Analytical spectroscopy method development to study mechanisms of Alzheimer's and tuberculosis diseases

2020 Spring.Includes bibliographical references.This dissertation covers the analytical method development created to study and enhance the knowledge of two specific disease mechanisms important to Alzheimer's disease and Mycobacterium tuberculosis. There are two parts in this dissertation where Part 1 is entitled Measurement of The Kinetic Rate Constants of Interpeptidic Divalent Transition Metal Ion Exchange in Neurodegenerative Disease. Part 2 is entitled The Electrochemistry of Truncated Menaquinone Electron Transporters with Saturated Isoprene Side Chains Important in Tuberculosis. These diseases appear on the World Health Organization's top 10 leading causes of death worldwide. The amyloid-beta (Aβ) peptides are associated with Alzheimer's disease, where neurodegeneration is caused by the aggregation of the peptide into senile plaques within neuronal synaptic cleft spaces. Alzheimer's disease currently has no cure due to its multi-causative pathology. One disease mechanism is the coordination of divalent metal ions to the peptide. Specifically, Aβ coordinates Cu(II) and Zn(II) ions that can enhance the aggregation of Aβ into plaques. These metal ions are highly regulated within the human body and are usually found bound to peptides and not as free ions. Therefore, the Aβ must sequester the metals from other proteins and peptides. The primary research in this dissertation advances fluorescence method development to measure interpeptidic Cu(II) exchange kinetics to be able to measure this type of disease mechanism. The small peptides GHK (Gly – His – Lys) and DAHK (Asp – Ala – His – Lys) both chelate Cu(II) with nM affinity, have biological relevance as they are motifs found in human blood like Aβ, and chelate Cu(II) with similar nitrogen-rich binding ligands as Aβ. By substituting non-coordinating lysine residues with fluorescent tryptophan, the interpeptidic exchange rates can be measured since tryptophan fluorescence is statically quenched when within 14 angstroms of a paramagnetic bound Cu(II). Thus Cu(II) transfer from Cu(H-1GHW) to either GHK or DAHK can be monitored by recovered GHW fluorescence as the Cu(II) is exchanged and second-order kinetic rate constants were determined. This methodology was then used to monitor the Cu(II) exchange from truncated Cu(Aβ1-16) and Cu(Aβ1-28) complexes to GHW and DAHW, where second-order reaction kinetic rate constants were determined. While in the exchanges between Cu(H-1GHW) with GHK/DAHK the second-order rate constants were on the magnitude of 102 or 101 M-1s-1, respectively, the exchanges from Cu(Aβ) complexes were 2-3 orders of magnitude larger, 104 M-1s-1 (to GHW and DAHW). These differences in rate constant magnitude arise from the fact that the affinity of GHW (KA = 1013 M-1) for Cu(II) is larger than Aβ (KA =1010 M-1). This method development is an important step to an accurate measurement of the interpeptidic exchange between Aβ peptides, including in their fibril and plaque formations. Since senile plaques are found in synaptic cleft spaces with nanometer distances between neurons, a model system was generated to study coordination reactions at the nanoscale. In order to do this, the metal ion would need to be released in a controlled manner. Studies of metal ion burst reactions through the use of photocages can simulate bursts of ions like those seen in the synaptic cleft. Zn(II) is often released in its ionic form within the synapse in its function as a neurotransmitter, so we simulated a burst of Zn(II) ions by using a photocage, [Zn(NTAdeCage)]- which releases Zn(II) when irradiated with light. The photocage was irradiated to release Zn(II) then we followed its reaction progress with an in situ chelator, Zincon, in reverse micelles and in bulk aqueous buffer. The coordination reaction was 1.4 times faster in an aqueous buffer than in reverse micelles, despite the Zn(II) and Zincon being closer in the nanoparticle. These observations suggested that there is an impact on coordination reactivity within a highly heterogeneous environment with a cell-like membrane, which is due to the partitioning of each ligand. We observe that the photocage stays in the water pool of the reverse micelle and the Zincon partitions into the membrane interface. Thus, the coordination reactivity is diminished, likely due to the need for Zn(II) to diffuse to the Zincon, crossing a highly organized Stern layer to encounter the Zincon. Whereas in aqueous buffer, these are free to encounter each other despite being hundreds of nanometers apart. These proof of concept studies are integral to studying initial binding dynamics of metal ions with peptides at the nanoscale present in cells and neuronal synapses. Tuberculosis is a pathogenic bacterium which despite having a curable medication, can be drug-resistant. Menaquinone (MK) analogs with regiospecific partial saturation in their isoprenyl side chain, such as MK-9(II-H2), are found in many types of bacteria, including pathogenic Mycobacterium tuberculosis and function as electron transport lipids cycling between quinone and quinol forms within the electron transport system. While the function of MK is well established, the role of regiospecific partial saturation in the isoprenyl side chain on MK remains unclear and may be related to the redox function. Recently, an enzyme in M. tuberculosis called MenJ was shown to selectively saturate the second isoprene unit of MK-9 to MK-9(II-H2). The knockout expression of this enzyme was shown to be essential to the survival of the bacterium. A series of synthesized truncated MK-n analogs were investigated using a systematic statistical approach to test the effects of regiospecific saturation on the redox potentials. Using principal component analysis on the experimental redox potentials, the effects of saturation of the isoprene tail on the redox potentials were identified. The partial saturation of the second isoprene unit resulted in more positive redox potentials, requiring less energy to reduce the quinone. While full saturation of the isoprene tail resulted in the most negative potentials measured, requiring more energy to reduce the quinone. These observations give insight into why these partially saturated menaquinones are conserved in nature

