This study concentrates on analysis of LongEZ aircraft data taken offshore of the Atlantic Coast of the United States. Due to the land structure of the region, it was possible to isolate the effect of narrow land on air as it flows offshore. The narrow land (Outer Banks) separates inland water from the sea. With greater land fetch, the internal boundary layer (IBL) over land grows deeper and the eddies presumably grow larger. Larger eddies typically decay more slowly than smaller eddies, and so the turbulence advected from land with a larger land fetch should survive longer over the sea and be greater in magnitude than that with smaller land fetch. The turbulence is studied using aircraft eddy correlation data as the flow is advected over the water. As expected, greater and longer-lasting turbulence is present downstream from greater land widths. Aircraft data taken over the Gulf Stream (GS) boundary are analyzed to study the effects of the sea surface temperature (SST) front on downstream boundary layer structure. Unstable and stable flows are studied in this region. The stable flow case is found to have an upside-down structure, with greater turbulence aloft causing stress convergence at the surface, which acts to accelerate the flow. The local thermally generated pressure gradient is important in the momentum budget across the GS front in both flow cases. A synthetic aperture radar (SAR) image is analyzed qualitatively in the region between the Atlantic Coast and the Gulf Stream front for intercomparison of data and to examine the influences of varying static stabilities and surface conditions upon the backscatter shown in satellite images. The growth rates of the internal boundary layer due to flow over a heterogeneous surface including flow from land over the water and flow between cooler water and warmer water are calculated. These results are compared to similar calculations of growth rates from previous experiments. It is found that the growth rate of an internal boundary layer is dependent on surface roughness, despite the inclusion of σ[subscript w] in the normalization of the growth rate. / Graduation date: 2003

Master of Science / Department of Biomedical Sciences / Sanjeev K. Narayanan / Fusobacterium necrophorum is an anaerobic Gram-negative non spore forming rod shaped bacteria that is a normal inhabitant of the alimentary tract of humans and animals. Two subspecies of F. necrophorum have been recognized- subspecies necrophorum and subspecies funduliforme. Subspecies necrophorum is an opportunistic pathogen in animals causing diseases such as bovine hepatic abscesses and sheep foot rot while as subspecies funduliforme is linked with human oral and hepatic infections such as sore throats, Lemierre’s syndrome and hepatic abscesses. The pathogenic mechanisms of F. necrophorum are complex and are not well understood or defined. Several virulence factors such as leukotoxin, haemolysin, haemagglutinin and adhesin have been described. One of the most important factors in F. necrophorum bacterial pathogenesis is the adhesion of the bacteria to the host cell. The adhesion of the bacteria to the host cell helps it colonize the host tissue and this is followed by intracellular multiplication with dissemination to other tissues, which could ultimately lead to septicemia and death. Bacteria use adhesins which are proteins found in the outer membrane which help them bind with host receptors and this helps with the adhesion of the bacteria to the host cell. Not much is known about F. necrophorum adhesins. Here, we describe and characterize a novel adhesin.

Lipopolysaccharides (LPS), major virulence determinants in Gram–negative bacteria, are responsible for many pathophysiological responses and can elicit strong immune responses. In order to better understand the role of LPS in host–pathogen interactions and elucidate the immunogenic properties of LPS outer core oligosaccharide, an all α–linked Escherichia coli R3 outer core pentasaccharide was first synthesized with a propyl amino linker at the reducing end. This oligosaccharide was also covalently conjugated to a carrier protein (CRM197) via the reducing end propyl amino linker. An immunological analysis demonstrated that this glycoconjugate can elicit specific anti-pentasaccharide antibodies with in vitro bactericidal activity. These findings will contribute to further exploring this pentasaccharide antigen as a vaccine candidate. Human milk oligosaccharides (HMOs) are a family of diverse unconjugated glycans that exist in human milk as one of the major components. Characterization, quantification and biofunctional studies of HMOs remain a big challenge due to their diversity and complexity. The accessibility of homogenous HMOs library is essential to solve these issues which have beset academia for several decades. In this study, an efficient chemoenzymatic strategy, namely Core Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse HMOs was reported. Based on 3 versatile building blocks and 4 robust glycosyltransferases, a library of 31 HMOs were chemoenzymatically synthesized and characterized by MS and NMR. CSEE indeed provides a practical approach to harvest structurally defined HMOs for various applications. Glycoproteins are extremely important for all life on the planet. Glycoproteins play important roles in various biological processes. Increasing evidences demonstrate that glycosylation of proteins could improve stability of proteins by stabilizing their tertiary structure and protecting them from proteolysis. Besides, glycosylation of proteins could provide targeting effects through glycan-lectin interaction. Furthermore, carbohydrates play crucial roles in humoral immunity in that many sugar epitopes are identified as antigens for antibodies. Glycoprotein could boost strong T cells mediated intercellular immune responses because homogeneous antigens present on the surface of proteins by multivalent bonds. In this study, the three advantages of glycoproteins, namely stabilizing proteins, targeting effects and eliciting immunological response, were extensively explored by broad collaboration with other groups.

