Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-02-17T18:26:18Z No. of bitstreams: 1 Maria Goreth Matos de Andrade Barberino... Distribuição clonal 2013.pdf: 2055064 bytes, checksum: da2e73dad9d911837f5434c00617e1b0 (MD5) / Made available in DSpace on 2014-02-17T18:26:18Z (GMT). No. of bitstreams: 1 Maria Goreth Matos de Andrade Barberino... Distribuição clonal 2013.pdf: 2055064 bytes, checksum: da2e73dad9d911837f5434c00617e1b0 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / A infecção do trato urinário (ITU) é considerada a segunda infecção mais comum em humanos, estima-se que ocorram cerca de 150 milhões de casos de ITUs por ano no mundo. O aumento das taxas de resistência aos antimicrobianos entre os uropatógenos tem tornado mais difícil o tratamento das ITUs. Objetivo: Determinar a distribuição clonal das cepas de E. coli isoladas em pacientes com ITU adquirida na comunidade de acordo com o perfil de susceptibilidade aos antimicrobianos e avaliar o papel dos grupos clonais na disseminação e persistência da resistência nestas infecções. Métodos: Foram isoladas 874 cepas de E. coli em pacientes com ITU, procedentes de unidades ambulatoriais em três hospitais na cidade de Salvador–Ba, no período de 2001 a 2009. O perfil de susceptibilidade foi determinado por microdiluição em placa (Microscan-Siemens®). Nas amostras selecionadas para genotipagem (n=275), a identificação dos grupos clonais foi realizada pela comparação dos padrões de PFGE, utilizando os critérios de Tenover (1995). Em todas as etapas do estudo foi utilizada como controle de qualidade a cepa ATCC E. coli 25922. Resultado: Entre os antibióticos testados, a maior prevalência de resistência foi encontrada para ampicilina (AMP) (49%), cefalotina (12-33%) e sulfametoxazol-trimetropin (SXT) (36-42%). A taxa de resistência à ciprofloxacina (CIP) variou de 9 a 14%. Na análise da distribuição clonal, segundo os fenótipos de resistência aos antimicrobianos, encontramos maior predomínio de um grupo clonal CgA (63%) entre as cepas consideradas multidroga resistentes. Diferentemente das amostras com algum grau de resistência ou multi-sensíveis, nas quais observamos diversidade clonal. Conclusão: A alta prevalência de resistência a SXT, AMP e cefalotina contraindica o uso destes antimicrobianos no tratamento empírico das ITU adquiridas na comunidade. A taxa de resistência à CIP relativamente alta, alerta para o aumento e disseminação de resistência a este antimicrobiano na comunidade. Isto irá dificultar e onerar o tratamento destas infecções. Observamos a surgimento de um grupo clonal (CgA) no período final do estudo (2008 a 2009) associado às cepas multidroga resistentes. Este achado sugere que a expansão de determinados clones pode ter um papel importante na disseminação de resistência bacteriana em ITUs adquiridas na comunidade. / Urinary tract infection (UTI) is considered the second most common infection in humans. It is estimated that there are about 150 million cases of UTIs per year worldwide. Increasing rates of antimicrobial resistance among uropathogens challenges UTI treatments. Objective: To determine the distribution of clonal strains of E. coli isolated from patients with community-acquired UTI according to the profile of antimicrobial susceptibility; and to evaluate the role of clonal groups in the spread and persistence of resistance in these infections. Methods: Eight hundred seventy four strains of E. coli were isolated from patients with UTI, coming from outpatient clinics in three hospitals in the city of Salvador - Bahia, from 2001 to 2009. The susceptibility profile was determined by broth microdilution method (Siemens - Microscan ®). The samples selected for genotyping (n = 275) were identified for clonal groups by comparing the patterns of PFGE, using the criteria of Tenover (1995). All study stages were quality control by strain E. coli ATCC 25922. Results: Among the antibiotics tested, the highest prevalence of resistance was for ampicillin (AMP) (49%) followed by trimethoprim - sulfamethoxazole (SXT) (36-42%) and for cephalothin (12-33%). The rate of resistance to ciprofloxacin (CIP) ranged between 9-14 %. In the Clonal Analysis distribution, performed according to antimicrobial resistance phenotypes, we found a higher prevalence of a clonal group CgA (63%) among multidrug resistant strains. This result differs from samples with some degree of resistance or multi-sensitive in which we observed clonal diversity. Conclusion: The high prevalence of resistance to SXT, AMP, and cephalothin contraindicate the use of these antibiotics in the empirical treatment of community-acquired UTI. The relatively high rate of resistance to CIP, raises attention to the increase and spread of antimicrobial resistance in this community and potentially complicate and encumber the treatment of these infections. We observe the emergence of a clonal group (CgA) in the final period of the study (2008-2009) associated with multidrug resistant strains. This finding suggests that the expansion of particular clones may have an important role in the spread of bacterial resistance in community-acquired UTI.

