L'obésité est une maladie complexe dont la prévalence est en constante augmentation à travers le monde. A l'instar de nombreux médicaments qui ont pour origine des molécules végétales, de nouveaux principes actifs ont été recherchés dans les plantes. Parmi eux, les phytoecdystéroïdes, notamment la 20-hydroxyecdysone présente dans le quinoa, ont montré des effets pharmacologiques bénéfiques, et seraient donc des candidats potentiellement efficaces contre l'obésité. L'objectif de cette thèse est de mettre en évidence les propriétés d'un extrait de quinoa enrichi en 20-hydroxyecdysone sur le surpoids et l'obésité et d'en caractériser les effets.Dans un premier temps, un apport alimentaire d'extrait de quinoa enrichi en 20-hydroxyecdysone chez la souris soumise à un régime hyperlipidique a permis de limiter le développement du tissu adipeux, sans modifier la prise alimentaire. Dans le tissu adipeux, ces effets ont été associés à une diminution de la quantité de transcrits de gènes impliqués dans le stockage lipidique et l'inflammation. L'analyse du bilan énergétique a mis en évidence un effet de l'extrait de quinoa sur l'augmentation de la dépense énergétique liée à une augmentation de l'oxydation des glucides, ainsi que sur la diminution de l'absorption intestinale des lipides. Ensuite, chez le sujet en surpoids ou obèse, l'extrait de quinoa a montré une tendance à la prévention de la reprise de poids et de masse adipeuse suite à une restriction énergétique. Cet effet s'est accompagné d'un maintien de l'amélioration de l'insulino-sensibilité. Enfin, des mesures de biodisponibilité de la 20-hydroxyecdysone chez la souris ont permis de quantifier sa teneur circulante et de suivre l'apparition de certains de ses métabolites dont le rôle reste à identifier. Ces résultats montrent un bénéfice potentiel de l'extrait de quinoa et de son principe actif, la 20-hydroxyecdysone, sur la stabilisation du poids et de la masse adipeuse suite à une réduction pondérale. D'autres études seront nécessaires afin de poursuivre la caractérisation de ces effets. / Obesity is a complex disease which is dramatically increasing worldwide. Following the example of many drugs that were developed from plants molecules, new natural active principles are currently being evaluated in order to prevent or to treat metabolic disorders. Among them, phytoecdysteroids, and mainly 20-hydroxyecdysone present in quinoa, have displayed beneficial pharmacological effects. Thus, they would be potentially active candidates against obesity. The objective of this thesis is to highlight the properties of a quinoa extract enriched in 20-hydroxyecdysone on overweight and obesity and to characterize their effects.First, the ingestion of 20-hydroxyecdysone enriched quinoa extract by mice fed a high-fat diet limited adipose tissue development, without modifying food intake. In adipose tissue, these effects were associated with a reduction of the amount of transcripts of genes involved in lipid storage and inflammation. Energy balance analyses showed that quinoa extract increased energy expenditure via an increase in glucose oxidation and diminished lipid intestinal absorption. Then, in overweight and obese subjects, quinoa extract displayed a tendency to prevent weight and fat mass regain after energy restriction. This effect was accompanied by the maintenance of insulin sensitivity improvement. Lastly, 20-hydroxyecdysone bioavailability measurements in mice allowed its plasma level quantification and the identification of metabolites whose role is still to be identified. These results show a potential benefit of the quinoa extract and its active principle, 20-hydroxyecdysone, on weight and adipose mass stabilization following weight loss. More studies are required to characterize further these effects.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-10-10T14:41:13Z No. of bitstreams: 1 HEMERSON SILVA DA ROSA SET 2018.pdf: 5885122 bytes, checksum: 19b2f5a47b842bf54f4c2e9feacfcb4f (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-10-10T14:41:40Z (GMT) No. of bitstreams: 1 HEMERSON SILVA DA ROSA SET 2018.pdf: 5885122 bytes, checksum: 19b2f5a47b842bf54f4c2e9feacfcb4f (MD5) / Made available in DSpace on 2017-10-10T14:41:40Z (GMT). No. of bitstreams: 1 HEMERSON SILVA DA ROSA SET 2018.pdf: 5885122 bytes, checksum: 19b2f5a47b842bf54f4c2e9feacfcb4f (MD5) Previous issue date: 2017-08-10 / O presente estudo avaliou os efeitos antinociceptivos e anti-inflamatórios dos extratos obtidos de