La modification des profils spécifiques en acides gras (AG) dans des huiles végétales peut servir à l’amélioration de leur qualité nutritionnelle, mais également à l’émergence d’une industrie biosourcée où les AG peuvent représenter une alternative aux ressources carbonées fossiles. Cependant des difficultés à modifier le profil en AG des huiles végétales, partagées par l’ensemble des équipes de recherche impliquées subsistent. Bien que la biosynthèse des AG est aujourd’hui bien caractérisée chez les plantes, des incertitudes persistent concernant les voies de transfert des AG du pool d’acyl-CoA vers les précurseurs lipidiques, et les formes de transport entre les différents compartiments impliqués dans les synthèses lipidiques. La carnitine est un acteur essentiel du trafic intracellulaire des AG chez l’animal et la levure. La présence avérée chez la plante d’acyl-Carnitines au côté de la carnitine libre, et la mise en évidence d’activités enzymatiques associées, prouvent également le lien entre la carnitine et le métabolisme des AG. Le travail de cette thèse a donc porté sur un approfondissement des connaissances sur le rôle de la carnitine dans le métabolisme lipidique chez Arabidopsis thaliana. A ce titre, nous avons pu montrer par LC-QQQ-MS/MS dans nos extraits végétaux, que l’oléoyl-, la palmitoyl- et la stéaroyl-Carnitine sont les espèces majoritaires et correspondent aux 3 AG (C18:1, C16:0 et C18:0) principalement synthétisés et exportés des plastes, pour alimenter la synthèse lipidique de la voie eucaryotique. De plus, une augmentation significative des teneurs en acyl-Carnitines a été mesurée dans des processus développementaux nécessitant une forte synthèse de glycérolipides, tels que le développement post-Germinatif (2 j post-Imbibition) ou lors de l’organogenèse racinaire, en concomitance avec le niveau d’expression des gènes marqueurs de la synthèse lipidique eucaryotique. D’autre part, la comparaison des teneurs en acyl-Carnitines et en carnitine estérifiée a révélée un résultat inédit montrant qu’une grande fraction de la carnitine est estérifiée à des AG inhabituels ou des acides organiques que nous cherchons actuellement à identifier par LC-QTOF-MS/MS. Ainsi, l’ensemble de nos résultats suggèrent que la carnitine pourrait être impliquée dans le trafic intracellulaire d’AG pour la synthèse de lipides eucaryotiques et de lipides particuliers et/ou dans la prise en charge d’acides organiques encore non identifiés dans les plantes. / Specific modification of fatty acid profiles (FA) in vegetable oils can help to improve their nutritional quality but also to the development of a bio-Based industry where FA may represent an alternative to fossil fuel sources. However problems to modify the FA profile of vegetable oils still remain and are shared by all the teams involved. Although the biosynthesis of AG is now well characterized in plants, uncertainties remain about the tranfert of AG of acyl-CoA pool to lipid precursors and the forms of transport between different compartments involved in lipid synthesis. Carnitine plays an essential role in intracellular trafficking of FA in animals and yeast. The known presence of acyl-Carnitines in plant alongside the free carnitine, and measurement of associatied-Enzymatic activities also show the link between FA metabolism and carnitine. During this thesis we tried to improve our understanding of the role of carnitine in lipid metabolism in Arabidopsis thaliana. We have shown by LC-QQQ-MS / MS in our plant extracts that oleoyl-, palmitoyl- and stearoyl-Carnitine are the predominant species and correspond to the three main AG (C18:1, C16:0 and C18:0) synthesized and exported from the plastids to supply the lipid synthesis of the eukaryotic pathway. Moreover, a significant increase of the levels of acyl-Carnitines were measured in developmental processes requiring high glycerolipid synthesis, such as the post-Germinative growth (2 days post-Imbibition) during root organogenesis, concomitantly with the level of expression of the eukaryotic lipid synthesis marker genes. Furthermore, comparison of the levels of acyl-Carnitine and carnitine ester revealed an unpublished results showing that a large fraction of carnitine is esterified to unusual FA or organic acids that we are currently identifing by LC QTOF-MS/MS. Thus, all of our results suggest that carnitine could be involved in the intracellular traffic of FA for the synthesis of lipids and particular eukaryotic lipids and/or in the transport of yet unidentified organic acids in plants.

