Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization. In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization. It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice. Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly. Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses. Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation. In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.

Ces travaux de thèse avaient pour objectifs (i) d’étudier le comportement du fer et du nickel au cours de la biominéralisation de sulfures de fer par des bactéries sulfato-réductrices et (ii) de tenter une première estimation de l’influence de l’exploitation minière sur les communautés microbiennes des sédiments de mangrove de Nouvelle-Calédonie. Pour atteindre ces objectifs, des expériences d’incubation ont été conduites en anoxie avec une espèce unique de bactérie (thio)sulfato-réductrice (i.e. Desulfovibrio capillatus) et avec un consortium de bactéries sulfato-réductrices natives de sédiments de mangrove de Nouvelle-Calédonie. Ces expériences ont été réalisées avec différentes sources de Fe(III) (i.e. goethite, ferrihydrite et citrate-ferrique) et en présence de nickel structural ou en solution. Les résultats montrent que l’activité bactérienne sulfato-réductrice (qu’elle soit synergique ou issue d’une espèce unique) conduit, dans toutes les expériences, à la formation principale de mackinawite (FeS). Ce sulfure de fer précipite sous forme de cristallites nanométriques et dont la cristallinité augmente avec la durée d’incubation. Lorsque le nickel est présent en solution, la quasi-totalité de cet élément peut se substituer au fer (i.e. substitution 4% molaire) dans la structure de la mackinawite. Ainsi, la formation d’une faible proportion de mackinawite permet de fixer la quasi-totalité du nickel initialement en solution (e.g. ratio FeS:Ni de 1). Ce mécanisme semble stable sur le long terme (pas de relargage de nickel en solution) et il accélère la croissance cristalline de la mackinawite, ce qui engendre une stabilité accrue de ce minéral. Ces résultats soulignent le rôle efficace des bactéries sulfato-réductrices dans la formation des sulfures de fer de type mackinawite et dans le piégeage du nickel, suggérant une stabilisation de cet élément dans les sédiments de mangrove et la limitation de sa biodisponibilité. Ceci pourrait expliquer les résultats de l’étude comparative des consortiums bactériens autochtones qui ne permet pas de déceler d’impact de l’activité minière sur les communautés bactériennes sulfato-réductrices présentes en Nouvelle-Calédonie. / The aims of the present work were (i) to study the behavior of iron and nickel in the biomineralization of iron sulfides by (thio)sulfate-reducing bacteria and (ii) to estimate the influence of open-cut mining activities on microbial communities development in mangrove sediments in New Caledonia. To achieve these objectives, incubation experiments were conducted under anoxic conditions with the (thio)sulfate-reducing bacteria (i.e. Desulfovibrio capillatus) and a consortium of sulfate-reducing bacteria native mangrove sediments of New Caledonia. These experiments were carried out with different Fe(III) precursors (i.e. goethite, ferrihydrite and ferric citrate) and in the presence of structural or soluble nickel. The results show that the sulfate-reducing bacterial activity leads, in all experiments, to the formation of mackinawite (FeS). This iron sulfide precipitates as nanosized crystallites that increase in size with incubation time. When nickel is present in solution, the total soluble amount can be substituted to iron (i.e. replacing 4 mol%) in the structure of mackinawite. Thus, the formation of a small proportion of mackinawite scavenged total soluble amount of nickel initially present in solution (e.g. FeS:Ni ratio of 1). This sequestration mechanism appears to be stable over time (no nickel was released in solution) and accelerates the crystal growth of mackinawite, leading to the stabilization of this mineral. These results highlight the effective role of sulfate-reducing bacteria in the biomineralization of iron sulfides such as mackinawite and in the sequestration of nickel, suggesting a stabilization of this element in mangrove sediments and limitation of its bioavailability. These results could explain the absence of negative impact of open-cut mining activities on the sulfate-reducing bacterial communities present in New Caledonia.

