Ce mémoire d'HDR (Habilitation à Diriger des Recherches) représente une synthèse de mes activités d'enseignement et de mes travaux de recherche. Mon parcours de recherche a été centré sur les acides gras polyinsaturés de la famille oméga-3, notamment l'acide docosahexaénoïque (DHA), un nutriment bioactif indispensable au développement du système nerveux central et à la fonction rétinienne. Mon intérêt pour cette molécule fondamentale m'a amenée au cours de ces années à réaliser des recherches allant de la biosynthèse du DHA à partir de son précurseur (l'acide alpha-linolénique), à son ciblage spécifique au niveau de la sphère cérébro-vasculaire en passant par son métabolisme (peroxydation lipidique et métabolisme oxygéné).

The effects of peptides derived from β-lactoglobulin and α-lactalbumin on metabolic syndrome were studied. α-lactalbumin and β-lactoglobulin were hydrolyzed with trypsin, alcalase, flavourzyme, or a combination of alcalase and flavourzyme and fractionated. Angiotensin coverting enzyme inhibition of the < 1 kDa fraction of alcalase hydrolyzed β-lactoglobulin was 95 %. Antioxidant activity of the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme was 18 %. Stimulated adipocytes incubated with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with either trypsin or alcalase produced 30 pg/mL of interleukin 6. Adiponectin and glucose transporter type 4 secretions increased 1.1 and 0.86 fold respectively during incubation with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme. Results indicate that β-lactoglobulin peptides formed with alcalase and a combination of alcalase and flavourzyme influence markers associated with metabolic syndrome and may be useful as functional foods or nutraceuticals.

Pour étudier l'impact de la signalisation de la prolactine (PRL), une hormone impliquée dans la proliférationcellulaire sur l'adipocyte et la cellule β pancréatique, deux types cellulaires impliqués dans la balanceénergétique, nous avons caractérisé le phénotype de souris déficientes en récepteur de la PRL (R PRL-/-) sousdifférentes conditions physiopathologiques. Dans un premier temps, nous avons étudié l'impact du R PRL sur ledéveloppement d'une obésité induite par un régime obésogène. Dans un deuxième temps, nous nous sommesintéressés à l'impact du R PRL sur l'ontogenèse des cellules β durant les adaptations périnatales. Nous avonsaussi évalué son rôle sur la sécrétion d'insuline à l'âge adulte.Notre première étude montre que les souris R PRL-/- sous régime obésogène ont une prise de poids réduite etune augmentation de la dépense énergétique comparées à celles des souris sauvages. Nous montrons que desadipocytes beiges, une nouvelle classe d'adipocytes thermoactifs récemment caractérisés et exprimant laprotéine découplante UCP1, émergent dans le tissu adipeux blanc périrénal des souris R PRL-/- soumises à unrégime gras. Nous avons démontré que le R PRL contribue à l'apparition des adipocytes beiges en modulant lavoie de signalisation pRb/FoxC2 permettant la résistance à l'obésité induite par le régime gras.Notre deuxième étude montre que la souris R PRL-/- et le rat GK, un modèle de diabète de type 2, ont un défautd'adaptation de la masse des cellules β en période périnatale. Cette altération est corrélée à un défautd'expression d'igf2 (Insulin-like Growth Factor 2), une cible de la PRL. A partir d'îlots de Langherans de sourisadultes, nous avons confirmé que le R PRL est essentiel à la sécrétion d'insuline.Les résultats obtenus ont permis de mieux comprendre le rôle de la PRL sur la balance énergétique. Ces travauxouvrent des perspectives nouvelles pour le développement de stratégies thérapeutiques dans la lutte contrel'obésité et le diabète de type II.

The effects of peptides derived from β-lactoglobulin and α-lactalbumin on metabolic syndrome were studied. α-lactalbumin and β-lactoglobulin were hydrolyzed with trypsin, alcalase, flavourzyme, or a combination of alcalase and flavourzyme and fractionated. Angiotensin coverting enzyme inhibition of the < 1 kDa fraction of alcalase hydrolyzed β-lactoglobulin was 95 %. Antioxidant activity of the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme was 18 %. Stimulated adipocytes incubated with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with either trypsin or alcalase produced 30 pg/mL of interleukin 6. Adiponectin and glucose transporter type 4 secretions increased 1.1 and 0.86 fold respectively during incubation with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme. Results indicate that β-lactoglobulin peptides formed with alcalase and a combination of alcalase and flavourzyme influence markers associated with metabolic syndrome and may be useful as functional foods or nutraceuticals.