La présente étude avait pour objectif de déterminer la composition en acides gras, la teneur en caroténoïdes, en vitamine E, et en polyphénols des huiles extraites de quatre variétés de palmier à huile cultivées en Côte d'Ivoire (variétés parentales : HP1et HP2, variétés hybrides : HP3 et HP4), puis d'étudier les propriétés antioxydantes de leurs extraits polyphénoliques. Les résultats ont montré : une richesse particulière en acides gras insaturés totaux avec une prédominance en acide oléique des variétés HP1 (60%), HP3 (56%) et HP4 (57%) comparée à HP2 (48%). Les concentrations en vitamine E étaient de 864 à 1124 µg/g avec une forte teneur en α- et γ- tocotriénol dans la variété HP2 et les hybrides HP3 et HP4. La variété HP1 et les hybrides ont des taux de caroténoïdes totaux significativement plus élevés que la variété HP2. La teneur en polyphénols totaux était élevée dans les variétés HP1 (0,195mM EAG), HP3 et HP4 (0,191mM et 0,2 mM EAG). Les différents polyphénols identifiés étaient l'acide caféique, l'acide chlorogénique, l'acide coumarique, la rutine et un dérivé non identifié de la quercétine. Ces extraits polyphénoliques avaient un effet inhibiteur sur l'oxydation des LDL de manière dose-dépendante, dans le système d'oxydation au cuivre qu'à l'AAPH. Les activités antioxydantes étaient significativement plus importantes pour HP3 et HP4 comparées à HP1 et HP2. Ces extraits polyphénoliques ont inhibé la production d'O2•― par modulation de la NADPH oxydase au niveau des cellules THP-1 stimulées au PMA, de manière dose-dépendante. Cette action antioxydante a été confirmée par l'inhibition de l'expression de la sous unité p22phox de 74% pour HP1, 87% pour HP2, 69% pour HP3 et 75% pour HP4. L'huile de palme brute pourrait ainsi être considérée comme une source sûre de composés polyphénoliques, notamment l'huile issue des formes variétales HP3 et HP4, dont les effets protecteurs semblaient être plus marqués. / The purpose of the present study was to determine fatty acid, vitamin E, carotene and polyphenol contents in four oil palm species of Côte d'Ivoire (parental varieties: HP1et HP2, crossing varieties: HP3 and HP4), then to study their polyphenol antioxidant properties. The results showed that palm oil species were characterized by the special richness in polyunsaturated fatty acids composition, with a prevalence in oleic acid, especially the first variety, HP1 (60%), and the two hybrids ensuing from crossing HP3 (56%) and HP4 (57%) compared to HP2 (48%). Total vitamin E content was 864–1124 μg g−1, with a notable higher content of γ -and α-tocotrienol in variety HP2 and hybrids HP3 and HP4. Total carotene content of those varieties was higher, and the β-carotene and α-carotene levels were predominant in HP1, HP3 and HP4. The highest total phenolic contents were found for parental variety HP1, (0.195 mM GAE), the two crossing varieties HP3 and HP4 (0.191mM and 0.2 mM GAE). The main identified phenolic compounds were caffeic, chlorogenic, coumaric acids, rutin, and a non identified quercetin derivative. The antioxidative effects were monitored by Cu2+- or AAPH-induced generation of conjugated dienes (lag-time and oxidation rate) showed that these phenolic compounds inhibit human LDL oxidation in a synergistic manner. The antioxidant activities were significantly more important for HP3 and HP4 varieties compared to HP1 and HP2 varieties. All palm oil polyphenol extracts (compared to control) decreased PMA-stimulated O2•― production by THP-1 cells in a dose dependent manner in the modulation of NADPH oxidase. Using Western blot analysis, we further demonstrated that incubation for 24h of these polyphenol extracts with THP-1 cells led to a significant reduction in the expression of NADPH oxidase subunit p22phox (from 74% for HP1, 87% for HP2, 69% for HP3 and 75% for HP4). Crude palm oil could be considered as a good source of phenolic compounds, in particularly oil resulting from HP3 and HP4 varieties. These protective effects seemed to be more marked with the two crossing varieties in comparison with the two parental varieties.

