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Objectives: Fusobacteria are prominent participants in the maturation of subgingival dental plaque biofilms in humans. A number of various species belonging to the Fusobacterium genus have been recovered from the subgingival microbiota of chronic periodontitis patients. However, conventional Fusobacterium species identification is labor-intensive, time-consuming, and complicated by shortcomings in phenotypic-based classification schemes, where many fusobacteria display overlapping and non-distinguishing morphologic features and biochemical properties. In addition, molecular identification of fusobacteria is plagued with difficulties of validating the specificity of nucleic acid probes and primers to various Fusobacterium species that have closely-related interspecies genetic profiles. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and its associated analytic software, was recently approved for clinical microbiology diagnostic use by the United States Food and Drug Administration. MALDI-TOF mass spectrometry has the potential to rapidly identify cultivable clinical isolates to a species level for 4,613 different bacterial species based on mass spectra of their bacterial protein profiles, including many Fusobacterium species. The purpose of this study was to use MALDI-TOF mass spectrometry to rapidly identify the patient distribution of fusobacteria isolated from the subgingival microbiota of chronic periodontitis patients. Methods: A total of 34 chronic periodontitis patients provided 96 fresh subgingival cultivable fusobacteria isolates (one to seven isolates per patient), which were presumptively identified by their chartreuse-positive colony autofluorescence under long-wave ultraviolet light on anaerobically-incubated, non-selective, enriched Brucella blood agar primary isolation plates. Each of the presumptive fusobacteria clinical isolates were subjected to MALDI-TOF mass spectrometry analysis using a bench top mass spectrometer, Bruker FlexControl 3.0 software, and MALDI Biotyper 3.1 software (Bruker Daltonics, Billerica, MA, USA), which contains mass spectra for a variety of fusobacteria in its reference library of bacterial protein profiles. Each clinical isolate underwent on-target plate formic acid protein extraction, and was taxonomically classified with MALDI-TOF mass spectrometry within an approximately 30-45 minute time period from the point of colony harvesting from primary isolation culture plates. A MALDI Biotyper log score of equal to or larger than 1.7 was required for reliable taxonomic classification of the clinical fusobacteria isolates. Results: A majority (58.8%) of the chronic periodontitis patients yielded two or three different species of subgingival Fusobacterium on non-selective enriched Brucella blood agar primary isolation plates. Fusobacterium naviforme was identified by MALDI-TOF mass spectrometry analysis in 14 (41.2%) chronic periodontitis study patients, Fusobacterium nucleatum subspecies vincentii in 13 (38.2%) patients, Fusobacterium nucleatum subspecies polymorphum in 9 (26.5%) patients, Fusobacterium nucleatum and Fusobacterium species each in 6 (17.6%) patients, Fusobacterium nucleatum subspecies nucleatum in 4 (11.8%) patients, and Fusobacterium nucleatum subspecies animalis in 3 (8.8%) patients. Three patients additionally yielded subgingival isolates of Fusobacterium canifelinum, normally an inhabitant of the oral cavity of dogs and cats. 52 (54.2%) of the fusobacteria clinical isolates revealed MALDI Biotyper log scores of equal to or larger than 1.7, the threshold for reliable taxonomic classification, while in comparison, 44 (45.8%) had log scores less than 1.7, indicating a less reliable species identification. No other microbial species, other than one of the Fusobacterium species, was listed by the MALDI-TOF mass spectrometry analytic software as the most likely organism for the tested clinical isolates. Conclusions: These findings indicate that a variety of Fusobacterium species may be identified with MALDI-TOF mass spectrometry in the subgingival microbiota of chronic periodontitis patients. F. naviforme and F. nucleatum subspecies vincentii were the most frequently isolated subgingival fusobacteria species in the evaluated study patients. Three chronic periodontitis patients also unexpectedly revealed subgingival isolates of the animal species F. canifelinum, which is normally in the oral cavity of dogs and cats. MALDI-TOF mass spectrometry may facilitate rapid identification of cultivable fusobacteria in human subgingival dental plaque biofilms, and enhance understanding of bacterial community structure in periodontal pockets. / Oral Biology

