Memoria para optar el título de Bioquímico / Los organismos regulan su número de células a través de un balance entre la división y muerte celular. Con respecto a este último, existen diversos mecanismos de muerte celular, como la apoptosis, autofagia y necrosis, presentando cada uno características morfológicas y moleculares diferentes. Se ha considerado a la necrosis como un mecanismo de muerte celular “accidental” y, por tanto, menos regulado. Sin embargo existen evidencias de que la necrosis también puede ser considerada un tipo de muerte celular programada. Es así que las ceramidas, precursores de los esfingolípidos eucariontes, han sido reconocidas como importantes segundos mensajeros implicados en el gatillamiento de procesos apoptótico/necróticos en diversos tipos celulares. Recientemente se ha descrito que células de linfoma B humano (línea celular A20) al ser estimuladas con ligando de Fas en presencia de ceramidas van a un destino de muerte necrótico. Además de esto, se ha descrito una relación entre estrés oxidativo y la generación de ceramidas, relación que se encontraría íntimamente ligada a procesos de señalización de muerte celular. Con estos datos se postuló que las ceramidas intervienen en la decisión apoptóticonecrótica en células epiteliales. Utilizando métodos de citometría de flujo y ensayos enzimáticos se encontró que las ceramidas aumentaron la muerte por necrosis en el tiempo tanto para células HeLa como para HTC, este aumento es dosis dependiente. Ácido flufenámico, un inhibidor inespecífico de canales catiónicos no selectivos involucrados en regulación del volumen celular por estrés oxidativo, en conjunto con ceramidas no cambió el porcentaje de muerte por necrosis y aumentó la población apoptótica. Al reemplazar el Na+ extracelular por el catión monovalente NMDG+ no se observó diferencia entre los porcentajes de las distintas poblaciones celulares. La aplicación en conjunto de H2O2 con ceramidas aumentó la muerte celular, pero sólo a altas concentraciones (10 mM). El efecto de ceramidas sobre la apoptosis se ensayó utilizando el inhibidor genérico de caspasas zVAD-fmk. No hubo cambios significativos en los porcentajes de muerte celular. Efectos similares se observaron al depletar los depósitos intracelulares de calcio. Estos resultados permiten concluir que las ceramidas inducen muerte celular necrótica de manera tiempo y dosis dependiente / Organisms regulate their number of cells through fine a balance between cell division and cell death. In respect to cell death, diverse mechanisms exist, such as apoptosis, autophagia and necrosis, presenting each one different morphologic and molecular characteristics. Necrosis has been considered as a mechanism of "accidental" cell death and therefore, less regulated. Nevertheless, evidences exist of which necrosis can be also considered as a type of programmed cell death. For example, ceramides, precursors of sphingolipids in euchariotic cells, have been recognized as important second messengers implicated in the development of apoptotic/necrotic processes in diverse cell types. It has been recentely described that human lymphoma B cells (A20 cell line),upon stimulation with Fas ligand in the presence of ceramides, induce necrotic cell death. Also, it has been described a relationship between oxidative stress and the generation of ceramides which are intimately linked to cell death signaling processes. Based on this information, it was postulated that ceramides take part in the apoptotic-necrotic decision in epithelial cells. Using flow citometry and enzymatic assays we found that ceramides increased cell death by necrosis in HeLa and HTC cells in a dose-dependent manner. Flufenamic acid, a non specific inhibitor of nonselective cation channels involved in regulation of the oxidative stress-dependent cell volume, used in conjunction with ceramides did not produce changes in the percentage of necrotic cell death and increased the population of apoptotic cells. Upon replacement of external Na+ by the monovalent cation NMDG+, the percentage of different cell populations was not altered. The application of ceramides with H2O2 produced an increase in the cell death, but only at high concentrations of H2O2 (10 mM). The effect of ceramides upon apoptosis was assayed using the generic caspase inhibitor zVAD- fmk. No significant changes were observed in the percentage of cell death. Similar effects were observed when depleting the intracellular calcium stores. These results allow us to conclude that ceramides induces necrotic cell death in a time and dose-dependent manner

