Analysis of fracture healing in osteopenic bone caused by disuse: experimental study

Osteoporosis has become a serious global public health issue. Hence, osteoporotic fracture healing has been investigated in several previous studies because there is still controversy over the effect osteoporosis has on the healing process. The current study aimed to analyze two different periods of bone healing in normal and osteopenic rats. Sixty, 7-week-old female Wistar rats were randomly divided into four groups: unrestricted and immobilized for 2 weeks after osteotomy (OU2), suspended and immobilized for 2 weeks after osteotomy (OS2), unrestricted and immobilized for 6 weeks after osteotomy (OU6), and suspended and immobilized for 6 weeks after osteotomy (OS6). Osteotomy was performed in the middle third of the right tibia 21 days after tail suspension, when the osteopenic condition was already set. The fractured limb was then immobilized by orthosis. Tibias were collected 2 and 6 weeks after osteotomy, and were analyzed by bone densitometry, mechanical testing, and histomorphometry. Bone mineral density values from bony calluses were significantly lower in the 2-week post-osteotomy groups compared with the 6-week post-osteotomy groups (multivariate general linear model analysis, P<0.000). Similarly, the mechanical properties showed that animals had stronger bones 6 weeks after osteotomy compared with 2 weeks after osteotomy (multivariate general linear model analysis, P<0.000). Histomorphometry indicated gradual bone healing. Results showed that osteopenia did not influence the bone healing process, and that time was an independent determinant factor regardless of whether the fracture was osteopenic. This suggests that the body is able to compensate for the negative effects of suspension.

The role of organic solvent amount in the lipase-catalyzed biodiesel production

This research note addresses the role of organic solvent amount in the production of fatty acid ethyl esters from soybean oil. N-hexane was chosen as solvent and two commercial immobilized lipases as catalysts, Novozym 435 and Lipozyme IM. The reactions were conducted in 6 hours, varying the solvent to oil ratio from zero to 50 (v/wt) and adopting adopting for Novozym 435: 65 ��C, enzyme concentration (E, wt%) = 5, oil to ethanol molar ratio (R) = 1:10, water addition (H, wt%) = 0, and for Lipozyme IM: 35 ��C, E = 5 wt%, R = 1:3, H = 10 wt%. For Lipozyme IM, an increase in solvent amount is shown to lead to an enhancement of reaction conversion, while a negligible effect was found for Novozym 435. When using 30 mL of solvent the reaction conversions were 88% for Lipozyme IM and 15% for Novozym 435.

Biosensor for determination of glucose in real samples of beverages

A biosensor was developed for spectrophotometric determination of glucose concentrations in real samples of orange juice energetic drinks, and sport drinks. The biosensor consisted of glucose oxidase (GOD) and horseradish peroxidase (HRP) immobilized onto polyaniline activated with glutaraldehyde (PANIG). Immobilization parameters were optimized for GOD, and maximum immobilization yield was 16% when 5.0 mg of PANIG and 8.9 U prepared in 0.1 mol.L-1 sodium phosphate buffer (pH 7.0) reacted for 60 minutes at 4 ��C with gentle stirring. The linear operational range for glucose determination using optimized operational parameters was between 0.05 and 6.0 mg.mL-1 with a very good reproducibility of response. The results obtained in the biosensor were compared with those obtained using free enzymes (commercial kits) and then validated through statistical analysis using the Tukey test (95% confidence interval).