One of the most significant challenges faced when modeling mass transfer from contaminant source zones is uncertainty regarding parameter estimates. These rate parameters are of particular importance because they control the connectivity between a simulated contaminant source zone and the aqueous phase. Where direct observation has fallen short, this study attempts to interpret field data using an inverse modeling technique for the purpose of constraining mass transfer processes which are poorly understood at the field scale. Inverse modeling was applied to evaluate parameters in rate-limited models for mass transfer. Two processes were analyzed: (i) desorption of hydrophobic contaminants and (ii) multicomponent Non-Aqueous Phase Liquid (NAPL) dissolution. Desorption was investigated using data obtained from elution experiments conducted with weathered sediment contaminated with 2,4,6 trinitrotoluene (TNT) (Sellm and Iskandar, 1994). Transport modeling was performed with four alternative source models, but predictive error was minimized by two first-order models which represented sorption/desorption using a Freundlich isotherm. The results suggest that first-order/Freundlich models can reproduce dynamic desorption attributed to high-and-low relative energy sorption sites. However, additional experimentation with the inversion method suggests that mass constraints are required in order to appropriately determine mass transfer coefficients and sorption parameters. The final portion of this research focused on rate-limited mass transfer from multicomponent NAPLs to the aqueous phase. Previous work has been limited to bench and intermediate scale findings which have been shown to inadequately translate to field conditions. Two studies were conducted in which numerical modeling was used to reproduce dissolution from multicomponent NAPL sources. In the first study, a model was generated to reproduce dissolution of chloroform (TCM), trichloroethylene (TCE) and tetrachloroethylene (PCE) observed during an emplaced-source field experiment conducted within a flow cell (Broholm et al., 1999). In the second study, a methodology was developed for analyzing benzene, toluene, ethylbenzene and xylene (BTEX) data during a field-scale mass transfer test conducted within a vertically-smeared source zone (Kavanaugh, 2010). The findings suggest that the inversion technique, when provided appropriate characterization of site and source parameters and when given appropriate dataset resolution, represents a viable method for parameter determination. Furthermore, the findings of this research suggest that inversion-based modeling provides an innovative predictive method for determining mass transfer parameters for multicomponent mixtures at the field scale. / Ph. D.

The chemical adsorption and reaction of methyl fluoride, methyl chloride, methyl bromide, methyl iodide, methyl sulfide, dimethyl sulfide, dimethyl disulfide, and bis(trifluoromethyl) disulfide on clean, polycrystalline nickel surface (at 25°C) were investigated using an X-ray photoelectron spectrometer (XPS) and a thermal desorption system with mass spectrometric analysis (TD). The ion implantation of the methyl halide series on nickel was investigated by XPS. The nickel surface was cleaned by high temperature heating in vacuum or argon ion sputtering. An industrial lubricant, FC-43 (tri-hepta-fluorobutyl amine), was adsorbed on a gold-plated nickel surface at 25°C. Tne FC-43 adsorption system was investigated by TD. The adsorption of the methyl halide series on nickel was predominantly dissociative with several co-existing surface species being observed. The XPS results show that two carbon and two halogen species are formed. The possible structures for carbon are a CH₃-type and a CH₂-type or CH_nX (n = 0-3) adsorbed species. The halogen species appear to be neutral and ionic species. The TO results support the dissociative adsorption process for methyl halides on nickel. The adsorption of methyl fluoride and methyl chloride on nickel studied by TD resulted in the corresponding nickel halide being detected. The adsorption of methyl bromide and methyl iodide on nickel studied by TO resulted in the corresponding hydrogen halide, HX, being detected. The XPS results of the ion implantation of the methyl halides resulted in only one type of carbon that is similar to graphite. A halide type of halogen was also formed upon ion implantation. The XPS results for the adsorption of methyl sulfide, dimethyl sulfide, and dimethyl disulfide on nickel showed that only one carbon and one sulfur were observed. The possible structure for carbon is a CH₃-type of adsorbed specie. lhe sulfur species appears to be a sulfide S⁼-type adsorbed specie. The XPS results for the adsorption of bis(trifluoromethyl) disulfide on nickel showed that only sulfur was observed on the nickel surface. The TD results- for the methyl sulfide adsorption process was shown to be predominantly an associative type. The TO results for the dimethyl sulfide adsorption process were a mixture of dissociative and associative adsorption processes. The dimethyl disulfide adsorption process was determined by TD to be predominantly a dissociative type. The bis(trifluoromethyl} disulfide was determined by TD to be completely dissociative upon adsorption and reacted with nickel to form nickel sulfide. The TD results of FC-43 adsorption on gold-plated nickel showed that the predominant adsorption process was the associative type. / Ph. D.

