Orientador: Luís Guilherme de Oliveira / Banca: Simone Maria Massami Kitamura Martins / Banca: Lizandra Amoroso / Resumo: A infecção persistente ao vírus da diarreia viral bovina (BVDV) pode viabilizar a disseminação do vírus no rebanho, assim como interferir no controle da infecção. Ao mesmo tempo, em suínos, a presença de soropositivos para BVDV pode causar transtornos aos inquéritos sorológicos para a peste suína clássica (PSC). Este trabalho teve como objetivo determinar a condição de persistentemente infectado em leitões nascidos de porcas infectadas experimentalmente pelo vírus da diarreia viral bovina. Foram selecionadas seis porcas prenhes para este estudo que foram divididas em dois grupos, sendo um grupo inoculado com BVDV-2 (G1; n=4) aos 45 dias de gestação, e um grupo controle (G2; n=2). Foram realizadas avaliações clínicas nas porcas diariamente. Os neonatos foram monitorados durante 35 dias, em que foram realizadas avaliações clínicas rotineiras e colheita de suabes nasal dos leitões e de amostras de sangue venoso das porcas e dos leitões para obtenção de sangue total e soro a cada 72 horas. Foram realizados testes de RT-PCR para diagnóstico direto, e virusneutralização para avaliação sorológica. As porcas apresentaram soroconversão entre o 17ºdia pós-infecção (dpi) e o 22ºdpi, mas não foi detectada viremia. Nenhum leitão apresentou títulos de anticorpos ou viremia ao nascimento. Não ocorreu a transmissão transplacentária do vírus, portanto, não foi possível observar animais PI. / Abstract: The persistently infection to bovine viral diarrhea virus (BVDV) can enable the spread of virus in the herd, as well as interfere in the control of infection. Concurrently, the presence of seropositive pigs may interfere with serological surveys for classical swine fever (CSF). This project aimed to determine the condition of persistently infected in piglets born from gilts infected with bovine viral diarrhea. BVDV-2 was inoculated in four pregnant gilts (G1; n=4), and a placebo was administered in two gilts, which were the control group (G2; n=2). Clinical evaluations were daily performed in the gilts. The newborns were monitored during 35 days, with clinical evaluation and whole blood, serum and nasal swabs sampling every 72 hours. RT-PCR and virus neutralization tests (VN) were performed. The gilts presented seroconversion between 17º dpi and 22ºdpi, but no viremia was detected. No piglets presented antibody titers or viremia at birth. Transplacental transmission of the virus did not occur, therefore, PI animals could not be observed. / Mestre

Microfabricated thermal cyclers for nucleic acid amplification by using polymerase chain reaction (PCR) have been demonstrated by several groups over the last decade, with improved cycling speed and smaller volumes when compared to conventional bench-top cyclers. However, high fabrication costs coupled with difficulties in temperature sensing and control remain impediments to commercialization. In this study we have used a silicon-glass device that takes advantage of the high thermal conductivity of silicon but at the same time utilizes minimum number of fabrication steps to make it suitable for disposable applications. The thermal cycler is based on noncontact induction heating developed in this group. The microchip reaction kinetics is studied for the first time in-situ during PCR, using a real-time fluorescence block that is capable of data acquisition every 0.7 s from the microchip. The fluorescence information from SYBR green I dye is used to optimize microchip amplification reactions and confirm the product by melting curve analysis. We have also developed a novel non-contact temperature sensing technique using SYBR green fluorescence that can be used for miniaturized PCR devices. The thesis is organized into the following chapters. In chapter 1 we introduce the basic biology ideas that are required to understand DNA amplification. DNA based analysis requires amplification of low initial concentrations to above detectable limits using a technique known as polymerase chain reaction (PCR). In this process, the sample is cycled through three thermal steps for 3040 times to produce multiple copies of DNA. In microchip PCR, conventional polypropylene tubes using 2050 µL volume are replaced by miniaturized devices using ~1 µL sample volumes. The device response improves in terms of ramp rate and total analysis time due to the small volume and smart design of the materials. In this chapter we summarize some of the issues important for miniaturized PCR devices and compare them with commercial tube PCR systems. In chapter 2 we describe the induction heating technique that was developed by our group for miniaturized devices. Induction heating is a noncontact heating technique unlike resistive heating which has been commonly used for microchip PCR. Though resistive heating is very efficient in terms of heat transfer efficiency, it is not suitable for disposable devices and requires multi-step microfabrication. Other non-contact heating techniques such as hot air and IR heating require larger size arrangements that are not suitable for miniaturized devices. The heating was verified by using a thermocouple soldered at the back of the secondary plate that was also used for