The role of metal ions in selective and sustainable processes

Sustainability and recycling are core values in today���s industrial operations. New materials, products and processes need to be designed in such a way as to consume fewer of the diminishing resources we have available and to put as little strain on the environment as possible. An integral part of this is cleaning and recycling. New processes are to be designed to improve the efficiency in this aspect. Wastewater, including municipal wastewaters, is treated in several steps including chemical and mechanical cleaning of waters. Well-cleaned water can be recycled and reused. Clean water for everyone is one of the greatest challenges we are facing today. Ferric sulphate, made by oxidation from ferrous sulphate, is used in water purification. The oxidation of ferrous sulphate, FeSO4, to ferric sulphate in acidic aqueous solutions of H2SO4 over finely dispersed active carbon particles was studied in a vigorously stirred batch reactor. Molecular oxygen was used as the oxidation agent and several catalysts were screened: active carbon, active carbon impregnated with Pt, Rh, Pd and Ru. Both active carbon and noble metal-active carbon catalysts enhanced the oxidation rate considerably. The order of the noble metals according to the effect was: Pt >> Rh > Pd, Ru. By the use of catalysts, the production capacities of existing oxidation units can be considerably increased. Good coagulants have a high charge on a long polymer chain effectively capturing dirty particles of the opposite charge. Analysis of the reaction product indicated that it is possible to obtain polymeric iron-based products with good coagulation properties. Systematic kinetic experiments were carried out at the temperature and pressure ranges of 60B100��C and 4B10 bar, respectively. The results revealed that both non-catalytic and catalytic oxidation of Fe2+ to Fe3+ take place simultaneously. The experimental data were fitted to rate equations, which were based on a plausible reaction mechanism: adsorption of dissolved oxygen on active carbon, electron transfer from Fe2+ ions to adsorbed oxygen and formation of surface hydroxyls. A comparison of the Fe2+ concentrations predicted by the kinetic model with the experimentally observed concentrations indicated that the mechanistic rate equations were able to describe the intrinsic oxidation kinetics of Fe2+ over active carbon and active carbon-noble metal catalysts. Engineering aspects were closely considered and effort was directed to utilizing existing equipment in the production of the new coagulant. Ferrous sulphate can be catalytically oxidized to produce a novel long-chained polymeric iron-based flocculent in an easy and affordable way in existing facilities. The results can be used for modelling the reactors and for scale-up. Ferric iron (Fe3+) was successfully applied for the dissolution of sphalerite. Sphalerite contains indium, gallium and germanium, among others, and the application can promote their recovery. The understanding of the reduction process of ferric to ferrous iron can be used to develop further the understanding of the dissolution mechanisms and oxidation of ferrous sulphate. Indium, gallium and germanium face an ever-increasing demand in the electronics industry, among others. The supply is, however, very limited. The fact that most part of the material is obtained through secondary production means that real production quota depends on the primary material production. This also sets the pricing. The primary production material is in most cases zinc and aluminium. Recycling of scrap material and the utilization of industrial waste, containing indium, gallium and geranium, is a necessity without real options. As a part of this study plausible methods for the recovery of indium, gallium and germanium have been studied. The results were encouraging and provided information about the precipitation of these valuables from highly acidic solutions. Indium and gallium were separated from acidic sulphuric acid solutions by precipitation with basic sulphates such as alunite or they were precipitated as basic sulphates of their own as galliunite and indiunite. Germanium may precipitate as a basic sulphate of a mixed composition. The precipitation is rapid and the selectivity is good. When the solutions contain both indium and gallium then the results show that gallium should be separated before indium to achieve a better selectivity. Germanium was separated from highly acidic sulphuric acid solutions containing other metals as well by precipitating with tannic acid. This is a highly selective method. According to the study other commonly found metals in the solution do not affect germanium precipitation. The reduction of ferric iron to ferrous, the precipitation of indium, gallium and germanium, and the dissolution of the raw materials are strongly depending on temperature and pH. The temperature and pH effect were studied and which contributed to the understanding and design of the different process steps. Increased temperature and reduced pH improve the reduction rate. Finally, the gained understanding in the studied areas can be employed to develop better industrial processes not only on a large scale but also increasingly on a smaller scale. The small amounts of indium, gallium and germanium may favour smaller and more locally bound recovery.

A comparative study of protoheme and heme d catalases : role of the heme and the heme pocket in catalysis and ligand binding

