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La germination des spores est une étape essentielle dans le cycle de vie de la majorité des champignons filamenteux. Les champignons mycorhiziens à arbuscules (CMA) forment un certain nombre de propagules infectieuses différentes qui augmentent leur potentiel à coloniser les racines. Parmi elles se trouvent les spores extraracinaires et intraracinaires. La paroi cellulaire des spores joue un rôle majeur dans la survie de ces propagules en étant une barrière physique et osmotique. Puisque une cellule peut faire des ajustements considérables dans la composition et la structure de sa paroi, en réponse aux conditions environnementales, il est possible que les parois des spores intraracinaires et extraracinaires montrent des propriétés mécaniques et osmotiques différentes affectant leur germination et leur survie. Pourtant, contrairement à la connaissance de la génétique moléculaire et de la formation de la paroi cellulaire des CMA, peu d’information est disponible au sujet de ces propriétés mécaniques. Les informations sur la germination des CMA dans des conditions hypertoniques sont aussi rares, et les modèles expérimentaux ne séparent généralement pas les effets directs de la forte pression osmotique externe sur la germination des champignons et les effets attribuables aux plantes. Cette étude avait pour but de répondre à deux importantes séries de questions concernant le comportement des spores mycorhiziennes. Nous avons d'abord déterminé la relation entre la composition de la paroi cellulaire, la structure et les propriétés mécaniques du champignon modèle Glomus irregulare (isolat DAOM 197198). La micro-indentation a été utilisée pour mesurer quantitativement les propriétés mécaniques de la paroi cellulaire. La composition (contenu de chitine et de glomaline) de la paroi cellulaire a été quantifiée par immunofluorescence tandis que la microscopie optique a été utilisée pour mesurer l'épaisseur de la paroi cellulaire. La densité locale en glomaline et l’épaisseur de la paroi étaient significativement plus élevées pour les parois des spores extraracinaires alors que la densité locale en chitine et la rigidité n’ont pas montré de variations entre les spores extraracinaires et intraracinaires. La grande variabilité dans les paramètres étudiés nous a empêchés de cibler un facteur principal responsable de la force totale de la paroi lors de la compression. La diminution des concentrations de chitine et de glomaline a été corrélée à l'évolution de la paroi du champignon au cours de son cycle de vie. On a aussi observé une composition différentielle des couches de la paroi: les polymères de chitine et de glomaline furent localisés principalement dans les couches externes et internes de la paroi, respectivement. Dans la deuxième partie de notre travail, nous avons exploré les effets directs d'engrais, par rapport à leur activité de l'eau (aw), sur la germination des spores et la pression de turgescence cellulaire. Les spores ont été soumises à trois engrais avec des valeurs de aw différentes et la germination ainsi que la cytorrhyse (effondrement de la paroi cellulaire) des spores ont été évaluées après différents temps d'incubation. Les valeurs de aw des engrais ont été utilisées comme indicateurs de leurs pressions osmotiques. L'exposition des spores de Glomus irregulare au choc osmotique causé par les engrais dont les valeurs de aw se situent entre 0,982 et 0,882 a provoqué des changements graduels au niveau de leur cytorrhyse et de leur germination. Avec l'augmentation de la pression de turgescence externe, la cytorrhyse a augmenté, tandis que le taux de germination a diminué. Ces effets ont été plus prononcés à des concentrations élevées en éléments nutritifs. La présente étude, bien qu’elle constitue une étape importante dans la compréhension des propriétés mécaniques et osmotiques des spores de CMA, confirme également que ces propriétés dépendent probablement de plusieurs facteurs, dont certains qui ne sont pas encore identifiés. / Spore germination is an essential developmental stage in the life cycle of many filamentous fungi. Arbuscular mycorrhizal fungi (AMF) form a number of different infectious propagules that increase their potential to colonize roots. Among them are extraradical and intraradical spores. The spore cell wall plays a major role in the survival of these propagules by being a physical and osmotic barrier. Because a cell can make considerable adjustments to the composition and structure of its wall in response to environmental conditions, it is possible that intraradical and extraradical spore walls show different mechanical and osmotic properties affecting their survival and germination. However, in contrast to the knowledge on the genetics and molecular composition of AMF cell wall, little is known about its mechanical properties. Information on the germination of AMF under hypertonic conditions is scarce, and experimental designs and methodologies have generally not allowed the direct effects of high external osmotic pressure on fungal germination to be separated from plant-mediated effects. This study had the goal to address two important sets of questions regarding the behavior of mycorrhizal spores. We first determined the relationship between cell wall composition, structure and mechanical properties of the model fungus Glomus irregulare. Micro-indentation was used to quantitatively measure the cell wall mechanical properties. Cell wall composition (chitin and glomalin content) was studied by immunofluorescence whereas optical microscopy was used to measure the cell wall thickness. Glomalin local density and wall thickness were both significantly higher for extraradical spore walls while chitin local density and rigidity were unaffected by origin of spores. High variability in results prevented us from identifying a primary factor responsible