Fatty acid desaturase 2 (FAD2) enzymes are phosphatidylcholine desaturases occurring as integral membrane proteins in the endoplasmic reticulum membrane and convert monounsaturated oleic acid into polyunsaturated linoleic acid. The major objective of this research was to study the expression and function of two cotton FAD2 genes (the FAD2-3 and FAD2-4 genes) and their possible role in plant sensitivity to environmental stress, since plants may increase the polyunsaturated phospholipids in membranes under environmental stress events, such as low temperature and osmotic stress. Two FAD2 cDNA clones corresponding to the two FAD2 genes have been isolated from a cotton cDNA library, indicating both genes are truly expressed in cotton. Model yeast cells transformed with two cotton FAD2 genes were used to study the chilling sensitivity, ethanol tolerance, and growth rate of yeast cells. The expression patterns of the two FAD2 genes were analyzed by reverse transcription polymerase chain reactions (RT-PCR) and Western blot analyses in cotton plants under different treatment conditions. The coding regions of both FAD2 genes were inserted downstream from the CaMV 35S promoter in the pMDC gateway binary vector system. Five different FAD2/pMDC constructs were transformed into the Arabidopsis fad2 knockout mutant background, and multiple potential transgenic Arabidopsis plant lines harboring the cotton FAD2 genes were generated. The cotton FAD2 genes were amplified by the polymerase chain reaction (PCR) from the genomic DNAs isolated from the transgenic Arabidopsis T1 plant lines. Complementation of the putative transgenic Arabidopsis plants with the two cotton FAD2 genes was demonstrated by gas chromatography analyses of the fatty acid profiles of leaf tissues. The cellular localization of cotton FAD2-4 polypeptides with N-terminal green fluorescence protein (GFP) was visualized by confocal fluorescence microscopy. The phenotype of transgenic Arabidopsis plants transformed with the cotton FAD2-4 gene was compared to Arabidopsis knockout fad2 mutant plants and wild type Arabidopsis plants regarding their sensitivity to low temperature, and the size and height of the plants.

Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing a class of lipid mediators, N-acylethanolamines (NAEs). Recent identification of an Arabidopsis FAAH homologue (AtFAAH) and several studies, especially those using AtFAAH overexpressing and knock-out lines suggest that a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, I provide evidence to support this concept by identifying candidate FAAH cDNA sequences in diverse plant species. NAE amidohydrolase assays confirmed that several of the proteins encoded by these cDNAs indeed catalyzed the hydrolysis of NAEs in vitro. Kinetic parameters, inhibition properties, and substrate specificities of the plant FAAH enzymes were very similar to those of mammalian FAAH. Five amino acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the plant FAAH sequences. Site-directed mutation of each of the five putative catalytic residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli. Contrary to overexpression of native AtFAAH in Arabidopsis that results in enhanced seedling growth, and in seedlings that were insensitive to exogenous NAE, overexpression of the inactive AtFAAH mutants showed no growth enhancement and no NAE tolerance. However, both active and inactive AtFAAH overexpressors displayed hypersensitivity to ABA, suggesting a function of the enzyme independent of its catalytic activity toward NAE substrates. Yeast two-hybrid screening identified Arg/Ser-rich zinc knuckle-containing protein as a candidate protein that physically and domain-specifically interacts with AtFAAH and its T-DNA knock-out Arabidopsis was hypersensitive to ABA to a degree similar to AtFAAH overexpressors. Taken together, AtFAAH appears to have a bifurcating function, via NAE hydrolysis and protein-protein interaction, to control Arabidopsis growth and interaction with phytohormone signaling pathways. These studies help to functionally define the group of enzymes that metabolize NAEs in plants, and further will expand the knowledge-base of lipid metabolism and signaling for manipulation of various physiological processes important to plant growth and responses to environmental stress.