Metal oxide and metal oxide-supported metal nanoparticles can adsorb and decompose chemical warfare agents (CWAs) and their simulants. Nanoparticle activity depends on several factors including chemical composition, particle size, and support, resulting in a vast number of materials with potential applications in CWA decontamination. Current instrumentation in our laboratory used to investigate fundamental gas-surface interactions require extensive time and effort to achieve operating conditions. This thesis describes the design and construction of a high-throughput, high vacuum surface analysis instrument capable of studying interactions between CWA simulants and nanoparticulate surfaces. The new instrument is small, relatively inexpensive, and easy to use, allowing for expeditious investigations of fundamental interactions between gasses and nanoparticulate samples. The instrument maintains the sample under high vacuum (10?⁷-10?⁹ torr) and can reach operating pressures in less than one hour. Thermal control of the sample from 150-800 K enables sample cleaning and thermal desorption experiments. Infrared spectroscopic and mass spectrometric methods are used concurrently to study gas-surface interactions. Temperature programmed desorption is used to estimate binding strength of adsorbed species. Initial studies were conducted to assess the performance of the instrument and to investigate interactions between the CWA simulant dimethyl methylphosphonate (DMMP) and nanoparticulate silicon dioxide. / Master of Science

Scanning Electrochemical Microscopy (SECM) is most commonly used to spatially resolve reaction rates, image surface topography and surface reactivity. In this research, SECM is applied to various chemical systems in order to resolve local reaction chemistry and to produce patterns with dimensions of tens of microns in n-alkanethiol passivated gold substrates. Upon completing construction of the instrumentation, SECM was applied to capillary electrophoresis to accurately and reproducibly place the electrode directly above a very small capillary opening. Feedback SECM was then used to image and pattern surfaces, effectively distinguishing between insulating and conductive domains. Finally, the size of desorbed features patterned on a passivated gold substrate were studied as a function of both applied potential and ionic strength. Electrochemical detection in capillary electrophoresis requires decoupling the voltage applied to the working electrode from the separation voltage applied across the capillary. End-capillary electrochemical detection achieves this by placing the electrode just outside the ground end of the separation capillary. Obtaining adequate signal-to-noise in this arrangement requires using small inner diameter capillaries. Decreasing the inner diameter of the separation capillary, however, increases the difficulty of aligning the microelectrode with the open end of the capillary. Using SECM, the position of the capillary opening is determined while electroactive material is continuously emerging from the end of the capillary. The SECM instrument is then used to place the electrode at the position of maximum current for subsequent separations. Subsequent measurements found that the best signal-to-noise is obtained when the detection electrode is placed directly opposite the capillary opening and just outside of the capillary opening. When the electrode is further above the opening (but still opposite the capillary opening), the signal-to-noise does not dramatically decrease until the electrode is more than 30 μm above the 10 μm inner-diameter capillary. Limits of detection for 2,3-dihydroxybenzoic acid were found to be 8.2 fmol when aligned manually, and 3.8 fmol when the SECM is used to automatically align the microelectrode. SECM was then used to image a series of multi-disk electrode arrays in order to demonstrate the ability of the instrument to discriminate between conductive and insulating domains. Upon demonstrating the capacity of the SECM to image very small domains of conductor on an insulating substrate, n-alkanethiol passivated