for overall wall strength during compression. Decreases of chitin and glomalin concentrations were correlated to the development of the fungal wall throughout its life-cycle. There was also differential association within the wall layers: The chitin and glomalin polymers were localized mostly in the outer and inner walls, respectively. In the second part of our work, we explored the direct effects of fertilizers, in relation to their water activity (aw), on spore germination and cellular turgor pressure. Spores were exposed to three fertilizers with different aw and spore germination and cytorrhysis of spores were assessed after different times of incubation. Water activities of the fertilizers were used as indicators of their osmotic pressures. Osmotic shock exposure of the Glomus irregulare spores to fertilizers at aw values between 0.982 and 0.882 caused gradual changes in cytorrhysis and germination. With the increase of external turgor pressure, cytorrhysis increased while the rate of germination decreased. These effects were most pronounced at high nutrient concentrations. The present investigation, while likely representing a significant step forward in understanding the mechanical and osmotic properties of AMF spores, also confirms that they might depend on many, as yet unidentified factors. Future research should examine differences in the physiology to discern reasons for such differences in spore properties.

<p>Modeling microbial inactivation has a great influence on the optimization, control and design of food processes. In the area of food safety, modeling is a valuable tool for characterizing survival curves and for supporting food safety decisions. The modeling of microbial behavior is based on the premise that the response of the microbial population to the environment factors is reproducible. And that from the past, it is possible to predict how these microorganisms would respond in other similar environments. Thus, the use of mathematical models has become an attractive and relevant tool in the food industry.</p> <p>This research provides tools to relate the inactivation of microorganisms of public health importance with processing conditions used in nonisothermal and non-thermal food processing technologies. Current models employ simple approaches that do not capture the realistic behavior of microbial inactivation. This oversight brings a number of fundamental and practical issues, such as excessive or insufficient processing, which can result in quality problems (when foods are over-processed) or safety problems (when foods are under-processed). Given these issues, there is an urgent need to develop reliable models that accurately describe the inactivation of dangerous microbial cells under more realistic processing conditions and that take into account the variability on microbial population, for instance their resistance to lethal agents. To address this urgency, this dissertation focused on mathematical models, combined mathematical tools with microbiological science to develop models that, by resembling realistic and practical processing conditions, can provide a better estimation of the efficacy of food processes. The objective of the approach is to relate the processing conditions to microbial inactivation. The development of the modeling approach went through all the phases of a modeling cycle from planning, data collection, formulation of the model approach according to the data analysis, and validation of the model under different conditions than those that the approach was developed.</p> <p>A non-linear ordinary differential equation was used to describe the inactivation curves with the hypothesis that the momentary inactivation rate is not constant and depends on the instantaneous processing conditions. The inactivation rate was related to key process parameters to describe the inactivation kinetics under more realistic processing conditions. From the solution of the non-linear ordinary differential equation and the optimization algorithm, safety inferences in the microbial response can be retrieved, such as the critical lethal variable that increases microbial inactivation. For example, for nonisothermal processes such as microwave heating, time-temperature profiles were modeled and incorporated into the inactivation rate equation. The critical temperature required to increase the microbial inactivation was obtained from the optimization analysis. For non-thermal processes, such as cold plasma, the time-varying concentration of reactive gas species was incorporated into the inactivation rate equation. The approach allowed the estimation of the critical gas concentration above which microbial inactivation becomes effective. For Pulsed Electric Fields (PEF), the energy density is the integral parameter that groups the wide range of parameters of the PEF process, such as the electric field strength, the treatment time and the electrical conductivity of the sample. The literature has shown that all of these parameters impact microbial inactivation. It has been hyphothesized that the inactivation rate is a function of the energy density and that above a threshold value significant microbial inactivation begins. </p> <p>The differential equation was solved numerically using the Runge-Kutta method (<i>ode45</i> in MATLAB ®). The<i> lsqcurvefit</i> function in MATLAB ® estimated the kinetic parameters. The approach to model microbial inactivation, whether when samples were subjected to nonisothermal or to non-thermal food processes, was validated using data published in the literature and/or in other samples and treatment conditions. The modeling approaches developed by this dissertation are expected to assist the food industry in the development and validation process to achieve the level of microbial reduction required by regulatory agencies. In addition, it is expected to assist the food industry in managing food safety systems through support food safety decision-making, such as the designation of the minimal critical parameter that may increase microbial inactivation. Finally, this dissertation will contribute in depth to the field of food safety and engineering, with the ultimate outcome of having a broad and highly positive impact on human health by ensuring the consumption of safe food products.</p>

