Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A doença hepática gordurosa não alcoólica é uma desordem multifatorial causada principalmente por excesso nutricional e resistência à insulina, com prevalência estimada de 20-40% nos países ocidentais. A dieta hiperlipídica e/ou rica em sacarose pode influenciar no desenvolvimento da esteatose hepática associada à obesidade e a resistência à insulina. O fígado, por assumir papel central no controle metabólico, é um órgão alvo nos casos de excesso alimentar, ocasionando, principalmente, acúmulo de gotículas de gordura nos hepatócitos. Este trabalho teve como objetivo avaliar o início das alterações morfológicas e metabólicas no fígado e no tecido adiposo de camundongos suíços machos alimentados com dieta hiperlipídica e/ou rica em sacarose. Camundongos suíços machos aos três meses de idade foram divididos em quatro grupos nutricionais: dieta padrão (SC), dieta hiperlipídica (HF), dieta rica em sacarose (HSu) e dieta hiperlipídica rica em sacarose (HFHSu). Os animais receberam as respectivas dietas durante quatro semanas. A massa corporal, a ingestão alimentar e a tolerância oral à glicose foram avaliados. Ao sacrifício, o fígado e os depósitos de gordura corporal foram removidos e processados para análises histomorfométricas e moleculares. As amostras de sangue foram obtidas para análises bioquímicas plasmáticas. Os dados foram expressos como média e erro padrão da média e as diferenças foram testadas por one-way ANOVA com pós-teste de Holm-Sidak, e foi considerado o nível de significância de p<0,05. Os grupos HF e HFHSu apresentaram-se mais pesados quando comparados aos grupos SC e HSu. Os animais dos grupos HF, HSu e HFHSu apresentaram intolerância à glicose, esteatose hepática e aumento de triglicerídeos hepáticos quando comparados ao grupo SC (p<0,0005). Adicionalmente, houve elevação na expressão hepática das proteínas transportador de glicose 2 (GLUT-2), proteína de ligação ao elemento regulador do esterol 1-c (SREBP1-c), fosfoenolpiruvato carboxiquinase (PEPCK), glicose -6- fosfatase (G6PASE), substrato do receptor da insulinaI-1 (IRS-1) e proteína quinase B (AKt/ou PKB) e redução da expressão no fígado do receptor ativador de proliferação peroxissomal (PPAR-&#945;) nos grupos experimentais em comparação com o grupo SC (p<0,0005). A administração de dieta hiperlipídica e/ou rica em sacarose promoveu intolerância à glicose e danos hepáticos (hepatomegalia, esteatose, redução da beta-oxidação, aumento na lipogênese e na produção de glicose) em camundongos machos adultos. / The non-alcoholic fatty liver disease is a multifactorial disorder caused mainly by excess nutritional and insulin resistance, with an estimated prevalence of 20-40% in Western countries. The diet can to influence in the development of fatty liver associated with obesity and insulin resistance. The liver plays a central role in metabolic control, is a target organ, in case of excess of food, mainly causing accumulation of fat droplets in hepatocytes. This study aimed to evaluate the early morphological and metabolic changes in the liver and adipose tissue of male Swiss mice fed high-fat and/or high-sucrose diets. Twenty three-month-old male Swiss Webster mice were divided into 4 groups: standard chow (SC), high-fat diet (HF), high-sucrose diet (HSu) and high-fat-high-sucrose diet (HFHSu). Animals received the respective diets for 4 weeks; throughout the experiment, body mass, food intake and oral glucose tolerance were evaluated. After the mice were euthanized, the liver was removed and processed for histomorphometrical and molecular analysis. Blood samples were obtained for serum analysis. The data were tested by one-way ANOVA with a Holm-Sidak post-hoc test and were expressed as the mean standard error of the mean; the significance level was set at p < 0.05. The HF and HFHSu groups were heavier than the SC and HSu groups. Animals from the HF, HSu and HFHSu groups presented glucose intolerance, hepatomegaly, liver steatosis and augmented hepatic triglycerides when compared to the SC group (p<0.0005). Additionally, there was an elevation in glucose transporter 2 (GLUT-2), sterol regulatory element binding protein-1c (SREBP-1c), phosphoenolpyruvate carboxykinase (PEPCK), glucose 6 phosphatase (G6PASE), insulin receptor substrate 1 (IRS-1), protein kinase B (AKT/ or PKB) protein expression and a reduction in peroxisome proliferator-activated receptor alpha (PPAR-alpha) expression in liver from the experimental groups compared to those of the SC group (p<0.0005). Administration of high-fat and/or high-sucrose diets promoted glucose intolerance and liver damage (hepatomegaly, steatosis, reduced beta-oxidation, increased lipogenesis and glucose production) in adult male mice.