Magíster en Bioquímica, área de especialización Bioquímica Toxicológica y Diagnóstico Molecular / Memoria para optar al Título de Bioquímica / La obesidad y la diabetes son condiciones altamente prevalentes que representan un importante factor de riesgo para el desarrollo de patologías cardiovasculares, principal causa de muerte entre los pacientes diabéticos. La lipotoxicidad y las alteraciones metabólicas juegan un papel fundamental en la resistencia a la hormona insulina y el daño cardiaco en estos pacientes. Los cardiomiocitos son las unidades funcionales del corazón y poseen un alto requerimiento energético que depende en su mayor parte de la función mitocondrial. Las mitocondrias forman una red dinámica que se remodela constantemente por eventos de fisión y fusión. La mantención de una morfología mitocondrial balanceada es fundamental para mantener una funcionalidad adecuada de este organelo. El objetivo de este trabajo fue investigar el efecto de las ceramidas, que derivan del metabolismo lipídico, en la señalización de la insulina y la dinámica mitocondrial en cultivos primarios de cardiomiocitos de rata. La señalización de insulina se evaluó mediante Western blot para Akt fosforilada y la morfología mitocondrial por microscopía confocal en células teñidas con Mitotracker Green. El tratamiento de los cardiomiocitos con C2-ceramida (40 μM, 3 h) disminuyó la fosforilación de Akt basalmente y en respuesta a insulina y favoreció la fisión mitocondrial, aumentando la translocación de la proteína de fisión Drp-1 hacia este organelo. Para evaluar si ambos efectos estaban relacionados, se inhibió la actividad de Drp-1 mediante el uso de un dominante negativo y de un inhibidor químico, antes del tratamiento con C2-ceramida. La inhibición de Drp-1 mediante ambas herramientas previno la fisión mitocondrial causada por C2-ceramida y rescató la fosforilación de Akt en respuesta a insulina. Trabajos previos de nuestro laboratorio muestran que el tratamiento de los cardiomiocitos con palmitato 500 μM durante 3 h también induce fisión de la red mitocondrial. En este trabajo se mostró que al inhibir la síntesis de ceramidas a partir de palmitato se previene, en parte, los efectos de este ácido graso sobre la dinámica mitocondrial de los cardiomiocitos. En conclusión, la fragmentación de la red mitocondrial inducida por ceramidas es necesaria para la disminución de la señalización de insulina en los cardiomiocitos. Además, la fisión mitocondrial inducida por palmitato en este modelo depende en parte de la generación de ceramidas. / Obesity and diabetes are highly prevalent conditions that represent an important risk factor for the development of cardiovascular diseases, the main cause of death in diabetic patients. Lipotoxicity and metabolic alterations take part in insulin resistance and heart damage in these patients. Cardiomyocytes are the functional basic units of the heart and have a high energy requirement that depends largely on mitochondrial function. Mitochondria form a dynamic network that is constantly remodelled by fission and fusion events. The maintenance of a balanced mitochondrial morphology is critical to maintain a proper functionality of this organelle. The aim of this study was to investigate the effect of ceramides, derived from lipid metabolism, in insulin signalling and mitochondrial dynamics in primary cultures of rat cardiomyocytes. Insulin signalling was assessed by Western blot for phosphorylated Akt and mitochondrial morphology by confocal microscopy in Mitotracker Green-stained cells. Treatment of cardiomyocytes with C2-ceramide (40 μM, 3 h) decreased the phosphorylation of Akt at baseline and in response to insulin and induced mitochondrial fission, increasing the translocation of the fission protein Drp-1 to this organelle. To assess whether both effects were related, Drp-1 activity was inhibited by using a dominant negative and a chemical inhibitor, before treatment with C2-ceramide. The inhibition of Drp-1 by both tools prevented mitochondrial fission caused by C2-ceramide and rescued Akt phosphorylation in response to insulin. Previous work in our laboratory showed that treatment of cardiomyocytes with palmitate 500 μM for 3 h also induces mitochondrial fission. We showed that inhibiting the synthesis of ceramides from palmitate prevented in part the effects of this fatty acid on mitochondrial dynamics in cardiomyocytes. In conclusion, the mitochondrial network fragmentation induced by ceramides is required for the decrease of insulin signalling in cardiomyocytes. Furthermore, palmitate-induced mitochondrial fission in this model depends in part on the generation of ceramides. / Fondap, Fondecyt