La germination des spores est une étape essentielle dans le cycle de vie de la majorité des champignons filamenteux. Les champignons mycorhiziens à arbuscules (CMA) forment un certain nombre de propagules infectieuses différentes qui augmentent leur potentiel à coloniser les racines. Parmi elles se trouvent les spores extraracinaires et intraracinaires. La paroi cellulaire des spores joue un rôle majeur dans la survie de ces propagules en étant une barrière physique et osmotique. Puisque une cellule peut faire des ajustements considérables dans la composition et la structure de sa paroi, en réponse aux conditions environnementales, il est possible que les parois des spores intraracinaires et extraracinaires montrent des propriétés mécaniques et osmotiques différentes affectant leur germination et leur survie. Pourtant, contrairement à la connaissance de la génétique moléculaire et de la formation de la paroi cellulaire des CMA, peu d’information est disponible au sujet de ces propriétés mécaniques. Les informations sur la germination des CMA dans des conditions hypertoniques sont aussi rares, et les modèles expérimentaux ne séparent généralement pas les effets directs de la forte pression osmotique externe sur la germination des champignons et les effets attribuables aux plantes. Cette étude avait pour but de répondre à deux importantes séries de questions concernant le comportement des spores mycorhiziennes. Nous avons d'abord déterminé la relation entre la composition de la paroi cellulaire, la structure et les propriétés mécaniques du champignon modèle Glomus irregulare (isolat DAOM 197198). La micro-indentation a été utilisée pour mesurer quantitativement les propriétés mécaniques de la paroi cellulaire. La composition (contenu de chitine et de glomaline) de la paroi cellulaire a été quantifiée par immunofluorescence tandis que la microscopie optique a été utilisée pour mesurer l'épaisseur de la paroi cellulaire. La densité locale en glomaline et l’épaisseur de la paroi étaient significativement plus élevées pour les parois des spores extraracinaires alors que la densité locale en chitine et la rigidité n’ont pas montré de variations entre les spores extraracinaires et intraracinaires. La grande variabilité dans les paramètres étudiés nous a empêchés de cibler un facteur principal responsable de la force totale de la paroi lors de la compression. La diminution des concentrations de chitine et de glomaline a été corrélée à l'évolution de la paroi du champignon au cours de son cycle de vie. On a aussi observé une composition différentielle des couches de la paroi: les polymères de chitine et de glomaline furent localisés principalement dans les couches externes et internes de la paroi, respectivement. Dans la deuxième partie de notre travail, nous avons exploré les effets directs d'engrais, par rapport à leur activité de l'eau (aw), sur la germination des spores et la pression de turgescence cellulaire. Les spores ont été soumises à trois engrais avec des valeurs de aw différentes et la germination ainsi que la cytorrhyse (effondrement de la paroi cellulaire) des spores ont été évaluées après différents temps d'incubation. Les valeurs de aw des engrais ont été utilisées comme indicateurs de leurs pressions osmotiques. L'exposition des spores de Glomus irregulare au choc osmotique causé par les engrais dont les valeurs de aw se situent entre 0,982 et 0,882 a provoqué des changements graduels au niveau de leur cytorrhyse et de leur germination. Avec l'augmentation de la pression de turgescence externe, la cytorrhyse a augmenté, tandis que le taux de germination a diminué. Ces effets ont été plus prononcés à des concentrations élevées en éléments nutritifs. La présente étude, bien qu’elle constitue une étape importante dans la compréhension des propriétés mécaniques et osmotiques des spores de CMA, confirme également que ces propriétés dépendent probablement de plusieurs facteurs, dont certains qui ne sont pas encore identifiés. / Spore germination is an essential developmental stage in the life cycle of many filamentous fungi. Arbuscular mycorrhizal fungi (AMF) form a number of different infectious propagules that increase their potential to colonize roots. Among them are extraradical and intraradical spores. The spore cell wall plays a major role in the survival of these propagules by being a physical and osmotic barrier. Because a cell can make considerable adjustments to the composition and structure of its wall in response to environmental conditions, it is possible that intraradical and extraradical spore walls show different mechanical and osmotic properties affecting their survival and germination. However, in contrast to the knowledge on the genetics and molecular composition of AMF cell wall, little is known about its mechanical properties. Information on the germination of AMF under hypertonic conditions is scarce, and experimental designs and methodologies have generally not allowed the direct effects of high external osmotic pressure on fungal germination to be separated from plant-mediated effects. This study had the goal to address two important sets of questions regarding the behavior of mycorrhizal spores. We first determined the relationship between cell wall composition, structure and mechanical properties of the model fungus Glomus irregulare. Micro-indentation was used to quantitatively measure the cell wall mechanical properties. Cell wall composition (chitin and glomalin content) was