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No. of bitstreams: 1 silva_nc_dr_jabo.pdf: 1682545 bytes, checksum: ca3b83a01f9c9a072da78273e4da0fca (MD5) Previous issue date: 2016-10-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Foram conduzidos três experimentos com os objetivos de determinar 1) a melhor dose e tipo de inoculante, homo ou heterofermentativo, para grão de milho reidratado; 2) os efeitos do tempo de estocagem e do uso de inoculante sobre o perfil fermentativo, estabilidade aeróbia e degradabilidade in situ de silagem de milho grão úmido e silagem de grão de milho reidratado; 3) as alterações nos parâmetros ruminais, digestibilidade e desempenho de bovinos Nelore, consumindo dietas contendo milho seco moído ou silagem de milho grão úmido ou silagem de grão de milho reidratado sem e com o uso de inoculante. No experimento 1 foi avaliado silagem de grão de milho reidratado sem inoculante (C), com L. plantarum + P. acidilactici (LPPA) e com L. buchneri (LB), ambos nas doses de 1 ×105, 5 ×105 e 1 ×106 ufc/g, estocadas por 124 dias. As silagens inoculadas com LB em relação ao C apresentaram maior (P < 0,01) contagem de bactérias ácido láticas (BAL), concentração de ácido acético, ácido propiônico e 1,2-propanodiol e menor (P < 0,01) pH, leveduras e fungos filamentosos nos 12 dias de exposição ao ar e maior (P < 0,01) estabilidade aeróbica, isso ocorreu já na primeira dose (1 ×105 ufc/g). O uso de LB também aumentou (P < 0,05) a quebra de prolamina e a degradabilidade in situ das silagens. Uso de LPPA não apresentou melhorias na fermentação e piorou a deterioração aeróbia em comparação ao controle. No experimento 2 foi avaliado um esquema fatorial 2 × 2, sendo o primeiro fator dois tipos de ensilagem, silagem de milho úmido (MU) e silagem de milho reidratado (MR); e o segundo fator o uso de inoculante, sem (Controle) e com L. buchneri (LB; 1 × 105 ufc/g), com aberturas aos 15, 30, 60, 90, 120, 180, 240 e 300 dias de estocagem. A MR apresentou maior concentração total de ácidos, enquanto as silagens com LB apresentaram maior concentração de ácido acético e 1,2-propanodiol e menor de ácido lático e ácido butírico. Ao longo dos tempos de estocagem, MU LB e MR LB apresentaram menor contagem de leveduras e maior contagem de BAL e estabilidade aeróbia. A MR com tamanho de partícula original apresentou maior degradabilidade in situ que a MU. O tempo mínimo para atingir ganhos altos de degradabilidade foi entre 30 e 70 dias de estocagem. No experimento 3 foram avaliadas cinco dietas contendo: silagem de milho grão úmido (MU), silagem de milho grão úmido com L. buchneri (MUB), silagem de grão de milho reidratado (MR; 1 × 105 ufc/g), silagem de grão de milho reidratado com L. buchneri (MRB; 1 × 105 ufc/g) e milho seco moído (MSC). No experimento de metabolismo foram utilizados 10 animais fistulados, agrupados em dois quadrados latinos (5 × 5), enquanto no experimento de desempenho foram utilizados 180 animais Nelore (310±17 kg), sendo 6 animais por baia e 6 baias por tratamento. Os animais que consumiam silagens apresentaram maior (P < 0,05) digestibilidade da MS, MO e PB; maior concentração de propionato e ácidos graxos de cadeia curta totais; menor (P < 0,01) CMS e pH ruminal; e maior (P < 0,01) eficiência alimentar, ELm e ELg da dieta em relação aos animais consumindo MSC. O uso de L. buchneri aumentou a estabilidade aeróbia, e o tempo de estocagem aumentou a degradabilidade das silagens. Recomenda-se o uso de silagens de grãos de milho por propiciar melhor digestibilidade e eficiência alimentar. A silagem de grão de milho reidratado é uma alternativa à silagem de milho grão úmido. / Three trials were conducted with the objective to determine 1) the best dose and type of inoculant, homo or hetero fermentative, for reconstituted corn grain silages; 2) the effects of storage time and the use of inoculant on the fermentation profile, aerobic stability and in situ degradability of high moisture and reconstituted corn grain silages; 3) changes in ruminal fermentation, digestibility and performance of Nellore steers fed diets containing dry ground corn or high moisture or reconstituted corn grain silages without and with inoculant. In trial 1, treatments were: reconstituted corn grain silages without inoculant (C), L. plantarum + P. acidilactici (LPPA) and L. buchneri (LB), both at doses of 1 × 105, 5 × 105 or 1 × 106 cfu/g, stored for 124 days. Silages inoculated with LB compared to C had greater (P < 0.01) count or lactic acid bacterial (LAB), acetic acid, proprionic acid and 1,2-propanediol concentrations and lower (P < 0.01) pH, yeasts and molds for 12 days of exposure to air and higher (P < 0.01) aerobic stability, this occurs already at the first dose (1 x 105 cfu/g). The use of LB also increased (P < 0.05) proteolysis and in situ degradability of silages. The LPPA showed no improvements in fermentation and increased mold counts during the aerobic stability. The trial 2 was designed in a 2 × 2 factorial arrangement: two types of silage, high moisture corn (HMC) or reconstituted corn grain silages (RCS); and without (control) or with L. buchneri (LB; 1 × 105). Silos were opened after 15, 30, 60, 90, 120, 180, 240 and 300 days of storage. The RCS showed higher overall concentration of acids, while silages inoculated with LB had greater concentrations of acetic acid and 1,2-propanediol and lower content of lactic and butyric acids. Along the storage time, HMC LB and RCS LB showed lower yeast count and higher BAL count and aerobic stability. The RCS with original particle size showed higher in situ degradability than HMC. The minimum length of storage to reach high degradability of gains is between 30 and 70 days of storage. In trial 3 were evaluated five diets: high moisture corn (HMC), HMC with L. buchneri (HMCLB), reconstituted corn grain silages (RCS), RCS with L. buchneri (RCSLB) and dry ground corn (DGC). We used 10 cannulated bulls in a metabolism experiment, assigned in two latin squares (5 × 5), and 180 Nellore bullocks (310±17 kg) in the performance experiment, with 6 animals per pen and 6 pens per treatment. Animals fed silages had higher (P < 0.05) DM, OM and CP digestibility, greater concentrations of propionato and total short-chain fatty acids, and lower (P < 0.01) DMI, rumen pH and higher (P < 0.01) feed efficiency, NEm and NEg for the diet for the animals consuming DGC. The use of L. buchneri increases aerobic stability and storage time increases the degradability of silages. It is recommended the use of corn grain silages by providing better digestibility and utilization of diet. Reconstituted corn grain silages is an alternative to the high moisture corn silage. / FAPESP: 2013/16720-2

Resumo: O tumor venéreo transmissível canino (TVTC) é uma neoplasia transmitida entre cães saudáveis pelo contato direto de pele e/ou mucosas lesionadas. Face aos escassos estudos relacionados aos eventos celulares envolvidos nas fases de crescimento do TVTC, o presente estudo teve por objetivo identificar a presença do gene TP53 e o RNAm referente a proteína codificada, além de detectar a expressão das proteínas p53, Bcl-2 e p63 em cortes histológicos de 13 amostras de TVTC. Com relação à evolução da neoplasia, 46% das amostras foram consideradas em fase de progressão e 54% no estágio de regressão. Foram utilizadas as técnicas de hibridização in situ (ISH) e RT-PCR in situ, que demonstrou a presença do DNA homólogo ao TP53 e seu respectivo RNAm em 92,30% das amostras. A expressão das proteínas p53, p63 e Bcl-2 foram detectadas em 50%, 70% e 100% das amostras, respectivamente. A p63 foi expressa de forma evidente nas amostras em regressão, porém a p53 e a Bcl-2 não apresentaram relação com o estágio evolutivo do tumor e provavelmente não podem ser analisados como fatores de prognóstico do TVTC. Observou-se, nesse estudo que, através das técnicas de ISH e RT-PCR in situ foi possível detectar o DNA do TP53 e seus transcritos, porém esse fato não significou a transcrição da p53, devido aos baixos níveis de expressão nas análises quantitativa e qualitativa nas amostras de TVTC / Abstract: The canine transmissible venereal tumor (CTVT) is transmitted by direct contact of skin or mucosal presenting lesions. In fact, few reports have been found describing the cellular immune response related to the evolution of the tumor. The objective of this study was to identify the TP53 gen and its transcription in CTVT in different stages of evolution, collected from dogs (N=13) examined at veterinary school, UNESP, Aracatuba, SP, Brasil. In addition, it was also evaluated the expression of p53, p63 