Ces travaux de thèse pluridisciplinaires à l‘interface entre biocatalyse et nanomatériaux visent la conception de matériaux biohybrides innovants par assemblage dans des conditions douces de matériaux inorganiques de type hydroxydes doubles lamellaires (HDL) avec des enzymes. La première partie de ce mémoire est consacrée à la caractérisation des interactions physico-chimiques entre les HDL et la fructose-6-phosphate aldolase (FSA) catalysant la formation stéréosélective de liaisons C-C pour conduire à des polyols chiraux. Les structures lamellaires HDL permettent un confinement efficace de systèmes enzymatiques grâce à leur structure bidimensionnelle poreuse, leurs propriétés physico-chimiques favorables à l‘échange ionique et leur biocompatibilité. Différentes stratégies d‘immobilisation de la FSA dans des matrices d‘HDL ont été explorées, le taux d‘immobilisation et l‘activité biocatalytique étant fortement dépendant de la méthode d‘assemblage et de la nature des phases HDL. Le taux d‘immobilisation de l‘enzyme obtenu par coprécipitaton est supérieur à celui obtenu par adsorption. Dans une deuxième partie, un bioréacteur a été élaboré par un assemblage hiérarchisé constitué de la FSA, de nanoplaquettes d‘HDL et de billes de polysaccharide, ce dernier jouant le rôle de matrice macrostructurante. De façon notable, le taux d‘encapsulation de l‘enzyme dans la matrice macroscopique est amélioré lorsque le biocatalyseur est pré-encapsulé dans les nanoplaquettes d‘HDL. Ceci est attribué aux interactions électrostatiques favorables entre les chaînes de polysaccharide et les HDL, facilitant une charge de matière plus importante. L‘efficacité catalytique du bioréacteur obtenu et sa recyclabilité ont été démontrés. Dans la troisième partie de cette thèse, nous décrivons pour la première fois la conception de bionanoréacteurs enzymes@HDL par co-immobilisation de systèmes bi- ou tétra-enzymatiques dans les HDL permettant de réaliser des cascades multienzymatiques biomimétiques. L‘immobilisation des différentes enzymes prises séparément a d‘abord été optimisée afin de déterminer les conditions de co-immobilisation et de réaliser les cascades biocatalytiques en phase hétérogène. Ces bionanoréacteurs, dont nous avons démontré la recyclabilité, ont été appliqués pour la synthèse de sucres phosphorylés de série D. Enfin, une cascade multienzymatique a été conçue de novo en solution aqueuse et optimisée pour synthétiser différents sucres phosphorylés rares de série L. / This multidisciplinary thesis at the biocatalysis/nanomaterial interface perfectly aims at designing innovative biohybrid materials by the assembly of inorganic materials the Layered Double Hydroxides (LDH) with enzymes under mild conditions. The first part of this thesis is devoted to the characterization of physico-chemical interactions between the LDH and the fructose-6-phosphate aldolase (FSA) catalyzing the stereoselective C-C bond formation to provide chiral polyols. LDH structures allow the effective confinement of enzymatic systems thanks to their opened two-dimensional structure as well as their chemical surface properties at the nanoscale and their biocompatibility. The FSA immobilization in different LDH matrices by different methods was studied. Biocatalytic activity is highly dependent on the method of assembling, modulating the final amount of FSA. The retaining activity rate of co-precipitated material was higher than that obtained for the adsorbed enzyme. In a second part, a bionanoreactor was developed based on a hierarchized assembly of FSA, LDH nanoplatelets and polysaccharide beads acting as a macrostructuring matrices. Significantly, the encapsulated enzyme rate in the beads was improved when the biocatalyst was pre-encapsulated in LDH nanoplatelets. This is attributed to favorable electrostatic interactions between the polysaccharide chains and LDH, facilitating a higher catalyst loading. The catalytic efficiency of the prepared bioreactor and its recyclability were demonstrated. In the third part of this thesis, we describe for the first time the design of bionanoreactors ―enzymes@LDH‖ by co-immobilisation of two and four enzymes in LDH allowing biomimetic multienzymatic cascades. We first studied the immobilization of the different enzymes taken separately. Then we worked on the optimization of the biocatalytic cascades in heterogeneous phase. These bionanoreactors, for which we have shown the recyclability, have been applied to the synthesis of D-series phosphorylated sugars. Finally, a multienzymatic cascade was de novo designed in aqueous homogeneous solution. It was optimized for the synthesis of rare L-phosphorylated sugars.

