ABSTRACT Objectives: Flowable composites are characterized by lower filler loading and a greater proportion of diluent monomers in their formulation. These composites were traditionally created by retaining the same small particle size of the conventional hybrid composites, but reducing the filler content and allowing the increased resin to reduce the viscosity of the mixture However, their various mechanical properties such as flexural strength and wear resistance have been reported to be generally inferior compared to those of the conventional composites. Dental restorative materials are in continuous contact with fluids and saliva in the patient’s mouth. Consequently, the water sorption and solubility of these materials are of considerable importance. Resin based materials demonstrate water sorption in the oral cavity, which is the amount of water absorbed by the material on the surface and into the body while the restoration is in service. The water intrusion in the dental material can lead in a deterioration of the physical/mechanical properties, decreasing the life of resin composites. Water uptake can promote breakdown causing a filler-matrix debonding. Water sorption affects the physical and mechanical properties of resin composite such as dimensional change, decrease in surface hardness and wear resistance, filler leaching, change in color stability, reduction in elastic modulus, and an increase in creep and a reduction in ultimate strength, fracture strength, fracture toughness, and flexural strength. In addition, penetration of water into the composite may cause release of unreacted monomers (solubility) which may stimulate the growth of bacteria and promote allergic reactions. The effect of water sorption on conventional composites has been extensively studied and reviewed in the dental literature. However , there are no published studies on the water sorption of flowable composites. Water sorption increases as the amount of resin matrix increases and filler content decreases, since the filler particles do not absorb water. Thus, it is of utmost importance to study the water sorption of flowable composite. Hence the aim of this study was to evaluate and compare water sorption and solubility values of different light-activated flowable composite materials in solutions with varying pH values. And, since water filled porosities in the flowable composites may form small incubation chambers, a second related objective was to compare and correlate water sorption values of the various flowables to their ability to form Streptococcus mutans and Streptococcus sanguis single species biofilms in/on their surfaces. Methods: In this study, water sorption and solubility tests were performed according to the ISO standards (International Organization for Standardization specification 4049:07-2009- Dentistry- Polymer Based Restorative Materials [available at http://www.iso.org/iso/home/store.htm]). Three disc-shaped specimens of each flowable composite were made in a jig consisting of a Teflon mold (15 mm in diameter by 1 mm in thickness) compressed between 2 glass slabs with mylar strips used as separating sheets. The flowable resin was inserted in the Teflon mold in a single increment. All specimens were cured with a light-emitting diode curing unit. According to the ISO standard, discs were weighted every day for 35 days using the same balance, with a repeatability of 0.1 mg, until a constant mass (M1) was obtained. Once a constant M1 was obtained, the volume (V) was then calculated in cubic millimeters as follow: V =π(d/2)2h, where π=3.14; d is the mean diameter of the specimen; and h is the mean thickness of the specimen. After M1 was achieved, each flowable composite resin group of 3 discs was placed into buffers of pH = 4.0,5.5 and 7.0. After 24 hrs, specimens were wiped free of excess buffer with absorbent paper and weighed. This cycle was repeated at one week , one month, and six months. When a constant mass was achieved it was designated M2. Mass gain (Mg) was defined as follows: (M2 –M1). Per cent mass gain (%Mg) was defined as follows: (M2-M1/M1). Finally, the specimens were reconditioned to constant mass, once again following the above-mentioned procedure. This constant mass was recorded as M3. Water sorption (Wsp) was calculated in micrograms per cubic millimeter for each of the specimens by using the following equation provided by ISO 4049 standard: Wsp=(M2-M3)/V, where M2 is the mass of the specimens in micrograms after immersion in buffer for 30 days; M3 is the reconditioned mass of the specimen, in micrograms; and V is the volume of the specimen in cubic millimeters. Water solubility (Wsl) was calculated in micrograms per cubic millimeter for each of the specimens, using the following equation, provided by ISO 4049 standard: Wsl=(M1-M3)/V, where M1 is the conditioned mass of the specimen in micrograms before immersion in buffer; M3 is the reconditioned mass of each specimen in micrograms, and V is the volume