Made available in DSpace on 2014-08-20T14:29:21Z (GMT). No. of bitstreams: 1 Carolina Galarza Vargas.pdf: 213067 bytes, checksum: 1dde22113f4a4fa1a427c0f78f4df93f (MD5) Previous issue date: 2012-05-10 / A crescente morbimortalidade relacionada às doenças crônico-degenerativas não transmissíveis como câncer, diabetes, hipertensão, doenças coronarianas e obesidade representa uma forte ameaça à saúde humana. Essas doenças têm sido apontadas como as maiores causas de morte no mundo, sendo responsáveis por um número estimado de 35 milhões de mortes a cada ano, em torno de 60% de todas as mortes a nível mundial, dados que apontam para a necessidade da adoção de medidas que visam a prevenção e a cura das mesmas. Tendo em vista as propriedades funcionais da linhaça e as diferentes formas de consumo da mesma, este estudo terá por objetivo verificar o efeito da suplementação de dietas hipercolesterolêmicas com óleo, farelo desengordurado e semente de linhaça triturada, em duas concentrações, sobre os parâmetros fermentativos e metabolismo lipídico de ratos.

Orientador: Helena Coutinho Franco de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T05:23:31Z (GMT). No. of bitstreams: 1 Raposo_HelenaFonseca_D.pdf: 7490061 bytes, checksum: b7d93ea4b63d773168cdfcbe34c70392 (MD5) Previous issue date: 2013 / Resumo: A proteína de transferência de colesteril éster (CETP) transfere colesterol esterificado (CE) das HDL para as lipoproteínas que contêm apo B, em troca de triglicérides. Assim, a CETP promove a redução dos níveis de HDL-colesterol no plasma, aumentando o risco de aterosclerose. Recentemente, nosso grupo verificou que camundongos transgênicos que expressam a CETP (CETP-Tg) apresentam menor adiposidade que os controles não transgênicos (NTg). Neste trabalho investigamos possíveis mecanismos que expliquem a redução da adiposidade. Camundongos CETP-Tg e controles NTg (C57/BL6 background) foram tratados com dieta padrão desde o desmame até os 5 meses de idade. Camundongos CETP-Tg apresentaram redução nos depósitos de tecido adiposo (~30%). Essa redução da adiposidade não foi compensada por deposição ectópica de lipídeos no fígado ou músculo, nem foi consequência de alteração do balanço de gordura (ingestão vs. excreção). Lipogênese in vivo e em adipócitos isolados (estimada por 3H2O e 14C-acetato) foram similares entre os grupos. A retenção de lipídeos exógenos e a captação de glicose pelo fígado, músculo e tecidos adiposos (estimada após dose oral de 3H-trioleina e injeção IP de 3H-2- deoxiglicose, respectivamente) não evidenciaram nenhuma diferença entre os genótipos. No estado alimentado, a expressão da CETP aumentou (~50%) a lipólise basal in vivo e estimulada por isoproterenol em adipócitos isolados. Coerentemente, os níveis de RNAm para HSL (hormone sensitive lipase) e ATGL (adipose triglyceride lipase) estavam aumentados, assim como FATP1 (fatty acid transport protein 1). No tecido adiposo marrom, verificou-se elevação nos níveis de ATGL, FATP1 e UCP1 (uncoupling protein 1). Além disso, o conteúdo protéico do receptor beta 3 adrenérgico também estava aumentado nos extratos de membrana celular de tecido adiposo visceral e marrom dos animais CETP-Tg. A expressão da CETP também elevou em cerca de 10% a taxa metabólica corporal (VO2 e gasto energético). Conclui-se que a CETP altera a expressão e/ou função de proteínas relacionadas à lipólise e termogênese, aumentando o gasto energético global e reduzindo a massa de gordura corporal. Em conjunto, esses resultados sugerem uma nova função antiadipogênica para a CETP / Abstract: Cholesteryl ester transfer protein (CETP) is a plasma protein that mediates the exchange of triglycerides for esterified cholesterol from HDL to the apoB containing lipoproteins. In this way, CETP promotes reduction of plasma HDLcholesterol and, thus, increases the risk of atherosclerosis. Recently, we found that CETP expressing mice (CETP-Tg) presented less adipose tissue mass than nonexpressing controls. In this work we investigated possible mechanisms to explain these findings. CETP-Tg and non-transgenic (NTg) control mice (C57/BL6 background) were fed with chow diet