feedback to the comparator circuit for control. The simple on-off circuit was able to control within ±0.1 ◦C with heating and cooling ramp rates of 25 ◦C/s and 2.5 ◦C/s respectively. In this chapter, we also describe the design and fabrication of the silicon-glass microchip fabricated in our lab. We have used silicon-glass hybrid device for PCR in which glass with a 2 mm drilled hole is anodically bonded to an oxidized silicon surface. The hole formed the static reservoir for 3 µL volume of amplification solution. During PCR, the solution needs to be cycled to high temperature of ~95 ◦C. Hence it was necessary to seal the tiny droplet of liquid against evaporation at this temperature. The devices after being filled by sample were covered by 4 µL of mineral oil to serve as an evaporation barrier. It was easy to recover the whole sample after amplification for further testing. Chapter 3 describes the development of a fluorescent block for SYBR green I dye (SG) used for real-time monitoring of the amplification. The block contains a blue LED for excitation, a dichroic beamsplitter, and silicon photodiode along with filters and focusing optics. Signal levels being weak, we incorporated lock-in detection technique. A TTL at 190 Hz was used to pulse the excitation source and detect the emission at the same frequency using a commercial lock-in amplifier. The block was first characterized using a commercial thermal cycler and polypropylene tubes with different dilution of initial template copy number, and the results crosschecked with agarose gel electrophoresis. Performing continuous monitoring every 0.7s within cycles, we discovered interesting features during extension which have not been studied previously. During the constant temperature extension step, the fluorescence shows a rise and then saturates until the temperature is cycled to the next set point. We have confirmed the same behavior in single cycle extension control experiments and established its connection with polymerase extension activity. We were thus able to extract the activity rate for two different kinds of polymerase in-situ during PCR. By monitoring PCR reactions with different fixed extension times, we were able to determine the optimum conditions for tube PCR. Chapter 4 implements the ideas of fluorescence monitoring from tube that was explained in the previous chapter for the silicon-glass microchip. Since the microchip uses parameters such as sample volume, ramp rates, stay time etc. which are different from tube PCR, we performed several initial test experiments to establish key capabilities such as low volume detection, 3 µL amplification, surface passivation of silicon-glass etc. The same fluorescence block was used to obtain DNA melting point information by continuously monitoring ds-DNA with SG while the temperature is ramped slowly (melting curve analysis). Depending on ds-DNA present, the fluorescence gives a melting temperature (TM ), which was used to calibrate the mix temperature with respect to the thermocouple sensor. After successfully calibrating the microchip, we confirmed complete chip PCR in silicon-glass devices using induction heater. The continuous monitoring of chip PCR gave similar curves as obtained previously for tubes except that the signal level was lower in silicon devices. Extension fluorescence information was used to find an optimum temperature for microchip that shows a maximum activity rate. Similarly the reaction time was optimized in-situ during PCR by using continuous fluorescence data in a feedback experiment. The commercial lock-in amplifier was also replaced by a homemade circuit to successfully pickup fluorescence signal from the microchip during melting curve analysis. In chapter 5, we describe a novel technique to sense the temperature from the microchip without touching the sample volume. Usually the temperature is monitored by a sensor spatially separated from the mix and it has always been challenging to measure the exact temperature accurately. Most of the sensors are not biocompatible and too large in size to be placed inside the small volume of liquid. We have developed a protocol that involves SG fluorescence with addition of excess sensor DNA to the amplification solution. The sensor DNA added into the mix is non specific to the primer used for amplification of the template. It therefore does not participate in the amplification and its number remains unchanged throughout the 3040 cycles of PCR. If the amount of sensor DNA is titrated accurately, it will saturate the fluorescence envelope which then shows very reproducible thermal response with cycling. We have used this thermal response of the fluorescence for feedback as a temperature sensor. The fluorescence feedback was shown to produce identical amount of product in comparison to thermocouple feedback. The product can also be verified by melting curve analysis if the sensor DNA is chosen carefully depending on the product. In this chapter we also discuss some preliminary experiments with smart devices that will use dye based temperature sensor and control along with fluorescence based amplification monitoring. Chapter 6 summarizes the thesis and discusses some of the future areas which can be explored in the field of microchip PCR devices.

