The Shigella species are Gram-negative enteropathogens that produce severe diarrhea, cramping, and dehydration in millions of people annually. The pathogens most commonly infect children under the age of 5 years in developing nations, where the rise of multidrug-resistant species is increasingly problematic. Despite several attempts to develop a vaccine against these pathogens, no successful vaccine has been produced. In order to achieve this goal, several characteristics of Shigella must be further elucidated. Namely, we must better understand the mechanisms Shigella employs in order to circumvent the immune response. A key way in which Shigella circumvents the innate defenses of the host is through resistance to bile salts, the principal component of bile, a substance found in the small intestine that is required for digestion. One such bile salt resistance mechanism of Shigella involves lipopolysaccharide (LPS), an extracellular structure composed of three regions: a transmembrane lipid, a polysaccharide core, and an O-antigen. LPS and LPS modifications have been implicated in bile salts resistance in other enteropathogens. Thus, the goal of this study was to build from preliminary findings to understand the role of LPS in conferring bile salts resistance in Shigella. Two Shigella flexneri mutants were studied to understand the roles of the polysaccharide core and O-antigen on bacterial growth and LPS modifications during exposure to bile salts. Growth comparisons of the mutants relative to wild type bacteria in the presence of bile salts were performed, including analysis of growth with exposure to bile salts and with varying levels of environmental glucose. Additionally, LPS was extracted from wild type and mutant bacteria grown in these conditions for analysis by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The growth curves demonstrated that both the O-antigen and polysaccharide core mutants exhibited slow growth with exposure to bile salts, while the SDS-PAGE analyses revealed changes in the LPS profile of wild type and both LPS mutants when grown in bile salts. These data indicate that the O-antigen likely has an important role in conferring bile salts resistance and that the polysaccharide core may also facilitate resistance. This study allows us to better understand how LPS contributes to bile salts resistance in S. flexneri, which may enhance efforts to develop an effective vaccine against this pathogen. / 2023-12-10T00:00:00Z

This study evaluates the ecology of Streptococcus pneumoniae (SP) nasopharynx (NP) colonization in response to the pneumococcal conjugate vaccines, specifically 7-Valent Pneumococcal Conjugate Vaccine (2000-2009), 13-Valent Pneumococcal Conjugate Vaccine (2010-2023) and 20-Valent Pneumococcal Conjugate Vaccine (2023-future date). It is anticipated that the replacement of PCV13 with PCV20, a pneumococcal conjugate vaccine with 7 additional polysaccharide conjugates to CRM197 will enhance the protection against non-vaccine serotypes which are in circulation in communities. The project will evaluate the dynamic changes in pneumococcal colonization over the 5-year time line from 2021-2026. Pneumococcal nasopharynx colonization is detected through nasopharyngeal culture and molecular techniques. The primary source of pneumococcal transmission occurs among the pediatric population and between children and adults. The impact of PCV7 and 13 on pneumococcal colonization over the prior 20 years created a herd effect that resulted in a reduction in pneumococcal disease in unimmunized children and adults. Studies of NP colonization has led to a deeper understanding of pneumococcal conjugate vaccine (PCV) effectiveness and the role of herd immunity in protecting the population, the emergence of replacement serotypes, the variation in invasive capability of each serotype and evolution of antimicrobial resistance resulting from the evolving ecology. In this 5-year-study, researchers at the Pelton Lab in Boston Medical Center set out to understand the prevalence of NP carriage of 13vPnC serotypes, the 7 unique 20vPnC serotypes and NVST (non-vaccine serotypes) within the pediatric population prior to and subsequent to the introduction of PCV 20 (Fall 2023).