and Bcl-2 in histological sections by the use of immunohistochemystry assay. The p53, p63 and Bcl-2 were evident in 50, 70 and 100% of analyzed samples. Regarding to tumor evolution, 6 out of 13 were considered in a progressive stage (46%), was list 7 out of 13 were classified in a regressive stage (54%). The use of in situ hybridization and reverse transcriptase polymerase chain reaction in situ (RT-PCR), revealed that TP53 was present in all samples and P63 was more expressed than p53. Take all results together, the real role of those marker are extremely important to understand the biological behavior and to improve therapeutic procedures for CTVT / Orientador: Maria Cecília Rui Luvizotto / Coorientador: Tereza Cristina Cardoso / Banca: Alexandre Lima de Andrade / Banca: Paula Rahal / Mestre

Submitted by Caroline Periotto (carol@ufscar.br) on 2016-10-10T15:06:40Z No. of bitstreams: 1 DissGRF.pdf: 3437375 bytes, checksum: 6462dc22eaf7cf1e73ecf9875bbef4d9 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T19:50:19Z (GMT) No. of bitstreams: 1 DissGRF.pdf: 3437375 bytes, checksum: 6462dc22eaf7cf1e73ecf9875bbef4d9 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T19:50:26Z (GMT) No. of bitstreams: 1 DissGRF.pdf: 3437375 bytes, checksum: 6462dc22eaf7cf1e73ecf9875bbef4d9 (MD5) / Made available in DSpace on 2016-10-20T19:50:32Z (GMT). No. of bitstreams: 1 DissGRF.pdf: 3437375 bytes, checksum: 6462dc22eaf7cf1e73ecf9875bbef4d9 (MD5) Previous issue date: 2016-06-22 / Outra / The Lear's Macaw (Anodorhyncus leari) is an endemic parrot of northeastern Bahia state, classified as endangered mainly due to the destruction of their habitats and wildlife trade. It was recently discovered in the wild, and few studies are known for this species. Understanding behavioral patterns of species threatened by extinction is essential for developing effective conservation strategies. Here we describe the behavioral repertoire of a group of this species maintained by the São Paulo Zoological Park Foundation. We carried out observations between October 2014 and February 2015, totaling 348 hours of sampling effort through ad libitum continuous record, in two different environmental conditions, both in in pairs and in flock. We described 60 behavioral states, grouped in categories maintenance, locomotion, feeding, social, stereotyped, reproductive and alert. We also found five patterns of vocalization (alarm, contact, cohesion, reproduction and imitation). Most of the behaviors described for other species is also reported here to A. leari, more similar to A. hyacinthinus. We discuss the differences in order to contribute to the formulation of a behavioral profile for this species, and with information that may assist in maintaining normal behavior in captivity. / A arara-azul-de-lear (Anodorhyncus leari) é um psitacídeo endêmico da caatinga do nordeste do estado da Bahia, classificada como em perigo de extinção em decorrência principalmente da destruição do seu habitat e do tráfico de animais silvestres. Foi descoberta na natureza a relativamente pouco tempo, sendo escassos estudos sobre a espécie. A compreensão de padrões comportamentais de espécies ameaçadas é fundamental para a elaboração de estratégias conservacionistas eficientes. O presente trabalho teve como objetivo o levantamento do repertório comportamental de um grupo de indivíduos da espécie mantidos pela Fundação Parque Zoológico de São Paulo. As observações ocorreram entre outubro de 2014 e fevereiro de 2016, totalizando 348 horas de esforço amostral através de amostragem ad libitum de registro contínuo com caráter qualitativo em duas condições ambientais distintas, tanto na manutenção dos indivíduos em pares quanto em bando, resultando na descrição de 60 condutas comportamentais dentro das categorias manutenção, locomoção, alimentação, social, estereotipado, reprodutivo e alerta, além de cinco padrões de vocalização (alarme, contato, coesão, reprodução e imitação). A maioria dos comportamentos descritos para outras espécies é relatada para A. leari, com maior semelhança com A. hyacinthinus. Porém as diferenças são discutidas com o objetivo de contribuir com a formulação de um perfil comportamental para a espécie e com informações que possam auxiliar na manutenção de comportamentos naturais em cativeiro.

