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Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: a) the optimization of the current industrial-scale extraction and purification of Horseradish peroxidase from horseradish at BBI Enzymes, focussing on: a. Raw material quality, b. Extraction, c. Ultra-filtration, d. Salt fractionation, e. Diafiltration, f. Ion Exchange Chromatography, b) developing an new in-process microtitre plate calorimetric assay, c) characterization of main groups of HRP relevant to BBI Enzymes by SDS-PAGE- and HPLC analysis. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: a) die optimisering van die huidige industriële-skaal ekstraksie en suiwering van peperwortelperoksidase vanuit peperwortel by BBI Enzymes, deur te focus op: a. Rou material kwaliteit, b. Ekstraksie, c. Ultra-filtrasie, d. Sout fraksionering, e. Diafiltrasie, f. Ioon-uitruilchromatografie b) Ontwikkeling van ‘n nuwe in-proses mikro-titer gebaseerde kalorimetriese toetsmetode c) die karakterisering van die hoof groepe peperwortel-peroksidase belangrik vir BBI Enzymes.

Phenols are priority pollutants that are commonly found in a large number of industrial wastewaters. Different processes are currently available for the elimination of phenol from wastewater but present some disadvantages like low efficiency, high energy-consumption, the necessity of acclimatisation of the sludges or the limitation of the treatment capacity. The need to find alternatives has made the enzymatic processes a good option. In the last two decades, several processes were implemented with different enzymes from plants and microorganisms, including peroxidases from several sources, as the horseradish peroxidase.Also, different enzyme configurations, free or immobilised enzyme and different supports for immobilisation have been studied. Substantial attention has been devoted to the covalent immobilisation of enzymes on porous insoluble supports such as glass, alumina, silica, and chitosan.The main novelty of this work is the utilisation of a torus reactor for the removal of organic contaminants from wastewaters. This reactor, which can be considered as a loop reactor, presents some advantages over other stirred tank reactors.The goal of this work is the study of the hydrodynamics of a torus reactor for its further application in the enzymatic elimination of phenol and the coupling of the kinetics and the modelisation.In a first step, the enzymatic elimination of phenol was experimentally studied in the torus reactor. In order to compare the performances, several assays were also carried out with a stirred reactor. A high degree of conversion was obtained for the enzymatic elimination of phenol in both reactors with the tested quantities of phenol. It was concluded that, keeping a ratio of 1:1 between the phenol and the H2O2 initial molar quantities, the highest final reaction conversion was obtained. Using the torus reactor was obtained 97% of phenol conversion when the optimal concentrations of substrates were usedIn order to improve economically the process, the enzyme should be used in a continuous regime over a long time period to exploit it completely. For this reason it was necessary to immobilise the enzyme. This work presents a new configuration that has never been tested: the horseradish peroxidase supported on Eupergit.In a second step, the characterisation using the CFD of the flow-field in a torus reactor of 100 ml, similar to the experimental reactor, was carried out for two different configurations, batch and continuous operating modes. Moreover, the scale-up of the volume of the torus reactor was carried out using CFD for a 300 ml reactor.Finally, the enzymatic reaction of phenol with the HRP was modelled using the CFD coupled to the kinetic model of the enzymatic reaction to the flow simulation. These results allowed the possibility of optimising and scaling-up the process using the CFD modelisation. / Los compuestos fenólicos son contaminantes prioritarios que se encuentran comúnmente en una gran cantidad de efluentes industriales. Diferentes procesos están disponibles actualmente para la eliminación de fenol desde dicho efluentes pero los mismos presentan algunas desventajas como pueden ser una baja eficiencia, un mayor consumo de energía, la producción de lodos conteniendo hierro o limitaciones en la capacidad de tratamiento. La necesidad de encontrar alternativas a estos problemas ha hecho del proceso enzimático una buena opción. En