A menopausa, fenômeno fisiológico que ocorre em todas as mulheres, em média, aos 51 anos, é acompanhada em cerca de 80% dos casos de sintomas como fogachos, secura vaginal, irritabilidade e insônia, que interferem na qualidade de vida e na produtividade socioeconômica das mulheres, além de predispô-las a doenças crônico-degenerativas, como arteriosclerose, obesidade e distúrbios cardiovasculares. A terapia de reposição hormonal à base de estrógenos, que visa reduzir os incômodos da menopausa, está associada ao aumento do risco de câncer de mama e do endométrio, como foi demonstrado em estudos científicos. Considerando que as mulheres orientais, consumidoras de soja, apresentam doenças crônico-degenerativas e câncer em taxas inferiores às dos países ocidentais, as isoflavonas da soja têm sido testadas em estudos clínicos e experimentais, porém com obtenção de dados até contraditórios. O presente estudo investigou o efeito da administração crônica de isoflavonas de soja no útero, mamas e tecidos adiposo e ósseo de ratas ovariectomizadas. Quarenta ratas Wistar adultas foram distribuídas em quatro grupos experimentais: a) ovariectomizadas: grupo ISO, recebendo isoflavonas de soja (100mg/kg/dia/v.o.); b) ovariectomizadas: grupo BE, recebendo benzoato de estradiol (10g/kg/dia/s.c.); c) ovariectomizadas: grupo OVX, recebendo salina (0,1ml/100g/dia/v.o.); d) controles: grupo FO, recebendo salina (0,1ml/100g/dia/v.o.). Antes e durante os 90 dias de tratamento, foram analisados os esfregaços vaginais, para acompanhamento do ciclo estral, determinação do peso corporal e do consumo de ração semanal. Após esse período, os animais foram anestesiados e o sangue coletado para análise de estradiol e progesterona séricos, por radioimunoensaio; e lipidograma e glicose, por espectrofotometria. Posteriormente, os animais foram sacrificados e necropsiados, coletando-se o útero, mamas, gordura intra-abdominal e fêmur para macroscopia e pesagem. Os tecidos selecionados para o estudo foram corados em HE, analisados por microscopia óptica e histomorfometria, visando investigar alterações do crescimento celular (software V.S NIH Image-J; imagens digitais-optronicos CCD). No grupo tratado com isoflavonas, o peso corporal diminuiu em relação OVX, no qual ocorreu aumento de peso em comparação aos animais falso-operados. O exame macroscópico revelou que o útero diminuiu de peso nas ratas do grupo ISO, semelhante às do OVX. Além disso, a histopatologia das glândulas endometriais não mostrou alterações entre os grupos ISO e OVX. Contudo, o grupo BE apresentou proliferação glandular, pseudoestratificação epitelial, frequentes mitoses típicas, metaplasia escamosa, infiltrado eosinofílico e hidrométrio. A concentração de estradiol no grupo ISO foi semelhante à do OVX. Porém, no grupo BE, o estradiol e o peso uterino apresentaram-se aumentados em relação ao OVX. Não foram observadas diferenças na histomorfometria mamária entre os grupos. Houve redução no peso do tecido adiposo abdominal no grupo ISO, comparado com o OVX, sem identificação de alterações morfológicas significativas, apenas hipotrofia celular, confirmada pela histomorfometria. Apesar de não ter havido diferenças na concentração de glicose, colesterol total e triglicerídeos, entre os grupos, o colesterol-HDL apresentou aumento no grupo ISO. Não houve diferença na densitometria do fêmur entre os grupos avaliados. Esses resultados indicam que o tratamento crônico com isoflavonas de soja, na dose testada, não induz mudanças significativas no útero, mamas e tecidos adiposo e ósseo, sugerindo segurança no tratamento, sem risco para o desenvolvimento de câncer. / Menopause, physiological phenomenon that occurs in all women, average age 51, is accompanied by about 80% of cases symptoms such as hot flashes, vaginal dryness, irritability and insomnia, which affect the quality of life and socioeconomic productivity women's, and predispose them to chronic degenerative diseases such as atherosclerosis, obesity and cardiovascular disorders. The hormone replacement therapy based on estrogen, which aims to reduce the discomforts of menopause is associated with an increased risk of endometrial and breast cancer, as has been shown in scientific studies. Whereas that women, consuming soy, had lower rates of chronic degenerative diseases and cancer at rates lower than those of Western countries, soy isoflavones have been tested in clinical and experimental studies, but with contradictory results. The present study investigated the effect of chronic administration of soy isoflavones on the uterus, breast, bone and adipose tissues from ovariectomized rats. Forty