The present study investigated the expression profiles of nine genes involved in immune defense of the Pacific white shrimp Litopenaeus vannamei and their responses to oral administration of beta-1,3-glucan. The nine immune related genes were beta-glucan binding protein-high density lipoprotein (BGBP-HDL), lipopolysaccharide/beta-glucan binding protein (LGBP), hemocyanin, prophenoloxidase (proPO), transglutaminase (TGase), penaeidin-3 (PEN-3), crustin, cytosolic manganese superoxide dismutase (cMnSOD), and lysozyme. A series of experiments were carried out in the study including: (1) cDNA cloning and characterization of proPO and LGBP; (2) tissue mRNA expressions of the nine genes in adult shrimp; (3) expression and localization of the nine genes during larval and postlarval ontogenic development; (4) the effects of dietary beta-1,3-glucan on the expression of the nine genes. The cDNA cloning study showed that the proPO cDNA contains an open reading frame of 2061 bp and encodes a 686 amino-acid peptide. The protein sequence of the proPO has a similarity of 85% with those of Penaeus monodon and P. semisulcatus and has an identity of between 58 and 77% with other crustaceans. Northern blot analysis revealed that proPO was constitutively expressed mainly in hemocytes. Its transcripts were observed in hemocytes and many other tissues when detected with RT-PCR. The results of in situ hybridizations showed that the hemocytes that infiltrated in tissues were responsible for the positive signals. The LGBP cDNA contains an open reading frame of 1104 bp and encodes a 367 amino-acid protein with a 17 a. a. signal peptide. The protein sequence of the LGBP has a similarity of 97% with LGBP of L. stylirostris, >90% identity with BGBP of P. monodon and LGBP of Fenneropenaeus chinensis and has an identity of between 63 and 86% with other crustaceans. Northern blot analysis revealed that LGBP was constitutively expressed mainly in hepatopancreas. The results of in situ hybridizations showed that the hepatopancreatic F cells might be the major cell type for LGBP production. Using the complete cDNAs of proPO and LGBP and partial fragments of the other seven genes, their tissue expressions were analyzed by conventional RT-PCR, quantitative real time PCR (qPCR) and in situ hybridization. The results demonstrated that BGBP-HDL, LGBP and hemocyanin were mainly expressed in the hepatopancreas and their expressions levels were about 10 to 30% those of

No description available.

Submitted by Beatriz Batista Trigo (beatrizbtrigo@hotmail.com) on 2018-08-21T18:41:25Z No. of bitstreams: 1 Dissertacao Beatriz Batista Trigo.pdf: 1261932 bytes, checksum: 24b3472ef232d399d51c17b258886009 (MD5) / Approved for entry into archive by Ederson Vasconcelos Pereira null (edersonpereira@fmva.unesp.br) on 2018-08-21T19:12:51Z (GMT) No. of bitstreams: 1 trigo_bb_me_araca_int.pdf: 1261932 bytes, checksum: 24b3472ef232d399d51c17b258886009 (MD5) / Made available in DSpace on 2018-08-21T19:12:51Z (GMT). No. of bitstreams: 1 trigo_bb_me_araca_int.pdf: 1261932 bytes, checksum: 24b3472ef232d399d51c17b258886009 (MD5) Previous issue date: 2018-08-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As diferentes espécies de Leishmania causam amplo espectro de manifestações clínicas sendo genericamente classificadas em leishmaniose cutanêa (LC), mucocutânea (LMC) e leishmaniose visceral (LV), acometendo tanto crianças como adultos, e várias espécies de mamíferos. Para a identificação da espécie faz-se necessário o isolamento do protozoário a partir de amostras biológicas e o diagnóstico em laboratórios de referência. Atualmente, o “padrão ouro” para identificação de cepas de Leishmania spp. em estudos epidemiológicos é o multilocus enzyme electrophoresis (MLEE), a qual é uma técnica laboriosa e de aplicação restrita à amostras em cultivo. As técnicas baseadas na Reação em Cadeia da Polimerase (PCR) têm sido bastante utilizadas para caracterizar e diferenciar as espécies de Leishmania. Tal diferenciação pode ser importante para a determinação do tratamento correto aos pacientes, diminuindo a letalidade, e para estudos epidemiológicos com o mapeamento do padrão de distribuição das leishmanioses, auxiliando assim na priorização das medidas de controle desta zoonose. A presente pesquisa teve como objetivo avaliar in silico os oligonucleotídeos iniciadores já utilizados no diagnóstico molecular e validar, por PCR em Tempo Real, sua especificidade em cepas de referência cultivadas in vitro. / Different species of Leishmania cause a wide spectrum