This study investigated In situ biodiesel production with supercritical methanol. A micro-algae based feedstock was used and obtained from a local water treatment plant situated just outside of Bethal, South Africa (S 26° 29’ 19.362” E 29° 27’ 11.552”). The wet feedstock was used as harvested with only the excess moisture being removed. Characterisation of the feedstock showed that a wide variety of macro-algae, micro-algae, cyanobacteria and bacterial species were present in the feedstock. The main algal species isolated from the feedstock were Nostoc sp. and Chlamydomonas. The feedstock was found to have a higher heating value (HHV) of 22 MJ.kg-1 and a lower heating value (LHV) of 16.03 MJ.kg-1 with an inherent moisture content of 270g.kg-1 feedstock. The protein and fat content of the feedstock was determined by the Agricultural Research Council (ARC) and found to be 370.1 g.kg-1 and 61.6 g.kg-1 on a moisture free basis respectively. The high protein and fat content gives a theoretical bio-yield of 430 wt%. The low lignin content and high cellulose and hemi-cellulose content indicated that the feedstock would be suitable for energy production. Three experimental sets were performed to determine the effect certain reaction parameters will have on the bio-char, bio-oil and biodiesel yields. The first set entailed hydrothermal liquefaction without the addition of methanol. The second set involved in situ biodiesel production with supercritical methanol, while both supercritical methanol and an acid catalyst were used during in situ biodiesel in the third set. For the first set of experiments the effect of temperature (240°C to 340°C in intervals of 20°C) on the crude bio-oil and bio-char yields were investigated. The highest bio-char yield was found to be 336g g char.kg-1 biomass at 280°C, while the highest crude bio-oil yield was 470.7 g crude bio-oil per kg biomass at 340°C. In the second set of experiments the dry biomass loading was kept constant at 500 g.kg-1 and the temperature varied (240°C to 300°C in intervals of 20°C) along with methanol to dry biomass ratio (1:1, 3:1 and 6:1). The optimum bio-oil yield of 597.1 g bio-oil per kg biomass for this set was found at 500 g.kg-1 biomass loading, 300°C and 3:1 methanol to dry biomass ratio. The highest bio-char yield was found to be 382.6 g bio-char.kg-1 biomass for a 1:1 methanol to dry biomass weight ratio set with 500 g.kg-1 biomass loading at 280°C. An increase in methanol ratio also led to an increase in crude bio-oil yields however the 3:1 methanol to dry biomass mass ratio was found to give the highest bio-oil yield and the purest biodiesel, with less unsaturated FAME. The 6:1 methanol to dry biomass mass ratio did however increase the FAME yield, which tends to show completion of the in situ production of biodiesel. This was also seen in the amount fatty acid methyl esters (FAME) present in the crude bio-oil as the degree of transesterification starts to increase with an increase in methanol. The FAME content was determined using gas chromatography (GC) and gas chromatography coupled to mass spectrometry (GC-MS). During the last set of experiments the temperature (260°C to 300°C in intervals of 20°C) and methanol to dry biomass ratio (1:1, 3:1 and 6:1) was varied at a constant catalyst loading of 1 wt% of the dry biomass. The optimum yields achieved were 627 g crude bio-oil per kg biomass and 376 g bio-char per kg biomass at 300°C and 280°C, respectively. These yields were achieved at 500 g.kg-1 biomass loading and 6:1 methanol ratio. Compared to the experiments where no catalyst was used, a slight increase in the yield was observed with the addition of an acid catalyst. This might be due to the base metals present in the feedstock that can lead to saponification during transesterification without the addition of an acid catalyst. An overall improvement in the extraction of crude bio-oil was observed with in situ production compared to hydrothermal liquefaction. During in situ liquefaction, the bio-oil yield increased by 150 g crude bio-oil per kg biomass higher, while the bio-char yields did not significantly vary at the optimum point of 280°C this finding has a significant value for green coal research. The highest HHV for the bio-char of 27 MJ.kg-1 +/- 0.17 MJ.kg-1 was found at 280°C and a 3:1 methanol ratio. The HHV of the bio-char decreases with an increase in temperature as more of the hydrocarbons are dissolved and form part of the bio-crude make-up. The highest HHV recorded for the crude bio-oil was 42 MJ.kg-1 at a 6:1 methanol ratio, a temperature of 300°C and an acid