female Wistar rats were divided into four groups: a) ovariectomized: ISO group receiving soy isoflavones (100mg/kg/dia/vo); b) ovariectomized: EB group receiving estradiol benzoate (10&#956;g/kg/dia/sc); c) ovariectomized: OVX group receiving saline (0.1 ml/100g/dia/vo); d) controls: FO group, receiving saline (0.1 ml/100g/dia/vo). Before and during the 90 days of treatment vaginal smears were analyzed to monitor the estrous cycle, given the body weight and food intake monitored weekly. After this period, the animals were anesthetized and blood was collected for analysis of serum estradiol and progesterone serum by radioimmunoassay, and lipid and glucose by spectrophotometry. Subsequently, the animals were euthanized and necropsied, collecting the uterus, breasts, intra-abdominal fat and femur for macroscopic exam and weighing. The tissues selected for the study were stained with HE and analyzed by light microscopy and histomorphometry, in order to investigated possible changes in cell growth (NIH Image software VS-J;-optronics CCD digital images). In the group treated with isoflavones a decrease in body weight decreased compared OVX in which there was an increase in weight compared to the false-operated animals. Macroscopically, the uterus weight was lower in ISO group, similar to the OVX group. Furthermore, no change was showed in the histopathology of endometrial glands between ISO and OVX groups. The EB group showed glandular proliferation, pseudo-stratified epithelium, frequent mitoses typical squamous metaplasia, and eosinophilic infiltrate and hidrometry. The estradiol concentration in the ISO group was similar to OVX. However, EB group showed increase in estradiol and uterine weight in relation to OVX. No differences in mammary histomorphometry were observed among the four groups. Fat abdominal tissue weight was lower in ISO group compared with OVX group but, no morphological changes were seen on microscopy, only a cellular hipertrophy confirmed by histomorphometry. Although there were no differences in the glucose concentration, total cholesterol and triglycerides among groups, the cholesterol-HDL was increased in the group ISO. There was no difference in femur density among the groups. These results indicate that chronic treatment with soy isoflavones, at the tested dose did not induce significant changes in the uterus, breast, bone and adipose tissues, suggesting safety in handling without risk for development of cancer.

Submitted by Natalia Cristine Dias Dos Santos null (santos.nataliacris@gmail.com) on 2018-01-04T14:17:17Z No. of bitstreams: 1 Dissertação.pdf: 3720898 bytes, checksum: 25acdc7e7879412ae2dcd73e6d9407ea (MD5) / Approved for entry into archive by Laura Akie Saito Inafuko (linafuko@assis.unesp.br) on 2018-01-08T15:30:42Z (GMT) No. of bitstreams: 1 santos_ncd_me_assis.pdf: 3720898 bytes, checksum: 25acdc7e7879412ae2dcd73e6d9407ea (MD5) / Made available in DSpace on 2018-01-08T15:30:42Z (GMT). No. of bitstreams: 1 santos_ncd_me_assis.pdf: 3720898 bytes, checksum: 25acdc7e7879412ae2dcd73e6d9407ea (MD5) Previous issue date: 2017-11-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Vários estudos têm apontado que a interação de plasma rico em plaquetas (PRP) com células-tronco mesenquimais de tecido adiposo humano (CT-TAs) é capaz de otimizar o processo regenerativo, estimulando a angiogênese e o recrutamento de células para o local da lesão. O avanço no conhecimento sobre os aspectos físico-químicos do gel de PRP, bem como na interação e no comportamento das CT-TAs quando associadas ao gel poderão resultar em aplicações práticas em processos de regeneração tecidual. Em função disso, objetivou-se analisar diferentes parâmetros relativos a aspectos morfológicos, físico-químicos e estrutural do gel de PRP isolado ou em associação com CT-TAs (PRP/CT-TAs). As análises dos géis foram realizadas por meio da microscopia eletrônica de varredura (MEV) e da técnica de espectroscopia no infravermelho com transformada de Fourier (FTIR). Também foi avaliada a viabilidade e migração das CT-TAS após associação ao gel de PRP. Os resultados mostraram que as CT-TAs continuam viáveis, mantendo suas propriedades, assim como o gel de PRP, após a associação. Não foram observadas alterações estruturais nos géis de PRP isolado e PRP/CT-TAs, preservando os perfis físico-químicos das amostras. Concomitantemente com estes resultados, observou-se que o gel de PRP pode proporcionar um ambiente favorável às células, permitindo proliferação e migração celular. Os resultados obtidos permitem concluir que o gel de PRP pode ser usado como um potencial scaffold, mantendo as