Die trockene, offene Erhitzung von Inulin im Bereich zwischen 100 und 220 °C resultiert in einem Abbau der Fructanketten. Dabei entsteht in Abhängigkeit vom pH-Wert ein weites Spektrum an Abbauprodukten, primär Mono- und Disaccharide, vorrangig Fructose und verschiedene Di-D-Fructose Dianhydride (DFDA). Da bisher nur ein DFDA als kommerzieller Standard erhältlich ist, wurden aus einem Inulinkaramell nach Aufreinigung per Flash-Chromatographie und semipräparativer HPLC-RI vier DFDA isoliert und identifiziert. Dabei handelt es sich um α-D-Fruf-1,2´:2,3´-β-D-Fruf (DFA III), α-D-Fruf-1,2´:2,1´-α-D-Fruf (DFA VII), β-D-Fruf-1,2´:2,1´-β-D-Fruf und α-D-Fruf-1,2´:2,1´-β-D-Fruf (DFA I). Eine fünfte Verbindung wurde als α-D-Fruf-1,2´-β-D-Fruf identifiziert, welches vermutlich aus α-D-Fruf-1,2´:2,6´-β-D-Fruf (DFA V) freigesetzt wird. Während unter sauren Bedingungen vor allem Fructose bestimmt wurde, kommt es unter basischen Erhitzungsbedingungen verstärkt zur Bildung löslicher, farbiger Verbindungen. Durch einen basischen Zusatz kann außerdem die antioxidative Wirksamkeit der Inulinkaramelle gesteigert werden, wobei wahrscheinlich besonders die farbigen Verbindungen für die antioxidative Wirkung verantwortlich sind. Die präbiotische Wirkung kann durch die Erhitzung nicht gesteigert werden, wird jedoch bei moderaten Erhitzungsbedinungen (etwa 160 °C) nicht verrringert. Durch eine gezielte Erhitzung kann Inulin so verändert werden, dass sich neue Eigenschaften und damit auch neue Einsatzmöglichkeiten in der Lebensmittelindustrie ergeben.

Thesis (PhDAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Stuck alcoholic fermentations are a major enological problem for the international winemaking industry. Incomplete wine fermentations are frequently characterized by high residual fructose concentrations and the near-absence of residual glucose, a fact that is due to the glucophilic character of the wine yeast Saccharomyces cerevisiae. Wines with high contents of post fermentation sugar are very susceptible for microbial spoilage since residual fructose and/or glucose can be metabolized by bacteria and yeast to undesired by-products such as volatile acid and off-flavours, resulting in wine spoilage and considerable economic losses. It has been reported that stuck fermentations are usually caused by several synergistically acting inhibition factors, and the glucose to fructose ratio (GFR) is thought to play an important role in this context. This study is aimed at contributing towards a better understanding of this industrial problem, and at finding industrially applicable solutions. In a first part, this study describes the isolation of two appropriate strains of the fructophilic yeast Zygosaccharomyces bailii from the natural microflora of grapevine, followed by trials in small scale test fermentations using stuck industrial fermentations as model media. These experiments were expanded to also investigate large scale industrial fermentations. As a result, a strategy for the treatment of stuck fermentations was developed and successfully applied in several wineries with fermentation problems. This methodology represents an entirely novel and industrially applicable solution to high residual fructose levels. In a second part, the data contributes to elucidating the molecular nature of the fructophilic phenotype of Z. bailii by characterizing some of the genes and proteins that may be responsible for the fructophilic character. In particular, the investigation focused on the first two steps of hexose metabolism, the transport of sugar into the cell by permeases and sugar phosphorylation by hexokinases, which combined are thought to be primarily responsible for sugar preference. One result of this study was Fructoferm W3©, a dry yeast product which is commercially available. Fructoferm W3 was awarded with the innovation medal for enological products at Intervitis/Interfructa, Stuttgart, Germany in 2007. / AFRIKAANSE OPSOMMING: Die voorkoms van steek alkoholiese fermentasies is ‘n ernstige problem in die internasionale wyn industrie. Onvolledige fermentasies word dikwels gekenmerk deur hoë residuele fruktose konsentrasies en die veitlike afwesigheid van residuele glukose. Die kenmerke kan meestal toegeskryf word aan die glukofilliese kakakter van die wyngis Saccharomyces cerevisiae. Wyne met ‘n hoë suiker inhoud na die afloop van fermentasie is vatbaar vir mikrobiese bederf aangesien residuele fruktose en/of glukose gemetaboliseer kan word deur bakterië en gis om ongewenste byprodukte soos vlugtige sure en bygeure te vorm wat kan lei tot wyn bederf en aansienlike ekonomies verlies. Dit is vasgestel dat steek fermentasies gewoonlik veroorsaak word deur verskeie sinergisties werkende inhibisie faktore, waartoe die glukose/fruktose verhouding ‘n noemenswaardiege bydrae lewer. Die mikpunt van hierdie studie was om ‘n bydrae te lewer tot die begrip van steek fermentasies en die daarstelling van moontlike industriële oplossings. Die eerste deel van die werk beskryf die isolasie van twee rasse van die gis Zygosaccharomyces baillie uit die natuurlike wingerd mikroflora, gevolg deur steekproewe in die vorm van kelinskaalse fermentasies met steek industriële fermentasies gebruik as model media. Hierdie ekserimente is vervolgens uitgebrei om grootskaalse industriële steek fermentasies te bestudeer. Die uitkoms van hierdie werk