The use of nitrogen (N) fertilizers is both costly to farmers and contributes to environmental degradation. N applied to wheat accounts for 18% of N applied to farmland globally, making it a prime target for reducing and optimizing N application. Chapter I is a review on nitrogen use efficiency (NUE) in wheat, with emphasis on breeding efforts and genetic resources available to increase NUE. The concept of effective use of nitrogen (EUN) as yield per unit N applied as a measure of N use, is also introduced. Chapter II is a study using two bi-parental double haploid families to evaluate genetic variability of both the genetic main effects (intercept) and linear response to N (slope) and determine the feasibility of selection for EUN in wheat. Using cross validation, a genomic prediction accuracy of 0.68 for intercept and 0.50 for slope was found, indicating that EUN is under genetic control and can be selected for. The prospect of breeding for EUN under limited resources, i.e., using fewer N rates and fewer experimental plots, is also explored. It was found that two different N treatments can be used to produce accurate predictions of intercept and slope as high as 0.98 and 0.95, respectively. Chapter III uses the same population described in chapter II to further investigate feasibility of selection for EUN using a normalized difference vegetation index (NDVI) obtained from multi-spectral aerial images gathered throughout the growing season. Cumulative photosynthesis across the growing season was estimated by integration across the NDVI curve, and compared to grain yield estimates to determine the efficacy of aerial imaging to identify high EUN lines. NDVI values and the area under the NDVI curve were able to predict yield and had the strongest ability to predict yield in moderate to low N treatments, with R2 values as high as 0.81 and 0.78 respectively. / Master of Science / Chapter I is a review on nitrogen use efficiency (NUE) in wheat, with emphasis on breeding efforts and genetic resources available to increase NUE. The concept of effective use of nitrogen (EUN) defined as grain yield per unit N applied, is contrasted to NUE as a more economic breeding goal. Chapter II uses two bi-parental mapping populations to evaluate genetic variability of both the genetic main effects and the linear response to N and determine the feasibility of selection for EUN in wheat. The efficacy of genomic prediction for EUN is explored and the prospect of breeding for EUN under limited resources is also explored. Chapter III uses the same populations described in chapter II to further investigate the feasibility of selection for EUN using a normalized difference vegetation index (NDVI) obtained from multi-spectral aerial images gathered throughout the growing season. Cumulative photosynthesis across the growing season was estimated and found to be predictive of grain yield estimates at accuracies ranging from 0.31 to 0.78.