studied by immunofluorescence whereas optical microscopy was used to measure the cell wall thickness. Glomalin local density and wall thickness were both significantly higher for extraradical spore walls while chitin local density and rigidity were unaffected by origin of spores. High variability in results prevented us from identifying a primary factor responsible for overall wall strength during compression. Decreases of chitin and glomalin concentrations were correlated to the development of the fungal wall throughout its life-cycle. There was also differential association within the wall layers: The chitin and glomalin polymers were localized mostly in the outer and inner walls, respectively. In the second part of our work, we explored the direct effects of fertilizers, in relation to their water activity (aw), on spore germination and cellular turgor pressure. Spores were exposed to three fertilizers with different aw and spore germination and cytorrhysis of spores were assessed after different times of incubation. Water activities of the fertilizers were used as indicators of their osmotic pressures. Osmotic shock exposure of the Glomus irregulare spores to fertilizers at aw values between 0.982 and 0.882 caused gradual changes in cytorrhysis and germination. With the increase of external turgor pressure, cytorrhysis increased while the rate of germination decreased. These effects were most pronounced at high nutrient concentrations. The present investigation, while likely representing a significant step forward in understanding the mechanical and osmotic properties of AMF spores, also confirms that they might depend on many, as yet unidentified factors. Future research should examine differences in the physiology to discern reasons for such differences in spore properties.

La décontamination microbienne est sujet majeur de préoccupation du secteur agroalimentaire. Des nouvelles technologies physiques de décontamination, dites athermiques, sont d'un emploi croissant. La Lumière Pulsée, utilisée pour décontaminer les surfaces et les liquides clairs, en fait partie. Elle utilise des flashes de lumière blanche riches en UV, et délivrés en moins d'une milliseconde. La plupart des traitements par lumière pulsée sont définis dans la littérature par des paramètres spécifiques à l'équipement utilisé. Le but de cette étude a été dans un premier temps de caractériser le traitement par lumière pulsée par les grandeurs physiques appropriées (fluence, tension aux bornes de la lampe, etc...), en reliant une dose de lumière à niveau de décontamination microbienne. L'équipement pilote de la société CLARANOR a révélé des réduction logarithmiques allant jusqu'à plus de 5 unités sur des spores de B. subtilis, et de plusieurs autres espèces de bactéries sporulées, avec des fluences inférieures à 1,5 J/cm², appliquée en un seul flash La mise au point d'une méthode d'inoculation par spray à permis d'évaluer l'efficacité décontaminante de la lumière sur différentes surfaces, y compris des hydrophobes, par pulvérisation des microorganismes en couches formées d'une seule épaisseur de cellules. L'application de la technologie sur des surfaces inertes comme le polystyrène a montré une décontamination notamment sur des spores de B. subtilis, et d'A. niger, supérieures à 4 cycles logarithmiques en utilisant des fluences inférieures à 1 J/cm². L'influence des facteurs liés au système d'éclairage a montré une importance capitale des longueurs d'onde UV, mais ne permettent pas de réduire l'efficacité à la seule action de la dose UV-C. L'efficacité de la technologie a permis de réaliser une étude concernant la décontamination de sirop de sucre dans une optique d'application industrielle. Une réduction supérieure à 3 cycles logarithmiques de spores d'A. acidoterrestris dans du sirop de saccharose a été obtenue en flux continu, sur une épaisseur de 10 mm de liquide

Plant form is a product of emergent processes of cell division, patterning and morphogenesis. These fundamental processes remain poorly characterised in plants. However, engineering approaches can provide new tools and frameworks for the study and manipulation of plant development. This dissertation describes the development of engineering frameworks for reprogramming of the early diverging land plant Marchantia polymorpha (Marchantia). I describe the generation of genomic and transcriptomic datasets for Marchantia, which has provided the basis for the compilation of a gene-centric registry of DNA parts for engineering (MarpoDB). I describe the development of Loop assembly, an efficient and standardised DNA assembly system based on Type IIS restriction enzymes for recursive fabrication of DNA circuits with high efficiency. MarpoDB was used to mine new DNA parts compatible with Loop assembly which were used to generate plant transformation vectors for labelling of cellular features to study aspects of growth and development. I performed image analysis of genetic markers for segmentation and quantification of cellular properties in germinating gemmae. I implemented high-efficiency Cas9-mediated mutagenesis in Marchantia for use in functional molecular genetics studies. Furthermore, I produced inducible systems for expression of heterologous elements by transactivation which showed negligible levels of basal activity. It was possible to use this system for induction of gene expression in single cells. Finally, these new frameworks were applied to study the gametophytic meristem in Marchantia gemmae. I mapped the expression of several putative candidate homologues for higher plant meristem regulators, performed overexpression and loss-of-function studies for homologues of WUSCHEL, CLAVATA3 and SHOOT MERISTEMLESS. A strategy for misregulation of endogenous genes was developed using inducible transactivation, and was used with cellular markers for WUSCHEL and CLAVATA3 homologues in Marchantia.