The advantages associated with the use of sewage sludge in agricultural land have motivated many countries to use sewage sludge for soil amendment purposes. South Africa’s deteriorated agricultural soil could benefit from this nutritional and cost effective product. However, the major shortcoming of sewage sludge is the presence of various pathogenic microorganisms. This raised concern amongst researchers with regard to public safety. The focus of this study, was to investigate the prevalence of pathogens in a crop grown in soil enriched with sewage sludge and to determine risk of infection thereof and to suggest appropriate management practice for sewage sludge use. Potato (Solanum tuberrosum), which is a high risk crop was used, to simulate a worst case scenario. Both the low metal sludge (LMS) and high metal sludge (HMS) were found to have associated diverse numbers of bacteria. Using culture-based technique, E.coli and Salmonella spp were found to persist in soil throughout the experimental period. One treatment option (LMS 16 tons/ha) showed a prevalence of these microorganisms in potatoes. Subsequent molecular studies based on amplification of 16S rRNA gene, yielded limited contamination of potatoes with enteric pathogens, however diverse types of opportunistic, pathogens (mostly environmental pathogens) were isolated from the potatoes. Enteric pathogens were isolated from the sewage treated soil in which these potatoes were grown. This study has indicated that growing even high risk crops, may lead to limited infestation of produce with primary pathogens. However, proper treatment of sewage sludge prior to use in agriculture is recommended to ensure public safety. The management requirements indicated in this study serve as recommended actions that can be implemented to ensure human safety with regard to sludge application to agricultural land. / Thesis (PhD (Water Utilisation))--University of Pretoria, 2006. / Chemical Engineering / unrestricted

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2009. / Water is used for various purposes and contamination can have severe implications if untreated. One of the most common and cost effective water disinfectants, especially used in developing countries, is chlorine. However, microorganisms have developed different mechanisms in response to environmental stress conditions, such as the viable but nonculturable (VBNC) effects possibly displayed in this study, enabling them to survive. Chlorine may also exert several effects on microorganisms, such as the expression of multi-substrate efflux pumps, decreased membrane permeability and transport inhibition that may lead to chlorine tolerance and antimicrobial resistance. In a descriptive and comparative study, the molecular characteristics of E. coli strains isolated from environmental waters in the Western Cape and the possible relationship between chlorination and antimicrobial resistance were investigated. Water and biofilm samples were exposed to chlorine, as well as efflux pump inhibitor (EPI) concentrations, and surviving E. coli strains were tested for their phenotypic characteristics including antimicrobial susceptibility profiles and morphological types. Candidate genes possibly involved in resistance to antimicrobials, disinfection and efflux pumps were detected with polymerase chain reaction (PCR) and sequenced. Sequencing analysis and homology searches were done and E. coli strains were typed as either Enteropathogenic E. coli strains (EPEC) or Enterotoxigenic E. coli strains (ETEC) on the presence of virulence genes. All water and biofilm sources examined were heavily polluted with E. coli, and a high enumeration level of this indicator organism of faecal contamination was recorded. Chlorine tolerance was found to be associated with antimicrobial resistance. Addition of EPI with exposure to chlorine decreased enumeration levels of these organisms, suggesting that efflux pumps may play a role in tolerance to chlorine. Several morphological patterns were described amongst the E. coli strains and a change in this was recorded after exposure to chlorine. Highly resistant antibiograms displayed by the isolated strains included ampC β-lactamase producing E. coli strains and extended spectrum β-lactamases (ESBLs). Amplification of the candidate genes