Most bacteriophages release progeny virions at the end of the infection cycle by lysis of the host. Large phages with double-stranded DNA genomes use a multigene strategy based on holins, small membrane proteins, and bacteriolytic enzymes, or endolysins. Holins mediate the control of endolysin activity and thus the timing of lysis. Phages with small genomes only encode a single protein for cell lysis. There are three known unrelated single protein lysis systems: the ?X174 E protein, the MS2 L protein, and the Q? A2 protein. None of these phages encodes a cell wall degrading activity, and previous work has shown that the lytic activity of E stems from its ability to inhibit the host enzyme, MraY, which catalyzes the formation of lipid I, the first lipid intermediate in cell wall synthesis. The purpose of the work described in this dissertation was to characterize the ?X174 E-mediated inhibition of MraY using genetic and biochemical strategies. A fundamental question was why no large phages use the single gene system. This was addressed by constructing a recombinant phage, ?E, in which the holin-endolysin based lysis cassette of ? was replaced with E. ?E was compared with ? in genetic and physiological experiments, with the results indicating that the holin-endolysin system increases fitness in terms of adjusting lysis timing to environmental conditions. Using ?E, physiological experiments were conducted to characterize the interaction between E and MraY in vivo. Transmembrane domains (TMD) 5 and 9 have been identified as the potential E binding site by isolating MraY mutants resistant to E inhibition. The five Eresistant MraY mutants were found to fall into three classes, which reflect the apparent affinity of the mutant proteins for E. Finally, an assay for MraY activity employing the dansylated UDP-MurNAc-pentapeptide and phytol-P, was used to demonstrate the inhibition of MraY by purified E protein. It was determined that E is a non-competitive inhibitor for MraY in respect with both substrates. A model for E-mediated inhibition of MraY was proposed, in which E binds to TMDs 5 and 9 in MraY and thus inactivates the enzyme by inducing a conformational change.

No description available.

Baculoviruses are lytic insect viruses. Upon internalisation, the viral genome orchestrates a sequential expression process ultimately leading to lysis of the infected cell. Release of progeny capable of infecting other cells during the process completes the infection cycle. Studies of the infection cycle in cell culture are typically conducted by synchronous infection, i.e. near simultaneous infection of all cells, by means of high virus concentrations. The behaviour of the synchronously infected culture, such as the timing of onset of progeny release, is considered representative for the infection progression within individual cells. In reality, however, the synchronously infected culture only reflects the average behaviour of all infected cells. The infection progresses in individual cells display large variability; this is most obvious in the observation that within the same culture some cells undergo cell lysis at two days post infection while others remain viable up to four days post infection. Such variabilities or asynchronies observed in synchronously infected culture is the topic of this thesis. Using a simple phenomenological model, it is demonstrated that cell death and associated intracellular product release is adequately described assuming that the waiting time from infection to cell death follows a Gaussian distribution with a mean of 59 hours post infection (hpi) and a standard deviation of 15hpi. Unlike other deterministic models developed over the last decade (Licari and Bailey 1992; Nielsen 2000), this stochastic model does not make the biologically inconsistent assumption that cells continue to be metabolically active following loss of membrane integrity. While elegant in its simplicity, the model provides no explanation for the underlying stochasticity. Investigations into the cause of this dispersion of cell death highlighted further asynchronies in the specific recombinant protein yield, in viral DNA content, in virus budding rate, and in cell volume increase instead of clarifying the issue. A modelling framework developed by Licari & Bailey (1992) and later Hu & Bentley (2000) incorporates the number of