La GPEC se construit de plus en plus à l’échelle territoriale. Des acteurs institutionnels d’horizons divers et des entreprises de taille variable réfléchissent et travaillent ensemble pour mettre en place des actions qui répondent aux problématiques liées à l’emploi, à la formation, et aux compétences. Ces démarches se font tantôt à « chaud », tantôt à « froid » selon les circonstances, les territoires et les acteurs. L’extension de l’échelle de construction de la GPEC de l’entreprise au territoire peut se justifier par la prise en compte de plusieurs facteurs : internes ou externes aux entreprises, politiques, conjecturaux, socio-économiques, etc. Ainsi à travers des volontés convergentes, de multiples acteurs ambitionnent de lever les limites et insuffisances consubstantielles à la GPEC d’entreprise en recourant à une GPEC-Territoriale. Cette nouvelle approche de construction et d’analyse de la GPEC pose néanmoins des interrogations. Parmi celles-ci, nous avons réfléchi, à cinq questions : comment faire travailler ensemble les acteurs ? Quel diagnostic permet de fédérer les acteurs autour de la GPEC-Territoriale ? Comment se construit cette GPEC-Territoriale en termes de phasage ? Comment les acteurs se mettent-ils d’accord sur la construction et le contenu des actions de la GPEC-Territoriale ? Comment mobiliser les acteurs dans de telles démarches collectives ? Ces questions sont issues de la question principale de notre recherche : quel est le processus de construction d’une GPEC-Territoriale impliquant des acteurs institutionnels et des entreprises ? Nous avons abordé et discuté ces questions sur la base de données empiriques collectées dans deux cas : la GPEC-Territoriale dans la Communauté de communes du Cher à la Loire et la GPEC-Territoriale dans la filière Bois dans le département du Loir-et-Cher. Ces données sont collectées à partir d’observations, d’entretiens qualitatifs, d’études quantitatives et documentaires. Les théories de l’interaction, de la traduction, du choix rationnel et de la mobilisation nous ont servi de grille d’analyse. Au croisement de ces approches et de ces analyses, il en est ressorti que la GPEC-Territoriale se construit à partir de quelques nécessités : capacité du pilote à faire travailler ensemble plusieurs acteurs, établissement d’un diagnostic préalable et partagé se basant sur les problématiques et enjeux des entreprises et des territoires, mobilisation des acteurs à travers des incitations sélectives et l’analyse des catégories de priorités des acteurs. En outre, il est apparu que le contenu de la GPEC-Territoriale est continûment traduit et s’obtient sous un consensus relatif. Enfin et malgré l’aspect sui generis de chaque cas, une modélisation, en phases, de sa construction est possible. / Nowadays HRP is built increasingly on a territorial scale. Institutional actors from different backgrounds and varying size businesses work together to put in place actions that address issues related to employment, training, and skills. These approaches are sometimes in "hot", sometimes in "cold" depending on the circumstances, territories and stakeholders. The extension of the building of the HRP across a territory can be justified by taking into account several factors: internal or external to enterprises, policies, situational, socio-economic, etc. Thus through converging wills, multiple actors aspire to lift the limits and shortcomings related to HRP by using a HRP-Territorial. This new construction approach and analysis of HRP nevertheless raises several questions. Among the many questions we reflected about five of them: how do actors work together? What diagnosis allows to unite stakeholders around HRP-Territorial? How is this HRP-Territorial built in terms of phasing? How do actors agree on the construction and content of the actions of HRP-Territorial? How to mobilize actors in such collective approaches? These questions are taken from the main issue of our research: What is the process of building a HRP-Territorial involving institutional actors and businesses? We discussed and debated these issues on the basis of empirical data collected in two cases: HRP-Territorial in the Community of communes of Cher à la Loire and the HRP-Territorial in the timber Industry in Loir-et-Cher. Those data are collected by observation, qualitative interview, quantitative studies and documentaries. Theory of interaction, actor network theory, rational choice theory and mobilization theory served as our analytical framework. At the intersection of these approaches and these analyzes, it appears that the HRP-Territorial be built from a few necessities : the ability of the pilot to work together several actors, establishing a prior and shared diagnosis that rely on problem and challenges for companies and territory, mobilization of actors through selective incentives and analysis of priority categories of actors. Furthermore, it appears that the contents of the HRP-Territorial is continuously translated and obtained by relative consensus. Finally, and despite the particular case of each situation, a modeling phase of this construction is possible.