Einleitung: Tiermodelle spielen für die Aufklärung pathophysiologischer Mechanismen und die Entwicklung erfolgsversprechender Therapieoptionen zentralnervöser Bewegungsstörungen eine unverzichtbare Rolle. Die Identifizierung von Gendefekten für die Parkinson-Krankheit und Dystonien ermöglichte die Generierung von Tiermodellen mit einer hohen „construct validity“. Weibliche transgene Thy1-aSyn Mäuse sowie DYT1 Knock-in (KI) Mäuse zeigen jedoch keine motorischen Störungen. In der vorliegenden Arbeit sollten zur Aufdeckung sensomotorischer Beeinträchtigungen, die bei Parkinson- und Dystoniepatienten beobachtet werden, detaillierte Untersuchungen des Verhaltens an diesen beiden Mausmodellen durchgeführt werden. Zielstellung: Zunächst sollte ein sensitiver Verhaltenstest konstruiert und entwickelt werden, bei dem sich ändernde sensorische Stimuli während der Ausübung der motorischen Aufgabe impliziert werden. Bei der Etablierung dieses sogenannten „adaptiven rotierenden Balkentests“ (ARB-Test) sollte auch der Einfluss des genetischen Hintergrunds bei Wildtyp-Mäusen evaluiert werden. Daraufhin sollte überprüft werden, ob dieser Test den Endophänotyp der weiblichen Thy1-aSyn Mäuse aufdecken kann. In dem DYT1 KI Mausmodell sollte der Frage nachgegangen werden, ob die Tiere Verhaltensdefizite in spezifischen Tests zeigen, die sensomotorische Verschaltungen untersuchen. Material und Methoden: Die mRNA-Expression von α-Synuclein in der Substantia nigra bei männlichen und weiblichen Thy1-aSyn Mäusen wurde mithilfe der quantitativen Echtzeit-PCR (qPCR) ermittelt. Im Anschluss an die Entwicklung des neuen Verhaltensapparates für den ARB-Test wurden Thy1-aSyn Tiere beider Geschlechter in diesem Versuch getestet und ihre Leistung den Ergebnissen auf etablierten motorischen Verhaltenstests („challenging beam test“, „pole test“) gegenübergestellt. Um den Einfluss des Hintergrundstammes auf das Verhalten der Tiere auf dem ARB-Test zu untersuchen, wurden Wildtypen der reinen C57BL/6J-Linie sowie Hybrid-Tiere des Stammes C57Bl/6J × DBA2 (BDF1) allen drei o. g. Versuchen unterzogen. Bei den Mäusen des DYT1 KI Modells wurde der „adhesive removal test“ und der ARB-Test zur Analyse der Sensomotorik durchgeführt. Im Vergleich dazu wurden vielfältige Verhaltensparameter in einer Reihe vorwiegend motorischer (Offenfeld-Test, „challenging beam test“, „pole test“, Zylinder-Test, Block-Test, Nestbau-Test) und kognitiver („y-maze test“) Verhaltenstests ausgewertet. Ergebnisse: Bei den weiblichen Thy1-aSyn Mäusen wurde eine geringere Expression des Transgens im Vergleich zu den männlichen Tieren festgestellt. Der neue ARB-Test wurde erfolgreich etabliert und konnte signifikante Verhaltensdefizite der weiblichen und männlichen Mutanten des Parkinson-Modells im Vergleich zu den Kontrolltieren aufdecken. Der genetische Hintergrund beeinflusste die Leistung der Wildtypen auf diesem Balkentest. Während die DYT1 KI Tiere in den rein motorischen und kognitiven Versuchen keine Beeinträchtigungen des Verhaltens zeigten, konnten der „adhesive removal test“ sowie der neue ARB-Test signifikante sensomotorische Defizite der KI Mäuse im Unterschied zu den Wildtypen zum Vorschein bringen. Schlussfolgerung: Im Thy1-aSyn Mausmodell konnte die Bedeutung der sensomotorischen Integration für die Ausprägung motorischer Defizite sowie für eine mögliche Kompensation solcher motorischen Beeinträchtigungen demonstriert werden. Hierfür hat sich der neu entwickelte, sensitive ARB-Test als geeignet herausgestellt. Die Aufdeckung von Beeinträchtigungen der Sensomotorik spricht auch bei den DYT1 KI Tieren für den Einfluss einer gestörten sensomotorischen Integration bei der Ausprägung der Symptomatik. Damit eignet sich dieses Mausmodell für die Untersuchung weiterer Parameter, die Auswirkungen auf die Aufdeckung des Phänotyps und die Penetranz der Erkrankung haben sowie um die zugrunde liegenden pathophysiologischen Mechanismen zu erforschen.

