Student Number: 0110826H - MSc research report - School of Occupational Therapy - Faculty of Health Sciences / A comparative study was undertaken to assess the reliability of the MVPT-3 when the instructions were translated into Afrikaans and the scores were then compared to the normative data obtained during the standardization process on a normal population of American children. The study was undertaken by testing 80 randomly selected, normally distributed, Afrikaans first language speaking eight year old children from the eastern suburbs of Pretoria, South Africa. The study confirmed that the MVPT-3 is reliable when the instructions are given in Afrikaans with a Chonbach’s alpha score of 0.79 compared to 0.83 obtained for the American population. The item analysis revealed some anomalies that suggest that the test may have a cultural bias as many items had a negative sign in the item analysis. Afrikaans girls and boys performed differently on the test, suggesting that the test may need to be adjusted for differing skills in the visual perception. There were a high number of non-contributing items that suggest that some items in the test may not be valid for South African Children.

Background: Many cancer patients who require external beam radiotherapy such as breast cancer patients, present with irregular surface topographies and tissue inhomogenieties in the treatment field. Such irregularities give rise to unacceptable dose non-uniformity. Standard fields cannot be applied without compensation for missing tissue. 1-D and 2-D missing tissue compensators can be used but they have limitations. 3-D compensators are the most effective but they are normally fabricated using very expensive automated systems. Objectives: To study the variation of linear attenuation coefficients of different materials in megavoltage photon beams, select a tissue equivalent compensating material and commission a local 3-D manual missing tissue compensator cutter. Methods and materials: Linear attenuation coefficients were measured for tin, River sand mix, Lincolnshire bolus and dental modelling wax for different energy megavoltage photon beams. Measurements were done in a water phantom using a cylindrical ionisation chamber at varying depths. The CT numbers and densities of the materials were also measured. Negative plaster of paris moulds of the breast and head and neck areas were made using a RANDOTM Alderson anthropomorphic phantom from typically simulated fields. 3-D missing tissue compensators were then fabricated on the manual cutter and were tested for their effectiveness during treatment delivery. Results: Linear attenuation coefficients were dependent on photon beam energy, the thickness and density of the attenuator, but independent of the depth of measurement for compensator thickness of more than 2 cm. Lincolnshire bolus and dental modelling wax with CT numbers of –78 ± 9 and -88 ± 18 and densities of 1.4 ± 0.0 g/cm3 and 0.9 ± 0.0 g/cm3 respectively can be regarded as tissue equivalent materials. The fabricated 3-D missing tissue compensators were effective in correcting for dose non-uniformities compared to fields with no beam-modifying devices or wedges (1-D compensators). Conclusions: The 3-D missing tissue compensators were effective in correcting for dose non-uniformities in treatment fields involving very irregular surface topographies compared to 1-D and 2-D methods. They can be fabricated cheaply using a 3-D manual missing tissue compensator cutter. Quality control procedures need to be followed during fabrication.

