Browsing by Author "Dr. Dennis Brown, Committee Member"

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Characterization of In Vitro Selected RNA Aptamers for the RB69 RegA Translational Repressor Protein and Optimization of in vitro Selection Utilizing Complex Targets
(2003-01-30) Dean, Timothy Richard; Dr. Dennis Brown, Committee Member; Dr. Eric Miller, Committee Chair; Dr. Michael Hyman, Committee Member; Dr. James Brown, Committee Member
The purpose of the research has been to characterize the nucleic acid sequence requirements necessary for recognition by the translational repressor protein RegA. RegA, from bacteriophage RB69 and T4 related bacteriophages, is a translational repressor protein that is highly conserved and capable of repressing translation of numerous transcripts. The mRNA binding sites for RegA are devoid of structure and are AU rich. in vitro selection was carried out for 5 rounds generating specific sequences capable of binding to RB69 RegA. Characterization of those sequences included sequencing, dissociation constant (Kdapp) determination, and use of the selected sequences to search the RB69 genome for potential binding sites. The second aspect of the research focused on optimization of conditions for completing successful in vitro selection experiments against complex biological targets. Bacteriophage R17 is an F+ coliphage infecting E. coli found in the intestinal tracts of swine. Development of a rapid diagnostic technique capable of identifying R17 would enable for detection of faeces in ground water. Generation of RNA aptamers against R17 was undertaken with the goal of optimizing selection conditions against a viable viral particle. Two selections were carried out one utilizing nitrocellulose to partition bound RNA from unbound RNA and the second using R17 immobilized on a agarose bead matrix. The results initiated the generation of specific considerations for initiating a selection experiment against a protein target.
Molecular and Structural Characterization of Proteins Involved in Bacterial Adaptive Responses
(2008-04-22) Sullivan, Daniel Michael; Dr. William Miller, Committee Member; Dr. David Bird, Committee Member; Dr. Dennis Brown, Committee Member; Dr. John Cavanagh, Committee Chair
NMR Structural Studies of Proteins Involved in the Competence and Sporulation Pathways of Bacillus subtilis and Pasteuria penetrans.
(2006-12-21) McLaughlin, Patrick Daniel; Dr. John Cavanagh, Committee Chair; Dr. Mike Goshe, Committee Member; Dr. Dennis Brown, Committee Member; Dr. Charles Opperman, Committee Member; Dr. Ronald Venters, Committee Member
The ability of bacteria to adapt to environmental stress is essential to their existence. Several responses to environmental triggers are known. Two of these, Competence and Sporulation, are widespread in the bacterial world. Competence and Sporulation are best studied in the organism Bacillus subtilis and the proteins involved in these pathways are well known. The purpose of this research is to gain insight into the regulation of these cellular processes by determining the NMR solution structures and looking at functional aspects of the ClpC R-domain, involved in bacterial competence, and the L66A mutant of the Spo0F protein involved in the regulation of sporulation. In addition to these studies attempts to purify a Spo0F homolog from the endospore-forming bacteria, Pasteuria penetrans, were made with the intention of determining the metal binding characteristics of the protein.
Structural and Functional Characterization of Proteins Involved in Calcium Binding and Sporulation.
(2007-11-30) Kordys, David Ronald; Dr. John Cavanagh, Committee Chair; Dr. Dennis Brown, Committee Member; Dr. Michael B. Goshe, Committee Member; Dr. Eric Miller, Committee Member; Dr. Ronald A. Venters, Committee Member