selected for heatshock, oxidative stress genes and efflux pump were most frequently detected while the structural genes involved in fluoroquinolones (FQs) resistance were detected less frequently in the selected strains. Sequencing of these amplified candidate genes demonstrated various changes in amino acid sequences, including one common mutational pathway taken by E. coli when exposed to stress conditions. Further homology searches of the sequenced candidate genes illustrated similarities in 19 pathogenic and 14 non-pathogenic E. coli as well as 3 Shigella strains. Detection of virulence genes found three EPEC strains (bfpA, eaeA), two EPEC (eaeA), ten EPEC (bfpA) and one ETEC strain (st) amongst the isolates. This study underlines the need for monitoring our water sources, which poses a public health risk due to incomplete chlorination, antimicrobial resistance and the spread of clinically relevant pathogenic strains.

Ces dernières années, les énormes progrès réalisés en nanotechnologie ainsi qu’en science des matériaux ont conduit à la préparation de nombreuses nouvelles nanoparticules sans réellement connaître l’ensemble des propriétés associées à leurs dimensions. La première partie de notre travail vise à évaluer les risques et les problèmes associés aux nanomatériaux, en termes de toxicité, en utilisant des nanoparticules de ZnO. Nous avons tout d’abord étudié la capacité de ces nanoparticules à générer des espèces réactives d’oxygènes (EROs) sous irradiation UV en utilisant trois types des quantum dots (QDs) comme modèles, ZnO, ZnO dopé avec des ions Cu2+ et ZnO avec des ions Cu2+ adsorbés à sa surface. Les trois types des QDs ont montré une forte capacité à générer des EROs mais ceux modifiés par les ions Cu2+ en périphérie sont les plus producteurs. Ces QDs inhibent également le plus fortement la croissance de la bactérie E. coli. La toxicité n’est cependant pas dépendante des EROs photo-produits ni du zinc (+2) libéré par les QDs et montre qu’un mécanisme plus complexe doit être considéré. Dans une second partie, nous avons tenté d’améliorer l’activité photocatalytique de nanobâtonnets de ZnO en les associant à de l’oxyde de graphène réduit (rGO). Des nanocomposites ZnO/rGO ont été préparés par voie solvothermale et utilisés pour la phototodégradation du colorant Orange II comme modèle de polluant. Les résultats obtenus montrent que le photocatalyseur ZnO/rGO est très efficace sous irradiation solaire ou visible et qu’il est peu sensible à des variations de pH ou à la présence de perturbateurs dans le milieu. Finalement, le photocatalyseur est très stable et peut être réutilisé plus de dix fois sans perte notable d’activité. / In recent years, tremendous advances in nanotechnology and materials science have led to the synthesis of many new nanoparticles without really knowing all the properties associated with their dimensions. The first part of our work aims to evaluate the risks and problems associated with nanomaterials, in terms of toxicity, using ZnO nanoparticles. We first studied the ability of these nanoparticles to produce reactive oxygen species (ROS) under UV irradiation using three ZnO-based quantum dots (QDs) as models, ZnO, ZnO doped with Cu2+ ions and ZnO with chimisorbed Cu2+ ions at their periphery. The three QDs have a strong capacity of generating ROS but those modified with Cu2+ at their surface were found the be the highest producers. These dots were also found to inhibit more markedly the growth of the E. coli bacteria. The toxicity does neither depend on the amount of photo-generated ROS nor on the amount of Zn(+2) leaked by the QDs, thus indicating that a more complex mechanism should be considered. In a second part, we tried to improve the photocatalytic efficiency of ZnO nanorods by associating these nanomaterials with reduced graphene oxide (rGO). ZnO/rGO composites were prepared by a solvothermal method and applied for the photodegradation of Orange II used as model pollutant. Results obtained demonstrate that the ZnO/rGO photocatalyst is highly efficient under solar and under visible light irradiation and weakly sensitive to pH changes and to the presence of perturbators in the reaction medium. Finally, the photocatalyst is stable and can be reused up to ten times without significant loss of catalytic activity.