Le cancer, principale cause de mortalité dans le monde, est un problème majeur de santé publique. Malgré les nombreux traitements disponibles, il est nécessaire de développer de nouvelles thérapies plus efficaces et moins envahissantes. Aujourd’hui la connaissance du génome humain a dirigé la recherche vers de nouvelles approches: il est possible de moduler la réponse biologique en contrôlant l'accès aux informations génétiques via la régulation épigénétique.L’épigénétique est l’ensemble des modifications de l’expression des gènes n’entraînant pas de modifications dans la séquence d’ADN, qui mènent à un phénotype héritable et stable. Chez les eucaryotes, la régulation épigénétique implique des modifications covalentes de l'ADN (méthylation) et des histones (acétylation, méthylation…). Ces phénomènes modifient la structure de la chromatine, aboutissant à une configuration "ouverte" ou "fermée" permettant la transcription ou la répression de gènes. Dans une situation cancéreuse, le profil épigénétique est modifié ; la méthylation anormale de l’ADN ou des histones mène à la répression de certains gènes comme des gènes suppresseurs de tumeur, ou à l’expression des oncogènes. Contrairement aux changements génétiques irréversibles, les aberrations épigénétiques sont des modifications chimiques réversibles. Ainsi, des molécules capables de rétablir l'équilibre épigénétique représentent des outils thérapeutiques potentiels contre le cancer.La méthylation et l’acétylation sont les modifications épigénétiques les plus étudiées. La méthylation de l’ADN est catalysée par les ADN méthyltransférases (DNMTs), et la méthylation des histones par les histones méthyltransférases (HMTs).Le sujet de ce projet doctoral est porté sur les HMTs et en particulier sur DOT1L (DOT1 like, disruptor of telomericsilencing), responsable des méthylations du résidu Lys79 de l’histone 3 (H3K79), conduisant à la transcription des oncogènes. En effet, des études ont montré que DOT1L est liée à la leucémie et se révèle être une cible intéressante à inhiber. DOT1L comme les DNMT ont un même cofacteur : le SAM (S-adénosyl-L-méthionine). Certains de leurs inhibiteurs présentent un mécanisme d'inhibition commun : ils entrent en compétition avec SAM.Nous présentons la conception basée sur des études de modélisation moléculaire, et la synthèse multi-étapes des séries des molécules formées par 3 motifs principaux : a) un motif aminopyrimidine, b) un motif de type benzimidazole ou phénylurée, liés par c) un groupement phényle ou hétérocyclique. L’activité des composés synthétisés sur DOT1L a été évaluée et des relations structure-activité (RSA) ont été établies. L’activité sur DNMT et d’autres HMTs a été déterminée également afin d’étudier la spécificité de nos composés.Différents structures ont été identifiées comme point de départ pour aboutir à des inhibiteurs sélectives de DOT1L ou à des inhibiteurs mixtes DOT1L/DNMT. Ces molécules sont considérées comme des outils thérapeutiques intéressants dans le traitement du cancer. / Cancer is a serious issue of public health as it is one of the main causes of mortality worldwide. Despite the multiple available treatments, it is necessary to develop more efficient and less invasive therapies against cancer. The knowledge of the human genome and epigenome has directed research to new cancer treatment approaches: it is possible to modulate the biological outcome by controlling the access to the genetic information by means of the epigenetic regulation.Epigenetics are the changes happening on the genome without modifying its DNA sequence, leading to a heritable andstable phenotype. In the eukaryotic chromatin, epigenetic regulation implies covalent modifications of DNA and histones. These chemical modifications remodel the chromatin structure leading to an “opened” or “closed” configuration, which is related to the expression or repression of genes. The epigenetic landscape is altered in cancers; for example, abnormal methylation leads to the silencing of certain genes (such as tumor suppressor genes), or to the over-expression of oncogenes. Unlike genetic alterations that are irreversible, epigenetic aberrations are reversible. Thus, molecules that can reestablish the epigenetic balance represent potent therapeutic tools for cancer treatment.Methylation and acetylation are the most studied epigenetic modifications. DNA methylation is carried out by the DNAmethyltransferases (DNMTs) and histone methylation by the histone methyltransferases (HMTs).This PhD project was focused on the histone methyltransferase DOT1L (DOT1 like, disruptor of telomeric silencing), responsible of methylation of residue Lys79 of histone 3 (H3K79), which leads to the transcription of some oncogenes. Recent studies have shown that DOT1L is implicated in MLL-rearranged leukemia (MLL-r, Myeloid-Lymphoid Leukemia) thus it is a potent target in cancer. As DOT1L and DNMTs share the same cofactor, S-adenosyl-L-methionine (SAM), DNMT and DOT1L inhibitors can present a common inhibition mechanism by competing with SAM.We present herein the in silico – based design, and the multi-step synthesis of some series of molecules containing 3 main moieties: a) an aminopyrimidine motif and b) a benzimidazole or phenylurea motif, linked by c) a phenyl or heterocycle motif. DOT1L activity was determined for the different compounds synthesized and structure-activity relationships (SAR) were established. The activity on DNMT and other HMTs was determined as well, in other to study the DOT1L specificity of our compounds.Different scaffolds were identified to obtain DOT1L-selective or DOT1L/DNMT dual inhibitors. These molecules are interesting therapeutic tools for cancer treatment.