of clinical manifestations which are generically classified as cutaneous leishmaniasis (CL), mucocutaneous (MCL) and visceral leishmaniasis (VL), affecting children and adults, as well as several species of mammals. Species identification requires isolating the protozoan from biological samples and the use of diagnostic techniques done in reference laboratories. Currently, the "gold standard" for identifying strains of Leishmania spp. in epidemiological studies is the multilocus enzyme electrophoresis (MLEE), which is a laborious technique and restricted used in cultured samples. Techniques based on the Polymerase Chain Reaction (PCR) are being more frequently used in order to characterize and differentiate Leishmania species. Such differentiation is important to indicate the adequate treatment for the patients minimizing lethality and for epidemiological purposes as mapping its distribution pattern, in order to help prioritizing the control measures for this zoonosis. The present research aimed to evaluate the currently used primers for molecular diagnosis and validate them by Real Time PCR for their specificity in reference strains cultivated in vitro.

Orientador: Cáris Maroni Nunes / coorientadora: Silvana de Cássia Paulan / Banca: Valérioa Marçal Felix de Lima / Banca:Adam Taiti Harth Utsunomiya / Resumo: As diferentes espécies de Leishmania causam amplo espectro de manifestações clínicas sendo genericamente classificadas em leishmaniose cutanêa (LC), mucocutânea (LMC) e leishmaniose visceral (LV), acometendo tanto crianças como adultos, e várias espécies de mamíferos. Para a identificação da espécie faz-se necessário o isolamento do protozoário a partir de amostras biológicas e o diagnóstico em laboratórios de referência. Atualmente, o "padrão ouro" para identificação de cepas de Leishmania spp. em estudos epidemiológicos é o multilocus enzyme electrophoresis (MLEE), a qual é uma técnica laboriosa e de aplicação restrita à amostras em cultivo. As técnicas baseadas na Reação em Cadeia da Polimerase (PCR) têm sido bastante utilizadas para caracterizar e diferenciar as espécies de Leishmania. Tal diferenciação pode ser importante para a determinação do tratamento correto aos pacientes, diminuindo a letalidade, e para estudos epidemiológicos com o mapeamento do padrão de distribuição das leishmanioses, auxiliando assim na priorização das medidas de controle desta zoonose. A presente pesquisa teve como objetivo avaliar in silico os oligonucleotídeos iniciadores já utilizados no diagnóstico molecular e validar, por PCR em Tempo Real, sua especificidade em cepas de referência cultivadas in vitro. / Mestre

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Early childhood caries (ECC) is considered a serious public health issue among children all over the world. However, many aspects should be explored about the oral microbiota related to the ECC progression and how the bacterial community modifies according to the dentine lesion activity. This thesis, composed by two chapters, aimed to investigate and quantify with quantitative polymerase chain reaction (qPCR) the following bacteria Actinomyces naeslundii, Bifidobacterium spp., Lactobacillus acidophilus, Streptococcus gordonii, Streptococcus mutans, as well as members of the groups Lactobacillus casei and Mitis on biofilm from pre-school children with different stages of early childhood caries progression (Chapter 1) and on active and inactive dentine carious lesions (Chapter 2), and also to verify the association of these microorganisms on the process of health or disease. The sample consisted on preschool children aged between 2 and 5 years from nurseries and public preeschools in Fortaleza-CE. The children were examined for caries diagnosis with ICDAS II index (International Caries Detection Assessment System), and the Nyvad criteria, in order to evaluate prevalence and caries activity. The supragingival biofilm collection was taken from 75 children, who were divided in three groups according to the ICDAS II: CF (caries free) (n=20), ECL (presence of enamel caries lesion) (n=17) and DCL (presence of dentine caries lesion) (n=38). Samples of carious dentine were collected under rubber dam isolation of 56 lesions of dentine affected by caries, being 17 inactive and 39 active. The DNA of all the collected samples was extracted and purified, then tested for the presence of the formerly mentioned bacterial species/groups through qPCR. The quantity of bacteria was compared through the Kruskal-Wallis and Mann-Whitney tests. Besides, the