Tuberculosis (TB) is an important disease worldwide. Currently, one-third of the world’s population is infected with TB, and it is a leading cause of death among people living with HIV. Immediate but also accurate diagnosis is required for disease control, yet available diagnostics cannot do both simultaneously. Therefore, designing a technique that can diagnose the disease correctly in the shortest possible time is in great demand in order to stop its spread. Diffraction-based sensing is a novel technique for measuring of biomolecular interaction that has potential for disease diagnosis. In this study, diffraction-based sensing successfully demonstrated its usefulness for diagnostics of TB using recombinant TB antigen, or by detection of interferon-γ that is produced from white blood cells when the immune system activates. The feasibility of the technology was also evaluated in terms of providing real time observation, reducing diagnostic duration, and increasing sensitivity of detection.

Tuberculosis (TB) is an important disease worldwide. Currently, one-third of the world’s population is infected with TB, and it is a leading cause of death among people living with HIV. Immediate but also accurate diagnosis is required for disease control, yet available diagnostics cannot do both simultaneously. Therefore, designing a technique that can diagnose the disease correctly in the shortest possible time is in great demand in order to stop its spread. Diffraction-based sensing is a novel technique for measuring of biomolecular interaction that has potential for disease diagnosis. In this study, diffraction-based sensing successfully demonstrated its usefulness for diagnostics of TB using recombinant TB antigen, or by detection of interferon-γ that is produced from white blood cells when the immune system activates. The feasibility of the technology was also evaluated in terms of providing real time observation, reducing diagnostic duration, and increasing sensitivity of detection.

Vagal sensory afferent innervation corresponds to regulation of neurogenic inflammation in the airways. Capsaicin is mostly used for stimulation of sensory nerves that induce pain and inflammatory responses. It can specifically stimulate sensory afferent nerves, inducing neurogenic inflammation in the airways. According the past studies, we have found the right thoracic vagus nerve (RTVN) and right recurrent laryngeal nerve (RRLN); branches of right thoracic vagus trunk (RTVT) mediate different degree of neurogenic inflammation by intraenous injection of capsaicin (300 nmol/ml/kg). In order to investigate the innervation from the RTVN and RRLN of rat tracheobronchi and their involvement in plasma exudation, we injected 3 £gl of capsaicin (10 mg/ml) into RTVT and denervated the RRLN or RTVN and used India ink as tracer dye to label the leaky microvessels. Our observation indicated that injection of capsaicin into the RTVT coud induce obvious plasma exudation in trachea (area density of leaky blood vessels was about 22%), but plasma exudation was significantly decreased after denervation of RRLN. The left upper side of trachea was decreased by 77.6% and the right upper side decreased by 84.5%. This phenomenon was not caused by denervation of RTVN. The results suggest that vagal nerve innervation of upper trachea mostly came from the RLN. Otherwise, capsaicin injection into the RTVT also induced neurogenic inflammation in the larynx. Experimental denervation of both superior and recurrent laryngeal nerves resulted in a decrease of plasma extravasation by 84.98%. Denervation of either RTVN or RRLN also decreased the plasma extravasation in the larynx. The evidence suggest that sensory fibers in the superior laryngeal nerve, recurrent laryngeal nerve, and thoracic vagus nerve might come from the same population of vagal ganglion sensory neurons.

The Plasmodium falciparum parasite, carried by Anopheles mosquitoes, is currently a global problem due to the rising incidence of resistance of the parasite to available antimalaria drugs. Resistance and difficult treatment groups, including pregnant woman and young children, are pressing for the development of new, safe and effective prophylactic and treatment antimalarials. Because of the extensive process of developing new drugs, researchers and health care professionals have turned to combination therapy where a fast acting antimalarial is combined with slower acting drugs, such as antibiotics. The macrolide antibiotics, erytbromycin and azithromycin, have been studied to a limited extent for their potential antimalarial effect. Certain advantages, such as their safety profile (especially that of azithromycin) in pregnancy and administration to young children, motivates continual research into the advancement of the effect these drugs exude on malaria. Drug delivery systems contribute to the efficacy of medicines, conquering several difficulties of treatment with oral medication. Pheroid™ technology is a patented drug delivery system, mainly consisting of plant and essential fatty acids, and has been demonstrated to entrap, carry and deliver pharmacologically active compounds and other useful molecules. This study compared the in vitro effects of the macrolide antibiotics on the growth of a chloroquine-resistant strain (RSA 11) of Plasmodium falciparum to the effects of the macrolides entrapped in Pheroid™ vesicles on the same strain over and extended observation period of 144 hours. ELISA assays were conducted