This research investigated the mechanical properties of laboratory manufactured, fire retardant treated (FRT) flakeboard in an effort to help establish proper manufacturing procedure and testing protocol for these types of composite products. Two sets of fire retardant treated and untreated panels were manufactured and tested to gauge the initial effects of treatment, as well as the effects of high temperature exposure on both panel types. Testing was also conducted on different bending specimen widths in an attempt to find the specimen size most suitable for testing flakeboard composites. Results indicated that FR treatment had an adverse effect on panel mechanical properties from both an initial standpoint as well as after the panels had been exposed. Results from the specimen width investigations revealed that using a wider dimension specimen for ASTM bending tests would reduce the amount of variation and error that can be found when testing smaller size samples.

<p> Diabetes mellitus is a major independent risk factor for cardiovascular disease (CVD) and stroke, however the cellular mechanisms by which diabetes contributes to vascular dysfunction are not fully understood. In recent decades, multiple molecular mechanisms have been implicated in hyperglycemia-associated vascular damage and CVD [1]. It is well established that hyperglycemia promotes intracellular glucose flux through the hexosamine pathway where the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFAT) produces glucosamine-6-phosphate [2,3]. We have shown that elevated levels of intracellular glucosamine cause ER stress and activation of the UPR in multiple cell types [4]. Additionally, we have previously shown that ER stress is associated with lipid accumulation, activation of inflammatory pathways, and is associated with atherosclerotic plaque formation in hyperglycemic mice [ 4,5]. We hypothesize that the accumulation of intracellular glucosamine, observed in conditions of hyperglycemia, promotes atherogenesis via a mechanism that involves the hexosamine pathway, protein glycosylation and ER stress.</p> <p> Using in vitro over-expression studies, we investigated the role of GFAT in hyperglycemia-associated ER stress. We developed methods to increase GFAT expression in both HepG2 cells and HASMC. However, we found that GFAT over-expression is insufficient to induce an ER stress response. Further investigation of this system suggests that the over-expressed GFAT does not increase intracellular glucosamine levels to sufficiently promote ER stress.</p> <p> We have also investigated the role of protein glycosylation in glucosamine-induced ER stress. We have shown that O-linked glycosylation plays a role in ER stress induction. We have also shown that N-linked protein glycosylation is affected by elevated cellular glucosamine levels. Thus, dysregulated glycosylation of newly synthesized proteins may contribute to the accumulation of unfolded protein in the ER and lead to the activation of the UPR.</p> / Thesis / Master of Science (MSc)

Phosphate (PO4-3 or Pi) is an essential molecule necessary for sustaining life and it plays important roles in nucleic acid and cell membrane integrity. However, phosphate is found in growth-limiting concentrations in most environments. Bacteria have developed a diverse set of transport systems to uptake and scavenge phosphate from their environment for use in cellular processes. In the soil bacterium, Sinorhizobium meliloti, one such Pi transport system is the Pap-Pit system. Pit is a membrane transporter for Pi and is associated with a cytosolic protein of unknown function known as Pap (Pit-accessory protein). Interestingly, the stop codon of pap overlaps with the start codon of pit by a single nucleotide. In previous work, the pap gene appeared to be required immediately upstream of pit in an operon for functional Pi transport. Thus, in a pap deletion mutant, when pap is present in trans, there is no Pi transport. This suggests a possible translational coupling mechanism between Pap and Pit, in which the translation of Pap is required for the translation of Pit. Here, an alkaline phosphatase (phoA/lacZ) and a β-glucuronidase (gusA) translational reporter were fused to Pit as a measure of its translation and to understand the role of translational coupling in the Pap-Pit system. Growth complementation experiments with a conditionally Pi transport deficient S. meliloti mutant carrying various mutations in both pap and pit have also been performed in an attempt to determine the function of Pap in Pi uptake. The results presented here provide evidence that pap and pit are translationally coupled, and this is necessary for functional Pi transport via Pap-Pit. / Thesis / Master of Science (MSc) / Microbes require phosphorus in the form of inorganic phosphate (Pi) as an essential nutrient, but it is often found in growth-limiting concentrations in the environment. Bacteria have developed a diverse set of Pi transport systems to scavenge and take up phosphate from the environment. In the soil bacterium, Sinorhizobium meliloti, one such Pi transport system is the Pap-Pit system. Pit is a membrane transporter for Pi and is associated with a cytosolic protein of unknown function known as Pap. Various mutations in both pap and pit have been constructed in an attempt to determine the function of Pap in Pi uptake via Pit. The pap gene appears to be required immediately upstream of pit in an operon for functional Pi transport. The pap and pit genes overlap by a single nucleotide and this may suggest a translational coupling mechanism that is required for functional Pi transport via Pap-Pit.