células viáveis quando encapsuladas, preservando os aspectos físico-químicos das CT-TAs e do PRP quando em associação. Além disso, pode-se propor que gel de PRP exerce efeitos positivos sob as CT-TAs, estimulando a proliferação e a viabilidade celular. / Several studies have shown that the platelet-rich plasma (PRP) in association with human adipose-derived mesenchymal stem cells (h-AdMSCs) is able to optimize the regenerative process, stimulating angiogenesis and the recruitment of cells to the lesion site. Advancing in the knowledge of the physico-chemical aspects of PRP-gels, as well as in the interaction and behavior of h-AdMSCs when associated to the gel will result in practical applications in tissue regeneration processes. Therefore, the aim of this study was to evaluate different morphological, physic-chemical and structural parameters of the isolated PRP-gel or in association with h-AdMSCs (PRP/h-AdMSCs). The PRP-gels were submitted to scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy technique (FTIR). The h-AdMSCs viability and migration after association with the PRP-gel were also evaluated. The results showed that as h-AdMSCs remain viable, retaining their properties, as the PRP gel, after association. There were no structural changes in the PRP gels isolated and PRP/h-AdMSCs, so maintaining the physical-chemical profiles of the samples. Additionally to these results, it was observed that the PRP-gel can provide a favorable environment for cells, allowing its proliferation and migration. In summary, it is possible to conclude that PRP-gel can be used as a potential scaffold, maintaining cell viability when encapsulated and preserving the physical-chemical aspects of h-AdMSCs and PRP when in association. In addition, it may be proposed that PRP gel exert chemotatic effects for h-AdMSCs, stimulating cell proliferation and viability.

Submitted by DANIELE LOPES PEREIRA null (danielelopesp@hotmail.com) on 2016-05-11T22:02:11Z No. of bitstreams: 1 dissertação maio - 2016.doc: 36656128 bytes, checksum: 544a8bb80add41fb7e4e0faa6dc3b70c (MD5) / Rejected by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: A versão final da dissertação/tese deve ser submetida no formato PDF (Portable Document Format). O arquivo PDF não deve estar protegido e a dissertação/tese deve estar em um único arquivo, inclusive os apêndices e anexos, se houver. E também, o arquivo submetido está sem a folha de aprovações providenciada pela Pós-graduação. Lembramos que a versão submetida por você é considerada a versão final da dissertação/tese, portanto não poderá ocorrer qualquer alteração em seu conteúdo após a aprovação. Corrija estas informações e realize uma nova submissão contendo o arquivo correto. Agradecemos a compreensão. on 2016-05-13T16:38:51Z (GMT) / Submitted by DANIELE LOPES PEREIRA null (danielelopesp@hotmail.com) on 2016-08-12T21:37:08Z No. of bitstreams: 1 Terapia celular em fibrose pulmonar.pdf: 14681718 bytes, checksum: ddd650e002e44e6ba3817e66564c1581 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-15T19:52:38Z (GMT) No. of bitstreams: 1 pereira_dl_me_assis.pdf: 14681718 bytes, checksum: ddd650e002e44e6ba3817e66564c1581 (MD5) / Made available in DSpace on 2016-08-15T19:52:38Z (GMT). No. of bitstreams: 1 pereira_dl_me_assis.pdf: 14681718 bytes, checksum: ddd650e002e44e6ba3817e66564c1581 (MD5) Previous issue date: 2016-03-15 / A Fibrose Pulmonar (FP), também denominada Alveolite Fibrosante, é uma pneumopatia idiopática, crônica e irreversível, que acomete o tecido pulmonar promovendo a deposição de tecido fibroso cicatricial e consequente remodelamento da arquitetura pulmonar. Considerando a inexistência de um tratamento clínico curativo que não seja apenas paliativo, a terapia com células-tronco desponta como alternativa promissora no tratamento da fibrose pulmonar e diversas outras patologias. Neste contexto, este projeto propôs o emprego da terapia celular com células-tronco mesenquimais de tecido adiposo humano (CTMTA) e “pool” de células mononucleares da medula óssea (BMMC), isoladas ou conjuntamente, em modelo experimental de fibrose pulmonar. Foram utilizados 36 ratos Wistar divididos em 6 grupos. O grupo controle recebeu instilação intratraqueal de tampão fosfato. Cinco grupos foram instilados com bleomicina (2U/animal) para a indução da doença e, após 14 dias, submetidos ou não a diferentes tratamentos utilizando células mononucleares da medula óssea e/ou células-tronco mesenquimais de tecido adiposo, sendo que um grupo recebeu tratamento placebo com tampão fosfato. A análise histológica evidenciou o desenvolvimento de fibrose nos animais instilados