from weaning to 5 month of age. CETP-Tg mice had reduced visceral and subcutaneous fat depots (~30%) that was not compensated by lipid ectopic deposition and could not be explained by differences in fat intake and excretion. Lipogenesis rates in vivo and in isolated adipocytes (estimated using 3H2O and 14C-acetate) were similar in both CETP-Tg and control mice. Lipid retention and glucose uptake by liver, muscle and adipose tissues (estimated after an oral dose of 3H-triolein and IP injection of 3H-deoxiglucose) showed no significant differences between groups. In the fed state, CETP group showed higher (~50%) basal lipolysis rates in vivo and in isoproterenol stimulated lipolysis rates in isolated adipocytes. In accordance, visceral adipose hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) mRNA expression were elevated, as well as fatty acid transport protein (FATP1). In brown adipose tissue, ATGL, FATP1 and uncoupling protein 1 (UCP1) were increased. Furthermore, beta 3 adrenergic receptor proteins mass were upregulated in visceral and brown adipose tissues membrane extracts. In addition, whole body energy expenditure (measured by respirometry) was found to be elevated in CETP-Tg mice (10%). In conclusion, CETP change expression and function of proteins related to lipolysis and thermogeneis, thus, increasing whole body energy expenditure and resulting in less body fat content. These findings disclose a novel anti-adipogenic role for CETP / Doutorado / Fisiologia / Doutora em Biologia Funcional e Molecular

Orientadores: Anibal Eugênio Vercesi, Helena Coutinho Franco de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T11:59:13Z (GMT). No. of bitstreams: 1 Marques_AnaCarolina_M.pdf: 1109688 bytes, checksum: 76a66df390130e764ca54b415b0756b4 (MD5) Previous issue date: 2014 / Resumo: Os camundongos hipercolesterolêmicos (LDLr-/-) provenientes do Jackson Laboratory são modelos experimentais valiosos para o estudo da aterosclerose. Estes animais apresentam elevadas taxas de lipogênese, processo que consome grandes quantidades de NADPH. Pesquisas recentes revelaram que esta linhagem possui, além da deleção do gene do receptor de LDL, uma mutação no gene da NADP- transidrogenase (NNT). A falta da NNT pode gerar estresse oxidativo devido ao fornecimento deficiente de NADPH mitocondrial. O objetivo deste trabalho foi investigar a participação da elevação da lipogênese e da deficiência de NNT sobre o estado redox mitocondrial e suscetibilidade à transição de permeabilidade mitocondrial (TPM). Para tanto foram comparadas três linhagens de camundongos: LDLr-/- (deficiente do receptor de LDL e da NNT), C57BL6/J (deficiente apenas da NNT) e C57BL6/JUnib (controle). Foram avaliados: o controle respiratório mitocondrial (consumo de oxigênio), o estado redox de NAD(P) (fluorimetria), a susceptibilidade à transição de permeabilidade mitocondrial induzida por cálcio (inchamento e dissipação do potencial elétrico de membrana (??) sensíveis à ciclosprina A e a geração de peróxido de hidrogênio (H2O2) (Amplex red®) em mitocôndrias isoladas de coração e fígado. Observamos que não houve diferenças significativas nos parâmetros respiratórios mitocondriais nos dois tecidos das três linhagens estudadas. Como esperado, as mitocôndrias dos camundongos LDLr-/- e C57BL6/J não podem sustentar o estado reduzido de NADPH in vitro, uma vez que são deficientes de NNT. Observamos que houve diferenças significativas entre as 3 linhagens quanto à TPM da seguinte maneira: LDLr-/- > C57BL6/J > C57BL6/JUnib (controle) em mitocôndrias isoladas de fígado (inchamento e dissipação de ??) e em mitocôndrias de coração (??). Além disso, a produção de H2O2 por mitocôndrias hepáticas seguiu o mesmo padrão, sendo LDLr-/- > C57BL6/J > C57BL6/JUnib (controle). Em conjunto, estes resultados indicam que a maior suscetibilidade à TPM das mitocôndrias de camundongos LDLr-/- está correlacionada com diminuição de NADPH, tanto por aumento de consumo (devido a elevada lipogênese) quanto por diminuição de sua produção (deficiência em NNT) / Abstract: Hypercholesterolemic LDL receptor knockout mice (LDLr-/-) from Jackson Laboratory are valuable