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BACKGROUND: Vibrio cholerae and Enterotoxigenic E. Coli (ETEC) are enteropathogens that are global causes of cholera and traveler’s diarrhea which are responsible for millions of diarrhea cases every year. ETEC and cholera are primarily found in Sub-Saharan Africa and Asia, particularly in nations with inadequate sanitation systems or little access to clean water. Infants and children are most vulnerable to these diseases, as severe infections can lead to stunting and death. The incidence of cholera and ETEC diarrhea have increased, due in part to changing weather patterns. At present, robust animal models for studies of ETEC colonization are lacking to study colonization and bottlenecks. The only licensed vaccines against cholera in endemic countries are killed whole cells, however, new live attenuated oral cholera vaccines (OCV) are in development and offer significant advantages. PanChol is a live attenuated OCV entering phase I trials. SPECIFIC AIMS: To propel studies of ETEC pathogenesis, I attempted to create a suckling mouse model of this globally important pathogen. To accomplish this goal, I constructed barcoded ETEC libraries that enabled me to determine founding population sizes along with intestinal ETEC burdens. To better understand PanChol, a new live attenuated OCV, I studied the shedding of the vaccine in the first 3 human volunteers to ingest this novel agent. METHODS: Triparental mating of donor strains MFDλpir pJMP1039 and MFDλpir pSM1 with recipient ETEC strains enabled construction of barcoded libraries. Neonatal CD-1 and C57BL/6 mice were infected with 104-107 CFU of wild-type ETEC to develop an infant mouse model. Founding population sizes of ETEC strains were compared via sequencing and STAMPR analysis while CFU burdens were determined via plating. Shedding of PanChol was done through enumeration of serial dilutions of fecal samples. Serotyping of shed PanChol was carried out using anti-Ogawa and anti-Inaba antisera. RESULTS: There were marked differences in ETEC small intestinal colonization in different mouse strains. Outbred CD-1 suckling mice only colonized with a 107 dose. In contrast, colonization of ETEC was approximately 106 CFU/small intestine at inocula sizes of 105 or greater in C57BL/6 mice. Laboratory studies using simulated bottlenecks made by serial dilutions established that the barcoded libraries accurately reflect founding population sizes up to 105 CFU. There was no difference in founding population sizes at the same inoculum size between WT ETEC and a hypervesiculation ∆mlaE mutant, though the founding population size increased with increasing input. PanChol retained the Hikojima serotype and shedding occurred in all volunteers with maximum colonization occurring 3 days post administration of 106 CFU. CONCLUSIONS: C57BL/6 P5 mice can serve as a new model to study ETEC intestinal colonization. Hypervesiculating ETEC did not produce a difference in founding population or colonization at the same input as WT ETEC strains. PanChol shows great promise as a viable OCV with shedding at 106 input and no serotype reversion.

Uma das estratégias mais promissoras para a biotecnologia de vacinas é o desenvolvimento de linhagens precisamente atenuadas, e que possam ser usadas como carregadoras de antígenos heterólogos. Mutantes de <i}>Salmonella Typhimurium têm sido extensivamente utilizados com essa fmalidade. A flagelina, monômero constituinte do filamento flagelar, vem sendo empregada como carregadora de antígenos heterólogos, inseridos na região central, hipervariável (região IV). Inserções nessa região são freqüentemente funcionais, e levam à exposição do epitopo na superfície do filamento. O presente trabalho explora o potencial de outras regiões da molécula para a inserção de epitopos. Nós inserimos a mesma seqüência usada anteriomente (epitopo da proteína M de S. pyogenes, Tipo 5) em regiões com diferentes níveis de homologia (III e VI), e em região totalmente conservada (VIII). Também foram feitas inserções duplas em regiões que se mostraram toleráveis (III e IV; IV e VI). Todas as proteínas híbridas foram sintetizadas pela Salmonella, como demonstrado em imunoblots, usando anticorpo contra a flagelina e contra o peptídeo. Todas as regiões, exceto a VIII, aceitaram a inserção sem perda de motilidade, apesar de, em alguns casos, ela ter sido extremamente reduzida. A imunogenicidade foi avaliada pela imunização de camundongos com bactérias vivas, inativadas ou, quando possível, flagelina purificada. Os resultados foram similares aos descritos na literatura para inserções envolvendo a região IV, obtendo-se um elevado título de anticorpos contra flagelina. Um baixo nível de anticorpo contra o peptídeo também foi detectado para todas as novas linhagens testadas. Nossos resultados com imunização de bactérias vivas sugerem uma resposta levemente melhor ao peptídeo quando duas cópias estão presentes, mas os dados não são conclusivos. / One of the most promising strategies for the biotechnology of vaccines is the development of