Thesis advisor: Robert Meyerhoff / Thurston showed that for all but a finite number of Dehn Surgeries on a cusped hyperbolic 3-manifold, the resulting manifold admits a hyperbolic structure. Global bounds on this number have been set, and gradually improved upon, by a number of Mathematicians until Lackenby and Meyerhoff proved the sharp bound of 10, which is realized by the figure-eight knot exterior. We improve this result by proving a stronger version of Gordon’s conjecture: that excluding the figure-eight knot exterior, cusped hyperbolic 3-manifolds have at most 8 non-hyperbolic Dehn Surgeries. To do so we make use of the work of Gabai et. al. from a forthcoming paper which parameterizes measurements of the cusp, then uses a rigorous computer aided search of the space to classify all hyperbolic 3-manifolds up to a specified cusp size. Their approach hinges on the discreteness of manifold points in the parameter space, an assumption which cannot be made if the manifolds have infinite volume. In this paper we also show that infinite-volume manifolds, which must be Free Bicuspid, can have cusp volume as low as 3.159. As such, these manifolds are a concern for any future expansion of the approach of Gabai et. al. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Mathematics.

Photochemistry studies of phenyl substituted-1,2,4-thiadiazoles have revealed that 5-phenyl-1,2,4-thiadiazoles 31, 90, 98, 54 and 47 undergo a variety of photochemical reactions including photofragmentation, phototransposition, and photo-ring expansion while irradiation of 3-phenyl-1,2,4-thiadiazoles 46, 105 and 106 leads mainly to the formation of photofragmentation products. The formation of the phototransposition products has been suggested to arise from a mechanism involving electrocyclic ring closure and sigmatropic sulfur migration via a bicyclic intermediate: phenyl-1,3-diaza-5-thiabicyclo[2.1.0]pentene (BC). 15N-Labeling experiments confirm that sulfur undergoes sigmatropic shifts around all four sides of the diazetine ring. Thus, irradiation of 31-4-15N or 54-4-15N leads to the formation of 31-2-15N or 54-2-15N and to an equimolar mixture of 46-2-15N and 46-4-15N or 57-2-15N and 57-4-15N. Work in this laboratory on 15N-labeling of 46-2-15N also shows that 46 does not undergo electrocyclic ring closure but reacts exclusively by photofragmentation of the thiadiazole ring. 15N-Scrambling in the photofragmentation products observed after irradiation of 31-4-15N or 54-4-15N is greater than 15N-scrambling in the starting thiadiazoles suggesting that these products cannot arise only from direct fragmentation of the thiadiazole rings. An additional pathway for the formation of these products is required. The formation of phenyltriazines, the photo-ring expansion products 39 and 40 or 65 and 66 from photolysis of 31 or 54 is proposed to arise via phenyldiazacyclobutadienes (CB), generated from elimination of atomic sulfur from the bicyclic intermediates. It is suggested that phenyldiazacyclobutadienes then undergo [4+2] cycloaddition self-coupling resulting in the formation of unstable tricyclic intermediates which finally cleave to give phenyltriazines and nitriles. The observed 15N distribution in the phenyltriazine photoproducts formed after photolysis of 31-4-15N or 54-4-15N and the formation triazine 72 after irradiation of a mixture of 31+54 are consistent with this mechanism. The formation of nitriles by this pathway would account for the additional 15N-scrambling in the photofragmentation products. The photochemically generated phenyl-1,3-diaza-5-thiabicyclo[2.1.0]pentenes are the key intermediates in this suggested mechanism. In the presence of furan, these intermediates are expected to be trapped as Diels-Alder adducts. Irradiation of phenylthiadiazoles 31, 54 and 47 in furan solvent lead to increased consumption of these thiadiazoles, to quenching of the known photoproducts, and to the formation of new products suggested to result from furan trapping of the thiadiazoles followed by elimination of sulfur. Irradiation of 46 in furan solvent leads only to the formation of the photofragmentation product; no furan trapping adduct is observed. This result is consistent with the 15N-labeling experiment indicating that 46 does not undergo electrocyclic ring closure after irradiation. The photoreactivity of these phenylthiadiazoles in acetonitrile is substantially decreased when the phenyl ring at position 4 is substituted with an electron donating or withdrawing group. However, they are more photoreactive in cyclohexane solvent than in acetonitrile. The fluorescence emission spectra of these (4¢-substituted)phenyl-1,2,4-thiadiazoles exhibit moderate - large Stokes' shifts in acetonitrile. The magnitudes of these Stokes' shifts decrease in cyclohexane. This suggests a charge transfer character associated with the excited states of these thiadiazoles. In acetonitrile, these charge transfer excited states would be stabilized and become the lowest energy excited state. These charge transfer excited states would not be photoreactive and, thus, fluorescence emission becomes an effective deactivation process. In cyclohexane solvent, the charge transfer excited states would be less stabilized and, thus, the relaxed S1 would, then, become the lowest excited state. The relaxed S1 would be the state from which the observed photoproducts originate and the observed fluorescence with the smaller Stokes' shifts compared with the Stokes' shifts observed in acetonitrile.