association between the presence of bacteria and ECC was analyzed through the Chi-square test, with a 5% significance level and the multiple logistic regression was applied. Bacteria from the group L. casei and L. acidophilus presented low detection on biofilm of all evaluated groups. The presence of S. mutans and Bifidobacterium spp. showed a strong association with dental caries progression on the biofilm from children with dentine lesions with odds ratio of 21,5 and 5,9; respectively. On active dentine lesions, concentrations of Bifidobacterium spp. and species from the Lactobacillus casei group were significantly higher when compared to the inactive lesions (p<0.05). The levels of Actinomyces naeslundii, Streptococcus gordonni and species from Mitis group were not significantly different among biofilm groups as well as comparing dentine lesions. In conclusion, the microbial profile from biofilm samples presented differences on the proportion of acidogenic and aciduric bacteria with dental caries progression. The presence of Bifidobacterium spp. and S. mutans presented a strong association with the development of the more advanced stages of ECC. Regarding the activity of dentine lesions, higher detection levels of the group L. casei and Bifidobacterium spp. showed an important role of these bacteria in the dentine caries activity. / A cÃrie precoce da infÃncia (CPI) Ã considerada um grave problema de saÃde pÃblica em crianÃas prÃ-escolares em todo mundo. No entanto, muitos aspectos ainda devem ser explorados acerca da microbiota oral relacionada com a progressÃo da CPI e como a comunidade bacteriana se modifica de acordo com a atividade da lesÃo dentinÃria. Esta tese, constituÃda de dois capÃtulos, teve como objetivo identificar e quantificar atravÃs da tÃcnica de reaÃÃo em cadeia da polimerase quantitativa (qPCR) as bactÃrias Actinomyces naeslundii, Bifidobacterium spp., Lactobacillus acidophilus, Streptococcus gordonii, Streptococcus mutans, bem como espÃcies dos grupos Lactobacillus casei e Mitis em biofilme de crianÃas prÃ-escolares com diferentes estÃgios de progressÃo da cÃrie precoce da infÃncia (CapÃtulo 1) e em lesÃes cariosas dentinÃrias ativas e inativas (CapÃtulo 2) e ainda verificar a associaÃÃo destes microrganismos ao processo de saÃde ou de doenÃa. A amostra consistiu em prÃ-escolares com idade entre 2 e 5 anos de idade que frequentavam creches e escolas pÃblicas de Fortaleza-CE. As crianÃas foram examinadas com o uso de Ãndices visuais ICDAS II (International Caries Detection Assessment System) e Nyvad, a fim de avaliar a prevalÃncia e atividade de cÃrie. A coleta de biofilme supragengival foi realizada em 75 crianÃas, as quais foram agrupadas de acordo com Ãndice ICDAS II em trÃs grupos: CF (livres de cÃrie) (n=20), ECL (presenÃa de lesÃes de cÃrie em esmalte) (n=17) e DCL (presenÃa de lesÃes de cÃrie em dentina) (n=38). Amostras de dentina cariada foram coletadas sob isolamento absoluto de 56 lesÃes cariosas dentinÃrias, sendo 17 inativas e 39 ativas. O DNA de todas as amostras coletadas foi extraÃdo e purificado e, em seguida, testado para a presenÃa das espÃcies/grupos bacterianos acima citados atravÃs de qPCR. A quantidade das bactÃrias foi comparada pelos testes Kruskal-Wallis e Mann-Whitney. AlÃm disso, a associaÃÃo da presenÃa de bactÃrias e CPI foi analisada atravÃs do teste Qui-quadrado, com nÃvel de significÃncia de 5% e aplicado a regressÃo logÃstica mÃltipla. BactÃrias L. acidophilus e do grupo L. casei apresentaram baixa detecÃÃo no biofilme de todos os grupos avaliados. A presenÃa de S. mutans e Bifidobacterium spp. mostrou forte associaÃÃo com a progressÃo da doenÃa no biofilme de crianÃas com lesÃes dentinÃrias com âodds ratioâ de 21,5 e 5,9, respectivamente. Em lesÃes dentinÃrias ativas, concentraÃÃes de Bifidobacterium spp. e bactÃrias do grupo L. casei foram significativamente maiores quando comparadas Ãs lesÃes inativas (p<0.05). Os nÃveis de A. naeslundii, bactÃrias do grupo Mitis e S. gordonni nÃo apresentaram diferenÃa significativa entre os grupos de biofilme, assim como nas lesÃes dentinÃrias. Conclui-se que as amostras de biofilme apresentaram alteraÃÃo na proporÃÃo de bactÃrias acidogÃnicas e acidÃricas com a progressÃo da doenÃa cÃrie. A presenÃa de Bifidobacterium spp. e S. mutans apresentou forte associaÃÃo com os estÃgios mais avanÃados da CPI. Em relaÃÃo Ãs lesÃes dentinÃrias, o aumento da concentraÃÃo de bactÃrias Bifidobacterium spp. e do grupo L. casei evidenciou um papel importante destas bactÃrias na atividade de lesÃes dentinÃrias.

