Reverse transcriptase (RT) is a crucial enzyme for retrovirus replication, and its presence in the virion is indispensable for infectivity. This thesis illustrates the use of RT activity assays as tools for quantitation and characterization of different retroviruses, particularly HIV. A non radioactive assay, using microtiter plates, for the RT of Moloney murine leukemia virus (MMuLV) was developed. Assay conditions for MMuLV and HIV-1 RT, together with isozyme specific RT activity blocking antibodies, were shown useful for discrimination between RTs from different retrovirus genera. RT activity assay for HIV-1 was found to quantitate different subtypes more equally efficient than p24 antigen assays did. Viral load (VL), the amount of HIV particles in the blood, is an important marker of the clinical status of an infected person. A method for VL determination based on RT activity (ExaVir Load) was developed. After plasma pretreatment, to inactivate cellular DNA polymerases, virions in patient plasma were immobilized on a gel, which was washed to remove disturbing factors. The virions were lysed with a detergent containing buffer and the lysate eluted. Finally, the RT activity in the lysate was determined and found to correlate strongly to VL by RNA according to a PCR based standard method (Roche Amplicor 1.5). The second version of the method was able to measure VL down to approximately 400 HIV-1 RNA copies/ml. The usefulness of RT from the VL procedure for determination of susceptibility towards anti-HIV drugs was demonstrated, and the results were in agreement with genotypic data. Due to its technical simplicity, and ability to detect a broad range of HIV-1 subtypes, ExaVir Load and the drug susceptibility application are interesting for clinical use, particularly but not only in resource limited settings. The concept is also potentially useful for research purposes, e.g. in combination with specific RT assay conditions.

L’objectif principal de cette recherche est de contribuer au développement de biocapteurs commerciaux utilisant des surfaces de papier comme matrices d’immobilisation, capables de produire un signal colorimétrique perceptible dans les limites sensorielles humaines. Ce type de biocapteur, appelé papier bioactif, pourrait servir par exemple à la détection de substances toxiques ou d’organismes pathogènes. Pour atteindre l’objectif énoncé, ce travail propose l’utilisation de systèmes enzymatiques microencapsulés couchés sur papier. Les enzymes sont des catalyseurs biologiques dotés d’une haute sélectivité, et capables d'accélérer la vitesse de certaines réactions chimiques spécifiques jusqu’à des millions des fois. Les enzymes sont toutefois des substances très sensibles qui perdent facilement leur fonctionnalité, raison pour laquelle il faut les protéger des conditions qui peuvent les endommager. La microencapsulation est une technique qui permet de protéger les enzymes sans les isoler totalement de leur environnement. Elle consiste à emprisonner les enzymes dans une sphère poreuse de taille micrométrique, faite de polymère, qui empêche l’enzyme de s’echapper, mais qui permet la diffusion de substrats à l'intérieur. La microencapsulation utilisée est réalisée à partir d’une émulsion contenant un polymère dissous dans une phase aqueuse avec l’enzyme désirée. Un agent réticulant est ensuite ajouté pour provoquer la formation d'un réseau polymérique à la paroi des gouttelettes d'eau dans l'émulsion. Le polymère ainsi réticulé se solidifie en enfermant l’enzyme à l'intérieur de la capsule. Par la suite, les capsules enzymatiques sont utilisées pour donner au papier les propriétés de biocapteur. Afin d'immobiliser les capsules et l'enzyme sur le papier, une méthode courante dans l’industrie du papier connu sous le nom de couchage à lame est utilisée. Pour ce faire, les microcapsules sont mélangées avec une sauce de couchage qui sera appliquée sur des feuilles de papier. Les paramètres de viscosité i de la sauce et ceux du couchage ont été optimisés afin d'obtenir un couchage uniforme répondant aux normes de l'industrie. Les papiers bioactifs obtenus seront d'abord étudiés pour évaluer si les enzymes sont toujours actives après les traitements appliqués; en effet, tel que mentionné ci-dessus, les enzymes sont des substances très sensibles. Une enzyme très étudiée et qui permet une évaluation facile de son activité, connue sous le nom de laccase, a été utilisée. L'activité enzymatique de la laccase a été évaluée à l’aide des techniques analytiques existantes ou en proposant de nouvelles techniques d’analyse développées dans le laboratoire du groupe Rochefort. Les résultats obtenus démontrent la possibilité d’inclure des systèmes enzymatiques microencapsulés sur papier par couchage à lame, et ce, en utilisant des paramètres à grande échelle, c’est à dire des surfaces de papier de 0.75 x 3 m2 modifiées à des vitesses qui vont jusqu’à 800 m/min. Les biocapteurs ont retenu leur activité malgré un séchage par évaporation de l’eau à l’aide d’une lampe IR de 36 kW. La microencapsulation s’avère une technique efficace pour accroître la stabilité d’entreposage du biocapteur et sa résistance à l’exposition au NaN3, qui est un inhibiteur connu de ce biocapteur. Ce projet de recherche fait partie d'un effort national visant à développer et à mettre sur le marché des papiers bioactifs; il est soutenu par Sentinel, un réseau de recherche du CRSNG. / The main objective of this research is the development of a commercial biosensor immobilized on paper surfaces, able to produce a