Catalase dismutes H20 2 to O2 and H20. In successive twoelectron reactions H20 2 induces both oxidation and reduction at the heme group. In the first step the protoheme prosthetic group of beef liver catalase forms compound I, in which the heme has been oxidized from Fe3+ to Fe4+=0 and a porphyrin radical has been created. Compound II is formed by the oneelectron reduction of comp I. It retains Fe4+=0 but lacks the porphyrin radical and is catalytically inert. Molecular structures are available for Escherichia coli Hydroperoxidase II, Micrococcus Iysodeiktus, Penicillium vitale and beef liver enzymes, which contain different hemes and heme pockets. In the present work, the pockets and substrate access channels of protoheme (beef liver & Micrococcus) and heme d (HPII of E. coli and Penicillium) catalases have been analysed using Quanta��� and CharmMTM molecular modeling packages on the Silicon Graphics Iris Indigo 2 computer. Experimental studies have been carried out with two catalases, HPII (and its mutants) and beef liver. Fluoride and formate' are inhibitors of both enzymes, and their binding is modulated by the heme and by distal residues N201 & H128. Both HPII and beef liver enzymes form compound I with H202 or peracetate. The reduction of beef liver enzyme compound I to II and the decay of compound II are accelerated by fluoride. The decay of compound II is also accelerated by formate, and this reagent acts as a 2-electron donor towards compound I of both enzymes. It is concluded that heme d enzymes (Penicillium and HPII of E. coli) are formed by autocatalytic transformation of protoheme in a modified pocket which contains a characteristic serine residue as well as a partially occluded heme channel. They are less active than protoheme enzymes but also do not form the inactive compound II species. Binding of peroxide as well as fluoride and formate is prevented by mutation of H128 and modulated by mutation of N201.

Investigations in flow injection analysis (FIA): conventional FIA determination of chloride in water; sequential injection (SI)-FIA determination of mercury in water

Flow injection analysis (FIA) was applied to the determination of both chloride ion and mercury in water. Conventional FIA was employed for the chloride study. Investigations of the Fe3 +/Hg(SCN)2/CI-,450 nm spectrophotometric system for chloride determination led to the discovery of an absorbance in the 250-260 nm region when Hg(SCN)2 and CI- are combined in solution, in the absence of iron(III). Employing an in-house FIA system, absorbance observed at 254 nm exhibited a linear relation from essentially 0 - 2000 Jlg ml- 1 injected chloride. This linear range spanning three orders of magnitude is superior to the Fe3+/Hg(SCN)2/CI- system currently employed by laboratories worldwide. The detection limit obtainable with the proposed method was determin~d to be 0.16 Jlg ml- 1 and the relative standard deviation was determined to be 3.5 % over the concentration range of 0-200 Jig ml- 1. Other halogen ions were found to interfere with chloride determination at 254 nm whereas cations did not interfere. This system was successfully applied to the determination of chloride ion in laboratory water. Sequential injection (SI)-FIA was employed for mercury determination in water with the PSA Galahad mercury amalgamation, and Merlin mercury fluorescence detection systems. Initial mercury in air determinations involved injections of mercury saturated air directly into the Galahad whereas mercury in water determinations involved solution delivery via peristaltic pump to a gas/liquid separator, after reduction by stannous chloride. A series of changes were made to the internal hardware and valving systems of the Galahad mercury preconcentrator. Sequential injection solution delivery replaced the continuous peristaltic pump system and computer control was implemented to control and integrate all aspects of solution delivery, sample preconcentration and signal processing. Detection limits currently obtainable with this system are 0.1 ng ml-1 HgO.

ORGANOSILICON BIOTECHNOLOGY: A BIO-INSPIRED APPROACH TO THE HYDROLYSIS OF ALKOXYSILANES and THE LIPASE-CATALYZED SYNTHESIS OF SILOXANE-CONTAINING POLYESTERS AND POLYAMIDES

The first part of this thesis studied the capacity of amino acids and enzymes to catalyze the hydrolysis and condensation of tetraethoxysilane and phenyltrimethoxysilane. Selected amino acids were shown to accelerate the hydrolysis and condensation of tetraethoxysilane under ambient temperature, pressure and at neutral pH (pH 7��0.02). The nature of the side chain of the amino acid was important in promoting hydrolysis and condensation. Several proteases were shown to have a capacity to hydrolyze tri- and tet-ra- alkoxysilanes under the same mild reaction conditions. The second part of this thesis employed an immobilized Candida antarctica lipase B (Novozym-435, N435) to produce siloxane-containing polyesters, polyamides, and polyester amides under solvent-free conditions. Enzymatic activity was shown to be temperature dependent, increasing until enzyme denaturation became the dominant pro-cess, which typically occurred between 120-130���C. The residual activity of N435 was, on average, greater than 90%, when used in the synthesis of disiloxane-containing polyesters, regardless of the polymerization temperature except at the very highest temperatures, 140-150���C. A study of the thermal tolerance of N435 determined that, over ten reaction cycles, there was a decrease in the initial rate of polymerization with each consecutive use of the catalyst. No change in the degree of monomer conversion after a 24 hour reaction cycle was found.