las últimas dos décadas, varios procesos han sido implementados utilizando diferentes enzimas extraídas de plantas y microorganismos como pueden ser las peroxidasas de diversas fuentes, incluyendo la horseradish peroxidasa.Diferentes configuraciones de enzimas, libre e inmovilizada y diferentes soportes para la inmovilización han sido también estudiados. Sustancial atención ha sido dedicada a la inmovilización de enzimas por enlace covalente sobre soportes porosos insolubles tales como vidrio, aluminio, sílice y chitosan. El objetivo de este trabajo es el estudio de la hidrodinámica dentro de un reactor tórico para su posterior aplicación en la eliminación enzimática de fenol y el acople entre las cinéticas y la modelización.En una primera etapa, la eliminación enzimática de fenol es estudiada experimentalmente en el reactor tórico. Con el objetivo de comparar el rendimiento de dicho reactor, varios ensayos se realizaron en un reactor agitado tradicional. Un alto grado de conversión de fenol ha sido obtenido para la eliminación enzimática de fenol en ambos reactores para las cantidades estudiadas de fenol. Ha sido observado que es necesario mantener una relación de 1:1 entre la concentración inicial de fenol y la de peróxido de hidrógeno para lograr la mayor conversión de fenol. Usando el reactor tórico ha sido obtenido un 97% de conversión de fenol cuando las concentraciones óptimas de substratos y enzimas fueron utilizados. Con el objetivo de mejorar económicamente el proceso y hacerlo factible para su uso a escala industrial, la enzima debería ser utilizada en un proceso en continuo sobre un largo período de tiempo para explotarla completamente. Por esta razón, ha sido necesario inmovilizar la enzima. Este trabajo muestra una nueva configuración que no ha sido aún probada: la horseradish peroxidase soportada en Eupergit. Asimismo, la caracterización usando CFD del campo de flujo de un reactor tórico similar al experimental de 100 ml ha sido realizada para un reactor trabajando de forma batch y continua. Un escalado en el volumen del reactor tórico ha sido realizado utilizando CFD para un reactor de 300 ml. Finalmente, la reacción enzimática de fenol con HRP has sido modelada acoplando el modelo cinético obtenido experimentalmente con las simulaciones del campo de flujo dentro del reactor. Estos resultados permitirán la optimización y el escalado del proceso usando CFD.

¡iAbstract¡j Vagal sensory neurons play an important role in the neural control of airway and other visceral organs. Regional distribution of vagal sensory neurons in the vagal ganglia that correspond to different viscera is uncertain. Horseradish peroxidase (HRP) was applied to the right thoracic vagus nerve and recurrent laryngeal nerve to be retrogradely transported to the neurons in vagal nodose and jugular ganglia. Labeling of neuronal cell bodies was visualized with diaminobenzidine reaction. Ricinus communis agglutinin-60 (RCA-60) was injected into the right thoracic vagus nerve and transported retrogradely to cause destruction of the ribosome in the vagal ganglia neuron cell bodies. The magnitude of neurogenic plasma extravasation induced by capsaicin was measured by the area density of the India ink-labeled leaky blood vessels in the trachea and bronchi. The present study demonstrated that there was no distinct localization of HRP- labeled neurons, except at the level of pharyngeal nerve that was connected to the middle of the nodose ganglion. There were approximately 42.2 % of labeled neurons in the nodose ganglion and 30.5 % in the jugular ganglion 3 days after application of HRP in the thoracic vagus nerve. After application of HRP to the recurrent laryngeal nerve, 13.3 % of labeled neurons was found in the nodose ganglion and 8.3 % in the jugular ganglion. One to two weeks after RCA injection into the thoracic vagus nerve, many cell bodies of neurons had a striking degenerative alteration and the cytoplasmic density was markedly reduced. Nissl¡¦s bodies obviously disappeared and vacuoles were the usual feature. Application of RCA-60 also inhibited the neurogenic plasma extravasation in the right bronchial tree that were reduced by 71- 89 %. It is suggested that RCA- 60 selectively destroyed the vagal sensory neurons that innervated the ipsilateral branchial tree. It is concluded that vagal nodose and jugular ganglia supplied different number of sensory neurons to the vagal branches and regulated the physiological function of the visceral organs.