infectious particles each individual cell receives as a possible source of the dispersions in the host cell responses. However, this was found NOT to be the cause of the observed asynchronies under non-substrate limiting conditions. The timing of cell death, cell volume increase, recombinant product yield, viral DNA content, and virus budding rate is identical in Sf9 cell cultures infected at multiplicities of infection of ~5, ~15, and ~45 infectious particles per cell. Cell cycle variation has previously been suggested as a possible cause for observed asynchronies in baculovirus infections (Brown and Faulkner, 1975). The cell cycle phase is indirectly linked to the cell volume, because a G_2-phase cell prior to division is inherently twice the cell volume of a G_1- phase cell after cell division. By the same logic, it is also apparent that a G_2-phase cell possesses twice the number of ribosomes of a G_1-phase cell and thus a doubled protein production capacity. The effect of the cell cycle or cell volume on the baculovirus infection was determined by splitting an exponentially growing Sf9 cell culture into 5 cell size dependent fractions by centrifugal elutriation. The subsequent infection of these fractions showed (1) no dependency of the timing of cell lysis and cell volume increase and (2) approximately twofold increase of a) recombinant protein yield, b) viral DNA concentration, and c) budded virus yield. The recombinant protein yield showed a strong proportionality to the initial cell volume and the total RNA concentration during the late phase of the infection. As argued in chapter 6, these proportionalities suggest that the observed differences in the responses of the cell fractions to the baculovirus infection are more likely caused by the difference in the protein production capacity than by cell cycle specific molecules. This investigation gave also reason to speculate that infected cells cannot progress beyond the G_2/M phase, and cell cycle progression continues undisturbed until ~8hpi when all cellular DNA replication appears to cease. Resuspended, infected Sf9 cells synchronised by centrifugal elutriation showed an identical cell cycle distribution as the non-infected control cultures for the first ~8hpi; G_1 and G_2/M phase cell proportions remained unchanged, whereas S phase cells progress to G_2/M phase. Subsequently, the non-infected control cells resumed normal cycling whereas all infected cells remained at the same cell cycle phase from 8 to 11hpi. The initial cell cycle arrests in G_2/M phase in both infected and non-infected cultures were caused through medium exchange.

Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-04T12:47:50Z No. of bitstreams: 1 arquivototal.pdf: 3356476 bytes, checksum: 04bc24e3e4cfbdf2557866eb0f6e7739 (MD5) / Made available in DSpace on 2017-09-04T12:47:50Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3356476 bytes, checksum: 04bc24e3e4cfbdf2557866eb0f6e7739 (MD5) Previous issue date: 2017-04-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Snake venoms are complex mixtures of components that act upon the disturbance of various physiological mechanisms. The composition and biological activities of the venom may vary in snakes according to age, sex and geographic region. The present work aimed to evaluate the protein composition and biological activities triggered, in vitro, by male and female venoms of the Bothrops leucurus snake. The protein profile was obtained by high performance liquid chromatography (HPLC) and one-dimensional electrophoresis under denaturing conditions (SDS-PAGE). The biological effects evaluated were oxidative stress, antioxidant, coagulant and hemolytic (direct and indirect) activities. A chromatographic and electrophoretic analysis presented similarities between the protein profiles of males and females venoms. The evaluated venoms did not present hemolytic effect (direct or indirect) on erythrocytes. Significant erythrocyte haemagglutination was observed, it was caused by all the measurements of the venoms evaluated. B. leucurus venoms did not present oxidative effect on hemoglobin. In the presence of both venoms studied, were observed a reduction of the oxidative effect on hemoglobin caused by phenylhydrazine; suggesting the presence of antioxidant components in the composition of the venoms. The evaluation of coagulating effect