Il existe deux populations d’adipocytes thermogéniques, les adipocytes bruns du tissu adipeux bruns et les adipocytes ‘’brites’’ qui apparaissent au sein du tissu adipeux blanc. Récemment, la caractérisation d’adipocytes bruns et brites fonctionnels chez l’homme adulte a permis d’envisager de nouvelles approches nutritionnelles et thérapeutiques pour traiter l’obésité. Mon projet de thèse a porté sur l’étude des effets des acides gras polyinsaturés ω6 et ω3 d’origine alimentaire sur la conversion des adipocytes blancs en brites, d’abord chez l’homme in vitro puis chez les rongeurs in vivo. Nous avons pu ainsi démontrer que l'acide arachidonique ω6 à l’origine de nombreux métabolites oxygénés, exerce 1) un effet inhibiteur sur la formation des adipocytes brites grâce aux prostaglandines E2 et F2α, et 2) un effet inducteur via la prostacycline. En effet, celle-ci active la conversion des adipocytes blancs en brites par une voie impliquant le récepteur membranaire IP et les récepteurs nucléaires PPARs. En nous basant sur les recommandations nutritionnelles actuelles chez l’Homme, qui ont pris en considération l’insuffisance de l’apport en acides garsω3 par rapport à l’excès des ω6, nous avons pu montrer chez la souris qu’une supplémentation en acides gras ω3 dans le régime alimentaire était capable d’inhiber l’effet néfaste des acides gras ω6 et d’activer le tissu adipeux brun. Nos résultats démontrent l’importance de la biodisponibilité de l’acide arachidonique dans la biologie du tissu adipeux et permettent de renforcer l’idée que le rééquilibrage du ratio ω6/ω3 est un outil de choix dans la prévention du surpoids et de l’obésité et les maladies métaboliques associées / There are two types of thermogenic adipocytes able to use fatty acids and glucose to produce heat. We distinguish brown adipocytes from the brown adipose tissue and ‘’brite’’ adipocytes which occur into the white adipose tissue. Recently, the characterization of functional brown and brite adipocytes in adult humans has led to the consideration of their use to treat obesity by increasing energy expenditure. My thesis project was to study the effect of dietary polyunsaturated fatty acids, on the conversion of white into brite adipocytes, in vitro and in vivo, in humans and rodents respectively. We demonstrated that arachidonic acid ω6, precursor of prostaglandins, has 1) an inhibitory effect on the recruitment of brite adipocytes via prostaglandins E2 and F2α and 2) an activatory effect via prostacyclin. In fact, prostacyclin induces the conversion of white into brite adipocytes through the IP receptor and the PPARs signaling pathways. Based on human present nutritional recommendations, we demonstrated that a supplementation of ω3 fatty acids in mice diet was able to inhibit the negative effect of ω6 fatty acids and activate brown adipose tissue. Our data highlights the importance of arachidonic acid bioavailability on the biology of adipose tissue and reinforce the idea that an equilibrate ω6/ω3 ratio is a tool that can be used to prevent overweight obesity and associated metabolic disorders

