Hang Chen. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1935. / Includes bibliographical references (leaf 91). / by Tzu Ching Huang. / Ph.D.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1994. / Includes bibliographical references (leaves 52-53). / by Georg Karl-Heinz Füllen. / M.S.