Los esfingolípidos de las células germinales y espermatozoides de mamífero son inusuales por contener una alta proporción de ácidos grasos poliinsaturados de muy larga cadena (VLCPUFA). En las gametas de rata, la esfingomielina (SM) y la ceramida (Cer) contienen especies con ácidos tetraenoicos y pentaenoicos de la serie n-6 de hasta 32 carbonos, en versiones no hidroxiladas (n-V) y 2-hidroxiladas (h-V). Estudios previos habían mostrado que las SM con VLCPUFA se forman en las células espermatogénicas, que la relación entre h-V SM y n-V SM aumenta con la diferenciación celular, y que ambas terminan localizadas específicamente en las cabezas espermáticas. Posteriormente se vio que estas SM disminuyen a medida que aumentan las Cer en las gametas de rata durante reacciones que ocurren en etapas claves de la fertilización, en especial durante la reacción acrosomal. El objetivo de este trabajo fue contribuir al conocimiento de las propiedades biofísicas de estos inusuales esfingolípidos y a determinar cómo estas propiedades cambian como resultado de dichos eventos bioquímicos. Nuestro primer desafío fue obtener estas relativamente minoritarias especies moleculares en suficiente cantidad y aceptable pureza. Para ello tomamos ventaja de su estructura química. Así, fracciones conteniendo especies de n-V SM y h-V SM se separaron de las mayoritarias SM saturadas y monoenoicas teniendo en cuenta el alto número de dobles ligaduras de sus ácidos grasos. Luego, de la primera fracción se obtuvieron especies de SM con n-28:4, n-30:5 y n-32:5, y de la segunda especies con h-28:4, h-30:5 y h-32:5, aprovechando las diferencias de hidrofobicidad entre estos ácidos grasos. Estas fracciones y especies se incluyeron en sistemas modelo de bicapa y monocapa, y se estudiaron aplicando metodologías e instrumentación previamente validadas para especies de SM con 16:0, 18:1, o 24:1. Un enfoque similar se aplicó para estudiar las especies de las fracciones n-V Cer y h-V Cer. Al estudiarlas por espectroscopía de fluorescencia, tanto la polarización generalizada (GP) del Laurdan como la anisotropía de fluorescencia (r) del DPH mostraron que la mayoría de las n-V SM y h-V SM compartieron la característica de tener temperaturas de transición (Tt) muy bajas, por debajo de 5 °C, salvo n-32:5 SM y h-32:5 SM, cuyas Tt fueron cercanas a los 22 °C. El aumento en la Tt que podría esperarse debido al inusual largo de la cadena acílica, es pues contrarrestado por la presencia de varias dobles ligaduras en estos ácidos grasos. Estos estudios indicaron que las cadenas acilicas de estas especies se encuentran en un estado desordenado a temperatura fisiológica. Las h-V SM exhibieron mayores valores de GP y de r que las correspondientes especies n-V SM, indicando que el grupo hidroxilo en las primeras tiende a promover las interacciones intermoleculares, probablemente a través de la formación de uniones tipo puente hidrógeno. Las interacciones entre las SM con VLCPUFA y otros lípidos se investigaron empleando sistemas modelo multicomponente en forma de bicapa. En bicapas formadas por dimiristoíl-PC y estas SM, se concluyó que éstas últimas jugarían el rol de lípidos “fluidificantes”, en discrepancia con la noción generalmente aceptada de que en las membranas celulares las SM son las que restringen la movilidad de otros lípidos. En sistemas conteniendo dioleoil-PC, colesterol y n-V o h-V SM, ninguna de éstas tendió a segregarse en dominios condensados enriquecidos en SM/colesterol a temperatura ambiente, tal como sí lo hizo claramente la 16:0 SM. Las dos estrategias experimentales sugirieron que a temperatura fisiológica y en membranas naturales donde las SM con VLCPUFA coexisten con otros lípidos, como es el caso de la membrana plasmática de la cabeza espermática, estas especies no se concentrarían en áreas de la membrana ricas en colesterol, sino en áreas ricas en glicerofolípidos poliinsaturados, esto es, en dominios más “desordenados” de la membrana. Una valiosa información acerca de las propiedades de superficie de las SM en estudio se obtuvo de los experimentos realizados en monocapas de Langmuir. Analizando los valores de la presión de superficie (π) y el potencial de superficie (ΔV), así como los datos de la microscopía de ángulo de Brewster (BAM) en función de la compresión aplicada, se caracterizaron las interacciones intermoleculares durante el empaquetamiento de los lípidos y los cambios termodinámicos implicados en ellas. Las SM con VLCPUFA formaron films con un espesor considerablemente menor al esperado, encontrándose en un régimen líquido-expandido (LE) con una alta libertad conformacional. Sus valores de área molecular media (MMA) fueron llamativamente grandes con respecto a la de las SM conocidas, lo cual indica que un mismo número de moléculas ocupa un área significativamente mayor en la membrana. Las h-V SM mostraron menores MMA que las equivalentes n-V SM, confirmando que el grupo hidroxilo en las primeras favorece las interacciones intermoleculares, permitiendo así un mayor grado de empaquetamiento durante el proceso de compresión, lo que explica que ocupen una menor área en el plano de la membrana. Cuando se obtuvieron las isotermas a 7 °C de las seis especies de SM en estudio, sólo n-32:5 SM y h-32:5 SM mostraron transiciones de fase LE → LC (líquido condensado) de primer orden, otro hallazgo consistente con el comportamiento