No description available.

Some organophosphorus (OP) esters induce a central-peripheral distal axonopathy called organophosphorus ester-induced delayed neurotoxicity (OPIDN). In the chicken model neurological deficits and microscopic lesions develop 7-21 days after exposure. Neurotoxic esterase (NTE) is thought to be the initial target in OPIDN. Evidence indicates that neuropathic OP esters have to bind NTE and chemically ?age? for OPIDN induction. It was hypothesized that phenyl saligenin phosphate (PSP), a neuropathic OP ester that essentially irreversibly inhibits NTE as it undergoes the chemical aging process, results in changes in spinal cord gene expression that do not occur with phenylmethylsulfonyl fluoride (PMSF), a non-neuropathic compound that inhibits NTE without aging. This hypothesis was tested in Gallus gallus in experiments designed to detect differences in spinal cord gene expression between PSP, PMSF and vehicle-treated birds 24 hours after exposure. Two approaches were used. Targeted display was developed and used to screen approximately 15000 gel bands. Three candidate genes were identified by targeted display. One, designated P1 has 100% homology with expressed sequence tag pgp1n.pk010.m23, another, P2, is homologous to human KIAA1307, and a third, P3, is unidentified. Northern blotting was used to measure spinal cord expression of a-tubulin and other genes previously reported to be differentially expressed following exposure to di-isopropryl phosphorofluoridate, another agent causing OPIDN. Only expression of a-tubulin was altered in PSP-treated hens. Time course experiments were undertaken to determine spinal cord expression changes of P1, P2, P3 and a-tubulin transcripts at 12, 24, 36 and 48 hours post-exposure. Findings indicated decreases and increases, respectively, of P1 (22%, p=0.0011) and P2 (26%, p=0.0055) transcripts at 12 hours in PSP treated hen spinal cord compared to DMSO controls. An ~2.5 kb a-tubulin transcript was decreased across most time points with maximum change at 48 hours (33%, p=0.0479); an ~4.5 kb a-tubulin transcript was upregulated at 12 hours (38%, p=0.0125) and down regulated at 48 hours (28%, p=0.0576). Responses to PMSF were different than responses to PSP. Spinal cord in-situ hybridization experiments revealed, 1.) mainly neuronal expression of P1, P2 and a-tubulin transcripts, and, 2.) decreased expression of neuronal P1 and a-tubulin transcripts at 12 and 48 hours, respectively. Results indicate that PSP can induce changes in gene expression distinct from those induced with the non-neuropathic NTE inhibitor, PMSF. However, expression changes were low in frequency and magnitude, and their mechanistic importance remains to be fully established. / Ph. D.

Baker's yeast (Saccharomyces cerevisiae) undergoes major biochemical and structural rearrangements in order to survive cycles of desiccation and rehydration, yet a firm understanding of the response is lacking. The purpose of this study was to examine the response of S. cerevisiae to desiccation and rehydration at both the physiological and molecular levels. Transmission electron microscopy was used to show that loss of vacuolar structure, enlarged nuclear boundaries, as well as cell wall thickening were all associated with the desiccation response. Molecular analysis focused on glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.13), a multifunctional protein that is involved in several cellular processes other than glycolysis, including nuclear translocation under stress and intracellular sensing of oxidative stress during apoptosis. Here, GAPDH was studied primarily to determine its potential role in mediating the changes in cell wall physiology identified through our structural studies. GAPDH appears to be shuttled between the cell wall and the cytoplasm during the desiccation/rehydration process. Western analyses in combination with the use of inhibitors of translation (cycloheximide) suggest that the shuttling process does not require de novo protein synthesis. Western analyses also identified an immuno-reactive peptide in the cell wall and cytoplasmic fractions of lower molecular mass than native GAPDH (27 KDa vs. 37 KDa). This lower molecular weight peptide exhibited the translocation process similar to that of the full length GAPDH. Studies with GAPDH deletion strains suggested that the 27 kDa fragment is encoded by tdh3. The importance of this lower molecular weight form is yet to be determined. / Master of Science in Life Sciences