het gelei tot die ontwikkeling van ‘n strategie vir die behandeling van steek fermentasies wat susksesvol toegepas is in verskeie wynmakerye. Die metodiek bring ‘n nuwe en industrieel toepasbare oplossing vir hoë residuele fruktose vlakke. Die data aangebied in die tweede afdeling dra by tot die verheldering van die molekulêre natuur van die fruktofilliese fenotipe van Z. baillie deur die tipering van gene en protiëne wat moontlik verantwoordelik is vir die fruktofilliese karakter van die gis. Die ondersoek het spesifiek op die eerste twee stappe van heksose metabolisme, naamlik die invoer van suiker in die sel deur permeases en suiker fosforilering deur heksokinases, gekonsentreer. Die kombinasie van die twee prosesse is vermoedelik verantwoordelik vir die regulering van suiker voorkeur. ‘n Gevolg van die studie was die ontwikkeling van ‘n droë gisproduk, Fructferm W3©, wat kommersieel beskikbaar gestel is. Fructoferm W3 is in 2007 toegeken met die innovasie medalje vir wynkundige produkte by Intervittis/Interfructa in Stuttgart, Duitsland.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology / Solid-state fermentation (SFF) has emerged as a good method for the production of microbial enzymes such as inulinases. The use of low-cost agricultural plants and agro-industrial residues as substrates in SSF processes provides a value adding alternative to these otherwise under/or un-utilised vegetation. Production of inulinases, using various inulin-containing plant materials as carbon sources was studied using pure and mixed cultures of yeast strains. All substrates resulted in different levels of enzyme activity. A mixed culture of Kluyveromyces marxianus and Saccharomyces cerevisiae produced an extracellular exoinulinase when grown on different types of inulin-containing plant materials. Initial inulinase production was achieved as follows: 10 IU/gds (garlic cloves), 15 IU/gds (parsnips), 10 IU/gds (wheat bran) and 7 IU/gds (amadumbe) by K. marxianus and S. cerevisiae in a mixed culture. The production of inulinases by a mixed culture of K. marxianus and S. cerevisiae under SSF was further optimized by investigating initial moisture content, temperature, carbon source, nitrogen source, inoculum volume and inoculum ratio. The highest inulinase activity attained was in garlic cloves (85 IU/gds), followed by parsnips (65 IU/gds), wheat bran (37 IU/gds) and amadumbe (25 U/gds). The activities yielded 5.6 fold higher inulinase than in preliminary studies. The optimum pH and temperature of the crude enzyme were 5.0 and 50 oC, respectively. The pH and temperature stability of the enzyme was steady for 1 hour retaining about 64% activity. The average inulinase/invertase activity (I/S) ratio of 1.0 by crude inulinases was also observed after 48 hours. The crude extracellular enzyme extracts from the garlic cloves, parsnips, amadumbe and wheat bran were partially purified by ammonium sulphate precipitation and showed a specific activity of 9.03 U/mg, 0.08 U/mg, 4.12 U/mg and 0.133 U/mg respectively. The Km and Vmax values of the inulinase were 21.95 mM and 2.09 μM/min; 19.79 mM and 1.38 μM/min; 31.59 mM and 0.51 μM/min; and 25.74 mM and 0.23 μM/min, respectively. All extracts demonstrated potential for large-.scale production of inulinase and fructose syrup.

Em 2013, a população idosa mundial era de 841 milhões e espera-se que aumente mais de três vezes até 2050. Neste sentido, a expectativa de vida das mulheres tem sido maior do que a dos homens. Apesar das doenças cardiovasculares (DCV) se desenvolverem mais tarde nas mulheres do que nos homens ela ainda é a principal causa de morte em mulheres. Adicionalmente, o aumento do consumo calórico, especialmente carboidratos refinados e frutose, tem sido correlacionado com o aumento de síndrome metabólica (SM). Estes dados confirmam que a idade, maus hábitos alimentares e o gênero têm uma importância significativa na incidência de risco cardiovascular. Evidências consistentes sobre os benefícios do treinamento físico nas alterações cardiovasculares, metabólicas e autonômicas associadas a DCV, têm levado muitos pesquisadores a sugerirem o treinamento físico regular como um procedimento não farmacológico importante na prevenção e tratamento de diferentes patologias. Neste sentido, o objetivo deste estudo foi investigar o papel do treinamento físico sobre os efeitos do envelhecimento e da ingestão de frutose em parâmetros metabólicos, cardiovasculares, inflamatórios e de estresse oxidativo em ratas submetidas à privação dos hormônios ovarianos. Métodos: Os experimentos foram realizados ratos Wistar fêmeas (n=56), jovens (3 meses) e idosas (22 meses) divididas nos seguintes grupos: jovem controle sedentária (JCS), jovem ooforectomizada sedentária (JOS), idosa controle sedentátia (ICS), idosa ooforectomizada sedentária (IOS), idosa ooforectomizada frutose (IOF), idosa ooforectomizada treinada (IOT) e idosa ooforectomizada frutose treinada (IOTF). A ovariectomia foi realizada por remoção dos ovários de forma bilateral. Os grupos frutose foram tratados com D-frutose (100g / L) na água de beber durante 10 semanas. O treinamento físico foi realizado em esteira durante 8 semanas. A morfometria e a função cardíaca foram avaliadas por ecocardiografia. A pressão arterial (PA) e a frequência cardíaca (FC) foram avaliadas pelo registro direto através de um sistema de PA para