of B. leucurus venoms were performed on citrated human plasma and clotting times were shorter in the presence of female venom and, both venoms presented clot formation time inversely proportional to the quantities evaluated. However, both protein profile and hemolytic (direct and indirect), oxidant and antioxidant effects observed in B. leucurus venom were similar for male and female venoms. Otherwise, the sexual variation linked to the coagulant effect may justify the differences in type and/or intensity of symptoms observed in ophidian accidents. / As peçonhas ofídicas são complexas misturas de componentes que atuam na perturbação de diversos mecanismos fisiológicos. A composição e atividades biológicas das peçonhas podem variar entre serpentes da mesma espécie de acordo com fatores como idade, sexo e região geográfica. O presente trabalho teve por objetivo avaliar a composição proteica e atividades biológicas desencadeadas, in vitro, pelas peçonhas de machos e fêmeas da serpente Bothrops leucurus. O perfil proteico foi obtido por cromatografia líquida de alta eficiência (HPLC) e eletroforese unidimensional em condições desnaturantes (SDS-PAGE). Os efeitos biológicos avaliados foram o estresse oxidativo, atividades antioxidante, coagulante e hemolíticas direta e indireta. A análise cromatográfica e eletroforética apresentaram semelhanças entre os perfis proteicos das peçonhas de machos e fêmeas. As peçonhas avaliadas não apresentaram efeito hemolítico (direto ou indireto) sobre eritrócitos. Foi observada uma expressiva hemaglutinação eritrocitária causada por todas as quantidades das peçonhas avaliadas. As peçonhas de machos e fêmeas de B. leucurus não apresentaram efeito oxidativo sobre hemoglobinas. Na presença de ambas as peçonhas estudadas, observou-se redução do efeito oxidativo sobre hemoglobinas provocado pela fenilhidrazina, sugerindo a presença de componentes antioxidantes na composição das peçonhas. A avaliação do efeito coagulante das peçonhas de B. leucurus foi realizada sobre o plasma humano citratado e os tempos de obtenção dos coágulos foram menores na presença da peçonha de fêmeas e, ambas as peçonhas apresentaram o tempo de formação dos coágulos inversamente proporcional às quantidades avaliadas. Contudo, tanto o perfil proteico como os efeitos hemolíticos (direto e indireto), oxidantes e antioxidantes observados nas peçonhas de machos e fêmeas de B. leucurus foram semelhantes. No entanto, a variação sexual obtida pela análise do efeito coagulante pode justificar as diferenças no tipo e/ou intensidade dos sintomas observados nos acidentes ofídicos.

Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-08T12:54:48Z No. of bitstreams: 1 arquivototal.pdf: 1516121 bytes, checksum: 772d5ba3351de06ed346dc46430f7188 (MD5) / Made available in DSpace on 2017-09-08T12:54:48Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1516121 bytes, checksum: 772d5ba3351de06ed346dc46430f7188 (MD5) Previous issue date: 2016-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The first-line drugs in Brazil for leishimaniasis treatment is the pentavalent antimonial, considered a prodrug because it is converted in trivalent (SbIII) during the treatment which presents many limitations, including the rising of the resistant parasite. Seeking to understand of these resistance effects in Leishmania (L.) amazonensis, promastigotes forms were cultured in glowing concentrations of SbIII then 2 mutants were selected, SbIII 1 and SbIII2 that gain resistance, respectively, 15,8 and 22,5 times bigger than wild cultures'. The morphological analysis show significant diferences in mutant's form and size when compared with the wild ones, once it has a long and slender cell body and with a long flagellum, while the majority mutants, with a resistance level 6,42 (SbIII 1) and 5,62 (SbIII 2) times bigger, presented a rounded cell body, with long flagellum. Curiously, when the mutants obtain a resistance level 15,8 (SbIII 1) e 22,5 (SbIII 2) times bigger, the morphological diference diminishes. The resistance phenotype to the SbIII manifested itself also in the amastigote like derived of both mutants. The SbIII 2 mutant presented a more stable profile of resistance than SbIII 1 one's when cultured in the absence of the drug, which denote diferences in the resistance mechanism. There was not observed cross-resistance to Amphotericin