previamente observado en bicapa. Las seis especies moleculares mayoritarias de Cer con VLCPUFA también fueron separadas y estudiadas en monocapas de Langmuir. Las isotermas de compresión presentaron una transición de fase LE  LC de primer orden a temperatura ambiente, con segregación de dominios durante esta transición que pudo ser seguida mediante BAM. En ambos estados de fase, todas ellas mostraron valores mucho más altos de MMA y ΔV que las especies conocidas usadas como referencia. El empaquetamiento molecular y el grado de ordenamiento de las cadenas fue relativamente menor para las n-V Cer que para las h-V Cer. Estas últimas formaron membranas de mayor espesor, ocuparon una menor área de superficie, y presentaron transiciones de fase LE  LC más pronunciadas y cooperativas que las n-V Cer. Tomados en conjunto, los parámetros termodinámicos y de superficie permiten concluir que estas Cer se organizan con sus ácidos grasos en una conformación curvada, formando monocapas con un espesor menor al esperado, siendo esta característica aún más marcada en las n-V Cer que en las h-V Cer. Mientras las propiedades reológicas de la h-28:4 SM fueron similares a las de otras especies de SM, las de la h-28:4 Cer fueron atípicas. Así, la fase LC de ésta fue altamente compresible, lo que es consistente con una gran libertad conformacional de sus cadenas hidrofóbicas, pero la difusión en ella, estuvo muy restringida, presentando un coeficiente de difusión (D) 100 veces más bajo que su contraparte h-28:4 SM. Las propiedades de mezclas en que coexisten h-28:4 SM y h-28:4 Cer fueron estudiadas mediante la pre-mezcla de distintas proporciones de ambas y mediante la generación de la última por acción de una esfingomielinasa colocada en la subfase de un film de h-28:4 SM. Ambos enfoques evidenciaron que la Cer tiende a separarse de la fase fluida en que se encuentra la SM, formando dominios ricos en Cer a medida que aumenta la relación molar Cer/SM. A temperatura fisiológica, estos dominios mostraron una morfología y una organización estructural sobre la superficie del film que difirieron marcadamente de los que exhibió la 16:0 Cer. Tomadas en conjunto, las presentes observaciones podrían ser relevantes para la reorganización estructural que se sabe ocurre sobre la cabeza espermática durante la reacción acrosomal, al completarse la cual la mayor parte de las SM con VLCPUFA preexistentes se convierten en las correspondientes Cer. / The sphingolipids of mammalian germ cells and spermatozoa are unique in that they are made up by a high proportion of very long chain polyunsaturated fatty acids (VLCPUFA). In the rat gametes, sphingomyelin (SM) and ceramide (Cer) contain species with tetraenoic and pentaenoic fatty acids of the n-6 series with up to 32 carbon atoms, in non-hydroxylated (n-V) and 2-hydroxylated (h-V) forms. Previous studies had shown that the SM with VLCPUFA are formed in spermatogenic cells, that the ratio between h-V SM and n-V SM increases with germ cell differentiation, and that both up localized specifically on the sperm heads. Then it was seen that these SM decrease as Cer increase in rat gametes during reactions that take place in key stages of fertilization, especially during the acrosomal reaction. The aim of this work was to contribute to the knowledge of the biophysical properties of these unusual sphingolipids and to determine how such properties change as a result of these biochemical events. Our first challenge was to obtain these relatively minor molecular species in sufficient amounts and with acceptable purity. To this aim we took advantage of their chemical structure. Thus, fractions containing n-V SM and h-V SM species were separated from the major saturated and monoenoic SM taking into account the high number of double bonds of their fatty acids. Then, SM species containing n-28:4, n-30:5 and n-32:5 from the first fraction, and SM species with h-28:4, h-30:5 and h-32:5 from the second one, were obtained by making use of the differences in hydrophobicity among their fatty acids. These fractions and species were included in bilayer and monolayer model systems and studied by applying methodologies and instrumentation previously validated for SM species with 16:0, 18:1 and 24:1. A similar approach was applied to study the species in the n-V Cer and h-V Cer fractions. When studied by fluorescence spectroscopy, both the generalized polarization (GP) of Laurdan and the fluorescence anisotropy (r) of DPH showed that most of the n-V SM and h-V SM shared the characteristic of having very low transition temperatures (Tt), both below 5 °C, except n-32:5 SM and h-32:5 SM, whose Tt values were around 22 °C . The increase in Tt that could have been expected to result from the unusual length of the acyl chains is thus counteracted by the presence of several double bonds in these fatty acids. These studies indicated that