Effective communication in English between its two billion users (Crystal, 2008), requires comprehension of others’ English and a willingness to accept differences in English. While some studies have attempted to measure the attitudes of Inner Circle (IC) (Kachru, 1985) respondents towards IC Englishes, and other studies have focused on attitudes of Outer Circle (OC) and Expanding Circle (EC) respondents to IC English, there is a dearth of research on OC and EC respondents’ attitudes to non-IC English. Therefore, this study addressed the need for further research focusing on OC respondents’ attitudes to EC users’ English. Specifically, this study of 31 Singaporeans attempted to gain an understanding of their attitudes towards Expanding Circle Accents of English (ECAE). This study drew on direct and indirect approaches in language attitude research, involving a verbal-guise task using semantic differential scales to elicit attitudes to speakers on a range of solidarity and status traits, and interviews. Descriptive statistics derived from mean scores were used for quantitative analysis of the data from the verbal-guise task, while coding procedures were used for qualitative analysis of the interview data. The findings show the respondents displayed predominantly negative attitudes to eight of the eleven ECAE and slightly positive attitudes to three. Phonological features common to the ECAE, notably mispronunciation of particular phonemes and vowels added to consonant clusters, affected the respondents’ attitudes. Moreover, certain prosodic features and the perceived degree of attractiveness and assertiveness affected attitudes to the ECAE. These findings indicate accent can affect listeners’ attitude to speakers. The implications of this study have relevance to the discussions on World Englishes and English as an International Language to the extent that notions of attitude and intelligibility are central to both. Furthermore, the findings suggest attitude might be of greater significance than intelligibility when evaluating others’ English.

For various values of n and m we investigate homomorphisms from Out(F_n) to Out(F_m) and from Out(F_n) to GL_m(K), i.e. the free and linear representations of Out(F_n) respectively. By means of a series of arguments revolving around the representation theory of finite symmetric subgroups of Out(F_n) we prove that each homomorphism from Out(F_n) to GL_m(K) factors through the natural map p_n from Out(F_n) to GL(H_1(F_n,Z)) = GL_n(Z) whenever n=3, m < 7 and char(K) is not an element of {2,3}, and whenever n>5, m< n(n+1)/2 and char(K) is not an element of {2,3,...,n+1}. We also construct a new infinite family of linear representations of Out(F_n) (where n > 2), which do not factor through p_n. When n is odd these have the smallest dimension among all known representations of Out(F_n) with this property. Using the above results we establish that the image of every homomorphism from Out(F_n) to Out(F_m) is finite whenever n=3 and n < m < 6, and of cardinality at most 2 whenever n > 5 and n < m < n(n-1)/2. We further show that the image is finite when n(n-1)/2 -1 < m < n(n+1)/2. We also consider the structure of normal finite index subgroups of Out(F_n). If N is such then we prove that if the derived subgroup of the intersection of N with the Torelli subgroup T_n < Out(F_n) contains some term of the lower central series of T_n then the abelianisation of N is finite.