?rea de concentra??o: Qu?mica Anal?tica. / Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-18T17:29:43Z No. of bitstreams: 1 delton_martins_pimentel.pdf: 3429904 bytes, checksum: 0bdb59431143a44261723bfd797ed6b3 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-18T17:56:59Z (GMT) No. of bitstreams: 1 delton_martins_pimentel.pdf: 3429904 bytes, checksum: 0bdb59431143a44261723bfd797ed6b3 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-18T18:02:22Z (GMT) No. of bitstreams: 1 delton_martins_pimentel.pdf: 3429904 bytes, checksum: 0bdb59431143a44261723bfd797ed6b3 (MD5) / Made available in DSpace on 2014-12-18T18:02:22Z (GMT). No. of bitstreams: 1 delton_martins_pimentel.pdf: 3429904 bytes, checksum: 0bdb59431143a44261723bfd797ed6b3 (MD5) Previous issue date: 2013 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (Capes) / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / No presente trabalho fabricou-se um material comp?sito ? base de polianilina (PAni), grafeno(rGO) e Horseradish peroxidase (HRP), visando o desenvolvimento de uma nova plataforma para detec??o de per?xido de hidrog?nio (H2O2) com uma alta sensibilidade. O grafeno utilizado foi produzido a partir da redu??o do ?xido de grafite (GO) por redu??o em forno de micro-ondas, que por sua vez foi sintetizado pelo m?todo de Laura J. Cote et al. Posteriormente, o nanomaterial foi modificado quimicamente com O,O?-Bis(2-aminoetil)-polietilenoglicol, produzindo assim grafeno funcionalizado com grupo amino (rGO-PEG-NH2). Ap?s esta etapa, foi produzido o comp?sito PAni/rGO-PEG-NH2 por via potenciodin?mica e o mesmo foi utilizado como substrato para fabricar um sensor para H2O2 atrav?s da imobiliza??o da enzima HRP sobre o pol?mero produzindo-se assim um material comp?sito. Os experimentos eletroqu?micos foram realizados em uma cela eletroqu?mica com capacidade para 10 mL com tr?s eletrodos, sendo o eletrodo de refer?ncia Ag/AgCl, contra eletrodo de fio platina, e eletrodo de trabalho de Au (0,071 cm2). Estudos comparativos entre os processos redox da PAni e PAni/rGO-PEG-NH2 realizados por voltametria c?clica e imped?ncia eletroqu?mica deixaram evidente que o material comp?sito apresenta uma maior densidade de corrente bem como uma menor resist?ncia ? transfer?ncia de carga em altos e baixos potenciais. Por fim, com o prop?sito de avaliar o comportamento eletroqu?mico do sistema a duas dimens?es 2D, foram realizadas imagens eletroqu?micas da PAni bem como da PAni/rGO-PEG-NH2 por Microscopia Eletroquimica de Varredura (SECM, do ingl?s Scanning Electrochemical Microscopy) utilizando uma c?lula eletroqu?mica de 3 mL com quatro eletrodos. Conforme observado, a PAni/rGO-PEG-NH2 apresenta regi?es com alta densidade de corrente devido ao maior feedback positivo em compara??o ? PANI na aus?ncia do grafeno, al?m de proporcionar um transporte mais efetivo de pr?tons para dentro e para fora da matriz polim?rica; tornando assim o material bastante promissor para imobiliza??o de esp?cies com carga formal positiva. Ap?s a inser??o da HRP ao material comp?sito os estudos realizados com o biossensor resultou em uma sensibilidade de 44 ?A mM?1 e LOD de 0,31 ?M e LOQ de 0,94 ?M para a detec??o de H2O2, sendo estes valores muito superiores aos de outros trabalhos presentes na literatura e muito superior ao obtido pelo pol?mero condutor aplicado isoladamente com a enzima. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2013. / ABSTRACT In the present work a composite material based on polyaniline (PAni), graphene (rGO) and Horseradish peroxidase (HRP) was fabricated, aiming the development of a new platform for the detection of hydrogen peroxide (H2O2) with high sensitivity. The graphene used in this work was produced from the reduction of graphite oxide, using a microwave oven. The graphite oxide was synthesized by the method of J. Laura Cote et al. Subsequently, the nanomaterial was chemically modified with O, O'-Bis (2 - aminoethyl) polyethylene glycol, thus producing graphene functionalized with amino groups (rGO / PEG). After this step, the composite PAni/rGO-PEG-NH2 was produced by potentiodynamic method and it was used as a substrate to fabricate a sensor for H2O2 by immobilizing the enzyme HRP over the polymer, thus producing a composite material. Electrochemical