Studies regarding the feasibility of ovalbumin (OVA) as a bone scaffold material have found its cost, availability, interaction with cells, and ability to degrade in the body into safe byproducts to be ideal for such an application. However, weak mechanical properties cause hesitation in the use of OVA as a scaffolding material in much stronger native tissue. To enhance the mechanical strength of the OVA scaffolds without compromising in vitro cellular performance, Ca-P crystals were grown on unmodified OVA and phosphonated OVA (p-OVA) samples via biomineralization processes using 5x-concentrated simulated body fluid (5x SBF). Electron microscopy (ESEM/EDS) data confirm the formation of Ca-P crystals on the surface of OVA and p-OVA scaffolds. Mechanically, rheology data measured a minimum of a three-fold increase in each mineralized scaffold's complex shear modulus over unmineralized counterparts. Degradation in a PBS+collagenase XI environment showed that mineralization extended total time to degradation. It was also shown that the formation of the Ca-P crystals had no negative effects on in vitro cell studies. To measure cellular response, a live/dead assay was conducted to confirm cell viability after 24 hours. In conclusion, improvements were made to mechanical strength without compromising in vitro cell-scaffold response. While it remains unknown whether the increase in strength is adequate for use as a bone scaffold, future work should focus on gathering necessary information to study OVA scaffolds in animal models for eventual consideration as a bone graft substitute material. / Master of Science

Biodegradable polyesters, such as poly(DL-lactic-co-glycolic acid) (PLGA), have been used to fabricate porous bone scaffolds to support bone tissue development. These scaffolds allow for cell seeding, attachment, growth and extracellular matrix production in vitro and are replaced by new bone tissue when implanted into bone sites in vivo. Hydroxyapatite (HAP) and μ-tricalcium phosphate (μ-TCP) ceramics have been incorporated into PLGA bone scaffolds and have been shown to increase their osteoconductivity (support cell attachment). Although HAP, μ-TCP, and biodegradable polyesters are osteoconductive, there is no evidence that these scaffold materials are osteoinductive (support cell differentiation). Calcium and phosphate ions, in contrast, have been postulated to be osteogenic factors that enhance osteoblast differentiation and mineralization. Recently, a zirconia-hybridized pyrophosphate stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized which permits controlled release of calcium and phosphate ions and thus is hypothesized to be osteoinductive. Incorporation of Zr-ACP into a highly porous poly(DL lactic-co-glycolic acid) (PLGA) scaffold could potentially increase the osteoinductivity of the scaffold and therefore promote osteogenesis when implanted in vivo. To determine the osteoinductivity of Zr-ACP, a MC3T3-E1 mouse calvarial-derived osteoprogenitor cell line was used to measure cell response to Zr-ACP. To accomplish this objective, Zr-ACP was added to cell culture at different stages in cell maturation (days 0, 4 and 11). DNA synthesis, alkaline phosphatase (ALP) activity, osteopontin synthesis and collagen synthesis were determined. Results indicate that culture in the presence of Zr-ACP significantly increased cell proliferation, ALP activity and osteopontin synthesis but not collagen synthesis. To determine the feasibility of incorporating Zr-ACP into a PLGA scaffold, PLGA/Zr-ACP composite foams (5% or 10% (w/v) polymer:solvent with 25 wt% or 50 wt% Zr-ACP) were fabricated using a thermal phase inversion technique. Scanning electron microscopy revealed a highly porous structure with pores ranging in size from a few microns to about 100 μm. The amorphous structure of the Zr-ACP was maintained during composite fabrication as confirmed by X-ray diffraction measurements. Composite scaffolds also showed significantly greater compressive yield strengths and moduli as compared to pure polymer scaffolds. The results of this study indicate that Zr-ACP enhances the osteoblastic phenotype of MC3T3-E1 cells in vitro and can be incorporated into a porous PLGA scaffold. Porous PLGA/Zr-ACP composites are promising for use as bone scaffolds to heal bone defects. / Master of Science