Sida tuberculata (ST), popularmente conhecida como “guanxuma”, em modelos agudo e crônico em camundongos. Além disso, foram feitas caracterizações fitoquímicas e análises do potencial antioxidante e citotóxico in vitro. De acordo com os dados encontrados, os extratos das folhas e raízes apresentaram um significativo efeito antioxidante (p<0.05) a partir das doses de 0.015 e 0.03 mg.mL-1 respectivamente, porém o extrato das folhas teve uma ação mais pronunciada comparado ao extrato das raízes (<IC50) sendo selecionado para os testes in vivo. Através das análises químicas, foi desenvolvido um método analítico por UHPLC para determinação do marcador fitoquímico, 20-hydroxyecdysone (20-HE), e também foram identificados oito compostos, sendo 20HE o majoritário juntamente com um derivado de Canferol. Nos testes de citotoxicidade in vitro, os extratos metanólicos das folhas (Sida tuberculata leaf extract - STLE) e raízes (Sida tuberculata root extract - STRE) demonstraram uma ação antiproliferativa contra as linhas celulares tumorais HepG2 e MCF-7 (IC50 entre 543.6 - 593.4 μg.mL-1 para STLE, e 397.1 - 493.9 μg.mL-1 para STRE). Também, STLE diminuiu a viabilidade em leucócitos humanos a partir da dose de 10 μg.mL-1. Já os resultados dos ensaios de dor e inflamação in vivo demonstraram que o STLE (10-300 mg.kg-1) administrado pela via oral (v.o.), 1h antes do teste, inibiu significativamente a nocicepção das fases neurogênica e inflamatória para o modelo de formalina (31,3 e 40,1% respectivamente). Do mesmo modo, STLE diminui significativamente as contorções induzidas por ácido acético, chegando a 71,8% de inibição na dose de 100 mg.kg-1. Os experimentos mostraram que, pelo menos em parte, o mecanismo de ação de STLE envolve os sistemas opióides e adenosinérgicos uma vez que seu efeito foi revertido pela Naloxona (inibidor não seletivo dos receptores opióides) e pelo DPCPX (inibidor seletivo do receptor de adenosina A1) no modelo de ácido acético. A avaliação por Docking computacional demonstrou que os compostos Canferol e 20HE, presentes no extrato, podem interagir principalmente com o receptor opióide μ. O pré-tratamento com STLE (100 mg.kg-1) também reduziu a migração celular total, o número de neutrófilos, a atividade da MPO, e o níveis das citocinas IL-1β, IL-6 e TNF-α no fluído peritoneal de animais com peritonite induzida por carragenina. No mesmo modelo foi verificado uma redução dos níveis de TBARS e um aumento nos níveis de NPSH. Além disso, o tratamento com STLE a 100 mg.kg-1 uma vez ao dia também apresentou efeito antinociceptivo no modelo de dor inflamatória crônica induzida por CFA (i.pl.), como ainda diminuiu o edema de pata após o quinto dia de tratamento. As doses administradas diariamente (100 mg.kg -1 durante 15 dias) não apresentaram alteração macroscópica e nem sobre o peso absoluto e o peso relativo dos órgãos vitais (coração, pulmão, fígado, baço e rins). Portanto, o conjunto de dados apresentados sugerem que ST possui uma significativa atividade antinociceptiva e anti-inflamatória frente a modelos agudo e crônico em camundongos, com mecanismo de ação envolvendo, parcialmente, sua ação antioxidante e a interação sobre o sistema opióide e adenosinérgico, e também ação redutora de mediadores pró-inflamatórios. Desta forma, a espécie ST apresenta uma atividade potencialmente terapêutica que corrobora com seu uso popular. Porém mais estudos são necessários a fim de elucidar mais precisamente seu mecanismo de ação e se o mesmo é atribuído a um ou mais compostos do extrato. / The present study evaluated the antinociceptive and anti-inflammatory effects of extracts obtained from Sida tuberculata (ST), popularly known as "guanxuma", on acute and chronic models in mice. In addition, were performed a phytochemical characterization and antioxidant and cytotoxic analysis in vitro. According to the data, leaf and root extracts presented a significant antioxidant effect (p <0.05) from the 0.015 and 0.03 mg.mL-1 doses respectively, however the leaves extract had a more pronounced action compared to the leaves root extract (<IC50) being selected for in vivo tests. Through the chemical analyzes, an analytical method was developed by UHPLC to determine the phytochemical marker, 20-hydroxyecdysone (20-HE), and eight compounds were identified, being 20HE the major compounds together with a Kaempferol derivative. In cytotoxicity tests, S. tuberculata leaf extract (STLE) and S. tuberculata root extract (STRE) demonstrated antiproliferative