com bleomicina (p<0,0001) e uma melhora do quadro fibrótico nos animais submetidos ao tratamento, independentemente do tipo celular utilizado (p<0,001). A ventilação pulmonar basal revelou a presença de esforço respiratório nos animais instilados com bleomicina (p=0,0260), porém nenhum tipo de terapia foi capaz de reverter esse quadro. Não houve diferença significativa entre o perfil de leucócitos encontrados no lavado broncoalveolar dos animais sadios e doentes, entretanto a terapia com CTMTA promoveu a redução da porcentagem de linfócitos e o aumento de macrófagos nos animais doentes. O tratamento com BMMC e CTMTA, realizado de modo isolado ou em conjunto, mostrou remissão significativa do quadro de fibrose instalado. / Pulmonary Fibrosis (PF), also known as fibrosing alveolitis, is an idiopathic, chronic, irreversible lung disease that affects the lung tissue by promoting the deposition of fibrous scar tissue and subsequent lung architecture remodeling. Considering the need for curative medical treatment that is not only palliative, stem cell therapy is emerging as a promising alternative for the treatment of pulmonary fibrosis and many other diseases. ln this context, it is proposed in this study the cell therapy with mesenchymal stem cells derived from human adipose tissue (CTMTA) in association with the pool of mononuclear bone marrow cells (BMMC) in an experimental model of pulmonary fibrosis. The disease was induced in 4 groups of Wistar rats (n = 6) by intratracheal bleomycin (2U/animal). 14 days after instillation, each group was subjected to a specific treatment with BMMC and CTMTA, isolated or combined mode, while a fourth group was treated with phosphate buffer. The control group was instilled and treated with phosphate buffer. Histological analysis showed the development of disease in animais instilled with bleomycin (p<0,0001) and untreated, and improvement in the fibrous framework of animais subjected to cell therapy (p<0,001 ). The basal ventilation revealed the presence of respiratory effort in animais instilled with bleomycin, submitted or not to therapy (p=0,0260). Treatment with BMMC and CTMTA, performed alone or in conjunction mode showed similar results, since both promoted remission of pulmonary fibrosis.

Les premières utilisations du tissu adipeux comme produit de comblement en chirurgie plastique remontent à la fin du 19ème siècle. Depuis quelques décennies, la greffe de tissu adipeux a bénéficié d'un regain d'intérêt utilisant un procédé chirurgical rigoureux. Devant la démonstration de la survie cellulaire et les bons résultats cliniques obtenus, l'utilisation de cette technique s'est élargie à tous les domaines de la chirurgie plastique. Cette technique est simple et efficace et représente actuellement le meilleur moyen de restaurer les défauts de contours et de volume. Récemment, de nouvelles indications utilisant les capacitésrégénératrices du tissu adipeux ont été décrites. Elles concernent la cicatrisation des plaies chroniques et l'amélioration des dystrophies cutanées. Mais la limite de la greffe de tissu adipeux est l'absence de site donneur disponible au prélèvement. Le tissu adipeux est aujourd'hui reconnu comme la source la plus abondante de cellules souches mésenchymateuses multipotentes. Cela a donné un nouvel essor à la médecine régénérative pour réparer, remplacer ou régénérer les tissus et organes endommagés à partirdes cellules souches. Cette régénération se fait soit in-situ après administration des cellules souches, soit après développement in-vitro d'un tissu par ingénierie. Après une présentation du tissu adipeux et ses cellules souches ainsi que leurs applications actuelles en chirurgie plastique, le but de ce travail était de : 1. de clarifier les facteurs influençant les résultats de la greffe adipeuse pour une optimisation de cette technique. 2. d'explorer les possibilités offertes par les cellules souches adipeuses pour la médecine régénérative et l'ingénierie tissulaire, en vue d'une utilisation en chirurgie plastique / The first uses of adipose tissue as filler in plastic surgery started in the late 19th century. In recent decades, the adipose tissue transplantation has received renewed interest using a rigorous surgical procedure. Before the demonstration of cell survival and good clinical results, the use of this technique was extended to all areas of plastic surgery. This technique is simple and effective and is currently the best way to restore the defects of contour and volume. Recently, new indications using the regenerative capacity of adipose tissue have been described. They concern the healing of