Les recherches sur les interactions porte-greffe/greffon chez la vigne en relation avec l’environnement perdurent depuis plusieurs décennies, mais les mécanismes physiologiques sous-jacents de la vigueur conférée sont toujours incompris. Ce manque de connaissance constitue un frein dans le développement des porte-greffes existants pour contrôler la vigueur et la productivité, ou dans la recherche de nouveaux génotypes de porte-greffe mieux adaptés aux conditions futures de production. L’objectif de ce travail est de comprendre par une approche de biologie intégrative couplant expérimentation et modélisation comment le porte-greffe interagit spécifiquement avec son greffon (et vice versa) pour modifier dès les premières étapes du greffage, les caractéristiques physiologiques de la plante entière afin de coordonner le développement et la croissance des parties aériennes avec celle des parties racinaires. L’azote étant considéré comme un élément-clef de contrôle de la croissance et de l’allocation de la biomasse au sein d’une plante, un accent particulier est porté sur le rôle de la nutrition azotée dans le contrôle trophique de la croissance du couple porte-greffe/greffon. Un travail expérimental en serre a été mené pour caractériser par marquage isotopique les flux d’azote (15N) et de carbone à l’échelle de la plante entière au sein de deux combinaisons de porte-greffe/greffon au stade végétatif : l’une conférant une forte vigueur (CS/1103P), l’autre une faible vigueur (CS/RGM), en réponse à une variation de la disponibilité externe en nitrate. Cette étude sur le couplage entre fonctions d’acquisition et d’utilisation des ressources azotées et carbonées a été complétée par un phénotypage dynamique de la croissance aérienne, de la répartition de biomasse entre les organes et de la composition biochimique et minérale des principaux organes de la plante. Nous avons ainsi pu appréhender les signaux de communication entre la partie aérienne et la partie racinaire de la vigne greffée, ce qui a abouti à l’élaboration d’un modèle conceptuel simplifié du fonctionnement de la vigne greffée. Une première version d’un modèle mécaniste basé sur un formalisme source-puits prenant en compte l’acquisition et l’allocation de C et N au sein de deux compartiments aérien et racinaire, ainsi que leur plasticité vis-à-vis de la disponibilité exogène et endogène en ressources a été élaborée. A terme, le modèle devrait permettre d’identifier des paramètres génétiques clef au niveau racinaire explicitant les différences de croissance et vigueur conférée observées selon les combinaisons porte-greffe/greffon / Research on rootstock/scion interactions in grapevine in relation to the environment persisted for several decades, but the physiological mechanisms determining the rootstock effect on scion vigour are still misunderstood. This lack of knowledge hampers the development of existing rootstocks to control the vigor and productivity, or research new rootstock genotypes better adapted to future conditions of production. The objective of this work is to understand by an integrative biology approach coupling experimentation and modeling how the rootstock interacts specifically with the scion (and vice versa) to change in the early stages of grafting , the physiological characteristics of the whole plant to coordinate the development and growth of the aerial parts with the root parties. Nitrogen is considered a key element in the control of the growth and the biomass allocation within a plant, and a particular emphasis is placed on the role of nitrogen nutrition in the nutritional control of the grafted grapevine growth. Experimental work was conducted in a greenhouse to characterize by isotopic labeling nitrogen (15N) and carbon flow within the whole plant for two rootstock/scion combinations at vegetative stage : one giving a strong vigour (CS/1103P), the other a low vigour (CS / RGM), in response to a change in the external nitrate availability. This study on the coupling between acquisition functions and use of nitrogenous and carbonaceous resources was completed by a dynamic phenotyping aerial growth, the distribution of biomass between the organs and the biochemical and mineral composition of the principal organs of plant. We were able to understand the communication signals between the aerial part and the root part of grafted vines, which led to the development of a simplified conceptual model of the functioning of the grafted vines. A first version of a mechanistic model based on a source-sink formalism taking into account the acquisition and allocation of C and N in both aerial and root compartments and their plasticity to the availability of exogenous and endogenous resource was developed.

Crop improvement is necessary for food security as the global population is expected to exceed 9 billion by 2050. Limitations in water resources and more frequent droughts and floods will make it increasingly difficult to manage agricultural resources and increase yields. Therefore, we must improve our ability to monitor agronomic research plots and use the information they provide to predict impacts of moisture stress on crop growth and yield. Towards this end, agronomists have used reductions in leaf expansion rates as a visible ‘plant-based’ indicator of moisture stress. Also, modeling researchers have developed crop models such as AquaCrop to enable quantification of the severity of moisture stress and its impacts on crop growth and yield. Finally, breeders are using Unmanned Aircraft Systems (UAS) in field-based High-Throughput Phenotyping (HTP) to quickly screen large numbers of small agronomic research plots for traits indicative of drought and flood tolerance. Here we investigate whether soybean canopy cover and color time series from high-resolution UAS ortho-images can be collected with enough spatial and temporal resolution to accurately quantify and differentiate agronomic research plots, pinpoint the timing of the onset of moisture stress, and constrain crop models such as AquaCrop to more accurately simulate the timing and severity of moisture stress as well as its impacts on crop growth and yield. We find that canopy cover time series derived from multilayer UAS image ortho-mosaics can reliably differentiate agronomic research plots and pinpoint the timing of reductions in soybean canopy expansion rates to within a couple of days. This information can be used to constrain the timing of the onset of moisture stress in AquaCrop resulting in a more realistic simulation of moisture stress and a lower likelihood of underestimating moisture stress and overestimating yield. These capabilities will help agronomists, crop modelers, and breeders more quickly develop varieties tolerant to moisture stress and achieve food security.