action against HepG2 and MCF-7 tumor cell lines (IC50 between 543.6 - 593.4 μg.mL-1 for STLE, and 397.1 - 493.9 mg.mL-1 for STRE). Besides, STLE decreased human leukocytes viability from dose of 10 μg.mL-1. In vivo assays demonstrated that STLE (10-300 mg.kg-1) given orally (p.o.) 1 h before the test, significantly inhibited the neurogenic and inflammatory phases in the formalin model (31.3 and 40.1% respectively). Likewise, STLE significantly decreased the contortions induced by acetic acid, reaching 71.8% inhibition at the dose of 100 mg.kg-1. The experiments showed, at least in part, the STLE mechanism of action involves the opioid and adenosinergic systems since its effect was reversed by Naloxone (non-selective opioid receptor inhibitor) and DPCPX (selective A1 adenosine receptor inhibitor) in acetic acid model. The evaluation by computational docking demonstrated that Kaempferol and 20HE compounds might interact primarily with the μ opioid receptor. Pretreatment with STLE (100 mg.kg-1) also reduced total cell migration, neutrophil number, MPO activity, and levels of IL-1β, IL-6 e TNF-α cytokines in the peritoneal fluid of animals submitted to carrageenan-induced peritonitis. In the same model, there was a reduction of TBARS levels and an increase in NPSH levels. In addition, treatment with STLE at 100 mg.kg-1 once daily also presented antinociceptive effect in chronic inflammatory pain model induced by CFA (i.pl.), and paw edema decreased after the fifth day of treatment. The doses administered daily (100 mg.kg-1 for 15 days) did not present macroscopic alteration and did not change the absolute weight and organs relative weight (heart, lung, liver, spleen and kidneys). Therefore, the data presented suggest that ST has a significant antinociceptive and anti-inflammatory activity against acute and chronic models in mice, with a mechanism of action involving, partially, its antioxidant action and the interaction on the opioid and adenosinergic system, as well as reducing action of pro-inflammatory mediators. Thus, the ST species presents a potential therapeutic action that corroborates with its popular use. However, more studies are needed in order to elucidate more precisely its mechanism of action and whether it is attributed to one or more compounds in the extract.

In Aedes aegypti, development and reproduction are regulated by juvenile hormone III (JH). This master regulatory hormone is synthesized by the corpora allata (CA), a pair of endocrine glands with neural connections to the brain. JH titers are largely determined by the rate of biosynthetic activity of the CA and are regulated by inhibitory and stimulatory factors. Like JH, the ecdysteroid 20-hydroxyecdysone (20E) is a key hormonal regulator and has been proposed as an allatoregulator in other insects. However, its part in the regulation of JH biosynthesis of mosquitoes was unknown. The specific aims of this dissertation were to (1) evaluate if 20E plays a role in the activation of the late pupal CA and (2) evaluate if 20E plays a role in the reactivation of JH synthesis in blood-fed females. To this end, we evaluated if 20E could prematurely activate JH biosynthesis in the CA of an early pupa (24h prior to eclosion or -24h). Remarkably, in vitro stimulation with 20E at -24h initiated JH synthesis at a time when transcript levels for most JH biosynthetic enzymes are low. Moreover, the application of 20E correlated with an increase in the enzymatic activity of juvenile hormone acid methyltransferase (JHAMT), a critical enzyme of the biosynthetic pathway. Additionally, separation of the CA from the brain increased JH synthesis. Together, these results indicate that 20E acts as a developmental mediator of CA maturation which overrides an inhibitory effect of the brain. In our previous aim we demonstrated that 20E mediates activation of the pupal CA which ensures the development of ovarian follicles of the newly emerged female. For mosquitoes, a blood-meal is required to complete vitellogenesis and results in suppression of CA activity. However, the CA must be reactivated to initiate the second gonotrophic cycle. Our findings show that in vitro stimulation with 20E at 24h post blood feeding reactivates the gland. Again, stimulation with the ecdysteroid resulted in increased activity of another key enzyme, farnesal dehydrogenase (FALDH). These results suggest a stimulatory role of 20E on the biosynthetic activity of the CA in the blood fed female.

No description available.