Células-tronco mesenquimais derivadas de medula óssea (CTMs-MO) e de tecido adiposo (CTMs-TA) são uma ferramenta atrativa para a reparação do tecido ósseo baseada na terapia celular. No presente estudo, foi investigado o potencial osteogênico e adipogênico de CTMs-MO e CTMs-TA, assim como o efeito da intercomunicação entre osteoblastos e adipócitos na expressão do fenótipo celular. CTMs-MO e CTMs-TA de ratos foram cultivadas em meios de crescimento, osteogênico e adipogênico para avaliar a diferenciação osteoblástica e adipocítica. Adicionalmente, osteoblastos e adipócitos foram cocultivados de forma indireta para investigar o efeito dos adipócitos sobre os osteoblastos e vice versa. CTMs-MO e CTMs-TA apresentaram potencial tanto osteogênico quanto adipogênico em condições não indutoras de diferenciação. No entanto, quando expostas ao meio osteogênico, as CTMs-MO exibiram maior expressão gênica de RUNX2, fosfatase alcalina e osteocalcina, expressão proteíca de RUNX2 e maior formação de matriz extracelular mineralizada comparadas às CTMs-TA. Por outro lado, em condições adipogênicas, as CTMs-TA apresentaram maior expressão gênica de PPAR&gamma;, proteína adipocítica 2 e resistina, expressão proteíca de PPAR&gamma; e maior formação de acúmulo lipídico comparadas às CTMs-MO. A presença de adipócitos em coculturas indiretas inibiu a expressão do fenótipo osteoblástico, enquanto os osteoblastos não apresentaram um efeito marcante sobre a expressão do fenótipo adipocítico. Em conclusão, o presente estudo mostrou que as CTMs-MO são mais osteogênicas enquanto as CTMs-TA são mais adipogênicas. Adicionalmente, foi observado que a intercomunicação entre osteoblastos e adipócitos pode afetar negativamente o reparo ósseo. Assim, postulamos que o maior potencial osteogênico das CTMs-MO as tornam a escolha mais adequada para a indução do reparo ósseo baseado na terapia celular. / Mesenchymal stem cells from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are attractive tools for cell-based therapies to repair bone tissue. In the present study, we investigated the osteogenic and adipogenic potential of BM-MSCs and AT-MSCs as well as the effect of crosstalk between osteoblasts and adipocytes on cell phenotype expression. Rat BM-MSCs and AT-MSCs were cultured either in growth, osteogenic or adipogenic medium to evaluate osteoblast and adipocyte differentiation. Also, osteoblasts and adipocytes were indirectly cocultured to investigate the effect of adipocytes on osteoblast differentiation and vice versa. BM-MSCs and AT-MSCs exhibit osteogenic and adipogenic potential under non-differentiation-inducing conditions. However, when exposed to osteogenic medium, BM-MSCs exhibited higher gene expression of RUNX2, alkaline phosphatase and osteocalcin, RUNX2 protein expression and more extracellular matrix mineralization compared with AT-MSCs. Conversely, under adipogenic conditions, AT-MSCs displayed higher gene expression of PPAR&gamma;, fatty acid binding protein 4 and resistin, PPAR&gamma; protein expression and more lipid accumulation compared with BM-MSCs. The presence of adipocytes as indirect coculture repressed the expression of osteoblast phenotype while osteoblasts did not exert remarkable effect on adipocyte phenotype expression. In conclusion, the present study showed that BM-MSCs are more osteogenic while AT-MSCs are more adipogenic. Also, we observed that the crosstalk between osteoblasts and adipocytes may negatively impact bone repair. Thus, we postulate that the higher osteogenic potential of BM-MSCs makes them the first choice for inducing bone repair in cell-based therapies.