aquisição de dados. A sensibilidade do barorreflexo (SBR) foi avaliada pelas respostas de taquicardia (RT) e bradicardia (RB) às reduções e aumentos da PA, respectivamente. O. controle autonômico foi avaliado pelo bloqueio farmacológico por atenolol e atropina. O stress oxidativo foi medido em tecido cardíaco e hepático e o perfil inflamatório avaliado no plasma. Os resultados foram apresentados em 2 protocolos. No primeiro, os efeitos do envelhecimento e da ooforectomia em animais jovens e idosos foram descritos descritos. No segundo, foram avaliados os efeitos do treinamento físico em ratas idosas ooforectomizadas. Resultados do primeiro protocolo: O envelhecimento ou a privação dos hormônios ovarianos promoveram aumento no peso corporal, na gordura e na concentração de triglicérides, uma redução da sensibilidade à insulina e na capacidade de exercício, disfunção diastólica do VE e aumento no índice de desempenho miocárdico (IDM). O envelhecimento e a privação dos hormônios aumentaram o tônus simpático e diminuíram o tônus vagal. A PAM aumentou nos grupos ooforectomizados jovens (117±2 vs 107±1 mmHg) e envelhecidos (119±2 vs 110±2 mmHg) e a FC não se modificou enquanto a variabilidade da FC estava reduzida nos grupos velhos. O envelhecimento se caracterizou por maior concentração de algumas citocinas inflamatórias (IL-6 e TNF- ? ). As enzimas antioxidantes estavam aumentadas nos grupos ooforectomizados e o dano a proteínas foi maior nas idosas ooforectomizadas. O aumento do tecido adiposo nos grupos jovens e idosos ooforectomizados ou não, se correlacionou com o aumento da IL-6 e do efeito simpático assim como com a redução da sensibilidade a insulina. O TNF- ? foi inversamente associado com a razão GSH/GSSG e diretamente com a função cardíaca global (IDM) que foi inversamente associada com a capacidade física. Resultados do segundo protocolo: A associação da síndrome metabólica (tratamento com frutose) com ooforectomia induziu disfunção exacerbada de alguns parâmetros como, aumento do peso corporal, tecido adiposo, efeito simpático, balanço simpato vagal, variabilidade da PAS e estresse oxidativo. No entanto, quando foi realizado o treinamento físico, ouve uma diminuição do tecido adiposo (IOT: 3,94 ± 0,44; IOTF: 5,28 ± 0,66 g) e da resistência à insulina (IOT: 4,89 ± 0,14; IOTF: 5, 12 ± 0,43 mg / dl / min) em comparação com os grupos sedentários (IOS: 6,27 ± 0,62, IOFS: 10,7 ± 0,61 g), (IOS: 3,18 ± 0,31 ; IOFS: 3,59 ± 0,55 mg / dl / min). O treinamento físico aumentou a capacidade física (IOT: 19,99 ± 0,89, IOTF: 17,55 ± 1,05 vs. IOS: 10,52 ± 0,87; IOSF: 10,34 ± 0,59 min). Os resultados hemodinâmicos demonstraram que o treinamento físico atenuou o aumento da PAM induzido por ovariectomia e / ou sobrecarga de frutose (IOT: 103,3 ± 1,0; IOTF: 107 ± 1,1 vs .: IOS: 119,1 ± 1, 86; IOSF: 119,1 ± 2,7 mmHg) e reduziu a FC basal (IOT: 302,1 ± 13,20 IOTF: 306,40 ± 8,2 vs: IOS: 389,53 ± 20,10, IOSF: 348 , 93 ± 17,55 bpm). O tônus simpático foi menor nos grupos treinados (IOT: 62, 2 ± 3,1; IOTF: 51,2 ± 7,1) em relação aos grupos com sobrecarga de frutose e ou ooforectomizadas (IOS: 102,6 ± 12,3; IOSF: 85, 39 ± 3,75 batimentos / min). O tônus vagal aumentou apenas no grupo treinado sem frutose (IOT: 44, 76 ± 5,87 vs IOFT: 22,87 ± 3,38; IOS: 17,14 ± 4,21; IOSF: 9,21 ± 2, 82 batidas / min). Os grupos IOT e IOTF apresentaram bradicardia reflexa melhorada em relação aos grupos sedentários (IOT: -1,74 ± 0,12; IOFT: -1,77 ± 0,15 vs 0,93 ± IOS-0,07 ; IOSF: -1,21 ± 0,12 bpm / mmHg). A modulação simpática da PAS estava reduzida nos grupos treinados (IOT: 3,33 ± 0,50; IOFT: 4,60 ± 0,65 vs: IOS: 6,03 ± 0,95; IOSF: 7,07 ± 0, 49 mmHg). Finalmente, os grupos IOT e IOTF apresentaram melhor função diastólica com menor tempo de relaxamento isovolumétrico (TRIV) (IOS: 3,08 ± 0; 21; IOSF: 2,9 ± 0,24; IOT: 1,98 ± 0,15; IOTF: 2,72 ± 0,2 ms), relação E / A (IOS: 1,60 ± 0,06 IOSF: 1,62 ± 0,05; IOT: 1,41 ± 0,17; IOTF: 1,66 ± 0,08 ms), e função global cardíaca (MPI) (IOS: 0,40 ± 0,06; IOSF: 0,46 ± 0,10; IOT: 0,14 ± 0,03; IOTF: 0 , 29 ± 0,04). O peso do tecido adiposo se associou positivamente com os níveis plasmáticos de IL-1B e com o efeito simpático e inversamente com a sensibilidade à insulina e com a sensibilidade barorreflexa. O tônus vagal foi positivamente relacionado com a razão redox da glutationa e com a função sistólica. De forma semelhante, os níveis de IL-6 foram positivamente associados com o índice de função cardíaca global. A melhora da capacidade física foi associada com a melhora autonômica e da função cardíaca. Nossos resultados demonstraram que o envelhecimento potencializou os efeitos deletérios cardíacos e funcionais da privação dos hormônios ovarianos em ratas, provavelmente associados à disfunção autonômica, à inflamação e ao estresse oxidativo. No entanto, o treinamento físico após a privação dos hormônios ovarianos, com ou sem sobrecarga de frutose, foi capaz de modular favoravelmente a função autonômica, reduzindo marcadores de inflamação e estresse oxidativo, e consequentemente induzindo melhora na função cardíaca e na capacidade física / In 2013 the world elderly population was 841 million and it is expected to increase more than three times in 2050. In this sense, women\'s life expectancy has been higher than men. In addition CVD develops later in women than in men and is still the major cause of death