catalyst. The crude bio-oil HHV, which increased with an increase in temperature, is well within the specifications of the biodiesel standard (SANS, 1935). The highest FAME yield of 39.0 g.kg-1 was obtained using a 6:1 methanol ratio and a temperature of 300°C in the presence of an acid catalyst. The crude oil contained 49.0 g.kg-1 triglycerides with alkenes (C13, C15 and C17) making up the balance. The purest biodiesel yield was achieved at 3:1 methanol to dry biomass mass ratio, as it had the lowest yield unsaturated methyl esters. The overall FAME yield increased with an increase in methanol ratio. The derivatised FAME yields were the highest during hydrothermal liquefaction (55.0 g.kg-1 biomass). The in situ production of biodiesel from waste water clarifier effluent stream was found to be possible. Further investigation is needed into sufficient harvesting methods, including the optimum harvesting location, as this will result in fewer impurities in the stream and subsequent higher yields. / MIng (Chemical Engineering), North-West University, Potchefstroom Campus, 2015

This study investigated In situ biodiesel production with supercritical methanol. A micro-algae based feedstock was used and obtained from a local water treatment plant situated just outside of Bethal, South Africa (S 26° 29’ 19.362” E 29° 27’ 11.552”). The wet feedstock was used as harvested with only the excess moisture being removed. Characterisation of the feedstock showed that a wide variety of macro-algae, micro-algae, cyanobacteria and bacterial species were present in the feedstock. The main algal species isolated from the feedstock were Nostoc sp. and Chlamydomonas. The feedstock was found to have a higher heating value (HHV) of 22 MJ.kg-1 and a lower heating value (LHV) of 16.03 MJ.kg-1 with an inherent moisture content of 270g.kg-1 feedstock. The protein and fat content of the feedstock was determined by the Agricultural Research Council (ARC) and found to be 370.1 g.kg-1 and 61.6 g.kg-1 on a moisture free basis respectively. The high protein and fat content gives a theoretical bio-yield of 430 wt%. The low lignin content and high cellulose and hemi-cellulose content indicated that the feedstock would be suitable for energy production. Three experimental sets were performed to determine the effect certain reaction parameters will have on the bio-char, bio-oil and biodiesel yields. The first set entailed hydrothermal liquefaction without the addition of methanol. The second set involved in situ biodiesel production with supercritical methanol, while both supercritical methanol and an acid catalyst were used during in situ biodiesel in the third set. For the first set of experiments the effect of temperature (240°C to 340°C in intervals of 20°C) on the crude bio-oil and bio-char yields were investigated. The highest bio-char yield was found to be 336g g char.kg-1 biomass at 280°C, while the highest crude bio-oil yield was 470.7 g crude bio-oil per kg biomass at 340°C. In the second set of experiments the dry biomass loading was kept constant at 500 g.kg-1 and the temperature varied (240°C to 300°C in intervals of 20°C) along with methanol to dry biomass ratio (1:1, 3:1 and 6:1). The optimum bio-oil yield of 597.1 g bio-oil per kg biomass for this set was found at 500 g.kg-1 biomass loading, 300°C and 3:1 methanol to dry biomass ratio. The highest bio-char yield was found to be 382.6 g bio-char.kg-1 biomass for a 1:1 methanol to dry biomass weight ratio set with 500 g.kg-1 biomass loading at 280°C. An increase in methanol ratio also led to an increase in crude bio-oil yields however the 3:1 methanol to dry biomass mass ratio was found to give the highest bio-oil yield and the purest biodiesel, with less unsaturated FAME. The 6:1 methanol to dry biomass mass ratio did however increase the FAME yield, which tends to show completion of the in situ production of biodiesel. This was also seen in the amount fatty acid methyl esters (FAME) present in the crude bio-oil as the degree of transesterification starts to increase with an increase in methanol. The FAME content was determined using gas chromatography (GC) and gas chromatography coupled to mass spectrometry (GC-MS). During the last set of experiments the temperature (260°C to 300°C in intervals of 20°C) and methanol to dry biomass ratio (1:1, 3:1 and 6:1) was varied at a constant catalyst loading of 1 wt% of the dry biomass. The optimum yields achieved were 627 g crude bio-oil per kg biomass and 376 g bio-char per kg biomass at 300°C and 280°C, respectively. These yields were achieved at 500 g.kg-1 biomass