ENZYME-FREE UBIQUITIN LIGATION. FROM NATIVE CHEMICAL LIGATION AND SPIN LABELING TO DIMERIZATION BY INTER-UBIQUITIN MIMICKING LINKAGE AND PH-DEPENDENT CONFORMATIONAL SWITCH

The delicate balance between the production and disposal of proteins is vital for the changes required in the cell to respond to given stimulus. Ubiquitination is a protein modification with a range of signaling outcomes when ubiquitin is attached to a protein through a highly ordered enzymatic cascade process. Understanding ubiquitination is a growing field and nowadays the application of chemical reactions allows the isolation of quantitative materials for structural studies. Therefore, in this dissertation it is described some of these suitable chemical methodologies to produce an isopeptide bond toward the polymerization of ubiquitin bypassing the enzymatic control with the purpose of showing if these chemical modifications have a direct impact on the structure of ubiquitin. First, the possibility of incorporating non-natural lysine analogs known as mercaptolysines into the polypeptide chain of Ubiquitin was explored when they were attached to ubiquitin by native chemical ligation at its C terminus. The sulfhydryl group was used for the attachment of a paramagnetic label to map the surface of ubiquitin. Second, the condensation catalyzed by silver nitrate was used for the dimer assembly. In particular, the main focus was on examining whether orthogonal protection and deprotection of each monomer have an impact on the reaction yield, since the synthetic strategy has been previously attempted successfully. Third, the formation of ubiquitin dimers was approached by building an inter-ubiquitin linkage mimicking the isopeptide bond with two approaches, the classic disulfide exchange as well as the thiol-ene click reaction by thermal initiation in aqueous conditions. After assembling the dimeric units, they were studied by Nuclear Magnetic Resonance, in order to establish a conformational state profile which depends on the pH conditions. The latter is a very important concept since some ligands have a preferred affinity when the protein-protein hydrophobic patches are in close proximity.

The acquisition of adjunct control: grammar and processing

This dissertation uses children’s acquisition of adjunct control as a case study to investigate grammatical and performance accounts of language acquisition. In previous research, children have consistently exhibited non-adultlike behavior for sentences with adjunct control. To explain children’s behavior, several different grammatical accounts have been proposed, but evidence for these accounts has been inconclusive. In this dissertation, I take two approaches to account for children’s errors. First, I spell out the predictions of previous grammatical accounts, and test these predictions after accounting for some methodological concerns that might have influenced children’s behavior in previous studies. While I reproduce the non-adultlike behavior observed in previous studies, the predictions of previous grammatical accounts are not borne out, suggesting that extragrammatical factors are needed to explain children’s behavior. Next, I consider the role of two different types of extragrammatical factors in predicting children’s non-adultlike behavior. With a new task designed to address the task demands in previous studies, children exhibit significantly higher accuracy than with previous tasks. This suggests that children’s behavior has been influenced by task- specific processing factors. In addition to the task, I also test the predictions of a similarity-based interference account, which links children’s errors to the same memory mechanisms involved in sentence processing difficulties observed in adults. These predictions are borne out, supporting a more continuous developmental trajectory as children’s processing mechanisms become more resistant to interference. Finally, I consider how children’s errors might influence their acquisition of adjunct control, given the distribution in the linguistic input. I discuss the results of a corpus analysis, including the possibility that adjunct control could be learned from the input. The kinds of information that could be useful to a learner become much more limited, however, after considering the processing limitations that would interfere with the representations available to the learner.