B. The promastigote forms of both mutants showed theirselves significantly more resistant to lysis throught the complement system, compared to the wild one's, sugesting that the parameter to the resistance to the antimonials and virulance might be correlated in Leishmania (L.) amazonensis. / A droga de primeira escolha para o tratamento das leishmanioses no Brasil é um antimonial pentavalente, considerado uma pró-droga por ser convertido na forma trivalente (SbIII) durante o tratamento, o qual apresenta várias limitações, incluindo o crescente surgimento de parasitos resistentes. Buscando compreender os efeitos desta resistência em Leishmania (L.) amazonensis, formas promastigotas foram cultivadas em concentrações crescentes de SbIII e selecionados dois mutantes, SbIII 1 e SbIII 2, que adquiriram, respectivamente, um nível de resistência 15,8 e 22,5 vezes maior que o apresentado pelas culturas selvagens. A análise morfológica mostrou diferenças significantivas no tamanho e formato dos mutantes em relação ao tipo selvagem, visto que estas apresentaram corpo celular alongado, delgado e com flagelo longo, enquanto que a maioria dos mutantes, com um nível de resistência 6,42 (SbIII 1) e 5,62 (SbIII 2) vezes maior, apresentaram um corpo celular arredondado, com flagelo alongado. Curiosamente, quando os mutantes adquiriram um nível de resistência 15,8 (SbIII 1) e 22,5 (SbIII 2) vezes maior, esta diferença morfológica diminuiu. O fenótipo de resistência ao SbIII se manifestou também nas formas amastigotas axênicas derivadas dos dois mutantes. O mutante SbIII 2 apresentou um perfil de resistência mais estável que o SbIII 1 quando cultivado na ausência da droga, o que pode indicar diferenças nos mecanismos de resistência. Não foi observada resistência cruzada com o fármaco Anfotericina B. As formas promastigotas dos dois mutantes mostraram-se significativamente mais resistentes à lise pelo sistema complemento, comparado aos selvagens, sugerindo que os parâmetros resistência a antimoniais e virulência podem estar correlacionados em Leishmania (L.) amazonensis.

O uso das técnicas de análise multivariada está reservado aos grandes centros de pesquisa, µas grandes empresas e ao ambiente acad^emico. Essas técnicas s~ao muito interessantes porque utilizam simultaneamente todas as variáveis respostas na interpretação teórica do conjunto de dados, levando em conta as correlações existentes entre elas. Uma das principais barreiras para a utilização dessas técnicas é o seu desconhecimento pelos pesquisadores interessados na pesquisa quantitativa. A outra dificuldade é que a grande maioria de softwares que permitem esse tipo de análise (SAS, MINITAB, BMDP, STATISTICA, S-PLUS, SYSTAT, etc.) não são de domínio público. A disseminação do uso das técnicas multivariadas pode melhorar a qualidade das pesquisas, proporcionar uma economia relativa de tempo e de custo, e facilitar a interpretação das estruturas dos dados, diminuindo a perda de informação. Neste trabalho, foram confirmadas algumas vantagens das técnicas multivariadas sobre as univariadas na análise de dados de expe- rimentos agropecuários. As análises foram realizadas com o auxílio do software R, um software aberto, \"amigável\" e gratuito, com inúmeros recursos disponíveis. / The use of the techniques of multivariate analysis is restricted to large centers of research, the higher companies and the academic environment. These techniques are very inte- resting because of the use of all answers variables simultaneously in theoretical interpretation of the data set, considering the correlations between them. One of the main obstacle to the usage of these techniques is that researchers interested in the quantitative research do not know them. The other di±culty is that most of the software that allow this type of analysis (SAS, MINITAB, BMDP, STATISTICA, S-PLUS, SYSTAT etc.) are not in public domain. Publishing the use of Multivariate techniques can improve the quality of the research, decrease the time spend and the cost, and make easy the interpretation of the structures of the data without cause damage of the information. In this report, were con¯rmed some advantages of the multivariate techniques in a univariate analysis for data of agricultural experiments. The analysis were taken with R software, a open software, \"friendly\" and free, with many statistical resources available.