Made available in DSpace on 2015-03-26T13:52:02Z (GMT). No. of bitstreams: 1 texto completo.pdf: 443818 bytes, checksum: 511a55303b3b59b278ccd2fab938970a (MD5) Previous issue date: 2006-04-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The mango (Mangifera indica Linn.) is one of the most important tropical fruits. It is very appreciated due to its flavor and nutritional characteristics. The fruits are easily spoiled because of intrinsic factors, which makes difficult its commercialization in natura. An alternative to reduce the losts is the conservation of the mango as pulp, juice and nectar. The thermal treatment is the most used process to conservate these products. However, the complete inativation of microorganisms during the thermal processing adopted by most industries of fruit juices and fruit pulps is limited by the presence of spore formers and termorresistents microorganisms. This work aimed to isolate and to identificate spoilage bacteria from thermally processed mango pulp, and to characterize the fermentative activity of these microorganisms. Samples of 25 lots of spoiled mango pulp from two industrial units processing mango pulp were evaluated in plate count agar, added with 20 % of steril mango pulp. A number of 104 bacteria were isolated and were characterized as Gram-positive rods, spore formers and mostly strict anaerobic, indicating that belonged to the genus Clostridium. Four isolated named LMA 45, LMA 63, LMA 72 and LMA 104, provenient of different lots of mango pulp from two industrial units, were identified by biochemical tests and by the cell membrane fatty acids profile as Clostridium tyrobutyricum. These four isolates were selected for use in the experiments that followed. The isolates were cultivated in BHI broth within a pH range of 3.0 to 7.0 and temperatures between 7 °C to 50 °C. C. tyrobutyricum grew in the pH range of 4.0 to 7.0, except for isolate LMA 104, that did not grow in pH 4.0 up to 48 hours of incubation. None of the isolates was able to grow in pH 3.5. The specific growth rate (&#956;) was higher in pH values between 6.5 and 7.0 and varied from 0.18 to 0.31 h-1. The growth was observed in a temperature range from 10 °C to 40 °C, with an optimum between 35 °C and 40 °C. The fermentative activity of C. tyrobutyricum LMA 45, LMA 63, LMA 72 and LMA 104 was evaluated in mango pulp with pH varying from 3.0 to 5.0. Gas production was detected in pH of 4.0, 4.5 and 5.0 before 50 hours of the incubation at 30 ºC, except for isolate LMA 104 which produced gas only after 100 hours of incubation. The other main products that resulted from mango pulp fermentation in pH values between 4.0 and 4.5 were acetate and butyrate, which was determined by high performance liquid chromatography (HPLC). The consumption of sucrose, glucose and frutose during mango pulp fermentation in pH 4.0 and 4.5 was also determined using HPLC. The C. tyrobutyricum isolates evaluated could grow at high sugar concentrations, as they fermented the mango pulp containing 729,2 mM of sugars, but did not use all the substrate. The growth of the isolates was inhibited in mango pulp at pH 3.5. This results suggest the adoption of the acidification strategy by the industries, wich generally process mango pulp with pH 4.0. This strategy might ensure the conservation of the fruit product, without changes in the thermal treatment. / A manga (Mangifera indica Linn.) é uma das mais importantes frutas tropicais, muito apreciada pelo sabor, aroma e por suas propriedades nutricionais. Os frutos são perecíveis em razão dos fatores intrínsecos, dificultando a sua comercialização in natura. Uma alternativa para reduzir as perdas é a conservação da manga na forma de polpa, suco ou néctar. O tratamento térmico é o processo mais empregado para a conservação desses produtos. Entretanto, a inativação completa de microrganismos durante o processamento térmico normalmente adotado pelas indústrias processadoras de sucos e polpas é limitada pela presença de organismos formadores de esporos e de termorresistentes. Este trabalho teve como objetivos isolar e identificar as bactérias deterioradoras de polpa de manga tratada termicamente e caracterizar a atividade fermentativa desses microrganismos. Amostras de 25 lotes de polpa de manga deteriorada provenientes de duas unidades industriais processadoras de polpa de manga foram analisadas em ágar padrão para contagem, enriquecido com 20 % de polpa de manga esterilizada. Um total de 104 isolados foi obtido e essas bactérias foram caracterizadas como bastonetes, Gram-positivas, formadoras de esporos sendo a grande maioria constituída de anaeróbias estritas, do gênero Clostridium. Destes, os isolados LMA 