Tissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.

Studies had shown that leucine supplementation increases insulin sensitivity and it has been studied that n-3 PUFA may have an anti-inflammatory effect in adipocytes. However, the extent to which dietary sources such as leucine and/or n-3 PUFA act through PFKFB3/iPFK2 to suppress adipocyte inflammatory response has not been studied; PFKFB3/iPFK2 is a regulator that links adipocyte metabolism and inflammatory responses. In this study, the involvement of PFKFB3/iPFK2 in the effects of insulin sensitizing and anti-inflammatory effect of leucine and/or n-3 PUFA are explored using cultured 3T3-L1 adipocytes including wild-type cells, PFKFB3-control cells (iPFK2-Ctrl) and PFKFB3-knockdown cells (iPFK2-KD). In iPFK2-Ctrl cells, leucine supplementation appears to have insulin-sensitizing effects through improving p-Akt/Akt insulin signaling, but have no effect on adiponectin expression, and appear to have limited anti-inflammatory effects. n-3 PUFA supplementation appears to have limited effects on both insulin sensitizing and anti-inflammatory effects in iPFK2-Ctrl. In contrast, n-3 PUFA exhibit pro-inflammatory expression in iPFK2-KD. The results of this study support the hypothesis that PFKFB3/iPFK2 is critically involved in insulin-sensitizing effects of leucine. This role of PFKFB3/iPFK2, however, appears to be independent of anti-inflammatory responses. Given this, it is likely that PFKFB3/iPFK2 only account, in part, for the beneficial effects of leucine. n-3 PUFA stimulate PFKFB3/iPFK2 activity in wild-type adipocytes. However, PUFA do not exhibit anti-inflammatory and insulin-sensitizing effects in controls. In contrast, n3-PUFA exhibit proinflammatory effects in iPFK2-KD cells. Taken together, PFKFB3/iPFK2 is involved, at least in part, in the effects of insulin sensitization of leucine and appears to protect adipocytes from inflammatory responses, which could be exacerbated by n-3 PUFA when PFKFB3/iPFK2 is disrupted.

Bei der vorliegenden Arbeit handelt es sich um eine experimentelle Untersuchung zum Einfluss von zwei Ko-Transmittern des autonomen Nervensystems, Neuropeptid Y und dem Pituitary Adenylate Cyclase-activating Polypeptide (PACAP), auf den intrazellulären Kalziumspiegel und die Insulinsensitivität von 3T3-L1 Adipozyten. Mittels Polymerasekettenreaktion und Western Blot Analyse konnte die Expression des NPY-1 (Y1) Rezeptors als auch die der PACAP Rezeptoren PAC1 und VPAC2 nachgewiesen werden. Die Aktivierung des Y1 oder des PAC1 Rezeptors durch ihre Agonisten führte zur Erhöhung des intrazellulären Kalziumspiegels. Im Weiteren führte NPY nach Ko-Applikation mit Insulin zu einer abgeschwächten Insulinsensitivität der Adipozyten, da sowohl die insulin-stimulierte Translokation von Glukosetransporter 4 zur Zelloberfläche als auch die Glukoseaufnahme durch NPY abgeschwächt wurde. Dieser Effekt konnte als Y1 spezifisch beschrieben werden. Diese Ergebnisse gewähren somit neue Einblicke über den peptidergen Einfluss auf den Adipozytenstoffwechsel und erlauben Rückschlüsse über die Rolle des autonomen Nervensystems in der Entwicklung von Adipositas und Diabetes mellitus Typ 2.

Diss. (sammanfattning) Göteborg : Univ., 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2004. / Härtill 4 uppsatser.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.112-113

Dissertation (Ph.D.)--University of Toledo, 2010. / "Submitted to the Graduate Faculty as partial fulfillment of the requirement for the Doctoral of Philosophy Degree in Biomedical Sciences." Title from title page of PDF document. "A Dissertation entitled"--at head of title. Bibliography: p. 80-98.

Thèse (Ph. D.)--Université Laval, 2004. / Bibliogr. Publié aussi en version électronique.