experimental models to study atherosclerosis development. These mice exhibit high rates of lipogenesis, a process that consumes large amounts of NADPH. It was recently discovered that the mice strain used to produce the LDLr-/- also carries a homozygous NADP-transhydrogenase (NNT) mutation. Loss of NNT may cause oxidative stress due to a poor supply of mitochondrial NADPH. The objective of this study was to investigate the role of elevated lipogenesis and NNT deficiency on the mitochondrial redox status and susceptibility to mitochondrial permeability transition (MPT). Three mice strains were compared: LDLr-/- mice (deficient of both LDL receptor and NTT), C57BL6/J (deficient in NNT only) and the wild type control mice C57BL6/JUnib. We evaluated the mitochondrial respiratory control (oxygen consumption), the NAD(P) redox status (fluorimetry), the susceptibility to calcium induced mitochondrial permeability transition (swelling and dissipation of membrane potential (??) sensitive to cyclosporin A) and the generation of H2O2 (Amplex red®) in isolated heart and liver mitochondria. We observed no significant differences in mitochondrial respiratory parameters in both tissues of the three mice strains studied. As expected, the mitochondria of LDLr-/- and C57BL6/J mice cannot maintain the NADP in the reduced state in vitro, since they are deficient in NNT. Regarding the susceptibility to MPT, we observed significant differences among mitochondria from the 3 strains, as follows: LDLr-/- > C57BL6/J > C57BL6/JUnib (control) in isolated liver mitochondria (swelling and potential dissipation) and in heart mitochondria potential dissipation). Furthermore, the production of H2O2 by the liver mitochondria followed the same MPT pattern: LDLr-/- > C57BL6/J > C57BL6/JUnib (control). Together, these results indicated that the greater susceptibility of LDLr-/- mitochondria to MPT is correlated with decreased NADPH which is explained by both increased consumption (due to high lipogenesis) and decreased production (deficiency NNT) / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestra em Ciências

Estudos anteriores em nosso laboratório demonstraram que pacientes portadores de DAC apresentam diferenças no metabolismo do CL e CE de uma nanoemulsão artificial rica em colesterol (LDE), nos quais o CL apresentou maior remoção plasmática e depósito arterial. Dando continuidade a esta linha de pesquisa, neste trabalho foram avaliadas a cinética plasmática, representada pela taxa fracional de remoção (TFR), e a captação do 3H-colesterol livre (3H - CL) e 14C - colesterol esterificado (14C - CE) da LDE por segmentos arteriais e por órgãos de coelhos normais (n=17) e coelhos submetidos à indução de aterosclerose por dieta rica em colesterol (1%) (n=13). Além disso, avaliou-se a captação in vitro do 3H CL e do 14C CE da LDE por células endoteliais aórticas de coelhos. Por último, foi avaliada a influência da inibição da enzima lecitina-colesterol aciltransferase (LCAT), e indiretamente, a esterificação do CL em ratos normais (n=9) e tratados com diazepam (n=9). Em coelhos que receberam dieta normal, não houve diferença entre a remoção plasmática do 3H - CL e do 14C - CE. Em coelhos que desenvolveram hiperlipidemia e aterosclerose através de dieta rica em colesterol, o 3H - CL foi removido mais rapidamente da circulação do que o 14C - CE (p<0,05), entretanto houve maior captação de 14C - CE do que de 3H - CL no arco aórtico (p<0,05). Em ambos os grupos, os principais órgãos captadores de colesterol da LDE foram fígado, pulmão, adrenais e baço (p<0,05). Tanto a TRF quanto a captação hepática de 3H - CL e 14C - CE foram menores no grupo que recebeu a dieta rica em colesterol. Em células endoteliais aórticas de coelhos, a captação de 3H - CL foi maior que a de 14C CE independente da massa de LDE incubada (p<0,01). Em ratos, não houve diferença entre a captação das duas formas de colesterol da LDE pela aorta no grupo controle, entretanto, quando a atividade da LCAT foi diminuída pelo tratamento com diazepam, a captação arterial de 3H - CL foi maior do que a de 14C - CE (p< 0,01). A hiperlipidemia e distúrbios no