The objectives of this study were to investigate the long-term effects of feeding increasing supplemental levels (20, 120, or 220 mg/kg) of dietary copper (Cu) as tribasic copper chloride (TBCC) or copper sulfate (CuSO4) on performance, antioxidant status, nutrient digestibility, and trace mineral deposition of sows and piglets; as well as to assess nursery dietary Cu levels on growth performance and response to immunological challenge in nursery pigs from sows fed either high or low Cu diets. In the long-term sow experiment, sows fed TBCC diets had greater adjusted weaning weight for litter and piglet (P < 0.10), as well as adjusted litter and piglet weight gain (P < 0.10) when compared to sows that received CuSO4 diets. Increasing dietary Cu level linearly increased live born piglet weight (P = 0.06). Sows fed TBCC diets had lower apparent total tract digestibility (ATTD) of ether extract (P = 0.01) during late gestation, but greater ATTD of dry matter, nitrogen, and phosphorous during lactation (P < 0.05). Increasing Cu levels linearly increased dry matter digestibility in lactating sows (P = 0.02). Milk from sows fed TBCC diets had a greater concentration of protein (P = 0.02) than that from sows fed CuSO4 diets. Increasing Cu levels increased levels of milk fat and Cu (linear, P < 0.05); but linearly decreased lactose and Zn levels (P < 0.05). Lactating sows fed TBCC diets had a greater activity of Cu/Zn superoxide dismutase (SOD) and ceruloplasmin in serum than those fed CuSO4 diets (P < 0.05). Increasing dietary Cu levels increased total and Cu/Zn SOD activity for lactating sows (linear, P < 0.05). Sows fed TBCC diets had lower concentrations of Cu (P = 0.04), but higher concentrations of iron and manganese (P < 0.05) in the liver, when compared to those fed with CuSO4 diets. In addition, liver Cu concentrations increased with increasing dietary Cu levels (linear and quadratic, P < 0.05). Increasing dietary Cu levels resulted in the elevation of concentrations and contents of Cu in the liver of weanling piglets (linear, P < 0.0001). In the nursery pig experiment, pigs from sows fed 120 mg/kg Cu diets had greater ADG from d 0 to 14 (P < 0.05), and tended to have greater ADG in the overall period (P < 0.08), when compared to pigs from sows fed 20 mg/kg Cu diets. During the lipopolysaccharide challenge period, the challenged pigs from sows fed 120 mg/kg Cu had a greater overall rectal temperature than those from sows fed 20 mg/kg Cu (P = 0.01). Also, the challenged pigs fed with 220 mg/kg Cu diets had greater serum tumor necrosis factor-alpha concentration over time as compared to those fed 20 mg/kg Cu diets (P = 0.03). In summary, the TBCC may be a superior Cu source compared to CuSO4 regarding reproductive performance, and higher dietary Cu levels result in greater birth weight of piglets; furthermore, high Cu levels in sow and nursery diets promote growth performance of nursery pigs and affects their responses to immunological challenge.

Semi-volatile lower chlorinated PCBs (LC-PCBs), comprised of mostly tetra or lower chlorinated congeners, are detected in old buildings and outdoors in high concentrations. PCBs cause endocrine disruption, neurotoxicity and many other adverse effects, and are human carcinogens. Epidemiological studies so far have relied upon serum concentrations of PCBs or OH-PCBs as markers of exposure. Despite the detection of LC-PCBs in high levels in buildings and other environments, only a few studies have reported LC-PCBs in human serum. One of the reasons for low serum detection of LC-PCBs could be the further biotransformation and excretion of OH-PCBs from the body. Therefore, the objective of this dissertation research was to study the metabolism of one of the LC-PCBs in rats and to identify a suitable metabolite marker of inhalation exposure for a future epidemiological study in humans. We chose PCB3 (4-chlorobiphenyl), a mono chlorinated PCB found in high concentrations in many buildings, as a model to study the metabolism. The first aim was to identify final metabolites of PCB3 in vivo. Male Sprague-Dawley rats were held in metabolism cages following exposure to PCB3 via i.p. injection. Blood, urine and feces were collected, and an analytical method for extraction of PCB3 metabolites was developed. By identifying the metabolites by LC/MS, a complete biotransformation pathway for PCB3 was elucidated. Major urinary metabolites of PCB3 were sulfates and mercapturates, while glucuronides and free phenolic forms were minor. The second aim was to study the disposition and toxicity of phenolic and sulfate metabolites after inhalation exposure to PCB3 in female Sprague-Dawley rats. Airborne PCB3 vapor was generated in a flask and passed through an inhalation exposure system, where rats were exposed via nose-only inhalation. Both hydroxylated and sulfated metabolites were detected in serum, liver, lungs, and brain. The serum clearance half-lives of these metabolites were less than two hours. Serum chemistry parameters were similar in PCB3 exposed and control rats. As a marker of bioactivation of PCB3 to electrophilic species, 8-oxo-dG was quantified in urine, but the difference was not statistically significant between control and exposed rats. The third aim was to study the routes of excretion of metabolites after inhalation to airborne PCB3. Bile cannulated and intact rats were exposed to PCB3 via nose only inhalation. Metabolites detected in bile were mostly sulfates and some glucuronide. Fecal metabolites were exclusively phenols despite very low concentration of free phenolic forms in bile. Peak excretion of metabolites in feces and urine occurred within 24 h, and over sixty percent of the dose was recovered within 24 h. The major route of excretion of PCB3 was urine in the form of sulfated metabolites. Sulfated metabolites were stable in urine for a month without aid of any preservatives. This dissertation shows that PCB3 is rapidly metabolized to phenols and conjugated mostly to sulfates. Conjugated metabolites, elaborated into bile, are either reabsorbed or hydrolyzed in the gut and excreted in feces as phenolic forms. Serum concentrations of sulfates are higher than free phenolic forms at any time after exposure, and are also detected in lungs and brain. PCB3 sulfates are stable metabolites and can serve as a metabolite marker of inhalation exposure to PCB3.