The objectives of this study were to carry out research to better understand of the formation, stability and properties of multilayer emulsions containing nano-laminated biopolymer coatings, and to utilize this information to develop food-grade delivery systems. The effect of various preparation parameters on the formation and stability of multilayer emulsions was investigated: droplet concentration; mean droplet diameter; droplet charge; biopolymer concentration. β-lactoglobulin (β-Lg) stabilized emulsions (0.5 – 10 wt% oil) containing different pectin concentrations (0 to 0.5 wt%) were prepared at pH 7 (where lipid droplets and pectin molecules were both anionic) and pH 3.5 (where lipid droplets were cationic and pectin molecules anionic) and “stability maps” were constructed. At pH 3.5, pectin adsorbed to the droplet surfaces, and the emulsions were unstable to bridging flocculation at intermediate pectin concentrations and unstable to depletion flocculation at high pectin concentrations. At certain droplet and pectin concentrations stable multilayer emulsions could be formed consisting of protein-coated lipid droplets surrounded by a pectin layer. An in situ electro-acoustic (EA) technique was introduced to monitor the adsorption of charged polysaccharides onto oppositely charged protein-coated lipid droplets. The possibility of controlling interfacial and functional characteristics of multilayer emulsions by using mixed polysaccharides (pectin/carrageenan or pectin/gum arabic) was then examined. Emulsions containing different types of polysaccharides had different interfacial characteristics and aggregation stabilities: carrageenan had the highest charge density and affinity for the protein-coated lipid droplets, but gave the poorest emulsion stability. The possibility of assembling protein-rich coatings around lipid droplets was examined using the electrostatic deposition method, with the aim of producing emulsions with novel functionality. Protein-rich biopolymer coatings consisting of β-Lg and pectin were formed around lipid droplets using the electrostatic deposition method. The composite particles formed had relatively small diameters (d < 500 nm) and were stable to gravitational separation. They also remained stable after they were heated above the thermal denaturation temperature of the globular protein and had better stability to aggregation at high salt concentrations (50 – 200 mM NaCl) than conventional emulsions stabilized by only protein. The effect of a polysaccharide coating on the displacement of adsorbed globular proteins by non-ionic surfactants from lipid droplet surfaces was examined to simulate situations where competitive adsorption occurs. Oil-in-water emulsions stabilized by β- Lg were prepared containing either no pectin (1º emulsions) or different amounts of pectin (2º emulsions). At pH 3.5, where pectin forms a coating around the β-Lg stabilized lipid droplets, the amount of desorbed protein was much less for the 2º emulsion (3%) than for the 1º emulsion (39%), which indicated that the pectin coating inhibited protein desorption by surface active agents. Knowledge gained from this research will provide guidelines for rationally designing emulsion-based delivery systems that are resistant to environmental stresses or with controlled release properties. These delivery systems could be used to encapsulate, protect and release functional components in various industrial products, such as foods, pharmaceuticals, cosmetics, and personal care products.

Thesis advisor: Jia Niu / Thesis advisor: Jim Morken / Polysaccharides make up one of the largest classes of nature’s macromolecules. However, they are severely understudied relative to other biomolecules such as proteins and DNA sequences. This is because discrete polysaccharides are difficult to isolate from nature or synthesize in laboratories in large enough quantities for thorough research. Polymerization is an efficient route to polysaccharides, yet has historically suffered from harsh conditions and lack of control. Herein, we investigate recent developments in the field of living polymerization as strategies towards synthesis of precision polysaccharides from 1,2- anhydrosugars. We specifically focus on cationic ring opening polymerization (ROP) and reversible addition-fragmentation chain transfer (RAFT) ROP polymerization of 1,2-O-Bn-3,4,6-anhydromannose and 1,2-O-Bn-3,4,6-anhydroglucose. Our research screens various catalyst/initiating systems. Our findings demonstrate that cationic ROP and RAFT polymerization are unsuccessful in the living ROP of 1,2-anhydrosugars. / Thesis (MS) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

This research presents a critical evaluation of chitin- and chitosan-based materials as innovative treatment alternatives for water contaminated with insensitive munitions (IMs) compounds. Specifically, chitin, chitosan, amineunctionalized chitin (AFC) were evaluated for adsorptive removal of these compounds. Cellulose and cellulose triacetate were evaluated for adsorptive performance for comparison. Chitosan-graphene oxide (CSGO) composite membranes were evaluated for removal via adsorption and filtration and compared against nanofiltration and reverse osmosis membranes in the current market. Insensitive munitions evaluated include nitrotriazolone (NTO), nitroguanidine (NQ), and 2,4-dinitroanisole (DNAN); 2,4,6-trinitrotoluene (TNT) was also studied as a traditional munition for comparison. AFC is an effective adsorbent for NTO, DNAN, and TNT. Cellulose triacetate was the only commercially available biopolymer adsorbent effective at removing munitions compounds from solution; only DNAN and TNT were removed. CSGO membranes effectively removed NTO, DNAN, and TNT, but removal performance degraded with time. Overall, this research shows that the materials studied are viable options for removing IM and traditional munitions from water.

No description available.

No description available.