Orientador: Edgard Graner / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T15:46:52Z (GMT). No. of bitstreams: 1 Almeida_LucianaYamamotode_D.pdf: 2870342 bytes, checksum: 136928121fd9ee8f8c8b29bf904ee111 (MD5) Previous issue date: 2015 / Resumo: O carcinoma espinocelular (CEC) é a neoplasia epitelial maligna mais frequente na cavidade oral, que pode apresentar vários graus de diferenciação escamosa e emite metástases em linfonodos regionais precocemente. Ocorre predominantemente na quinta e sexta décadas de vida em pacientes etilistas e tabagistas. O prognóstico é dependente do tamanho do tumor primário e da presença de linfonodos positivos e/ou metástases à distância no momento do diagnóstico. A cirurgia é a principal modalidade terapêutica para o CEC oral, no entanto, em estágios avançados, os pacientes são incluídos em protocolos radioterápicos e quimioradioterápicos. 5-fluorouracil (5-FU), cisplatina (CDDP) e paclitaxel (PTX) são drogas usadas em regimes quimioterapêuticos de várias neoplasias, dentre as quais o CEC oral. Entretanto, independente da modalidade de tratamento, a taxa de sobrevida global em 5 anos ainda está próxima a 50%. Diversos estudos mostram que a enzima metabólica ácido graxo sintase (FASN), responsável pela biossíntese endógena de ácidos graxos de cadeia longa, apresenta expressão aumentada em neoplasias malignas, incluindo o CEC oral, no qual está aparentemente associada a um pior prognóstico. Orlistat (ORL) é uma droga antiobesidade que também possui propriedades antineoplásicas por inibir de maneira irreversivel FASN. Em nosso laboratório, demonstramos que os camundongos BALB/c nude inoculados de maneira ortotópica com células SCC-9 ZsGreen LN1 e tratados com ORL apresentam CECs de língua menores e redução significativa (43%) no número de metástases para linfonodos cervicais. Até o momento, não existem estudos investigando o efeito de ORL como adjuvante nos protocolos de quimioterapia em uso clínico. Portanto, no presente trabalho, investigamos os efeitos da associação de ORL com 5-FU, CDDP e PTX, em seus respectivos IC50, na linhagem celular metastática SCC-9 ZsGreen LN1, sobre a proliferação, morte, migração (em monocamada e em transwell) e invasão celular. As combinações ORL + CDDP (OC) e ORL + PTX (OP) foram as únicas capazes de aumentar significativamente os índices apoptóticos em 48 h de tratamento, o que foi precedido por acúmulo das células nas fases S e G2/M do ciclo celular, respectivamente. A terapia ORL + 5-FU (OF) não foi capaz de induzir alterações significativas na progressão do ciclo ou na morte celular. OP reduziu a expressão de ciclina B1, mas sem causar alterações significativas em CDK1 ou na fosfatase Cdc25C, os quais constituem complexos proteicos envolvidos diretamente na transição G2/M. Já OC, apesar de não alterar os níveis proteicos de CDK1, reduziu a expressão de ciclina B1 e de Cdc25C. OC e OP aumentaram a expressão de FASN mas não alteraram significativamente os níveis do receptor ERBB2, envolvido na regulação desta enzima. O fenótipo invasivo foi reduzido pelo tratamento com OC e OP, mas a capacidade migratória das células foi alterada principalmente pela ação de PTX. Em conjunto, estes achados sugerem que as combinações OC e OP têm efeitos sinérgicos nas células metastáticas SCC-9 ZsGreen LN-1 e potencial aplicação no tratamento de CECs orais / Abstract: Squamous cell carcinoma (SCC) is the most frequent malignant epithelial tumor of the oral cavity, which presents variable degree of squamous differentiation and early regional lymph node metastases. It is most frequent in alcohol and tobacco users at the fifth and sixth decades of life. The prognosis of oral SCC depends on the size of the primary tumor, presence of positive lymph nodes and / or distant metastases at the moment of diagnosis. Surgery is the main treatment modality of choice for oral SCC, however, in advanced stages, patients are included in radiotherapy and chemoradiotherapy protocols. 5-fluorouracil (5-FU), cisplatin (CDDP) and paclitaxel (PTX) are used in chemotherapeutic regimens of several neoplasms, including OSCC. However, regardless the treatment modality, the overall 5-year survival rate is approximately 50%. Several studies demonstrate that the metabolic enzyme fatty acid synthase (FASN), responsible for the endogenous biosynthesis of long-chain fatty acids, is highly expressed in malignant neoplasms, including oral SCC, in which it seems to be associated with a worse prognosis. Orlistat (ORL) is an anti-obesity drug that also has antineoplastic properties by irreversibly inhibiting FASN. In our laboratory, we previously demonstrated that BALB/c nude mice orthotopically inoculated with SCC-9 ZsGreen LN1 cells and treated with ORL have smaller primary tumors as well as significant less cervical lymph node metastases (43%). To date, there are no studies investigating the effect of ORL as an adjuvant for chemotherapy of OSCC. In the present study, we describe