Made available in DSpace on 2019-03-30T00:21:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-30 / Obesity and Its co morbidity has become world endemic health problem. Among several triggers to this condition, evolution in diet composition with large amounts intake of energetic foods, added to limited physical activities due to the modern people habits, are notables factors. Change in ratio omega-6/omega-3 fatty acids intake is an important obesity cause and can make the co morbidity become worst. Many researches evidence the role of the fatty acids omega-6/ômega3 ratio as effective health promoter by, modulating its bio-active derivatives; the eicosanoids and cytokines. The aim of this study is to evidence the anti-obesity effect of a diet supplementation with omega-3 fatty acids, and, specific aims are following up rats corporal weight, adipocytes size and blood levels of total cholesterol, high density lipoprotein (HDL) and triglycerides. This was an experimental study with five Wistar male rats groups (n=6), with body weight upper 260g. One group was euthanized at day one; two groups received intake of water by gavage during 45 and 90 days, respectively, and two groups received intake of 0,1mg/kg/weigh/day of high purified omega-3 (EPA 350 mg/ml + DHA 220 mg/ml) for 45 and 90 days. All animals received intake of water and food at libitum during the experience. .The results showed decreasing in blood total cholesterol, and triglycerides levels (p<0,05), increase in HDL blood level (p<0,05), decrease in weight and had reduced its adipocytes area (p< 0,01) with omega-3 intake. The group which received omega-3 for 45 days compared to the 90 days group choose less parameter reductions. None of the rats presented any sign of side effects during the study. Conclusion: the current study evidenced ante obesity effect, in rats, of omega-3 fatty acid intake. This effect was timedependent. Keywords: PUFAS n-3; Obesity; Metabolic Syndrome; low grade Inflammation. / Obesidade tornou-se um problema endêmico mundial de saúde, com graves repercussões decorrentes de suas comorbidades. Entre os fatores predisponentes, mudanças nos padrões alimentares é o mais agravante. A inversão na composição alimentar da relação entre ácidos graxos ômega-3 e ômega-6, é fator indutor de obesidade e síndrome metabólica. Há evidências na literatura científica de que o balanço desses ácidos graxos pode ser uma poderosa arma na promoção de saúde, por modular ação dos derivados bioativos do grupo dos eicosanoides e citocinas, além de interferir com outros fatores indutores de obesidade e síndrome metabólica. O objetivo principal desta pesquisa foi investigar o efeito ante obesidade da suplementação com ômega-3 em ratos, utilizando como metas específicas observar evolução do peso, tamanho dos adipócitos e níveis sanguíneos de colesterol total, lipoproteína de alta densidade e triglicerídeos dos ratos sob o efeito do ômega-3. Foi uma pesquisa experimental com 5 grupos (n=6) de ratos Wistar machos, com peso corporal superior a 250g, fornecidos pelo Núcleo de Biologia Experimental da Universidade de Fortaleza. Um grupo controle foi sacrificado no primeiro dia utilizado como parâmetro basal dos animais. Dois grupos receberam água por gavgem 45 e 90 dias, e dois grupos receberam por 45 e 90 dias, 1mg /kg ao dia de gel de ômega-3, com ácido eicosapentaenoico (EPA, 350 mg/ml + ácido docosahexaenoico (DHA, 220 mg/ml). Todos os ratos receberam água e alimento ad libitum. Os resultados mostraram efeito, tempo dependente, reduzindo níveis de colesterol total e triglicerídeos (p<0,05), aumentando níveis de HDL (p<0,05), e evitando ganho de peso nos ratos (p<0,05), além de conter o aumento dos adipócitos (p<0,01) no grupo que recebeu ômega-3 por 90 dias, comparados aos grupos que receberam água. O grupo que recebeu ômega-3 por 45 dias apresentou menores índices na redução dos parâmetros citados. Nenhum dos ratos apresentou sinal de efeitos adversos durante a pesquisa. Como conclusão, verificamos que a suplementação com ômega-3 em apresenta evidente ação ante obesidade. Novas pesquisas devem ser feitas visando efeito emagrecedor em humanos e a possível utilização no controle e prevenção da obesidade. Palavras-chave: Ômega-3; Obesidade; Síndrome Metabólica; Inflamação de baixo grau.

Dans cette étude on décrit une méthode reproductible et efficace pour isoler des cellules microgliales et leurs exosomes. Les résultats montrent qu'il n'y a pas de différences morphologiques entre les cellules microgliales issues d'origines tissulaires différentes e.g. cortex et moelle épinière. D'autre part, en utilisant une plateforme de protéomique à grande échelle, nous démontrons que les microglies dérivées du cortex et de la moelle épinière des rats expriment des phénotypes différents tant dans les conditions physiologiques normales ou inflammatoires. Cette différence a été confirmée également au niveau des exosomes qu’elles sécrètent.Des essais biologiques in vitro démontrent que les exosomes dérivées de microglies testées sur des sphéroïdes 3D de gliomes de rat étaient capables d'inhiber l'invasion tumorale. Ces résultats ont permis de mettre en évidence que des exosomes dérivées de la microglie pouvaient être utilisés comme agents nano thérapeutiques vis-à-vis des gliomes. Sur la base des études précédentes conduites au laboratoire, nous avons montré que les EVs isolées à partir de macrophage KD PC1/3 traitées avec Paclitaxel inhibaient la croissance de la lignée C6 de gliome de rat. Nous avons isolé les exosomes et nos résultats mettent en valeur le potentiel d'une stratégie thérapeutique combinant Paclitaxel et inhibition de PC1/3 et utilisation des exosomes produits par ces cellules comme agents thérapeutiques. / In present study, we present a reproducible and an efficient method for isolating microglial cells and their exosomes. The results show that there are no morphological differences between microglial cells from different tissue origins e.g. cortex and spinal cord. On the other hand, using a large-scale proteomic platform, we demonstrate that microglia derived from the cortex and spinal cord of rats express different phenotypes under both normal and inflammatory physiological conditions. This difference has also been confirmed at the level of the exosomes they secrete.In vitro bioassays demonstrate that microglia-derived exosomes tested on 3D spheroids of rat gliomas were able to inhibit tumor invasion. These results made it possible to demonstrate that exosomes derived from microglia could be used as nano-therapeutic agents vis-à-vis gliomas.Based on previous studies conducted in the laboratory, we have shown that EVs isolated from KD PC1/3 macrophage treated with Paclitaxel inhibited the growth of the rat glioma C6 line. We isolated the exosomes and our results highlight the potential of a therapeutic strategy combining Paclitaxel and PC1/3 inhibition and use of the exosomes produced by these cells as therapeutic agents.

O objetivo deste estudo foi avaliar a influência de dietas suplementadas com semente de linhaça (ricas em ácido alfa-linolênico, LNA, ômega3) e antioxidantes naturais, provenientes do orégano e do alecrim, sobre o nível de incorporação dos ácidos graxos poliinsaturados ômega 3 (PUFA ômega3) em ovos e tecidos de aves. Para isto, 192 galinhas poedeiras da linhagem comercial Babcock de 22 semanas, foram alimentadas durante 30 dias com dietas constituídas de 0 (zero) e 5% de óleo de linhaça. Foram definidos 8 tratamentos: 4 grupos com 5% de óleo de linhaça (controle/sem antioxidante; BHA+BHT, 100+100 ppm; orégano, 200 ppm; alecrim, 200 ppm) e 4 grupos sem óleo de linhaça, mas utilizando os mesmos antioxidantes. A amostragem dos ovos foi realizada durante o experimento nos períodos 0, 10, 20 e 30 dias e dos tecidos das aves (sobrecoxa, coxa, asa, peito, coração, tecido adiposo e fígado) no tempo final do experimento. Os ácidos graxos foram determinados por cromatografia gasosa e o grau de oxidação lipídica através do teste do ácido tiobarbitúrico (TBA). De acordo aos resultados obtidos, verificou-se aumento significativo dos ácidos graxos alfa-linolênico (LNA) e docosahexaenóico (DHA) nas gemas de ovo das aves que receberam 5% de óleo de linhaça nos tratamentos controle, BHA+BHT, orégano e alecrim, nos diferentes tempos (10, 20 e 30 dias), quando comparados com a dieta 0% linhaça. Além disso, a incorporação máxima dos ácidos LNA e DHA nas gemas de ovo foi obtida aos 20 dias de alimentação das aves, com um índice de incorporação de 15 a 30 e de 2,5 a 4,5 vezes o grupo controle, respectivamente. Na avaliação do grau de oxidação lipídica nas gemas de ovo, foi verificada diferença significativa na redução dos valores de absorbância nas 2 dietas, em todos os tratamentos com antioxidantes, quando relacionados ao seu respectivo controle. Com relação aos tecidos das aves, também houve incorporação significativa dos ácidos LNA e DHA, quando comparadas as duas dietas, sendo o fígado o tecido que apresentou a maior concentração destes ácidos graxos. Também foi possível verificar a eficácia dos antioxidantes naturais na proteção contra a oxidação lipídica nos tecidos sobrecoxa, coxa, asa e peito. Portanto, os extratos das especiarias, alecrim e orégano, podem ser utilizados satisfatoriamente para se obter ovos e tecidos de aves enriquecidos com PUFA ômega-3, melhorando a estabilidade lipídica. Considerando que os PUFA ômega-3 têm um interesse considerável na saúde humana, o fornecimento de dietas ricas em ácido alfa-linolênico presente na linhaça, para galinhas poedeiras, permitiu a obtenção de ovos e tecidos enriquecidos, os quais tornam-se uma fonte alternativa de PUFA ômega-3. / The aim of this work was to evaluate the influence of diets containing flaxseed (rich in alpha-linolenic acid, LNA, omega-3) and natural antioxidants from oregano and rosemary on the level of incorporation of the polyunsaturated fatty acids omega 3 (PUFA omega-3) in eggs and tissues of poultry. For this purpose, 192 laying hens at 22 weeks of age, of commercial lineage Babcock, were fed for 30 days with diets containing 0 (zero) or 5% of flaxseed oil. The hens were divided in 8 groups: 4 groups received diets with 5% of flaxseed oil (control / no antioxidant; BHA+BHT, 100+100 ppm; oregano, 200 ppm; rosemary, 200 ppm) and 4 groups received no flaxseed oil, but the same antioxidants. The sampling of the eggs was accomplished in 4 periods (0, 10, 20 and 30 days) and of the tissues of poultry (upper thigh, thigh, wing, breast, heart, adipose tissue and liver) at the end of experiment. The fatty acids were analysed by gas chromatography and the lipid oxidation was determined by thiobarbituric acid test (TBA). The results showed that the levels of alpha-linolenic (LNA) and docosahexaenoic (DHA) acids increased in egg yolks from hens of the 4 groups fed diets with 5% flaxseed oil after 10, 20 and 30 days, when compared with diets 0% flaxseed oil. In addition, the maximum incorporation of LNA and DHA in egg yolks was obtained after 20 days, with an index of incorporation ranging from 15 to 30 and from 2.5 to 4.5, respectively. Also, a significant decrease of lipid oxidation in egg yolks for all groups receiving antioxidants was observed, when related with control. In the tissues of the hens, there was also significant incorporation of the LNA and DHA acids, when comparing the 2 diets, with the liver presenting the major concentration of these fatty acids. It was also possible to verify the effectiveness of the natural antioxidants in the protection against lipid oxidation in upper thigh, thigh, wing and breast tissues. Therefore, rosemary and oregano can be used satisfactorily to obtain eggs and tissues of poultry enriched with PUFA omega-3, improving the lipid stability. Considering that the PUFA omega-3 have considerable interest in the human health, the administration of diets rich in alpha-linolenic acid from flaxseed to laying hens allows the eggs and tissues enrichment as an alternative source of PUFA omega-3.