L'obésité est une maladie complexe dont la prévalence est en constante augmentation à travers le monde. A l'instar de nombreux médicaments qui ont pour origine des molécules végétales, de nouveaux principes actifs ont été recherchés dans les plantes. Parmi eux, les phytoecdystéroïdes, notamment la 20-hydroxyecdysone présente dans le quinoa, ont montré des effets pharmacologiques bénéfiques, et seraient donc des candidats potentiellement efficaces contre l'obésité. L'objectif de cette thèse est de mettre en évidence les propriétés d'un extrait de quinoa enrichi en 20-hydroxyecdysone sur le surpoids et l'obésité et d'en caractériser les effets.Dans un premier temps, un apport alimentaire d'extrait de quinoa enrichi en 20-hydroxyecdysone chez la souris soumise à un régime hyperlipidique a permis de limiter le développement du tissu adipeux, sans modifier la prise alimentaire. Dans le tissu adipeux, ces effets ont été associés à une diminution de la quantité de transcrits de gènes impliqués dans le stockage lipidique et l'inflammation. L'analyse du bilan énergétique a mis en évidence un effet de l'extrait de quinoa sur l'augmentation de la dépense énergétique liée à une augmentation de l'oxydation des glucides, ainsi que sur la diminution de l'absorption intestinale des lipides. Ensuite, chez le sujet en surpoids ou obèse, l'extrait de quinoa a montré une tendance à la prévention de la reprise de poids et de masse adipeuse suite à une restriction énergétique. Cet effet s'est accompagné d'un maintien de l'amélioration de l'insulino-sensibilité. Enfin, des mesures de biodisponibilité de la 20-hydroxyecdysone chez la souris ont permis de quantifier sa teneur circulante et de suivre l'apparition de certains de ses métabolites dont le rôle reste à identifier. Ces résultats montrent un bénéfice potentiel de l'extrait de quinoa et de son principe actif, la 20-hydroxyecdysone, sur la stabilisation du poids et de la masse adipeuse suite à une réduction pondérale. D'autres études seront nécessaires afin de poursuivre la caractérisation de ces effets.

Made available in DSpace on 2014-12-17T15:01:23Z (GMT). No. of bitstreams: 1 GracianaCD_DISSERT.pdf: 963468 bytes, checksum: 406e6de21c3be72fd5a9ff01dcdda958 (MD5) Previous issue date: 2010-08-02 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Among the pests that attack corn crop in Brazil, there is Spodoptera frugiperda (JE Smith, 1797) (Lepidoptera: Noctuidae), known as fall armyworm, which is the major corn pest. Due to genetic instability during serial passage of baculoviruses in insect cell culture, the viral bioinseticides in vitro production development is the greatest challenge for mass production of this bioproduct. Successive passages of virus using extracellular viruses (BVs), necessary during viral bioinseticides production scaling up, leads to the appearance of aberrant forms of virus, a process so called as "passage effect ". The main consequence of passage effect is the production of occlusion bodies (OB) decrease, preventing its production using in vitro process. In this study, it was carried out a serial passage of baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus, isolate 18, using Sf21 cells. A decrease in the production of occlusion bodies from 170 to 92 in the third to fourth passage was observed. A factorial experimental design (22) was employed to verify the influence of two input variables, concentration of the hormone 20 - hydroxyecdysone (CH) and cholesterol (CC) on the values of response variables (volumetric and the specific OB production) of the process, seeking to define the optimum operating ranges trying to reverse or minimize the passage effect. The result indicated a negative influence of the cholesterol addition and positive effect in the hormone supplementation which the optimum range found for the concentrations studied were 8 to 10&#956;g/mL and 5 to 6.5 mg / mL, for cholesterol and hormone concentrations respectively. New experiments were performed with addition of hormone and cholesterol in order to check the influence of these additives on the OB production independently. While the best result obtained from the factorial experiment was 9.4 x 107 OB/mL and 128.4 specific OB/cell, with the addition of only 6&#956;g/mL 20-hydroxyecdysone these concentrations increased to 1.9 x 108 OB/mL and 182.9 OB/cell for volumetric and specific OB production, respectively. This result confirms that the addition of the hormone 20-hydroxyecdysone enhances the SfMNPV in vitro production process performance using Sf21 cells / Dentre as pragas que atacam a cultura do milho no Brasil, destaca-se a Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), conhecida no est?dio larval como lagarta-do-cartucho, considerada a praga chave da cultura, alimentando-se da planta em todas as suas fases de crescimento, principalmente dos cartuchos de plantas jovens. Para seu controle, tem-se empregado inseticidas