Orientador: Mary Luci de Souza Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T23:46:23Z (GMT). No. of bitstreams: 1 Pereda_MariaDelCarmenVelazquez_D.pdf: 4069355 bytes, checksum: 2eba0c803662fe7e920cab4f907dc527 (MD5) Previous issue date: 2009 / Resumo: Clinicamente, a celulite é definida como uma alteração na topografia da pele que ocorre principalmente em mulheres, na região pélvica, abdômen e membros inferiores, caracterizando-se pelo aspecto de "casca de laranja". Etiologicamente, celulite é definida como uma desordem metabólica localizada do tecido subcutâneo que provoca uma alteração na forma corporal, onde muitas estruturas são alteradas na derme, na micro circulação e, também, no tecido adiposo. Este fenômeno, por sua vez, está associado com modificações morfológicas, histoquímicas e bioquímicas na pele e resultam nas alterações que levam ao desconforto estético e à aparência clínica da celulite. Partes destas alterações decorrem do acúmulo de lipídeos no interior de adipócitos, células chave no equilíbrio lipólise-lipogênese, o qual é visto hoje como uma unidade glandular funcional capaz de promover uma relação direta com o sistema nervoso central. Pesquisas atuais indicam que a busca por substâncias capazes de promover a homeostase dermo-hipodérmica através de uma ação lipolítica direta, aliada ao estímulo da produção de fatores de crescimento, especialmente TGF-? e GM-CSF, bem como de proteínas da matriz extracelular, como colágeno, elastina e GAGs, além de substâncias reguladoras do metabolismo adipocitário. Tendo em vista a complexidade clínica e estética da celulite, bem como as alterações bioquímicas dérmicas e hipodérmicas, buscamos neste trabalho avaliar os efeitos da associação entre o extrato oleoso dos grãos verdes de café (Coffea arabica L.) (OC) e os fitoesteróis de canola (Brassica campestris L.) (F), cuja combinação resulta no produto Slimbuster® L (SBL). Para isto, avaliamos neste estudo os efeitos in vitro (fibroblastos e adipócitos humanos), ex-vivo (fragmentos de pele humana) e clínicos (voluntários humanos) de SBL, OC e/ou F, utilizando metodologias e modelos experimentais que possibilitassem a visão global do processo celulítico. Os resultados demonstram que SBL foi capaz de estimular significativamente a lipólise, a síntese de leptina em cultura de adipócitos humanos, a produção de GM-CSF, TGF-?, colágeno, elastina e GAGs em cultura de fibroblastos humanos, a contração de fibroblastos em gel de colágeno, bem como promover a melhora das características histológicas gerais da pele (ex-vivo). As frações isoladas (OC e F) também demonstraram efeitos importantes e distintos, sendo estes mais pronunciados para OC e potencializados quando ambos são associados (SBL). Os resultados clínicos demonstram que o produto Slimbuster® L foi bem apreciado pelas voluntárias quanto aos itens "facilidade de aplicação", "pele mais lisa" e "pele mais macia", "atenuação de celulite" e "redução de gordura", especialmente após 60 dias de utilização de uma loção corporal contendo 3,0% (p/p) do produto. Os resultados das medidas instrumentais obtidos demonstram que o produto foi capaz de reduzir significativamente as medidas na região da coxa (superior, mediana e inferior), sendo esta diminuição centimétrica observada em aproximadamente 30% do painel em D30 e em 50% do painel em D60. As demais áreas avaliadas - abdome, pernas e quadris - não apresentaram significância estatística, porém devem ser levadas em consideração, pois apresentaram forte tendência após 60 dias de aplicação da loção corporal, indicando uma possível melhora significativa com o uso prolongado. A análise adipométrica das voluntárias demonstrou também forte tendência de redução da taxa de gordura localizada nas regiões do abdome, supra-ilíaca, supra-espinal e coxa proximal. A espessura da hipoderme através da ultrassonografia (ecografia) possibilitou observar uma redução superior a 2% nas regiões da coxa e abdome em aproximadamente 70% do painel em todos os tempos experimentais avaliados (D30 e D60). De acordo com todos os resultados obtidos neste trabalho, podemos concluir que o produto Slimbuster® L pode ser considerada uma ferramenta segura e eficaz na prevenção e tratamento tópico da gordura localizada e celulite / Abstract: Cellulite is an alteration of the topography of the skin that occurs mainly in women on the pelvic region, lower limbs and abdomen. It is characterized by a padded or 'orange peel' appearance. In according with its etiology, cellulite is defined as a metabolic located disorder of the subcutaneous tissue that provokes an alteration in the physical form, where many structures are alterated in the dermis, in the microcirculation and within the adipocytes. This phenomenon is associated to morphological, histochemical and biochemical modifications in the skin, culminating in the alterations that lead to the aesthetic discomfort and to the clinical appearance of the cellulitis. These alterations are consequence, in parts, from the lipid accumulation into the adypocites, crucial cells in the lypolisis-lipogenesis balance, and it is considerate as a glandular functional unity able to promote a fine-tune