the acyl chains of these species are in a disordered state at physiological temperature. The h-V SM exhibited higher GP and r values than the corresponding n-V SM species, indicating that the hydroxyl group in the former tends to promote intermolecular interactions, probably through the formation of hydrogen-bond type of unions. The interactions between SM with VLCPUFA and other lipids were investigated by employing multicomponent lipid systems in bilayer form. In bilayers formed by dimiristoyl-PC and these SM, it was concluded that the latter played the role of “fluidifying” lipids, in discrepancy to the generally accepted notion that in cell membranes the SMs are lipids that restrict the mobility of other lipids. In systems containing dioleoyl-PC, cholesterol and n-V SM or h-V SM, neither of these tended to segregate in SM/cholesterol-rich domains at room temperature, as clearly did 16:0 SM. Both experimental strategies suggested that at physiological temperature and in natural membranes were VLCPUFA-containing SM coexist with other lipids, as is the case of the plasma membrane of the sperm head, these species would not concentrate in cholesterol-rich membrane areas, but in areas rich in polyunsaturated fatty acids, namely in more “disordered” domains of the membrane. Valuable information about the surface properties of the SM under study was obtained from the experiments performed in Langmuir monolayers. By analyzing the values of surface pressure (π) and surface potential (ΔV), and the data from Brewster angle microscopy (BAM) as a function of the compression applied, the intermolecular interactions during the lipid packing and the thermodynamic changes implied in these interactions were characterized. The VLCPUFA-containing SM formed films with a considerably smaller thickness than that expected, and they were found in a liquid-expanded (LE) state with a high conformational freedom. The values of their mean molecular areas (MMA) were markedly high compared with those of best-known SM species, indicating that the same number of molecules occupies an area significantly larger in the membrane. The h-V SM species displayed lower MMA values than the equivalent n-V SM, confirming that the hydroxyl group in the former facilitates intermolecular interactions, thereby allowing a higher degree of packing during the compression process, and explaining that they occupy a smaller area in the plane of the membrane. When the isotherms of the six SM under study were obtained at 7 °C, only n-32:5 SM and h-32:5 SM exhibited first order LE → LC phase transitions, another finding consistent with the behavior previously observed in bilayer. The six major molecular species of Cer with VLCPUFA were also separated and studied in Langmuir monolayers. The compression isotherms showed that they underwent a first-order LE → LC (liquid condensed) phase transition at room temperature, the segregation of domains during that transition being clearly followed by means of BAM. In both phase states, all of them had MMA and ΔV values far higher than the Cer species used as reference. The molecular packing and degree of ordering of the chains were relatively lower for the n-V Cer than for the h-V Cer. The latter formed thicker films, occupied lower surface areas, and presented more pronounced and cooperative LE → LC phase transitions than the n-V Cer. Taken together, the thermodynamic and surface behavior parameters allow to conclude that these Cer species organize with their fatty acids in a curved conformation, forming monolayers with a lower than expected thickness, this feature being more marked for the n-V Cer than for the h-V Cer. Whereas the rheological properties of h-28:4 SM were similar to those of other SM species, those of h-28:4 Cer were atypical. Thus, the LC phase of the latter was highly compressible, in agreement with a high conformational freedom of their hydrophobic chains, but the diffusion in such phase was highly restricted, showing a diffusion coefficient (D) 100-fold lower than that of h-28:4 SM. The properties of mixtures in which h-28:4 SM and h-28:4 Cer coexist were studied by mixing them in different proportions and by generating the latter by the action of a sphingomyelinase placed in the subphase of an h-28:4 SM film. Both approaches evidenced that Cer tends to separate from the fluid phase of SM, forming Cer-rich domains as the molar Cer/SM ratio increases. At physiological temperature, these domains had a morphology and structural organization on the film surface that markedly differed from those displayed by 16:0 Cer. Taken together, the present observations could be relevant to the structural reorganization of the membrane that is known to take place on the sperm head during the acrosomal reaction, after the completion of which a major part of the preexisting VLCPUFA-rich SM are converted into the corresponding Cer.