processo de estabilização do colesterol, favorecem a dissociação entre o CL e o CE das lipoproteínas, e podem elevar o risco de desenvolvimento da aterosclerose, assim como agravar o processo de aterogênese. / I n previously studies, it was shown that free cholesterol (FC) and cholesterol ester (CE) of a cholesterol-rich nanoemulsion (LDE) behaves differently in patients with coronary artery disease (CAD). The FC plasma clearance and arterial deposition is greater than CE. In the present study we evaluate the plasma kinetics, estimated by the fractional clearance rate (FCR), and the tissue uptake of 3H-free cholesterol (3H FC) and of 14C cholesterol ester (14C - CE) of LDE by arterial segments and organs of rabbits with (n=13) and without atherosclerosis (n=17). Furthermore, it was evaluated the in vitro uptake of 3H FC and 14C - CE by rabbit aortic endothelial cells. Finally, it was evaluated the inhibition of the enzyme lecithin-cholesterol acyltransferase (LCAT), and indirectly, the FC esterification in rats non-treated (n=9) and treated with diazepam (n=9). In rabbits without atherosclerosis that received an standard diet there was no difference between the plasma clearance of 3H FC and 14C CE. In rabbits with hyperlipidemia and atherosclerosis induced by the cholesterol-rich diet the 3H - FC was removed faster than 14C - CE (p<0.05), however the arch aortic uptake of 14C CE was greater than of 3H - FC (0p<0.05). In both groups, liver, lungs, adrenals and spleen were the principal sites of LDE cholesterol uptake. The FCR and tissue uptake were smaller in rabbits with than those without atherosclerosis. In rabbit aortic endothelial cells the 3H - FC uptake was greater than 14C CE independently of incubated LDE mass (p<0.01). In control rats there was no difference on the arterial uptake of both cholesterol forms of LDE, but when the LCAT activity was diminished by the diazepam treatment, the arterial uptake of 3H FC were greater than 14C CE (p< 0.01). The hyperlipidemia and cholesterol stability alterations may lead to dissociation between lipoproteins FC and CE. This dissociation may increase the risk for atherosclerosis and likewise enhance the severity of atherosclerosis.

Hábitos de vida saudáveis e uma dieta balanceada aliados ao alto consumo de frutas e vegetais, estão associados a redução do risco de doenças e à manutenção da saúde. O óleo de abacate possui em sua composição substâncias bioativas capazes de prevenir e controlar as dislipidemias. Como existem poucas pesquisas científicas avaliando o potencial deste óleo para consumo humano, o presente trabalho estudou os processos de extração e refino do óleo de abacate, bem como suas propriedades funcionais. Os resultados mostraram que os processos de extração e refino do óleo a partir da variedade Margarida são tecnicamente viáveis, o que o torna excelente matéria-prima para a indústria alimentícia. Além disso, possui um perfil de ácidos graxos muito semelhantes ao azeite de oliva, predominando em ambos o ácido oléico, que em conjunto com os esteróis vegetais e a vitamina E presentes, é capaz de influenciar positivamente o controle metabólico do colesterol, prevenindo ou retardando as doenças cardiovasculares. / Healthy life habits and an equilibrate diet, associated with a high fruit and vegetable intake, are joined with the prevention of diseases and health maintenance. The avocado oil has in these composition bioactives substances that can help in the prevention and control of hyperlipidemia. As there are a few scientific researches evaluating the oil potential for human consumption, the present proposal studied the extraction and refining process of avocado oil and its functional properties. The results have been showed that extraction and refining process of Margarida's variety are technically possible, became it an excellent raw material for food industry. Besides, the fatty acid composition of avocado oil are similar to olive oil, predominating in both, the oleic acid, that together of sterols and vitamin E presents, can to influence to metabolic control of cholesterol, preventing or delaying the cardiovascular disease.