the effects of ORL combined with 5-FU, CDDP and PTX at their respective IC50 on proliferation, death, migration and invasion on metastatic SCC-9 ZsGreen LN-1 cells. ORL + CDDP (OC) and ORL + PTX (OP) significantly increased apoptosis after 48 h, which was preceded by the accumulation of cells in the S and G2/M phases of the cell cycle, respectively. ORL + 5-FU therapy (OF) did not change cell cycle progression or cell death. OP decreased cyclin B1 production without significant changes in CDK1 and Cdc25C levels, which are responsible for G2/M transition. Although CDK1 were not changed by OC, this combination reduced the expression of cyclin B1 and Cdc25C. OC and OP increased FASN but not changed ERBB2, a cell surface receptor involved in the regulation of the former. The invasive phenotype was reduced by treatment with OC and OP, however, PTX was the main drug responsible for the inhibition of cell migration. Together, these findings suggest that OC and OP combinations show synergistic effects in the metastatic SCC-9 ZsGreen LN-1 cells and may have potential application for the oral SCC treatment / Doutorado / Patologia / Doutora em Estomatopatologia

Orientador: Edgard Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-28T00:41:43Z (GMT). No. of bitstreams: 1 Melo_EstevaoAzevedo_M.pdf: 1050744 bytes, checksum: b28d2d7f1bf1683ed75a9b3e47166ad2 (MD5) Previous issue date: 2015 / Resumo: A ácido graxo sintase (FASN) é a enzima responsável pela biossíntese endógena de ácidos graxos e apontada como uma oncoproteína metabólica, por favorecer a proliferação e sobrevivência das células tumorais nas quais sua expressão é elevada. Vários são os compostos capazes de inibir a atividade de FASN, dentre eles o orlistat (Xenical®), que possui efeitos antiproliferativos previamente mostrados em células de câncer de mama, próstata, boca e melanoma. O sistema imunológico apresenta um importante papel na prevenção e defesa do organismo contra neoplasias malignas. As células do sistema imune que se infiltram nos melanomas são produtoras de uma vasta gama de citocinas, dentre elas interleucina 12 (IL-12) e interferon gama (IFN-?) que favorecem uma resposta imune bem sucedida contra os tumores, porém, as células dos melanomas possuem capacidade de produzir interleucina 10 (IL-10) e fator de crescimento transformante beta (TGF-?), capazes de inibir as células imunocompetentes, favorecendo a progressão tumoral e disseminação metastática. O objetivo deste estudo foi avaliar a secreção das citocinas IL-10, IL-12, IFN-? e TGF-? pelas células de melanoma murino B16-F10 após tratamento com orlistat. Para isto, inicialmente determinou-se a dosagem de orlistat capaz de inibir a proliferação celular em 50% (IC50). Em seguida, as células foram tratadas por 24 e 48 horas, quando realizou-se a quantificação da secreção das citocinas por ELISA. Após 24 horas de tratamento, observou-se aumento da secreção de IL-10 e IL-12, no entanto, após 48 horas de tratamento não foi detectada diferenças estatisticamente significantes na secreção de ambas as citocinas, quando comparadas aos seus controles. IFN-? e TGF-? não foram detectáveis. Assim, os resultados desta pesquisa mostram que o tratamento com orlistat alterou a produção das citocinas IL-10 e IL-12, sugerindo que o tratamento promove um equilíbrio entre estas citocinas pró e anti-inflamatórias nas células estudadas / Abstract: Fatty acid synthase (FASN) is the enzyme responsible for the endogenous biosynthesis of fatty acids suggested as a metabolic oncoprotein by promoting proliferation and survival of cancer cells. Several compounds are known to inhibit FASN activity, including orlistat (Xenical®), which has antiproliferative effects in breast, prostate, and oral cancer as well as melanoma cells. Melanoma is an aggressive malignant tumor of melanocytes with high propensity for metastatic spread and resistant to chemotherapy. The immune system plays an important role in the prevention and defense against malignant neoplams. In fact, immune cells that infiltrate melanomas produce a wide range of cytokines, such as interleukin 12 (IL-12) and interferon gamma (IFN-?), which favor a successful immune response against the tumor. However, melanomas cells are able to produce interleukin 10 (IL-10) and transforming growth factor beta (TGF-?) and in turn inhibit immunocompetent cells, favoring tumor progression and metastatic spread. The aim of this research was to evaluate the effect of the FASN inhibitor orlistat on the secretion of the cytokines IL-10, IL-12, IFN-? and TGF-? by B16-F10 mouse melanoma cells. For this purpose, we first searched for the IC50 Of orlistat in B16-F10 cells. Then, cells were treated for 24 and 48 hours with the drug and the secretion of cytokines quantified by ELISA. After 24 hours of treatment the secretion of IL-10 and IL-12 was increased, however, after 48 hours there were no statistically significant changes in the secretion of both cytokines, compared to their controls. IFN-? and TGF-? were not detectable. Thus, the results of this study show that the treatment with orlistat change the production of IL-10 and IL-12, suggesting a balance between the secretion of pro- and anti-inflammatory cytokines / Mestrado / Estomatopatologia / Mestre em Estomatopatologia