Whole-cell biocatalysts are preferred in many biocatalysis applications. However, cell envelope often represents a formidable permeability barrier. As a result, reactions catalyzed by whole-cells are reportedly orders of magnitude slower than those of by their free enzyme counterparts. The present research addresses this critical issue by using membrane engineering approaches. Two E. coli strains with genetically altered outer membrane structures were used in the study, a lipopolysaccarides (LPS) mutant SM101 and a Braun's lipoprotein mutant E609L. The effects of outer membrane mutation on the permeability of substrates differing substantially in size and hydrophobicity were investigated by combining the mutant cells with model enzymes. The reduction of the outer membrane permeability barrier by these mutations led to significant accelerations (2 to 14 fold) in reaction rates of all whole-cell catalyzed reactions investigated. In the case of tetrapeptide, LPS mutation of the outer membrane can render the outer membrane completely permeable to substrate, a barrier-less condition that maximizes the reaction rate. For reaction rates of toluene dioxygenase (TDO)-catalyzed reactions, a dramatic increase of up to six fold was observed with the lipoprotein mutant for each of the three small, hydrophobic substrates tested. Mutations in either the LPS or in the Braun's lipoprotein are effective for accelerating reactions with UDP-glucose, resulting in a striking acceleration (up to 14 fold) of reaction rate. The magnitude of reaction rate acceleration was found to be dependent upon the substrate concentrations, the enzyme expression level, and on the nature of the mutations and substrates. In addition, the mutations have been demonstrated to be far more superior to common permeabilization procedures like freeze-thaw (FT) or treatment with the chelating agent EDTA (ethylene diamine tetraacetic acid). Importantly, lipoprotein mutant E609L exhibited a normal growth rate and expressed the recombinant multi-component enzyme as well as the isogenic parent. The exact nature of lpp lipoprotein mutation in E609L was further studied and deletion of lpp was successfully introduced into E. coli strain with different genetic background for whole-cell biocatalysis applications. An example was provided by introducing an lpp deletion into an E. coli O44K74 strain to achieve a higher yield for L-carnitine production. This research and the results outlined in this dissertation demonstrate a valid strategy for addressing permeability issues in whole-cell biocatalysis. The work also highlights a need for accessing substrate permeabilities in biocatalysis research and development.

This thesis reports the characterisation of a novel outer membrane protein (OMP) from M. catarrhalis, designated M35, with a molecular mass of 36.1 kDa. This protein is structurally homologous to classic Gram-negative porins, such as OMP C from E. coli and OMP K36 from K. pneumoniae, with a predicted structure of 8 surface loops connecting 16 antiparallel -sheets. Comparison of the DNA sequences of the M35 genes from 18 diverse clinical isolates showed that the gene was highly conserved (99.6-100 % of nucleotides) with only one isolate (ID78LN266) having base variations that resulted in amino acid substitutions. A single amino acid mutation in the 3rd external loop of M35 in isolate ID78LN266 significantly affected antibody recognition, indicating that loop 3 contains an immunodominant B-cell epitope. The reduction in antibody-binding to M35 from ID78LN266 was similar to that caused by complete removal of loop 3. Since loop 3 folds into the porin channel in the classic structure, the antibody specificity to loop 3 was hypothesised to be a potential mechanism for evasion of host immune responses targeted to M35, potentially explaining the high degree of conservation across isolates. A series of recombinant proteins were constructed to analyse the binding to M35 of antibodies specificity for loop 3 or the remainder of the protein. It was found that loop 3- specific antibodies were not able to bind to M35 on the surface of M. catarrhalis and that this corresponds both with a lack of ability to enhance opsonophagocytosis in vitro and bacterial clearance in vivo. Additionally, antibodies raised against a version of M35 lacking loop 3 and M35 from the variant isolate ID78LN266 were both no less effective than the full consensus M35 by both these measures. It therefore appears that while the majority of antibodies raised against M35 are specific for loop 3 these antibodies do not mediate anti-M. catarrhalis actions. Two deletion mutant strains of M. catarrhalis that do not contain the outer membrane protein M35 were created by insertional inactivation of the M35 gene. Growth comparisons between these mutant strains and their wildtype parent strains initially led to the hypothesis that M35 is necessary for efficient glutamic acid uptake by M. catarrhalis, however this hypothesis was later shown to be incorrect. Efficient uptake of glutamic acid seemed to be mediated by a novel 40 kDa protein that was up-regulated in the deletion mutant strains, presumably to compensate for the lack of M35. M35 was also found to be essential for in vivo survival of M. catarrhalis in the nasal cavities of mice, indicating that it is an essential functional protein for colonisation of the mucosal surface.