in women. Additionally increased caloric consumption, especially refined carbohydrates and fructose, has been correlated with the metabolic syndrome (MS) increase. These data, confirm that the age, habits and gender have a significant importance in the incidence of cardiovascular risk. Constant evidences of cardiovascular, metabolic and autonomic benefits of chronic exercise training have led many researchers to suggest a regular physical training as an important non-pharmacological procedure in the prevention and treatment in pathologies conditions. In this sense, the objective of this study was to investigate the effects of aging and fructose on metabolic, cardiovascular, inflammatory and oxidative stress parameters in female rats submitted to ovarian hormone deprivation (OVX), as well as the role of exercise training in this condition. Methods: experiments were performed on 56 female rats. Sixteen young rats with 3 months of age and forty old rats with 22 month of age (n = 8 in each group) were divided into: adult control (JCS), ovariectomized sedentary (IOS), aged sedentary control (ICS), aged ovariectomized sedentary (IOS), aged ovariectomized fructose (IOF), aged ovariectomized trained (IOT) and aged ovariectomized trained fructose (IOTF). Ovariectomy was performed by bilateral ovaries removal. Fructose-fed rats received D-fructose (100g/L) in drinking water for 10 weeks. The exercise training was performed on a treadmill for 8 weeks. Cardiac morphometric and function were evaluated by echocardiography. Blood pressure (BP) and heart rate (HR) were evaluated by recording direct through a PA system for data acquisition. The baroreflex sensitivity (SBR) was evaluated by tachycardic (RT) and bradycardic (RB) responses. Autonomic control was assessed by vagal and sympathetic tonus and effect. Oxidative stress was measured in cardiac and hepatic tissue and inflammatory profile in plasma. Results: Aging or OVX promoted an increase in body and fat weight, triglyceride concentration and a reduction in insulin sensitivity and exercise capacity. Left ventricular diastolic dysfunction and increased cardiac overload (IPM) were observed in old compared to young groups. Aging and OVX lead to increase in sympathetic tonus, vagal tonus was lower just in old groups. TNF-? was higher in ICS when compared to JCS. IL-6 was increased in old compared young groups. Glutathione redox balance was reduced in JOS, ICS and IOS groups when compared to JCS, indicating increased oxidative stress. The association of metabolic syndrome with ovariectomy induced exacerbation of some dysfunctions, such as increase in body weight, adipose tissue, sympathetic effect, LF/HF balance, VARR PAS and stress oxidative. However when the animal did exercise training decreased the adipose tissue (IOT: 3.94± 0.44; IOTF: 5.28± 0.66 g) and insulin resistance (IOT: 4.89±0.14; IOTF: 5.12±0.43 mg/dl/min) compared with the sedentary groups (IOS: 6.27 ±0.62, IOFS: 10.7 ±0.61 g), (IOS: 3.18±0.31; IOFS: 3.59 ±0.55 mg/dl/min). Exercise training increased physical capacity (IOT: 19.99 ±0.89, IOTF: 17.55 ±1.05 vs. IOS: 10.52± 0.87; IOSF: 10.34± 0.59 Min). Hemodynamic results demonstrated that the exercise training attenuated the increase in MBP induced by ovariectomy and/or overload of fructose (IOT: 103.3 ± 1.0; IOTF: 107±1.1 vs.: IOS: 119.1± 1.86; IOSF: 119.1±2.7 mmHg) and reduced the basal HR (IOT: 302.1±13.20; IOTF: 306.40±8.2 vs: IOS: 389.53±20.10, IOSF: 348.93±17.55 bpm). The sympathetic tonus was lower in exercise training groups (IOT: 62. 2± 3.1; IOTF: 51.2±7.1) compared to ovaryectomized and fructose overload groups (IOS: 102.6±12.3; IOSF: 85.39±3.75 beats/min). Vagal tonus was increased only in the trained group without fructose (IOT: 44.76± 5.87 vs IOFT: 22.87± 3.38; IOS: 17.14± 4.21; IOSF: 9.21±2.82 beats/min). IOT and IOTF groups presented reflex bradycardia similar and was observed to be higher than the sedentary groups (IOT: -1.74±0.12; IOFT: -1.77±0.15 vs IOS-0.93±0.07; IOSF: -1.21±0.12 bpm/mmHg). Sympathetic modulation of SAP was reduced in exercise training groups (IOT: 3.33±0.50; IOFT: 4.60 ±0.65 vs: IOS: 6.03±0.95; IOSF: 7.07 ±0.49 mmHg). Finally, the groups T and TF showed better diastolic function with lower isovolumetric relaxation time (IVRT) (IOS:3.08±0.21; IOSF:2.9±0.24; IOT: 1.98±0.15; IOTF: 2.72±0.2 ms), E/A ratio (IOS:1.60±0.06; IOSF:1.62±0.05; IOT:1.41±0.17; IOTF:1.66±0.08 ms), and cardiac global function by the myocardial performance index MPI (IOS:0.40±0.06; IOSF:0.46±0.10; IOT: 0.14±0.03; IOTF: 0.29±0.04). Our findings demonstrated that aging potentialized the deleterious cardiac and functional effects of OVX in rats, probably associated with exacerbated autonomic dysfunction, inflammation and oxidative stress. However, exercise training after ovarian hormone deprivation, with or without fructose overload, was able to positively modulate the autonomic function, reducing inflammatory and oxidative stress markers, consequently inducing improvement on cardiac function and physical capacity