Submitted by MIRIANE DE OLIVEIRA null (miriane.deoliveira@yahoo.com.br) on 2016-03-14T13:42:50Z No. of bitstreams: 1 Tese Miriane de Oliveira 2016 II.pdf: 8269819 bytes, checksum: 06d6147cbc170e832f61a251756885a2 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-03-14T18:06:26Z (GMT) No. of bitstreams: 1 oliveira_m_dr_bot.pdf: 8269819 bytes, checksum: 06d6147cbc170e832f61a251756885a2 (MD5) / Made available in DSpace on 2016-03-14T18:06:26Z (GMT). No. of bitstreams: 1 oliveira_m_dr_bot.pdf: 8269819 bytes, checksum: 06d6147cbc170e832f61a251756885a2 (MD5) Previous issue date: 2016-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os hormônios tireoidianos (HT) são essenciais para a sobrevivência, estando envolvidos nos processos de desenvolvimento, crescimento e metabolismo. As ações dos HT são distintas para diferentes órgãos alvos, e podem ocorrer por meio de seus receptores TRα e TRβ, ou por vias alternativas, as quais podem envolver a integrina αvβ3, as proteínas quinases ativadas por mitógenos (MAPK) e a fosfatidil inositol 3 quinase (PI3K). O tecido adiposo (TA) representa um importante alvo dos HT; produzem várias substâncias biologicamente ativas com diferentes funções fisiológicas, denominadas adipocinas, entre elas a leptina e adiponectina. A leptina é considerada um sinal adipostático ao cérebro e está envolvida na regulação do balanço energético. Adiponectina está envolvida em importantes efeitos metabólicos, como estimulação da oxidação de ácidos graxos, redução da gliconeogênese e aumento da termogênese. Vários estudos têm procurado relacionar a síntese de leptina e adiponectina com os HT; entretanto, os resultados obtidos são controversos. O objetivo desse trabalho foi avaliar o envolvimento da via PI3K nos efeitos da triiodotironina (T3) sobre a expressão gênica de leptina, adiponectina e do TRα em cultura de adipócitos, 3T3-L1. Para isso utilizamos inibidor da via PI3K, LY294002 e inibidor da síntese proteica (ciclohexamida - CHX), para verificar se ação do T3 é direta ou indireta. Após a diferenciação das células 3T3-L1 elas foram incubadas por uma hora com T3 na presença ou ausência dos inibidores citados acima. Posteriormente ao período de tratamento foi realizada a análise de expressão gênica de mRNA (leptina, adiponectina e TRα) por RT- PCR e de proteina (leptina) pela técnica de Western Blot. Os resultados para leptina, assim como para TRα, demonstraram a ativação da via de sinalização PI3K para ação do T3 na expressão gênica desses genes, sendo que essa ação pode ser indireta ou direta, dependendo da dose de hormônio administrada. Em relação a adiponectina, a dose suprafisiológica de T3 (sem a ativação da via PI3K) ou desativação da via PI3K eleva os níveis dessa adipocina, e constatamos que a ação do T3, nessa dose, sobre os níveis de mRNA de adiponectina é indireta em adipócitos, 3T3-L1. Em resumo, durante uma hora de tratamento, diferentes doses de T3 aumentam os níveis de expressão das adipocinas, leptina e adiponectina, e do TRα em cultura celular de adipócitos, 3T3-L1, e a via de sinalização PI3K se faz importante para a ação do T3. / The thyroid hormones (HT) are essential for survival, being involved in the processes of development, growth and metabolism. Process actions are distinct for different organs, and may occur either directly through its receptors TRα and TRβ, or indirectly through alternative pathways, which may involve integrin αvβ3, the mitogen-activated protein kinases (MAPK) or phosphatidyl inositol 3 kinase (PI3K). Adipose tissue (TA) is an important target of HT; it produces various biologically active substances with different physiological functions, called adipokines, including leptin and adiponectin. Leptin is considered an adipostatic signal to the brain and is involved in the regulation of energy balance. Adiponectin is involved in important metabolic effects, such as stimulating fatty acid oxidation, reducing gluconeogenesis and increasing thermogenesis. Several studies have sought to relate the leptin and adiponectin synthesis with HT, however the results are controversial. The present study aimed to examine the effects of triiodothyronine (T3), on the modulation of leptin, adiponectin and TRα expression and the involvement of the PI3K signaling pathway in adipocytes, 3T3-L1, cell culture. Will be used pathway inhibitors PI3K, LY294002, and protein synthesis inhibitors (cycloheximide - CHX), to examine whether directly or indirectly T3 action. After the differentiation of the cells 3T3-L1 will be incubated by one hour with T3 in the presence or absence of the inhibitors mentioned above. After hormone treatment was measured the of leptin, adiponectin and TRα gene expression using RT-PCR in real time, it was also assessed the expression of leptin by Western blot. The results to leptin, as well as TRα, demonstrated that the activation of the PI3K signaling pathway has a role in TH-mediated direct or indirect gene expression, depending on the dose administered. Regarding adiponectin, T3 supraphysiological dose (without activation of the PI3K pathway) or deactivation of the PI3K pathway raises the levels of this adipokine in adipocytes, 3T3-L1. In summary, during an hour of treatment, different doses of T3 increase of adipokines levels of expression, leptin and adiponectin, and TRα in cell culture of adipocytes 3T3-L1 and PI3K signaling pathway becomes important for action T3.