[EN] During the last 25 years, eel populations have declined considerably, this species is considered outside safe biological limits. The life cycle of the eel is quite complex including transoceanic migration whose conditions are still so unknown that even the natural spawning process has never been observed. Eels cease feeding during migration and that is why their energy reserves such as lipids play a crucial role and thus their in deep study is essential to obtain gametes (eggs and sperm) quality as the first step for their reproduction in captivity. In the last years, many efforts have been directed at the study of the influence of fatty acids on the reproductive performance in females, but not in male eels. The present manuscript describes the results obtained in two experiments. The first experiment (which led to the three first articles included in the memory) focused on the effect of fatty acids along maturation, their relationship with steroid hormones and their effect on sperm quality parameters. The second experiment involved the comparison of different diets designed, considering the previously acquired insights into the most influential fatty acids on male eel reproduction, and the assessment of their influence on the reproductive performance of males. The first experiment was used to assess the changes in the fat content, as well as fatty acids, in different tissues of male eels hormonally induced to sexual maturation under different thermal regimes (two variables and one constant). Males finished spermatogenesis earlier with a constant temperature of 20 ºC, suggesting that eel spermatogenesis is tightly regulated by temperature. The fat content did not change significantly in the muscle, but was increased in the liver and decreased in testes during testicular development. With regard to fatty acids, during sperm maturation, the liver was highlighted as the main site of synthesis. Finally, EPA, ARA and DHA remained constant in testes, while the level of the rest of fatty acids decreased significantly. Using samples from the same experiment, the correlation between the main steroid hormones and fatty acids at different stages of testicular development were investigated. Similarly as occurs in mammals, EPA and DHA were highlighted as possible modulators of androgen synthesis. The set of the results obtained suggests new perspectives concerning the functions and interactions between fatty acids and steroids in fish spermatogenesis. Also, with samples from the first experiment, a study was carried out seeking to establish the possible correlations between different fatty acids and several sperm quality parameters. Correlations between several highly unsaturated fatty acids with parameters such as volume, the percentage of motile sperm and sperm velocity were found. Finally, with the acquired knowledge so far, a second experiment was carried out designing feeds with different fatty acid percentages in order to evaluate the influence of the diets on sperm quality. The results showed that high levels of DHA and EPA in the diets induce higher volumes and high sperm motility. This study allowed obtaining results applicable to the design of optimum broodstock diets for this species. / [ES] Durante los últimos 25 años las poblaciones de anguila han disminuido considerablemente, de modo que la especie se considera fuera de los límites biológicos de seguridad. El ciclo vital de la anguila es bastante complejo incluyendo una migración transoceánica cuyas condiciones son todavía tan desconocidas que incluso el proceso natural de desove no se ha observado nunca. Las anguilas dejan de alimentarse durante la migración y es por ello que sus reservas energéticas en forma de lípidos tienen un papel crucial y su estudio en profundidad es importante para obtener gametos (huevos y esperma) de calidad como primer paso para su reproducción en cautividad. En los últimos años se han dirigido muchos esfuerzos al estudio de la influencia de los ácidos grasos en el rendimiento reproductivo de las hembras, pero no en el de los machos de anguila. El presente documento describe los resultados obtenidos en dos experimentos. El primer experimento (que dio lugar a los tres primeros artículos incluidos en la memoria) se centró en el efecto de los ácidos grasos durante la maduración, su relación con las hormonas esteroideas y su efecto sobre los parámetros de calidad espermática. El segundo experimento consistió en la comparación de diferentes dietas, diseñadas considerando los conocimientos adquiridos previamente sobre los ácidos grasos más influyentes en la reproducción de machos de anguila, y en la