interaction with the central nervous system. Recent studies indicate the search for active substances with the ability of promote the dermo-hypodermic homeostasis through lipolytic action, allied to the stimulus of the production of growth factors, mainly TGF-? and GM-CSF, proteins of extracellular matrix, such as collagen, elastin and glycosaminoglycans, as well as substances ables to regulate the adipocyte metabolism. According to the clinical and aesthetic complexity of the cellulitis, as well as the biochemical alterations in the dermis and hypodermis, in this work we evatuate the effects of green coffee seed oil (Coffea arabica L.) and canola phytoesterols (Brassica campestris L.) (F), whose association is the product Slimbuster L (SBL). In this way, we evaluate in this study the effects of these compounds through in vitro, ex-vivo and clinical assessment, using methodologies and experimental models that show the global vision of the process of cellulites. The results demonstrate that SBL was able to stimulate significantly the lipolysis, the synthesis of leptin in human adipocyte cell culture, the production of GM-CSF, TGF-?, collagen, elastin and GAGs in human fibroblast cell culture, the contraction of fibroblastos in collagen gel matrix, as well as promoting the improvement of skin general histologic aspect. The separated fractions (OC and F) also demonstrated important and different effects, being this more pronounced for OC and when both (OC e F) are associated (SBL). The clinical results demonstrate that Slimbuster L was well appreciated by the volunteers for the items "facility of application", "more smooth skin" and "shear it more me" reduction of cellulite" and "reduction of fat accumulation", specially after 60 days use of a body lotion containing 3.0% (w/w) of SBL. The results of the instrumental assessment obtained demonstrate that SBL was able to reduce significantly the measures in the region of the thigh (superior, medium and inferior), being this centrimetric reduction observed in approximately 30% of the panel at D30 and in 50% of the panel at D60. The other evaluated areas - abdome, legs and hips - did not present statistical significance, however they must be taken into account since they presented strong tendency after 60 days of application of the body lotion, indicating a possible improvement with the time of use. The adipometric analysis of the volunteers demonstrated a strong tendency of accumulated fat reduction in the areas of abdomen, supra-iliac, supra-spinal and proximal thigh. The thickness of hypodermis measured by ultrasonography (ecography) showed a reduction superior to 2% in the areas of the thigh and abdomen in approximately 70% of the panel in the all experimental evaluated times (D30 and D60). In according to the results presented in this work, we can conclude that the product Slimbuster L can be considered a safe and efficient tool in the prevention and topical treatment of the located fat and cellulite / Doutorado / Farmacologia / Doutor em Farmacologia

Visando novos estudos com a proteína amilóide sérica A (SAA), propusemos a produção de seu recombinante humano em Escherichia coli, mais especificamente, a isoforma encontrada na fase aguda (A-SAA1) e da constitutivamente expressa (C-SAA4). Realizamos a expressão, identificação e purificação das proteínas recombinantes. Concomitantemente, também avaliamos o efeito da hipóxia na expressão e produção da proteína SAA nativa em linhagens de pré-adipócitos murinos 3T3-L1, não diferenciados e diferenciados e adipócitos humanos. Aparentemente quanto maior o grau de diferenciação celular, maior a expressão e produção da proteína. Para os adipócitos humanos, o perfil de expressão de mRNA da SAA mostra que SAA1>SAA2>SAA4 nas relações 500:150:1. Na hipóxia, há um aumento na expressão de SAA, entretanto não associamos esta expressão a um aumento da concentração da proteína. A importância do reconhecimento de que SAA pode ser umas das proteínas induzidas pela hipóxia em adipócitos é discutida em relação ao seu papel pró-inflamatório. / In order to provide more complex studies with the protein serum amyloid A (SAA), this study proposed the production of its human recombinant in bacteria (Escherichia coli), more specifically, the synthesis of the main isoform found in the acute phase (A-SAA1) and the constitutively expressed (C-SAA4). The expression, identification and purification of the recombinants proteins was performed. Concurrently, we also did a study evaluating the effect of hypoxia in the expression and protein production of native SAA in undifferentiated and differentiated murine preadipocytes 3T3-L1 and humans adipocytes. Apparently, the protein expression and production increases with the cell differentiation degree. For human adipocytes, we demonstrated the mRNA expression profile of SAA, in which SAA1 > SAA2 > SAA4 (500:150:1). In hypoxia, there is an increased expression of SAA, but we could not link it to an increase of protein concentration. The importance of recognizing that SAA may be one of the proteins induced by hypoxia in adipocytes is discussed in relation to the proinflammatory role of this protein.