INTRODUÇÃO: A exposição materna à frutose durante a gestação e lactação pode contribuir para o desenvolvimento de doenças crônicas na vida adulta da prole. OBJETIVO: Estudar os efeitos da ingestão de frutose durante a gestação e lactação sobre a vida pós-natal no que diz respeito às alterações metabólicas, de expressão gênica e ao comportamento alimentar em ratos machos. METODOLOGIA: Foram utilizados ratos Sprague-Dawley, provenientes de mães alimentadas com ração controle e de mães alimentadas com ração rica em frutose (60%), durante a gestação; a lactação e gestação/lactação. Após o desmame, a prole foi alimentada com ração controle até os 91 dias de vida. Foram analisados os parâmetros: biométricos, dietéticos, metabólicos quanto a glicemia, sensibilidade periférica à insulina, leptina e insulina (séricas e vias de sinalização), neuropeptídios envolvidos no controle do comportamento alimentar, e deposição hepática de gordura. RESULTADOS: Houve redução do índice de Lee no nascimento e hipoleptinemia aos 90 dias de vida, intolerância à glicose na vida pós-natal desde o desmame, concomitante com alterações na sensibilidade à ação da insulina na vida adulta, hiperinsulinemia, dislipidemia, deposição hepática de TAG, estímulo hedônico do comportamento alimentar alterado aos 14 dias de vida pós-natal via núcleo accumbens shell com maior expressão de CART. CONCLUSÃO: As proles possuem possível marca epigenética que pode contribuir para o prejuízo na produção e ação da insulina, ocasionando alterações na homeostase do metabolismo glicídico e lipídico hepáticos, levando a deposição hepática de gordura e alterações no perfil lipídico, com redução da leptinemia na vida adulta, e no controle hedônico central do comportamento alimentar apenas aos 14 dias de vida pós-natal. / INTRODUCTION: Maternal exposure to fructose during gestation and lactation can contribute to the development of chronic diseases in the adult life of offspring. OBJECTIVE: To study the effects of ingestion of fructose during gestation and lactation on postnatal life with respect to metabolic changes, gene expression and feeding behavior in male rats. METHODS: Male Sprague-Dawley rats from mothers fed with control rat chow (AIN-93) and from mothers fed a high fructose diet (60%). Both isocaloric, during gestation; lactation and gestation/lactation. After weaning, the offspring were fed with control rat chow until the 91st days of life. The following parameters were analyzed: biometric, dietary, metabolic for glycaemia, peripheral insulin sensitivity, leptin and insulin (serum and signaling pathways), neuropeptides involved in food behavior control, and hepatic deposition of fat. RESULTS: Reduction in the Lee index at birth and hypoleptinemia at 90 days of age (only in the offspring that the mothers consumed fructose during pregnancy), glucose intolerance in postnatal life since with changes in insulin sensitivity in adult life, hyperinsulinemia, dyslipidemia, hepatic deposition of GAD, hemodialysis of altered eating behavior at 14 days of age). CONCLUSION: The offspring from mothers who consumed fructose rat chow during gestation and lactation have a possible epigenetic mark that may contribute to the impairment in the production and action of insulin, leading to changes in hepatic glucose and lipid metabolism homeostasis, leading to hepatic fat deposition and alterations in the lipid profile, with reduction of leptinemia in adult life, and in the central hedonic control of dietary behavior only at the 14 days postnatal life.

Neste estudo testamos a hipótese que alterações no controle autonômico precedem alterações cardiometabólicas em animais espontaneamente hipertensos tratados com frutose (SHR). Adicionalmente avaliamos os efeitos do treinamento físico aeróbio (TFA) no curso temporal das disfunções observadas neste modelo de síndrome metabólica. Para isso, foram utilizados ratos machos Wistar e SHR divididos em grupos (n=8): Controle (C), Hipertenso (H), Hipertenso+Frutose (HF) e Hipertenso+Frutose+Treinamento Físico (HFT). A sobrecarga de frutose (100g/L) e o TFA (esteira: 1h/d., 5d/sem.) foram iniciados 30 dias após o nascimento dos animais. Os grupos experimentais foram divididos em subgrupos que foram avaliados após 7, 15, 30 e 60 dias de protocolo. A partir dos 7 dias de protocolo a associação da hipertensão ao consumo de frutose (grupo HF) induziu redução da sensibilidade barorreflexa para respostas bradicárdicas (RB) e taquicárdicas (RT) em comparação ao grupo C; observando-se redução adicional da RB no grupo HF em relação ao grupo H (7 dias: -40% e 15 dias: -36%). A reatividade vascular à fenilefrina (8ug/ml: 7 aos 60 dias) estava prejudicada nos grupos H e HF em relação ao C; com prejuízo adicional do grupo HF ao nitroprussiato de sódio na dose de 20ug/ml (15 aos 60 dias de protocolo vs. C). Além disso, aos 15 e 30 dias de protocolo, o grupo HF apresentou um aumento marcante do TNFalfa e IL-6 no tecido adiposo e de IL-1beta no baço em relação aos grupos C e H. Houve redução de nitritos plasmáticos no grupo HF em 15 (55%) e 30 dias (58%) vs. o grupo C, acompanhado de aumento do peróxido de hidrogênio em 30 dias vs. o grupo H (98%). Em relação as defesas antioxidantes, o grupo HF apresentou menor atividade da superóxido dismutase (SOD) vs. os grupos C (15 dias: 36%, 30 dias: 31% e 60 dias: 47%) e H (15 dias: 25%, 30 dias: 21% e 60 dias: 43%), sem alterações significantes na catalase e no potencial antioxidante total. O grupo HF teve maior lipoperoxidação e oxidação de proteínas vs. os grupos C (46% e 117%) e H (49% e 45%) somente aos 60 dias de protocolo. A partir de 15 dias de protocolo foram observadas alterações metabólicas no grupo HF, como o aumento dos