Tetrathiafulvalene and 2,2'-Bipyridine: Bridging both Worlds in the Pursuit of Novel Molecular Materials

The preparation and characterization of two families of building blocks for molecule-based magnetic and conducting materials are described in three projects. In the first project the synthesis and characterization of three bis-imine ligands LI - L3 is reported. Coordination of LI to a series of metal salts afforded the five novel coordination complexes Sn(L4)C4 (I), [Mn(L4)(u-CI)(CI)(EtOH)h (II), [CU(L4)(u-sal) h(CI04)2 (sal = salicylaldehyde anion) (III), [Fe(Ls)2]CI (IV) and [Fe(LI)h(u-O) (V). All complexes have been structurally and magnetically characterized. X-ray diffraction studies revealed that, upon coordination to Lewis acidic metal salts, the imine bonds of LI are susceptible to nucleophilic attack. As a consequence, the coordination complexes (I) - (IV) contain either the cyclised ligand L4 or hydrolysed ligand Ls. In contrast, the dimeric Fe3+ complex (V) comprises two intact ligand LI molecules. In. this complex, the ligand chelates two Fe(III) centres in a bis-bidentate manner through the lone pairs of a phenoxy oxygen and an imine nitrogen atom. Magnetic studies of complexes (II-V) indicate that the dominant interactions between neighbouring metal centres in all of the complexes are antiferromagnetic. In the second project the synthesis and characterization two families of TTF donors, namely the cyano aryl compounds (VI) - (XI) and the his-aryl TTF derivatives (XII) - (XIV) are reported. The crystal structures of compounds (VI), (VII), (IX) and (XII) exhibit regular stacks comprising of neutral donors. The UV -Vis spectra of compounds (VI) - (XIV) present an leT band, indicative of the transfer of electron density from the TTF donors to the aryl acceptor molecules. Chemical oxidation of donors (VI), (VII), (IX) and (XII) with iodine afforded a series of CT salts that where possible have been characterized by single crystal X -ray diffraction. Structural studies showed that the radical cations in these salts are organized in stacks comprising of dimers of oxidized TTF donors. All four salts behave as semiconductors, displaying room temperature conductivities ranging from 1.852 x 10-7 to 9.620 X 10-3 Scm-I. A second series of CT salts were successfully prepared via the technique of electrocrystallization. Following this methodology, single crystals of two CT salts were obtained. The single crystal X-ray structures of both salts are isostructural, displaying stacks formed by trimers of oxidized donors. Variable temperature conductivity measurements carried out on this series of CT salts reveal they also are semiconductors with conductivities ranging from 2.94 x 10-7 to 1.960 X 10-3 S em-I at room temperature. In the third project the synthesis and characterization of a series of MII(hfac)2 coordination complexes of donor ligand (XII) where M2+ = Co2+, Cu2+, Ni2+ and Zn2+ are reported. These complexes crystallize in a head-to-tail arrangement of TTF donor and bipyridine moieties, placing the metal centres and hfac ligands are located outside the stacks. Magnetic studies of the complexes (XV) - (XVIII) indicate that the bulky hfac ligands prevent neighbouring metal centres from assembling in close proximity, and thus they are magnetically isolated.

Animals without Borders: Farmed Animal Resistance in New York

While billions of farmed animals are immobilized within agribusiness, every year some of these animals manage to break free. This thesis examines the stories of those who flee slaughterhouses and the public response to these individuals. My objective is to understand how animals resist and the role that their stories play in disrupting the ways that humans, particularly as consumers, are distanced from the violence of animal enterprises. Included are six vignettes that allow for an in-depth case study of those who have escaped within New York State. Located in the interdisciplinary field of critical animal studies, my inquiry draws upon new animal geographies, transnational feminisms, and critical discourse analysis. This contribution provides discussion of farmed animal resistance in particular and compares experiences and representations of their resistance from both the ���view from below,��� which is learned through the animals��� caretakers, and a ���view from above,��� which is gleaned from their representations in corporate-driven mainstream media.