Le travail de thèse présenté dans ce manuscrit s’inscrit dans une dynamique d’intégration de matériaux non conventionnels en systèmes microfluidiques. Il vise à démontrer le potentiel du cPDMS, un matériau nanocomposite formé d’une matrice de polydiméthylsiloxane rendu conducteur par l’ajout de nanoparticules de carbone. Compatible avec les procédés technologiques habituels, le cPDMS peut être structuré dans une large gamme d’épaisseurs et de géométries mais présente surtout l’avantage de pouvoir être collé irréversiblement sur verre, PDMS et silicium. Son intégration est parfaitement étanche, rapide à mettre en oeuvre, et très économique. La première partie du manuscrit est consacrée à la caractérisation de ce matériau. Ses propriétés électriques et de surface, pouvant être critiques pour une utilisation en microfluidique, ont été particulièrement étudiées. Les champs électriques offrant de nombreuses possibilités pour réaliser des fonctions clés en microfluidique (détection, séparation, manipulation de fluides ou de particules), nous avons choisi d’évaluer l’intérêt d’électrodes de cPDMS dans deux types d’applications. Les aspects de détection ont d’abord été mis en évidence à l’aide de mesures électrochimiques. Cette méthode a permis à la fois de caractériser la surface du cPDMS tout en validant son utilisation potentielle pour des applications d’analyses électrochimiques. Dans la dernière partie du manuscrit, le matériau a été testé pour la manipulation de particules à travers l’observation de différents phénomènes électrocinétiques. Ceux-ci ont conduit à la mise au point de dispositifs microfluidiques (intégrant des lectrodes de cPDMS) dédiés à la lyse et à l’électrofusion de cellules. / The work presented in this thesis deals with the integration of non-conventional materials in microfluidic systems. It aims to demonstrate the potential of cPDMS, a conductive nanocomposite material made up of polydimethylsiloxane matrix mixed with carbon nanoparticles. Compatible with the usual technological processes such as soft lithography, cPDMS can be microstructured in a large range of thicknesses and geometries. Moreover, cPDMS can be quickly, irreversibly and perfectly sealed to glass, PDMS and silicon substrates, something that is not possible for conventional metallic electrodes. The first part of the manuscript is centered on the characterization of this material. Its electrical and surface properties that may turn out critical for microfluidic applications have been particularly studied. Electric fields present many opportunities to perform key functions in microfluidic (detection, separation, fluid or particles handling). We have chosen to assess the potential of cPDMS electrodes for two kinds of applications. Aspects of detection were first demonstrated using cyclic voltammetry measurements. This electrochemical method has enabled both to characterize the cPDMS surface while validating its potential as an electrochemical analysis tool. In the last part of this manuscript, cPDMS was tested for the electrokinetic manipulation of particles through thre study of different electrical fields with induced phenomena. This has led to the development of microfluidic devices (integrating cPDMS electrodes) designed for cell lysis and cells electrofusion.

The subject of the thesis Online Propagation of Web Portal is formulation of recom-mendations for online campaigns for selected business entity. In the first chapter are defined basic concepts of Internet advertising as part of marketing and terms of web analytics. In the second chapter, there are internet campaigns analyzed using web analy-tics. On the basis of this analysis are formulated specific recommendations in the third chapter. The fourth chapter deals with the evaluation of modified campaigns. The fifth chapter deals with the evaluation of the results and the formulation of final recommen-dations.

There is an ever growing demand for low cost biosensors in medical diagnostics. A well known commercially successful example is glucose biosensors which are used to diagonize and monitor diabetes. These biosensors use electrochemical analysis (electro analysis) as transduction mechanism. Electro analytical techniques involve application of electrical stimulus to the chemical/biochemical system under consideration and measurement of electrical response due to the oxidation and reduction reactions that occur because of the stimulus. They offer a lot of advantages in terms of sensitivity, selectivity, cost effectiveness and compatibility towards integration with electronics. Besides glucose, there are several biomolecules of significance for which electro analysis can potentially be used to develop low cost, rapid, easy to use biosensors. One such biomolecule is Glycated Hemoglobin (GHb). It is a post translational, non-enzymatic modification of hemoglobin with glucose and is a very good biomarker that indicates the average value of blood glucose over the past 120 days. It is always expresses as a percentage of total hemoglobin present in blood. Monitoring diabetes based on the value of percentage Glycated hemoglobin is advantageous as it gives an average value of glucose unlike