45, LMA 63, LMA 72 e LMA 104 obtidos de lotes diferentes de polpa de manga provenientes de duas unidades industriais, foram identificados por teste bioquímicos e pelo perfil de ácidos graxos de membrana como Clostridium tyrobutyricum e selecionados para a continuidade deste estudo. Os isolados foram cultivados em caldo BHI com pH variando de 3,0 a 7,0 e em temperatura entre 7 °C e 50 °C. O crescimento de C. tyrobutyricum ocorreu em valores de pH de 4,0 a 7,0 com exceção do isolado LMA 104 que não apresentou crescimento em pH 4,0 em até 48 horas de incubação. Não houve crescimento em pH 3,5. A velocidade específica de crescimento (&#956;) foi maior em pH entre 6,5 e 7,0 e alcançou valores entre 0,18 e 0,31 h-1. O crescimento foi observado em temperaturas de 10 °C a 40 °C, com ótimo entre 35 °C e 40 °C. A atividade fermentativa de C. tyrobutyricum LMA 45, LMA 63, LMA 72 e LMA 104 foi avaliada em polpa de manga com pH variando de 3,0 a 5,0. A produção de gás foi detectada em valores de pH de 4,0, 4,5 e 5,0 em menos de 50 horas de incubação a 30 ºC, com exceção do isolado LMA 104 cuja produção de gás foi detectada após 100 horas de incubação. Além de gás, os principais produtos da fermentação da polpa de manga com pH entre 4,0 e 4,5 foram acetato e butirato, determinados por cromatografia líquida de alta eficiência (HPLC). O consumo de sacarose, glicose e frutose durante a fermentação de polpa de manga com pH 4,0 e 4,5 foi determinado utilizando-se HPLC. Os isolados de C. tyrobutyricum avaliados apresentaram capacidade de crescer em altas concentrações de açúcar, pois fermentaram a polpa de manga com 729,2 mM de açúcares e não utilizaram todo o substrato durante a fermentação. A inibição do crescimento dos isolados em polpa de manga com pH 3,5 permite sugerir a adoção da estratégia de acidificação para garantir a conservação do produto pela indústria, que geralmente processa esse produto com pH 4,0.

Meiosis is a specialized type of cell division essential for the production of four normal haploid gametes. In early prophase I of meiosis, the intimate synapsis between homologous chromosomes, and the formation of chiasmata, is facilitated by a proteinaceous structure known as the synaptonemal complex (SC). Ultrastructural analysis of germ cells of a number of organisms has disclosed that SC is a specialized tripartite structure composed of two lateral elements, one on each homolog, and a central element, which, in turn, are linked by transverse elements. Genetic studies have revealed that defects in meiotic chromosome alignment and/or segregation result in aneuploidy, which is the leading cause of spontaneous miscarriages in humans, hereditary birth defects such as Down syndrome, and are also, associated with the development and progression of certain forms of cancer. The mechanism(s) underlying the alignment/pairing of chromosomes at meiosis I differ among organisms. These can be divided into at least two broad pathways: one is independent of DNA double-strand breaks (DSB) and other is mediated by DSBs. In the DSB-dependent pathway, SC plays crucial roles in promoting homolog pairing and disjunction. On the other hand, the DSB-independent pathway involves the participation of telomeres, centromeres and non-coding RNAs in the pre-synaptic alignment, pre-meiotic pairing as well as pairing of homologous chromosomes. Although a large body of literature highlights the central role of SC in meiotic recombination, the possible role of SC components in homolog recognition and alignment is poorly understood. Genetic screens for Saccharomyces cerevisiae mutants defective in meiosis and sporulation lead to the isolation of genes required for interhomolog recombination, including those that encode SC components. In S. cerevisiae, ten meiosis-specific proteins viz., Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8 have been recognized as bona fide constituents of SC or associated with SC function. Mutations in any of these genes result in defective SC formation, thus leading to reduction in the rate of recombination. HOP1 (Homolog Pairing) encodes a ̴ 70 kDa structural protein, which localizes to the lateral elements of SC. It was found to be essential for the progression of meiotic recombination. In hop1Δ mutants, meiosis specific DSBs are reduced to 10% of that of wild type level and it fails to produce viable spores. It also displays relatively high frequency of inter-sister recombination over inter-homolog recombination. Bioinformatics analysis suggests that Hop1 comprises of an N-terminal HORMA domain (Hop1, Rev7 and Mad2), which is conserved among Hop1 homologs from diverse organisms. This domain is also known to be present in proteins