qu?micos de amplo espectro, o que tem causado efeitos adversos ao homem e ao meio ambiente. Por essa raz?o, torna-se necess?ria a busca de alternativas mais eficientes, de baixo custo e de f?cil utiliza??o, como o uso de bioinseticidas, especialmente os baculov?rus. Devido ? instabilidade gen?tica durante a passagem seriada de baculov?rus em cultivo de c?lulas de inseto, o desenvolvimento da produ??o in vitro de bioinseticidas virais ? o maior desafio para a sua produ??o massal. Passagens sucessivas de v?rus usando v?rus extracelulares (BVs), necess?rias para o aumento de escala durante a produ??o de bioinseticida viral, leva ao aparecimento de formas aberrantes de v?rus, processo conhecido como efeito de passagem . A principal consequ?ncia do efeito passagem ? a diminui??o da produ??o de corpos de oclus?o (OB), inviabilizando economicamente sua produ??o pelo processo in vitro. Neste trabalho, foi realizada a passagem seriada do isolado 18 do baculov?rus Spodoptera frugiperda multiple nucleopolyhedrovirus em c?lulas Sf21. Foi observada uma queda na produ??o de OB de 170 para 92 da terceira para quarta passagem. Um planejamento experimental fatorial (22) foi empregado para verificar a influ?ncia de duas vari?veis de entrada, concentra??o de horm?nio 20- Hidroxiecdisona (CH) e concentra??o de colesterol (CC), sobre os valores das vari?veis de resposta do processo (produ??o volum?trica e produ??o espec?fica de OB), procurando definir as faixas ?timas de opera??o para reverter ou minimizar o efeito passagem. O resultado deste planejamento indicou influ?ncia negativa da adi??o do colesterol e positiva na adi??o de horm?nio, onde as faixas ?timas encontradas para as concentra??es estudadas foram: 8 a 10&#956;g/mL e 5 a 6,5&#956;g/mL, para as concentra??es de colesterol e horm?nio, respectivamente. Novos experimentos foram realizados com a adi??o individual de horm?nio e colesterol com a finalidade de verificar a influ?ncia destes adjuvantes na produ??o de OB de forma independente. No planejamento experimental obteve-se produ??o volum?trica de 9,4 x 107 OB/mL e espec?fica de 128,4 OB/c?lula. Quando se adicionou 6 &#956;g/mL de 20-Hidroxiecdisona, as concentra??es elevaram-se para 1,9 x 108 OB/mL e 182,9 OB/c?lula, indicando que a adi??o do horm?nio melhorou a efici?ncia da produ??o in vitro de produ??o de SfMNPV em cultivos de c?lulas Sf21

No description available.

The heat shock response (HSR) is a highly conserved mechanism that enables organisms to survive environmental and pathophysiological stress. In Drosophila, the HSR is regulated by a single transcription factor, heat shock factor (HSF). During stress, HSF trimerizes and binds to over 200 loci on Drosophila polytene chromosomes with only nine mapping to major heat shock (HS) inducible gene loci. The function of HSF binding to the other sites in the genome is currently unknown. Some of these sites may contain yet unidentified “minor” HS genes. Interestingly, the binding of HSF also coincides with puff regression at some sites. Two such sites contain the major developmentally regulated genes Eip74 and Eip75: key regulators in the response to 20-hydroxyecdysone (20E), the main hormone responsible for the temporal co-ordination of post-embryonic development in Drosophila. Previous work in our and other labs indicates that the regression of non-HS puffs during the HSR is dependent on the presence of functional HSF. Using chromatin immunoprecipitation (ChIP) followed by hybridization to genome tiling arrays (Chip), I have identified 434 regions in the Drosophila Kc cell genome that are bound by HSF during HS, and have determined that 57% of these sites are located within the transcribed regions of genes. By examining the transcriptional response to HS in Kc cells and third instar larvae using expression microarrays, I found that only about 10% of all genes within 1250 bp of an HSF binding site are transcriptionally regulated by HS and many genes whose transcript levels change during HS do not appear to be near an HSF binding site. Furthermore, genes with an HSF binding site within their introns are significantly enriched (modified Fisher Exact p-value between 2.0x10-3 and 1.5x10-6) in gene ontology terms related to developmental processes and reproduction. Using expression microarray technology, I characterized the transcriptional response to 20E and its structural analog ponasterone A. I have identified multiple HSF binding sites within Eip74 and Eip75, and show that induction of the HSR correlates with repression of these genes and all other 20E-inducible genes. Taken together, this work provides a basis for further investigation into the role of HSF binding to sites not associated with HS genes and its possible function as a repressor of gene transcription during conditions of stress and as a regulator of developmental genes under stress and non-stress conditions.

No description available.

No description available.