plasma glucose values which vary a lot on a day to day basis depending on the dietary habits and the stress levels of the individual. This thesis is focused on the development of a low coat, easy to use, disposable sensor for measuring percentage Glycated hemoglobin. The first challenge in developing such a sensor is isolation of hemoglobin. Unlike glucose which is present in blood plasma (liquid content of blood), hemoglobin resides inside red blood cells also known as erythrocytes. O isolate hemoglobin, these cells have to be broken or lysed. All the existing approaches rely on mixing blood with lysing reagents to lyse erythrocytes. Ideal biosensors should be devoid of liquid reagents. Keeping this in perspective, in this thesis, this challenge is addressed by developing two entirely buffer/reagentless techniques to lyse erythrocytes and isolate hemoglobin. In the first technique, cellulose acetate membranes are embedded with lysing reagents and are used for lysing reagents and are used for lysing application. In the second techniques, commercially available nylon mesh nets are modified with lysing reagents to lyse and isolate hemoglobin. These membranes or mesh nets can be easily integrated on top of a disposable strip. After isolating hemoglobin, the next challenge is to selectively detect Glycated hemoglobin. Boronic acid conjugates are known to bind Glycated hemoglobin. Using this principle, a new composite is sysnthesized to specifically detect glc\ycated hemoglobin. The composite (GO-APBA) is a result of functionalization of Graphene Oxide (GO) with 3-aminophenylboronic acide (APBA). Detection of Glycated hemoglobin is achieved by modifying screen printed electrode strips with the synthesized compound, thus taking a step forwards achieving the objective. Since Glycated hemoglobin is always expressed as a percentage of hemoglobin, the next challenge is to detect total hemoglobin. In this thesis a low cost way of detecting hemoglobin is achieved by using GO modified or surfactant modified screen printed electrode strips. Furthermore, the potential interferences that blood plasma can cause in these measurements are eliminated with the help of permselective coatings. Thus using the technologies developed in this thesis, measurements of percentage Glycated hemoglobin can be potentially made on handheld electronic devices akin to glucose meters by using just a drop of blood.

Sepsis, commonly referred to as blood poisoning, is a serious medical condition characterized by a whole-body inflammatory state caused by microbial infection. Rapid treatment is crucial, however, traditional culture-based diagnostics usually takes 2-5 days.  The overall aim of the thesis is to develop microfluidic based sample preparation strategies, capable of isolating bacteria from whole blood for rapid sepsis diagnostics.  Although emerging technologies, such as microfluidics and “lab-on-a-chip” (LOC) devices have the potential to spur the development of protocols and affordable instruments, most often sample preparation is performed manually with procedures that involve handling steps prone to introducing artifacts, require skilled technicians and well-equipped, expensive laboratories.  Here, we propose the development of methods for fast and efficient sample preparation that can isolate bacteria from whole blood by using microfluidic techniques with potential to be incorporated in LOC systems. We have developed two means for high throughput bacteria isolation: size based sorting and selective lysis of blood cells. To process the large blood samples needed in sepsis diagnostics, we introduce novel manufacturing techniques that enable scalable parallelization for increased throughput in miniaturized devices. The novel manufacturing technique uses a flexible transfer carrier sheet, water-dissolvable release material, poly(vinyl alcohol), and a controlled polymerization inhibitor to enable highly complex polydimethylsiloxane (PDMS) structures containing thin membranes and 3D fluidic networks. The size based sorting utilizes inertial microfluidics, a novel particles focusing method that operates at extremely high flow rates. Inertial focusing in flow through a single inlet and two outlet, scalable parallel channel devices, was demonstrated with filtration efficiency of >95% and a flowrate of 3.2 mL/min. Finally, we have developed a novel microfluidic based sample preparation strategy to continuously isolate bacteria from whole blood for downstream analysis. The method takes advantage of the fact that bacteria cells have a rigid cell wall protecting the cell, while blood cells are much more susceptible to chemical lysis. Whole blood is continuously mixed with saponin for primary lysis, followed by osmotic shock in water. We obtained complete lysis of all blood cells, while more than 80% of the bacteria were readily recovered for downstream processing. Altogether, we have provided new bacteria isolation methods, and improved the manufacturing techniques and microfluidic components that, combined offer the potential for affordable and effective sample preparation for subsequent pathogen identification, all in an automated LOC format. / QC 20120611