involved in processes like chromosome synapsis, repair and sex chromosome inactivation. Additionally, Hop1 harbors a 36-amino acid long zinc finger 348374 motif (CX2CX19CX2C) which is critical for DNA binding and meiotic progression, and a putative nuclear localization signal corresponding to amino acid residues from 588-594. Previous studies suggested that purified Hop1 protein exists in multiple oligomeric states in solution and displays structure specific DNA binding activity. Importantly, Hop1 exhibited higher binding affinity for the Holliday junction (HJ), over other early recombination intermediates. Binding of Hop1 to the HJ at the core resulted in branch migration of the junction, albeit weakly. Intriguingly, Hop1 showed a high binding affinity for G4 DNA, a non-B DNA structure, implicated in homolog synapsis and promotes robust synapsis between double-stranded DNA molecules. Hop1 protein used in the foregoing biochemical studies was purified from mitotically dividing S. cerevisiae cells containing the recombinant plasmid over-expressing the protein where the yields were often found to be in the low-microgram quantities. Therefore, one of the major limitations to the application of high resolution biophysical techniques, such as X-crystallography and spectroscopic analyses for structure-function studies of S. cerevisiae Hop1 has been the non-availability of sufficient quantities of functionally active pure protein. In this study, we have performed expression screening in Escherichia coli host strains, capable of high level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed +2 for expression and purification of S. cerevisiae Hop1 protein, using Ni-NTA and double-stranded DNA-cellulose chromatography. Recombinant S. cerevisiae Hop1 protein thus obtained was >98% pure and exhibited DNA binding activity with high-affinity for Holliday junction. The availability of the bacterial HOP1 expression vector and functionally active Hop1 protein has enabled us to glean and understand several vital biological insights into the structure-function relationships of Hop1 as well as the generation of appropriate truncated mutant proteins. Mutational analyses in S. cerevisiae has shown that sister chromatid cohesion is required for proper chromosome condensation, including the formation of axial elements, SC assembly and recombination. Consistent with these findings, homolog alignment is impaired in red1hop1 strains and associations between homologs are less stable. red1 mutants fail to make any discernible axial elements or SC structures but exhibit normal chromosome condensation, while hop1 mutants form long fragments of axial elements but without any SCs, are defective in chromosome condensation, and produce in-viable spores. Using single molecule and ensemble assays, we found that S. cerevisiae Hop1 organizes DNA into at least four major distinct DNA conformations: (i) a rigid protein filament along DNA that blocks access to nucleases; (ii) bridging of non-contiguous segments of DNA to form stem-loop structures; (iii) intra-and intermolecular long range synapsis between double-stranded DNA molecules; and (iv) folding of DNA into higher order nucleoprotein structures. Consistent with B. McClintock’s proposal that “there is a tendency for chromosomes to associate 2-by-2 in the prophase of meiosis involving long distance recognition of homologs”, these results to our knowledge provide the first evidence that Hop1 mediates the formation of tight DNA-protein-DNA nucleofilaments independent of homology which might help in the synapsis of homologous chromosomes during meiosis. Although the DNA binding properties of Hop1 are relatively well established, comparable knowledge about the protein is lacking. The purification of Hop1 from E. coli, which was functionally indistinguishable from the protein obtained from mitotically dividing S. cerevisiae cells has enabled us to investigate the structure-function relationships of Hop1, which has provided important insights into its role in meiotic recombination. We present several lines of evidence suggesting that Hop1 is a modular protein, consisting of an intrinsically unstructured N-terminal domain and a core C-terminal domain (Hop1CTD), the latter being functionally equivalent to the full-length Hop1 in terms of its in vitro activities. Importantly, however, Hop1CTD was unable to rescue the meiotic recombination defects of hop1null strain, indicating that synergy between the N-terminal and C-terminal domains of Hop1 protein is essential for meiosis and spore formation. Taken together, our findings provide novel insights into the molecular functions of Hop1, which has profound implications for