Os objetivos deste trabalho foram avaliar a sobrevivência de cepas probióticas incorporadas em queijo petit-suisse potencialmente probiótico e simbiótico no produto armazenado por até 28 dias e em ensaios de sobrevivência in vitro frente às condições encontradas no trato gastrintestinal, utilizando-se métodos independentes de cultivo, paralelamente aos métodos convencionais de semeadura em ágar seletivo, bem como avaliar características tecnológicas dos queijos elaborados. O delineamento experimental constituiu-se de 3 tratamentos de queijo petit-suisse: QP = queijo probiótico (com cultura ABT-4, composta por Lactobacillus acidophilus LA-5 e Bifidobacterium animalis subsp. lactis BB-12 e da cultura starter Streptococcus thermophilus); QS = queijo simbiótico, contendo os probióticos e prebióticos (cultura ABT-4 + inulina e fruto-oligossacarídeo) e QC = queijo controle, elaborado apenas com uma cultura starter de Streptococcus thermophilus. Os queijos foram armazenados a 4 ºC e as análises foram realizadas semanalmente, por 28 dias. A contagem dos micro-organismos probióticos foi realizada por técnicas independentes (qPCR) e dependentes de cultivo. Adicionalmente, foi monitorada a presença de contaminantes e avaliadas a aceitabilidade sensorial e a textura instrumental dos produtos ao longo de seu armazenamento. Os queijos petit-suisse apresentaram populações de L. acidophilus LA-5 e B. animalis BB-12 superiores a 7 log UFC/g, até o 28º dia de armazenamento. Em QS, observou-se maior estabilidade da população de L. acidophilus LA-5, ao longo do armazenamento e maior sobrevivência in vitro de B. animalis BB-12, até 14 dias de armazenamento (p < 0,05), com taxas médias de sobrevivência diminuindo de 88,0% (dia 1) para 59,6% (dia 28) em QS e de 80,0% (dia 1) para 59,8 (dia 28) em QP. Em contrapartida, para L. acidophilus LA-5, as taxas médias de sobrevivência diminuíram de 49,1% (dia 1) para 36,9% (dia 28) em QS e de 61,6% (dia 1) para 39,2% (dia 28) em QP, e foram maiores em QP nos dias 1 e 14 (p < 0,05). A adição de probióticos no queijo petit-suisse resultou em influência significativa nos parâmetros de textura, com uma diminuição da dureza, o que provavelmente contribuiu para maiores escores de aceitabilidade, nas formulações com probióticos (> 5,9), em relação ao produto controle (< 5,3). Adicionalmente, houve influência positiva da mistura prebiótica na aceitabilidade de QS (escore = 6,8), aos 21 dias de armazenamento (p < 0,05). Entre os métodos utilizados para a quantificação de micro-organismos, os valores obtidos através dos métodos convencional e qPCR foram semelhantes, porém observou-se uma tendência a superestimar a população dos micro-organismos, no método de qPCR. No ensaio de sobrevivência in vitro, observou-se valores semelhantes e um coeficiente de correlação de Pearson de 0,92 entre os método de qPCR com Propidium Monoazide (PMA) e convencional, para B. animalis subsp. lactis BB-12, enquanto que para L.acidophilus LA-5, os resultados mostraram-se promissores, particularmente na quantificação de células viáveis, porém não cultiváveis. Os resultados obtidos sugerem que a formulação QS foi a mais favorável em termos de estabilidade e sobrevivência dos probióticos, além de apresentar a melhor aceitabilidade aos 21 dias de armazenamento. Em relação aos métodos de quantificação, técnicas moleculares mostraram-se mais adequadas, particularmente com a