La menopausia es el cese permanente de la menstruación, determinado de manera retrospectiva, después de 12 meses consecutivos de amenorrea, sin causas patológicas. Durante la menopausia se observa generalmente una reducción en la producción de estrógenos y un incremento en la secreción de hormonas luteinizante (LH) y folículo estimulante (FSH). Estos cambios hormonales serían responsables de la aparición de los síntomas como bochornos, sudoración (picos elevados de LH), cambios del carácter y humor; así como de síntomas tardíos, entre los que podemos citar las alteraciones del revestimiento de la vagina (que causan sequedad del epitelio, ardor, prurito y dolor durante el coito), osteoporosis, insomnio, depresión, y otros relacionados con la audición, voz y visión. También se modifica el patrón lipídico con incremento del colesterol en sangre y de ahí una mayor tendencia al aumento de la presión sanguínea y un mayor riesgo a accidentes cardiovasculares. / Tesis

Introducción: Los centros comerciales han tenido una expansión rápida en Lima y tienen una oferta predominante de platos de comida rápida. Al no haber alternativas fuerzan a sus usuarios a comer platos con alta densidad calórica, baja disponibilidad de fibra y altos en colesterol. Objetivo. Evaluar la oferta y composición nutricional de las ensaladas ofrecidas como plato principal en los patios de comida de los centros comerciales de Lima. Materiales y Métodos. Se revisó las cartas de comida de todas las franquicias presentes en los patios de comida de los 11 centro comerciales de Lima, luego se evaluó la composición nutricional de las ensaladas ofertadas como plato principal calculando su contenido calórico, proteico, carbohidratos, grasas, colesterol, fibra y sodio y la adecuación de ingesta para una cena (30% de una dieta de 2000 kcal). Resultados. Las ensaladas como plato principal representaron el 4,7% de la oferta, solo 7/17 franquicias ofrecían al menos una ensalada. El costo promedio de las ensaladas fue superior a los otros platos ($5,3 vs 4,7; p<0,001). El contenido calórico promedio fue de 329kcal y de 2,7g de fibra, en relación a una cena se encontró un alto porcentaje de adecuación proteica (172,9%), de colesterol (121,0%), y bajo para calorías (54,8%), carbohidratos (23,1%) y fibra (36,4%). Conclusiones. Las ensaladas en patios de centros comerciales de Lima son escasas y más costosas, presentan poco aporte de fibra y altos contenidos de colesterol. Se deben revisar estrategias para mejorar la accesibilidad de ensaladas de calidad en escenarios donde solo ofertan comidas rápidas. / Introduction. Malls have had a rapid expansion at Lima and have a predominant number of fast food. Because of the few alternatives, costumers are forced to consume high density, low fiber and high cholesterol options. Objectives. Evaluate the availability and nutritional composition of salads offered as main courses in food courts found in malls at Lima. Material and Methods. The menus of all the franchises found in food courts of the 11 malls at Lima were revised. Then, the nutritional composition of the salads offered as a main course was evaluated, calculating its caloric, protein, carbohydrate, fat, cholesterol, fiber and sodium content and the intake adequacy for a dinner (30% of a 2000kcal diet). Results. The salads offered as a main course represented the 4,7% of the overall availability, only 7/17 franchises offered at least one salad. The average cost of a salad was superior to the other courses ($5,3 vs 4,7; p<0,001). The average caloric content was 329kcal and 2,7g of fiber. In relation to a dinner meal, a high protein adequacy was found (172,9%), cholesterol (121,0%), and low in calories (54,8%), carbohydrates (23,1%) and fiber (36,4%). Conclusions. The salads offered in malls at Lima are limited and more expensive; they present low quantities of fiber and high quantities of cholesterol. Strategies, which improve the availability of salads in places where fast food is offered, must be reviewed. / Tesis

Antecedentes y objetivos: Varios estudios han reportado el efecto de las dietas vegetarianas en el síndrome metabólico (SM) y/o sus componentes; Sin embargo, los resultados de estos estudios no son consistentes. El objetivo del presente estudio fue realizar una revisión sistemática y metanálisis de ensayos controlados aleatorios (ECA) y estudios observacionales que evaluaran la asociación entre dietas vegetarianas y SM y/o sus componentes (presión arterial sistólica, presión arterial diastólica, glucosa en ayunas, triglicéridos, circunferencia de cintura, y HDL-colesterol en adultos. Métodos: Se realize la búsqueda en PubMed, Web of Science, EMBASE, Scopus y Cochrane Library. Se utilizaron metanálisis de efectos aleatorios estratificados según el diseño de