Adipositas und die damit verbundenen Folgeerkrankungen sind eine der zentralen Gesund-heitsherausforderungen unserer Zeit. Dauerhafte Adipositas führt zu einer Dysregulation fettgewebseigener Peptidhormone. Diese sogenannten Adipokine stellen ein Verbindungsglied zwischen Fettgewebsakkumulation und den vielfältigen Adipositaskomplikationen des gesamten Organismus dar. Adipocyte fatty acid binding protein (AFABP) wurde in den letzten Jahren als zirkulierendes Adipokin mit diabetogenen, proinflammatorischen und proateriosklerotischen Effekten etabliert. Zu Beginn der Dissertation lagen unzureichende Erkenntnisse über die Elimination von AFABP sowie die Regulation des Adipokins bei eingeschränkter Nierenfunktion vor. Aus diesem Grund untersucht die vorliegende Arbeit die AFABP-Regulation in Abhängigkeit von der Nierenfunktion in 532 Patienten mit chronischer Niereninsuffizienz (Studienpopulation 1) und 32 Patienten mit akuter Nierenfunktionsverminderung nach Nephrektomie (Studienpopulation 2). In beiden Kohorten stiegen die medianen AFABP-Serumkonzentrationen mit abfallender Nierenfunktion an. Zudem waren Marker der Nierenfunktion in beiden Studienpopulationen die stärksten unabhängigen Prädiktoren für zirkulierendes AFABP. Untersuchungen aus der Arbeitsgruppe zur AFABP-Regulation in einem Rattenmodell der akuten Niereninsuffizienz unterstützen die klinischen Studienergebnisse. Zusammenfassend zeigen diese Ergebnisse zum ersten Mal signifikant steigende AFABP-Serumspiegel bei chronischer und akuter Nierenfunktionsstörung, sowie bei akutem Abfall der Nierenfunktion. Diese Befunde stützen die Hypothese, dass AFABP renal eliminiert wird. Inwiefern AFABP darüber hinaus in die Pathogenese der chronischen Niereninsuffizienz eingreift, muss in weiterführenden Studien beleuchtet werden.

Background: Facial fat grafting for rejuvenation is one of the most popular facial aesthetic procedures in plastic surgery. It is always challenging and since there are a lot of techniques for adipose tissue (AT) harvesting, there are no standard procedures that guarantee natural and long-lasting results. We developed the selective tissue engineering photo stimulation technique (One STEP™) in which we used a novel infrared 1210-nm wavelength laser diode for fat preserved harvesting and direct fat injection that we named PicoGraft™, with no fat manipulation. Methods: This is a retrospective descriptive study in which we included all senior author’s patients that got facial fat grafting using the One STEP™ technique. We compared the AT aspirated, after laser emission (STEP-PicoGraft) and the standard assisted liposuction samples (SAL) in cultures. We study the mitochondrial activity of the ASC between STEP and SAL in fresh samples and after 24 h. The evaluation of the results included subjective changes regarding wrinkles, grooves, palpebral bags, hyperchromic spots, and fat hypotrophy of our patients. Results: Between July 2013 and May 2018, a total of 245 patients underwent facial fat grafting using this novel technique. We observed adipocytes preserved after STEP harvesting comparing morphologic changes in SAL samples with a high concentration of inflammatory particles in cultures. ASC mitochondrial activity shows an important difference of more than 7 times in STEP samples in fresh analysis that increase 12 times in 24 h. The subjective results show a good improvement in the periorbital area. The changes on the skin and subcutaneous tissue are seen from the second month and continue to improve up to 12 months. Conclusions: Facial fat grafting using the PicoGraft™ obtained by One STEP™ technique gives excellent volumetric and regenerative results in a single treatment without volumetric hypercorrection, and it is a good alternative for facial rejuvenation. The fat graft obtained with this novel technique is homogenous, without lumps, and has high concentration of viable stimulated ADSC and a high number of viable adipocytes. Level of evidence: Level III, therapeutic study. / Revisión por pares