L’enginyeria metabòlica és una estratègia molt útil per produir molècules d’alt valor afegit mitjançant microorganismes. Molècules d’interès per la seva funció biològica, d’estructura complexa i amb dificultats en la seva obtenció i síntesi s’han obtingut de forma molt satisfactòria mitjançant aquesta metodologia. En el laboratori de Bioquímica de l’IQS s‘estudia la glicosiltransferasa de Micoplasma genitalium codificada pel gen mg517 i responsable de la síntesi de glicoglicerolípids (Andrés et al., 2010). S’ha vist que aquesta proteïna sobreexpressada en E.coli és funcional i acumula diferents glicoglicerolípids en la membrana plasmàtica. Aquests glicoglicerolípids mostren diferents punts d’interès. D’una banda, són tensioactius d’alt valor afegit que permeten la construcció de niosomes per l’alliberament controlat de fàrmacs i, d’altra banda, s’han relacionat com agents terapèutics amb inhibició de tumors cancerígens. Degut al creixent interès d’aquests productes,en el present treball s‘ha escollit E. coli com a microorganisme a modificar per enginyeria metabòlica per la producció de glicoglicerolípids, ja que per una banda, no presenta aquests lípids però sí sintetitza els seus precursors UDP-glucosa i diacilglicerol (DAG). S’han dissenyat diferents soques d’E.coli on se sobreexpressen la glicosiltransferasa MG517 i, a més, la uridiltransferasa GalU procedent d’E.coli JM109, que sintetitza el precursor UDP-glucosa a partir de glucosa 1-fosfat, i l’aciltransferasa PlsC involucrada en la biosíntesi del precursor DAG. En les soques on les proteïnes GalU i PlsC s’han sobreexpressat, les seves activitats han augmentat 220 i 80 vegades, respectivament. La glicosiltransferasa MG517 és activa en totes les soques però, sorprenentment, la seva activitat després de les cinc hores d‘inducció és10 vegades inferior quan es dóna la coexpressió de MG517 i PlsC. S’observa que la sobreproducció de UDP-glucosa no incrementa la quantitat total de glicoglicerolípids mentre que el DAG sí, de manera que la soca AbC amb els gens mg517 i plsC és la que sintetitza més glicoglicerolípids, arribant a nivells de 1059 nM per biomassa. Dels tres glicoglicerolípids formats, el diglucosilacilglicerol és sempre el més abundant i el seu percentatge varia entre 57 i 82% en funció de la coexpressió dels enzims. La producció d’aquests nous lípids en la membrana d’E. coli implica que el percentatge del fosfolípid fosfatidiletanolamina disminueixi un 20%, mentre els fosfolípids anionis es mantenen constants. Es conclou que la soca modificada d’E. coli AbC és una bona plataforma per la producció de nous glicolípids amb diferent estructura. / La ingeniería metabólica es una estrategia muy útil para producir moléculas de valor añadido mediante microorganismos. Moléculas de interés por su función biológica, de estructura compleja y con dificultades en su obtención y síntesis se han obtenido de forma muy satisfactoria con el uso de esta metodología. En el laboratorio de Bioquímica del IQS se estudia la glicosiltransferasa de Micoplasma genitalium codificada por el gen mg517 y responsable de la síntesis de glicoglicerolípidos (Andrés et al. 2011). Se ha observado que esta proteína sobreexpresada en E.coli es funcional y acumula estos lípidos en la membrana plasmática. Los glicoglicerolípidos muestran diferentes puntos de interés. Por una parte, son tensioactivos que permiten la construcción de niosomas para la liberación controlada de fármacos y, por otra parte, se han seleccionado como agentes terapéuticos con inhibición de tumores cancerígenos. Debido al creciente interés de estos productos, en el presente trabajo, se ha escogido E.coli como microorganismo a modificar por ingeniería metabólica para la producción de glicoglicerolípidos, ya que no presenta estos lípidos pero sí sintetiza sus precursores UDP-glucosa y diacilglicerol (DAG). Se han diseñado diferentes cepas de E.coli donde se sobreexpressa la glicosiltransferasa MG517 y, además, la uridiltransferasa GalU de E.coli JM109, que sintetiza