estudio. Resultados: Un total de 64 estudios (n = 45 345) cumplieron los criterios de inclusión (5 ECAs, 2 cohortes, 57 transversales). Las dietas vegetarianas no se asociaron con SM en comparación con la dieta omnívora (OR 0,96, IC del 95%: 0,50 a 1,85, p = 0,9). Del mismo modo, el metanálisis de los ECAs y los estudios de cohorte indicaron que el consumo de dietas vegetarianas no está asociado a los componentes del SM. Sin embargo, el metanálisis de estudios transversales mostró que las dietas vegetarianas se asociaron con niveles significativamente más bajos de presión arterial sistólica (DM -4,40 mmHg, IC del 95%: -6,16 a -2,63, p <0,00001), presión arterial diastólica (DM -2.99 mmHg, 95 % CI -5,00 a -0,98, p = 0,004), glucosa en ayunas (DM -0,31 mmol / l, IC del 95% -0,43 a -0,20, p <0,00001), y HDL-colesterol (CM -0,05 mmol / l, 95 % CI - 0,08 a -0,03, p = 0,0001) en comparación con la dieta omnívora. La heterogeneidad entre los estudios transversales fue alta y se mantuvo en el análisis por subgrupos realizado. Además, la mitad de los estudios incluidos tienen un alto riesgo de sesgo. Conclusiones: Las dietas vegetarianas están asociadas a un menor riesgo de SM en comparación con las dietas omnívoras. La asociación entre las dietas vegetarianas y los niveles más bajos de presión arterial sistólica, presión arterial diastólica, HDL-colesterol y glucosa en ayunas no es clara, debido a que existe una elevada heterogeneidad y severas limitaciones metodológicas en los estudios transversales. Se necesitan estudios prospectivos y controlados para evaluar la asociación entre las dietas vegetarianas y SM y sus componentes. / Background and aims: Several studies have reported the effect of vegetarian diets on metabolic syndrome (MetS) or its components; however, results are not consistent. The aim of this study was to carry out a systematic review and meta-analysis of randomized controlled trials (RCT’s) and observational studies that evaluate the association between vegetarian diets and MetS or its components (systolic blood pressure [SBP], diastolic blood pressure [DBP], fasting glucose, triglycerides, waist circumference [WC], HDL-cholesterol (HDL-C) in adults. Methods: PubMed, Web of Science, EMBASE, Scopus and the Cochrane Library were searched. Random effects meta-analyses stratified by study design were used. Results: A total of 64 studies (n=45 345) met the inclusion criteria (5 RCTs, 2 cohorts, 57 cross- sectional). Vegetarian diets were not associated with MetS when compared with omnivore diet (OR 0.96, 95%CI 0.50 to 1.85, p=0.9). Likewise, meta-analysis of RCTs and cohort studies indicated that consumption of vegetarian diets were not associated with MetS components. However, meta-analysis of cross-sectional studies showed that vegetarian diets were associated with significantly lower levels of SBP (MD -4.40 mmHg, 95%CI –6.16 to -2.63, p<0.00001), DBP (MD -2.99 mmHg, 95%CI –5.00 to -0.98, p=0.004), fasting glucose (MD -0.31 mmol/l, 95%CI –0.43 to -0.20, p<0.00001), and HDL-C (MD -0.05 mmol/l, 95%CI –0.08 to -0.03, p=0.0001) in comparison to omnivore diet. Heterogeneity of effects among cross-sectionals studies was high, and was maintained in most of the subgroup analysis performed. Furthermore, half of the included studies have high risk of bias. Conclusions: Vegetarian diets were not associated with lower risk of MetS in comparison to omnivore diets. The association between vegetarian diets and lower levels of SBP, DBP, HDL-C, and fasting glucose is unclear due to high heterogeneity and severe methodological limitations found in cross-sectional studies. Future prospective and controlled studies are needed to evaluate the association between vegetarian diets and MetS and its components. / Tesis

Evalúa el efecto hipocolesterolémico del extracto alcohólico de hojas de Desmodium molliculum (HBK) DC “manayupa” en ratas con dieta hipercolesterolémica. También se evaluó la presencia de metabolitos que puedan justificar la actividad biológica de los extractos alcohólicos de hojas de D. molliculum. Se colectó hojas de esta especie en el distrito de Baños de Inca, departamento de Cajamarca, se secó, se molió y se maceró con alcohol. Se trabajó con 22 ratas que se dividió en 4 grupos, un grupo control con alimento balanceado, y 3 grupos al que se alimentó con dieta hipercolesterolemica durante un mes con colesterol suspendido en aceite de oliva a una dosis de 200 mg/kg peso corporal. El tratamiento consistió en administrar a un grupo extracto etanólico seco suspendido en CMC al 0,5%, a la dosis de 500 mg /kg peso corporal, y otro grupo se trató con droga estándar atorvastatina a la dosis 2 mg/kg peso corporal, al grupo control negativo se le administró suero fisiológico y al grupo control positivo se le siguió administrando colesterol 200 mg/kg. El tratamiento duró 30 días. La evaluación química nos permitió confirmar la presencia de flavonoides, isoflavonas y saponinas en el extracto alcohólico. Los resultados del ensayo biológico mostraron que el extracto alcohólico de hojas de D. molliculum no posee una actividad hipocolesterolémica significativa a la dosis ensayada (p = 0,258). / Tesis

