Bonn, Univ., Diss., 2005. / Computerdatei im Fernzugriff.

Universiẗat, Diss., 2005--Bonn.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 229-236).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The overwhelming increase in antibiotic resistant bacterial strains poses a serious public health problem, with multiply-resistant strains becoming an important cause of mortality in hospitals. The predominant mechanism of resistance to aminoglycoside antibiotics involves enzymatic modification of the drug, rendering it ineffective. The crystal structure of the aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ii (AAC(6')-Ii) in complex with its cofactor, acetyl coenzyme A (AcCoA), was determined at 2.7 Å resolution by the multiwavelength anomalous diffraction technique. The resolution of this structure was subsequently extended to 2.15 Å by molecular replacement, with no significant changes in the topology of the complex. The enzyme was found to exhibit a novel CoA-binding fold, with the cofactor bound in a cleft between the N-and C-terminal arms of the protein molecule. Although the enzyme packs as a monomer in the I4₁32 crystal form, the most probable physiological dimer of the complex was determined through analysis of a number of symmetry-related molecules. The crystal structure of the AAC(6')-Ii•AcCoA complex was compared to the structures of three members of a large superfamily of GCN5-related 𝘕-acetyltransferases (GNATs), namely yeast histone acetyltransferase HAT1, 𝘕-myristoyltransferase, and aminoglycoside acetyltransferase(3)-Ia. Despite negligible sequence similarity between these GNAT superfamily members, a distinct folding pattern is conserved in all four structures. This establishes AAC(6')-Ii as a structural homolog of enzymes with protein acetylating activity, supporting the hypothesis that the enzyme may possess another physiological function in 𝘌𝘯𝘵𝘦𝘳𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘧𝘢𝘦𝘤𝘪𝘶𝘮. / Thesis / Master of Science (MS)

In 2106, there were 171,550 new cases of blood cancers and over one million people in the United States living with one of these disorders. Bone marrow transplants have good outcomes, but these procedures require a donor who is a perfect match, and thus many patients are unable to receive treatment. It is important to find patient-derived treatments, such as molecules which stimulate hematopoietic stem cell (HSC) formation without the need for a donor. Therefore, a study was initiated to use human-induced pluripotent stem cell (hiPSC) technology to make a patient-derived, personalized HSC. Epigenetic regulators are divided into readers, writers, and erasers, and each of these classes has shown some effect on HSC formation. Writers add functional groups to deoxyribonucleic acid (DNA) and histone proteins, whereas erasers remove them. Readers are groups on transcription factors which interpret these changes and increase or decrease the recruitment of transcriptional machinery accordingly. In this study, a screen of 12 different candidate molecules with distinct epigenetic targets in casper zebrafish was conducted at 36 hours postfertilization (hpf) to reveal increases or decreases in definitive HSC development. The two writer molecules, C646 (histone acetyltransferase, or HAT, inhibitor) and OICR9249 (WDR5 inhibitor), and the two eraser molecules, Ex-527 (Sirt1 inhibitor) and JIB-04 (bromodomain inhibitor), showed varying degrees of increasing HSC formation. Of these molecules, C646 created the most significant increase and was further tested in the zebrafish at 48 and 72 hpf and in a murine model using ex vivo technique and a colony-forming unit (CFU) assay. In contrast to these results, the two eraser molecules, entinostat (class I histone deacetylase, or HDAC, inhibitor) and vorinostat (general HDAC inhibitor), were found to decrease HSC formation in zebrafish. The overall findings of this study provide insight into specific epigenetic regulators in HSC development and identify particular epigenetic markers that could regulate HSC formation from endothelial cells. This discovery will be a stepping stone in utilizing patient-derived hemogenic endothelial cells as a novel source of bone marrow-independent HSCs to treat patients with leukemia, lymphoma, and bone marrow cancers. / 2019-07-11T00:00:00Z

Thioesters are highly reactive centers for acyl-CoAs which allows them to be utilized in a variety of differing enzyme chemistries. As a result of this reactivity, structure-function studies of enzymes using acyl-CoA substrates is difficult. When acyl-CoAs are used in structure-function studies, they often result in a hydrolyzed CoA substrate fragment bound in the active site or require only one of multiple substrates in order to be bound. This results in a lack of information regarding enzyme interactions with the key thioester and acyl chain. To overcome this challenging problem, I have synthesized acetyl- and malonyl-CoA analogs where the thioester has been replaced by an ester (oxygen), amide (nitrogen), or carbonyl (carbon) in a way that is easier, cheaper, and more efficient than performed previously. In addition, we used our synthetic analogs to study a enzymes which span different acyltransferase mechanisms in a combination of kinetics and structure. With this work, it was determined that the amide analogs were stable in all enzymes it was utilized for, while the ester analogs were mostly stable, except the acetyl analog in KasIII, where it acted as a pseudo substrate. As such, these synthetic analogs may have future potential in either type of enzyme for structure-function studies, albeit limited for the acetyl ester analog.

Cocaine- and amphetamine-regulated transcript (CART) peptide, a family of neuropeptides, is shown to inhibit food intake upon intracerebroventricular injection to the rat. CART peptide-immunoreactivity (irCART) was detected in neurons of the dorsal motor nucleus of the vagus (DMNV) of postnatal day one (P1) rats, the earliest day examined. The number of labeled DMNV neurons reached the peak between P5 and P8 rats and gradually declined thereafter. Few irCART neurons were noted in the DMNV between P22 and P90 rats. Double-labeling the medullary sections from P5 and P8 rats with CART-antiserum and choline acetyltransferase (ChAT)-antiserum revealed that irCART neurons in the DMNV were ChAT-immunoreactive (irChAT), but not all irChAT neurons were irCART. Intraperitoneal injection of the retrograde tracer Fluorogold to P3 and P5 rats labeled DMNV neurons, the majority of which were also irCART. The number of irCART neurons in other regions of the brain and spinal cord generally showed an increase in adult rats as compared to that of the same regions in immature rats. Our result suggests that expression of irCART in DMNV neurons undergoes developmental changes such that few neurons appear to contain irCART in mature rats. As a corollary, CART may be a signaling molecule to the gastrointestinal tract during the critical period of early development.

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