Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 144-151).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 167-181).

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 68-71). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (M.S.)--Mississippi State University. Department of Biological Sciences. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.

RNA:protein interactions are central in many cellular processes, including activation of innate immune responses against microbial infection. Their study is essential to better understand the diverse biological events that occur within cells. However, isolation of RNA:protein complexes is often laborious and requires specialized techniques. This thesis is concerned with attempts to develop an improved purification protocol to isolate specific RNA:protein complexes. Taking advantage of the specific interaction of the Pseudomonas aeruginosa PP7 protein with its cognate RNA binding site, termed the PP7 recognition sequence (PRS), the aim was to identify cellular proteins involved in activating cell-signalling pathways, including the interferon-induction cascade, following viral infection with stocks of parainfluenza virus 5 (PIV5) rich in copyback defective interfering (DI) particles. Copyback DI genomes are powerful inducers of IFN and, here, I show they also activate the induction of IL-6, IL-8 and TNFα; cytokines that also have antiviral properties. Following the successful cloning of the PRS into a copyback DI genome, we investigated conditions for optimal in vitro capture of DI-PRS:protein complexes by PP7 on Dynabeads. When tested, the protocol led to the successful capture of ILF3 and PKR, two dsRNA binding proteins induced by IFN. We further developed a tap-tagging system to minimize the presence of non-specifically bound proteins to Dynabeads that may interfere with future mass spectrometry analysis. To isolate DI-PRS RNA:protein complexes from infected cells, attempts were made to rescue replicating DI-PRS genomes in the context of wild type PIV5. Similarly, efforts were made to isolate influenza A virus RNPs that contained the PRS in the neuraminidase (NA) gene from infected cells using the PP7-based protocol developed. However, for reasons discussed, unfortunately RNA:proteins complexes were not successfully purified from infected cells in either case.

Developmental disorders including: autism, intellectual disability, and congenital abnormalities are present in 3-8% of live births and display a huge amount of phenotypic and genetic heterogeneity. Traditionally, geneticists have identified individual monogenic diseases among these patients but a majority of patients fail to receive a clinical diagnosis. However, the genomes of these patients frequently harbour large copynumber variants (CNVs) but their interpretation remains challenging. Using pathway analysis I found significant functional associations for 329 individual phenotypes and show that 39% of these could explain the patients’ multiple co-morbid phenotypes; and multiple associated genes clustered within individual CNVs. I showed there was significantly more such clustering than expected by chance. In addition, the presence of a multiple functionally-related genes is a significant predictor of CNV pathogenicity beyond the presence of known disease genes and size of the CNV. This clustering of functionally-related genes was part of a broader pattern of functional clusters across the human genome. These genome-wide functional clusters showed tissuespecific expression and some evidence of chromatin-domain level regulation. Furthermore, many genome-wide functional clusters were enriched in segmental duplications making them prone to CNV-causing mutations and were frequently seen disrupted in healthy individuals. However, the majority of the time a pathogenic CNV affected the entire functional cluster, where as benign CNVs tended to affect only one or two genes. I also showed that patients with CNVs affecting the same functional cluster are significantly more phenotypically similar to each other than expected even if their CNVs do not affect any of the same genes. Lastly, I considered one of the major limitations in pathway analysis, namely ascertainment biases in functional information due to the prioritization of genes linked to human disease, and show how the modular nature of gene-networks can be used to identify and prioritize understudied genes.

Les protéines à domaine PDZ, en très grand nombre dans le génome humain, sont impliquées dans des interactions protéine-protéine. Elles participent ainsi à véhiculer des signaux à l’origine de différentes pathologies (cancer, douleur….). L’interruption de l’interaction entre la protéine à domaine PDZ, PSD-95, et le récepteur de la sérotonine, 5-HT2A, entraîne une réduction de l’hyperalgie chez le rat neuropathique. Le développement de molécules capables d’inhiber cette interaction pourrait donc conduire à une nouvelle classe d’antalgiques.Nous avons réalisé, au cours de ces travaux, la synthèse de trois générations de ligands, comportant un noyau indolique, capables d’interagir avec le site S0, site très conservé des protéines à domaines PDZ. Dans un premier temps, nous avons préparé 15 biligands possédant un noyau indolique polysubstitué lié, via un espaceur de longueur variable (2 à 6 atomes de carbone), à différents acides aminés, dans le but d’interagir avec le site S1, montrant beaucoup de diversité en fonction du domaine. Nous avons ensuite, après une étude de relation structure/activité, développé deux autres générations d’indoles polysubstitués présentant notamment des substituants hydrophobes en position 5.Nous avons montré, par RMN HSQC 1H/15N et chromatographie d’affinité, que deux de ces composés sont des inhibiteurs de l’interaction PSD-95/5-HT2A et présentent de fortes interactions avec le site S0 de PSD-95. Ces molécules présentent également des propriétés antalgiques particulièrement intéressantes in vivo. Nous avons également déterminé, par RMN NOESY, la structure du complexe protéine/ligand pour ces deux composés. L’orientation d’une de ces molécules dans le site de la protéine nous permet d’envisager le développement d’une nouvelle génération d’indoles polysubstitués, pouvant interagir avec le site S1 de la protéine et permettant ainsi d’obtenir des inhibiteurs sélectifs de l’interaction PSD-95/5-HT2A. / Protein-protein interactions play a central role in the regulation of biological processes and represent a promissing class of therapeutic targets. It has been recently reported that disrupting the interaction between the PDZ protein PSD-95 and the serotonin receptor 5-HT2A induced an antihyperalgesic effect in diabetic rats. In this context, the development of original ligands capable to inhibit specifically this interaction could lead to a new class of analgesic compounds.We carried out the synthesis of three generations of ligands possessing an indole moiety in order to interact with the highly conserved carboxylate-binding loop (GLGF loop) of PSD-95. Two generations of compounds were developed to find out the position and the nature of the substituents furnishing the best interactions. One generation consists of a family of 15 biligands possessing a substituted indole moiety, coupled with a linker (having from 2 to 6 carbon atoms) via an amid function, ended with various amino acids to interact with the S1 site of the protein, in order to obtain specific ligands.By various biological evaluations, NMR HSQC 1H/15N, chromatography affinity assays and in vivo experiments, we identified two promising inhibitors of the interaction PSD-95/5-HT2A with strong interactions with S0 site of PSD-95. For these compounds, we determined the structure of the complex protein/ligand by NMR NOESY experiments. The orientation of one of these molecules in the S0 site allows us to envisage a new generation of ligands capable to interact with the S1 site of the protein.