Lipase-Mediated Enzymatic Catalysis For The Synthesis of New Chiral Polymers

Immobilized lipase B from Candida antarctica (N435) was investigated as a potential biocatalyst to generate silicone-based chiral polymers from monomers derived from the enzymatic dihydroxylation of bromobenzene. Several conditions and parameters have been investigated for this purpose and lipase transesterification preference to each of the free secondary alcohols in the chiral monomers was documented. The N435 was challenged with a series of substrates where the free alcohol moieties were systematically protected in order to study the substrate preference(s) for the transesterification reactions.

Cyclotetrasiloxane Frameworks for the Chemoenzymatic Synthesis of Oligoesters

Immobilized lipase B from Candida antarctica (Novozym�� 435, N435) was utilized as part of a chemoenzymatic strategy for the synthesis of branched polyesters based on a cyclotetrasiloxane core in the absence of solvent. Nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were utilized to monitor the reactions between tetraester cyclotetrasiloxanes and aliphatic diols. The enzyme-mediated esterification reactions can achieve 65��� 80% consumption of starting materials in 24���48 h. Longer reaction times, 72���96 h, resulted in the formation of cross-linked gel-like networks. Gel permeation chromatography of the polymers indicated that the masses were Mw �� 11 400, 13 100, and 19 400 g mol 1 for the substrate pairs of C7D4 ester/ octane-1,8-diol, C10D4 ester/pentane-1,5-diol and C10D4 ester/octane-1,8-diol respectively, after 48 h. Extending the polymerization for an additional 24 h with the C10D4 ester/octane-1,8-diol pair gave Mw �� 86 800 g mol 1. To the best of our knowledge this represents the first report using lipase catalysis to produce branched polymers that are built from a cyclotetrasiloxane core.

The Ligand and Membrane-binding Behaviour of the Phosphatidylinositol Transfer Proteins (PITPa & PITPb)

Human Class I phosphatidylinositol transfer proteins (PITPs) exists in two forms: PITP�� and PITP��. PITPs are believed to be lipid transfer proteins based on their capacity to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments in vitro. In Drosophila, the PITP domain is found to be part of a multi-domain protein named retinal degeneration B (RdgB��). The PITP domain of RdgB�� shares 40 % sequence identity with PITP�� and has been shown to possess PI and PC binding and transfer activity. The detailed molecular mechanism of ligand transfer by the human PITPs and the Drosophila PITP domain remains to be fully established. Here, we investigated the membrane interactions of these proteins using dual polarization interferometry (DPI). DPI is a technique that measures protein binding affinity to a flat immobilized lipid bilayer. In addition, we also measured how quickly these proteins transfer their ligands to lipid vesicles using a fluorescence resonance energy transfer (FRET)-based assay. DPI investigations suggest that PITP�� had a two-fold higher affinity for membranes compared to PITP��. This was reflected by a four-fold faster ligand transfer rate for PITP�� in comparison to PITP�� as determined by the FRET assay. Interestingly, DPI analysis also demonstrated that PI-bound human PITPs have lower membrane affinity compared to PC-bound PITPs. In addition, the FRET studies demonstrated the significance of membrane curvature in the ligand transfer rate of PITPs. The ligand transfer rate was higher when the accepting vesicles were highly curved. Furthermore, when the accepting vesicles contained phosphatidic acid (PA) which have smaller head groups, the transfer rate increased. In contrast, when the accepting vesicles contained phosphoinositides which have larger head groups, the transfer rate was diminished. However, PI, the favorite ligand of PITPs, or the presence of anionic lipids did not appear to influence the ligand transfer rate of PITPs. Both DPI and FRET examinations revealed that the PITP domain of RdgB�� was able to bind to membranes. However, the RdgB�� PITP domain appears to be a poor binder and transporter of PC.

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3��-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 �� 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.