Glycosaminoglycans (GAGs) are sulfated polysaccharides that mediate a variety of extracellular interactions. Heparan sulfate (HS) is one of the most prominent GAGs on human cell surfaces. Both endogenous proteins, such as growth factors, and exogenous proteins, such as pathogen surface proteins, recognize and bind GAGs to gain access to human cells. Oligosaccharides and other structural analogs of HS and GAGs have been evaluated for a variety of therapeutic targets including angiogenesis and infectious diseases. Development of compounds to block HS-protein interactions has primarily focused on optimizing the degree and orientation of anionic substituents on a scaffold, to mimic HS structure, but their utility is diminished by their large size and non-specific interactions with many proteins. To overcome these limitations, it has been demonstrated that replacing N-sulfo groups on heparin with non-anionic N-arylacyl groups increased affinity and selectivity for binding different heparin-binding proteins. However, the heparin-derived compounds in that work were heterogeneous polysaccharides. Strategies to obtain small, structurally-defined and lower charge ligands are needed to ultimately obtain specific bind-and-block antagonists of HS-binding proteins. This study addresses these challenges by synthesizing N-arylacyl O-sulfonated aminoglycosides as small molecule, structurally-defined ligands to identify novel structures that selectively bind to HS-binding proteins. This study details development of new HPLC and LC-MS methods to separate, characterize, and purify amphiphilic oligosaccharides. The development of these methods enabled the synthesis of a panel of N-arylacyl O-sulfonated aminoglycosides. The compounds in this panel were screened for affinity and selectivity in binding with HS-binding proteins. This work demonstrates for the first time the selective binding of small amphiphilic oligosaccharides with HS-binding proteins. Significantly, individual compounds demonstrate heparin-like affinity for binding with select HS-binding proteins. Structural differences between the N-arylacyl O-sulfonated aminoglycosides, including changing the aminoglycoside core or the structure of the N-arylacyl moiety, are shown to impart specificity for these compounds to selectively bind different HS-binding proteins.

Common methods for determining mineral absorption by the body are invasive and frequently utilize radioisotopes. In experiment one. rats were given a dose of radiolabeled ferrous sulfate. Relative absorption was estimated by changes in serum iron and by appearance of the radiotracer in the serum. There were no differences in relative absorption determined by the methods, although the low overall absorptions by the iron-replete rats may have decreased the sensitivity. In experiments two and three. iron and magnesium supplements were given orally to 12 women age 19-25. Each subject received iron and magnesium supplements once each week for 5 weeks. Blood samples were taken via venous catheter every 30 minutes for 2 hours and 30 minutes following dosing, and a urine sample was taken following the collection time period. Samples were analyzed for serum iron, serum magnesium, hematocrit, ferritin, urinary magnesium, and urinary creatinine. The increase in serum iron was evaluated from both the peak increase in serum iron and from the integrated increase in serum iron over the blood collection time. The data for each supplement were compared by analysis of variance. For the iron supplements. the taste-free iron supplement increased serum iron less than either the Ferrochel or the ferrous sulfate supplements. When the iron absorption was then compared to ferritin stores (low, medium, and high), the relative absorption of Ferrochel was higher in the low ferritin range (0-15 ng/ml) than in the upper ranges (P=.00l for peak and P=.0002 for area). Relative absorption from Ferrochel iron was also higher than the other supplements for subjects with low ferritin stores. Neither serum nor urinary magnesium values differed significantly among the three compounds examined. Serum magnesium values are stable in healthy individuals, and the urinary magnesium data were not evaluated over 24 h as in typical magnesium load tests. In summary, both the ferrous sulfate and Ferrochel supplements were absorbed more efficiently than the taste-free iron supplement. The Ferrochel was also absorbed more efficiently in individuals with low iron stores, demonstrating better regulation by the body than with the other supplements examined.