colorimetric signal detected by human sensorial limits. This kind of biosensor could be used, for example, in the detection of toxic substances or pathogens. To achieve this objective, microencapsulated enzymes fixed on paper are proposed. Enzymes are biological catalysts with a high selectivity that can accelerate the speed of some chemical reactions up to a million times. However, the enzymes are very sensitive substances that lose their functionality easily; it is therefore necessary to protect them from conditions that could damage them. Microencapsulation is a technique that protects the enzymes without totally isolating them from their environment. In fact, microencapsulation entraps the enzymes into a micron size sphere, made of a porous polymer which prevents the enzyme to be released but allows the diffusion of its substrate inside. The microencapsulation process consists in making an emulsion containing a polymer dissolved in an aqueous phase with the desired enzyme, and the wall of the microcapsule is formed by adding a crosslinking agent that forms a polymer network at the interface of the emulsion. The crosslinked polymer solidifies and it encloses the enzyme in the interior of the capsule. Thereafter, this kind of microcapsules are used to give biosensor properties to the paper. Blade coating technique is used in order to immobilize the enzyme capsules on paper because it is the most widely used method in the paper industry. The microcapsules are mixed with a coating suspension and applied on sheets of paper. The viscosity parameters of the suspension and those of the coating are optimized to obtain a uniform coating in order to meet the industry standards. Bioactive paper obtained is first studied to assess whether the enzymes are still active or not after all the treatments because, as described above, enzymes are iii very sensitive substances. An enzyme known as laccase is used, which allows an easy evaluation of its activity. Enzymatic activity was evaluated through existing analytical techniques or new analysis techniques developed in the Rochefort lab. The results demonstrate the possibility to transfer microencapsulated enzyme systems onto paper by blade coating, by using large scale settings, with paper surfaces of 0.75 x 3 m2 modified at speeds ranging up to 800 m/min. Biosensors retained their activity, despite a drying process by evaporation of water using an IR lamp of 36 kW. The microencapsulation technique proposed here is an effective technique to increase the storage stability of the biosensor and its resistance to exposure to NaN3, which is a known inhibitor of this biosensor. This research is part of a national effort in order to develop a commercial device called bioactive paper; it is supported by the NSERC research network Sentinel.

Awareness of detoxification, nowadays known as biotransformation, has become an integral part of our daily lives. It is a modern buzz word that is used to promote anything from health food to enhancement of performance in sports. Another lesser known application for detoxification is as a therapy for alleviating symptoms of inborn errors of metabolism. Detoxification is the process where endogenous and xenobiotic metabolites are transformed to less harmful products, in the liver and kidneys, in two phases. Phase 1 detoxification includes oxidation, hydroxylation, dehydrogenation metabolic reduction and hydrolysis. Phase 2 detoxification uses conjugation reactions to increase hydrophillicty of metabolites for excretion in bile and urine. Glycine N-acyltransferse (GLYAT; EC 2.3.1.13) is one of the amino acid conjugation enzymes. There are two variants of human GLYAT. I focused on the full-length mRNA human GLYAT isoform a, with a long term view of using it as a viable therapeutic enzyme for enhanced detoxification of harmful metabolites. I investigated if it is possible to clone and express a biologically active GLYAT. To achieve this goal I used three expression systems: traditional bacterial expression using the pET system; second generation cold shock bacterial expression using the pCOLDTF expression vector to improve solubility of the recombinant protein; and baculovirus expression in insect cells since therein some form of post translation glycosylation of the recombinant protein can occur which might improve solubility and ensure biological activity. The recombinant GLYAT expressed well in all three expression systems but was aggregated and no enzyme activity could be detected. A denature and renature system was also used to collect aggregated recombinant GLYAT and used to try to refold the recombinant protein in appropriate refolding buffers to improve solubility and obtain biological activity. The solubility of the recombinant GLYAT was improved but it remained biologically inactive. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