Orientador: Fernando Antonio Santos Coelho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T16:21:23Z (GMT). No. of bitstreams: 1 Yamakawa_NathaliaChristinaGoncalves_M.pdf: 16524766 bytes, checksum: 72cb2d423e322cd66bf985a8a162ae5a (MD5) Previous issue date: 2012 / Resumo: A epilepsia é uma das principais doenças do sistema nervoso central, caracterizada por uma alteração na atividade elétrica do cérebro, que leva a perdas de memória e convulsões. Essa doença afeta cerca de 50 milhões de pessoas no mundo, que são discriminadas e isoladas da sociedade. Existem vários medicamentos que podem ser utilizados, no entanto, muitas vezes é necessário a administração de associações desses que apresentam severos efeitos colaterais. Esse quadro justifica a necessidade da busca por substâncias mais eficientes para o tratamento dessa doença. Em 2008, Ahmed e cols. isolaram da esponja marinha Negombata corticata duas ceramidas que apresentaram importante atividade anti-convulsiva. Assim, este trabalho teve por objetivo estabelecer uma estratégia sintética para a preparação do fragmento polar de tais ceramidas. A rota foi baseada em um aduto de Morita-Baylis-Hillman (MBH), obtido com elevada diastereosseletividade a partir de uma reação entre o aldeído de Garner e o acrilato de etila. Uma reação de ozonólise na dupla ligação do aduto de MBH fornece um a-cetoéster, cuja carbonila cetônica e reduzida conduzindo a um único produto. A proteção das hidroxilas, permitiu a confirmação da estereoquímica relativa através de análise por difração de raios-X, que também evidenciou a ocorrência de racemização na reação de MBH. O diol também foi utilizado na preparação de um aza-açúcar, potencial inibidor de glicosidase, sendo esta síntese em apenas 4 etapas com 32% de rendimento global. A fração apolar da ceramida foi sintetizada a partir de uma reação de Grignard, e a junção dos fragmentos pode ser realizada utilizando-se uma reação de Wittig. Desta maneira, foi descrita uma nova estratégia sintética que pode ser aplicada na preparação de diversos análogos das ceramidas, desenvolvendo um antiepiléptico mais potente / Abstract: Epilepsy is a chronic brain disorder that affects around 50 million people all over the world. It is characterized by recurrent seizures - which are physical reactions to sudden excessive electrical discharges in a group of brain cells. The discrimination and social stigma that surround epilepsy worldwide are often more difficult to overcome than the seizures themselves. Because of this fact and the economical impacts of the disease, the research for new biologically active compounds is still necessary. In 2008, Ahmed et al. isolated from the Red Sea sponge Negombata corticata two ceramides, which exhibit in vivo anticonvulsant activity. This work is focused on establishing of a synthetic sequence to prepare the polar fragment present in both ceramides. The strategy was based on a Morita-Baylis-Hillman reaction between a Garner¿s aldehyde and ethyl acrylate that provided a functionalized intermediate in good diastereoselectivity. The major diastereoisomer was employed as substrate in an ozonolysis reaction, followed by a stereoselective reduction that afforded 1,2-diol as a single isomer. The acetonide derived from this 1,2-diol allowed us to determine through X-Ray diffraction analysis the relative stereochemistry of this compound as being 1,2- anti. To finish the synthesis of the polar fragment, the ester group present in the acetonide was reduced to the corresponding aldehyde. The diol also was applied in a high diastereoselective preparation of an azasugar in 4 steps and 32% yield overall. In this work we also describe the synthesis of a carbon chain of the ceramide, our route includes an approach to the apolar fragment obtained by a Grignard reaction; then a Wittig reaction can couple both fragments toward the finalization of the sphingosine¿s synthesis. Our synthetic route can also be used in the preparation of several analogues of the antiepiletic ceramides / Mestrado / Quimica Organica / Mestre em Química