As Hsp são utilizadas nos tratamentos de autoimunidades e tumores, mas seus mecanismos de ação não foram esclarecidos. Para verificar o efeito da Hsp65 no envelhecimento, analisamos leucócitos em ♀ HIII envelhecidas e, utilizando-se camundongos F1, investigou-se a influência genética na susceptibilidade. ♀HIII velhas apresentaram aumento de linfócitos T ativados e B no baço, e de APC no sangue. A sobrevida em ♀F1H injetadas foi menor e semelhante aos dados obtidos em ♀HIII. A produção de IgG aumentou nos ♂F1 tratados, e a de IgM foi maior nos animais inoculados independente do sexo. Nos híbridos, apenas ♀F1 envelhecidas apresentaram maior porcentagem de linfócitos T CD4 e CD8 ativados e de células CD11c. A Hsp65 induz, in vivo, ativação imune em ♀HIII e ♀F1 velhas; a susceptibilidade parece ser ligada a um efeito do sexo feminino e à influência genética da linhagem HIII. / Hsp are used for the development of autoimmunities and tumors treatments, however, the mechanism of action has not been fully enlightened. To verify the effect of Hsp65 in the aging, we analyzed aged ♀HIII leukocytes; using F1 hybrid mice we studied the genetic influence in Hsp65 susceptibility. Aged ♀HIII presented increase of T and B lymphocytes in the spleen and augmented APC in the blood. The survival of aged ♀F1H was reduced, similarly to aged ♀HIII previous results. The anti-Hsp65 IgG production increased in ♂F1 males, and the IgM was greater in all groups regardless of gender. In hybrid mice, only ♀F1 mice presented increased percentage of CD4 and CD8 activated T cells and of CD11c cells. The Hsp65 induces in vivo, activation of the immune system in aged ♀HIII and ♀F1 mice; the susceptibility to the protein can be given by specific gender effects and the genetic influence of the HIII strain.

Common bean is a major dietary component in several countries, but its productivity is negatively affected by abiotic stresses. Dissecting candidate genes involved in abiotic stress tolerance is a paramount step toward the improvement of common bean performance under such constraints. Thereby, this thesis presents a systematic analysis of the DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) gene subfamily, which encompasses genes that regulate several processes during stress responses, but with limited information for common bean. First, a series of in silico analyses with sequences retrieved from the P. vulgaris genome on Phytozome supported the categorization of 54 putative PvDREB genes distributed within six phylogenetic subgroups (A-1 to A-6), along the 11 chromosomes. Second, we cloned four novel PvDREB genes and determined their inducibility-factors, including the dehydration-, salinity- and cold-inducible genes PvDREB1F and PvDREB5A, and the dehydration- and cold-inducible genes PvDREB2A and PvDREB6B. Afterwards, nucleotide polymorphisms were searched through Sanger sequencing along those genes, revealing a high number of single nucleotide polymorphisms within PvDREB6B by the comparison of Mesoamerican and Andean genotypes. The nomenclature of PvDREB6B is discussed in details. Furthermore, we used the BARCBean6K_3 SNP platform to identify and genotype the closest SNP to each one of the 54 PvDREB genes. We selected PvDREB6B for a broader study encompassing a collection of wild common bean accessions of Mesoamerican origin. The population structure of the wild beans was accessed using sequence polymorphisms of PvDREB6B. The genetic clusters were partially associated with variation in latitude, altitude, precipitation and temperature throughout the areas such beans are distributed. With an emphasis on drought stress, an adapted tube-screening method in greenhouse conditions enabled the phenotyping of several drought-related traits in the wild collection. Interestingly, our data revealed a correlation between root depth, plant height and biomass and the environmental data of the location of the accessions. Correlation was also observed between the population structure determined through PvDREB6B and the environmental data. An association study combining data from the SNP array and DREB polymorphisms enabled the detection of SNP associated with drought-related traits through a compressed mixed linear model (CMLM) analysis. This thesis highlighted important features of DREB genes in common bean, revealing candidates for further strategies aimed at improvement of abiotic stress tolerance, with emphasis on drought tolerance / O feijoeiro