chronic wounds and the improvement of skin dystrophy. But the limit of the adipose tissue graft is the lack of available donor site for harvesting. Adipose tissue is now recognized as the most abundant source of multipotent mesenchymal stem cells. This gave a boost to regenerative medicine to repair, replace or regenerate damaged tissues and organs from stem cells. This regeneration is done either in-situ after administration of stem cells, after in-vitro development of tissue engineered. After a presentation of adipose tissue and stem cells and their current applications in plastic surgery, the aim of this study was to: 1. clarify the factors influencing the results of fat transplantation to optimize this technique. 2. explore the possibilities offered by ASCs for regenerative medicine and tissue engineering, for use in plastic surgery

Expansion of visceral adipose tissue correlates with the metabolic syndrome and increased cardiovascular risk. Hypertrophied visceral fat becomes inflamed, causing increased lipolysis, decreased triglyceride storage, and lipotoxicity in skeletal muscle and liver resulting in insulin resistance. Perivascular adipose tissue is a normal component of the adventitia of arteries in humans and animals. Whether or not perivascular adipose also becomes inflamed in obesity is an important question, as this may be an additional, direct mechanism by which obesity causes vascular inflammation and disease. Thus, for the first part of my thesis, we asked the question: does perivascular adipose in mice become inflamed with high fat feeding? In contrast to visceral adipose, macrophage gene expression was not increased in perivascular adipose in response to high fat diet, and this correlated with reduced F480 antigen positive cells as seen by immunohistochemistry and flow cytometry. Interestingly, perivascular adipose surrounding the thoracic aorta was similar to brown adipose tissue, a highly thermogenic fat depot, as shown by histology and DNA microarrays. Moreover, inter-scapular brown adipose was also resistant to diet induced inflammation in comparison to visceral adipose. These findings suggest that brown adipose in the perivascular niche may serve to protect the vasculature from diet induced inflammation, or from cold exposure, or both; whether or not brown perivascular adipose tissue exists in humans has yet to be determined. In the second part of my thesis, we evaluated the role of perivascular adipose tissue in the apolipoprotein E knockout mouse, which exhibits severe hyperlipidemia and atherosclerosis, but is resistant to diet induced obesity and glucose intolerance. We tested the hypothesis that in this model of severe atherosclerosis, inflammation of perivascular adipose does occur. However, we were surprised to find that macrophage specific gene expression, as determined by either microarray analysis or quantitative polymerase chain reaction, was not increased in either the perivascular or the visceral adipose of high fat diet fed apolipoprotein E knockout mice. While the visceral adipose of wild type mice had extensive alterations in gene expression in response to high fat diet, in particular, enrichment of inflammatory gene expression and broad down regulation of peroxisome proliferator activated receptor gamma target genes, apolipoprotein E knockout visceral adipose did not. Importantly, the apolipoprotein E knockout visceral adipose instead showed increased expression of genes encoding enzymes in fatty acid oxidation pathways. High fat diet fed apolipoprotein E knockout visceral adipose was also characterized by smaller adipocyte size. We conclude that, 1) inflammation in thoracic perivascular adipose does not occur in conjunction with diet induced obesity in normal animals nor with atherosclerosis in apolipoprotein E knockout mice, 2) thoracic perivascular adipose tissue is essentially identical to brown adipose tissue in mice, thus potentially protecting the vasculature from the cold, and 3) apolipoprotein E knockout mice remain lean on a high fat diet, despite hyperlipidemia and atherosclerosis, and the decreased adiposity correlates with decreased adipocyte size and adipose inflammation but increased oxidation of fatty acids. Consistent with previous work showing apolipoprotein E controls adipocyte uptake and deposition of triglyceride, its absence prevents adipocyte hypertrophy and resultant inflammation of visceral adipose tissue. Thus limiting adipocyte acquisition of fatty acids may be advantageous, provided that compensatory mechanisms to prevent sustained hyperlipidemia and peripheral organ lipotoxicity can be activated.

