The oligomerization of CPS from E. coli was investigated in order to examine the influence of this property on the catalytic activity. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (αβ)4 tetramer through disruption of the molecular binding interactions between monomeric units. The results are consistent with a model for the structure of the (αβ)2 dimer that is formed through molecular contact between two pairs of allosteric domains. No significant dependence of the specific catalytic activity on the protein concentration could be detected. The molecular tunnel within CPS was inspected in order to characterize the role on kinetic properties. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The kinetic data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain to yield a final product carbamoyl phosphate. In addition, a unique feature of the carbamate tunnel has been noted where five highly conserved glutamates are located on a particular interior face of the tunnel. It has been postulated that the negative charge stabilizes the acid-labile intermediate, and facilitates catalysis. Also, the proposed gate keeping residues, Arg-306 and Arg-848, have been mutated to alanines to test their roles. However, since the arginines directly interact with MgATP, the mutation appeared to interrupt the binding of the substrate. The ammonia tunnel has been engineered to contain a hole to further support the proposed role of the tunnel that it is utilized in guiding diffusion of ammonia from the site of glutamine hydrolysis to the subsequent active site in the large subunit. Triple mutant αP360A/αH361A/βR265A exhibited kinetic behaviors consistent with a model of an impaired channeling.

This is a story of the failing Texas foster care system, told by the people who work in it and more importantly, the children affected by it. Foster parents Jim and Kay Mayo and their foster children Brian and Heaven shared their troubled pasts and hopes for the future so more people could understand the problems facing them. There is no easy answer but hopefully some planned changes in policy will shape a better system. To delve deeper into the Mayo’s family dynamic and hear Brian tell his story in person, I invite you to see my video about Brian, Misael and Heaven here: “Far From Home” http://vimeo.com/41420290. The password is: foster care. / text

The oligomerization of CPS from E. coli was investigated in order to examine the influence of this property on the catalytic activity. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (αβ)4 tetramer through disruption of the molecular binding interactions between monomeric units. The results are consistent with a model for the structure of the (αβ)2 dimer that is formed through molecular contact between two pairs of allosteric domains. No significant dependence of the specific catalytic activity on the protein concentration could be detected. The molecular tunnel within CPS was inspected in order to characterize the role on kinetic properties. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The kinetic data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain to yield a final product carbamoyl phosphate. In addition, a unique feature of the carbamate tunnel has been noted where five highly conserved glutamates are located on a particular interior face of the tunnel. It has been postulated that the negative charge stabilizes the acid-labile intermediate, and facilitates catalysis. Also, the proposed gate keeping residues, Arg-306 and Arg-848, have been mutated to alanines to test their roles. However, since the arginines directly interact with MgATP, the mutation appeared to interrupt the binding of the substrate. The ammonia tunnel has been engineered to contain a hole to further support the proposed role of the tunnel that it is utilized in guiding diffusion of ammonia from the site of glutamine hydrolysis to the subsequent active site in the large subunit. Triple mutant αP360A/αH361A/βR265A exhibited kinetic behaviors consistent with a model of an impaired channeling.

Texas Child Protective Services has been struggling with keeping its workers. The turnover rate, which measures the frequency of workers quitting and entering the agency, has been extremely high since the 1980s. In 2012, CPS reported that about one-fourth of its skilled workers are leaving the agency. This puts extra burden on the remaining workers, eventually leading them to resign as well. To fix the turnover issue, the state of Texas initiated a major reform in CPS in 2006. Although the reform succeeded in improving the quality of work environment and other areas, it failed to bring down the turnover rate. To identify why the statewide reform had failed, former caseworkers share their experiences with the agency to reveal what causes the high turnover, the detrimental effects it has on children, and what should be done to reduce the rates. / text

Vibrio vulnificus is an opportunistic human and animal pathogen with the highest death rate of any foodborne disease agent. The capsular polysaccharide (CPS) is essential for virulence. Over 100 CPS types (carbotypes) have been identified among natural isolates, yet little is known about the genetic mechanisms that drive such diversity. Chitin, the second most abundant polysaccharide in nature, induces competence in Vibrio species. We found that transformation frequency varies by strain and (GlcNAc)2 was the shortest chitin-derived polymer capable of inducing competence. We confirmed that V. vulnificus can undergo chitin-dependent carbotype conversion following the uptake and recombination of complete cps loci from exogenous genomic DNA. The acquisition of a partial locus was also demonstrated when internal regions of homology between the endogenous and exogenous loci existed. Thus, the same mechanism governing the transfer of complete cps loci also contributes to their evolution by generating novel combinations of CPS biosynthesis genes.

Vibrio vulnificus is an opportunistic human and animal pathogen with the highest death rate of any foodborne disease agent. The capsular polysaccharide (CPS) is essential for virulence. Over 100 CPS types (carbotypes) have been identified among natural isolates, yet little is known about the genetic mechanisms that drive such diversity. Chitin, the second most abundant polysaccharide in nature, induces competence in Vibrio species. We found that transformation frequency varies by strain and (GlcNAc)2 was the shortest chitin-derived polymer capable of inducing competence. We confirmed that V. vulnificus can undergo chitin-dependent carbotype conversion following the uptake and recombination of complete cps loci from exogenous genomic DNA. The acquisition of a partial locus was also demonstrated when internal regions of homology between the endogenous and exogenous loci existed. Thus, the same mechanism governing the transfer of complete cps loci also contributes to their evolution by generating novel combinations of CPS biosynthesis genes.

Campylobacter jejuni bacterial pathogen is among the primary causes of food-borne acute gastroenteritis in North America and the world. It has also been linked to severe post-infection sequelae such as Guillain-Barré syndrome. Previous studies identified C. jejuni surface capsular polysaccharide (CPS) as a target for creation of a carbohydrate based vaccine in which the CPS is conjugated to a carrier protein. In this thesis, following sample purification, aspects of C. jejuni HS:5 CPS structure were characterized using numerous analytical techniques such as NMR and GC-MS. CPS is comprised of α-DD-Heptoses linked at C2 to the anomeric carbons of glucose. The α-Glucose molecules are linked though C4 to the α-DD-Heptose anomeric carbon. The α-DD-Heptose structure also has an occasional ring structured amino acid modification. Following characterization the CPS was oxidized and developed into a prototype glycoconjugate vaccine using TEMPO oxidation and EDC-CRM197 coupling methods. / The Natural Sciences and Engineering Research Council of Canada (NSERC)

The main aim of this dissertation is to address the security issues of the physical layer of Cyber Physical Systems. The network security is first assessed using a 5-level Network Security Evaluation Scheme (NSES). The network security is then enhanced using a novel Intrusion Detection System that is designed using Supervised Machine Learning. Defined as a complete architecture, this framework includes a complete packet analysis of radio traffic of Routing Protocol for Low-Power and Lossy Networks (RPL). A dataset of 300 different simulations of RPL network is defined for normal traffic, hello flood attack, DIS attack, increased version attack and decreased rank attack. The IDS is a multi-model detection model that provides an efficient detection against the known as well as new attacks. The model analysis is done with the cross-validation method as well as using the new data from a similar network. To detect the known attacks, the model performed at 99% accuracy rate and for the new attack, 85% accuracy is achieved. / Graduate

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