It is widely appreciated by the medical community that subtle deficits in intellect, academic skills and executive functioning exist in early treated phenylketonuria (PKU). In this study, we described the relationship between intellectual outcome and concentration/variation in blood phenylalanine (Phe) during specific developmental periods (0-5 years, 6-10 years, >10 years). We also examined the association between mean number of blood Phe samples and maintenance of Phe within treatment range (120-360 ìmol/L) and within one standard deviation (SD) of index of dietary control, defined as the mean of 12-month median Phe. Retrospective data was collected from 55 patients receiving treatment at the University of Utah Metabolic Clinic. Index of dietary control (IDC) and SD blood Phe steadily increased and mean number of samples decreased during each developmental period. The correlation between IDC during 6-10 years of life and perceptual reasoning was -.370 (p = 0.006). Using multivariate linear regression, IDC during 0-5 years and 6-10 years were associated with a 0.5-point decrease and 0.3-point decrease in perceptual reasoning scores for every 100 ìmol/L increase in blood Phe, though associations were nonsignificant (p = 0.067; 0.082). SD of Phe was not associated with any measure of intelligence. The likelihood of IDC >360 ìmol/L in those 6-10 years was 32.3% lower for each additional blood Phe sample per year (p = 0.001). The present study suggests frequent blood Phe monitoring during ages 6-10 years may reduce blood Phe and prevent deficits in perceptual reasoning later in life.

Current methods for studying RNA binding proteins (RBPs) combine the use of ultraviolet (UV) crosslinking and immunoprecipitation (CLIP) to analyze RNA-protein interactions. An underexplored alternative approach is using site-specific incorporation of photoactivatable non-canonical amino acids (ncAAs) to enhance the crosslinking efficiency of many CLIP protocols. This thesis describes the incorporation of the photo-crosslinking unnatural amino acid p-azido-L-phenylalanine (AzF) into the Hepatitis C Virus (HCV) non-structural protein 3 helicase (NS3h) for photo-crosslinking and in vitro analysis of the potential binding sites found within the HCV RNA genome. From the five potential sites identified from the NS3h crystal structure for AzF incorporation, two sites, E503AzF and Q580AzF, allowed for nucleic acid photo-crosslinking with fluorescently labelled DNA substrates. We further tested if these mutations adversely affected NS3h and binding activity through a molecular beacon helicase assay and fluorescence polarization methods. We found that E503AzF unexpectedly had a faster unwinding rate than wild type (WT) NS3h and managed to have a similar binding affinity to the tested DNA substrate. Finally, we found that there was a 5-fold increase in the photo-crosslinking efficiency of nucleic acids for E503AzF NS3h mutant compared to our WT NS3h at 254 nm UV light. We are currently working on methods for our CLIP-based protocol to ensure quality RNA footprint generation and purification from photo-crosslinked NS3h. Other work contained in this thesis consists of using Prevotella sp. P5-125 Cas13b (PspCas13b), a clustered regularly interspaced short palindromic repeats (CRISPR) RNA-targeting system, which has been previously shown to knockdown viral RNA and mRNA through designable guide CRISPR RNA (crRNA). Here we incorporated the photo-crosslinking ncAA AzF into PspCas13b to irreversibly bind the crRNA in an attempt to enhance knockdown efficiency and longevity of viral and mRNA targets. We were able to design a crRNA that produced significant knockdown targeting the luciferase mRNA of a luciferase rennilla reporter system. When targeting an HCV subgenomic replicon luciferase reporter system, knockdown was not observed. Additionally, the WT PspCas13b had photo-crosslinking to the bound crRNA and requires further optimization for future use.

Chan, Ka Man Carmen. / "October 2009." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 160-165). / Abstracts in English and Chinese. / Table of Contents --- p.I / Acknowledgements --- p.IV / Abstract --- p.V / Abstract (Chinese version) --- p.VIII / List of Figures --- p.X / List of Tables --- p.XVIII / Chapter Chapter One. --- Introduction / Chapter 1.1 --- Pulmonary drug delivery --- p.1 / Chapter 1.2 --- Inhalation drug delivery systems --- p.4 / Chapter 1.3 --- Dry powder inhalation aerosols --- p.5 / Chapter 1.3.1 --- Principle of operation of DPIs --- p.5 / Chapter 1.3.2 --- Aerodynamic diameter --- p.6 / Chapter 1.3.2.1 --- Fine particle fraction --- p.8 / Chapter 1.3.3 --- Dispersibility --- p.8 / Chapter 1.3.4 --- Factors that affect dispersibility --- p.9 / Chapter 1.3.4.1 --- Particle Size --- p.9 / Chapter 1.3.4.2 --- Particle Density and Morphology --- p.10 / Chapter 1.3.4.3 --- Interparticulate interactions一Cohesion and adhesion --- p.11 / Chapter 1.3.4.3.1 --- Surface energetics --- p.11 / Chapter 1.3.4.3.2 --- Effect of hygroscopicity and electrostatic charges --- p.12 / Chapter 1.4 --- Particle formation techniques for DPI formulation --- p.14 / Chapter 1.4.1 --- Spray-drying --- p.14 / Chapter 1.4.2 --- Surface modification --- p.16 / Chapter 1.5 --- Physical characterization --- p.17 / Chapter 1.5.1 --- Laser diffraction --- p.17 / Chapter 1.5.2 --- X-ray powder diffraction --- p.18 / Chapter 1.5.3 --- Thermal analysis --- p.19 / Chapter 1.5.4 --- Particle morphology and surface area --- p.20 / Chapter 1.5.5 --- In vitro aerosol performance --- p.21 / Chapter 1.6 --- Surface characterization --- p.21 / Chapter 1.6.1 --- X-ray photoelectric spectroscopy (XPS) --- p.21 / Chapter 1.6.2 --- Inverse gas chromatography --- p.22 / Chapter 1.7 --- Atomic force microscopy in pharmaceutical science --- p.23 / Chapter 1.7.1 --- Principle of operation --- p.24 / Chapter 1.7.1.1 --- Tapping mode --- p.27 / Chapter 1.7.1.2 --- Contact mode --- p.27 / Chapter 1.8 --- Scope of thesis --- p.29 / Chapter Chapter Two. --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Optimization of spray-drying parameters --- p.32 / Chapter 2.2.2 --- Preparation of spray-dried salbutamol sulphate powders containing different concentrations of amino acid additive --- p.33 / Chapter 2.2.3 --- Physical characterization of spray-dried powders --- p.34 / Chapter 2.2.3.1 --- Particle size and size distribution --- p.34 / Chapter 2.2.3.2 --- Specific surface area --- p.35 / Chapter 2.2.3.3 --- X-ray powder diffraction --- p.35 / Chapter 2.2.3.4. --- Scanning electron microscopy --- p.36 / Chapter 2.2.3.5. --- Thermal analysis --- p.36 / Chapter 2.2.3.5.1 --- Thermogravimetric analysis (TGA) --- p.36 / Chapter 2.2.3.5.2 --- Differential scanning calorimetry (DSC) --- p.36 / Chapter 2.2.3.6 --- Water vapour sorption isotherm --- p.37 / Chapter 2.2.3.7 --- Density measurements --- p.37 / Chapter 2.2.3.8 --- In vitro particle deposition (MSLI) --- p.38 / Chapter 2.2.4 --- Surface characterization of the spray-dried powders --- p.39 / Chapter 2.2.4.1 --- X-ray photoelectric spectroscopy (XPS) --- p.39 / Chapter 2.2.4.2 --- Surface energy measurement by inverse gas chromatography (IGC) --- p.40 / Chapter 2.2.4.2.1 --- Calculation of standard free energy of adsorption --- p.41 / Chapter 2.2.4.2.2 --- Dispersive component of surface free energy and related thermodynamic parameters --- p.42 / Chapter 2.2.4.2.3 --- Specific interactions and associated acid-base properties --- p.43 / Chapter 2.2.5. --- Atomic Force Microscopy (AFM) --- p.43 / Chapter 2.2.5.1. --- Imaging --- p.43 / Chapter 2.2.5.2. --- Force measurements --- p.44 / Chapter 2.2.5.2.1 --- Adhesion force measurements --- p.44 / Chapter 2.2.5.2.2 --- Force curve data conversions --- p.44 / Chapter Chapter Three. --- "Optimal Spray-drying Conditions, Physical Characterization and Aerosol Performance of Additive-modified Spray-dried Salbutamol Sulphate particles" / Chapter 3.1 --- Optimization of spray-drying conditions --- p.46 / Chapter 3.2 --- Effect of phenylalanine on the spray-dried SS particles --- p.52 / Chapter 3.2.1. --- Phenylalanine as the additive --- p.52 / Chapter 3.2.1.1 --- In vitro aerosol performance --- p.53 / Chapter 3.2.1.2 --- Particle morphology --- p.55 / Chapter 3.2.1.3 --- Crystallinity --- p.62 / Chapter 3.2.1.4 --- Particle size distribution and specific surface area --- p.63 / Chapter 3.2.1.5 --- Density --- p.65 / Chapter 3.2.1.6 --- Thermal analysis --- p.66 / Chapter 3.2.1.7 --- Water vapour isotherm --- p.70 / Chapter 3.3 --- Effect of leucine on the spray-dried SS particles --- p.77 / Chapter 3.3.1. --- L-Leucine as the additive --- p.77 / Chapter 3.3.1.1 --- In vitro aerosol performance --- p.78 / Chapter 3.3.1.2 --- Particle morphology --- p.80 / Chapter 3.3.1.3 --- Crystallinity --- p.86 / Chapter 3.3.1.4 --- Particle size distribution and specific surface area --- p.87 / Chapter 3.3.1.5 --- Density --- p.90 / Chapter 3.3.1.6 --- Thermal analysis --- p.92 / Chapter 3.3.1.7 --- Water vapour isotherm --- p.95 / Chapter Chapter Four. --- Surface Characterization of Additive-modified Spray-dried Salbutamol Sulphate Particles / Chapter 4.1 --- X-ray photoelectric spectroscopy --- p.103 / Chapter 4.1.1 --- Phenylalanine --- p.103 / Chapter 4.1.2 --- Leucine --- p.104 / Chapter 4.2 --- Inverse gas chromatography --- p.105 / Chapter 4.2.1 --- Phenylalanine --- p.105 / Chapter 4.2.2 --- Leucine --- p.107 / Chapter 4.3 --- Atomic force microscopy --- p.109 / Chapter 4.3.1 --- Surface topography --- p.109 / Chapter 4.3.2 --- Adhesive force measurements --- p.118 / Chapter Chapter Five. --- Conclusions and Suggestions for Future Works / Chapter 5.1 --- Conclusions --- p.139 / Chapter 5.1.1 --- Physical properties --- p.139 / Chapter 5.1.2 --- Surface characteristics and aerosol performance --- p.140 / Chapter 5.2 --- Future studies --- p.142 / Appendix --- p.143 / References --- p.160

La phénylcétonurie (PCU est une maladie métabolique génétique causée par une déficience d'activité phénylalanine hydroxylase (PAH). Une hypomyélinisation du cerveau a été documentée chez les patients non traités, mais sa pathophysiologie reste floue. Nous avons investigué l'influence de la phénylalanine (Phe), phénylpyruvate (PP) et phénylacétate (PA) sur les oligodendrocytes. Nous avons premièrement montré dans un modèle murin de PCU que le nombre d'oligodendrocytes n'était pas différent dans le corps calleux entre animaux PCU et sains. Ensuite, en utilisant la technique des co-cultures myélinisantes nous avons pu déterminer que Phe, PP et PA n'ont pas d'effet direct sur la synthèse des gaines de myéline. Ces données indiquent que ces trois composés n'exercent probablement pas de rôle direct dans l'hypomyélinisation du système nerveux central constatée dans le cadre de la PCU. Ces données suggèrent donc des mécanismes d'action indirects. De plus, nous avons investigué la faisabilité d'un modèle de thérapie génique pour la PCU. Celui-ci implique la transduction ex vivo d'hépatocytes ou cellules souches mésenchymateuses par un vecteur lentiviral puis leur implantation dans le foie de l'organisme receveur. Phenylketonuria (PKU) is a metabolic genetic disease characterized by deficient phenylalanine hydroxylase (PAH) enzymatic activity. Brain hypomyelination has been reported in untreated patients, but its mechanism remains unclear. We therefore investigated the influence of phenylalanine (Phe), phenylpyruvate (PP), and phenylacetate (PA) on oligodendrocytes. We fisrt showed in a mouse model of PKU that the number of oligodendrocytes is not different in corpus callosum sections from adult mutants or from control brains. Then, using enriched oligodendroglial cultures, we detected no cytotoxic effect of high concentrations of Phe, PP, or PA. Finally, we analyzed the impact of Phe, PP, and PA on the myelination process in myelinating cocultures using both an in vitro index of myelination, based on activation of the myelin basic protein (MBP) promoter, and the direct quantification of myelin sheaths by both optical measurement and a bioinformatics method. None of these parameters was affected by the increased levels of Phe or its derivatives. Taken together, our data demonstrate that high levels of Phe, such as in PKU, are unlikely to directly induce brain hypomyelination, suggesting involvement of alternative mechanisms in this myelination defect. Moreover, we investigated the feasibility of a gene therapy for phenylketonuria. This project involved the ex vivo transduction of hepatocytes and mesenchymal stem cells with lentivirus vector and the engraftment of these cells in the liver's recipient.

No description available.

Esta tese de doutorado descreve os estudos realizados para elucidar a biologia molecular da bioluminescência fúngica e sua relevância ecológica na natureza. A recente descoberta de que a luciferina fúngica é a 3-hidroxihispidina permitiu a caracterização do metabolismo secundário da fenilalanina nos genomas recém-sequenciados e transcriptomas de micélios das espécies luminescentes Panellus stipticus e Neonothopanus gardneri. Adicionalmente os genomas e transcriptomas de variedades não luminescente de P. stipticus e Lentinula edodes serviram como respectivos controles. Em geral, os genes envolvidos no metabolismo secundário da fenilalanina em amostras luminescentes tinham expressão igual ou superior àquela de espécies não luminescentes. Um agrupamento de genes relacionados com a biossíntese de fenilalanina foi encontrado em ambos os genomas luminescentes e não luminescentes de P. stipticus. A abundância de genes transcritos neste agrupamento foi semelhante para as espécies luminescentes e não luminescentes de P. stipticus, mas a policetídeo sintase tipo I em P. stipticus não luminescentes foi significativamente sub-regulada. Não foi encontrado agrupamento semelhante nos genomas de N. gardneri e L. edodes, sendo que os correspondentes homólogos estavam espalhados em diferentes loci. Extratos de fungos podem ser preparados in vitro, com a adição de 3-hidroxihispidina para produzir luz verde em abundância. A preparação de extratos proteicos de luciferase foi melhorada e a estrutura da luciferase, parcialmente purificada, foi investigada por espectrometria de massas. A presença de luciferase nos géis de purificação foi revelada usando-se luciferina e molécula similares à luciferina advindas de extratos de plantas. O nicho ecológico nas vizinhas de cogumelos bioluminescentes foi investigado de duas maneiras, armadilhas adesivas com cogumelos artificiais de acrílico, iluminados com luz LED verde e através da observação direta de cogumelos bioluminescentes com fotografia no infravermelho com lapso de tempo. Os estudos ecológicos foram conduzidos nos biomas da Mata Atlântica e da Mata dos Cocais, no Brasil. Baratas, aranhas, tesourinhas, grilo e vagalumes tec-tecs foram os animais mais comuns que interagiram com os cogumelos. Todos estes animais podem agir como dispersores de propágulos e, em alguns casos, como defensores dos cogumelos. / This PhD thesis describes the studies performed to elucidate the molecular biology of fungal bioluminescence and the ecological significance of the trait in the wild. The recent discovery that the fungal luciferin is 3-hydroxyhispidin has allowed for the characterization of phenylalanine secondary metabolism in the newly sequenced genomes and mycelium transcriptomes of luminescent Panellus stipticus and Neonothopanus gardneri, additionally the genomes and transcriptomes of a non-luminescent variety of P. stipticus and Lentinula edodes served as respective controls. In general the genes involved in phenylalanine secondary metabolism had greater or equal expression in luminescent samples than non luminescent. A cluster of genes related to the secondary metabolism of phenylalanine was found in both luminescent and non luminescent P. stipticus genomes. Transcript abundance of genes in this cluster was similar in both luminescent and non-luminescent Panellus stipticus, but the type I polyketide synthase in non luminescent Panellus stipticus was significantly down regulated. A similar gene cluster in the N. gardneri and L. edodes genomes was absent with corresponding homologues scattered at different genomic loci. Cell free fungal extracts can be combined in vitro with the addition of 3-hydroxyhispidin to produce abundant green light. Preparation of proteinaceous luciferase extracts was improved and partially purified luciferase samples were investigated by mass spectrometry. The presence of luciferase in the separation gel was also evidenced by using luciferin and luciferin-like molecules from plant extracts. The ecological niche surrounding bioluminescent mushrooms was investigated through two main means, glue traps with acrylic mushroom facsimiles that were internally illuminated with green LED lights and direct observation of bioluminescent mushrooms with infrared time lapse photography. Ecological studies were performed in the Atlantic rainforest (Mata Atlântica) and transitional Coconut Palm forest (Mata dos Cocais) biomes of Brazil. Cockroaches, spiders, earwigs, crickets, and luminescent click beetles were the most common animal interacting with mushrooms. All of these animals may be acting as fungal propagule dispersers and in some cases defense of the mushroom.

Nos últimos anos tem se intensificado o uso de produtos com a finalidade de aumentar a produtividade na cultura da soja. Dentre estes estão os bioestimulantes que podem possuir em sua constituição extratos de algas, aminoácidos e hormônios. No entanto, pouco se sabe sobre o efeito isolado de cada um destes constituintes. Frente a isto, esta pesquisa teve por objetivos avaliar o efeito da aplicação de aminoácidos isolados em plantas de soja. Para isto, o trabalho foi dividido em três experimentos. No primeiro, foi realizada a aplicação via sementes de glutamato, cisteína, fenilalanina e glicina em doses variáveis. Essa etapa foi conduzida em sistema de canteiros e foi avaliada a emergência, índice de velocidade de emergência, acúmulo de massa de matéria seca, metabolismo antioxidante (enzimas superóxido dismutase - SOD, catalase - CAT, peroxidase - POD, teor de peróxido de hidrogênio - H2O2, prolina e peroxidação lipídica - PL) e produtividade. A partir da seleção das melhores doses obtidas na primeira etapa, foi realizado o segundo experimento, conduzido em casa de vegetação. As aplicações desse experimento foram realizadas no tratamento de semente, via foliar ou em ambas as épocas, além disso, foi realizada a aplicação de todos os aminoácidos em associação. Nesse experimento foram avaliados metabolismo antioxidante, enzimas de resistência (polifenoloxidase - PFO e fenilalanina amônia-liase - PAL), metabolismo do nitrogênio (enzimas nitrato redutase e urease, teor de NO3-, NH4+, N-Aa, ureídeos e N-Total), variáveis de crescimento de raíz, acúmulo de massa de matéria seca e produtividade. Já o experimento III foi realizado em campo, utilizando os mesmos tratamentos do experimento II. Foram avaliados o metabolismo antioxidante, enzimas de resistência, metabolismo do nitrogênio, massa de matéria seca e produtividade. Todos os experimentos foram conduzidos em delineamento em blocos casualizados com quatro repetições para cada tratamento. Todos os aminoácidos proporcionaram efeito positivo em diversas variáveis fisiológicas analisadas. O uso de glutamato, fenilalanina, cisteína e glicina de forma isolada repercutiram em melhores efeitos quando a aplicação é realizada somente no tratamento de sementes. A partir da aplicação desses aminoácidos ocorreu incremento da assimilação de nitrogênio e no acúmulo de massa de matéria seca, o que levou a maior produtividade dessas plantas. O maior efeito na produtividade foi observado por meio da aplicação de fenilalanina em todos os experimentos, quando comparados com os demais aminoácidos. Com relação ao metabolismo antioxidante o uso de cisteína no tratamento de sementes proporcionou aumento da atividade das enzimas SOD e PAL e redução da PL. O uso de fenilalanina no tratamento de sementes induz ao incremento da CAT e SOD e o glutamato induz o aumento de PAL e SOD. A utilização de todos os aminoácidos em associação somente foi eficiente na aplicação foliar, o que proporcionou maior desenvolvimento de raíz, maior assimilação de nitrogênio, acúmulo de massa de matéria seca e produtividade. Portanto, foi possível perceber que o glutamato, cisteína, fenilalanina e glicina apresentam importante papel de sinalização em plantas, pois pequenas doses já são suficientes para induzir ao incremento de parâmetros fisiológicos e, consequentemente aumentar a produtividade. / In recent years, the use of products to increase productivity in soybean has been intensified. Bio-stimulants can have in their constitution algae extracts, amino acids and hormones. However, little is known about the isolated effect of each of these constituents. Facing this problem, this research aimed to evaluate the effect of the application of single amino acids to soybeans. For this, the work was divided into three experiments. In the first, the application of amino acids was performed via glutamate, cysteine, phenylalanine and glycine to seeds. This stage was carried out on planting beds and the following variables were evaluated: emergency, emergency speed index, dry matter accumulation, antioxidant metabolism (superoxide dismutase - SOD, catalase - CAT, peroxidase - POD, hydrogen peroxide - H2O2 - content, proline and lipid peroxidation - PL) and productivity. From the selection of the best rates obtained in the first stage, the second experiment was carried out in a greenhouse. The applications of this experiment were performed as seed treatments, foliar application and both procedures; furthermore, the application of all amino acids in combination was also performed. In this experiment, the following variables were evaluated: antioxidant metabolism, resistance enzymes (polyphenol oxidase - PFO and phenylalanine ammonia lyase - PAL), nitrogen metabolism (nitrate reductase and urease, NO3- content, NH4+, N-Aa, ureide and N-Total), root growth, dry matter accumulation and productivity. The third experiment was carried out in the field using the same treatments of the second experiment. The following variables were evaluated: antioxidant metabolism, resistance enzymes, nitrogen metabolism, dry matter and productivity. All experiments were carried out in a randomized block design with four replications for each treatment. All amino acids provided positive effect on several physiological variables. The use of glutamate, phenylalanine, cysteine, and glycine alone lead to the best effect when the application was done only as seed treatment. From the application of these amino acids, the nitrogen assimilation was increased and the dry matter accumulation, which led to higher productivity of the plants. The greatest effect on productivity was observed by application of phenylalanine in all experiments, when compared with other amino acids. Regarding the antioxidant metabolism, cysteine use in seed treatment increased SOD and PAL activity and PL reduction. The phenylalanine use in seed treatment increased CAT and SOD activities and glutamate induced an increase of PAL and SOD activities. The use of all amino acids in association was only effective in foliar application, which provided further development of root, greater assimilation of nitrogen, dry matter accumulation and productivity. So, it was possible to conclude that glutamate, cysteine, phenylalanine and glycine have an important signaling role in plants, because small rates are enough to induce the increase of physiological parameters and consequently increase productivity.

Made available in DSpace on 2017-07-10T19:24:47Z (GMT). No. of bitstreams: 1 Simone Maria Menegatti de Oliveira.pdf: 745176 bytes, checksum: 3a5240485b0941a0c7d894af6e3b5ade (MD5) Previous issue date: 2010-07-07 / 2-phenylethanol is the most used fragrance in the food, cosmetics and fragrance industry. It is mainly produced by chemical process, but with many impurities. The biotechnological process of production is feasible and is being studied with various microrganisms, among them, Geotrichum fragrans, Saccharomyces cerevisiae and Kluyveromyces marxianus. The use of cassava wastewater as substrate in this process makes it less expensive and reduce environmental pollution caused by it. The objective or this work was to evaluate the production of 2-phenylethanol in the cultivation of Geotrichum fragrans, Saccharomyces cerevisiae and Kluyveromyces marxianus in cassava wastewater, analyzing the effect of two carbon sources on production of the aroma, besides increasing the production of 2 - phenylethanol to the greater efficiency of conversion carbon source/2-feniletanol, varying concentrations of carbon source and phenylalanine at cassava wastewater. For this, the three microrganisms mentioned were cultured in 50mL cassava wastewater plus 50g.L-1 of glucose and 3g.L-1 of L-phenylalanine in sterile Erlenmeyer flasks, on shaker at 150 rpm, 24 ° C for 120 hours, to check the time of peak production of 2-phenylethanol and what microrganism largest producer. It was then checked the production of aroma with 50g.L-1 of glucose and fructose, while maintaining the same amount of L-phenylalanine. And finally, by fatorial design, were tested concentrations of glucose and L-phenylalanine, in order to obtain higher yield and bioconversion of 2-phenylethanol. The experimental model was validated. It was analyzed in the pH, COD, reducing sugars, L-phenylalanine and 2-phenylethanol. In the precipitate was quantified indirectly as biomass, the Volatile Suspended Solids. The results showed that S. cerevisiae was the best producer of 2-phenylethanol in the effluent of cassava starch, obtaining 0.74 g.L-1, followed by K. marxianus, with 0.19 g.L-1 and finally by G. fragrans with 0.08 g.L-1. Glucose was the best carbon source for obtention of 2- phenylethanol for S. cerevisiae and K. marxianus. There was no significant difference between the production of 2-phenylethanol using glucose or fructose for G. fragrans. The best combination of glucose and L-phenylalanine for production of 2-phenylethanol, was 20,0 and 5.5 g.L-1 respectively, obtaining production of 1.33 g.L-1of aroma in the validation. / O 2-feniletanol é a fragrância mais utilizada na indústria de alimentos, cosmética e de perfumes. É produzido principalmente por processo químico, porém com muitas impurezas. O processo biotecnológico de produção é viável e está sendo estudado com vários microrganismos, entre eles, o Geotrichum fragrans (GF), Saccharomyces cerevisiae (SC) e Kluyveromyces marxianus (KM). O uso de resíduo líquido de fecularia como substrato neste processo torna-o menos dispendioso, além de reduzir a poluição ambiental causada pelo mesmo. O objetivo deste trabalho foi avaliar a produção de 2-feniletanol no cultivo de GF, SC e KM em resíduo líquido de fecularia, analisando o efeito de duas fontes de carbono na produção do aroma, além de aumentar a produção de 2-feniletanol até a maior eficácia de conversão fonte de carbono/2-feniletanol, variando as concentrações da fonte de carbono e de fenilalanina no resíduo líquido de fecularia. Para isso, os três microrganismos citados foram cultivados em 50 ml de resíduo líquido de fecularia, acrescidos de 50 g.L-1 de glicose e 3 g.L-1 de L-fenilalanina, em frascos erlenmeyers estéreis, em agitador a 150 rpm, 24 ºC, por 120 h, para verificar o tempo de maior produção de 2-feniletanol e qual microrganismo maior produtor. Foi então verificada a produção de aroma com 50g.L-1 de glicose e frutose, mantendo a mesma quantidade de L-fenilalanina. E finalmente, através de planejamento fatorial, foram testadas concentrações de glicose e L-fenilalanina, a fim de se obter maior produção e bioconversão de 2-feniletanol. O modelo experimental foi validado. Analisou-se o pH, a DQO, os açúcares redutores, a L-fenilalanina e o 2-feniletanol. No precipitado foi quantificada a biomassa indiretamente como sólidos suspensos voláteis. Os resultados obtidos demonstraram que SC foi o melhor produtor de 2-feniletanol em água residual de fecularia de mandioca, obtendo 0,74 g.L-1, seguido pelo K. marxianus, com 0,19 g.L-1 e por último pelo G. fragrans, com 0,08 g.L-1. A glicose foi a melhor fonte de carbono para obtenção do 2-feniletanol para S. cerevisiae e K. marxianus. Não houve diferença significativa entre a produção de 2-feniletanol utilizando glicose ou frutose para o G. fragrans. A melhor conjugação dos níveis de glicose e L-fenilalanina para produção de 2- feniletanol foi de 20,0 e 5,5 g.L-1 respectivamente, obtendo produção de 1,33 g.L-1do aroma na validação.

Made available in DSpace on 2017-05-12T14:48:11Z (GMT). No. of bitstreams: 1 Simone Maria Menegatti de Oliveira.pdf: 745176 bytes, checksum: 3a5240485b0941a0c7d894af6e3b5ade (MD5) Previous issue date: 2010-07-07 / 2-phenylethanol is the most used fragrance in the food, cosmetics and fragrance industry. It is mainly produced by chemical process, but with many impurities. The biotechnological process of production is feasible and is being studied with various microrganisms, among them, Geotrichum fragrans, Saccharomyces cerevisiae and Kluyveromyces marxianus. The use of cassava wastewater as substrate in this process makes it less expensive and reduce environmental pollution caused by it. The objective or this work was to evaluate the production of 2-phenylethanol in the cultivation of Geotrichum fragrans, Saccharomyces cerevisiae and Kluyveromyces marxianus in cassava wastewater, analyzing the effect of two carbon sources on production of the aroma, besides increasing the production of 2 - phenylethanol to the greater efficiency of conversion carbon source/2-feniletanol, varying concentrations of carbon source and phenylalanine at cassava wastewater. For this, the three microrganisms mentioned were cultured in 50mL cassava wastewater plus 50g.L-1 of glucose and 3g.L-1 of L-phenylalanine in sterile Erlenmeyer flasks, on shaker at 150 rpm, 24 ° C for 120 hours, to check the time of peak production of 2-phenylethanol and what microrganism largest producer. It was then checked the production of aroma with 50g.L-1 of glucose and fructose, while maintaining the same amount of L-phenylalanine. And finally, by fatorial design, were tested concentrations of glucose and L-phenylalanine, in order to obtain higher yield and bioconversion of 2-phenylethanol. The experimental model was validated. It was analyzed in the pH, COD, reducing sugars, L-phenylalanine and 2-phenylethanol. In the precipitate was quantified indirectly as biomass, the Volatile Suspended Solids. The results showed that S. cerevisiae was the best producer of 2-phenylethanol in the effluent of cassava starch, obtaining 0.74 g.L-1, followed by K. marxianus, with 0.19 g.L-1 and finally by G. fragrans with 0.08 g.L-1. Glucose was the best carbon source for obtention of 2- phenylethanol for S. cerevisiae and K. marxianus. There was no significant difference between the production of 2-phenylethanol using glucose or fructose for G. fragrans. The best combination of glucose and L-phenylalanine for production of 2-phenylethanol, was 20,0 and 5.5 g.L-1 respectively, obtaining production of 1.33 g.L-1of aroma in the validation. / O 2-feniletanol é a fragrância mais utilizada na indústria de alimentos, cosmética e de perfumes. É produzido principalmente por processo químico, porém com muitas impurezas. O processo biotecnológico de produção é viável e está sendo estudado com vários microrganismos, entre eles, o Geotrichum fragrans (GF), Saccharomyces cerevisiae (SC) e Kluyveromyces marxianus (KM). O uso de resíduo líquido de fecularia como substrato neste processo torna-o menos dispendioso, além de reduzir a poluição ambiental causada pelo mesmo. O objetivo deste trabalho foi avaliar a produção de 2-feniletanol no cultivo de GF, SC e KM em resíduo líquido de fecularia, analisando o efeito de duas fontes de carbono na produção do aroma, além de aumentar a produção de 2-feniletanol até a maior eficácia de conversão fonte de carbono/2-feniletanol, variando as concentrações da fonte de carbono e de fenilalanina no resíduo líquido de fecularia. Para isso, os três microrganismos citados foram cultivados em 50 ml de resíduo líquido de fecularia, acrescidos de 50 g.L-1 de glicose e 3 g.L-1 de L-fenilalanina, em frascos erlenmeyers estéreis, em agitador a 150 rpm, 24 ºC, por 120 h, para verificar o tempo de maior produção de 2-feniletanol e qual microrganismo maior produtor. Foi então verificada a produção de aroma com 50g.L-1 de glicose e frutose, mantendo a mesma quantidade de L-fenilalanina. E finalmente, através de planejamento fatorial, foram testadas concentrações de glicose e L-fenilalanina, a fim de se obter maior produção e bioconversão de 2-feniletanol. O modelo experimental foi validado. Analisou-se o pH, a DQO, os açúcares redutores, a L-fenilalanina e o 2-feniletanol. No precipitado foi quantificada a biomassa indiretamente como sólidos suspensos voláteis. Os resultados obtidos demonstraram que SC foi o melhor produtor de 2-feniletanol em água residual de fecularia de mandioca, obtendo 0,74 g.L-1, seguido pelo K. marxianus, com 0,19 g.L-1 e por último pelo G. fragrans, com 0,08 g.L-1. A glicose foi a melhor fonte de carbono para obtenção do 2-feniletanol para S. cerevisiae e K. marxianus. Não houve diferença significativa entre a produção de 2-feniletanol utilizando glicose ou frutose para o G. fragrans. A melhor conjugação dos níveis de glicose e L-fenilalanina para produção de 2- feniletanol foi de 20,0 e 5,5 g.L-1 respectivamente, obtendo produção de 1,33 g.L-1do aroma na validação.

There has been limited investigation as to whether a predominantly forage-based diet can provide adequate amounts of limiting amino acids (AA) to horses. The first objective was to determine if AA supplementation of AA believed to be limiting to protein synthesis in forage-based diets would affect measures of whole-body protein metabolism in sedentary mature horses. The effect of forage type (timothy or alfalfa) and AA supplementation (lysine, threonine or histidine) on plasma urea nitrogen (PUN) and AA concentrations and measures of whole-body phenylalanine kinetics were evaluated. There was no effect of either forage type or AA supplement on rates of whole-body protein synthesis (P > 0.05). The second objective was to determine the effects of either timothy or alfalfa hay supplemented with either a high or low protein ration balancer on measures of whole-body protein metabolism in yearling horses. The effect of forage type and the ration balancer protein level on concentrations of PUN, plasma AA and measures of wholebody phenylalanine kinetics were evaluated. There was no effect of treatment on average daily gain (P = 0.18). When horses consumed the alfalfa-based diets, rates of phenylalanine flux, oxidation and use for protein synthesis were greater than when they consumed timothy-based diets (P < 0.05). Phenylalanine use for protein synthesis was not affected by the protein level of the ration balancer (P = 0.3). Yearling horses achieve greater rates of protein synthesis when fed alfalfa-based diets, compared to timothy-based diets, supplemented with a low protein ration balancer.