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The enzyme, deoxyribophosphoaldolase, reversibly cleaves deoxyribose- 5-P to glyceraldehyde-3-P and acetaldehyde. Until this exposition, it was not known to exist in human erythrocytes. However, the enzyme was postulated to occur in erythrocytes in order to account for the synthesis of glyceraldehyde-3-P observed in ghosts when deoxynucleosides were metabolized. Therefore, initial experiments were designed to detect acetaldehyde as an intermediate of deoxynucleoside metabolism by ghosts. Indeed, it was found in incubation mixtures containing deoxyinosine or deoxyadenosine as substrate. Accumulation of acetaldehyde was found to depend upon substrate concentration and no acetaldehyde was detected in the absence of substrate or in the presence of inosine or ribose-5-P. Acetaldehyde was identified spectrophotometrically by its reaction with yeast alcohol dehydrogenase and colorimetrically by its reaction with buffered semicarbazide solution. Confirmation of its identity was obtained by isolation of the 2,4 dinitrophenylhydrazone derivative of acetaldehyde aerated from incubation extracts. A direct relationship between acetaldehyde production and triosephosphate production to deoxyribose-5-P utilization by hemolysates prepared from human erythrocytes was shown. Conclusive evidence for the existence of deoxyribophosphoaldolase was obtained by isolating the enzyme from human erythrocytes. Two procedures were developed to isolate the enzyme. The first involved the use of Sephadex G-100 and DEAE-cellulose columns. The second procedure proved more fruitful and employed ammonium sulfate fractionation followed by elution from calcium phosphate gels. It was found that 26% of the original enzyme activity was recovered and purification was approximately 3,000 fold. Several characteristic properties of partially purified and purified preparations of the enzyme were studied. It was found that: 1. Enzyme activity decayed rapidly upon storage. Magnesium ion, cysteine HCl, beta-mercaptoethanol or reduced glutathione increased enzyme stability. In addition, the sulfhydryl containing compounds were able to partially reactivate previously inactivated enzyme. 2. The molecular weight of deoxyribophosphoaldolase was estimated by Sephadex gel fractionation to be slightly greater than 68,000, the molecular weight of hemoglobin. Because of its low affinity for DEAE-cellulose and high affinity for carboxy methyl cellulose, the enzyme appeared to be a very basic protein. 3. The reaction catalyzed by deoxyribophosphoaldolase favored cleavage of deoxyribose-5-P. At equilibrium, 60% of deoxyribose-5-P was converted to products. When acetaldehyde and glyceraldehyde-3-P were used as substrates, 40% of each was converted to deoxypentose product. 4. The enzyme showed a high specificity for each of its three substrates. No reaction of the enzyme occurred with ethyl alcohol, pyruvate, ribose-5-P, deoxyribose, lactate and dihydroxyacetone phosphate. 5. The enzyme reacted optimally at pH 6.5. 6. Deoxyribophosphoaldolase was activated by several carboxylic acids. The degree of activation was greater in the presence of citric acid than any dicarboxylic acid tested. An optimal activation of enzyme occurred when the concentration of citrate was varied between 3-15 umoles/ml. Citrate activation did not appear to reside in its ability to act as a chelator. Comparison of enzyme elution from Sephadex G-100 columns in the presence and absence of citrate suggested that citrate causes enzyme aggregation by a still unexplained mectanism. 7. The apparent Michaelis constants for deoxyribose-5-P cleavage were determined in the presence and absence of citrate and were found to be 6.4 x 10^-4 moles/liter and 24.0 x 10^-4 moles/liter respectively. The presence of deoxyribophosphoaldolase in human erythrocytes can clearly explain the production of triosephosphate and acetaldehyde from deoxynucleoside substrates and may, in fact, provide a major catabolic pathway for the deoxypentose moiety of these deoxynucleosides. The enzyme provides a simple mechanism for triosephosphate formation from deoxypentose and may be part of a pathway that converts deoxypentose phosphate to pentose phosphate. / 2999-01-01