experiments were performed in an electrochemical cell for up to 10 mL, with three electrodes: Ag / AgCl was used as the reference electrode; platinum wire as the counter electrode and the working electrode was an Au electrode with an area of 0.071 cm2. Comparative studies of the redox processes of PAni and PAni/rGO-PEG-NH2, performed by cyclic voltammetry and electrochemical impedance spectroscopy, made it clear that the composite material has a higher current density and a lower charge transfer resistance in high and low potentials. Finally, in order to evaluate the electrochemical behavior of the two dimensional (2D) system, electrochemical images were taken of PAni and PAni/rGO-PEG-NH2 by Scanning Electrochemical Microscopy (SECM) using an electrochemical cell with 3 mL and four electrodes. As noted, the PAni/rGO-PEG-NH2 presents regions with high current density due to the higher positive feedback compared to PAni in the absence of graphene, besides it promotes a proton transportation more effective, to the inside and the outside of the polymeric matrix. After insertion of the HRP enzyme to the composite material, the studies performed with the biosensor resulted in a sensitivity of 44 ?A mM-1, LOD 0.31 and LOQ 0.94 mM for the detection of H2O2, being these values much higher than other works found in the literature, and much higher than the one obtained with the conducting polymer used only with the enzyme.

Orientador: Carlos Alberto de Souza Costa / Resumo: O objetivo geral deste estudo foi avaliar a eficácia clareadora e toxicidade sobre células pulpares humanas (HDPCs) de dois produtos experimentais recomendados para terapia clareadora de consultório. Para isso, dois sistemas clareadores foram desenvolvidos: um gel clareador contendo um espessante catalisador (EP) e um primer polimérico catalisador (PR), indicado para aplicação sobre o esmalte previamente ao uso de agentes clareadores. Em ambos os produtos foi incorporada a enzima horseradish peroxidase (HRP) como agente catalisador do peróxido de hidrogênio (H2O2). Na primeira etapa deste estudo, géis clareadores com 10% e 20% de H2O2 foram preparados a partir de uma solução estoque de 35% de H2O2 combinada com um espessante contendo ou não as concentrações de 0,5, 1,0 e 2,0 mg/mL de HRP (HRP). Estes produtos foram avaliados quanto ao pH, temperatura, estabilidade de reação, formação de radicais-livres (EROs, sonda HORAC) e radicais hidroxila (HO•, sonda H2DCFDA), bem como eficácia clareadora (E). Um protocolo in vitro de pigmentação intrínseca de discos de esmalte/dentina bovinos foi usado para avaliar a citotoxicidade e a difusão trans-amelodentinária H2O2. De modo geral, o pH e a temperatura dos géis mantiveram-se constantes durante todo período de análise, sendo que a adição da enzima HRP ao EP acelerou a catálise do H2O2, estimulando a produção de EROs e HO• (ANOVA/Tukey; p<0,05). Além disso, a presença da HRP no EP aumentou o E do gel e reduziu a difusão trans-amelod... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The main objective of this study was to assess the bleaching effectiveness and cytotoxicity on human dental pulp cells (HDPCs) of two experimental products recommended in-office tooth bleaching therapy. For this, two bleaching systems were developed: a gel containing a catalyst thickener (CT) and a polymeric catalyst primer (CP) indicated for application on the enamel prior to the use of bleaching agents, were prepared. The horseradish peroxidase enzyme (HRP) was incorporated in both products as a catalyst for hydrogen peroxide (H2O2) molecules. In the first step of this study, 10% and 20% H2O2 bleaching gels were prepared from a 35% H2O2 stock solution combined with a thickener with or without concentrations of 0.5, 1.0 and 2.0 mg/mL of HRP (HRP). These products were evaluated concerning the pH, temperature, reaction stability, free radicals (ROS, HORAC probe) and hydroxyl radical (OH•, H2DCFDA probe) formation, as well as bleaching efficacy (E). An in vitro protocol for intrinsic pigmentation of bovine enamel/dentin discs was used to assess the cytotoxicity as well as the trans-enamel and trans-dentinal diffusion of H2O2. Overall, the pH and temperature of the gels remained constant during all the analysis period. The addition of HRP enzyme to the CT accelerated the H2O2 catalysis, stimulating the production of ROS and OH• (ANOVA/Tukey; p<0,05). Furthermore, the HRP added to the CT enhanced E of the gel and reduced the trans-enamel and trans-dentinal diffusion of residua... (Complete abstract click electronic access below) / Doutor