el precursor UDP-glucosa, y la aciltransferasa PlsC involucrada en la biosíntesis del precursor DAG. En las cepas donde las proteínas GalU y PlsC se han sobreexpressado, sus actividades han aumentado 220 y 80 veces, respectivamente. La glicosiltransferasa MG517 es activa en todas las cepas pero, sorprendentemente, su actividad después de inducir es 10 veces inferior cuando se da la coexpresión de MG517 y PlsC. Se observa que la sobreproducción de UDP-glucosa no incrementa la cantidad total de glicoglicerolípidos mientras que el DAG sí, de forma que la cepa AbC con los genes mg517 y plsC es la que sintetiza más glicoglicerolípidos, llegando a niveles de 1059 nM por biomasa. De los tres glicoglicerolípidos formados, el diglucosildiacilglicerol es siempre el más abundante y su porcentaje varía entre 57 y 82% en función de la coexpressión de las enzimas. La producción de los nuevos lípidos en la membrana de E.coli implica que el porcentaje del fosfolípido fosfatidiletanolamina disminuya un 20%, mientras los fosfolípidos aniónicos se mantienen contantes. Se concluye que la cepa modificada de E.coli AbC es una buena plataforma para la producción de nuevos glicolípidos con distinta estructura. / Metabolic engineering is a useful strategy to achieve target molecules using microorganisms. Molecules of high biological value, with complex estructure and difficulties to be obtained and synthesised, as for example, glycoconjugates, have been successfully obtained by this methodology (Ruffing i Chen, 2010). Our group studies the Mycoplasma genitalium glycosyltransferase encoded by mg517 gene and responsible of glycoglycerolipid synthesis. (Andrés et al., 2010). This protein overexpressed in E. coli is functional and accumulates the glycolipids in its plasma membrane. These glycoglycerolipids have different points of interest. On one hand, they are biosurfactants and evencan form niosomes for drug delivery systems. On the other hand, they have been related to inhibition of cancer tumors. Due to growing interest of these products, and in order to improve production of glycoglycerolipids, different metabolic engineered E. coli strains have been designed in this work. This microorganism has been chosen since on the one hand, it does not produce these lipids but its metabolism produces the glicoglicerolipids precursors, UDP-glucose and diacylglycerol (DAG). In these strains, the glycosyltransferase is coexpressed with genes related to biosynthesis of both precursors. Therefore coexpression of the glycosyltransferase MG517, the uridyl transferase GalU from E. coli JM109, which synthesizes the precursor Glc-UDP from glucose-1-phosphate, and the acyl transferase PlsC involved in the biosynthesis of the precursor DAG have been studied. Once modified strains were constructed, their phenotype have been analysed. On one hand, the three enzymatic activities have been determined in vitro from the cell extracts. When GalU and PlsC were overexpressed, their activities increased 220 and 80-fold, respectively, compared to the controls. The glycosyltransferase MG517 was active in these modified strains but, surprisingly, its activity decreases 10-fold when MG517 and PlsC were coexpressed. It is observed that overproduction of UDP-glucose does not increase total glycolipids amount while DAG have a positive impact on this production, being strain with mg517 and plsC genes which produces more glycolipids achieving 1059 nM per biomass. . Furthermore, the modified strains showed different glycoglycerolipids profiles. In all strains the disaccharide glycoglycerolipid is the most abundant but its percentage varies from 57% to 82% depending on enzyme coexpression. Production of these new lipids in E. coli membrane implies less synthesis of phosphatidylethanolamine phospholipid, which is characteristic of this microorganism. Our results show the modified E. coli strain with mg517 and plsC genes is a good platform microorganism for the production of new glicolipids with different structure.