é um componente essencial na dieta em diversos países, no entanto, sua produção é afetada negativamente por estresses abióticos. O estudo de genes candidatos envolvidos na adaptação aos estresses é uma etapa fundamental para o melhoramento da performance do feijoeiro sob tais estresses. Desse modo, esta tese apresenta uma análise sistemática da subfamília de genes DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB), que reúne genes envolvidos em diversos processos em resposta a estresses, mas pouco estudados no feijoeiro. Primeiramente, uma série de análises in silico com sequências de feijoeiro obtidas da plataforma Phytozome possibilitaram a categorização de 54 genes PvDREB putativos, distribuídos em seis subgrupos (A-1 até A-6) nos 11 cromossomos da espécie. Posteriormente, quatro novos genes PvDREB foram clonados e seus padrões de inducibilidade foram determinados. PvDREB1F e PvDREB5A foram induzidos por desidratação, baixa temperatura e salinidade, enquanto PvDREB2A e PvDREB6B foram predominantemente induzidos por desidratação e baixa temperatura. Polimorfismos de nucleotídeos foram buscados através de sequenciamento por método derivado de Sanger, revelando elevado número de SNP no gene PvDREB6B. A nomenclatura desse gene foi discutida detalhadamente ao longo da tese. A plataforma de marcadores SNP BARCBean6K_3 foi acessada para identificar o SNP mais próximo de cada um dos 54 PvDREB. O gene PvDREB6B foi selecionado para um estudo mais amplo, envolvendo uma coleção de acessos selvagens de origem Mesoamericana. A estrutura populacional destes genótipos foi analisada a partir de polimorfismos na sequência de PvDREB6B. Os grupos genéticos apresentaram associação parcial com variação da latitude, altitude, precipitação e temperatura das áreas em que os acessos naturalmente ocorrem. Com ênfase no estudo do déficit hídrico, uma plataforma de fenotipagem destes acessos em casa de vegetação, utilizando um sistema de tubos plásticos, foi elaborada para a análise de diversos parâmetros relacionados ao estresse por déficit hídrico. Os dados revelaram correlação entre profundidade de raízes, altura das plantas e a biomassa e as variáveis ambientais de cada local. A correlação também foi detectada entre a estrutura populacional estudada por PvDREB6B e os dados ambientais. Finalmente, um estudo de associação genética foi realizado entre os SNP da plataforma e ligados a DREB e os parâmetros fenotípicos, permitindo a identificação de marcadores SNP associados a caracteres específicos, usando um modelo linear misto (CMLM). Esta tese apresentou importantes aspectos sobre os genes DREB em feijoeiro, revelando candidatos para seu uso em estratégias de melhoramento para tolerância a estresses abióticos, com ênfase em déficit hídrico

Der ischämische Schlaganfall ist global eine der bedeutendsten Volkskrankheiten. Die derzeit verfügbaren kurativen Therapieoptionen werden vorrangig durch ein enges therapeutisches Zeitfenster limitiert. Ziel der aktuellen Schlaganfallforschung ist die Entwicklung von über dieses Zeitfenster hinaus wirksamen Therapien. Ein vielversprechender neuer Ansatz ist die experimentelle Behandlung mit humanen Nabelschnurblutzellen. Diese Arbeit erforscht das therapeutische Zeitfenster für die systemische Therapie des ischämischen Schlaganfalls mittels mononukleärer Nabelschnurblutzellen (hUCB MNC) in spontanhypertensiven Ratten nach permanentem Verschluss der Arteria cerebri media (pMCAO). Hierzu wurden die Therapiezeitpunkte 4, 24, 72, 120 Stunden und 14 Tage nach experimentellem Schlaganfall in einem komplexen Studiendesign inklusive neurofunktioneller Tests, magnetresonanztomographischer und immunhistochemischer Verfahren untersucht. In vitro wurde der Einfluss kokultivierter hUCB MNC auf Nekrose und Apoptose in neuralem Gewebe unter Sauerstoff-Glukose-Deprivation betrachtet. Die Studie ergab eine verbesserte funktionelle Rekonvaleszenz und eine geringere Ausprägung von Atrophie und Astroglianarbe bei Therapie innerhalb eines 72- Stunden-Zeitfensters. In vitro wurde eine signifikante Reduktion von Nekrose und Apoptose durch kokultivierte hUCB MNC beobachtet. Eine histologische Relokalisierung der intravenös applizierten Zellen war in keiner Therapiegruppe möglich. Die Integration der hUCB MNC ins Hirnparenchym stellt somit keine conditio sine qua non für die funktionelle Erholung nach Schlaganfall dar. Trotz des beobachteten erweiterten Zeitfensters ist die Translation dieses Therapieansatzes in