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Horses that are exposed to Sarcocystis neurona, a causative agent of equine protozoal myeloencephalitis, produce antibodies that are detectable in serum by western blot (WB). A positive test is indicative of exposure to the organism. Positive tests in young horses can be complicated by the presence of maternal antibodies. Passive transfer of maternal antibodies to S. neurona from seropositive mares to their foals was evaluated. Foals were sampled at birth (presuckle), at 24 hours of age (postsuckle), and at monthly intervals. All foals sampled before suckling were seronegative. Thirty-three foals from 33 seropositive mares became seropositive with colostrum ingestion at 24 hours of age, confirming that passive transfer of S. neurona maternal antibodies occurs. Thirty-one of the 33 foals became seronegative by 9 months of age, with a mean seronegative conversion time of 4.2 months. These results indicate that evaluation of exposure to S. neurona by WB analysis of serum may be misleading in young horses. Cerebrospinal fluid (CSF) samples from 15 neonatal (2-8 day) foals were examined for the presence of antibodies to S. neurona by WB analysis. Twelve of 13 foals that were seropositive were also CSF positive, suggesting that maternal antibodies to S. neurona cross the blood-CSF barrier in neonatal foals resulting in a positive CSF WB. Repeat taps were performed on 5 of the foals which showed that the immunoreactivity of the western blot decreases over time. Two of the 5 foals were CSF negative at 83 and 84 days of age, with 1 foal still positive at 90 days, and 2 foals positive at 62 days. These results indicate that maternal antibodies to S. neurona in the CSF can confound WB results in neonatal foals up to several months of age. / Master of Science

Chemokines are chemotactic cytokines, which mediate cell trafficking and play a key role in mobilisation of leukocytes. More recently, chemokines and their cognate receptors have been described as key players in different aspects of cancer biology contributing to proliferation, angiogenesis and metastasis. In particular, chemokines CCL19 and CCL21 acting on their associated receptor CCR7 are postulated to be key drivers of lymph node metastasis in a number of malignancies including breast, colon, gastric, & thyroid cancers. It has been reported that the cleavage of the pre-cysteine bridge N-terminal residues of CCL21 (SDGGAQD) and of CCL19 (GTNDAED) renders both peptides incapable of fully activating CCR7. However, little is known about the nature of the interactions that occur between the N-terminal residues of CCL19 or CCL21 and the CCR7 receptor, or the role they have in activation of CCR7. The aim of this study is to investigate the role of the residues in the N-terminus of CCL19 and in particular CCL21 in the context of CCR7 activation and to use this information in the discovery of novel CCR7 antagonists or agonists. To achieve this, we synthesised a number of short (three to seven amino acids) peptides and peptidomimetics inspired by the seven N-terminal amino acid residues of CCL19 and CCL21 and pharmacologically characterised their ability to activate CCR7 or block the activation of CCR7 using a number of in vitro assays such as calcium flux, trans-well (Boyden chamber), and Western blotting. We also carried out computational studies to better understand and predict the activity of these peptides. Our results demonstrate that some of these peptides are indeed capable of acting as agonists or antagonists of CCR7. / Kuwait Health Ministry and Kuwait Civil Services Commission

Amyloidosis is a group of diseases that originate from misfolded proteins. These proteins form fibrils which is stored in the tissue and organs. Approximately three to twelve per million people are diagnosed with amyloid light chain immunoglobulin amyloidosis (AL) each year and the median age is 65. The prognosis for AL amyloidosis is poor with a median survival rate of six months to three years. The patients may survive an additional 15-20 years with treatment such as stem cell transplantation.   The aim of this rapport was to optimize a monoclonal antibody named pwlam to diagnose AL λ amyloid with immunohistochemistry (IHC) when the amount of sample is small. In this rapport three different methods were used, pressure preparation, IHC and western blot. For both pressure preparation and IHC, colour development occurred in case of positive results. Western blot was used as a quality control for the tissue since they were all originally diagnosed using that method.    A total of 26 samples were tested, 16 positive AL λ samples, five AL κ samples and five negative samples. Pwlam is developed against AL λ so the AL κ samples function as a quality control for the antibody since it doesn’t bind to the κ light chains. A total of seven AL λ samples had a good binding with pwlam. More research is needed before pwlam can be used in IHC on pressure preparations as a supplementary method to diagnose samples when the amount of AL λ amyloid is small.