gold surfaces were patterned using site-selective desorption. A number patterns, potentially useful for enzyme deposition, were subsequently produced in the passivated gold substrate. The feature size of the desorbed domains was monitored as a function of applied potential and the ionic strength of the solution used for desorption. Results showed that applying a more negative potential or increasing the ionic strength of the solution increased the magnitude of the electric field at the surface of the passivated gold substrate and resulted in a more complete, larger desorption. Both ionic strength and applied desorption potential prove to be parameters useful for controlling the size of patterned features in site selective desorption. / Master of Science

Developing a fundamental understanding of the interactions of chemical warfare agents (CWAs) with surfaces is essential for the rational design of new sorbents, sensors, and decontamination strategies. The interactions of chemical warfare agent simulants, molecules which retain many of the same chemical or physical properties of the agent without the toxic effects, with amorphous silica were conducted to investigate how small changes in chemical structure affect the overall chemistry. Experiments investigating the surface chemistry of two classes of CWAs, nerve and blister agents, were performed in ultrahigh vacuum to provide a well-characterized system in the absence of background gases. Transmission infrared spectroscopy and temperature-programmed desorption techniques were used to learn about the adsorption mechanism and to measure the activation energy for desorption for each of the simulant studied. In the organophosphate series, the simulants diisopropyl methylphosphonate (DIMP), dimethyl methylphosphonate (DMMP), trimethyl phosphate (TMP), dimethyl chlorophosphate (DMCP), and methyl dichlorophosphate (MDCP) were all observed to interact with the silica surface through the formation of a hydrogen bond between the phosphoryl oxygen of the simulant and an isolated hydroxyl group on the surface. In the limit of zero coverage, and after defect effects were excluded, the activation energies for desorption were measured to be 57.9 ± 1, 54.5 ± 0.3, 52.4 ± 0.6, 48.4 ± 1, and 43.0 ± 0.8 kJ/mol for DIMP. DMMP, TMP, DMCP, and MDCP respectively. The adsorption strength was linearly correlated to the magnitude of the frequency shift of the ν(SiO-H) mode upon simulant adsorption. The interaction strength was also linearly correlated to the calculated negative charge on the phosphoryl oxygen, which is affected by the combined inductive effects of the simulants’ different substituents. From the structure-function relationship provided by the simulant studies, the CWA, Sarin is predicted to adsorb to isolated hydroxyl groups of the silica surface via the phosphoryl oxygen with a strength of 53 kJ/mol. The interactions of two common mustard simulants, 2-chloroethyl ethyl sulfide (2-CEES) and methyl salicylate (MeS), with amorphous silica were also studied. 2-CEES was observed to adsorb to form two different types of hydrogen bonds with isolated hydroxyl groups, one via the S moiety and another via the Cl moiety. The desorption energy depends strongly on the simulant coverage, suggesting that each 2-CEES adsorbate forms two hydrogen bonds. MeS interacts with the surface via a single hydrogen bond through either its hydroxyl or carbonyl functionality. While the simulant work has allowed us to make predictions agent-surface interactions, actual experiments with the live agents need to be conducted to fully understand this chemistry. To this end, a new surface science instrument specifically designed for agent-surface experiments has been developed, constructed, and tested. The instrument, located at Edgewood Chemical Biological Center, now makes it possible to make direct comparisons between simulants and agents that will aid in choosing which simulants best model live agent chemistry for a given system. These fundamental studies will also contribute to the development of new agent detection and decontamination strategies. / Ph. D.