Supporting Healthy Identity Development Using Text Selection and Instructional Activities in Secondary Language Arts Classes

Adolescence is a critical juncture in the process of identity development and has implications for secondary-level students. This project connects the topics of adolescent identity development with literature instruction at the secondary level, investigating the question “How can secondary language arts teachers support healthy identity development in adolescent learners using text selection and instructional activities?” A literature review provides a theoretical basis of adolescent identity development, including foundational theories such as Erik Erikson’s psychosocial development theory and contemporary ideas like Kimberlé Crenshaw’s work on intersectionality. The review provides a framework for understanding how text selection and instructional activities affect adolescent identity development and discusses research that guides educators toward curricular choices and texts that support healthy student identity development. Essential findings in the literature review include the importance of selecting texts that are culturally relevant and affirming, and the practice of pairing classic canon literature with more accessible and relevant young adult literature. A website for secondary language arts educators, Linking Literature for the Student Self, is based on this research and provides guidance for selecting texts and pairing classic and young adult literature

Save your energy with the four P\u27s

This patient education handout shares tips on using the four p\u27s of energy conservation

Tips to Save Your Energy

This patient education handout shares energy conservation tips

Operating Reserve Reductions from a Proposed Energy Imbalance Market with Wind and Solar Generation in the Western Interconnection

This paper considers several alternative forms of an energy imbalance market (EIM) proposed in the nonmarket areas of the Western Interconnection. The proposed EIM includes two changes in operating practices that independently reduce variability and increase access to responsive resources: balancing authority cooperation and sub-hourly dispatch. As the penetration of variable generation increases on the power system, additional interest in coordination would likely occur. Several alternative approaches could be used, but consideration of any form of coordinated unit commitment is beyond the scope of this analysis. This report examines the benefits of several possible EIM implementations--both separately and in concert

Western Interconnection Energy Imbalance Market Status and Prospects

This presentation describes how a new wholesale electricity market for energy imbalance ancillary services could be implemented and operated. Some conclusions of this presentation are: (1) Method for calculating additional reserve requirements due to wind and solar production; (2) EIM results in substantial reduction in reserves requirements and ramping demand; (3) Reduced participation reduces benefits for all but reduces the benefits to non-participants the most; (4) Full participation leads to maximum benefit across the Western Interconnection, up to 42% of total reserve requirement; and (5) Regional EIM implementations have smaller but substantial benefits

A NON-CLEAVABLE UmuD VARIANT THAT ACTS AS A UmuD' MIMIC

UmuD{sub 2} cleaves and removes its N-terminal 24 amino acids to form UmuD'{sub 2}, which activates UmuC for its role in UV-induced mutagenesis in E. coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD has been shown to bind to the beta processivity clamp (''beta'') of the replicative DNA polymerase, pol III. A possible beta-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with beta. We performed alanine-scanning mutagenesis of this motif (14-TFPLF-18) in UmuD and showed that it has a moderate influence on UV-induced mutagenesis but is required for the cold sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the beta-binding motif variant bind to beta with similar K{sub d} values as determined by changes in tryptophan fluorescence. However, this data also implies that the single tryptophan in beta is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is noncleavable, we find that cells harboring it exhibit phenotypes more consistent with the cleaved form UmuD', such as resistance to killing by UV light and failure to exhibit the cold sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD' mimic