by analysing the HRP II (histidine-rich protein) levels on a pre-coated microtitre plate. The effects of the type of formulation, concentration and time were compared. The in vitro difference between erythromycin alone and entrapped in Pheroid™ vesicles were found to be statistically significant (p = 0.000000) while the effects of both formulations did not seem to be concentration dependant (p = 0.628424). Prolonged exposure was also statistically meaningful (p = 0.008268), though it seems that exposure need not exceed 96 hours. The type of formulation, in the case of azithromycin (azithromycin alone vs. azitbromycin entrapped in Pheroid™ vesicles), proved statistically significant (P = 0.002572), while neither formulation seemed concentration dependant (P = 0.427731). Prolonged exposure was found to be statistically insignificant for azithromycin (P = 0.221941). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

The Plasmodium falciparum parasite, carried by Anopheles mosquitoes, is currently a global problem due to the rising incidence of resistance of the parasite to available antimalaria drugs. Resistance and difficult treatment groups, including pregnant woman and young children, are pressing for the development of new, safe and effective prophylactic and treatment antimalarials. Because of the extensive process of developing new drugs, researchers and health care professionals have turned to combination therapy where a fast acting antimalarial is combined with slower acting drugs, such as antibiotics. The macrolide antibiotics, erytbromycin and azithromycin, have been studied to a limited extent for their potential antimalarial effect. Certain advantages, such as their safety profile (especially that of azithromycin) in pregnancy and administration to young children, motivates continual research into the advancement of the effect these drugs exude on malaria. Drug delivery systems contribute to the efficacy of medicines, conquering several difficulties of treatment with oral medication. Pheroid™ technology is a patented drug delivery system, mainly consisting of plant and essential fatty acids, and has been demonstrated to entrap, carry and deliver pharmacologically active compounds and other useful molecules. This study compared the in vitro effects of the macrolide antibiotics on the growth of a chloroquine-resistant strain (RSA 11) of Plasmodium falciparum to the effects of the macrolides entrapped in Pheroid™ vesicles on the same strain over and extended observation period of 144 hours. ELISA assays were conducted by analysing the HRP II (histidine-rich protein) levels on a pre-coated microtitre plate. The effects of the type of formulation, concentration and time were compared. The in vitro difference between erythromycin alone and entrapped in Pheroid™ vesicles were found to be statistically significant (p = 0.000000) while the effects of both formulations did not seem to be concentration dependant (p = 0.628424). Prolonged exposure was also statistically meaningful (p = 0.008268), though it seems that exposure need not exceed 96 hours. The type of formulation, in the case of azithromycin (azithromycin alone vs. azitbromycin entrapped in Pheroid™ vesicles), proved statistically significant (P = 0.002572), while neither formulation seemed concentration dependant (P = 0.427731). Prolonged exposure was found to be statistically insignificant for azithromycin (P = 0.221941). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Doctor of Philosophy / Department of Civil Engineering / Alok Bhandari / Aquifers are frequently contaminated by phenolic compounds from spills, leaking underground storage tanks, or landfills. These compounds can be toxic to a variety of organisms including humans. Their disposal is restricted in many countries with strict limits for acceptable concentrations in drinking water. Phenols that are chlorinated have significantly greater toxicity and are resistant to aerobic biodegradation. Enzyme-mediated in situ stabilization has been advocated as an approach for the treatment of phenolic compounds in soils and groundwater. This research investigated the applicability of a luminol-based chemiluminescence assay to monitor transport of horseradish peroxidase (HRP) enzyme in saturated porous media. The chemiluminescence assay was optimized by varying solution conditions such as the concentration of luminol, p-iodophenol, hydrogen peroxide, ionic strength and pH. All assay components were found to affect the maximum chemiluminescene intensity. The study also evaluated the ability of HRP to mediate the removal of phenol from solution by catalyzing its oxidative polymerization in simulated aquifer conditions. HRP behaved as a conservative tracer in the column packed with Ottawa sand. The concentration of phenol in the column effluent was found to decrease by nearly 90% in the presence of HRP and H2O2 in the continuous flow system. HRP mediated oxidative polymerization of phenols resulted in the production of soluble and insoluble oligomeric products. Modification of porous media caused by the deposition of phenol polymerization products was studied and the impact of media modification on subsequent transport of phenolic contaminants was evaluated using 2,4-dichlorophenol (2,4-DCP) as a probe solute. The pore volume of the porous media was reduced due to the deposition of insoluble phenolic oligomers. The transport behavior of 2,4-DCP showed that the contaminant was retarded in the modified porous media.