loading and 6:1 methanol ratio. Compared to the experiments where no catalyst was used, a slight increase in the yield was observed with the addition of an acid catalyst. This might be due to the base metals present in the feedstock that can lead to saponification during transesterification without the addition of an acid catalyst. An overall improvement in the extraction of crude bio-oil was observed with in situ production compared to hydrothermal liquefaction. During in situ liquefaction, the bio-oil yield increased by 150 g crude bio-oil per kg biomass higher, while the bio-char yields did not significantly vary at the optimum point of 280°C this finding has a significant value for green coal research. The highest HHV for the bio-char of 27 MJ.kg-1 +/- 0.17 MJ.kg-1 was found at 280°C and a 3:1 methanol ratio. The HHV of the bio-char decreases with an increase in temperature as more of the hydrocarbons are dissolved and form part of the bio-crude make-up. The highest HHV recorded for the crude bio-oil was 42 MJ.kg-1 at a 6:1 methanol ratio, a temperature of 300°C and an acid catalyst. The crude bio-oil HHV, which increased with an increase in temperature, is well within the specifications of the biodiesel standard (SANS, 1935). The highest FAME yield of 39.0 g.kg-1 was obtained using a 6:1 methanol ratio and a temperature of 300°C in the presence of an acid catalyst. The crude oil contained 49.0 g.kg-1 triglycerides with alkenes (C13, C15 and C17) making up the balance. The purest biodiesel yield was achieved at 3:1 methanol to dry biomass mass ratio, as it had the lowest yield unsaturated methyl esters. The overall FAME yield increased with an increase in methanol ratio. The derivatised FAME yields were the highest during hydrothermal liquefaction (55.0 g.kg-1 biomass). The in situ production of biodiesel from waste water clarifier effluent stream was found to be possible. Further investigation is needed into sufficient harvesting methods, including the optimum harvesting location, as this will result in fewer impurities in the stream and subsequent higher yields. / MIng (Chemical Engineering), North-West University, Potchefstroom Campus, 2015

Cette thèse de doctorat s'inscrit dans le cadre de projets CRIAQ (Consortium de recherche et d'innovation en aérospatiale du Québec) orientés vers le développement d'approches embarquées pour la détection de défauts dans des structures aéronautiques. L'originalité de cette thèse repose sur le développement et la validation d'une nouvelle méthode de détection, quantification et localisation d'une entaille dans une structure de joint à recouvrement par la propagation d'ondes vibratoires. La première partie expose l'état des connaissances sur l'identification d'un défaut dans le contexte du Structural Health Monitoring (SHM), ainsi que la modélisation de joint à recouvrements. Le chapitre 3 développe le modèle de propagation d'onde d'un joint à recouvrement endommagé par une entaille pour une onde de flexion dans la plage des moyennes fréquences (10-50 kHz). À cette fin, un modèle de transmission de ligne (TLM) est réalisé pour représenter un joint unidimensionnel (1D). Ce modèle 1D est ensuite adapté à un joint bi-dimensionnel (2D) en faisant l'hypothèse d'un front d'onde plan incident et perpendiculaire au joint. Une méthode d'identification paramétrique est ensuite développée pour permettre à la fois la calibration du modèle du joint à recouvrement sain, la détection puis la caractérisation de l'entaille située sur le joint.Cette méthode est couplée à un algorithme qui permet une recherche exhaustive de tout l'espace paramétrique.Cette technique permet d'extraire une zone d'incertitude reliée aux paramètres du modèle optimal. Une étude de sensibilité est également réalisée sur l'identification. Plusieurs résultats de mesure sur des joints à recouvrements 1D et 2D sont réalisées permettant ainsi l'étude de la répétabilité des résultats et la variabilité de différents cas d'endommagement. Les résultats de cette étude démontrent d'abord que la méthode de détection proposée est très efficace et permet de suivre la progression d'endommagement. De très bons résultats de quantification et de localisation d'entailles ont été obtenus dans les divers joints testés (1D et 2D). Il est prévu que l'utilisation d'ondes de Lamb permettraient d'étendre la plage de validité de la méthode pour de plus petits dommages. Ces travaux visent d'abord la surveillance in-situ des structures de joint à recouvrements, mais d'autres types de défauts. (comme les disbond) et. de structures complexes sont également envisageables.

Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The cellular expression of the enzymes implicated in regulating sucrose metabolism and accumulation in sugarcane is poorly understood. The present study was therefore aimed at the development of an in situ hybridisation (ISH) technique to study differential gene expression among the various cell types of the sugarcane culm. This technique in conjunction with northern and western blotting was then used to determine the sites of cellular and tissue specific expression of the cytosolic enzymes, UDP-Glc dehydrogenase, pyrophosphate dependent phosphofructokinase and UDP-Glc pyrophosphorylase, involved in sucrose metabolism. This study revealed that the determination of the influencing parameters associated with the development of an ISH protocol was essential for the successful detection of the endogenous RNA sequences in sugarcane internodal tissues. The parameters that were investigated included the type of embedding medium, duration of fixation period, pre-treatment procedures and hybridisation temperature. It further revealed that fresh internodal tissue sections, fixed for a period of 24 h and thereafter exposed to pre-treatment and hybridisation, facilitated the analysis of cytological gene expression at all stages of sugarcane development. The second part of this study revealed very localised transcript expression for UDP-Glc DH, PFP and UGPase in the different internodal tissue and cell types. The UDP-Glc DH and UGPase transcripts were localised to the phloem elements, whilst xylem tissue only expressed the UDP-Glc DH transcript. Transcripts of UDP-Glc DH, PFP and UGPase were all expressed in the parenchyma cells that were associated with the vascular bundles and the stem storage compartment, suggesting that the parenchyma cells distributed throughout the stem in the different tissue types complement each other in function for the purposes of phloem loading, unloading and assimilate transport processes. Complimentary northern and western hybridisations demonstrated that internode 7 represents a shift in the sink from utilisation to storage. This is evident by the observed decline in both the relative transcript and protein abundances of UDP-Glc DH, PFP and UGPase at this stage of development. The relative mRNA and protein abundances for the three enzymes showed a similar trend. Higher levels of the gene transcripts and translated products were observed in the younger sucrose importing tissues, than in the older sucrose accumulating internodes. At a cellular level, it was found that the sites of cellular UDP-Glc DH, PFP and UGPase expression differed marginally. Whilst UDP-Glc DH was expressed in the phloem, xylem and parenchyma cells of the vascular complex and in storage parenchyma cells, PFP was expressed exclusively in parenchyma cells that were associated with the vascular bundles and those serving a storage function in the stem pith and UGPase was found to be localised in the phloem and parenchyma of the vascular bundles and the storage parenchyma cells. Such findings have demonstrated an increase in resolution with which gene expression can be examined at a cellular level. Hence, the results from this study have demonstrated that the knowledge of metabolic compartmentation between different tissue and cell types is a requisite to understanding the function(s) of individual enzymes within complex structures such as the sugarcane culm. / AFRIKAANSE OPSOMMING: Die sellulêre lokalisering van die ensieme wat geïmpliseer word in die regulering van sukrose metabolisme is onbekend. Met dit in gedagte, was hierdie studie gefokus op die ontwikkeling van 'n in situ hibridisasie (ISH) tegniek om differensiële geenuitdrukking in die verskillende seltipes van die suikerrietstingel te ondersoek. Hierdie tegniek, tesame met RNA-en proteïen gel blots, is volgens aangewend om die areas van sellulêre-en weefselspesifieke uitdrukking van die sitosoliese ensieme UDP-glukose dehydrogenase, pirofosfaat-afhanklike fosfofruktokinase en UDP-glukose pirofosforilase, wat almal betrokke is by sukrosemetabolisme, te bepaal. Dit het duidelik geword gedurende die studie dat die bepaling van die optimale parameters van die ISH protokol vir suikerriet van deurslaggewende belang sou wees vir die opsporing van endogene RNA volgordes. Die parameters wat ondersoek is het ingesluit die tipe inbeddingsmedium, die tydsduur van fiksering, vooratbehandelings- en hibridisasiemetodes. Dit het duidelik geword dat vars internodale weefselsnitte wat vir 24 h gefikseer is en daarna voorafbehandeling en hibridisasie ondergaan het, die bepaling van geenuitdrukking tydens alle fases van suikkerrietontwikkeling moontlik