Synth��se de brosses de poly��lectrolytes par polym��risation initi��e depuis une surface de mica et ��tude de leur r��ponse en fonction du pH et de la force ionique

Cette th��se rapporte le greffage chimique de brosses de polym��res neutres de poly(acrylate de tert-butyle) (PtBA) et de brosses charg��es d���acide polyacrylique (PAA) sur des substrats de mica afin d�����tudier leur conformation en fonction de la densit�� de greffage, du pH et de la force ionique. Le greffage est r��alis�� par polym��risation contr��l��e par transfert d���atome (ATRP) initi��e depuis la surface de mica afin de contr��ler la croissance du polym��re et sa densit�� de greffage. L�����tude de la conformation des brosses de PtBA et de PAA a ��t�� men��e avec la technique AFM en mesurant les ��paisseurs des films �� sec et gonfl��s sous diff��rentes conditions de solvant, de pH et de force ionique. Une monocouche d���amorceurs est tout d���abord greff��e sur du mica porteur de groupes hydroxyles cr����s par plasma (Ar/H2O). Cette couche a ��t�� caract��ris��e par des mesures d���angle de contact et par la technique TOF-SIMS. L���amorceur greff�� a ensuite permis d���initier l���ATRP directement depuis la surface pour former des brosses neutres de PtBA li��s de fa��on covalente au mica. La croissance lin��aire de l�����paisseur du film avec la masse molaire du polym��re en solution et le taux de conversion montre que la polym��risation est contr��l��e. De plus, la r��-initiation des cha��nes greff��es atteste du caract��re vivant de la polym��risation. L���hydrolyse des brosses de PtBA, confirm��e par des mesures d���angle de contact, d�����paisseur et par FT-IR, conduit �� des brosses de PAA. Les diff��rentes couches greff��es sont stables �� l���air, en milieu organique et en milieu aqueux et leur gonflement est r��versible. Le degreffage de la couche de PAA est observ�� suite �� une longue exposition �� pH basique. Cette ��tude repr��sente le premier exemple de brosses greff��es chimiquement sur du mica par polym��risation initi��e depuis la surface. La variation des param��tres de la r��action de greffage de l���amorceur, tels que la concentration et la dur��e de r��action, a permis de contr��ler le taux de recouvrement de l���amorceur et la densit�� de greffage du polym��re. Une grande gamme de taux de recouvrement de l���amorceur est accessible et se traduit par un intervalle de densit��s de greffage allant de faibles �� ��lev��es (e.g. 0,04 cha��ne/nm2 �� 0,5 cha��ne/nm2). L�����tude de la conformation des cha��nes de PtBA dans le DMF montre que cet intervalle de densit��s recouvre le r��gime cr��pe au r��gime brosse. Le gonflement de brosses de PAA et la variation de la hauteur de la brosse L ont ��t�� ��tudi��s en fonction de la densit�� de greffage, du pH et du sel ajout�� cs (NaCl). Une transition brusque de collaps��e �� ��tir��e est observ��e avec l���augmentation du pH, ind��pendamment de la densit�� de greffage. A pH neutre, les brosses sont collaps��es et se comportent comme des brosses neutres en mauvais solvant. A pH basique, les brosses sont gonfl��es et charg��es et se trouvent dans un r��gime de Pincus caract��ristique des poly��lectrolytes forts. En pr��sence de sel, les charges sont partiellement ��crant��es et les r��pulsions ��lectrostatiques dominent toujours dans la brosse. Cette ��tude contribue �� une meilleure compr��hension du comportement complexe des brosses de poly��lectrolytes faibles et apporte un soutien exp��rimental �� la th��orie sur le comportement de ces brosses.

Marquage fluorescent des prot��ines pour ��tudier les enzymes prot��olytiques solubles et immobilis��es par la cartographie peptidique ��lectrophor��tique