A obesidade, doença multifatorial, ocasiona inúmeros distúrbios no metabolismo lipídico e energético, provocando disfunção na bioenergética mitocondrial. A partir deste fato, o presente estudo teve como hipóteses que: o desequilíbrio na bioenergética mitocondrial e as alterações metabólicas causadas pela obesidade são terapeuticamente modificados com o treinamento físico. Dessa maneira, o objetivo do estudo foi avaliar a capacidade oxidativa e conteúdo mitocondrial em tecido adiposo branco, marcadores de doenças cardiosculares (esfingolipídios e N-óxido de trimetilamina, TMAO) e as alterações na composição corporal, desempenho físico e taxa metabólica de repouso (TMR) de mulheres com obesidade submetidas a treinamento físico combinado. A casuística do presente trabalho foi composta de 14 mulheres adultas jovens com diagnótico clínico de obesidade (IMC 33±3 kg/m² e idade 35±6 anos). Foram submetidas a um programa de treinamento físico combinado (exercícios aeróbios e força alternadamente, 55 min à 75-90% da frequência cardíaca máxima, 3 vezes por semana, durante 8 semanas). Todas as participantes foram avaliadas antes e após a intervenção com o treinamento, quanto a: composição corporal, TMR, oxidação de substratos (carboidrato e lipídios) e coeficiente respiratório (QR), desempenho físico, capacidade oxidativa (respiração acoplada: VADP/VOLIGO, e respiração desacoplada: VOLIGO/VCCCP) e conteúdo mitocondrial (enzima citrato sintase, CS) em tecido adiposo branco, nível de esfingolípidios, TMAO e precursores plasmáticos. Os dados foram analisados pelo test t pareado ou Wilcoxon (as pacientes foram consideradas controle de si próprio), após determinação da normalidade da amostra, considerado nível de significância p<= 0,05. Após a intervenção (treinamento físico combinado), houve o aumento da TMR, oxidação de lipídios e desempenho físico, com redução da oxidação de carboidratos e QR, mas não houve perda de peso e alteração da composição corporal. Após o treinamento combinado houve, o aumento da atividade da enzima CS (marcador de conteúdo mitocondrial) e redução à respiração desacoplada (VOLIGO/VCCCP). No plasma, o treinamento físico foi capaz de reduzir os níveis de esfingolipídios e TMAO (fatores de risco cardiovasculares). Também foram encontradas correlações positivas entre TMR, oxidação de lipídios e desempenho físico com CS e negativamente correlacionado com respiração desacoplada. Concluindo, o treinamento físico em mulheres com obesidade aumentou o metabolismo energético, com aumento da TMR, conteúdo e grau de acoplamento mitocondrial, aumentou o desempenho físico e reduziu fatores de risco cardiovasculares (TMAO), independente da perda de peso. / The obesity, a multifactorial disease, causes various metabolic disorders in lipid and energy metabolism, may induce mitochondrial bioenergetic dysfunction. From this, the present study hypothesized that: mitochondrial bioenergetics dysfunction and metabolic problems caused by obesity are be therapeutically modified with physical training. Thus, the objective of study was to evaluated: the oxidative capacity and mitochondrial content in white adipose tissue, markers of cardiovascular diseases (sphingolipids and trimethylamine N-oxide, TMAO) and changes of body composition, physical performance and resting metabolic rate (TMR) of obese women submitted to combined physical training. The present study was composed of 14 young women with obesity (BMI 33 ± 3 kg/m² and age 35 ± 6 years old). They underwent a combined physical training program (aerobic exercises and strength alternately, 55 min at 75-90% of maximal heart rate, 3 times a week, for 8 weeks). All participants were evaluated before and after the intervention: body composition, TMR, substrates oxidation (carbohydrate and lipids) and respiratory coefficient (RQ), physical performance, oxidative capacity (by mitochondrial respiration - Couple: VADP/VOLIGO; Uncoupling: VOLIGO/VCCCP) and Citrate Sinthase activity in white adipose tissue, level of sphingolipids, TMAO and precursors from plasma. Data analysis were made by paired t test or Wilcoxon, after normality determination of the sample, with level of significance p <0.05. After intervention with combined physical training, there was an increase in TMR, lipid oxidation and physical performance, reduced carbohydrate oxidation and RQ, but did not cause weight loss and changes of body composition. In adipose tissue, physical activity increased CS activity (mitochondrial content marker) and reduced uncoupling respiration (VOLIGO/VCCCP). In plasma, physical training was able to reduce levels of sphingolipids and TMAO (cardiovascular risk factors). In addition, positive correlations were found between, TMR, lipid oxidation and physical performance with CS and negatively correlation with uncoupling respiration. Therefore, physical training in obese women improve energy metabolism, with increased TMR, content and degree of mitochondrial coupling, increased physical performance and reduced cardiovascular risk factors, regardless of weight loss.