Heme-substituted horseradish peroxidases and myoglobins were reconstituted from the apoenzyme using mesoheme and diacetyldeuteroheme. X-ray absorption spectroscopy was used to determine the dimensions of the active sites of these heme-substituted proteins, and were compared with those of the proto-hemeproteins. The change in the active-site structure corresponded with the electron withdrawing and donating effects of the different side chains. The oxidation-reduction potentials of Fe4+/Fe3+ couples of the heme-substituted proteins were measured at pH 7 with K2IrC16. The oxidation-reduction potential sequence for compound I/compound II was diacetyldeutero-> proto-> meso-in horseradish peroxidase. The oxidation-reduction potential sequence for compound II /ferric was meso-> proto-> diacetyldeutero-in both HRP and myoglobin. These results indicate that the oxidation of ferric to ferryl form may be related to a radical mechanism. A net charge theory was also proposed to explain these results.

Made available in DSpace on 2017-07-20T12:40:18Z (GMT). No. of bitstreams: 1 Rosana M.pdf: 3183755 bytes, checksum: ceaa632c444d6daec2678d0f7fe428db (MD5) Previous issue date: 2016-02-23 / This thesis describes the use of self-assembled monolayers (SAM) for the development of biosensors based on horseradish peroxidase enzyme - HRP. In the first chapter, the enzyme was immobilized on self-assembled monolayers of thiolactic acid (Au-TLA) and mixed SAM composed of 11-mercaptoundecanoic acid (MUA) together with the TLA (SAMmix). The steps of construction and characterization of biosensors have been carried out by electrochemical techniques and morphological ones. The enzyme immobilization method on SAM which provided higher stability was by covalent bond. The Au/TLA/HRP biosensor was used for the determination of H2O2 by chronoamperometry, yielding a detection limit (DL) of 5.46 μmol L-1 and quantification (QL) of 18 μmol L-1. Despite the good results for the H2O2 detection presented by this biosensor, the device was stable for only 6 days. Therefore, in order to increase the stability of the biosensor, SAM containing both the mercaptoundecanoic acid molecule (MUA) and TLA was obtained, to the formation of SAMmix (Au/MUA:TLA). In this system, the electron transfer rate can be considerably affected because while the TLA enables an increase of SAMmix conductivity due to formation of “islands”, the MUA provides greater stability for HRP immobilization, although it partially blocks the surface. The biosensor Au-SAMmix-HRP prepared in the ratio 0.5: 1.0 MUA/TLA showed higher sensitivity compared to other modifications ratio, with an apparent Michaelis-Menten constant = 0.40 mmol L-1. This biosensor has been applied to the determination of hydroquinone (HQ) in the presence of a fixed amount of [H2O2] = 0.3 mmol L-1. By differential pulse voltammetry technique (DPV), the biosensor exhibited excellent electrocatalytic activity for HQ in the range 3-30 μmol L-1, with good sensitivity, DL = 1.26 μmol L-1 and QL = 4.23 μmol L -1. The SAMmix increased the stability of the biosensor for at least 15 days, which is more stable when compared to the biosensor Au/TLA/HRP. In the second chapter of this thesis, another strategy was used for immobilization of the HRP enzyme based on the formation of TLA monolayer on the top of gold nanoparticles (AuNPs) stabilized in the inorganic polymer, 3-n-propylpyridinium silsesquioxane chloride (SiPy+Cl-). The presence of AuNPs-SiPy+Cl- was confirmed by UV-Vis spectroscopy from the plasmon band at 521 nm. The AuNps showed good distribution with approximate size between 4-18 nm, evidenced by transmission electron microscopy (TEM) and by dynamic light scattering (DLS), with good stability (ζ = + 38.5 mV). The AuNPs were deposited on the glassy carbon electrode (GCE) and modified with the TLA for immobilization of HRP enzyme. The formation of this biosensor was confirmed by electrochemical impedance spectroscopy (EIS) and morphologically by field-effect scanning electron microscopy (SEM-FEG). The GCE/AuNPs/TLA/HRP biosensor was used for the detection of H2O2 with = 0.46 mmol L-1, which was similar to the Au-SAMmix-HRP biosensor. This biosensor has been applied to the determination of catechol (CT) in presence of [H2O2] = 0.03 mmol L-1. Using the DPV technique, the biosensor showed an excellent electrocatalytic activity