utilização de PMA, embora requeiram otimizações. / This study aimed to evaluate the survival of probiotic strains incorporated into petit-suisse cheese in the product stored for up to 28 days and under in vitro gastrointestinal tract simulated resistance assay, using culture-independent and culture-dependent methods. Furthermore, sensory acceptability and instrumental texture of the cheeses studied were evaluated. Experimental design involved the evaluation of three trials of petit-suisse cheese, as follows: PC = probiotic cheese (with the ABT-4 culture, containing Lactobacillus acidophilus LA-5, Bifidobacterium animalis subsp. lactis BB-12, and Streptococcus thermophilus, as starter culture), SC = synbiotic cheese, containing both the probiotics and the prebiotics (with the ABT-4 culture + inulin and fructooligosaccharides), and CC = control cheese (containing only the starter culture of Streptococcus thermophilus). Cheeses were stored at 4°C and the microbial counts in the products, as well as the in vitro survival assays were conducted weekly up to the 28th day. The probiotic counts in the product and in the in vitro assays were conducted through culture-independent (qPCR) and dependent methods. Additionally, the presence of contaminants was monitored and the sensory acceptability and instrumental texture of the products were evaluated during storage at 4°C. The petit-suisse cheeses presented L. acidophilus LA-5 and B. animalis BB-12 populations above 7 log CFU/g, up to the 28th day of storage. For SC, a higher L. acidophilus LA 5 population stability during the shelf-life and a higher B. animalis BB-12 in vitro survival up to 14 days of storage (p < 0.05) were observed. BB-12 presented mean survival rates decreasing from 88.0% (day 1) to 59.6% (day 28) in SC and from 80.0% (day 1) to 59.8% (day 28) in PC. In contrast, for L. acidophilus LA-5, the mean survival rates decreased from 49.1% (day 1) to 36.8% (day 28) in SC and from 61.6% (day 1) to 39.2% (day 28) in PC, which presented higher survival rate on days 1 and 14 (p < 0.05). The addition of probiotics in petit-suisse cheese influenced texture parameters significantly, leading to decreased hardness (p < 0.05), which probably contributed for the higher acceptability scores of both cheeses supplemented with probiotics (> 5.9), compared to the control product (< 5.3). Additionally, there was a positive influence of the prebiotic mixture on the SC acceptability (score = 6.8) on day 21 of storage (p < 0.05). Regarding the methods used for the enumeration of microorganisms, the results for the qPCR and the conventional method were quite similar. However, a trend towards overestimating microbial populations with the qPCR method was observed. For the in vitro survival assays, we observed similar results for the qPCR method with Propidium Monoazide (PMA) and the conventional method for B. animalis subsp. lactis BB-12 and a Pearson correlation coefficient of 0.92, whereas for L. acidophilus LA-5, the results were promising, especially in the quantification of viable, but not cultivable cells. The results suggest that the synbiotic cheese was the most favorable in terms of stability and probiotics survival, besides presenting the highest acceptance at 21 days of storage. Regarding quantification methods, molecular techniques have proved to be more suitable, particularly with PMA, but an optimization is required.