Metallothioneins (MTs) are small, non-enzymatic proteins that are involved in cellular detoxification and metal homeostasis because of their high cysteine content. MTs have also been identified as one of the vast number of adaptive responses to mitochondrial respiratory chain (RC) deficiencies. Aside from this, numerous other studies have linked MTs to several mitochondrion-linked components, including reactive oxygen species (ROS) and oxidative stress, apoptosis, glutathione, energy metabolism and nuclear- and mitochondrial DNA transcription regulation. However, most of the reports concerning the putative link between MTs and mitochondria are from in vitro studies and relatively little supportive in vivo evidence has been reported. Information on the involvement of MTs with respiratory chain function is especially limited. Is was therefore the aim of this study to investigate the involvement of MTs in mitochondrial respiration and respiratory chain enzyme function by using an MT knockout (MTKO) mouse model, which was treated with the irreversible complex I inhibiting reagent, rotenone. The aim was achieved by implementing three objectives: firstly, the RC function was investigated as a complete working unit; secondly, the functional and structural properties of single units (enzymes) of the RC were investigated utilising enzyme activity assays and BN- PAGE/western blot analysis; and thirdly, the possible effect of MTs on mtDNA copy number was investigated. While some tendencies of variation in RC enzyme activity and expression were identified, no significant effect on the overall mitochondrial respiratory function, or any significant differences in the relative mtDNA copy number of MTKO mice were observed. Thus it is concluded, while MTs have in this study revealed relatively small changes in respiratory chain function, which may still prove to have biological ignificance in vivo, the exact nature of the putative role of MTs in mitochondrial respiration or oxidative phosphorylation remains undefined. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2012.

Awareness of detoxification, nowadays known as biotransformation, has become an integral part of our daily lives. It is a modern buzz word that is used to promote anything from health food to enhancement of performance in sports. Another lesser known application for detoxification is as a therapy for alleviating symptoms of inborn errors of metabolism. Detoxification is the process where endogenous and xenobiotic metabolites are transformed to less harmful products, in the liver and kidneys, in two phases. Phase 1 detoxification includes oxidation, hydroxylation, dehydrogenation metabolic reduction and hydrolysis. Phase 2 detoxification uses conjugation reactions to increase hydrophillicty of metabolites for excretion in bile and urine. Glycine N-acyltransferse (GLYAT; EC 2.3.1.13) is one of the amino acid conjugation enzymes. There are two variants of human GLYAT. I focused on the full-length mRNA human GLYAT isoform a, with a long term view of using it as a viable therapeutic enzyme for enhanced detoxification of harmful metabolites. I investigated if it is possible to clone and express a biologically active GLYAT. To achieve this goal I used three expression systems: traditional bacterial expression using the pET system; second generation cold shock bacterial expression using the pCOLDTF expression vector to improve solubility of the recombinant protein; and baculovirus expression in insect cells since therein some form of post translation glycosylation of the recombinant protein can occur which might improve solubility and ensure biological activity. The recombinant GLYAT expressed well in all three expression systems but was aggregated and no enzyme activity could be detected. A denature and renature system was also used to collect aggregated recombinant GLYAT and used to try to refold the recombinant protein in appropriate refolding buffers to improve solubility and obtain biological activity. The solubility of the recombinant GLYAT was improved but it remained biologically inactive. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

Metallothioneins (MTs) are small, non-enzymatic proteins that are involved in cellular detoxification and metal homeostasis because of their high cysteine content. MTs have also been identified as one of the vast number of adaptive responses to mitochondrial respiratory chain (RC) deficiencies. Aside from this, numerous other studies have linked MTs to several mitochondrion-linked components, including reactive oxygen species (ROS) and oxidative stress, apoptosis, glutathione, energy metabolism and nuclear- and mitochondrial DNA transcription regulation. However, most of the reports concerning the putative link between MTs and mitochondria are from in vitro studies and relatively little supportive in vivo evidence has been reported. Information on the involvement of MTs with respiratory chain function is especially limited. Is was therefore the aim of this study to investigate the involvement of MTs in mitochondrial respiration and respiratory chain enzyme function by using an MT knockout (MTKO) mouse model, which was treated with the irreversible complex I inhibiting reagent, rotenone. The aim was achieved by implementing three objectives: firstly, the RC function was investigated as a complete working unit; secondly, the functional and structural properties of single units (enzymes) of the RC were investigated utilising enzyme activity assays and BN- PAGE/western blot analysis; and thirdly, the possible effect of MTs on mtDNA copy number was investigated. While some tendencies of variation in RC enzyme activity and expression were identified, no significant effect on the overall mitochondrial respiratory function, or any significant differences in the relative mtDNA copy number of MTKO mice were observed. Thus it is concluded, while MTs have in this study revealed relatively small changes in respiratory chain function, which may still prove to have biological ignificance in vivo, the exact nature of the putative role of MTs in mitochondrial respiration or oxidative phosphorylation remains undefined. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2012.