Well functioning circadian system is crucial component of healthy organism and its disruption can result in impairment of metabolic functions with consequential development of obesity and type 2 diabetes mellitus. Obesity is in general caused by enhanced migration of pro- inflammatory polarized macrophages (M1) into adipose tissue. We have shown, that interaction of this type of macrophages with adipose tissue had significant effect on rhythmic expression of clock genes in adipocytes. We further investigated effect of high fat diet and diet enriched by omega-3 fatty acids on circadian oscillations in WAT and differently polarized macrophages. This diet affected oscillations in adipose tissue and in M0 and M2 polarized macrophages. These results support previous findings of effect of omega-3 fatty acids on metabolism and suggest their effect on circadian system as well. Key words: circadian rhythms, adipose tissue, macrophages, omega-3 fatty acids, high fat diet

L’obésité est un facteur de risque pour de nombreuses maladies telles que le diabète de type 2 et les maladies cardiovasculaires. La prokinéticine-2 a été caractérisée comme une hormone anorexigène qui joue un rôle dans la régulation de l’appétit et le métabolisme énergétique via une action sur le récepteur PKR2 au niveau centrale.Cette étude consiste à étudier l’implication de la PK2 et de PKR1 dans le tissu adipeux et dans la physiopathologie de l’obésité. Les souris PKR1-/- dans lesquelles le gène codant pour PKR1 est totalement inactivé, présentent une obésité par hyperplasie, du fait de la prolifération des préadipocytes. Les souris PKR1-/- présentent un état diabétique, avec une insensibilité à l’insuline accrue et une forte intolérance au glucose. Les souris aP2-PKR1-/- dans lesquelles PKR1 est spécifiquement inactivé dans le tissu adipeux, présentent, elle aussi, une obésité hyperplasique, due de la prolifération des préadipocytes. In vitro, nos résultats montrent que la PK2 est capable d’inhiber la différenciation des préadipocytes en inhibant la prolifération des préadipocytes lors de la phase d’expansion clonale mitotique. Nos résultats permettent d’envisager un rôle de la PK2 et de son récepteur PKR1 dans le traitement de l’obésité. / Obesity is a risk factor for various disorders such as type 2 diabetes and cardiovascular diseases. The prokineticins, prokineticin-1 and prokineticin-2 bind two similar G protein-coupled receptors, PKR1 and PKR2. Prokineticin-2 is an anorexigenic hormone that plays a role in appetite regulation and energy metabolism, via a direct hypothalamic mechanism. Since adipocytes express mainly PKR1, we investigated the role of PKR1 in adipocyte functions. PKR1-null mutant mice exhibit increased body weight that is due to an increased visceral fat mass. Mutant adipose tissue is characterized by adipocyte hyperplasia due to an increase in number of proliferating preadipocyte. Mutant adipocytes exhibit downregulation of insulin signaling that is associated with glucose and insulin tolerance. Adipocyte-specific aP2-PKR1 knockout mice present also an increased visceral adipose tissue that lead to a slight increased body weight. Fat mass is also characterized by an hyperplasia and an increased preadipocyte proliferation. This mice present slight metabolic changes. Utilizing 3T3-L1 murine preadipocytes, our study reveals that prokineticin-2 exerts an antiadipogenic function in murine cells. Inhibition of adipogenesis mediated by prokineticin-2 involved PKR1. Prokineticin-2 also inhibits proliferation of preadipocytes. These results suggest that prokineticin-2 via PKR1 signaling plays a crucial role in adipogenesis and adipose tissue hyperplasia.

L’imagerie du petit animal est incontournable pour le développement des recherches dans les secteurs de labiologie, de la médecine et de l’industrie pharmaceutique. Parmi les différentes modalités d’imagerie développéeschez l’humain et adaptées à l’animal, l’imagerie tomographique à rayons X est devenue une référencepour l’analyse des caractères anatomiques et phénotypiques chez la souris. Elle permet de réaliser des étudeslongitudinales in vivo et des analyses haute résolution ex vivo de façon non invasives et en 3D. L’analyse deces images 3D nécessite des outils spécifiques à chaque problématique.Dans ce contexte, notre travail de thèse a permis d’apporter des contributions sur les thématiques suivantes :1. le développement d’outils d’analyse, à la fois quantitatifs et qualitatifs, pour l’imagerie des tissusminéralisés et adipeux2. l’application des méthodologies développées à des problématiques de recherche biomédicale3. l’étude comparative et "multi-échelle" de différentes technologies de tomographie X pour l’imagerie dupetit animal4. la mise au point d’une méthode originale par résonnance paramagnétique électronique pour la dosimétried’un acte d’imagerie X chez le petit animalEn conclusion, les outils d’imagerie 3D que nous avons développés représentent un nouvel apport pour la dissectionvirtuelle de l’animal de laboratoire, permettant l’exploration de nombreux tissus et organes et rivalisantavec les méthodes d’histologie et de microscopie électronique.L’application de ces méthodes d’imagerie pour la recherche fondamentale et pré-clinique ouvre la perspectived’une alternative nouvelle dans l’expérimentation animale. / Small animal imaging is highly necessary for the development of biomedical research and pharmaceuticalapplications. Amongst various available imaging methods, X-Ray tomography is now considered as a goldstandard for anatomical and phenotypical analysis in mice. CT imaging allows non invasive longitudinal studiesin vivo and high resolution analysis ex vivo. The 3D image analysis requires the development of specific toolsdepending on the biomedical problematics.In this context, we have investigated the following research areas :1. Development of 3D image tools for qualitative and quantitative image analysis of mineralized andadipose tissues in murin models2. Application of our tools to biomedical investigations3. Comparative and multi-scale analysis of various tomography technologies for small animal imaging4. Development of an original method using Electronic Paramagnetic Resonance (EPR) for X-ray dosimetryin miceIn conclusion, our 3D imaging methods are potentially of high interest for the virtual dissection of laboratoryanimals, allowing extended analysis of various tissues and organs complementary to standard histological andmicroscopic approaches.