die klinische Realität kritisch zu diskutieren, da weiterführende Studien unserer Arbeitsgruppe eine limitierte Wirksamkeit unter sehr praxisnahen Bedingungen (z.B. Einsatz kryokonservierter hUCB MNC) gezeigt haben. / Experimental treatment strategies using human umbilical cord blood mononuclear cells (hUCB MNCs) represent a promising option for alternative stroke therapies. An important point for clinical translation of such treatment approaches is knowledge on the therapeutic time window. Although expected to be wider than for thrombolysis, the exact time window for hUCB MNC therapy is not known. Our study aimed to determine the time window of intravenous hUCB MNC administration after middle cerebral artery occlusion (MCAO). Male spontaneously hypertensive rats underwent MCAO and were randomly assigned to hUCB MNC administration at 4h, 24h, 72h, 120h or 14d. Influence of cell treatment was observed by magnetic resonance imaging on days 1, 8 and 29 following MCAO and by assessment of functional neurological recovery. On day 30, brains were screened for glial scar development and presence of hUCB MNCs. Further, influence of hUCB MNCs on necrosis and apoptosis in post-ischemic neural tissue was investigated in hippocampal slices cultures. Transplantation within a 72h time window resulted in an early improvement of functional recovery, paralleled by a reduction of brain atrophy and diminished glial scarring. Cell transplantation 120h post MCAO only induced minor functional recovery without changes in the brain atrophy rate and glial reactivity. Later transplantation (14d) did not show any benefit. No evidence for intracerebrally localized hUCB MNCs was found in any treatment group. In vitro hUCB MNCs were able to significantly reduce post-ischemic neural necrosis and apoptosis. Our results for the first time indicate a time window of therapeutic hUCB MNC application of at least 72 hours. The time window is limited, but wider than compared to conventional pharmacological approaches. The data furthermore confirms that differentiation and integration of administered cells is not a prerequisite for poststroke functional improvement and lesion size reduction.

Made available in DSpace on 2015-12-06T22:54:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2009 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Neste estudo, 151 isolados brasileiros de Sporothrix schenckii foram caracterizados quanto as suas características fenotípicas e genotípicas. Quanto às características fenotípicas, a maioria dos isolados apresentou conídios ovais, sendo que os de origem animal são maiores que os de origem humana (animal: 2,96 ± 1,07 versus forma linfocutânea: 2,37 ± 0,43; forma fixa: 2,33 ± 0,53). Os isolados provenientes da região Norte do Brasil são os mais termotolerantes e os isolados do Nordeste são os mais termossensíveis. Os isolados de origem animal são mais tolerantes à temperatura quando comparados com isolados obtidos de pacientes. Os isolados brasileiros apresentaram grande variabilidade genética quando avaliados por RAPD. Por Southern Blot, utilizando a enzima APA I para o estudo do polimorfismo das regiões ITS, três genótipos foram obtidos. Não houve correlação entre o perfil obtido por RAPD ou Southern Blot com a origem clínica ou geográfica dos isolados. A secreção de proteínas/glicoproteínas é variável entre os isolados, sofrendo influência direta do meio de cultura. Os isolados brasileiros avaliados são produtores das enzimas DNAse e urease, 15,78% produzem gelatinase, 26,31% produzem proteinase e 21,05% produzem caseinase. Todos os isolados avaliados toleram pressão osmótica de 16,6% de glicerol, 89,47% toleram 20% e todos são inibidos a 23% e 28,5 % de glicerol. Todos os isolados avaliados toleram pressão salina de 6% e 8%; 42,10% toleram pressão de 9% e 10% de sal e todos são inibidos a 12% de sal. Todos os isolados avaliados crescem na faixa de pH de 2,2 a 12,5. A patogenia e virulência são variáveis entre os isolados, podendo estar relacionada ao genótipo do isolado. Diferentes proteínas são reconhecidas pelos soros dos camundongos infectados, sendo a molécula de 60 kDa, a mais reconhecida neste sistema. Em relação à esporotricose humana, as moléculas de 70 kDa e 38 kDa são comumente reconhecidas por soros de pacientes com esporotricose fixa e linfocutânea. O antígeno bruto do isolado Ss 118 e a fração SsCBF são eficientes para diagnóstico da esporotricose felina através da técnica de ELISA. A fração