Glycosaminoglycans (GAGs), a large family of complex, unbranched polysaccharides, display a variety of essential physiological functions. The structural complexity of GAGs greatly impedes their availability, thus making it difficult to understand the biological roles of GAGs and structure-property relationships. A method that can access GAGs and their analogs with defined structure at relatively large scales will facilitate our understandings of GAG biological roles and biosynthesis modulation. Cellulose is an abundant and renewable natural polymer. Applications of cellulose and cellulose derivatives have drawn increasing attention in recent decades. Chemical modification is an efficient method to append new functionalities to the cellulose backbones. This dissertation describes chemical modification of cellulose and cellulose derivatives to prepare unsulfated and sulfated GAG analogs. Through these studies, we have also discovered novel chemical reactions to modify cellulose. Systematic study of these novel chemistries is also included in this dissertation. We first demonstrated our preparation of two unsulfated GAG analogs by chemical modification of a commercially available cellulose ester. Cellulose acetate was first brominated, followed by azide displacement to introduce azides as the GAG amine precursors. The resulting 6-N3 cellulose acetate was then saponified to liberate 6-OH groups, followed by subsequent (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation of the liberated primary hydroxyl groups to carboxyl groups. Finally, the azides were reduced to amines using a novel reducing reagent, dithiothreitol (DTT). Alternatively, another process utilized thioacetic acid to reduce azides to a mixture of amine and acetamido groups. Through pursuing these GAG analogs, we applied novel azide reductions by DTT and thioacetic acid that are new to polysaccharide chemistry. We systematically investigated the scope of DTT and thioacetic acid azide reduction chemistry under different conditions, substrates, and functional group tolerance. Selective chlorination is another interesting reaction we discovered in functionalization of cellulose esters. We applied this chlorination reaction to hydroxyethyl cellulose (HEC). We then utilized the chlorinated HEC as a substrate for displacement reactions with different types of model nucleophiles to demonstrate the scope of its utility. Overall, we have designed a novel synthetic route to two unsulfated GAG analogs by chemical modification of cellulose acetate. Through exploration of GAG analogs synthesis, we discovered novel methods to modify polysaccharide and polysaccharide derivatives, including azide reduction chemistry and selective chlorination reactions. Successful synthesis of various types of GAG analogs will have great potential biomedical applications and facilitate structure-activity relationship studies. / Doctor of Philosophy / Polysaccharides are long chains of natural sugars. Glycosaminoglycans (GAGs) are an important class of polysaccharides which have complicated chemical structures and play critical roles in many biological processes, including regulation of cell growth, promotion of cell adhesion, anticoagulation, and wound repair. Current methods to obtain these GAGs and GAG analogs are expensive, lengthy, and limited in capability. Novel methods to access these GAGs and their analogs would be promising and would facilitate understanding of biological activities of GAGs. Cellulose is an abundant polymer on earth and provides structural reinforcement in plant cell walls. Cellulose can be further chemically modified to tailor its physiochemical properties. Cellulose and cellulose derivatives have been widely used in many industries for various applications, such as textiles, plastic films, automotive coatings, and drug formulation. This dissertation focuses on modifying inexpensive, abundant cellulose and its derivatives to GAGs and GAG analogs. We start from the simple plant polysaccharide cellulose and obtain structurally complicated analogs of animal-sourced GAGs and GAG analogs. We reached our goal by designing a carefully crafted synthetic route, finally successfully obtaining two types of novel GAG analogs. During this process, we discovered two useful chemical reactions. We systematically investigated these chemical reactions and demonstrated their utility for polysaccharide chemical modification. These successful chemical syntheses of GAGs and their analogs will accelerate our understanding of their natural functions and have potential biomedical applications. The novel chemical methods we discovered will be helpful in chemical modification of polysaccharides.

Sludge dewatering and chemical conditioning requirements were examined from the perspective of biopolymer and cation release from activated sludge flocs. Both aerobic and anaerobic digestion processes were considered from two different activated sludge sources at a temperature of 20° C. Polymer demand and specific resistance to filtration increased with an increase in total soluble biopolymer concentration for all temperature ranges. In anaerobic digestion, the protein release was three times greater than the polysaccharide release. Conversely, aerobic digestion of the same sludge resulted in a greater release of polysaccharides than proteins. Polymer conditioning requirements in the anaerobic digestors were an order of magnitude higher than in the aerobic digestors; proteins were considered to be the biopolymer fraction responsible for the high polymer conditioning requirements and poor dewatering properties. Biopolymer is released to the supernatant as colloids bound by divalent cations. Peptidase and glucosidase activity were used to monitor enzymatic activity relative to biopolymer release and degradation. The reasons for the increases and decreases in hydrolase activity are unknown. / Master of Science

The biomass from a pilot-scale two-stage (carbon oxidation first stage, ammonia oxidation second stage) fixed-film biological aerated filter (BAF) was divided in three fractions depending on their attachment strength to the media: detached, easily detachable and strongly attached. VSS measurement showed that the detached and easily detachable fractions accounted for 25 to 40% of the biomass in the bed and are present even after backwash. Protein was the major constituent of all fractions of the biomass. The ratio of carbohydrate to protein differed between fractions and between type of biofilms, with a larger value for detached and detachable fractions and a lower value for a largely heterotrophic biofilm, implying a difference in the composition of the biomass matrix that could be related to the attachment state of the biomass. The biomass did not appear to be substrate-limited anywhere in the system, although the specific activity of the biomass was dependent upon the position in the column. Activity of the strongly attached biomass was less than 70% of the total activity, even after backwash. A mass balance on VSS showed that the backwash flushed a mass equivalent to less than 35% to 45% of the detached and detachable fractions, which was less than 15% of the total biomass present in the system. Data also suggested that during backwash, part of the strongly attached biomass was sheared off the media and regenerated the mass of biomass in the detached phase. In conclusion, it can be stated that a non-negligible part of the biomass in a BAF is in a detached state. Actual mechanistic BAF models based solely on biofilm modeling may be overlooking the role of that biomass, especially in the performance recovery of BAF systems after backwash. / Master of Science