The specific interactions of macromolecules along with the activity of enzymes are central to all aspects of biology. It is well recognized that when the relative concentration or activity of macromolecules is perturbed, it can lead to human diseases. Thus, the development of simple methods for the detection of macromolecules and the activity of enzymes in complex environments is important for understanding biology. Moreover, the development of methods for measuring interactions allows for the testing of inhibitors that can be used as tools or drugs for improving human health. Towards this goal, a promising new method has been developed, which is the focus of this thesis, called split-protein reassembly or protein fragment complementation. In this method, a protein reporter, such as the green fluorescent protein or firefly luciferase, is dissected into two fragments, which are attached to designed adaptor proteins. The designed split-protein systems only produce a measurable signal, either fluorescence or luminescence, when a specific macromolecular interaction or activity is present. In this thesis, I have extended previous research on the direct detection of DNA using split-protein sensors utilizing a red fluorescent protein, dsRED from Discosoma that allows for multiplexed DNA detection. I have designed a new split-luciferase based sensor for detection of poly (ADP-ribose) or PAR, which plays a key role in the response to DNA damage and have applied it for monitoring the activity of poly (ADP-ribose) glycohydrolase that controls PAR levels in the cell. Furthermore, I have significantly expanded upon a three-hybrid split-luciferase system for identifying protein kinase inhibitors. I have designed and tested two orthogonal peptide based chemical inducers of dimerization based on BAD and p53mt conjugates. I have studied these chemically induced dimerization systems in detail in order to begin to provide a theoretical basis for the observed experimental results. Finally, in a less related area, I have developed methods for producing water soluble semiconductor nanoparticles called Quantum Dots (QDs), with potential application in biological imaging. I have developed methods for functionalizing the QDs with orthogonal peptides, which can be potentially used for the assembly of high affinity non-covalent QD targeted proteins.

Pathogenic bacteria use an arsenal of toxic protein virulence factors to cause disease in host cells. The mono-ADP-ribosyltransferase (mART) toxins are a family of exotoxins produced by pathogens which contribute to a wide range of diseases including cholera, diphtheria and whooping cough. Specifically, mART toxins act by transferring ADP-ribose from NAD+ to target proteins in host cells, altering or inhibiting target activity with deleterious downstream effects. Recently, in silico analyses have revealed two novel mARTs, Photox and Certhrax, from pathogenic organisms. Photox, from Photorhabdus luminescens was successfully expressed and purified from E. coli and was shown to target actin, specifically at Arg177. This covalent modification inhibits actin polymerization and leads to observed cytotoxicity in yeast cells. Photox has 35% identity with SpvB from Salmonella enterica, which allowed for a structural model to be built, showing the location of all characteristic mART active site components, and the binding site for potential inhibitors. Certhrax originates from Bacillus cereus G9241, implicated in a number of severe pneumonia cases. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we demonstrated that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. In vivo tests employing toxin gene expression in yeast, and receptor-mediated infection of mammalian, cells showed the extreme cytotoxicity of Certhrax (LD50 = 100 pg/mL against mouse macrophage cells), making it 60 times more toxic than its infamous counterpart, anthrax lethal factor. In vitro analysis indicated that Certhrax possesses NAD+ glycohydrolase activity, characteristic of many mART toxins, but we continue to search for the natural host protein target of this toxic enzyme. We determined the crystal structure of Certhrax to 2.2 Å, which illustrates a close structural similarity with anthrax lethal factor. Furthermore, we identified several small molecule inhibitors which show protection against Certhrax both in vitro and in vivo. We determined a 1.9 Å crystal structure of one inhibitor in complex with Certhrax. Through identification and characterization of novel mART enzymes, we seek to better understand this family of toxic enzymes to aid in the discovery and development of more potent therapeutics. / National Sciences and Engineering Research Council, Canadian Institutes of Health Research

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

DNA double strand breaks (DSBs) are primarily repaired in eukaryotic cells by two different mechanisms – non-homologous end joining (NHEJ) or homologous recombination (HR). In mammalian somatic cells the balance between the two highly favours NHEJ. Gene targeting is a technique that exploits HR repair to alter a defined gene locus. While it holds potential to be implemented as a treatment option for several diseases, the outlook for using it in a clinical setting has been obstructed by a low gene targeting efficiency. This has been coupled to the low frequency of HR in mammalian cells. With the intention of shifting the repair balance, antibodies against DSB repair proteins will be introduced into mammalian cells. It is predicted that by targeting key repair proteins with antibodies, a compensatory increase in the frequency of HR can be fostered, ultimately resulting in improved gene targeting. / xv, 168 leaves : ill. ; 29 cm

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available via the Internet as viewed 10 July 2008.

Freiburg i. Br., Univ., Diss., 2007.

Konstanz, Univ., Diss., 2008.

Thèse (Ph. D.)--Université Laval, 2008. / Bibliogr.: f. 250-258. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph.D.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2009/06/09). Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.