SsCBF é capaz de elicitar resposta imune do tipo HTT em camundongos experimentalmente infectados. Em geral, os resultados indicam que os isolados brasileiros são heterogêneos, apresentando características genéticas e fenotípicas individuais, independentes de sua origem. Somente a área conidial e a tolerância a temperatura foram características fenotípicas relacionadas à origem. / In this study, we analyzed 151 Brazilian S. schenckii isolates by phenotyping and genotyping aspects. About phenotyping aspects, most of isolates showed oval conidia and the mean conidial area of S. schenckii animal isolates was greater than clinical isolates (animal: 2.96 ± 1.07 versus lymphocutaneous form: 2.37 ± 0.43; fixed form: 2.33 ± 0.53). Isolates from the Northeast region exhibited the lowest thermotolerance and the Northern isolates exhibited the highest thermotolerance. Animal isolates are better thermotolerants than clinical isolates. The Brazilian isolates showed great genetic diversity when analyzed by RAPD. By Southern blot using the endonuclease APA I to study polymorphism of ITS regions, three genotypes were obtained. There was no association between RAPD and Southern profiles with clinical forms origin or geographic origin. The protein/glycoprotein secretion was variable among isolates and was directly influenced by the medium composition. All Brazilian isolates studied produced DNAse and urease exoenzymes, 15.78% produced gelatinase, 26.31% produced proteinase and 21.05% produced caseinase. All analyzed isolates grew at 16,6% of glycerol, 89,47% grew at 20% of glycerol and all of them were inhibited at 23% and 28,5 % of glycerol. All analyzed isolates grew at 6% and 8% of NaCl, 42.10% grew at 9% and 10% of NaCl and all of them were inhibited at 12% of NaCl. All of them were able to grow at pH 2.2 to 12.5. The pathogenicity and virulence were variable among isolates and it can be related to the genotype. Different proteins were recognized by sera of infected mice and the 60 kDa molecule was the most one recognized in murine system. In relation to human sporotrichosis, 70 kDa and 38 kDa molecules were common recognized by sera from patients with fixed and lymphocutaneous sporotrichosis. The crude exoantigen of Ss 118 isolate and the SsCBF fraction are able to be used in the diagnosis of feline sporotrichosis by ELISA. The SsCBF fraction is able to elicit DTH immune response in infected mice. In general, our results indicate that Brazilian isolates are heterogeneous, which present individual phenotyping and genotyping characteristics, independent of origin. Only the conidial area and thermotolerance were related to origin. / BV UNIFESP: Teses e dissertações

Doctor of Philosophy / Agronomy / Kevin Price / Stephen M. Welch / Plant phenotyping has been studied for decades for understanding the relationship between plant genotype, phenotype, and the surrounding environment. Improved accuracy and efficiency in plant phenotyping is a critical factor in expediting plant breeding and the selection process. In the past, plant phenotypic traits were extracted using invasive and destructive sampling methods and manual measurements, which were time-consuming, labor-intensive, and cost-inefficient. More importantly, the accuracy and consistency of manual methods can be highly variable. In recent years, however, photogrammetry and 3D modeling techniques have been introduced to extract plant phenotypic traits, but no cost-efficient methods using these two techniques have yet been developed for large-scale plant phenotyping studies. High-throughput 3D modeling techniques in plant biology and agriculture are still in the developmental stages, but it is believed that the temporal and spatial resolutions of these systems are well matched to many plant phenotyping needs. Such technology can be used to help rapid phenotypic trait extraction aid crop genotype selection, leading to improvements in crop yield. In this study, we introduce an automated high-throughput phenotyping pipeline using affordable imaging systems, image processing, and 3D reconstruction algorithms to build 2D mosaicked orthophotos and 3D plant models. Chamber-based and ground-level field implementations can be used to measure phenotypic traits such as leaf length, rosette area in 2D and 3D, plant nastic movement, and diurnal cycles. Our automated pipeline has cross-platform capabilities and a degree of instrument independence, making it suitable for various situations.