evaluación de su influencia en el rendimiento reproductivo de los machos. El primer experimento sirvió para evaluar los cambios en el contenido graso, así como en los ácidos grasos, en diferentes tejidos de machos de anguila hormonalmente inducidos a la maduración sexual bajo diferentes regímenes térmicos (dos variables y uno constante). Los machos concluyeron la espermatogénesis más temprano a una temperatura contante de 20 ºC, sugiriendo que la espermatogénesis de anguila está estrechamente regulada por la temperatura. El contenido en grasa no varió significativamente en el músculo, se incrementó en el hígado y bajó en el testículo a lo largo del desarrollo testicular. Con respecto a los ácidos grasos, durante la maduración del esperma, el hígado destacó como el principal sitio de síntesis. Finalmente, EPA, ARA y DHA permanecieron constantes en el testículo, mientras que el nivel del resto de ácidos grasos bajó significativamente. Usando muestras del mismo experimento, se investigaron las correlaciones entre las principales hormonas esteroideas y los ácidos grasos en los diferentes estadios del desarrollo testicular. De forma similar a lo que ocurre en mamíferos, EPA y DHA destacaron como posibles moduladores de la síntesis de andrógenos. El conjunto de los resultados obtenidos sugiere nuevas perspectivas sobre las funciones y las interacciones entre los ácidos grasos y los esteroides en la espermatogénesis de peces. También con muestras del primer experimento, se llevó a cabo un estudio que buscó las posibles correlaciones de los diferentes ácidos grasos con varios parámetros de calidad espermática. Se encontraron correlaciones entre varios ácidos grasos altamente insaturados con parámetros como el volumen, el porcentaje de espermatozoides móviles y la velocidad del esperma. Finalmente, con los conocimientos adquiridos hasta el momento, se realizó un segundo experimento para el que se diseñaron piensos con diferentes porcentajes de ácidos grasos con el fin de evaluar la influencia de las dietas sobre la calidad del esperma. Los resultados mostraron que niveles altos de DHA y EPA en las dietas inducen mayores volúmenes y una alta motilidad del esperma. Este estudio permitió obtener resultados aplicables al diseño de dietas óptimas para reproductores de esta especie. / [CAT] Durant els últims 25 anys les poblacions d'anguila han disminuït considerablement, de manera que l'espècie es considera fora dels límits biològics de seguretat. El cicle vital de l'anguila és bastant complex incloint una migració transoceànica les condicions de la qual són encara tan desconegudes que fins i tot el procés natural de fresa mai ha estat observat. Les anguiles deixen d'alimentar-se durant la migració i és per això que les seves reserves energètiques en forma de lípids tenen un paper crucial i el seu estudi en profunditat és important per a obtenir gamets (ous i esperma) de qualitat com a primer pas per a la reproducció en captivitat. En els últims anys s'han dirigit molts esforços a l'estudi de la influència dels àcids grassos en el rendiment reproductiu de les femelles, però no en el dels mascles d'anguila. El present document descriu els resultats obtinguts en dos experiments. El primer (que dona lloc als tres primers articles inclosos en la memòria) es va centrar en l'efecte dels àcids grassos durant la maduració, la seva relació amb les hormones esteroides i el seu efecte sobre els paràmetres de qualitat espermàtica. El segon experiment va consistir en la comparació de diferents dietes, dissenyades considerant els coneixements adquirits prèviament sobre els àcids grasos més influents en la reproducció dels mascles d'anguila, i en l'avaluació de la seva influència en el rendiment reproductiu dels mascles. El primer experiment va servir per avaluar els canvis en el contingut de gras, així com en els àcids grassos, en els diferents teixits de mascles d'anguila hormonalment induïts a la maduració sexual baix diferents règims tèrmics (dos variables i un constant). Els mascles van concloure la espermatogènesis més prompte a una temperatura de 20 ºC, suggerint que l'espermatogènesis d'anguila està estretament regulada per la temperatura. El contingut en greix, no va variar significativament en el múscul, es va incrementar en el fetge i va baixar en els testicles al llarg del desenvolupament testicular. Pel que fa als àcids grassos, durant la maduració de l'esperma, el fetge va destacar com al principal lloc de síntesi. Finalment, EPA, ARA y DHA van romandre constants en el testicle, mentre que el nivell de la resta d'àcids grassos va baixar significativament. Emprant mostres del mateix experiment, es van investigar les correlacions entre les principals hormones esteroides i els àcids grassos en els diferents estadis de desenvolupament testicular. De manera semblant al que ocorre en mamífers, EPA i DHA van destacar com a possibles moduladors de la síntesi d'andrògens. El conjunt dels resultats obtinguts suggerixen noves