Die rheumatoide Arthritis (RA) ist eine chronisch-entzündliche Bindegewebserkrankung mit bevorzugtem Befall der Gelenke. Es bestimmen Knorpel- und Knochendestruktionen das Krankheitsbild. Eine Schlüsselrolle in der Pathogenese nehmen proliferierende, synoviale Fibroblasten (RASF) durch Auflösung der extrazellulären Matrix (EZM), durch Interaktion mit immunkompetenten Zellen und durch Produktion pro-inflammatorischer Zytokine ein. Die vorliegende Arbeit zeigt die Migrationseigenschaften von RASF und zwei verschiedenen murinen (LS48) und humanen (TK188) Fibroblastenzelllinien in einem In-vitro-Migrationsassay. Es wird der Einfluss von Antikörpern sowie verschiedener EZM-Komponenten auf die Migration der Zelllinien untersucht. Die nachfolgende Analyse phänotypischer Charakteristika stellt dabei die besondere Rolle der genannten Fibroblastenzelllinien heraus, welche eine Reihe von Gemeinsamkeiten untereinander und mit RASF besitzen. Sie zeigen ebenso erhöhte Migrationsaktivität unter dem Einfluss eines Chemoattraktants und besitzen ähnliche Destruktionsmuster von Kollagenmatrizen. Beide Zellreihen exprimieren mehr RASF-typische Proteasen, Adhäsionsmoleküle und immunologisch agierende Proteine als nicht pathologisch transformierte Fibroblasten. Ebenso weisen sie eine gesteigerte Stoffwechselaktivität und Proliferationstätigkeit auf. Diese in vitro erbrachten Hinweise auf mögliche Knorpeldestruktionen sollten Anlass für weitere In-vivo-Studien zu den genannten Zelllinien geben.

Made available in DSpace on 2016-06-02T19:22:07Z (GMT). No. of bitstreams: 1 4217.pdf: 3961717 bytes, checksum: 3053793c39a626c9cf9d5fb8f9c0fa21 (MD5) Previous issue date: 2011-02-18 / Universidade Federal de Sao Carlos / The ventricular myocardial contractility of the matrinxã teleost, Brycon amazonicus, was analysed in previous experimentation with isometric multicellular preparations, in time effect and force-frequency relationship experiments, with and without exposure to the alkaloid ryanodine, a sarcoplasmic reticulum (SR) Ca2+ release blocker. In this study, different methodological approaches were used, such as isometric stress (EI, mN.mm-2), that permitted to identify the sensitivity of the myocardium to ryanodine, with the majority contribution of Ca2+ from the SR. The use of time to peak tension and time to half relaxation parameters (TPT and THR, ms) were ineffective to evaluate the contraction and relaxation performances during time effect experiments. New approaches such as contraction rates and initial relaxation rates (TC and TIR, mN.mm-2.s-1) demonstrated directly the impairment of the myocardium exposed to ryanodine. The negative staircase effect, characteristic of the teleost s forcefrequency relationship was evidenced by EI. The maximum rates of contraction and maximum rates of relaxation (TMC and TMR, mN.mm-2.s-1) and the average rates of contraction and average rates of relaxation (TMedC and TMedR, mN.mm-2.s-1) showed the impairment of the myocardium contractility exposed to ryanodine as well as the low sensitivity of frequency increments on the contractility when considered the values of TPT and THR. The TMedC and TMedR values indicated a greater possibility of heart rate regulation than the TMC and TMR values, probably due to these estimates consider the amount of instantaneous rate changes of contraction wave on calculation instead of just one point on the curve. The cardiac pumping capacity (CBC, mN.mm2.min-1) showed that the optimal range of frequency for isometric contraction is narrow and that the myocardium of the species should be working on the limit at rest. The analyses of integral of isometric tension (ITI, mN.mm- 2.s), and integral of isometric tension per minute (ITIPM, mN.mm-2.min), showed that these were unsuitable indexes for the assessment of cardiac contractility in the way they were calculated, as inconsistent interpretations were generated, probably by using information from the curve of contraction irrelevant to the isometric preparation. The contractility index (IC, mN.mm-2) created in this work, suggested that the myocardium contractility of the species is more sensitive to increases in frequency. On the other hand, the contractility index per minute (ICPM, mN.mm-2) showed that the optimum range of frequencies for the B. amazonicus myocardial contraction can be much wider and could allow performance reserve, such as reported in other studies of cardiac function in teleosts. The protein expression of SERCA2a and phospholamban (PLB) were analysed by Western Blot technique and their expression supported the findings of the SR functionality. The comparative analysis of these proteins, using amino acid sequences available in public databases (GenBank and UniProt), revealed levels of similarity between the SERCA2a and PLB in fish and other vertebrates, strengthened the findings of studies with Western Blot experiments. Taken together, the results suggest that B. amazonicus myocardium is dependent on SR Ca2+ stores under physiological frequencies and, despite the negative staircase pattern, must possess a performance reserve at supraphysiologic frequencies. / A contratilidade do miocárdio ventricular do teleósteo matrinxã, Brycon amazonicus, foi analisada com dados de experimentos prévios, realizados com preparações isométricas multicelulares e protocolos de efeito do tempo e de relação forçafrequência, com e sem exposição ao alcalóide rianodina, bloqueador dos canais de liberação de Ca2+ do retículo sarcoplasmático (RS). Nesse estudo foram utilizadas diferentes abordagens metodológicas de tratamento de dados, como o estresse isométrico (EI, mN.mm-2), a partir do qual foi possível identificar a sensibilidade do miocárdio à rianodina, com contribuição majoritária do Ca2+ proveniente do RS. A utilização dos tempos para o pico de tensão e para metade do relaxamento (TPT e THR, ms), se mostraram ineficazes para avaliar o desempenho da contração e relaxamento no experimento do efeito do tempo. Novas abordagens na análise dos dados, como a taxa de contração e taxa inicial de relaxamento (TC e TIR, mN.mm- 2.s-1) demonstraram explicitamente o comprometimento do miocárdio durante a exposição à rianodina. O efeito escada negativo, característico da relação forçafrequência de teleósteos foi evidenciado pelo EI. As taxas máximas de contração e de relaxamento (TMC e TMR, mN.mm-2.s-1) e as taxas médias de contração e de relaxamento (TMedC e TMedR, mN.mm-2.s-1) além de demonstrarem o comprometimento da contratilidade durante exposição à rianodina, indicaram que existe um menor comprometimento da tensão durante elevação da frequência, quando comparados aos valores de TPT e THR. A TMedC e a TMedR apresentaram resultados relacionados a uma frequência cardíaca com maior possibilidade de ajustes do que a TMC e a TMR, provavelmente por considerarem o conjunto de variação das taxas instantâneas da contração e não somente um único ponto da curva. A capacidade de bombeamento cardíaco (CBC, mN.mm-2.min-1) mostrou que a faixa ótima de frequência para contração isométrica é estreita e que o miocárdio da espécie deve estar trabalhando no limiar na condição de repouso. As análises da integral da tensão isométrica (ITI, mN.mm-2.s), e da integral da tensão isométrica por minuto (ITIPM, mN.mm-2.min) mostraram-se inapropriadas para a avaliação da contratilidade cardíaca pelo modo como foram calculadas, uma vez que geraram interpretações incoerentes, provavelmente por utilizarem informações da curva de contração irrelevantes para a preparação isométrica. O índice de contratilidade (IC, mN.mm-2) criado nesse trabalho, sugere que o miocárdio da espécie é mais sensível às elevações de frequência. Por outro lado, o índice de contratilidade por minuto (ICPM, mN.mm-2) mostrou que a faixa ótima de frequências para a contração cardíaca de B. amazonicus pode ser mais ampla, o que permitiria uma reserva de desempenho, assim como observado para outros teleósteos. A expressão das proteínas SERCA2a e fosfolambano (PLB) foram analisadas pela técnica de Western Blot e sua expressão reforçaram os achados de funcionalidade do RS. A análise comparativa dessas proteínas utilizando sequências de aminoácidos disponíveis em bancos de dados públicos (GenBank e UniProt) revelou os níveis de similaridade entre a SERCA2a e PLB de peixes e mamíferos, reforçando os achados dos estudos com Western Blot. Em conjunto, os dados sugerem que o miocárdio do B. amazonicus, apresenta uma nítida dependência do RS em frequências fisiológicas e, apesar de exibir um claro padrão escada negativo, deve apresentar uma reserva de desempenho para frequências suprafisiológicas.

Made available in DSpace on 2014-12-17T14:03:32Z (GMT). No. of bitstreams: 1 RaniereFMS.pdf: 3115798 bytes, checksum: 664aaedacd292e8b8c2d08a15e193378 (MD5) Previous issue date: 2010-02-25 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (?H and ??C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 ?g of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods, pharmaceuticals and chemical industries. / O sabugo de milho ? um subproduto agr?cola ainda pouco utilizado, isto se deve em parte ao baixo conhecimento do potencial biotecnol?gico de suas biomol?culas. Xilana de sabugo de milho (XSM) ? um polissacar?deo presente em maior quantidade na estrutura do vegetal e seu potencial biotecnol?gico ? pouco conhecido. Este trabalho teve como objetivo a extra??o, caracteriza??o qu?mica e avalia??o de atividades biol?gicas de XSM. Sabugos de milho foram limpos, cortados, desidratados e triturados, dando origem a uma farinha. Esta foi submetida a uma metodologia que combina o uso de meio alcalino com ondas de ultra-som. Ap?s precipita??o metan?lica, centrifuga??o e secagem obteve-se um rendimento de 40% (g/g de farinha). An?lises qu?micas indicaram um alto percentual de polissacar?deos na amostra (60%) e baixa contamina??o por prote?nas (0.4%) e compostos fen?licos (>0.01%). An?lises da composi??o monossacar?dica por cromatografia em papel e por cromatografia l?quida de alta performance (CLAE) indicaram a presen?a de xilose:glicose:arabinose:galactose:manose:?cido glucur?nico em uma propor??o molar de 50:20:15:10:2,5:2,5. A presen?a de xilana na amostra foi confirmada por resson?ncia magn?tica nuclear (13C e 1H) e por espectroscopia de infravermelho (IR). Testes foram realizados para avalia??o do potencial antioxidante de XSM. Esta mostrou uma capacidade antioxidante total de 48.45 EAA/g de amostra. Contudo, a mesma n?o mostrou atividade sequestradora de super?xido, radical hidroxila bem como poder redutor. Em contra partida, apresentou 70% de atividade quelante de ?ons de ferro na concentra??o de 2 mg/mL. A capacidade de XSM em influenciar a prolifera??o celular em cultura tamb?m foi avaliada. Este polissacar?deo n?o influenciou a prolifera??o de c?lulas fibrobl?sticas normais (3T3), entretanto, diminuiu a taxa de prolifera??o de c?lulas tumorais (HeLa) de maneira dose-dependente, chegando a uma inibi??o de aproximadamente 50% com concentra??o em torno de 2 mg/mL. Analisando prote?nas relacionadas ? morte celular, atrav?s de immunoblotting, XSM aumenta a quantidade de Bax, citocromo c e AIF e diminui Bcl-2 e procaspase- 3, indicando a indu??o de morte celular por apoptose dependente e independente de caspase. XSM n?o apresentou atividade anticoagulante pelo teste de PT. Todavia, no teste de tempo de tromboplastina parcial ativada (aPTT), XSM aumentou o tempo de coagula??o em cerca de 5 vezes utilizando 600 ?g de amostra, quando comparadas com o controle negativo. A presen?a de sulfato ligado a XSM foi descartada por eletroforese em gel de agarose e por IR. Ap?s carboxirredu??o de XSM a atividade anticoagulante diminuiu drasticamente. Os dados deste trabalho demonstram que XSM apresenta potencial como composto antioxidante, antiproliferativo e anticoagulante. Estudos futuros de caracteriza??o dessas atividades do XSM contribuir?o para aumentar o conhecimento sobre esta mol?cula extra?da de milho e permitir?o a sua utiliza??o em alimentos funcionais, produtos farmac?uticos e ind?strias qu?micas.

Depuis quelques années, les oligomères du peptide Aβ sont identifiés comme étant responsables du déclenchement de la pathologie alors que les dépôts amyloïdes sont des conséquences aggravantes de la pathologie. Cependant, les formes oligomériques d’Aβ impliquées dans la pathologie ainsi que l’origine de ces peptides sont toujours débattues. Notre objectif principal était d’identifier des signatures Aβ oligomériques cérébrales et vasculaires et de déterminer si nous pouvions interférer avec ces signatures pour modifier le décours de la pathologie. Nous avons réalisé des analyses biochimiques qualitative des formes d’Aβ dans des échantillons de cerveau et de vaisseaux issus de patients atteints de la MA et de souris transgéniques modèle de la MA. Nous avons montré qu’une même forme Aβ oligomérique (17-18 kDa) est impliquée dans le développement de la pathologie cognitive chez l’homme et chez la souris APP/PS1. Une signature Aβ oligomérique vasculaire spécifique a été observée dans les vaisseaux périphériques et plus particulièrement la veine porte hépatique des souris APP/PS1. De plus, un traitement pharmacologique ciblant l’expression des protéines de transport de l’Aβ a permis de restaurer les profils Aβ oligomériques contrôles dans le cerveau des souris APP/PS1 tout en « chargeant » la veine porte des mêmes souris en Aβ oligomérique. Ces résultats montrent que les signatures Aβ vasculaires et cérébrales sont intimement liées. De plus, nos travaux mettent l’accent sur une possible intervention thérapeutique agissant sur les formes Aβ cérébrales et vasculaires. / Alzheimer's disease (AD) is a complex disorder of the central nervous system that affects an increasing number of people worldwide due to the overall aging of the human population. Vascular factors and mechanisms have emerged as an area of key importance. Accumulating evidence indicates that pre-fibrillar aggregates, specifically the low-molecular weight oligomers of Aβ peptide, are responsible for the synaptic dysfunction and neuronal loss that occur in AD pathology. But, these oligomeric forms implicated in the pathology are currently under debate. Our primary goal was to identify cerebral and vascular oligomeric signatures. Secondly, we try to interfere with these signatures in order to modify the evolution of AD. We realize qualitative analyses of cerebral and vascular oligomers Aβ by western-blot. Vascular and cerebral tissues were extracted from AD patients and from a transgenicmouse model of AD. We demonstrate that the same oligomer Aβ (17-18 kDa) is implicated in the cognitive impairment for patients and APP/PS1 mouse. A specific vascular signature of oligomer Aβ was detected in peripheral vessels and particularly in portal vein from liver of APP/PS1 mouse. Moreover, pharmacological treatment targeting clearance of soluble Aβ restored the control signature of oligomer Aβ in the brain of APP/PS1 mouse. This configurational change was associated with an increase of oligomer Aβ in portal vein from liver. These results show that cerebral and vascular oligomeric signatures were closely linked. Finally, our work emphasizes potential therapeutic strategies for AD by targeting cerebrals and vasculars oligomers Aβ.

Orientador: Maeli Dal Pai Silva / Tese ( doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T12:24:51Z (GMT). No. of bitstreams: 1 Carani_FernandaRegina_D.pdf: 24872848 bytes, checksum: e4b7c794fa866be614e932ec5a33eaf6 (MD5) Previous issue date: 2011 / Resumo: O pirarucu (Arapaima gigas) caracteriza-se como uma espécie promissora para a Aqüicultura, devido principalmente às suas características de rápido crescimento e rusticidade. Sua criação em regime intensivo tem obtido enorme sucesso, podendo alcançar até 10 quilos de peso corporal em apenas um ano de criação. O pirarucu é considerado hoje uma das mais importantes espécies de peixes da bacia Amazônica e, por esta razão, é primordial que se investigue os mecanismos celulares e moleculares que controlam o rápido crescimento muscular, contribuindo com novas estratégias de criação e com a manutenção da espécie. O crescimento do músculo estriado esquelético nos peixes pode ocorrer por dois mecanismos: hipertrofia e/ou hiperplasia das fibras, a partir de células satélites ou mioblastos. Esse processo é controlado por Fatores Reguladores Miogênicos (MRFs) e pelo fator de crescimento Miostatina. O objetivo do presente estudo foi avaliar as características morfológicas e o crescimento muscular hipertrófico e hiperplásico, bem como analisar a expressão gênica e protéica da MyoD, da Miogenina e da Miostatina na musculatura esquelética do pirarucu (A. gigas), em diferentes fases de crescimento. Os animais utilizados no presente estudo foram provenientes de duas pisciculturas: na primeira, foram obtidos os "alevinos" (pós-larvas; 48 g); na segunda, os animais em diferentes estágios de crescimento, divididos em quatro grupos de acordo com o peso corporal. Grupo A (50 gramas, n=7), grupo B (420 gramas, n=7), grupo C (5,5 quilogramas, n=7) e grupo D (9,1 quilogramas, n=7). As amostras musculares foram coletadas, congeladas e submetidas à coloração HE, para avaliação do padrão morfológico das fibras, e à reação para a enzima NADH-TR, para avaliar o metabolismo oxidativo das fibras. Para avaliar o padrão de crescimento hiperplásico e hipertrófico da musculatura branca, foi calculado o menor diâmetro de uma população de fibras por animal, e estas foram distribuídas em classes, na dependência do seu diâmetro. A análise da expressão gênica de MyoD, miogenina e miostatina foi feita por Reação em Cadeia da Polimerase após Transcrição Reversa (RTqPCR); para análise da expressão protéica, foi utilizado o Western Blot. A distribuição das fibras em classes de diâmetro exibiu o seguinte padrão: o grupo A mostrou a maior parte das fibras na classe 20, o grupo B, na classe 50, o grupo C, nas classes 50 e 80, e o grupo D, na classe 80. Isso indica uma alta taxa de hiperplasia das fibras nos grupos menores (A e B) e alta hipertrofia das fibras nos grupos maiores (C e D). Para a análise da expressão gênica de MyoD e miogenina no músculo vermelho e branco dos "alevinos", não houve diferença estatística; para a miostatina, houve expressão diferencial, com os maiores níveis encontrados no músculo branco em comparação com o músculo vermelho. Na avaliação da expressão de MyoD e miogenina, tanto a expressão gênica como a expressão protéica não mostraram diferença significativa. Por outro lado, a expressão gênica da miostatina foi menor no grupo A e maior nos demais, e a expressão da proteína miostatina foi maior no grupo A, diminuindo nos demais grupos. Estes resultados refletem as características de crescimento muscular da espécie e sugerem que a expressão dos MRFs e da miostatina são responsáveis pelo balanço entre a hiperplasia e a hipertrofia das fibras, contribuindo para o rápido crescimento da espécie e a manutenção das características do filé / Abstract: Pirarucu (Arapaima gigas) is a promising fish species for Aquaculture programs mainly by the fast growing feature and rusticity. Their rearing under intensive conditions generated much successful results, as they reach up to 10 kilograms in just one year. Considered one of the most important fish species from Amazon basin, it is of primary interest to investigate the cellular and molecular mechanisms that control the fast muscle growth in pirarucu, providing information for new rearing strategies and species conservation. In most fish, skeletal muscle growth occurs by two mechanisms: hypertrophy and hyperplasia, from satellite cells or myoblasts. These process are under control by Myogenic Regulatory Factors (MRFs) and by the growth factor Myostatin. The animals were obtained from two pisciculture, where we got the alevin pirarucu (n=7; 48 grams weight), and the specimens at different growth stages, divided into groups according body weight. Group A (50 grams, n=7), group B (420 grams, n=7), group C (5,5 kilograms, n=7) and group D (9,1 kilograms, n=7). Muscle samples were collected, frozen and stained with HE for morphological analysis, and submitted to NADH-TR enzyme reaction for oxidative methabolism analysis. To evaluate hyperplasic and hypertrophic muscle growth, it was measured the smallest diameter from a set of fibers, which were grouped into diameter classes. Gene expression analysis of MyoD, Myogenin and Myostatin were performed by Quantitative Polimerase Chain Reaction after Reverse Transcription (RT-qPCR); protein content analysis was by Western Blot technique. Muscle fibers distribution in classes showed the following pattern: group A showed most fibers in class 20, group B, in class 50, group C, in classes 50 and 80, and group D, in class 80. This is an indicative of high fiber hiperplasia rate in groups A and B, and high hypertrophy in groups C and D. There was no statistical difference in MyoD and Myogenin genes expression in red and white muscles of pirarucu; however, Myostatin expression showed high levels in white muscle compared to red muscle. Evaluating MyoD and Myogenin expression in white muscle of pirarucu at different growth stages, both gene and protein levels were similar. Myostatin gene expression was low in group A and high in the other groups; on the other hand, Myostatin protein was high in group A and low in the other groups. These results reflect the muscle growth characteristics in pirarucu and suggest that the MRFs and Myostatin expression are controlling the balance between hyperplasic and hypertrophic mechanisms, promoting the fast rate feature of pirarucu and their fillet quality / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Six isoflavones derived from Millettia griffoniana namely, 4’-methoxy-7-O-[(E)-3-methyl-7hydroxymethyl-2,6 octadienyl]isoflavone (7-O-DHF), Griffonianone C (Griff C), 7-O-geranylformononetin (7-O-GF), 3’,4’-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO), Griffonianone E (Griff E), 4’-O-geranylisoliquiritigenin (4-O-GIQ) were tested for potential oestrogenic activities in three different oestrogen receptor alpha (ERα) dependent assays, namely a recombinant yeast assay, a reporter gene assay based on stably transfected MCF-7 cells (MVLN cells) and the induction of alkaline phosphatase in Ishikawa cells. The oestrogenic activities of isoflavones from Millettia griffoniana could be completely suppressed by the pure oestrogen antagonist, fulvestrant. The expression of Ki-67, proliferating cell nuclear antigen (PCNA) and cyclin D1 (CD1) mRNA used as indicator of cell proliferation in MCF-7 cells was assayed. Based on these in vitro results, Griff C was further tested in vivo. The main objective of this part of the work was to study the mechanistic basis of the oestrogenicity Three different doses of Griff C (2, 10, or 20 mg/kg BW) of Griff C in ovariectomised Wistar rats. 17β-oestradiol (E2: 10 µg/kg BW) was used as positive control. They were treated daily for three consecutive days and sacrificed 24 hours after receiving the last dose. The whole uterus was removed and weight. Liver and vena cava fragments were also collected and stored together with uteri in liquid nitrogen for subsequent real-time PCR to evaluate the effects of Griff C on the regulation of some relevant oestrogen–responsive genes in the uterus, the liver and the vena cava. The role of Griff C in apoptosis or in cell survival, through mediation of the phosphatidylinositol 3 kinase-Akt (PI3K-Akt) signaling pathway, was also investigated. Western blot analysis revealed that Griff C slightly increased the phosphorylation of Akt at its serine 473 residue. In this work, oestrogenic properties of the isoflavones derived from Millettia griffoniana are described using reporter gene assays and the oestrogen-inducible alkaline phosphatase Ishikawa model for the first time. These in vitro data were verified in vivo showing the regulation of the expression of various relevant oestrogen-responsive genes by Griff C. The spectrum of its activity was clearly similar to that of 17β-oestradiol on uterine hepatic and vena cava tissues of ovariectomised rats except for the proliferative response. However Griff C remained 100 to 1000 times less effective than oestradiol. These findings confirmed that some of the biological effects attributed to Millettia griffoniana are closely related to oestrogen-mediated action.