Magister Scientiae (Biodiversity and Conservation Biology) / Land-based abalone aquaculture in South Africa started in the early 1990s and is based on the local species Haliotis midae. This industry expanded with great success over the last decade. In 2006 abalone exhibiting typical clinical signs of tubercle mycosis was discovered for the first time in South African abalone culture facilities,posing a significant threat to the industry. Halioticida noduliformans, a fungus belonging to the Peronosporomycetes (formerly Oomycetes), has been identified as the causative agent of abalone tubercle mycosis (ATM). While diagnoses of this disease are currently done by gross observation and histopathology, these methods fail to be sensitive enough to identify the causative agent accurately and reliably.Molecular confirmation could provide for quicker more accurate diagnostic information. The aim of this study was to develop a DNA based molecular diagnostic test. Polymerase chain reaction (PCR) has been used to rapidly detect, characterise and identify a variety of organisms. Nucleotide sequences of the smalland large-subunit ribosomal ribonucleic acid (rRNA) and mitochondrial cytochrome oxidase subunit II (cox2) genes of H. noduliformans were compared with closely related Peronosporomycete gene sequences to identify potential PCR primer sites. H. noduliformans specific real-time quantitative PCR (Q-PCR) primer sets were designed and optimised for each of the selected genes. Results indicate that, although all tested primers sets could amplify fungal DNA, only the LSU and cox2 primer sets - v -demonstrated no cross-amplification with the closely related Peronosporomycete and non-fungal DNA tested in the present study. The H. noduliformans specific LSU primer set was chosen for further analysis and used for all subsequent real-time PCR assays. The lowest detection limit for the LSU primer set was evaluated by running Q-PCR on serial dilutions of known quantities of extracted H. noduliformans DNA.Serial dilutions were made in PCR grade water as well as in an abalone tissue matrix.The sensitivity of the Q-PCR reaction was determined to be 266 pg of H.noduliformans DNA per 25 μL reaction volume. However, inclusion of a nested PCR step, utilising universal fungal outer primers, followed by Q-PCR with the H.noduliformans LSU specific primers improved sensitivity to 0.266 pg of H.noduliformans DNA per 25 μL reaction volume. This equates to approximately 2.4spores per 25 μL reaction volume. DNA extraction protocols were optimised to ensure efficient and repeatable extraction of high quality fungal DNA from pure fungus and tissue samples spiked with known quantities of fungal DNA. PCR amplification efficiency and potential inhibition were examined for each extraction method. Results suggest that real-time PCR has great potential in monitoring and quantifying H. noduliformans on abalone culture facilities in South Africa.

Plant alkaloids are secondary metabolites that may be synthesized in an inducible defense response to herbivory (Baldwin 1999). Genetic engineering of secondary metabolic pathways in plants to enhance or reduce metabolite production is limited by the current understanding of these pathways and their regulation in response to environmental conditions. This study was intended to provide new insights into the mechanism and regulation of alkaloid biosynthesis in N. tabacum by identifying genes that are coordinately regulated during conditions that induce alkaloid biosynthesis and by comparing their expression in regulatory mutant backgrounds that differ at two quantitative alkaloid loci, A and B. In order to identify novel genes that are differentially expressed during alkaloid biosynthesis, the transcriptional profiling procedure, fluorescent differential display (FDD), was used to screen total RNA isolated from Burley 21 (WT, AABB) and LA21 (low alkaloid regulatory mutant, aabb) tobacco root cultures that were induced for alkaloid synthesis. Four of thirteen cloned FDD fragments showed sequence homology to genes with defense-related functions. The differential expression of genes represented by selected FDD gene fragments was confirmed by comparing Northern blots of transcripts of those genes to known alkaloid biosynthetic genes, putrescine methyl transferase (PMT3), ornithine decarboxylase (ODC3), arginine decarboxylase (ADC1), and quinolinate phosphoribosyltransferase (QPRT). The role of the A and B loci in differential expression of genes represented by FDD clones and of known nicotine biosynthetic genes was examined using quantitative real time polymerase chain reaction (QRT-PCR) to measure transcript levels of these genes in four tobacco genotypes differing in alkaloid content, Burley 21(AABB), HI21 (AAbb), LI21(aaBB), and LA21 (aabb). Results of this study suggest that the A/B regulon is not limited to alkaloid biosynthetic genes, but includes multiple genes with defense-related functions. QRT-PCR analysis of nicotine biosynthetic genes and genes represented by confirmed differentially expressed FDD clones showed increased mRNA accumulation in response to alkaloid induction in all the tested genotypes, which suggests that the A and B mutations affect overall mRNA accumulation levels, rather than gene inducibility, per se. Baldwin, I.T. 1999. Inducible nicotine production in native Nicotiana as an example of adaptive phenotypic plasticity. Journal of Chem. Ecol. 25: 3-30. / Master of Science