Probing The Active And Allosteric Binding Sites Of Soybean Lipoxygenase-1

Soybean lipoxygenase-1 is a model for lipoxygenase activity. While the mechanism of oxygenation is understood, the substrate binding mechanism has not yet been elucidated. Two putative binding mechanisms are the ¿head-first¿ and ¿tail-first¿ models, in which the carboxy-terminus or the methyl terminus of the fatty acid substrate is inserted into the active site while the remainder of the molecule protrudes from the surface, respectively. Previous work has demonstrated that derivatization of fatty acid substrates with D-tryptophan increases active site affinity. It has also been shown that while polyunsaturated fatty acids are the natural substrates of lipoxygenases, monounsaturated fatty acids can be oxygenated at a much slower rate. Starting with a monounsaturated fatty acid, oleic acid, as a platform, the molecule N-oleoyl-D-tryptophan (ODT) was synthesized with the anticipation of it being a potent competitive substrate-analogue inhibitor that could be used to discern the substrate binding mechanism. Inhibition kinetics demonstrated that this molecule functions as a partially competitive inhibitor, through an unknown mechanism. The implication behind partially competitive inhibition is that substrate and inhibitor molecules can bind simultaneously to the enzyme, which alludes to the presence of an allosteric binding domain. To investigate the possibility of an inhibitor binding site on the non-catalytic subunit, limited proteolysis was used to cleave the subunits apart which should have eliminated inhibition. Interestingly, it was observed that at high substrate concentrations the inhibitor was completely ineffective, but at low substrate concentrations the inhibitor maintained its standard efficacy. A satisfactory explanation for these results has not yet been determined.

Enhancing Sensitivity In Natural Product Nmr: Exploring Optimal Non-Uniform Sampling

Recent optimizations of NMR spectroscopy have focused their attention on innovations in new hardware, such as novel probes and higher field strengths. Only recently has the potential to enhance the sensitivity of NMR through data acquisition strategies been investigated. This thesis has focused on the practice of enhancing the signal-to-noise ratio (SNR) of NMR using non-uniform sampling (NUS). After first establishing the concept and exact theory of compounding sensitivity enhancements in multiple non-uniformly sampled indirect dimensions, a new result was derived that NUS enhances both SNR and resolution at any given signal evolution time. In contrast, uniform sampling alternately optimizes SNR (t < 1.26T2) or resolution (t~3T2), each at the expense of the other. Experiments were designed and conducted on a plant natural product to explore this behavior of NUS in which the SNR and resolution continue to improve as acquisition time increases. Possible absolute sensitivity improvements of 1.5 and 1.9 are possible in each indirect dimension for matched and 2x biased exponentially decaying sampling densities, respectively, at an acquisition time of ¿T2. Recommendations for breaking into the linear regime of maximum entropy (MaxEnt) are proposed. Furthermore, examination into a novel sinusoidal sampling density resulted in improved line shapes in MaxEnt reconstructions of NUS data and comparable enhancement to a matched exponential sampling density. The Absolute Sample Sensitivity derived and demonstrated here for NUS holds great promise in expanding the adoption of non-uniform sampling.

Aporias of Trauma in Joyce's Ulysses and Faulkner's Light in August

In this thesis, I explore the relationships between trauma, memory, and narrative, particularly the ways in which trauma simultaneously disrupts and engenders narrative structures. I consider various trauma theories by authors such as Cathy Caruth, Judith Herman, Ruth Leys, and Dominick LaCapra. I also consider how psychoanalytic theory and criticism, specifically the writings of Sigmund Freud, inform the study of traumatic experience from both literary and personal perspectives. Furthermore, I consider theories regarding the relationship between trauma and narrative by authors such as Peter Brooks and John Pilkington. James Joyce¿s Ulysses and William Faulkner¿s Light in August serve, for my purposes, as trauma-texts and reflect the ways in which trauma might complicate the simultaneous destruction and creation of narrative strategies. Reading Ulysses and Light in August as trauma-texts that are both in mourning and melancholic gives us complementary, and contradictory, reasons for why we enjoy them. Mourning constructs a relationship between victim and witness, in which we can hear the voice of trauma and engage it in discourse. Conversely, melancholia creates a relationship between performer and spectator, in which we experience, and are fascinated by, the spectacle of another¿s trauma. Laughter, perversity, sorrow, and respite engage the reader in both texts, and raise questions about how one `remembers-to-forget¿ traumatic experiences. The narratives of each text¿s characters offer unique performances of mourning and melancholia. Thus, while this thesis engenders more questions than answers, I hope to argue that Ulysses and Light in August are significant literary works because each engages the reader in traumatic discourse, entertains the reader with the traumatic spectacle, and enlightens the reader on the complex relationship between trauma and narrative.