of the specimen in cubic millimeters. For biofilm experiments, flowable discs were prepared as described above. Each disc was then sectioned into three equal portions using high speed and low speed handpieces , a diamond bur, and sandpaper discs, such that the three samples of each flowable had the same mass to within 0.3 mg. The samples were sterilized by dipping in 1.2% sodium hypochlorite (Chlorox), followed by rinsing with sterile distilled water, and then conditioning to a constant mass as described above, inside a desiccator that was wiped with 1.2 % Chlorox. Biofilm experiments were conducted as follows: three equal mass specimens of each flowable composite were placed in a series of wells of a sterile culture disc. Then sterile BHI broth (2 ml) was added to each well. One well served as control and no growing bacteria were added to it. To the other specimens was added 40 μl log phase S. mutans or S. sanguis cells. The culture dishes were then placed on a rotator at 37C for six hrs. Biofilm formation was measured by staining attached cells with crystal violet, destaining with 30% acetic acid, and measuring the satin spectrophotometically. Results: The pH of the solution influenced the % mass gain, as all samples gained more mass at pH 4.0 as compared to pH 5.5 and 7.0. The flowable resin SureFill showed the least % mass gain at each pH. However, there was no statistical difference in % mass gain based on pH of storage buffer for any of the flowable composites (P=.05) . Time had a significant influence on the % mass gain for the first week for all samples, with minor gains thereafter, and became steady after 1 month. Surefill showed the least water sorption when stored in buffer for 30 days, however it was not significant compared to the other flowables (P= 0.05). Filtek showed the least water solubility, but is not significant compared to the other flowables (P=0.05). The highest significant values (P< 0.05) for water sorption and solubility were observed for Virtuoso. Two trials indicated that strains of S. mutans and S. sanguis form biofilm readily on the surface of the composites, with S. sanguis having a higher predilection to form biofilm on all composites (Figure 6). However, no correlation was found between water sorption and solubility values of the flowable composites and biofilm formation. Conclusions: Within the limitations of this study the following is concluded: Time and storage conditions are important to the % mass gain due to water, with all flowable composites showing more mass gain at low PH. Due to its hydrophilic nature, as well as to the filler characteristics, the flowable composite Virtuoso exhibited significantly higher values of water sorption and water solubility than the other flowable composites that were tested. All flowable composites formed S. sanguis and S. mutans single species biofilm on their surfaces, with S. sanguis forming higher concentrations of biofilm on all samples. There was no clear correlation to water sorption and biofilm formation characteristics of the composites. / Oral Biology

Objectives: Cardiovascular disease (CVD), including heart attack, angina, and stroke, is ranked as the number one cause of mortality world wide. High blood cholesterol is linked to CVD and is an important risk factor. Statins – cholesterol lowering drugs- are first choice drugs for reducing the chance of suffering a CVD event. In the USA alone, approximately 32 million individuals take statins. Although randomized control trials of statins have demonstrated their efficacy in preventing CVD, much less information has been reported on their unintended effects. Although not thought of traditionally as antimicrobials, statins have been shown to have antimicrobial effects in vitro. The statins belong to a family of drugs that lower cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase, a rate limiting enzyme in the human mevalonate pathway of which cholesterol in the biosynthetic end product. The mevalonate pathway is an important cellular metabolic pathway present in many bacteria. Hence, the aim of this study was to assess the in vitro efficacy of statins against selected strains of oral streptococci, as determined by the minimum inhibitory concentration. A second related objective is to assess the in vitro effect of statins on single species biofilm formation , as determined by binding of the same streptococci to hydroxyapatite pegs. Methods: The effect of statins on S. mutans, S. sanguis, and S. salivarius was determined by finding the minimum inhibitory concentration (MIC) by broth dilution assays. Simvastatin, pravastatin atorvastatin, and rousuvastatin were used in this study. The minimum inhibitory concentration was considered to be the lowest concentration of statin that prevented bacterial growth, i.e. a clear test tube. Experiments were repeated twice for each bacterial species. The effect of simvastatin, atorvastatin, and pravastatin on the ability of S. mutans and S. sanguis to