Objetivo: Sabe-se que trabalhadores noturnos tem preferência por lanches com maior concentração de gordura saturada. Trabalhadores de turno, particularmente trabalhadores noturnos, apresentam mais frequentemente hipertrigliceridemia e hiperglicemia, bem como menores níveis de HDL-colesterol quando comparados a trabalhadores diurnos, devido a alterações circadianas e estilo de vida, sugerindo uma predisposição para o desenvolvimento de doenças cardiovasculares. Estas alterações são conhecidas como Síndrome Metabólica (SM). Portanto, para entender as conseqüências de uma dieta hiperlipídica é importante a padronização de dietas para modelos animais que mimetizem a alimentação do trabalhador de turno. Materiais e Método: estudo experimental com 20 ratos Wistar dos quais 10 eram controle (CG) e 10 submetidos à dieta hiperlipídica (HFD). Utilizaram-se três dos cinco critérios do NCEP-ATP III para diagnóstico de SM - glicose, triglicerídeos (TG) e HDL-col. A quantidade de tecido adiposo visceral (TAV) foi avaliada bem como o peso do fígado e das glândulas adrenais. O peso ponderal foi avaliado semanalmente e o a ingestão alimentar e hídrica diariamente. Foi utilizado o t-test de Student’s para amostras independentes. Resultados: não houve diferença significante entre os grupos para glicose, HDL-col e TG na medida basal. Após 15 semanas de intervenção, grupo HFD mostrou um aumento dos níveis de TG (p=0,01) e glicose (p=0,01) e diminuição de HDL-col (p=0,009) quando comparados com CG. Grupo HFD apresentou maior TAV (p=0,005) e peso do fígado (p=0,01). CG mostrou um aumento de ingestão alimentar e hídrica (p<0,001 e p<0,001 respectivamente) enquanto que o consumo energético foi maior no grupo HFD (p<0,001). Não foi encontrada diferença no peso das glândulas adrenais (p=0,07) e peso ponderal (p=0,63). Conclusão: os animais submetidos à dieta hiperlipídica apresentaram alterações metabólicas apesar da manutenção do peso corporal. Não foi encontrada correlação entre peso corporal e quantidade de TAV, sugerindo que o peso corporal não é preditor para quantidade de gordura corporal e que a composição da dieta influi diretamente nos marcadores de SM. / Background and Aim: Shift workers, particularly night workers, more frequently present metabolic changes when compared to day workers suggesting a predisposition for cardiovascular disease. These changes are known as Metabolic Syndrome (MS). Therefore, to increase understanding of the consequences of a high-fat diet it is important to standardize a diet for experimental animals that mimics the shift workers’ diet and its effects. Methods and Results: experimental study with 20 Wistar rats of which 10 were control group (CG) and 10 high-fat diet group (HFD). Three of five criteria for MS diagnosis from NCEP-ATP III were assessed. The amount of visceral adipose tissue was determined (VAT). Body weight was assessed weekly and food and water intake were measured daily. Student’s t-test for independent samples was used. The groups were similar at baseline. After fifteen weeks of intervention, HFD group showed an increase in serum TG (p=0.01) and glucose (p=0.01) levels and a decrease in HDL (p=0.009) compared to CG. HFD showed increased VAT (p=0.005) and liver weight (p=0.01). Food intake and water intake were higher in CG (p<0.001 and p<0.001 respectively) while energy intake was increased in HFD (p<0.001). No difference was found in adrenal glands weight (p=0.07) and body weight (p=0.63). Conclusion: Animals submitted to HFD present significant metabolic alterations in spite of the maintenance of body weight. Body weight is not a predictive factor for the amount of VAT and that the quality of diet composition direct influences MS markers.