triglicerídeos plasmáticos em relação aos grupos C (15 dias: 20%; 30 dias: 19%; 60 dias: 23%) e H (15 dias: 19%; 30 dias: 21%; 60 dias: 24%); aumento da insulina plasmática em comparação ao grupo C (30 dias: 160% e 60 dias: 260%); e redução da sensibilidade à insulina em relação aos grupos C (17%) e H (17%) em 60 dias de protocolo. Além das alterações metabólicas, houve aumento progressivo da pressão arterial (PA) nos grupos hipertensos, com aumento adicional da PA no grupo HF em relação ao grupo H em 30 dias (8%) e 60 dias de protocolo (11%). Por outro lado, o treinamento físico aeróbio atenuou parte destas disfunções. Nesse sentido, observamos aumento da sensibilidade barorreflexa no grupo HFT para as RB em 7 (100%), 15 (86%), 30 (76%) e 60 dias de protocolo (74%) e para as RT em 7 (34%), 15 (31%) e 30 dias de protocolo (49%) vs. grupo HF. Entretanto, o treinamento físico não atenuou o prejuízo na RT em 60 dias de protocolo e na reatividade vascular. Houve redução da razão entre TNF-alfa/IL-10 no tecido adiposo (40%) e da IL-1beta no baço (30%) no grupo HFT em relação ao grupo HF em 15 dias; todavia o TNF-alfa foi maior no baço no grupo HFT em 60 dias de protocolo em relação aos seus valores iniciais. Os nitritos plasmáticos estavam aumentados no grupo HFT quando comparado ao grupo HF em 7 e 15 dias de protocolo. Em relação ao estresse oxidativo, aos 60 dias de protocolo, observou-se aumento da SOD (74%) e redução da lipoperoxidação (46%) e do dano à proteína (60%) no plasma do grupo HFT em relação ao grupo HF. O treinamento físico reduziu a glicemia (15%) e aumentou a sensibilidade à insulina (31%) ao final do protocolo, apesar de não alterar os triglicerídeos sanguíneos ou a PA. Concluindo, os resultados demonstram que disfunções hemodinâmicas, autonômicas, inflamatórias e de estresse oxidativo são tempo dependente em animais SHR e que a associação ao consumo de frutose induz prejuízos adicionais. Além disto, a disfunção do barorreflexo precede as alterações hemodinâmicas, metabólicas, de inflamação e de estresse oxidativo nesse modelo. Por outro lado, o treinamento físico aeróbio foi eficaz em atenuar parte das disfunções neste modelo de síndrome metabólica / This study tested the hypothesis that changes in autonomic control precede cardiometabolic changes in spontaneously hypertensive rats (SHR) treated with fructose. Additionally, the effects of aerobic exercise training (AET) were evaluated in the time course of the dysfunctions observed in this metabolic syndrome model. Wistar and SHR rats were divided into groups (n=8/group): control (C), hypertensive (H), hypertensive + fructose (HF) and hypertensive + fructose + AET (HFT). The fructose overload (100g/l) and the AET (treadmill 1h/d, 5d/wk) was initiated at 30 days of life. The experimental groups were divided into subgroups, which were evaluated after 7, 15, 30 and 60 days of protocol. Since 7 day of protocol the association of hypertension and fructose consumption (HF group) induced a reduction in baroreflex sensitivity for bradycardic (BR) and tachycardia responses (TR) when compared to the C group; there was an additional reduction of BR in the HF group when compared to the H group (7 days: 40% and 15 days: 36%). The vascular reactivity to phenylephrine (8ug/ml: 7 to 60 days) was impaired in H and HF groups when compared to the C group; it was observed an additional impairment in the HF group to sodium nitroprusside at the dose of 20?g/ml (15 to 60 days of protocol vs. C). Additionally, at 15 and 30 day of protocol, the HF group showed an increase in TNFalfa and IL-6 in adipose tissue and in IL-1beta in the spleen when compared to C and H groups. There was a reduction in plasma nitrites in the HF group in 15 (55%) and 30 days of protocol (58%) vs. the C group, accompanied by an increase of the hydrogen peroxide in 30 days vs. H group (98%). Regarding the antioxidant defenses, the HF group showed reduced superoxide dismutase (SOD) activity as compared to C (15 days: 36%, 30 days: 31% and 60 days: 47%) and H groups (15 days: 25% 30 days: 21% and 60 days: 43%), without significant changes in catalase and in total antioxidant potential. The HF group had increased lipid peroxidation and protein oxidation vs. the C (46% and 117%) and H groups (49% and 45%) only at 60 days of protocol. Since 15 day of protocol, the metabolic changes were observed in the HF group, such as an increase in plasma triglycerides in HF group when compared to the C (15 days: 20%; 30 days: 19%; 60 days: 23%) and H groups (15 days 19%; 30 days: 21%, 60 days: 24%). It was observed an increase in plasma insulin in the HF group when compared to the C group (30 days: 160% and 60 days: 260%); and a decrease in insulin sensitivity in HF group when compared to the C (17%) and H groups (17%) at 60 days of protocol. In addition to metabolic changes, there was a progressive increase in blood pressure (BP) in hypertensive groups. There was a further increase in BP in the HF group when compared to the H group in 30 days (8%) and 60 days of protocol (11%). On the other hand, AET attenuated part of these dysfunctions. Accordingly, we observed an increase in baroreflex sensitivity in HFT group for BR at 7 (100%), 15 (86%), 30 (76%) and 60 days of protocol (74%) and for TR in 7 (34%), 15 (31%) and 30 days of protocol (49%) as compared to the HF group. However, the AET did not attenuate the