Made available in DSpace on 2018-08-01T23:26:47Z (GMT). No. of bitstreams: 1 tese_8177_65 - Juliany Morosini França.pdf: 1061932 bytes, checksum: b8255bdf847e632117bc64cfe166dd25 (MD5) Previous issue date: 2016-06-28 / O gênero Passiflora apresenta elevada diversidade genética, contudo os plantios comerciais se restringem a duas espécies, o que tem caracterizado uma baixa resistência as principais doenças no maracujazeiro. Nesse contexto objetivou-se analisar o vigor rizogênico e trocas gasosas na diversidade genética de Passiflora mucronata Lam. tratadas ou não com auxina, avaliar o número de segmentos apicais em lavouras comerciais de P. edulis Sims f. flavicarpa Deg. e testar dois métodos de enxertia entre P. edulis f. flavicarpa e P. mucronata através do uso de fixadores. Entre as espécies silvestres, a Passiflora mucronata apresenta características agronômicas relevantes à produção clonal, pela resistência à bacteriose nas folhas, a antracnose nos frutos e ramos e à fusariose. Neste cenário desenvolveram dois capítulos: o primeiro trata-se da avaliação da diversidade e vigor rizogênico de genótipos de P. mucronata tratados ou não com auxina. O delineamento utilizado foi o de blocos ao acaso, em esquema fatorial 9x2 [genótipos: 1; 2; 3; 4; 5; 6; 7; 8 e 9 x auxina (AIB ácido indol-3-butírico): ausência e presença (1000 mg kg-1)] com quatro repetições de 16 estacas. As características radiculares avaliadas foram emergência; índice de velocidade de emergência, tempo médio de emergência e massa seca, não obstante avaliou-se massa seca de parte aérea, fotossíntese líquida, condutância estomática, carbono interno, transpiração. O segundo objetivou-se avaliar enxertias entre P. edulis f. flavicarpa e P. mucronata. O delineamento empregado foi em blocos ao acaso, fatorial 3x2 [modalidade de enxertia: garfagem e encostia x fixadores: parafilme® micropore® e vedarosca®], com quatro repetições de seis enxertos. As características avaliadas foram o número de segmentos apicais em lavouras comerciais e porcentagem de pegamento. Os dados foram submetidos à análise de variância e as médias dos diferentes tipos de estacas, comparadas pelo teste de Tukey e por correlação fenotípica. A diversidade genética foi estudada de acordo com o método de agrupamento de Tocher, baseado na distância de Mahalanobis (D2 ii) e variáveis canônicas. Considerando as variáveis analisadas, é indicado à utilização do ácido-indol-3-butírico, nas estacas, na concentração de 1000 mg kg-1, devido ao aumento da porcentagem no vigor rizogênico, taxa fotossintética líquida, massa seca radicular e aérea em alguns genótipos quando comparado à ausência de auxina. A diversidade genética permitiu a formação detrês agrupamentos. As variáveis morfofisiológicas de maior contribuição relativa para a divergência genética na ausência e presença de auxina, foram, na ausência, IVER (38,75), E (20,27%) e MSRA (15,87 g) e na presença, gS (23,19 mol m-2 s-1), MSRA (20,91 g) e MSPA (19,66 g). As lavouras comerciais de P. edulis f. flavicarpa, podem contribuir expressivamente na minigarfagem, através da produção de segmentos apicais, sem prejuízo à planta matriz. Os tratamentos representados pelos métodos de enxertia (minigarfagem e encostia) e fixadores (parafilme®, micropore® e veda rosca®) apresentaram média de 94,90%, indicando enorme compatibilidade entre as espécies estudadas independentedo método de enxertia e dos fixadores na união P. edulis f. flavicarpa e P. mucronata. Indica-se o fixador parafilme® pela praticidade e facilidade de manuseio, recomendável para os dois métodos de enxertia.