Le cancer du sein est le cancer le plus fréquemment observé chez les femmes en France et dans le monde. Bien que le nombre de cas observés chaque année a tendance à diminuer depuis 2005, notamment grâce au dépistage organisé, cette maladie reste la première cause de décès par cancer chez les femmes. De nombreux travaux ont montré que la progression tumorale est dépendante des cellules tumorales mais également des cellules " saines " du microenvironnement (ou stroma) qui entourent la tumeur. Dans le cas du cancer du sein, les adipocytes, type cellulaire majeur du stroma mammaire, représentent des acteurs émergents dans la progression tumorale. Mon équipe est l'une des premières à avoir montré que les adipocytes péritumoraux étaient impliqués dans l'agressivité des cancers du sein. Du dialogue bidirectionnel qui s'instaure entre les adipocytes et les cellules cancéreuses mammaires résulte des modifications des deux types cellulaires : (i) les cellules tumorales " éduquées " par les adipocytes présentent des capacités invasives augmentées et une plus grande résistance aux traitements et (ii) les adipocytes co-cultivés avec les cellules tumorales acquièrent un phénotype activé avec des modifications spécifiques telles que délipidation, perte de marqueurs adipocytaires, surexpression de cytokines pro-inflammatoires et sécrétion de protéines de la matrice extracellulaire, qui nous ont amené à les nommer CAA pour " Cancer-Associated Adipocytes ". De façon intéressante, le dialogue paracrine entre les tumeurs et les adipocytes pourrait être amplifié dans l'obésité, où l'équilibre normal des protéines sécrétées par le tissu adipeux est perturbé. Dans le cancer du sein, l'obésité est associée à une augmentation des risques de survenue après la ménopause et une aggravation du pronostic indépendamment du statut ménopausique s'expliquant par une augmentation de la dissémination locale et à distance et par une réponse diminuée aux traitements, notamment par une résistance plus importante. L'objectif de ma thèse a été d'évaluer le rôle des adipocytes dans la chimiorésistance des cellules tumorales mammaires. En effet, la résistance est une limite majeure à l'efficacité des traitements et contribue à l'apparition de rechutes, lesquelles sont augmentée chez les patientes obèses. Au moyen d'un modèle de co-culture en 2D, nous avons montré que les adipocytes sont capables de promouvoir une résistance pléïotropique (doxorubicine, paclitaxel, 5-fluorouracile et cyclophosphamide) dans diverses lignées tumorales mammaires, indépendamment du type de tumeur. Grâce aux propriétés de fluorescence des anthracyclines, nous avons montré que cette résistance implique une augmentation de l'efflux de doxorubicine, l'empêchant d'agir au niveau de son site d'action nucléaire. Ce mécanisme d'efflux implique un processus original, qui fait intervenir la protéine de voûte majeure MVP / LRP (Major vault protein / Lung resistance protein), un transporteur nucléocytoplasmique dont la fonction reste à ce jour mal comprise. Suite à l'efflux nucléaire de drogue, celle-ci s'accumule dans des vésicules cytoplasmiques avant d'être effluée hors de la cellule via des vésicules extra cellulaires. Nous avons également pu montrer que cette résistance médiée par MVP s'explique par la sécrétion de facteurs solubles adipocytaires et est amplifiée en conditions d'obésité. En conclusion, nos résultats montrent que les adipocytes péritumoraux sont capables d'influencer la progression tumorale en favorisant la chimiorésistance via un mécanisme original impliquant la protéine MVP, qui pourrait potentiellement devenir un marqueur de résistance aux traitements. Ces travaux pourraient expliquer, au moins en partie, le mauvais pronostic des cancers du sein