Studies on human cancer variant and mycobacterial isocitrate dehydrogenases

Mutations in human IDH genes occur in cancer and result in active site IDH variants with a gain-of-function ability to reduce the normal 2-oxoglutarate (2-OG) product of IDH catalysis to 2-hydroxyglutarate (2-HG). As reviewed in Chapter 1, elevated 2-HG levels are proposed to promote tumorigenesis via chromatin remodelling. Efficient IDH1 variant inhibitors bind in an allosteric manner at the dimer-interface and hinder binding of 2-OG and Mg2+. Ivosidenib is an IDH1 variant inhibitor that is approved for acute myeloid leukaemia (AML) treatment; however, acquired second-site S280F mutations to IDH1 render cancer cells resistant to ivosidenib treatment. The research described in this thesis investigated the mechanism of action of the second site IDH1 mutation and how to overcome resistance due to it. Kinetic analyses show that the IDH1 S280F substitution not only leads to resistance against ivosidenib but results in a higher affinity for 2-OG and Mg2+, and consequently, more efficient turnover of 2-OG to 2-HG. 1H Nuclear magnetic resonance (NMR) studies reveal that IDH1 cancer variants can turn over D-isocitrate to 2-HG. The rate of conversion of D-isocitrate to 2-HG by S280F substituted variants is more efficient than for IDH1 wildtype or active site variants without the S280F substitution. Mechanistic studies on IDH1 variants provide insights into the influence of various R132 substitutions and the role of the dimer-interface in IDH1 catalysis. In addition to resistance enabled by more efficient 2-HG production, ivosidenib binding is hindered by the loss of a hydrogen bond to S280, steric hindrance due to the S280F substitution, formation of a new hydrophobic pocket at the dimer-interface, and higher enzymatic affinity for 2-OG and Mg2+. Certain IDH1 variant inhibitors were shown to retain activity against isolated IDH1 R132C S280F and R132H S280F, some with high potency. Non-denaturing mass spectrometry (MS) reveals that inhibitors retaining activity bind with a stoichiometry of two inhibitors per IDH1 variant dimer, in contrast to ivosidenib, which binds with a stoichiometry of one inhibitor per dimer. Several inhibitors reduce 2-HG levels in cell lines overexpressing IDH1 R132C S280F or R132H S280F. Some of these inhibitors are in phase 2 clinical studies (FT-2102, DS-1001B) indicating that S280F-mediated ivosidenib resistance can be overcome by using alternative inhibitors. Targeting metabolism has also been of long-standing interest in the antibacterial field, including for Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msm). After establishing production strategies and an activity assay for Mtb IDH1/IDH2 and Msm IDH, kinetic studies support the proposal that Mtb IDH2 is likely the essential IDH isoform for oxidative decarboxylation of isocitrate in Mtb metabolism. Mtb IDH2 activity is enhanced by several reactive carbonyl group containing metabolites. Most Hs IDH1 cancer variant inhibitors are not active against Mtb IDH1/IDH2 and Msm IDH but some exhibit weak activity. The overall results provide mechanistic insights into resistance to Hs IDH1 variant inhibitors and show how this can be overcome. The studies suggest that targeting IDH may be a viable strategy for mycobacterial treatment

The Effects of Laity Ministry Involvement on Transformational Discipleship

This study sought to determine the impacts of The Place Assessment process within four congregations that are a part of The Church of the Nazarene in central and Eastern Iowa. The Place Assessment process included structured teaching, completing a comprehensive assessment online, and structured coaching. The results of this process showed measurable increases in the areas of discipleship engagement and discipleship transformation

Exploring Sepsis Bundle Compliance in the Medical-Surgical/Telemetry Unit

Abstract Problem: Sepsis creates complications for patients and nursing staff. Increasing sepsis bundle compliance has been shown to decrease patient mortality. This quality improvement project utilized a self-administered questionnaire to better understand the opinions of nursing staff on the Medical-Surgical/Telemetry unit and explore ways to improve sepsis bundle compliance. Context: The microsystem is a 32-bed Medical-Surgical/Telemetry unit at Hospital X, a 244-bed, not-for-profit hospital serving the Bay Area of California. Interventions: A questionnaire distributed to nursing staff on the unit was the primary intervention. Through microsystem assessment, questionnaire results, and direct conversations with staff; active and passive data were obtained. Measures: The self-administered questionnaire distributed aimed to understand if staff had received sepsis bundle training and what their opinions were regarding the bundle, Rapid Response Team, eCART, and suggestions for improvement. Self-administered questionnaires were distributed over a one-month period. Results: This project achieved a 67% response rate. Of the nurses who responded, 16.7% of them indicated that they did not receive the sepsis bundle at any time during their employment at Hospital X. 50% of respondents rated the effectiveness of training between an 8 and 10, 36.1% rated is between 5 and 7, and 11.1% rated it between 0 and 4. 88.9% felt that rapid response was effective when managing the care of patients admitted with sepsis. Qualitative data was obtained from written responses that provided valuable insight into the lack of sepsis bundle compliance. Conclusion: Qualitative and quantitative data were obtained from the self-administered questionnaires, microsystem assessment, and conversations with staff. High response rate reflected staff willingness to improve their practice. Project findings contribute to improvements to be implemented by Hospital X. Results from the self-administered questionnaire support the need for repeat sepsis training, visual-aids, simplification of current sepsis protocol, and investigation into the transfer process beginning in the Emergency Department. Recommendations for improvement were provided to the leadership team for implementation. These recommendations are supported by the Nurses’ responses to the self-administered questionnaires and research on the best evidence-based practices. Keywords: sepsis, sepsis bundle compliance, mortality, eCART, quality improvemen

Factors Affecting Moisture Distribution in 290-Kilogram Stirred-Curd Cheddar Cheese Blocks

The purpose of this dissertation was to study factors affecting moisture distribution in 290-kilogram stirred-curd Cheddar cheese blocks cooled in stainless steel hoops. Uneven moisture distribution within blocks may create cheese with variable texture and flavor, which can be extremely costly to the producer. The effects of temperature, pH, and vacuum treatment on moisture distribution were investigated. Temperature, pH, moisture, and pressure profiles were presented. Also, comparisons were made between temperature profiles of 290- kilogram stirred-curd Cheddar cheese blocks cooled in stainless steel and in plywood hoops, as well as between temperature profiles of 66-kilogram Swiss cheese blocks cooled in cardboard and in plastic boxes. Moisture transferred from high to low temperature in the cheese blocks. Moisture may have transferred in response to thermally induced curd moisture-holding capacity gradients in the cheese blocks. Moisture also may have transferred in the cheese blocks by a mechanism similar to thermo-osmosis of liquids in porous solids. The cheese in the plywood or cardboard insulating materials cooled more uniformly than the cheese in the stainless steel or plastic containers. More uniform cooling of the cheese produced more uniform moisture distribution in the cheese blocks. Recommendations were made to help the cheesemaker produce cheese with even moisture distribution