Dentro de la superfamilia de los canales iónicos activados por ligando (LGIC) se encuentra la familia de los receptores Cys-loop que incluye a canales catiónicos, como los receptores nicotínicos de acetilcolina (nAChRs) y los receptores 5-HT3 de serotonina; y canales aniónicos, como los activados por GABA (GABAA) y glicina. Estos receptores revisten importancia tanto en la fisiología normal como así también en diversas y muy variadas patologías, entre las que se pueden mencionar las enfermedades neurodegenerativas como la enfermedad de Alzheimer (EA). Este hecho los posiciona como blancos moleculares importantes para el desarrollo de nuevos fármacos, y es por esto que el conocimiento de su función y modulación resulta un aspecto clave. Los receptores Cys-loop son proteínas transmembrana, por lo que su función se encuentra condicionada por su entorno lipídico y estímulos propios de la membrana celular, además de por distintos estímulos o factores externos a ella. Están formados por 5 subunidades organizadas pseudo-simétricamente alrededor de un poro o canal central. En estos receptores pueden distinguirse un dominio extracelular (ECD, del inglés extracellular domain), donde se encuentran los sitios de unión al agonista y/o antagonistas, un dominio transmembrana (TMD, del inglés transmembrane domain), que contiene el canal iónico y que se encuentra en extenso contacto con los lípidos de la membrana, y un dominio pequeño intracelular (ICD, del inglés intracellular domain), que contiene distintos sitios de regulación y señalización intracelular. En esta Tesis Doctoral se profundizó en el estudio de la función y modulación de 2 receptores de neurotransmisores pertenecientes a los receptores Cys-loop: los nAChRs, muscular y α7 neuronal, y los receptores 5-HT3. En el capítulo I se estudió la modulación del nAChR muscular por el entorno lipídico de la membrana en la que se encuentra. Se hizo foco especialmente en el colesterol (Col), lípido clave para el correcto funcionamiento del nAChR y que presenta distintas asimetrías laterales y transmembrana en diferentes momentos de la vida y en condiciones de salud o enfermedad. Se trabajó con distintos sistemas modelo: vesículas unilamelares grandes (LUVs), vesículas unilamelares gigantes (GUVs), membranas ricas en nAChR de T. californica o células BOCSC 23 expresando heterologamente el nAChR muscular. A cada uno se le realizaron distintos tratamientos para modificar las condiciones de Col con el fin de obtener sistemas que presenten: i) un aumento del contenido de Col, ii) un aumento de esteroles en la hemicapa externa (por agregado de colesterol hemisuccinato (CHEMS)), o iii) transformación de parte del Col a colestenona (por la enzima colesterol oxidasa (Cox)). Mediante estudios de fluorescencia utilizando las sondas DPH, TMA-DPH y Laurdan se determinó que el orden de membrana aumentó cuando se incorporó CHEMS. Mediante estudios de espectrofotometría utilizando la sonda cristal violeta (CrV) y la técnica de patch-clamp se detectaron, respectivamente, modificaciones conformacionales en el nAChR y cambios en la activación del mismo cuando los diferentes sistemas habían sido incubados con CHEMS o Cox, no así con Col. Tratando LUVs con Tritón, y posterior centrifugación y separación por SDS-PAGE, se determinó que luego de la incorporación de Col o CHEMS al sistema, el nAChR se localizó mayoritariamente en fracciones ordenadas, mientras que cuando se produjo la transformación del Col a colestenona el receptor se localizó principalmente en fracciones desordenadas. Por último, estudios de microscopía confocal utilizando GUVs mostraron que alteraciones en el Col causaron cambios significativos en la presencia y distribución de dominios ordenados y desordenados; y que la presencia de un componente proteico en el sistema, como es un péptido representativo del segmento transmembrana yTM4 del nAChR, alteró las características de la membrana, llevando a todo el sistema a un mayor orden. De acuerdo con estos resultados, distintas modificaciones del Col en la membrana llevan a cambios de su orden y asimetría, y al mismo tiempo a cambios en la localización del nAChR en un determinado entorno, hecho que repercute directamente en su funcionalidad. Su localización en dominios ordenados ocasiona una disminución de su actividad, mientras que su presencia en dominios más desordenados lleva a un aumento de su función. De esta