chez les patientes obèses et ouvrent donc, à plus long terme, des perspectives thérapeutiques intéressantes, destinées à interrompre le dialogue délétère entre adipocytes et cellules tumorales, en particulier chez les patients obèses. / Breast cancer is the most common cancer among women in France, as well as in the European Union and the United States. Although the number of cases observed each year has tended to decrease since 2005, notably due to organized screening, this disease remains the leading cause of cancer death in women. Many studies have shown that tumor progression is dependent on tumor cells but also on the "healthy" cells of the microenvironment (or stroma) that surround the tumor. In the case of breast cancer, adipocytes, the major cell type of the mammary stroma, represent emerging actors in tumor progression. My team is one of the first to have shown that peritumoral adipocytes were involved in the aggressiveness of breast cancers. From the bi-directional dialogue that takes place between adipocytes and mammary cancer cells results some changes in both cell types : (i) the tumor cells "educated" by the adipocytes have increased invasive capacities and greater resistance to treatments and (ii) the adipocytes co-cultured with the tumor cells acquire an activated phenotype with specific modifications such as delipidation, loss of adipocyte markers, overexpression of pro-inflammatory cytokines and secretion of proteins of the extracellular matrix, which led us to name them CAA for "Cancer-Associated Adipocytes". Interestingly, the paracrine dialogue between tumors and adipocytes could be amplified in obesity, where the normal balance of proteins secreted by adipose tissue is disrupted. In breast cancer, obesity is associated with an increased risk of occurrence after menopause and a worsening prognosis independent of menopausal status due to increased dissemination (local and remote) and decreased response to treatments, in particular by a greater resistance. The objective of my thesis was to evaluate the role of adipocytes in the chemoresistance of mammary tumor cells. Indeed, resistance is a major limit to the effectiveness of treatments and contributes to the onset of relapses, which are increased in obese patients. Using a 2D co-culture model, we have shown that adipocytes are able to promote pleiotropic resistance (doxorubicin, paclitaxel, 5-fluorouracil and cyclophosphamide) in various mammary tumor lines, irrespective of tumor type. By taking advantage of the fluorescence properties of anthracyclines, we have shown that this resistance implies an increase in the doxorubicin efflux, preventing it from acting at its site of nuclear action. This efflux mechanism implies an original process involving the major vault protein MVP / LRP (Major Vault Protein / Lung Resistance Protein), a nucleocytoplasmic transporter whose function remains poorly understood to date. Following nuclear drug efflux, it accumulates in cytoplasmic vesicles before before being expelled from the cell via extracellular vesicles. We also showed that this resistance mediated by MVP could be explained by the soluble factors secreted from adipocytes and is amplified in obesity conditions. In conclusion, our findings highlight that peritumoral adipocytes are able to influence tumor progression by promoting chemoresistance via an original mechanism involving the MVP protein, which could potentially become a marker of resistance to treatments. This work may explain, at least in part, the poor prognosis of breast cancers in obese patients and thus could provide interesting therapeutic perspectives, in order to interrupt the deleterious dialogue between adipocytes and tumor cells, particularly in obese patients.