precisely attenuated strains, which could be used as carriers of heterologous antigens. Mutants of Salmonella Typhimurium have been extensively explored to this effect, since the infection ofmice by S. Typhimurium mimics the infection of humans by S. Typhi, and the genetics of the species is extremely well known, making it easy the obtention of defined mutants with reduced pathogenicity. Mutants with auxotrofy in genes of the aromatic pathway are particularly attractive, since they need PABA and DHB to grow, and these compounds are unavailable in mammalian tissues. Flagellin, the monomer which constitutes the flagellar filament, has been used as a carrier for heterologous epitopes, inserted in a central, hypervariable region (region IV). Insertions in this region are often functional, and lead to exposition of the epitope at the filament\' s surface. The present work explored the potential of the other regions ofthe molecule for the insertion of epitopes. We inserted the same reporter sequence (MS epitope from S. pyogenes M protein) in regions with different levels of homology (III and VI), and totally conserved (VIII). We also made double insertions in regions shown to be permissive (III and IV; IV and VI). All hybrid proteins were synthesized by Salmonella, as demonstrated by immunoblots using antibody against flagellin and against the synthetic peptide. All regions, except the highly conserved region VIII, accepted the insertions without loss of motility, albeit, in some cases, motility was seriously reduced. Immunogenicity of the hydrids was evaluated by immunization with live bacteria, killed bacteria, and purified flagellin (when possible). Results obtained with the new constructs were similar to the ones published for insertions involving region IV, in the sense that antibody titers to the carrier protein were very high. A low level of antibody to the inserted peptide was also detected in all groups of animals. Our results with live immunization suggest a slightly better response to the peptide when two copies are present, but the data are not conclusive.

Production of and demand for Canadian canola meal have been increased yearly. In order to improve the competitiveness of canola meal domestically and internationally, as well as to develop potential markets for canola meal, it is necessary to develop canola meal-based products that have high feed values and can be easily transported. The objectives of this research were: 1) to investigate the effects of temperature and time of conditioning during pelleting process on the nutritive values of canola meal in terms of chemical profiles, protein and carbohydrate subfractions, and energy values, using the AOAC procedures, CNCPS v6.1 and NRC (2001), respectively; 2) to detect the effects of temperature and time of conditioning during the pelleting process on rumen degradation and intestinal digestion characteristics and predicted protein supply of canola meal, using the in situ procedure, the three-step in vitro procedure, and the NRC 2001 model; and 3) to determine pelleting-induced changes in spectral characteristics of molecular structures of canola meal using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) with univariate and multivariate analysis, and reveal the relationship between molecular structures of protein and carbohydrate and nutrient values, rumen degradation and intestinal digestion characteristics, and predicted protein supply of canola meal. Three different conditioning temperatures (70, 80 and 90ºC) and two different conditioning time (50 and 75 sec) were applied in this research. Two different batches of canola meal from a commercial feed company were selected. A randomized complete block design (RCBD) with 3 × 2 factorial arrangement was employed in this research. Molecular spectral functional groups related to protein, cellulosic compounds, and carbohydrates were used in the spectral study. This research indicated: 1) soluble crude protein (SCP) was decreased and neutral detergent insoluble CP (NDICP) was increased with increasing temperature; 2) the lowest protein rumen degradation of pellets was observed at conditioning temperature of 90 ºC and protein rumen degradation was increased by pelleting; 3) the amount of protein digested in the small intestine tended to increase with increasing conditioning temperature; 4) pelleting under different temperatures and time in the current study shifted the protein digestion site to the rumen, rather than to the small intestine; 5) with respect to predicted protein supply, based on the NRC 2001 model, increasing conditioning temperature tended to increase the metabolizable protein supply of canola meal pellets to dairy cattle; 6) changes in the molecular structure of canola meal induced by pelleting can be detected by ATR-FTIR; 7) not only protein molecular structure characteristics but also carbohydrate molecular structure characteristics play important roles in determining nutrient values, rumen degradation and intestinal digestion characteristics, and the predicted protein supply of canola meal.