gemaak het. Die tweede fase van hierdie studie het aangetoon dat al drie ensieme spesifiek gelokaliseerde uitdrukkingspatrone gehad het in verskillende internodale weefsels en seltipes. Al drie gene is konstitutief uitgedruk in internodes. Die UDP-glukose dehydrogenase en UDP-glukose pirofosforilase transkripte is gelokaliseer na die floeëm elemente, terwyl xileem slegs die UDP-glukose dehydrogenase transkripte bevat het. Al die gene is in die parenchiemselle uitgedruk wat geassosieer is met die vaatbondels en die stingel stoorkompartement, wat moontlik beteken dat die parenchiem selle wat deur die stingel versprei is 'n sentrale netwerk vorm wat direk of indirek koolstofassimileringsprosesse beïnvloed. RNA-en proteïen gel blots op dieselfde internodes het gewys dat internode sewe 'n verskuiwing, van koolstofverbruik na berging, verteenwoordig. Dit word gerllustreer deur die afname in beide transkrip en proteïen vlakke van die drie ensiem in hierdie stadium van ontwikkeling. Alhoewel beide mRNA en proteïen vlakke vir al die ensieme 'n soortgelyke tendens getoon het, het die sellulêre uitdrukking van die ensieme volgens ISH verskil, wat die krag van die tegniek illustreer. Die resultate van hierdie studie het gedemonstreer dat begrip van die kompartementalisasie van metabolisme tussen verskillende weefsel-en seltipes 'n voorvereiste is om die funksie/s van individuele ensieme in komplekse strukture soos die suikerrietstingel te bepaal.

published_or_final_version / Pathology / Master / Master of Medical Sciences

Multicolor fluorescence in-situ hybridization (M-FISH) techniques provide color karyotyping that allows simultaneous analysis of numerical and structural abnormalities of whole human chromosomes. Chromosomes are stained combinatorially in M-FISH. By analyzing the intensity combinations of each pixel, all chromosome pixels in an image are classified. Often, the intensity distributions between different images are found to be considerably different and the difference becomes the source of misclassifications of the pixels. Improved pixel classification accuracy is the most important task to ensure the success of the M-FISH technique. Along with a reliable pixel classification method, automation of the karyotyping process is another important goal. The automation requires segmentation of chromosomes, which not only involves object/background separation but also involves separating touching and overlapping chromosomes. While automating the segmentation of partially occluded chromosomes is an extremely challenging problem, a pixel classification method that satisfies both high accuracy and minimum human intervention has not been realized. The main contributions of this dissertation include development of a new feature normalization method for M-FISH images that reduces the difference in the feature distributions among different images, and development of a new decomposition method for clusters of overlapping and touching chromosomes. A significant improvement was achieved in pixel classification accuracy after the new feature normalization. The overall pixel classification accuracy improved by 40% after normalization. Given a cluster, a number of hypotheses was formed utilizing the geometry of a cluster, pixel classification results, and chromosome sizes, and a hypotheis that maximized the likelihood function was chosen as the correct decomposition. Superior decomposition results were obtained using the new method compared to the previous methods. Contributions also include development of a color compensation method for combinatorially stained FISH images (including M-FISH images) based on a new signal model for multicolor/multichannel FISH images. The true signal was recovered based on the signal model after color compensation. The resulting true signal does not have color spreading (channel crosstalk) among different color channels. Two new unsupervised nonparametric classification methods for M-FISH images are also introduced in this dissertation: a fuzzy logic classifier and a template matching method (a minimum distance classifier). While both methods produce an equivalent accuracy compared to a supervised classification method, their computation time is significantly less than a Bayes classifier. Highly sophisticated and practical algorithms have been developed through this research. Using the developed methods, the amount of human intervention required will be significantly reduced: chromosomes are reliably and accurately segmented from the background, pixels are accurately classified, and clusters of overlapping and touching chromosomes are automatically decomposed. / text

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