the assembly of mature SC, homolog synapsis and recombination. Several lines of investigations suggest that HORMA domain containing proteins are involved in chromatin binding and, consequently, have been shown to play key roles in processes such as meiotic cell cycle checkpoint, DNA replication, double-strand break repair and chromosome synapsis. S. cerevisiae encodes three HORMA domain containing proteins: Hop1, Rev7 and Mad2 (HORMA) which interact with chromatin during diverse chromosomal processes. The data presented above suggest that Hop1 is a modular protein containing a distinct N-terminal and C-terminal (Hop1CTD) domains. The N-terminal domain of Hop1, which corresponds to the evolutionarily conserved HORMA domain, although, discovered first in Hop1, its precise biochemical functions remain unknown. In this section, we show that Hop1-HORMA domain expressed in and purified from E. coli exhibits preferential binding to the HJ and G4 DNA, over other early recombination intermediates. Detailed functional analyses of Hop1-HORMA domain, using mobility shift assays, DNase I footprinting and FRET, have revealed that HORMA binds at the core of Holliday junction and induces marked changes in its global conformation. Further experimental evidence also suggested that it causes DNA stiffening and condensation. However, like Hop1CTD, HORMA domain alone failed to rescue the meiotic recombination defects of hop1 null strain, indicating that synergy between the N-and C-terminal domains of Hop1 is essential for meiosis as well as for the formation of haploid gametes. Moreover, these results strongly implicate that Hop1 protein harbours a second DNA binding motif, which resides in the HORMA domain at its N-terminal region. To our knowledge, these findings also provide the first insights into the biochemical mechanism underlying HORMA domain activity. In summary, it appears that the C-terminal (CTD) and N-terminal (HORMA) domains of Hop1 may perform biochemical functions similar (albeit less efficiently) to that of the full-length Hop1. However, further research is required to uncover the functional differences between these domains, their respective interacting partners and modulation of the activity of these domains.

This diploma thesis deals with a problem of spores venturia inaequlis recognition. These spores are captured on a special tape which is then analyzed using a microscope. The tape can be analyzed by a laboratorian or by the program Sporedetect v3. This program provides functions for complete picture processing and object recognition. In this diploma thesis, there are also described ways to automatically control a sliding stage of a microscope utilizing motorized translation stages and linear actuators. The information about automatic control of a microscope stage was obtained from catalogues of the companies Standa and Edmundoptics.

Indiana University-Purdue University Indianapolis (IUPUI) / Spore photoproduct lyase (SPL) is a radical SAM (S-adenosylmethionine) enzyme that is responsible for the repair of the DNA UV damage product 5-thyminyl-5,6-dihydrothymine (also called spore photoproduct, SP) in the early germination phase of bacterial endospores. SPL initiates the SP repair process using 5'-dA• (5'-deoxyadenosyl radical) generated by SAM cleavage to abstract the H6proR atom which results in a thymine allylic radical. These studies provide strong evidence that the TpT radical likely receives an H atom from an intrinsic H atom donor, C141 in B. subtilis SPL. I have shown that C141 can be alkylated in native SPL by iodoacetamide treatment indicating that it is accessible to the TpT radical. Activity studies demonstrate a 3-fold slower repair rate of SP by C141A which produces TpTSO2 - and TpT simultaneously with no lag phase observed for TpTSO2- formation. Additionally, formation of both products shows a Dvmax kinetic isotope effect (KIE) of 1.7 ± 0.2 which is smaller than the DVmax KIE of 2.8 ± 0.3 for the WT SPL reaction. Removal of the intrinsic H atom donor by this single mutation disrupts the rate-limiting process in the enzyme catalysis. Moreover, C141A exhibits ~0.4 turnover compared to the > 5 turnovers in the WT SPL reaction. In Y97 and Y99 studies, structural and biochemical data suggest that these two tyrosine residues are also crucial in enzyme catalysis. It is suggested that Y99 in B. subtilis SPL uses a novel hydrogen atom transfer pathway utilizing a pair of cysteinetyrosine residues to regenerate SAM. The second tyrosine, Y97, structurally assists in SAM binding and may also contribute to SAM regeneration by interacting with radical intermediates to lower the energy barrier for the second H-abstraction step.