nÃo hà / Este trabalho descreve a composiÃÃo quÃmica das espÃcies marinha Palythoa caribaeorum e Protopalythoa variabilis, coletadas no municÃpio de Paracuru-CE. O fracionamento cromatogrÃfico do extrato hexÃnico de P. caribaeorum, resultou no isolamento de quatro esterÃides tetracÃclico de esqueleto ergostano: 24(R)-ergost-5-en-3&#61538;-ol (P-1); 5&#61537;,8&#61537;-epidioxi-24(R)-ergost-6-en-3&#61538;-ol (P-2); 24(R)-ergost-5-en-3&#61538;,7&#61537;-diol (P-4) e 24(R)-7&#61537;-hidroperoxi-ergost-5-en-3&#61538;-ol (P-7), um derivado do glicerol, 1-O-hexadecilglicerol (P-3) e quatro ceramidas: N-(2S,3R,4E,8E,1,3-dihidroxi-4,8-octadecadieno)hexadecanamida (P-5); N-(2S,3R,4E,1,3-dihidroxi-4-octadeceno)-hexadecanamida (P-6), N-[2S,3R,4E,8E,1-(2â-metilamino-etanosulfonila)-3-hidroxi-4,8-octadecaÂdieno]hexadecanamida (P-8) e N-[2S,3R,4E,1-(2â-metilaminoetano-sulfonila)-3-hidroxi-4-octadeceno]hexadecanamida (P-9). Do fracionamento cromatogrÃfico do extrato etanÃlico, foi possÃvel isolar o esterÃide 24(R)-ergost-7-en-3&#61538;,5&#61537;&#61484;6&#61538;-triol (P-10) e o nucleosÃdeo 2-metil-timidina (P-11). Do estudo quÃmico do extrato hexÃnico de P. variabilis obteve-se os mesmos constituintes quÃmicos isolados de P. caribaeorum (P-1, P-2, P-3 e P-4) e as quatro ceramidas (P-5, P-6, P-8 e P-9). AlÃm destes compostos foi isolado um Ãster de cadeia alifÃtica, hexadecanoato de nonila (P-12) e o esterÃide Ãcido 24(R)-B-norergostan-3&#61538;-5&#61538;-diol-6&#61538;-carboxÃlico (P-13). O potencial citotÃxico e antifÃngico das ceramidas foi avaliado, entretanto, estas nÃo apresentaram atividade. Os compostos foram isolados atravÃs de cromatografia de adsorÃÃo em gel de sÃlica e cromatografia lÃquida de alta eficiÃncia. As estruturas dos compostos obtidos foram elucidadas utilizando tÃcnicas espectroscÃpicas e espectromÃtricas, tais como: espectrometria de massa acoplada a cromatografia gasosa (CG/EM); espectrometria de massa de alta resoluÃÃo (EMAR); espectroscopia na regiÃo do infravermelho (IV) e RessonÃncia MagnÃtica Nuclear (RMN 1H, 13C e 15N) atravÃs de sequÃncias de pulsos uni e bidimensionais e comparaÃÃo com dados de RMN na literatura. / This work describes the chemical composition of the marine species Palythoa caribaeorum and Protopalythoa variabilis, both collected at Paracuru beach, state of CearÃ. The cromatographic fractionation of the hexane extract from P. caribaeorum resulted in the isolation of four tetracyclic sterols possessing the ergostan skeleton: 24(R)-ergost-5-en-3b-ol (P-1); 5 a,8a-epidioxy-24(R)-ergost-6-en-3b-ol (P-2); 24(R)- ergost-5-en-3b,7a-diol (P-4) and 24(R)-7a-hydroperoxy-ergost-5-en-3b-ol (P-7), a glycerol derivative, 1-O-hexadecylglycerol (P-3) and four ceramides: N- (2S,3R,4E,8E,1,3-dihydroxy-4,8-octadecadienyl)hexadecanamide (P-5); N- (2S,3R,4E,1,3-dihydroxy-4-octadecenyl)hexadecanamide (P-6); N-[2S,3R,4E,8E,1-(2â- methylamino-ethanosulfonyl)-3-hydroxy-4,8-octadecaenyl]hexadecanamide (P-8) and N-[2S,3R,4E,1-(2â-methylaminoethano-sulfonyl)-3-hydroxy-4-octadecenyl]hexadecanamide (P-9). The cromatographic fractionation of the ethanol extract permited the isolation of a steroid, 24(R)-ergost-7-en-3b,5a,6b-triol (P-10) and a nucleoside 2- methyltimidine (P-11). Column chromatography of the hexane extract of P. variabilis led to the isolation of nonyl hexadecanoate (P-12), the sterol 24(R)-B-norergostan-3b- 5b-diol-6b-carboxylic acid (P-13) and the same chemical constituents previously isolated from P. caribaeorum (P-1, P-2, P-3 e P-4) including the four ceramides (P-5, P-6, P-8 e P-9). The citotoxic and antifungal properties of all ceramides were evaluated, nevertheless none of them showed any activity. All compounds were isolated through adsorption column cromatography over silica gel followed by high performance liquid chromatography. The structures of the isolated compounds were elucidated using spectrometric techniques, such as: GC/MS, HRESIMS, IR and NMR (1H, 13C and 15N) through 1D and 2D pulse sequences and, whenever the case, comparison with literature data