No description available.

The increased soil organic phosphorus (P) mineralization observed under trees compared to pasture has been attributed to the contrasting mycorrhizal associations of the two systems but to date, little work has been conducted comparing P dynamics under different tree species with contrasting mycorrhizal associations. This study investigated rhizosphere P dynamics and P acquisition of three tree species with contrasting mycorrhizal associations (ectomycorrhizal Pinus radiata, arbuscular mycorrhizal Cupressus macrocarpa and tripartite Ecualyptus nitens) using a combination of field and controlled environment studies. Short-term field studies revealed greater acid phosphomonoesterase activity and therefore greater potential organic P mineralization under radiata pine and eucalypt compared with macrocarpa, which correlated with ectomycorrhizal colonization. Related field work revealed that the presence of pasture understory and litter had a significant impact on P dynamics. Pasture acts to increase phosphatase activity and microbial activity within the soil but with a litter layer, the increased activities are mainly above the soil in the overlying litter. A glasshouse study investigated the separate influence of mycorrhizal hyphae and roots and results showed that ectomycorrhizal fungi associated with radiata pine and eucalypt stimulated microbial activity and increased rhizosphere phosphatase activity with a concomitant decline in soil organic P. Macrocarpa and eucalypt roots exuded higher concentrations of low molecular weight organic anions compared with radiata pine which stimulated microbial activity, increased rhizosphere phosphatase activity and decreased soil organic P. A series of experiments were then conducted to further investigate the precise role of mycorrhizae in P dynamics and results showed that ectomycorrhizal colonization increased rhizosphere acid phosphomonoesterase to a greater extent than arbuscular mycorrhizae. Fluorescent staining revealed that the mycorrhizae associated with all three species had similar phosphatase production capacities. The ability of the three species to utilize organic P was investigated in two exhaustive pot experiments. Radiata pine was shown to be the best adapted to utilize organic P, especially in soils with high relative organic P contents. Eucalypt appeared to enhance soil organic P mineralization but did not take up the released inorganic P, while macrocarpa caused limited organic P mineralization compared with radiata pine and eucalypt. This work shows that ectomycorrhizal trees are best adapted to utilize organic P through increased rhizosphere phosphatase activity and stimulated microbial activity. Root exudation of LMWOAs is of higher relative importance to arbuscular mycorrhizal trees to increase microbial activity and consequently phosphatase activities when fewer hyphae are in the rhizosphere are present to produce them.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2007. / Härtill 4 uppsatser.

Submitted by Suelen Cristina Nunes Alves (suelen_cris88@yahoo.com.br) on 2018-05-30T16:50:57Z No. of bitstreams: 1 SuelenCristinaNunesAlvesTeseRevisada.pdf: 2888189 bytes, checksum: 543ba9a57a76662fdd1500ad5ee48e03 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-06-05T18:24:30Z (GMT) No. of bitstreams: 1 alves_scn_dr_jabo.pdf: 2888189 bytes, checksum: 543ba9a57a76662fdd1500ad5ee48e03 (MD5) / Made available in DSpace on 2018-06-05T18:24:30Z (GMT). No. of bitstreams: 1 alves_scn_dr_jabo.pdf: 2888189 bytes, checksum: 543ba9a57a76662fdd1500ad5ee48e03 (MD5) Previous issue date: 2018-04-04 / O níquel (Ni) é um metal de ocorrência natural em rochas magmáticas. Pode ser adicionado ao solo por ações antrópicas, como a agricultura, disposição de resíduos industriais e deposições atmosféricas. Este elemento satisfaz critérios de essencialidade para as plantas, mas é considerado tóxico em altas concentrações. A hipótese deste trabalho é: doses variadas de Ni na forma de Ni(NO3)2 e Ni2O3 contribuem para maior absorção de Ni por plantas de sorgo e causam efeitos nas atividade biológicas e enzimáticas do solo. A degradação de plantas de sorgo contaminadas em solo que não foi adicionado Ni(NO3)2 e Ni2O3 não causam efeitos na atividade biológica e enzimática. O experimento foi instalado em casa de vegetação localizada no departamento de Tecnologia da FCAV/UNESP – Jaboticabal. O solo utilizado