Approved for public release; distribution is unlimited / Both the number of hosts using Internet Protocol version 6 (IPv6), and the volume of IPv6 traffic, has increased exponentially since 2012.With this adoption, the IPv6 routed infrastructure becomes an increasingly important component of global critical infrastructure and network policy. Unfortunately, the tools and techniques used to perform active network topology discovery were designed for Internet Protocol version 4 (IPv4), leading to a potentially opaque view of the IPv6 Internet. In this thesis, we extend nascent work on stateless high-speed IPv4 active topology probing to develop a new IPv6 traceroute method Yelling At Random Routers Progressively version 6 (Yarrp6). Yarrp6 randomly permutes the set of IPv6 targets and hop counts to distribute load, thereby helping to avoid IPv6 response rate limiting. Further, we encode state in the IPv6 payload to permit Yarrp6 to both match responses with probes and use different probe transport protocols. Via active experimentation on the public IPv6 Internet, we compare the results obtained from Yarrp6 against the current state-of-the-art IPv6 topology mapping tool. We show that Yarrp6 can discover topology at more than an order of magnitude faster than previously possible. Finally, we conduct a study of the effect of transport layer protocol on forward Internet Protocol (IP) path inference to determine what protocol is best used for active IPv6 topology discovery. / Outstanding Thesis / Information Systems Technician First Class, United States Navy

Advances in protein topology mapping methods are urgently needed to complement the wealth of interactome data that is presently being generated at a rapid pace. Chemical crosslinking followed by mass spectrometry (MS) has evolved over the last decade as an attractive method for protein topology and interface mapping, and holds great promise as a counterpart to modern interactome studies in the field of proteomics. Furthermore, stabilization of proteins and protein complexes with crosslinking offers many advantages over high-resolution structural mapping methods, including the ability to study protein topologies in vivo. The reliance on direct detection of crosslinked peptides, however, continues to pose challenges to protein topology and interface mapping with chemical crosslinking plus MS. The present body of work aimed to develop a novel generic methodology that utilizes chemical crosslinking, cyanogen bromide (CNBr) cleavage and MS for the low-resolution mapping of protein topologies and interfaces. Through such low-resolution mapping of crosslinked regions, this novel strategy overcomes limitations associated with the direct detection of crosslinked peptides. Following optimization of various steps, the present method was validated with the bacterial DNA-directed RNA polymerase core complex and was subsequently applied to probe the tetrameric assembly of yeast Skp1p-Cdc4p heterodimers. Further improvements were made through the enrichment of crosslinked CNBr-cleaved protein fragments prior to their identification via MS. Two enrichment strategies were developed which depended upon the conjugation of tags to CNBr-cleaved peptide C-termini followed by either tandem affinity purification or tandem reversed-phase HPLC purification. These strategies were successfully applied for the efficient purification of disulfide-linked peptides from peptide mixtures. It is expected that the potential to achieve sensitive mapping of topologies and interfaces of multi-subunit protein complexes in vivo, in combination with further enhancements to permit studies on complex protein samples, will extend the utility of this method to complement large-scale interactome studies.

Advances in protein topology mapping methods are urgently needed to complement the wealth of interactome data that is presently being generated at a rapid pace. Chemical crosslinking followed by mass spectrometry (MS) has evolved over the last decade as an attractive method for protein topology and interface mapping, and holds great promise as a counterpart to modern interactome studies in the field of proteomics. Furthermore, stabilization of proteins and protein complexes with crosslinking offers many advantages over high-resolution structural mapping methods, including the ability to study protein topologies in vivo. The reliance on direct detection of crosslinked peptides, however, continues to pose challenges to protein topology and interface mapping with chemical crosslinking plus MS. The present body of work aimed to develop a novel generic methodology that utilizes chemical crosslinking, cyanogen bromide (CNBr) cleavage and MS for the low-resolution mapping of protein topologies and interfaces. Through such low-resolution mapping of crosslinked regions, this novel strategy overcomes limitations associated with the direct detection of crosslinked peptides. Following optimization of various steps, the present method was validated with the bacterial DNA-directed RNA polymerase core complex and was subsequently applied to probe the tetrameric assembly of yeast Skp1p-Cdc4p heterodimers. Further improvements were made through the enrichment of crosslinked CNBr-cleaved protein fragments prior to their identification via MS. Two enrichment strategies were developed which depended upon the conjugation of tags to CNBr-cleaved peptide C-termini followed by either tandem affinity purification or tandem reversed-phase HPLC purification. These strategies were successfully applied for the efficient purification of disulfide-linked peptides from peptide mixtures. It is expected that the potential to achieve sensitive mapping of topologies and interfaces of multi-subunit protein complexes in vivo, in combination with further enhancements to permit studies on complex protein samples, will extend the utility of this method to complement large-scale interactome studies.

Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM, where Wzy polymerizes the subunits to lengths regulated by Wzz1/2. The Wzx and Wzy IM topologies were mapped using random C-terminal-truncation fusions to PhoALacZα, which displays PhoA/LacZ activity dependent upon its subcellular localization. Twelve transmembrane segments (TMS) containing charged residues were identified for Wzx. Fourteen TMS, two sizeable cytoplasmic loops (CL), and two large periplasmic loops (PL3 and PL5 of comparable size) were characterized for Wzy. Despite Wzy PL3–PL5 sequence homology, these loops were distinguished by respective cationic and anionic charge properties. Site-directed mutagenesis identified functionally-essential Arg residues in both loops. These results led to the proposition of a “catch-and-release” mechanism for Wzy function. The abovementioned Arg residues and intra-Wzy PL3–PL5 sequence homology were conserved among phylogenetically diverse Wzy homologues, indicating widespread potential for the proposed mechanism. Unexpectedly, Wzy CL6 mutations disrupted Wzz1-mediated regulation of shorter O-Ag chains, providing the first evidence for direct Wzy–Wzz interaction. Mutagenesis studies identified functionally-important charged and aromatic TMS residues localized to either the interior vestibule or TMS bundles in a 3D homology model constructed for Wzx. Substrate-binding or energy-coupling roles were proposed for these residues, respectively. The Wzx interior was found to be cationic, consistent with translocation of anionic O-Ag subunits. To test these hypotheses, Wzx was overexpressed, purified, and reconstituted in proteoliposomes loaded with I−. Common transport coupling ions were introduced to “open” the protein and allow detection of I− flux via reconstituted Wzx. Extraliposomal changes in H+ induced I− flux, while Na+ addition had no effect, suggesting H+-dependent Wzx gating. Putative energy-coupling residue mutants demonstrated defective H+-dependent halide flux. Wzx also mediated H+ uptake as detected through fluorescence shifts from proteoliposomes loaded with pH-sensitive dye. Consequently, Wzx was proposed to function via H+-coupled antiport. In summary, this research has contributed structural and functional knowledge leading to novel mechanistic understandings for O-Ag biosynthesis in bacteria. / Bookmarks within the document have been provided for ease of access to a particular section in the body of the thesis. Each entry in the Table of Contents, List of Tables, and List of Figures has been "linked" to its respective position and as such can be clicked for direct access to the entry. Similarly, each in-text Figure or Table reference has been "linked" to its respective figure/table for direct access to the entry. / 1.) Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award, 2.) CIHR Michael Smith Foreign Study Award, 3.) Cystic Fibrosis Canada (CFC) doctoral studentship, 4.) University of Guelph Dean's Tri-Council Scholarship, 5.) Ontario Graduate Scholarship in Science and Technology, 6.) Operating grants to Dr. Joseph S. Lam from CIHR (MOP-14687) and CFC