prespectives sobre les funcions i les interaccions entre els àcids grassos i els esteroides en l'espermatogènesi dels peixos. També amb mostres del primer experiment, es va dur a terme un estudi que va buscar les possibles correlacions dels diferents àcids grassos amb diversos paràmetres de qualitat espermàtica. Es van trobar correlacions entre diversos àcids grassos altament insaturats amb paràmetres com el volum, el percentatge d'espermatozoides mòbils i la velocitat de l'esperma. Finalment, amb els coneixements adquirits fins al moment, es va realitzar un experiment per al qual es van dissenyar pinsos mb diferents percentatges d'àcids grassos per tal d'avaluar la influència de les dietes sobre la qualitat de l'esperma. Els resultats van mostrar que nivells alts de DHA i EPA en les dietes indueixen a majors volums i una alta motilitat de l'esperma. Aquest estudi va permetre obtenir resultats aplicables al disseny de dietes òptimes per a reproductors d'aquesta espècie. / Baeza Ariño, R. (2015). Roles of lipids and fatty acids through the spermatogenesis of European eel (Anguilla anguilla) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/49988 / TESIS

Medium chain fatty acids (MCFAs) are essential molecules for a wide range of pharmaceutical, biotechnological, and industrial applications. These are naturally occurring saturated or unsaturated fatty acids containing 6-12 carbons with complex and pH sensitive aggregation. Medium chain fatty acids such as capric acid (C10) or lauric acid (C12) have additionally been shown to exhibit antibacterial activity. A number of studies have observed the aggregation behavior of long chain fatty acid using the titration curves by molecular dynamic (MD) simulations.  In this study, we performed constant-pH coarse-grained MD simulations to determine pKa values and titration behavior using a two-states model for C10 and C12. In the simulations, pH was varied between 2 to 8 and pKa values were determined using the Hill equation. The pKa for C10 (capric acid) was found to be 4.8 and for C12 (lauric acid) 5.4, in good agreement with the literature values (4.9 and 5.3, respectively).

Soybean [Glycine max (L.) Merr.] is the world’s most widely grown protein/oilseed crop and provides about 70% of global protein meal and 53% of vegetable oil in the United States. Soybean seed oil contains five major fatty acids, from which palmitic acid and stearic acid are two saturated fatty acids, oleic acid improves oxidative stability and linolenic acid is an essential fatty acid for human health. Soybean seed protein and oil are two important quality indices for soybean germplasm breeding. Soluble carbohydrates present in soybean meal provide metabolizable energy in livestock feed. To develop soybean germplasm with improved seed composition traits, it is important to discover novel source of seed fatty acid, protein, and carbohydrates traits. This dissertation aims to develop novel functional genomic technology coupled with an integrated approach for facilitating molecular soybean breeding. In this study, the first objective is to develop a high-throughput TILLING (Targeting Induced Local Lesions IN Genomes) by Target Capture Sequencing (TbyTCS) technology to improve the efficiency of discovering mutations in soybean. The robustness of this technology underlies the high yield of true mutations in genes controlling complex traits in soybean. Soybean mutagenized lines with modified fatty acids composition have been successfully developed to meet the different needs of end users. Altered fatty acids phenotypes have been associated with induced mutations in 3-ketoacyl-acyl carrier protein (ACP) synthase II (GmKASII), Delta-9-stearoyl-acyl carrier protein desaturase (GmSACPD), omega-6 fatty acid desaturase 2 (GmFAD2), and omega-3 fatty acid desaturase (GmFAD3) genes identified through TbyTCS. The second objective is to characterize the soybean acyl-ACP thioesterase gene family through a comprehensive analysis. The additional members have been discovered belonging to 16:0-ACP fatty acid thioesterase (GmFATB) gene family. The mutations at oleoyl-ACP fatty acid thioesterase (GmFATA1A) have been revealed to result in the high seed oleic acid content. The novel alleles of GmFATB genes have also been identified to confer low palmitic acid and high oleic acid phenotypes in soybean seeds. The third objective is to assess the phenotypic variations and correlation among seed composition traits in mutagenized soybean populations. Correlation analyses have been conducted among soybean carbohydrates, protein, and oil content of soybean mutagenized populations and germplasm lines. Chemical mutagenesis played an essential role in soybean breeding to generate novel and desired seed composition traits.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23088号 / 医博第4715号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 柳田 素子, 教授 岩田 想, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM