Observing the natural and heteromorphic organization of the human genome in nanoscale resolutiondissertationsHorrell, Jeremy ChristiancreatorNeretti, NicolaAdvisorSedivy, JohnAdvisorLarschan, EricaReaderFreiman, RichardReaderCreton, RobbertReaderNir, GuyReaderBrown University. Department of Molecular Biology, Cell Biology and Biochemistrysponsor2022xiv, 109 p.born digitalThesis (Ph. D.)--Brown University, 2022thesesThe human genome is organized in a hierarchical manner from higher order to lower order structures. At the highest level are chromosome territories that are each separated into functional transcriptionally active and transcriptionally non-active regions. These active, A, and non-active, B, regions are further segmented into self-interacting regions called topologically associating domains. These organizational features of the genome are conserved between almost all organisms. The detection of organization has relied on analysis of DNA sequences through chromosome conformation capture technology, most notably, HiC. The question remains, however, how these organizational structures occur in situ. The development of two technologies in genome targeting and microscopy has allowed for the interrogation of the 3-dimensional genome structure. Collectively, these technologies are known as OligoSTORM where the genome is targeted at super-resolution, beyond the diffraction limit of visible light. Here we use automated microfluidics and OligoSTORM to observe the genome of a human B-lymphocyte with chromosome territory, sub-compartment, and topologically associated domains (TAD) context at nano-scale resolution. This accomplished disambiguation of human homologs to reveal finite structural features that distinguish these homologs one from another in a TAD by TAD manner . In addition, a key finding was the observation of homolog "pairing," later termed homolog kissing, where we observed the close proximity of homologs in a somatic human cell type—to our know, the first in a mammalian cell.Chromatinnuclear architectureHigh resolution imaging3d fishOligopaintsstochastic optical reconstruction microscopyEnglishRPB20220706