Uma das estratégias mais promissoras para a biotecnologia de vacinas é o desenvolvimento de linhagens precisamente atenuadas, e que possam ser usadas como carregadoras de antígenos heterólogos. Mutantes de <i}>Salmonella Typhimurium têm sido extensivamente utilizados com essa fmalidade. A flagelina, monômero constituinte do filamento flagelar, vem sendo empregada como carregadora de antígenos heterólogos, inseridos na região central, hipervariável (região IV). Inserções nessa região são freqüentemente funcionais, e levam à exposição do epitopo na superfície do filamento. O presente trabalho explora o potencial de outras regiões da molécula para a inserção de epitopos. Nós inserimos a mesma seqüência usada anteriomente (epitopo da proteína M de S. pyogenes, Tipo 5) em regiões com diferentes níveis de homologia (III e VI), e em região totalmente conservada (VIII). Também foram feitas inserções duplas em regiões que se mostraram toleráveis (III e IV; IV e VI). Todas as proteínas híbridas foram sintetizadas pela Salmonella, como demonstrado em imunoblots, usando anticorpo contra a flagelina e contra o peptídeo. Todas as regiões, exceto a VIII, aceitaram a inserção sem perda de motilidade, apesar de, em alguns casos, ela ter sido extremamente reduzida. A imunogenicidade foi avaliada pela imunização de camundongos com bactérias vivas, inativadas ou, quando possível, flagelina purificada. Os resultados foram similares aos descritos na literatura para inserções envolvendo a região IV, obtendo-se um elevado título de anticorpos contra flagelina. Um baixo nível de anticorpo contra o peptídeo também foi detectado para todas as novas linhagens testadas. Nossos resultados com imunização de bactérias vivas sugerem uma resposta levemente melhor ao peptídeo quando duas cópias estão presentes, mas os dados não são conclusivos. / One of the most promising strategies for the biotechnology of vaccines is the development of precisely attenuated strains, which could be used as carriers of heterologous antigens. Mutants of Salmonella Typhimurium have been extensively explored to this effect, since the infection ofmice by S. Typhimurium mimics the infection of humans by S. Typhi, and the genetics of the species is extremely well known, making it easy the obtention of defined mutants with reduced pathogenicity. Mutants with auxotrofy in genes of the aromatic pathway are particularly attractive, since they need PABA and DHB to grow, and these compounds are unavailable in mammalian tissues. Flagellin, the monomer which constitutes the flagellar filament, has been used as a carrier for heterologous epitopes, inserted in a central, hypervariable region (region IV). Insertions in this region are often functional, and lead to exposition of the epitope at the filament\' s surface. The present work explored the potential of the other regions ofthe molecule for the insertion of epitopes. We inserted the same reporter sequence (MS epitope from S. pyogenes M protein) in regions with different levels of homology (III and VI), and totally conserved (VIII). We also made double insertions in regions shown to be permissive (III and IV; IV and VI). All hybrid proteins were synthesized by Salmonella, as demonstrated by immunoblots using antibody against flagellin and against the synthetic peptide. All regions, except the highly conserved region VIII, accepted the insertions without loss of motility, albeit, in some cases, motility was seriously reduced. Immunogenicity of the hydrids was evaluated by immunization with live bacteria, killed bacteria, and purified flagellin (when possible). Results obtained with the new constructs were similar to the ones published for insertions involving region IV, in the sense that antibody titers to the carrier protein were very high. A low level of antibody to the inserted peptide was also detected in all groups of animals. Our results with live immunization suggest a slightly better response to the peptide when two copies are present, but the data are not conclusive.

Independent control of the surface and bulk properties is advantageous for many applications such as adhesives, release coatings, and antimicrobial films. Traditional methods for achieving independent control typically require multiple processing steps such as wet-on-wet or wet-on-dry coating methods. Independent control over the surface properties can achieved in a single step utilizing the temporal and spatial control inherent to photopolymerization. Specifically, a co-photopolymerization of monomers with different reactivities in the presence of a light gradient is capable of producing a polymer film with a surface chemistry that differs from the bulk chemistry. The light gradient, produced via the concentration of photoinitiator in the formulation, results in a reaction gradient through the film with the higher rates of reaction occurring in the high light intensity regions of the film. The preferentially reacting monomer adds at a greater rate in the high light intensity regions resulting in non-uniform consumption yielding a concentration gradient. Consequently, diffusion of the preferentially reacting monomer from the bulk to the surface of the film and a counter-diffusion of the other monomer from the surface to the bulk of the film occurs from the non-uniform monomer consumption thus producing a film with a concentration gradient through the depth of the film with the preferentially reacting monomer enriching the high light intensity regions. A variety of kinetic differences capable of producing a stratified film will be presented including inherent monomer reactivity, number of functional groups per monomer, oxygen inhibition, thiol-ene chemistry, and Norrish type two initiation. Additionally, parameters that control the degree of stratification, such as methods of varying polymerization rate and the light gradient, will be examined. Changes in surface properties (such as contact angle, surface hardness, adhesion) and bulk properties (such as mechanical properties measured by dynamic mechanical analysis and polymer swelling) are studied as a function of stratification. Finally, a mathematical model which describes and predicts the production of stratified films via photopolymerization is presented. Photopolymerization allows for a facile, single step method of generating stratified films with controllable surface chemistries.