La cartographie peptidique est une m��thode qui permet entre autre d���identifier les modifications post-traductionnelles des prot��ines. Elle comprend trois ��tapes : 1) la prot��olyse enzymatique, 2) la s��paration par ��lectrophor��se capillaire (CE) ou chromatographie en phase liquide �� haute performance (HPLC) des fragments peptidiques et 3) l���identification de ces derniers. Cette derni��re ��tape peut se faire par des m��thodes photom��triques ou par spectrom��trie de masse (MS). Au cours de la derni��re d��cennie, les enzymes prot��olytiques immobilis��es ont acquis une grande popularit�� parce qu���elles peuvent ��tre r��utilis��es et permettent une digestion rapide des prot��ines due �� un rapport ��lev�� d���enzyme/substrat. Pour ��tudier les nouvelles techniques d���immobilisation qui ont ��t�� d��velopp��es dans le laboratoire du Professeur Waldron, la cartographie peptidique par CE est souvent utilis��e pour d��terminer le nombre total de peptides d��tect��s et leurs abondances. La CE nous permet d���avoir des s��parations tr��s efficaces et lorsque coupl��e �� la fluorescence induite par laser (LIF), elle donne des limites de d��tection qui sont 1000 fois plus basses que celles obtenues avec l���absorbance UV-Vis. Dans la m��thode typique, les peptides venant de l�����tape 1) sont marqu��s avec un fluorophore avant l���analyse par CE-LIF. Bien que la sensibilit�� de d��tection LIF puisse approcher 10-12 M pour un fluorophore, la r��action de marquage n��cessite un analyte dont la concentration est d���au moins 10-7 M, ce qui repr��sente son principal d��savantage. Donc, il n���est pas facile d�����tudier les enzymes des peptides d��riv��s apr��s la prot��olyse en utilisant la technique CE-LIF si la concentration du substrat prot��ique initial est inf��rieure �� 10-7 M. Ceci est attribu�� �� la dilution suppl��mentaire lors de la prot��olyse. Alors, afin d���utiliser le CE-LIF pour ��valuer l���efficacit�� de la digestion par enzyme immobilis��e �� faible concentration de substrat,nous proposons d���utiliser des substrats prot��iques marqu��s de fluorophores pouvant ��tre purifi��s et dilu��s. Trois m��thodes de marquage fluorescent de prot��ine sont d��crites dans ce m��moire pour ��tudier les enzymes solubles et immobilis��es. Les fluorophores ��tudi��s pour le marquage de prot��ine standard incluent le naphtal��ne-2,3-dicarboxald��hyde (NDA), la fluoresc��ine-5-isothiocyanate (FITC) et l���ester de 6-carboxyfluoresc��ine N-succinimidyl (FAMSE). Le FAMSE est un excellent r��actif puisqu���il se conjugue rapidement avec les amines primaires des peptides. Aussi, le substrat marqu�� est stable dans le temps. Les prot��ines ��tudi��es ��taient l������-lactalbumine (LACT), l���anhydrase carbonique (CA) et l���insuline cha��ne B (INB). Les prot��ines sont dig��r��es �� l���aide de la trypsine (T), la chymotrypsine (CT) ou la pepsine (PEP) dans leurs formes solubles ou insolubles. La forme soluble est plus active que celle immobilis��e. Cela nous a permis de v��rifier que les prot��ines marqu��es sont encore reconnues par chaque enzyme. Nous avons compar�� les digestions des prot��ines par diff��rentes enzymes telles la chymotrypsine libre (i.e., soluble), la chymotrypsine immobilis��e (i.e., insoluble) par r��ticulation avec le glutarald��hyde (GACT) et la chymotrypsine immobilis��e sur billes d���agarose en gel (GELCT). Cette derni��re ��tait disponible sur le march��. Selon la chymotrypsine utilis��e, nos ��tudes ont d��montr�� que les cartes peptidiques avaient des diff��rences significatives selon le nombre de pics et leurs intensit��s correspondantes. De plus, ces ��tudes nous ont permis de constater que les digestions effectu��es avec l���enzyme immobilis��e avaient une bonne reproductibilit��. Plusieurs param��tres quantitatifs ont ��t�� ��tudi��s afin d�����valuer l���efficacit�� des m��thodes d��velopp��es. La limite de d��tection par CE-LIF obtenue ��tait de 3,0���10-10 M (S/N = 2,7) pour la CA-FAM dig��r��e par GACT et de 2,0���10-10 M (S/N = 4,3) pour la CA-FAM dig��r��e par la chymotrypsine libre. Nos ��tudes ont aussi d��montr��es que la courbe d�����talonnage ��tait lin��aire dans la r��gion de travail (1,0��10-9-1,0��10-6 M) avec un coefficient de corr��lation (R2) de 0,9991.