The acetylcholinesterase (AChE) content of rat vagal efferent neurons was studied. Retrograde transport of horseradish peroxidase (HRP) by cut vagal axons provided a means for localizing efferent cell bodies; tissue sections were then processed for the simultaneous visualization of HRP and AChE. A dorsal vagal efferent column contained the dorsal motor nucleus of the vagus, as a primary component, and extended caudally into the upper cervical spinal cord. A ventral column contained neurons in the nucleus ambiguus and the surrounding reticular formation. Although most of the vagal efferent neurons stained with moderate to heave intensity for AChE there were some HRP-labeled cells that contained little AChE and a small percentage in which AChE was absent. In spite of the fact that AChE has been demonstrated in certain non-cholinergic neurons, it has also been found in all cholinergic neurons. Therefore, the presence of AChE has been regarded as a necessary (but not sufficient) component for identifying cholinergic neurons. The absence of AChE in a small percentage of the vagal efferent neurons indicates that some preganglionic parasympathetic fibers in the vagus nerve are not cholinergic.

The brainstem localization of acetylcholinesterase (AChE)-containing neurons projecting to the anteroventral thalamic nucleus (AVN) was studied in rats. The AVN is one of several forebrain regions innervated by the AChE-containing dorsal tegmental pathway described by Shute and Lewis. In the present study, horseradish peroxidase (HRP) was injected into the region of the AVN to determine the brainstem origin of afferent projections. Alternate sections of tissue were stained for HRP or AChE. HRP-labeled neurons were found in the laterodorsal tegmental nucleus (LTN) and the locus coeruleus. Examination of adjacent sections revealed AChE-containing neurons in both of these nuclear regions. Combined HRP/AChE histochemistry demonstrated that transported HRP and AChE were in the same cells. In further experiments, unilateral lesions of the LTN were found to cause a decrease in AChE staining of the ipsilateral AVN. Destruction of the locus coeruleus had no effect. In combination with available evidence, the present findings suggest that cholinergic neurons in the LTN innervate the AVN. © 1980, All rights reserved.

Indiana University-Purdue University Indianapolis (IUPUI) / The extracellular matrix (ECM) governs many cellular processes through biochemical and mechanical cues. Particularly, the effect ECM mechanical properties on cells fate has been well established over the years. Many hydrogel systems have been used to mimic the dynamic stiffening processes occurring in ECM. However, changes in ECM stiffness does not fully recapitulate the mechanics of native ECM, as viscoelasticity is also a major factor contributing to ECM dynamic property. This thesis describes the design and characterization of an enzyme-crosslinked hydrogel system that is not only capable of being stiffened on demand, but also can be tuned to obtain viscoelasticity. The first objective of this thesis was to utilize horseradish peroxidase (HRP) to crosslink thiol-norbornene hydrogel and use mushroom tyrosinase (MT) to create secondary DOPA-dimer crosslinks that stiffened the hydrogel. The cytocompatibility of HRP-mediated thiol-norbornene gelation and the effect of stiffening on cell fate was evaluated. The second objective of this thesis represented the first step towards developing a hydrogel system whose viscoelasticity could be dynamically tuned. Thiol-norbornene hydrogel was designed to yield dynamically adaptable boronic ester bonds via partial enzymatic reaction. Thiol-norborne hydrogel was made to contain hydroxyl phenol as well as boronic acid residues within its network. MT, in this case was used to oxidize the hydroxy phenol moieties into DOPA, which then complexed with boronic acid, created dynamic bonds, introducing viscoelasticity to an initial elastic hydrogel.