for CT in the range 6-46 μmol L-1, with good sensitivity, DL = 0.852 μmol L-1 and QL = 2.84 μmol L -1. The stability of this device was approximately 25 days, being superior to other biosensors developed (TLA-HRP and SAMmix-HRP), which can be attributed to the three-dimensional immobilization of HRP molecules in this device. / Esta tese descreve a utilização das monocamadas auto-organizadas (SAM) para o desenvolvimento de biossensores enzimáticos a base da enzima horseradish peroxidase – HRP. No primeiro capítulo, diferentes biossensores foram desenvolvidos utilizando a SAM de ácido tioláctico (Au/TLA) e também a SAM mista composta por ácido 11-mercaptoundecanóico (MUA) juntamente com o TLA (SAMmista). As etapas de construção e caracterização dos biossensores foram realizadas pelas técnicas eletroquímicas e também morfológicas. O método de imobilização da enzima sobre a SAM que proporcionou maior estabilidade foi pela ligação covalente. O biossensor Au/TLA/HRP foi utilizado para a detecção do H2O2 por cronoamperometria, obtendo-se um limite de detecção (LD) de 5,46 μmol L-1 e de quantificação (LQ) de 18 μmol L-1. Apesar dos bons resultados na detecção do H2O2 apresentados por este biossensor, o dispositivo foi estável por apenas 6 dias. Portanto, no intuito de aumentar a estabilidade do biossensor utilizou a molécula de ácido mercaptoundecanóico (MUA) juntamente com TLA, para a formação da SAMmista (Au/MUA:TLA). Neste sistema, a velocidade de transferência eletrônica é sensivelmente afetada, pois enquanto o TLA possibilita o aumento da condutividade da SAMmista devido a formação de “ilhas”, o MUA confere maior estabilidade à monocamada para a imobilização da enzima HRP, apesar de bloquear parcialmente a superfície. O biossensor Au/SAMmista/HRP preparado na razão 0,5:1,0 MUA:TLA apresentou a melhor sensibilidade em comparação as outras modificações, com uma constante de Michaelis-Menten aparente, = 0,40 mmol L-1. Este biossensor foi aplicado na detecção da hidroquinona (HQ) na presença de uma quantidade fixa de [H2O2] = 0,3 mmol L-1. Pela técnica de voltametria de pulso diferencial (VPD), o biossensor exibiu uma excelente atividade eletrocatalítica para HQ na faixa de 3 a 30 μmol L-1, com boa sensibilidade, LD = 1,26 μmol L-1 e o LQ = 4,23 μmol L-1. A SAMmista aumentou a estabilidade do biossensor para 15 dias, sendo este mais estável quando comparado ao biossensor Au/TLA/HRP. No segundo capítulo da tese, outra estratégia para a imobilização da enzima HRP sobre o TLA foi realizada a partir de nanopartículas de ouro (AuNps) estabilizadas no cloreto de 3-n-propilpiridínio silsesquioxano (SiPy+Cl-). A presença das AuNps-SiPy+Cl- foi confirmada pela Espectroscopia UV-Vis a partir da banda plasmon em 521 nm, as quais apresentaram boa distribuição com tamanho aproximado entre 4 a 18 nm, constatado pela microscopia eletrônica de transmissão (MET) e por espalhamento dinâmico de luz (DLS), apresentando boa estabilidade (ζ = + 38,5 mV). As AuNps foram incorporadas sobre o eletrodo de carbono vítreo (ECV) e modificadas com o TLA para imobilização da enzima HRP. A formação deste biossensor foi comprovada pela espectroscopia de impedância eletroquímica (EIE) e morfologicamente pela microscopia de varredura de efeito de campo (MEV-FEG). O biossensor ECV/AuNps/TLA/HRP foi utilizado na detecção do H2O2 com = 0,43 mmol L-1, sendo próxima ao biossensor Au-SAMmista-HRP. Este biossensor foi aplicado na detecção do catecol (CT) na presença de [H2O2] = 0,03 mmol L-1. Pela técnica de VPD, o biossensor exibiu uma excelente atividade eletrocatalítica para CT na faixa de 6 a 46 μmol L-1, com boa sensibilidade, LD = 0,852 μmol L-1 e o LQ = 2,84 μmol L-1. A estabilidade deste dispositivo é de 25 dias, sendo superior aos outros biossensores desenvolvidos (TLA/HRP e SAMmista/HRP) devido a imobilização ocorrer tridimensionalmente aumentando a quantidade de moléculas da TLA e HRP no dispositivo.

Magister Scientiae - MSc / The main hypothesis in this study is the development of a nanocomposite mediated amperometric biosensor for detection of hydrogen peroxide. The aim is to combine the electrochemical properties of both polyaniline and ferrocenium hexafluorophosphate into highly conductive nano composites capable of exhibiting electrochemistry in non acidic media; shuttling electrons between HRP and GCE for biosensor applications. / South Africa