form single species biofilm was assayed using sterile microplates and the MBEC Biofilm Inoculator (Innovatech). Results: Two trials indicated that the MIC of simvastatin against the selected oral bacteria was determined to be 15.6 μg/ml for S. mutans and S. sanguis, and 7.8 μg/ml for S. salivarius. The MIC of rosuvastatin and atorvastatin was determined to be 100 μg/ml against all three streptococci, whereas the MIC of pravastatin was even higher (200 μg/ml) against all three streptococci. Likewise, two trials indicated that statins decreased single species biofilm formation by S. mutans and S. sanguis. For simvastatin, biofilm formation was decreased by concentrations eight fold below the MIC . The results were substantiated by spectrophotometric assay . For atorvastatin and pravastatin, biofilm formation was decreased by concentrations 3-4 fold below the MIC. Conclusions: These experiments demonstrate the in vitro antimicrobial effect of statins on S. mutans, S.sanguis, and S. salivarius. The data indicate that the statins inhibit growth of the test organisms with MIC’s ranging from 7.8-200 μg/ml. Simvastatin has in vitro efficacy against the specific strains of bacteria used in this study at concentrations slightly less than the observed MIC’s of 15.6-7.8 μg/ml . The MIC’s for atorvastatin, pravastatin, and rosuvastatin are much higher than simvastatin, in the range of 100-200 μg/ml . The effects of statins on biofilm parallels the effect on growth of the bacteria. / Oral Biology

Ever since traditional medicine developed thousands of years ago, humans have looked to natural substances as remedies for maladies. Today, many isolation and natural product chemists have begun revisiting ancient folk medicines in an attempt to isolate the compound(s) responsible for effective treatment. In addition to the examination of traditional remedies, the secondary metabolites of many newly discovered species, especially bacteria, get tested against a wide array of pathogenic cells. Isolated from the myxobacterium Sorangium cellulosum, the secondary metabolite carolacton was discovered to be lethal to Streptococcus mutans cells transitioning to the biofilm state. This was a significant finding since S. mutans is the main causative agent of dental caries, the most prevalent chronic childhood and adolescent disease worldwide. Herein, our efforts to design, synthesize, and biologically evaluate a 16-member library of carolacton-inspired analogs is described. In addition to natural product inspired research, two projects borne from a target-oriented templated approach are also described. The first, the synthesis of the antitumor compound FRAX-1036, was completed as part of a collaboration with the Chernoff group at Fox Chase Cancer Center to provide them with more material for murine testing. The second, the synthesis of macrocycles for the formation of atomically precise membranes, was conducted using spiroligomer building blocks and unnatural amino acids to furnish a triangle-shaped macrocycle via solution and solid phase techniques. This dissertation highlights the usefulness of the techniques of diverted total synthesis and building block synthesis in organic chemistry. / Chemistry

Histophilus somni is a commensal of the mucosal surfaces of respiratory and reproductive tracts of cattle and sheep. However, as an opportunistic pathogen, H. somni can cause diseases such as pneumonia, myocarditis, abortion, arthritis, and meningo-encephalitis. Previously, several virulence factors/mechanisms had been identified in H. somni of which the phase-variable lipooligosaccharide, induction of host cell apoptosis, intraphagocytic survival, and immunoglobulin Fc binding proteins were well characterized. To further understand the biological properties of H. somni, the genomes of pneumonia strain 2336 and preputial strain 129Pt have been sequenced. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, putative genes that encode proteases, restriction-modification enzymes, hemagglutinins, glycosyltransferases, kinases, helicases, and adhesins have been identified in H. somni. Most of the H. somni strain-specific genes were found to be associated with prophage-like sequences, plasmids, and/or transposons. Therefore, it is likely that these mobile genetic elements played a significant role in creating genomic diversity and phenotypic variability among strains of H. somni. Functional characterization of H. somni luxS in the genomic context revealed that the gene encodes S-ribosylhomocysteinase that can complement biosynthesis of AI-2 quorum sensing signal molecules in Escherichia coli DH5alpha. It was also found that several pathogenic isolates of H. somni form a prominent biofilm and that luxS as well as phosphorylcholine expression can influence biofilm formation by H. somni. In conclusion, comparative analyses of the genomes and functional characterization of putative genes have shed new light on the versatility and evolution of H. somni. / Ph. D.

This work represents an interdisciplinary effort to investigate microbiological and chemical manganese (Mn) cycling in drinking water systems using concepts and tools from civil and environmental engineering, microbiology, chemistry, surface science, geology, and applied physics. Microorganisms were isolated from four geographically diverse drinking water systems using selective Mn-oxidizing and -reducing culture media. 16S rRNA gene sequencing revealed that most are bacteria of the Bacillus spp. (i.e., Bacillus pumilus and Bacillus cereus). These bacteria are capable of performing Mn-oxidation and -reduction under controlled laboratory conditions. Pseudo-first order rate constants obtained for microbiological Mn-oxidation and -reduction (aerobic and anaerobic) of these isolates ranged from 0.02 - 0.66 days⁻¹. It is likely that spores formed by Bacillus spp. protect them from chlorine and other disinfectants applied in drinking water systems, explaining their ubiquitous presence. A new method was developed using X-ray photoelectron spectroscopy (XPS) to identify Mn(II), Mn(III), and Mn(IV) on the surfaces of pure oxide standards and filtration media samples from drinking water treatment plants. A necessary step for the comprehensive analysis of Mn-cycling in drinking water systems is to characterize the chemical properties of filtration media surfaces. Analyses of filtration media samples show that, while Mn(IV) was predominant in most samples, a mixture of Mn(III) and Mn(IV) was also identified in some of the filtration media samples studied. The use of both the XPS Mn 3s multiplet splitting and the position and shape of the Mn 3p photo-line provide added confidence for the determination of the oxidation state of Mn in complex heterogeneous environmental samples. XPS was applied to investigate Mn(II) removal by MnOx(s)-coated media under experimental conditions that closely resemble situations encountered in drinking water treatment plants in the absence and presence of chlorine. Macroscopic and spectroscopic results suggest that Mn(II) removal in the absence of chlorine was mainly due to adsorption, while in the presence of chlorine was due to oxidation. Mn(IV) was predominant in all the XPS analyses while Mn(II) was detected only in samples operated without chlorine. Future research should apply XPS under different experimental conditions to understand the specific mechanisms affecting Mn(II) removal by MnOx(s)-coated media. / Ph. D.

The goal of this project is to decipher the quorum sensing (cell-to-cell signaling) abilities of Shewanella oneidensis MR-1, a Gram-negative bacterium well known for its ability to use geologic substrates, such as Fe and Mn oxides, for respiratory purposes. Overall our results show that S. oneidensis cannot utilize either an acyl-homoserine lactone (AHL) or AI-2 quorum sensing signal, despite previous work that indicated that it produced an AHL that would enhance it ability to growth in certain anaerobic environments. Using a variety of quorum sensing signal sensors, no evidence could be found that S. oneidensis has a typical AHL signal. An in silco analysis of the genome also produced little evidence that S. oneidensis has the genes to accept or relay an AHL signal. S. oneidensis can produce a luminescence response in the AI-2 reporter strain, Vibrio harveyi MM32. This luminescence response is abolished upon deletion of luxS, the gene responsible for catalyzing AI-2. Deletion of luxS also affected biofilm formation. Within 16 hours of growth in a biofilm flow-through reactor, the luxS mutant had an inhibited ability to initiate biofilm formation. After 48 hours of growth, the mutant's biofilm had developed similarly to wild-type. The addition of synthetic AI-2 did not restore the mutant's ability to initiation biofilm formation, which led to the conclusion that AI-2 is not likely used as a quorum sensing signal in S. oneidensis for this phenotype. Because of the involvement of LuxS in the activated methyl cycle (AMC) in other organisms, growth on various sulfur sources was examined. A mutation in luxS produced a reduced ability to growth with methionine as the sole sulfur source. Methionine is a key metabolite used in the AMC to produce a methyl source in the cell and homocysteine. This data suggests that LuxS is important in metabolizing methionine and the AMC in S. oneidensis. / Ph. D.

Bacterial type IV pili (T4P) are filamentous surface appendages with a variety of functions including motility, surface attachment, and biofilm formation. In many species of bacteria a clear understanding of how the functions of T4P in lifestyle switching are regulated remains to be elucidated. Here, we focus on understanding the regulation of the T4P assembly ATPase PilB. We examined its interactions with the secondary messenger cyclic-di-GMP (cdG). Specifically we investigated how cdG binding regulates PilB functions not only as the assembly ATPase, but also as an EPS signaling molecule in Myxococcus xanthus biofilm regulation. Chapter 2 focuses on the development of a microplate-based biofilm assay for M. xanthus. This new assay allows for the analysis of the M. xanthus submerged biofilms under vegetative conditions in a high throughput format which has been absent in the published literature. M. xanthus biofilm formation tightly correlates with EPS production, suggesting that the assay can be used as a convenient method of examining EPS production. Chapter 3 examines the regulation of M. xanthus PilB (MxPilB) by cdG binding in vivo. We carried out a mutational analysis of the MshEN cdG binding domain in MxPilB. Mutations were created that either diverge with or converge from the MshEN consensus sequence. These two classes of MxPilB variants are expected to either decrease or increase cdG binding affinity, respectively. We examined the motility, EPS production, and piliation phenotypes of these mutants. Our results were consistent with a model where the function of MxPilB is altered in response to cdG binding, and suggesting that PilB responds to different thresholds of cdG concentration. In Chapter 4, we examine the ligand binding to the N-terminal cdG binding domain and C-terminal ATPase domain of Chloracidobacterium thermophilum PilB (CtPilB) in vitro. Our results confirm that these two domains bind to their respective ligands specifically, and demonstrate these domains communicate with each other in response to ligand binding. The results from all of the studies help us to establish a model where cdG binding fine tunes the functions of PilB to regulate the switch of bacteria between the motile and planktonic states. In addition to their roles in motility and biofilm formation, T4P are key virulence factors in many significant human pathogens. Antivirulence chemotherapeutics are considered to be a promising alternative to antibiotics, as they target disease processes rather than bacterial viability. Because PilB is essential for T4P biogenesis, we sought to identify PilB inhibitors for the development of antivirulence therapies. In Chapter 5, we describe the development of the first high throughput screen (HTS), for PilB inhibitors. This assay is uses the reduction of the binding of a fluorescent ATP analog to CtPilB in vitro, leading to the discovery of the plant flavonoid quercetin as a PilB inhibitor. Using M. xanthus as a model a bacterium, quercetin was found to inhibit T4P-dependent motility and T4P assembly in vivo. Builds on this initial success with CtPilB, Chapter 6 describes the development and implementation of a second HTS based on the inhibition of CtPilB as an ATPase. Screening a large chemical library led to the identification of benserazide and levodopa as CtPilB inhibitors. We show that both compounds inhibit T4P assembly in M. xanthus without any detrimental effects on bacterial growth. Furthermore we demonstrate that both levodopa and benserazide inhibit T4P-mediated motility in Acinetobacter nosocomialis, a human pathogen, providing the first evidence that the compounds identified with CtPilB can be effective against a pathogenic bacterium. Both of these studies validate the effectiveness not only of our HTSs, with of CtPilB as a model protein for the identification of PilB inhibitors. / Doctor of Philosophy / Bacteria can be motile or sessile. Motile bacteria can use hair like structures on their surface, called pili, to move in their natural environment, whereas sessile bacteria produce intricate structures attached to solid surfaces known as biofilms. Bacteria are able to switch between being motile and sessile states depending on their environment conditions. However, it isn't clear how this switch is controlled in bacteria that use pili to move. To answer this question, we studied how PilB the protein that assembles pili, might control this switching process. We specifically investigated PilB because it has two known roles. The first is that it can assemble pili, to enable pili-mediated motility. The second is that it can stimulate or promote biofilm formation. This places PilB at the intersection of these two lifestyles, suggesting that this protein may play a key role in deciding whether a bacterium is to be motile or sessile. Another important reason to understand how PilB functions is because pili are used by some antibiotic resistant pathogenic bacteria. Since PilB is essential for the formation of pili, if the actions of PilB could be blocked, bacteria would be unable to make pili. This could stop bacteria from causing disease. By searching for new chemicals which stop PilB from creating pili, we can potentially identify new drugs to treat bacterial infections.

Adherent microbial communities, known as biofilms, are a major contributing factor in the incidence of healthcare-associated infections (HCAIs). HCAIs are responsible for annually causing 100,000 deaths and medical expenses estimated to be $35-45 billion. Physical and chemical surface modification techniques are thought to be critical in the fight against biofilm formation within medical settings. Nanoscale structural features have been found to have significant effects on bacterial adhesion and biofilm formation, but their effects on fungal pathogens are less explored. This thesis systematically explores the effect of surface topography in the form of nano and microscale polymeric fibers (~0.4-1.2 µm in diameter) on biofilm formation and virulence of a common HCAI-causing fungal pathogen, Candida albicans. We show that both C. albicans attachment density and differentiation to its virulent phenotype significantly vary with fiber diameter and spacing on polymeric fiber-coated surfaces. We further show that high throughput and high content techniques, such as Raman spectroscopy, can be used to track environmental and physical effects on the organism's resulting morphology and associated virulence. Findings from this thesis will inform the design of antifouling surfaces including implantable medical devices. In a prototypical example, we demonstrate the use of fiber coating to modulate C. albicans attachment on polyurethane, silicone, and latex catheters. / Master of Science / Microbial communities that adhere to surfaces, known as biofilms, are largely associated with incidence of healthcare-associated infections (HCAIs). HCAIs are responsible for annually causing 100,000 deaths and medical expenses estimated to be $35-45 billion. Modification of surfaces using physical and chemical techniques is believed to be critical in the fight against biofilm formation on surfaces within medical settings. Nanoscale structural features have been found to have significant effects on bacterial adhesion and biofilm formation, but their effects on fungal pathogens have not been extensively studied. This thesis focuses on the effect of surface topography, or textures, in the form of nano and microscale polymeric fibers (~0.4-1.2 µm in diameter) on biofilm formation and disease-causing ability, or virulence, of a common HCAI-causing fungal pathogen, Candida albicans. We show that both C. albicans attachment density and differentiation to its virulent form significantly vary with fiber diameter and spacing on polymeric fiber-coated surfaces. We also show that high throughput and detailed imaging techniques, such as Raman spectroscopy, can be used to track environmental and physical effects on the organism's resulting morphology and associated virulence. Findings from this thesis can be used to aid in the design of surfaces that discourage biofilm formation, including implantable medical devices. We demonstrate the use of fiber coating to vary C. albicans attachment on polyurethane, silicone, and latex catheters in one of our studies.

Bacterial biofilms attached to food contact surfaces are an ongoing concern for the food industry due to the resistance of bacteria within biofilms to detergents and sanitizers. Within food manufacturing facilities, stainless steel is a common food-contact surface in which microbial cell attachment and biofilm formation may occur. Identifying methods to prevent and remove biofilms during standard cleaning and sanitation practices could prove useful, as mature biofilms can release planktonic cells into an aqueous environment, causing continual low-level contamination. Dental studies involving delmopinol hydrochloride, a cationic surfactant, have found a preventative and dissociating affect on biofilms, where food applications have scarcely been researched. This study demonstrates the prevention and removal of Listeria monocytogenes 1/2a and S. enterica Agona biofilms on stainless steel with pre- and post-exposures of delmopinol hydrochloride. Stainless steel blanks (#304, 16 gauge, 2cm x 2cm, finish #4) were submerged in a 0.2% or 0.5% delmopinol solution before or after biofilm formation. Treatment times were 1, 5 or 10 minutes, whereas controls were not exposed to the delmopinol solution. Disinfected stainless steel blanks were spot-inoculated with 20µL of a 10⁹ CFU/mL liquid culture, and pre-exposed blanks were additionally submerged in delmopinol and dried prior to inoculation. Biofilms were exclusively formed on the finished and inoculated side by placing the surface face-down on TSA. After cell attachment and biofilm development for 24 hours at 25°C, blanks were rinsed with phosphate buffer. Post-exposed blanks were submerged in 0.2% or 0.5% delmopinol for 1, 5 or 10 minutes before all blanks were individually vortexed for 90 seconds to dislodge films. Bacterial populations were determined by surface plating onto TSA followed by incubation at 32°C for L. monocytogenes and 37°C for S. Agona for 48 hours. Treatments were in-duplicate and repeated three times for each microorganism. Pre-exposure of 0.2% delmopinol resulted in a significant decrease in L. monocytogenes concentration at 1, 5 and 10 minute exposures (P < 0.05). Pre-exposures with the 0.5% solution had no significant effect on L. monocytogenes biofilm populations (P > 0.05), whereas all post-exposures lead to a significant decline in biofilm concentrations (P < 0.0001). Post-exposures of 10 minutes exhibited a mean log₁₀ reduction of 5.59 and 6.40 log₁₀ for 0.2% and 0.5% delmopinol solutions, respectively. For S. Agona, 0.2% pre-exposure resulted in no significant log10 reduction (P > 0.05), while the 10 minute 0.5% pre-exposure exhibited a minimal reduction in bacterial growth (P < 0.05). Post-exposures of 10 minutes exhibited a mean log10 reduction of 7.65 and 7.75 log10 for 0.2% and 0.5% delmopinol solutions, respectively. For L. monocytogenes and S. Agona, post-exposure to delmopinol hydrochloride caused a notable log10 reduction. The removal effect of delmopinol on biofilms is significantly greater the preventative effect. / Master of Science in Life Sciences

Clostridium perfringens is a Gram-positive bacterium that secretes a biofilm matrix material. The goal of these experiments was to identify pilin mutants that are needed for secretion of the biofilm matrix and develop a functional model for a type II secretion system (T2SS) in C. perfringens. Protein tagging, western blot, and slot blot experiments were done to quantify protein secretion. After performing experiments using a CPE0515-FLAG construct, it was concluded from immunoblot densitometry data that, except for the pilA1 deletion mutant, none of the 18 tested pilin mutants had a statistically significant difference from the wild type (WT) with regard to protein secretion. From slot blot densitometry assays, it was concluded that the pilA1 and CPE2280 mutants showed statistically significant lower values than the WT but the pilA2 and CPE1841 mutants had values that were higher than the wild type. Testing the construct containing only CPE0514 and CPE0515-FLAG showed that CPE0516 and CPE0517 are not needed for secretion of the protein CPE0515. HA-tagged CPE0516 qualitative immunoblots showed that, unlike CPE0515, oligomerization of CPE0516 is not occurring, and that this protein likely forms a heat stable dimer. Overall, the data did not allow us to construct a T2SS model, since there were not enough proteins revealed to be involved to create a complete Type II secretion system. / Master of Science / The methods by which C. perfringens can persist and survive in environmental conditions is something that would be useful to learn more about. One of the methods that many bacteria use to survive is by creating a biofilm matrix material, which provides protection for the bacteria from environmental stresses. In this study, the goal was to determine which specific proteins are needed for the secretion of the biofilm matrix material. Using molecular biology techniques, the proteins thought to be involved in biofilm formation quantified. The results showed that while two proteins ultimately appeared to be needed for secretion, there were not enough proteins involved to create a complete model for a functional secretion system in C. perfringens.