impairment in TR at 60 days of protocol nor the vascular reactivity dysfunction. There was a reduction in the relation of TNF-alfa/IL-10 in fat tissue (40%) and IL-1beta in the spleen (30%) in the HFT group when compared to the HF group in 15 days of protocol. However, the TNF-alfa in spleen was higher in the HFT group at 60 days of protocol when compared to its initial values. The nitrites in plasma were increased in HFT group as compared to the HF group at 7 and 15 days of protocol. Regarding oxidative stress in plasma at 60 days of protocol, there was an increase in SOD activity (74%) and a decrease in lipid peroxidation (46%) and in protein oxidation (60%) in the HFT group when compared to the HF group. The AET reduced blood glucose (15%) and increased insulin sensitivity (31%) at the end of the protocol, although it did not alter blood triglycerides or BP. In conclusion, the results show that hemodynamic, autonomic, inflammatory and oxidative stress disorders are time dependent in SHR and that the association of fructose overload induces additional impairments. Furthermore, the baroreflex dysfunction precedes hemodynamic, metabolic, inflammatory and oxidative stress disorders in this model. On the other hand, the aerobic exercise training was effective in attenuate disorders in this model of metabolic syndrome

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A superativação do eixo ECA/AT1r está intimamente relacionada à síndrome metabólica e no organismo tem grande relação com o quadro de inflamação. A administração de frutose, seja por dieta ou pela água, tem sido usada como um modelo para a indução da superatividade desse eixo e para o estudo das vias inflamatórias relacionadas ao AT1r. Com isso, o objetivo deste trabalho foi avaliar se a administração de GW510156 poderia diminuir a superativação do eixo ECA/AT1r e consequentemente diminuir os danos causados pela dieta rica em frutose. Para isso foram utilizados camundongos machos C57Bl/6 que receberam uma dieta contendo 47% de frutose durante oito semanas ou uma dieta controle. Após oito semanas, os grupos foram redivididos aleatoriamente para o início da administração do GW501516 durante três semanas, totalizando quatro grupos experimentais. Os animais tratados apresentaram uma melhora da pressão arterial sistólica e também dos parâmetros urinários como proteinúria e ácido úrico. Houve ainda uma melhora dos triglicerídeo e ácido úrico plasmáticos. No tecido adiposo branco, o GW501516 foi capaz de diminuir a expressão dos componentes do eixo ECA/AT1r e também amenizou a inflamação causada pela dieta rica em frutose. No fígado, não houve alterações significativa do eixo, porém a fosforilação de JAK2 dependente de AT1r foi diminuída e consequentemente houve uma menor ativação das células estreladas no grupo que recebeu o GW501516. Além disso, as proteínas e genes relacionados à β-oxidação foram aumentados com o tratamento e aqueles relacionados à lipogênese de novo, diminuídos o que resultou em menor esteatose no parênquima hepático. Os rins apresentaram uma melhora da inflamação induzida pelo eixo, apesar de o eixo também não ter apresentado diferenças significativas com o tratamento. Também não foram encontradas diferenças significativas na expressão proteica e gênica das proteínas antioxidantes. Com esses resultados podemos concluir que a curta administração do GW501516 pôde aliviar os efeitos inflamatórios e a esteatose hepática causada pela dieta rica em frutose, podendo ser pensado como uma nova ferramenta terapêutica no tratamento da superativação do eixo ECA/AT1r. / High-activation of ACE/AT1r axis is closely linked to metabolix syndrome and low-grade inflammation state. Fructose administration in water or in diet has been proposed as a model to study the high-activity of this axis and AT1r-related inflammatory pathways. In this view, we aimed to evaluate if GW501516 administration could decrease the high-activation of ACE/AT1r axis and consequently fructose damage. To this males mice C57Bl/6 were fed a high-fructose diet (47%) during eight weeks or standard-chow diet. After eight weeks, the groups were randomly divided to start treatment with GW501516, totaling four experimental groups. Animals treated with GW501516 presented an improvement of systolic blood pressure and in urinary parameters, as proteinuria and uric acid. Also was verified an improvement in plasmatic triglycerides and uric acid. In white adipose tissue, GW501516 was able to decrease the components of this axis and improved inflammation as well. In liver, it was not found differences in axis, but JAK2 phosphorylation AT1r-dependent was decreased and consequently it was found a diminished activations of hepatic stellate cells. In addition, proteins and genes related to β-oxidation were increased with GW501516 and those related to lipogenesis de novo, were diminished, improving hepatic parenchyma. Kidneys presented an improvement of inflammation state, although it was not found differences in axis with treatment. Also, it was not found differences in gene and protein expression in relation to anti-oxidants proteins. These results show that short-administration of GW501516 could alleviate inflammatory effects and hepatic steatosis caused by high fructose diet, suggesting that GW501516 could be a new therapeutic option to treat the high activation of ACE/AT1r axis.