No description available.

The research developed a UV Taylor Couette reactor for disinfecting milk as a model opaque fluid. The principal of the reactor was to generate laminar vortices to support efficient mixing and homogenous UV photon distribution. The UV reactor parameters were optimized to generate laminar vortices that were stabilized by modification of the unit with baffles. A model was developed to predict the UV dose required to inactivate model microbes in milk. Through verification trials it was noted the predicted UV dose underestimated that required to support a 5 log cfu reduction of microbes. It was subsequently identified that the deviation from predicted values could be attributed to fat content that enhances the UV inactivation of microbes in milk with proteins providing protection to microbes. In conclusion, the UV Taylor Couette reactor has strong potential for disinfecting opaque fluids although matrix effects need to be considered when undertaking validation trials.

To unravel the mysteries of complex biological processes carried out by biomolecules it is necessary to adopt a multifaceted approach, which involves employing a wide variety of tools both computational and experimental. In order to gain a clear understanding of the function of biomolecules their three dimensional structure is required. X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy are the only two methods capable of providing high-resolution three-dimensional structure of biomolecules. NMR has the advantage of allowing the study of structure of biomolecules in solution and is better equipped to characterize the dynamics of the protein. Protein structure determination by NMR spectroscopy consists of recombinant expression of isotopically labeled proteins, purification, data collection, data processing, resonance assignment, distance restraint and angular restraint generation, structure calculation and structure validation. Apart from 3D structure determination of biomolecules NMR has become the method of choice for studying transient protein-protein interactions, which are notoriously difficult to study at higher resolution by other methods. Mass spectrometry plays an important role in enabling rapid identification of biomolecules and their modifications. The high sensitivity and resolution mass spectrometry offers makes it the method of choice for studying post-transitional modification of proteins. Use of computers in biology has played an essential role in elucidating those structure function relationships in biomolecules that are not possible to study by experimental techniques. The first chapter of this thesis deals with the introduction of methods used in this study. A brief introduction about the theory of Nuclear Magnetic Resonance (NMR) spectroscopy is given. Protein NMR methods used for structure determination of medium sized proteins are discussed. A part of this chapter discusses about the application of mass spectrometry in biochemistry and the use of tandem MS/MS experiments in identification of proteins and peptide fragments. Finally, the last part of this chapter gives an introduction about the theory of molecular dynamics and techniques used in the post processing of MD trajectories to elucidate the dynamics of proteins. The second chapter of this thesis is concerned with NMR characterization of a novel protein-protein interaction between the glycolytic enzyme Triosephosphate isomerase and the redox protein Thioredoxin. Chemical shift perturbation studies have been done to map the binding interfaces of these proteins. The structure of the complex was then modeled using NMR restraints based docking using the known 3D structure of these proteins. The docked complex reveals crucial insights into the glutathione mediated redox regulation of Triosephosphate isomerase and the role of thioredoxin as a deglutathionylating agent. Enzyme activity assays of Triosephosphate isomerase were done to show the inhibitory effects of s-glutathionylation of Cys217 and the role of thioredoxin as a deglutathionylating agent. The third chapter of the thesis is aimed to address some important issues related to the inhibition of Plasmodium falciparum Triosephosphate isomerase by S-glutathionylation. Oxidative stress induces protein glutathionylation which is a reversible post translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione. Mass spectrometric analysis of the kilnetics of glutathionylation along with enzyme activity assays clearly show that gluthionylation of either Cys-13 (situated in the dimmer interface) or Cys-217 (situated in Helix G) can render the enzyme inactive. Molecular dynamics simulations provide a mechanistic basis of inhibition and predict that glutathionylation at Cys217 allosterically induces loop 6 disorder. The fourth chapter of this thesis addresses the stabilizing effect of introduction of a cross-strand disulfide bond across a non-hydrogen bonded position of an antiparallel beta sheet. Multidimensional heteronuclear NMR experiments have been used to get the backbone and side-chain resonance assignments, distance and angular restraints. In addition RDC based restraints have been used to calculate the structure of oxidixed form of L79C, T89C thiroedoxin. The observation of predominantly –RH staple conformation among the NMR ensemble in typical of cross-strand disulfides. The fifth chapter of this thesis deals with the dynamics of thioredoxin using computational methods.In this chapter analysis of known complexes of thiroedoxin was done to determine binding hot spot residues using free energy calculations. The physicochemical basis for the multispecificity of thioredoxin is probed using molecular dynamics simulations. In this chapter it has been shown that conformational selection plays a very important role in thioredoxin target recognition.