foi o Latossolo Vermelho distrófico (LVd). O experimento foi conduzido em duas etapas. A primeira etapa diz respeito ao desenvolvimento de plantas de sorgo em solo contaminado por Ni(NO3)2 e Ni2O3. O delineamento experimental foi o fatorial 2 x 3 +1. Nessa etapa foram testadas 2 fontes de Ni [Ni(NO3)2 e Ni2O3] em 3 doses, com 3 repetições, mais um tratamento controle com adubação mineral sem níquel. As doses de Ni foram 35, 70 e 140 mg kg-1 solo. Nesta etapa foram realizadas análises de Ni pseudototal e extraível, Ni nas folhas diagnose, planta inteira e grãos, assim como, quantificação do carbono na biomassa microbiana, respiração basal do solo, hidrólise do diacetato de fluoresceína e cálculo de quociente metabólico, além da produtividade da cultura. A contaminação do solo com Ni(NO3)2 e Ni2O3 influencia as atividades biológica e enzimática, pois provocou estresse na comunidade microbiana. O Ni(NO3)2 ocasionou maior absorção de Ni na planta e a absorção dos elementos Cu, Zn, Mn e Mg também foi influenciada pelo Ni no solo. A produtividade foi menor no tratamento com plantas contaminadas. A segunda etapa foi desenvolvida em delineamento experimental inteiramente casualizado, em que foi avaliada a liberação de Ni pela biodegradação das plantas de sorgo, e sua influência do Ni nas atividades biológica e enzimática do solo no decorrer do tempo, com 4 tratamentos e 5 repetições. Foram adotados os tratamentos, todos com o mesmo solo: controle absoluto sem plantas (CABS), controle com plantas não contaminadas com Ni (CONT), plantas contaminadas com Ni(NO3)2 (NN) e Ni2O3 (OX). Foram realizadas amostragens aos 0, 15, 30, 60 e 120 dias após a instalação do experimento, para verificar as mudanças no decorrer do tempo. Foram analisados o níquel extraível, o carbono na biomassa microbiana e a hidrólise do diacetato de fluoresceína. / Nickel (Ni) is a naturally occurring metal in magmatic rocks. Can be added to the soil by anthropogenic actions, such as agriculture, industrial waste disposal and atmospheric deposition. This element satisfies criteria for essentiality to the plants, but is considered toxic at high concentrations. The hypothesis of this paper is: varied doses of Ni in the form of Ni(NO3)2 and Ni2O3 contribute to greater absorption of Ni for sorghum plants and cause effects on biological and enzyme activity of soil. The degradation of sorghum plants contaminated in soil that has not been added Ni(NO3)2 and Ni2O3 do not cause effects on biological and enzyme activity. The experiment was installed in greenhouse located in the Department of technology of UNESP/FCAV-Jaboticabal. The soil used was Red Latosol distrófic (LVd). The experiment was conducted in two stages. The first stage concerns the development of sorghum plants in contaminated soil by Ni(NO3)2 and Ni2O3. The experimental design was factorial 2 x 3 +1. In this step 2 Ni sources were tested [Ni(NO3)2 and Ni2O3] into 3 doses, with 3 replications, more control treatment with mineral fertilization without nickel. The doses of Ni were 35, 70 and 140 mg kg-1 soil. Analyses were performed at pseudototal and extractable Ni, Ni on diagnosis, entire plant and grain, as well as quantification of microbial biomass carbon, soil basal respiration, hydrolysis of fluorescein diacetate and calculation of Metabolic Quotient, in addition to the productivity of the crop. Soil contamination with Ni(NO3)2 and Ni2O3 influences the biological and enzymatic activities, because it caused stress on microbial community. The Ni(NO3)2 caused increased absorption of Ni in the plant and the absorption of the elements Cu, Zn, Mn and Mg was also influenced by Ni in soil. Productivity was lower in the treatment with contaminated plants. The second step was developed in completely randomized experimental design, in which it was evaluated the release of Ni by the biodegradation of sorghum plants, and your influence of biological and enzymatic activities Ni of the soil over time, with 4 treatments and 5 repetitions. The treatments were adopted, all with the same soil: absolute control without plants (CABS), with control plants contaminated with Ni (CONT), contaminated plants with Ni(NO3)2 (NN) and Ni2O3 (OX). Samplings were carried out at 0, 15, 30, 60 and 120 days after installation of the experiment, to check the changes over time. We analyzed the extractable nickel, carbon in microbial biomass and fluorescein diacetate hydrolysis.