Resumo: A presente dissertação permitiu quantificar a presença de glicerol-3-fosfato oxidase (GPO, sn-glicerol-3-fosfato: oxigênio 2-oxidorredutase, EC 1.1.3.21) em extratos de levedura seca de panificação por dois métodos: polarográfico e colorimétrico. A melhor metodologia de purificação da GPO foi obtida por rompimento celular com esferas de vidro, em homogenizador do tipo Bead Beater (Biospec products, USA), por 15 minutos, com 27,6% de eficiência de lise celular. O extrato celular bruto foi tratado com 1% de sulfato de estreptomicina antes da precipitação com igual volume de solução 30% (p/v) de polietilenoglicol 3350, dialisado e a sua atividade otimizada por método colorimétrico. A determinação das características da GPO foi possível em ensaios contendo: 250 mM de glicerol-3-fosfato em tampão 0,1 M Tris-HCl pH 8,0 contendo 0,1% Triton X-100; 0,0133% de 4-aminoantipirina; 0,0266% de fenol; cerca de 0,40 unidade de peroxidase (PO) e água destilada para completar o volume de ensaio. A reação foi iniciada pela adição de 15 æL de extrato enzimático diluído 10 vezes seguido de uma incubação de 2 horas a 60°C e interrompida pela adição de solução 10% de SDS e a coloração desenvolvida foi medida a 500 nm. A GPO apresentou alta estabilidade térmica, pH de estabilidade entre 7,0 - 8,0 e a presença de azida de sódio na concentração de 0,05% manteve a atividade da enzima por 21 dias a 40°C. Este método permitiu também quantificar glicerol-3- fosfato, importante metabólito intermediário da biossíntese lipídica e glicolítica, na faixa de 56 - 250 mM. / Abstract: The present dissertation allowed to quantify the presence of glycerol-3- phosphate oxidase (GPO, sn-glycerol-3-phosphate: oxygen 2-oxidoreductase, EC 1.1.3.21) in baker’s yeast extract by two methods: polarographic and colorimetric. The best methodology of purification of GPO was obtained by cell debris with glass beads, in a Bead Beater homogenizator (Biospec products, USA), for 15 minutes, with 27.6% of efficiency of cellular lysis. The crude cellular extract was treated with 1% of streptomycin sulfate before the precipitation, with equal volume of a solution of 30% (w/v) polyethylene glycol 3350, dialysed and its activity was optimized by colorimetric method. The determination of the characteristics of GPO was possible in assays containing: 250 mM of glycerol-3-phosphate in 0.1 M Tris-HCl buffer, pH 8.0 containing 0.1% Triton X-100; 0.0133% of 4-aminoantipyrine; 0.0266% of phenol; about 0.40 unit of peroxidase (PO) and water distilled to complete the volume. The reaction was started by the addition of 15 ìL of enzymatic extract diluted 10 times, followed by incubation of 2 hours at 60°C, interrupted by the addition of solution 10% of SDS, and the developed coloration was measured at 500 nm. GPO presented high thermal stability, pH of stability among 7.0 - 8.0, and the presence of sodium azide in the concentration of 0.05% maintained the activity of the enzyme for 21 days at 40°C. This method also allowed to quantify glicerol-3- phosphate, important intermediate metabolite of lipid biosynthesis and glycolysis, in the range 56 - 250 mM. / Orientador: Edwil Aparecida de Lucca Gattás / Coorientador: Maristela de Freitas Sanches Peres / Banca: Valdir Augusto Neves / Banca: Maria de Lourdes T. de Moraes Polizeli / Mestre