Bacteriophages are viruses that specifically infect bacteria. The bactericidal nature of lytic bacteriophages has been exploited by scientists for decades with the hope to utilise them in the fight against bacterial infections and antibiotic resistant bacteria in medical settings. More recently, the potential applications of bacteriophages for biocontrol in the agrifood and environmental sectors have been investigated in an attempt to develop ‘natural’ antimicrobial products. Bacteriophages have a couple of decisive advantages over conventional methods of controlling pathogenic bacteria, such as high host specificity, the ability to self-replicate, and the ability to evolve with their hosts. However, more research is needed to optimise the parameters for phage applications, including the impact of environmental conditions on lysis efficiency, multiplicity of infection, and to significantly minimise the emergence of bacterial resistance to phages. Temperature plays a key role in every biological activity in nature. It is also assumed that temperature has an effect on phage lysis efficiency. A comprehensive study of it and how it affects both the host cells and their corresponding phages is crucial to ensure the efficient removal of bacterial pathogens. In this thesis, temperature (as selected parameter) was investigated to determine its influence on the lysis effectiveness of the three different phages belonging to the family of the Myoviridea that were isolated and purified from a single water sample taken from a brook receiving treated wastewater. We used the multiplicity of infection of 1 in all of our study in this project. Temperature was found to have a significant impact on phage-mediated lysis efficiency. Both the temperature of incubation of the phage-bacteria mixture (incubation temperature) and the temperature history of bacterial hosts were found to have profound effects on plaque sizes as well as plaque numbers. Plaque size and number decreased with increasing temperature. For the phages examined, bacterial lysis was more efficient at 20°C compared to 30 or 37°C. Phages were suggested to be well adapted to the environment where they were isolated from with general implications for use in biological disinfection. Furthermore, the temperature history of the bacteria (prior to phage encounter) was found to have a modulating effect on their susceptibility to lysis. A second part of this study compared the performance of the three phages in regard to bacterial resistance. The emergence of bacterial resistance is a major obstacle to the success of bacteriophages applications. The use of multiple phages is typically recommended and has proven better than the use of a single phage. However, the bestway to perform phage treatment is still very unclear. This study therefore compared simultaneous addition of multiple phages (in form of a cocktail) with the sequential addition of the individual phages at different time points in trying to delay the emergence of bacterial resistance. The data obtained from this work suggest that lysis effectiveness can be adjusted to optimize any treatment goal. For fast initial bacterial clearance the use of a single phage with short time maximal lysis efficiency proved most efficient, while the simultaneous addition of phages in the form of a cocktail was most successful strategy in our study. Addition of selected phages sequentially can be normalized in such a way that is just as effective as a cocktail. A third part of this thesis looked into the susceptibility of bacteria that had undergone sublethal disinfection. We addressed the question whether bacteria subjected to sublethal doses of chlorine and UV are still susceptible to phage-mediated lysis. The chlorine treatments indicated the development of a phage-insensitive phenotype for a critical chlorine dose in the transition zone between live and dead. The remaining live (and culturable) bacteria were shown insensitive to the selected phage. The lowest UV exposure at 2.8 mJ/cm2 eliminated bacteria susceptibility to the phages. This phage- resistant phenotype may have serious consequences for the application of phages on foods or water that have previously undergone a weak disinfection regime.