No / N-Linked glycans were ionized from several matrices with a Shimadzu-Biotech AXIMA-QIT matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer. [M+Na]+ ions were produced from all matrices and were accompanied by varying amounts of in-source fragmentation products. The least fragmentation was produced by 2,5-dihydroxybenzoic acid and the most by -cyano-4-hydroxycinnamic acid and 6-aza-2-thiothymine. Sialic acid loss was extensive but could be prevented by formation of methyl esters. Fragmentation produced typical low-energy-type spectra dominated by ions formed by glycosidic cleavages. MSn spectra (n = 3 and 4) were used to probe the pathways leading to the major diagnostic ions. Thus, for example, an ion that was formed by loss of the core GlcNAc residues and the 3-antenna was confirmed as being formed by a B/Y rather than a C/Z mechanism. The proposed structures of several cross-ring cleavage ions were confirmed and it was shown that MS3 spectra could be obtained from as little as 10 fmol of glycan

No / A novel ion trap time-of-flight hybrid mass spectrometer (qIT-TOF MS) has been applied for peptide sequencing in proteolytic digests generated from spore mixtures of Bacilli. The method of on-probe solubilization and in situ proteolytic digestion of small, acid-soluble spore proteins has been recently developed in our laboratory, and microorganism identification in less than 20 min was accomplished.1 In this study, tryptic peptides were generated in situ from complex spore mixtures of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki, and B. cereus T, respectively. MALDI analysis of bacterial peptides generated was performed with an average mass resolving power of 6200 and a mass accuracy of up to 10 ppm using a trap-TOF tandem configuration. Precursor ions of interest were usually selected and stored in the quadrupole ion trap with their complete isotope distribution by choosing a window of ±2 Da. Sequence-specific information on isolated protonated peptides was gained via tandem MS experiments with an average mass resolving power of 4450 for product ion analysis, and protein and bacterial sources were identified by database searching.

I takt med att behovet av att minska utsläppen av växthusgasen ökar, ökar även intresset för negativa utsläpp. En lovande teknik för att uppnå negativa utsläpp är koldioxidlagring från biomassa, även kallad BECCS (Bio-Energy Carbon Capture and Storage). Trots teknologins mognad är de stora energibehoven vid lösningsmedelsregenerering ett hinder för storskalig implementering. I den här studien utforskas den relativt okända kinetiken för lösningsmedelsregenerering av kaliumkarbonat i ett steg för att optimera processen. Dessutom undersöks möjligheten att använda vanadin(V)oxid som katalysator för att förbättra desorptionshastigheten. Experimentella analyser utfördes i en sats-reaktor och gick ut på att undersöka förändringen av lösningsmedlets loading över tid genom regelbundna titreringar. Utöver detta undersöks den påverkan som temperatur och omrörning har på desorptionshastigheten. Experimenten utförs vid atmosfärstryck och temperaturer från 80°C till 100°C. Resultaten visade på god repeterbarhet trots svårigheter med temperaturöverskridningar. Desorptionshastigheten var lägre vid 80°C och 90°C än vid 100°C, men de logaritmiska hastighetskonstanterna följde inte en linjär relation mot temperaturinverserna vilket antyder att reaktionen är begränsad av massöverföring. Vidare påverkade inte användandet av en katalysator desorptionskinetiken märkbart, vilket än en gång antyder ett massöverföringsberoende. Slutligen visades ingen märkbar skillnad i desorptionshastighet trots olika omrörningshastigheter. Detta beror troligen på den redan höga massöverföringen som sker vid kokpunkten. Sammanfattningsvis bidrar denna studie med insikter för att förbättra effektiviteten hos regenereringen av lösningsmedel vid BECCS, vilket är avgörande för att motverka utsläppen och möta utmaningarna med klimatförändringarna.