nitrogen has commonly been a deficient element in the cultivated soils of the world since the beginning of agriculture. The general acceptance of the practice of using manures as a means of increasing plant growth, as shown by the records of ancient civilizations, attest to this fact. Since the time of von Liebig there has been an increasing awareness of the importance of this deficiency in soils. As a result of a better understanding of the problem and the increasing availability of commercial forms of nitrogen, a rapid increase in the use of nitrogen fertilizers has taken place in the last few decades. this increasing use of commercial forms of nitrogen is accompanied by the need for more information concerning the proper use of these materials in order to accomplish the greatest benefit. For example, with the advent of increasing use of ammonium fertilizer to improve soil productivity, there has arisen a possibility of lengthening the period from the date of the fertilizer application to the time of utilization by the crop. this advanced application, especially in the fall of the year, has many advantages and is widely advocated. The following questions need to be answered in connection with efficiency of such a practice: Will the ammonium form of nitrogen remain unoxidized in the soil over the winter months? Could it be only partially oxidized and result in an accumulation of nitrites that may cause toxicity or be lost from the soil in gaseous forms such as nitrous oxide? What are the chance for significant losses by volatilization before it is oxidized? How does the amount of moisture and the prevailing temperature affect these transformations? The study reported here is an endeavor to contribute to more complete answers to some of these questions. Although much research has been conducted relating to the effects of moisture or temperature on nitrogen transformations in the soil, more information is needed covering greater variations in the moisture and temperature levels along with the interactions of these. Accordingly, experiments were conducted under carefully controlled conditions to measure the changes occurring in the inorganic soil nitrogen from an applied ammonium source, at various moisture and temperature levels.

The objective of this investigation was to assess the influence of the SO3 content on the durability and strength of portland cement. Four portland cements were used in this study. The cements had a variable tricalcium silicate, tricalcium aluminate, and alkali contents, as well as differences in the amount and form of calcium sulfates. The SO3 content of the cements was increased by replacing part of the cement by gypsum according to ASTM C 452-95. Mortar bars and cubes were prepared for the as-received as well as for the cements with an SO3 content of 3.0% and 3.6%. The durability of the as-received and doped cements was determined by measuring the length change of the mortar bars that were exposed to sodium sulfate environment. The compressive strength of the mortar cubes prepared for the same mixes was measured at different ages for sets of cubes cured both in sodium sulfate solution and in saturated lime solution. It was concluded at the end of this study that there is an optimum SO3 content for the lowest expansion that is different from that determined for the highest compressive strength. Optimum values also differed from one cement to another and from one age to another for the same cement. The results also indicate the dependence of SO3 content on tricalcium aluminate and alkali content of cements. In addition, for all cements examined in this study with alkali content of less than 0.60%, increasing the SO3 content above 3.0% had negative effects on durability assessed by strength or expansion measurements. For the cement with highest alkali and tricalcium aluminate content, increasing the SO3 content from 3.0% to 3.6% delayed the onset of strength drop; however, at 360 days the strength drop experienced by both doping levels was the same.

In an effort to make structures more sustainable and durable, supplementary cementitious materials are often used to replace cement. Ground granulated blast furnace slag, for instance, is an industrial by-product of iron refinement and is frequently used in concrete mixture design to not only reduce cost, but also increase later-age strength as well as durability. However, published literature indicates that slags with a high alumina content may have a detrimental effect when concrete is exposed to a sulfate environment. ASTM standard C989 does not suggest any information or guidelines regarding using slags with an alumina content between 11-18%. Therefore, the objective of this study was to fill in the gap of this standard by studying slags of variable alumina content as high as 16 percent. This study presents data collected for compressive strengths of mortar cubes exposed to lime and 5 percent sodium sulfate solution at ages of 7, 28, 91, and 182 days from the date of mixing as well as expansion data for mortar bar specimens exposed to 5 percent sodium sulfate solution up to 120 days. Slag replacement levels used here were 0, 30, 50, and 70%. Mortar bar specimens showing deterioration were analyzed using x-ray diffraction coupled with Rietveld refinement to assess the mechanism of deterioration. Cubes were stored in lime and sulfate solutions abiding by ASTM C1012 in order to analyze the resistance to sulfate attack. Sulfate resistance was measured in terms of decalcification of the CSH gel as well as expansion. The results suggest using high alumina slags at a low percentage adversely affects sulfate resistance since the acquired strength at 182 days fell below that of 28 day strength, which is often used in the industry as the parameter which constitutes whether a mixture is adequate. It was also seen that increasing alumina content of the slag resulted in increased expansion. X-ray diffraction analysis indicates that the mechanism of deterioration, of the control as well as the blended mortar, is due to secondary gypsum and secondary ettringite formation. Therefore, it is recommended that slags having a high alumina content should be further analyzed in laboratory tests to examine their performance especially if concrete will be subjected to a sulfate environment during its service life.

Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.