Heterogeneity in immunodominant outer membrane proteins has been proposed as a significant factor in the failure of an NTHi infection to induce immune protection against subsequent infections. This study has examined the vaccine potential of three outer membrane proteins in an attempt to identify conserved regions that could be targeted by an immune response after vaccination. The three proteins investigated were: TbpB, P5 and P48 (HI0164). The optimal route of immunisation in clearing a bolus inoculum of NTHi to the lung in the rat has been shown to be a combination of gut sensitisation with a respiratory boost and this regime was used in the present study. A panel of NTHi isolates was assessed to determine the frequency with which strains were able to bind transferrin and thus be targeted by a TbpBspecific immune response. A high proportion of strains was able to bind transferrin with similar frequencies in isolates associated with infection and those from normal throat swabs. A protocol was developed to purify nonlipidated recombinant TbpB from NTHi using a glutathione-Stransferase (GST)-rTbpB fusion protein and Glutathione-Sepharose affinity chromatography. Mucosal-directed immunisation with rTbpB significantly enhanced clearance of an NTHi challenge to the lung, however, whilst rTbpB-specific antibodies were cross-reactive on Western immunoblots, the cross-reactivity was variable in both transferrin binding inhibition assays and bactericidal activity. This suggested that the rTbpB-specific humoral response would be variable in the recognition of heterologous NTHi isolates. The secondary structure of P5 has been controversial with several reports suggesting that P5 was a fimbrin protein composed of coiled coils. In this present study the interstrain variation in P5 amongst isolates from diverse anatomical sites, as well as computer prediction methods and spectrophotometric analysis, generated a model of P5 based on the homologous E. coli protein, OmpA. This model suggested a B-barrel conformation with no evidence of coiled coils. Synthetic peptides corresponding to conserved regions of P5 that were thought to be surface exposed, as well as a region (H3) with some homology to a protective epitope in the P. aeruginosa protein, OprF, were then combined with a "promiscuous" T cell epitope from the measles virus F protein (MVF) and used for immunisation studies. Whilst variable protection was seen with the peptides, the MVF/H3 peptide was the most efficacious of the antigens assessed in this study in enhancing clearance of NTHi. This occurred in the absence of detectable peptide- or PS-specific antibody leading to the suggestion that cell mediated responses may have played an important role in enhancing clearance in this model. The highly conserved nature of the region in P5 represented by the H3 peptide suggests that further study should be focused on this peptide as a potential NTHi vaccine candidate. The last antigen, P48, is homologous to a A. pleuropneumoniae antigen, AopA, which has been proposed to have potential as a vaccine component against pleuropneumonia in pigs. Sequence analysis of the gene encoding P48 from several isolates showed that this protein was well conserved. Recombinant P48 was purified from a GST-rP48 fusion protein and used for immunisation, which also conferred significant protection. However, immunisation with rP48 was not as efficacious as immunisation with the MVF/H3 peptide. Whilst immunisation with rP48 induced high antibody titres, no bactericidal activity could be detected indicating that bactericidal antibody had not contributed to the observed clearance. In addition, the rP48- specific serum IgG was predominantly of the IgG2a isotype suggesting that Thl cell mediated responses had been induced by immunisation with rP48.

The outer membrane protein OmpA is one of the major outer membrane proteins in many species of bacteria, including the Yersiniae. Our goal was to explore the role of OmpA in Y. pestis. This encompasses the ability of Yersinia to infect and survive within macrophages, as well as to resist antimicrobial compounds. Our laboratory found that a delta ompA mutant is impaired in a macrophage-associated infectivity assay. We also found that OmpA might play a role in the ability of the bacteria to resist antimicrobial peptides, specifically polymyxin B. Aditionally, we assessed the differences in OmpA of Y. pestis and E. coli, and determined that the characteristics we have observed in Y. pestis are unique compared to what has previously been described in E. coli. Our results indicate that Y. pestis OmpA might act through known pathways of antimicrobial resistance such as the PhoPQ two-component regulatory system, although further experiments are needed to determine the precise mechanism of function OmpA. Overall, our project characterizes the different functions of OmpA in Y. pestis, both as a key player in intracellular survival and as a necessary component in conferring resistance to antimicrobial peptides.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 77-93).