It is generally acknowledged that a variety of problems affecting aesthetics, health, and corrosivity of potable water can arise during installation of building plumbing systems. These include 'blue water', microbial infestation, and rapid loss of disinfectant residual, among other things. Frequently cited causes of the problems include metallic fines left in the plumbing lines from deburring, cutting and product fabrication; solder flux residuals (water soluble and petroleum based flux); and solvents for CPVC. Mechanistically, some materials such as flux contain high chloride, high ammonia and cause low pH, which can increase the corrosivity of water held in the lines. Indirect effects are also suspected to be important. For example, ammonia from flux and organic carbon from flux or PVC solvents can spur microbial growth, which in turn can reduce pH or otherwise increase corrosivity. Recent work has also demonstrated that problems with lead leaching to water from brass in modern plumbing can actually be worse in PVC/plastic than in copper systems, if certain types of microbes such as nitrifiers proliferate and drop pH. Some of the problems initiated by construction practices can persist indefinitely, causing higher levels of lead and copper in water, or longer term, contributing to failures of the plumbing system. Blue water from high copper concentrations is a confounding problem that continues to arise in some locales of the United States. One public elementary school in Miami Dade County is experiencing blue water issues as manifested by blue ice cubes and sink staining. In addition to the aesthetic problems, copper levels are above the EPA's Copper Action Level of 1.3 ppm. Bottled water has been substituted for tap water consumption, which has created a financial burden. The pH of the school's water ranges from 7.15 - 7.5 and the school itself is located 1 ½ miles off the main distribution line resulting in a very low chlorine residual of between 0.06 mg/L Cl2 and 0.18 mg/L Cl2. On site water was shipped to Virginia Tech from Miami to be used in this study. Preliminary testing showed that an increase in the pH of the water would decrease copper leaching. Several pH's were tested which revealed that increasing the pH of the water to 8.5 would drop copper below 1.3 mg/L. When these recommendations were implemented at the school, the high alkalinity and calcium rich water caused calcite scales to form which clogged the chemical feed nozzles. Further bench scale testing indicated that adding 2 mg/L orthophosphate corrosion inhibitor would effectively decrease copper to a level that would comply with the EPA's Copper Action Limit. Orthophosphate corrosion inhibitors are used by utilities to limit lead and copper corrosion from consumer's plumbing. An evaluation comparing the effects of both 100% orthophosphate inhibitor and orthophosphate/polyphosphate inhibitor blends was performed to study the effects they have on galvanic corrosion, metallic corrosion, microbial growth and the decay of chloramine disinfectant. On site water was sent to Virginia Tech from UNC for use in this bench scale study. The results from this study indicated that 100% orthophosphate inhibitor was the most effective corrosion inhibitor at decreasing metallic corrosion. It has long been known that microbial activity can have significant effects on water quality. This study evaluated nitrifying and heterotrophic bacterial growth in water systems containing copper pipes, a common plumbing product, and flux which is used in soldering copper pipes together in new construction. There are several types of commercially available fluxes which are often used when soldering new pipes together. Flux ingredients vary and can include extremely high concentrations of ammonia, zinc, chloride, tin, copper and TOC. Flux containing high amounts of ammonia can be detrimental to water quality because it can accelerate the occurrence of nitrification, thus creating a cascading set of problems including, but not limited to, pH decrease and copper corrosion. The results from this case study indicated that flushing a pipe system can effectively decrease the high concentrations of flux present in a new construction system; however, high levels of ammonia from flux can create an environment in which nitrifiers may proliferate within the system. Many water utilities in the United States are switching disinfection type from chlorine to chloramine due to the increased stability, longer residual time, and overall safety benefits of chloramine. Although chloramines have been found to be a desirable means for disinfection, chloramine decay is an issue of great concern because if the chloramine residual decays, it can leave a water system unprotected against microbial infestation. A preliminary examination of this issue was performed in a laboratory setting to evaluate the many components that effect the stability of chloramine decay, including alkalinity, phosphate, temperature, and various pipe materials. The results from this experiment revealed that temperature increase, pH increase, and aged tygon tubing all accelerated the rate of chloramine decay. / Master of Science