Made available in DSpace on 2015-04-17T15:03:08Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 760407 bytes, checksum: 36ff52186c6a7e31beb2bd5ddc4a7fa0 (MD5) Previous issue date: 2011-03-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This study aimed to isolate strains of Staphylococcus aureus in ricotta cheeses and to characterize them respecting phenotypic markers of lipolytic activity, and bacteriocin typing and resistance typing. For isolation, were acquired 11 samples of different brands of ricotta cheese sold in supermarkets in João Pessoa - Paraíba. Among the 41 strains, 82.9% proved positive lipase (Lip+) and 17.1%, negative lipase (Lip-). In relation to bacteriocin typing, 9.76% showed bacteriocin producing against a strain of Staphylococcus aureus isolated from surfaces processing food and 55% were susceptible to a strain of Staphylococcus aureus isolated from ricotta cheese. For resistance typing, 87.8% of strains were resistant to streptomycin, 26.59% to penicillin and 19.51% to erythromycin. None of the strains were resistant to tetracycline. Variability was observed between the minimal inhibitory concentrations (MIC), both in resistant strains as sensitive ones, so the variability showed more prevalent with penicillin (ranging from 0.015625 to 16 mg / mL) and the variability between strains of the same cheese. Only erythromycin-resistant strains were subjected to "D-test" to determine the type of resistance, of which all presented inductive resistant. From this results obtained, can conclude that although the phenotypes show an adaptive answer on related to the peculiarities of local environmental pressures, it is clear the importance of these genetic markers to discern new strains of S. aureus for those endemic, as well as to investigate possible epidemiological impacts and on food safety. / Este estudo teve como objetivo isolar cepas de Staphylococcus aureus de queijos ricota e caracterizá-las com relação a marcadores fenotípicos de atividade lipolítica, bacteriocinotipagem e resistotipagem. Para o isolamento, foram adquiridas 11 amostras de queijo ricota de diferentes marcas comercializadas em supermercados da cidade de João Pessoa-PB. Entre as 41 cepas isoladas, 82,9% revelaram-se lipase positiva (Lip+) e 17,1%, lipase negativa (Lip-). Com relação à bacteriocinotipagem, 9,76% mostraram-se produtoras de bacteriocinas frente a uma cepa de Staphylococcus aureus isolada de superfícies de processamento de alimentos e 55% mostraram-se sensíveis a uma cepa de Staphylococcus aureus isolada de queijo ricota. Para resistotipagem, 87,8% das cepas apresentaram-se resistentes a estreptomicina, 26,59% a penicilina e 19,51% a eritromicina. Nenhuma das cepas foi resistente a tetraciclina. Observou-se a variabilidade entre as Concentrações Inibitórias Mínimas, tanto em cepas resistentes como sensíveis, sendo tal variabilidade mais predominante com penicilina (variação de 0,015625 a 16 μg/mL), bem como a variabilidade entre cepas do mesmo queijo. Apenas cepas resistentes a eritromicina foram submetidas ao D-teste a fim de determinar o tipo de resistência, das quais todas as cepas mostraram-se com resistência indutiva. A partir dos resultados obtidos, pode-se concluir que embora os fenótipos revelem uma resposta adaptativa relacionada às peculiaridades das pressões ambientais locais, é evidente a importância destes marcadores genéticos a fim de se distinguir novas linhagens de S. aureus daquelas endêmicas, bem como investigar possíveis impactos epidemiológicos e na segurança alimentar.