Biochemistry electronic theses and dissertations (MU)
https://hdl.handle.net/10355/5232
The electronic theses and disserations of the Division of Biochemistry.2024-03-29T09:33:36ZAdvances in aptamer evolution and engineering
https://hdl.handle.net/10355/60401
Advances in aptamer evolution and engineering
Alam, Khalid Kamal
[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Aptamers are single-stranded nucleic acids that fold into unique three-dimensional shapes that allow them to bind with high affinity and specificity to targets of interest. They are selected through the process of in vitro evolution, wherein large libraries of randomized sequence are iteratively partitioned and amplified to enrich for high-fitness, functional molecules. Selected libraries are sequenced and individual aptamers are characterized for their structure and function. Aptamers have found use as research tools, diagnostics, and therapeutics and in the control of biological systems. The work described herein presents several advancements to the selection and application of aptamers. I first describe an aptamer bioinformatics platform, FASTAptamer, which performs the primary sequence tasks common to all combinatorial selection techniques. I then describe a poly-target selection approach that leverages high-throughput sequencing, the aptamer bioinformatics platform, and parallel selections against a family of related targets to identify the first RNA aptamers capable of potent broad-spectrum inhibition of HIV reverse transcriptase. Finally, this work describes the engineering and in vitro validation of a bifurcated aptamer, Split-Broccoli, for direct visualization of RNA:RNA processes.
2016-01-01T00:00:00ZAdvancing single molecule fluorescence microscopy through the study of dynamic macromolecules
https://hdl.handle.net/10355/63672
Advancing single molecule fluorescence microscopy through the study of dynamic macromolecules
Menke, Drew Edwin
We took a two pronged approach to our investigation of protein synthesis by the ribosome: 1) the generation of a novel fluorescence enhancing substrates 2) the development of a method for the generation of fluorescent ribosome constructs. The development and application of fluorescence based diagnostics platforms for the detection of diseases represents a promising field of research. In collaboration with another lab, we established a method for the generation of a fluorescence enhancing substrate that does not require the use of expensive imaging equipment. During further testing of the fluorescence enhancing substrate with biologically relevant samples, our method proved promising for addition applications. Understanding how and why the ribosome synthesizes protein is important to all living organisms. One known challenge for the study of the ribosome has been the generation of complex samples. We developed a process for the generation of ribosomes that is quicker, easier, and cheaper than existing methods. We then applied our methods for the generation of ribosome constructs to investigate how the ribosome, the molecule responsible for protein synthesis, is coordinated during protein synthesis. Our experiments determined that specific interactions play different roles throughout protein synthesis.
Field of study: Biochemistry.; Dr. Peter V. Cornish, Dissertation Supervisor.; Includes vita.; "December 2017."
2017-01-01T00:00:00Z[alpha]A-crystallin derived peptide, [alpha]A66-80 induced aggregation and precipitation of soluble [alpha]-crystallin : a contribution to age-related cataract formation
https://hdl.handle.net/10355/40172
[alpha]A-crystallin derived peptide, [alpha]A66-80 induced aggregation and precipitation of soluble [alpha]-crystallin : a contribution to age-related cataract formation
Kannan, Rama, 1975-
[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Age-related cataract is the result of aggregation of lens proteins, crystallins. The formation of aggregates in the aging lens has been shown to correlate with progressive accumulation of specific low molecular weight (LMW) peptides (<3.5-kDa) derived from crystallins. Prominent among LMW peptides is [alpha]A66-80, a peptide derived from [alpha]A-crystallin and present in increased concentrations in the water-insoluble fractions of the aging lens. We investigated the characteristics of the peptide and its interactions with soluble [alpha]-crystallin. The [alpha]A66-80 peptide has amyloid-like properties and preferentially insolubilizes [alpha]-crystallin from soluble lens fractions. The peptide interacts with [alpha]B-crystallin at multiple sites, suppresses [alpha]-crystallin chaperone activity, increases the surface hydrophobicity, decreases the solubility and induces its aggregation. A sequential proline scan studies shows those residues 71-77 critical for the activity of the peptide. Lens extracts exhibit protease activity that generates [alpha]A66-80. Thus, the [alpha]-crystallin-derived peptide could play a role in the pathogenesis of cataract formation.
2013-01-01T00:00:00ZAmyloid beta induces cPLA2 activation by an NADPH oxidase-dependent mechanism in neurons
https://hdl.handle.net/10355/7205
Amyloid beta induces cPLA2 activation by an NADPH oxidase-dependent mechanism in neurons
Shelat, Phullara B., 1978-
[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Alzheimer's disease (AD) is a neurodegenerative disorder affecting more than 20 million people worldwide. An increase in production of amyloid beta peptides (A[Beta]) and their aggregation to the oligomeric form is thought to contribute to neurotoxicity in the AD brain. cPLA2 is an enzyme responsible for hydrolysis of fatty acids in the sn-2 position of membrane phospholipids. Substantial attention has been paid to arachidonic acid (AA) because this lipid mediator is not only used for prostanoid synthesis but is also regarded as a retrograde transmitter. cPLA2 has been implicated in the pathogenesis of a number of neurodegenerative diseases including AD. Recent studies provided evidence for the production of reactive oxygen species (ROS) in association with glutamate excitotoxicity and involvement of NMDA receptors in A[Beta]-induced neurotoxicity. This study is focused on elucidating the role of NADPH oxidase, a superoxide producing enzyme, on the signaling pathway leading to cPLA2 activation and AA release in cortical neurons. Results show that A[Beta] and NMDA can produce ROS in neurons via NADPH oxidase and that this pool of ROS is important for ERK1/2 phosphorylation, cPLA2 phosphorylation and subsequent AA release. The study presented here identifies for the first time the importance of the ROS producing enzyme NADPH oxidase in the signaling pathway leading to A[Beta]-induced and NMDA receptor-mediated cPLA2 activation in neurons. This novel mechanism may contribute to a better understanding of the oxidative mechanism underlying the pathogenesis of AD.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.; Title from PDF of title page (University of Missouri--Columbia, viewed on April 29, 2010).; Vita.; Thesis advisor: Grace Y. Sun.; "May 2008"; Ph. D. University of Missouri-Columbia 2008.
2008-01-01T00:00:00Z