manera, la localización del nAChR en uno u otro dominio podría explicar los cambios en la señal colinérgica reportados en situaciones de envejecimiento y/o patologías como la EA. Existen numerosas evidencias de interacciones funcionales entre los péptidos β-amiloide (Aβ), involucrados en la enfermedad de Alzheimer, y el nAChR α7. En el capítulo II se estudió la modulación del nAChR α7 neuronal por oligómeros de Aβ1-40 y Aβ1-42. Se ha postulado que la acumulación excesiva de estos péptidos en el cerebro provoca la formación de placas seniles, directamente relacionadas con el proceso de neurodegeneración e inflamación característico de esta enfermedad. Sin embargo, evidencias más recientes sugieren que las especies más neurotóxicas serían las formas oligoméricas de Aβ. Mediante estudios de fluorescencia utilizando la sonda CrV se demostró que los oligómeros de Aβ provocan cambios conformacionales en el receptor α7. Utilizando la técnica de patch clamp se determinó que dichos oligómeros son capaces de activar al receptor α7 en muy bajas concentraciones y que, a concentraciones más altas, disminuyen la potenciación de α7 por moduladores alostéricos positivos (PAMs, positive allosteric modulators). Estos resultados muestran un rol dual de los oligómeros de Aβ dependiente de su concentración, como agonistas y como moduladores negativos de α7. El efecto agonista, potenciador de la señal colinérgica, podría tener un rol importante en la fisiología normal del individuo, mientras que el efecto inhibitorio, dado por su exacerbada producción, podría contribuir a la deficiencia en la memoria y cognición asociada a la EA. Teniendo en cuenta las características multifactoriales de la EA, la estrategia de búsqueda de nuevas moléculas apunta al descubrimiento y desarrollo de “multitarget drugs”, es decir de moléculas activas en varios blancos moleculares, por lo que el capítulo III se centró en el descubrimiento y optimización de nuevas moléculas bifuncionales que sean capaces de activar al nAChR e inhibir a la acetilcolinesterasa (ACE). Partiendo de un screening de extractos obtenidos de distintas plantas medicinales, mediante técnicas cromatográficas y de resonancia magnética nuclear se identificó a la molécula de cafeína como una molécula sumamente interesante para la EA. Además de confirmar su ya conocida actividad inhibidora de la ACE, utilizando técnicas de espectrofotometría con la sonda fluorescente CrV y de patch-clamp, en este trabajo se postula su actividad agonista sobre los nAChRs muscular y α7 neuronal. Caracterizada la actividad de la cafeína sobre el sistema colinérgico, se obtuvieron luego 5 análogos sintéticos de esta molécula mediante diseño racional. Se determinó que todos los análogos presentan actividad sobre ambos blancos moleculares, la ACE y los nAChRs, y cuya potencia es mayor a la de la cafeína. Mediante estudios de modelado molecular y docking se identificaron los sitios de inhibición de los análogos de la cafeína en la ACE y de interacción con el nAChR permitiendo postular a estos compuestos como agonistas parciales. Estos hallazgos resultan prometedores en la búsqueda de nuevos fármacos como una estrategia terapéutica para la EA. El otro receptor estudiado en este trabajo de Tesis fue el receptor 5-HT3, uno de los menos conocidos de la familia de los Cys-loop, el cual a nivel presináptico participa de la finalización de la señal nerviosa, tornándose un blanco molecular novedoso tanto en la EA como en otras patologías neurodegenerativas que requieran una potenciación de dicha señal. En el capítulo IV, utilizando el receptor de alta conductancia 5-HT3AHC, se descifraron por primera vez las diferencias entre la activación ortostérica del mismo y su activación alostérica por timol y carvacrol. Mediante la técnica de patch clamp, combinando registros de corrientes macroscópicas en la configuración whole-cell y de canal único en la configuración cell-attached, se caracterizó en primer lugar la activación del receptor humano 5-HT3AHC por los agonistas ortostericos 5-HT y triptamina, y luego la activación alostérica mediada por 2 terpenoides, carvacrol y timol. Se confirmó que tanto el carvacrol como el timol se comportan macroscópicamente como agonistas menos eficaces que 5-HT. Sin embargo, a nivel de canal único, estos 2 terpenoides activan a los receptores 5-HT3A con mayor eficacia que 5-HT. Más aún, se determinó que además de actuar como agonistas, el carvacrol y el timol actúan como modulares alostéricos positivos del receptor 5-HT3AHC humano, y que el efecto potenciador es evidente a concentraciones menores que el efecto agonista. De esta manera, los resultados de este capítulo permitieron definir las bases mecanísticas que subyacen a las diferencias entre la activación y potenciación alostérica u ortostérica del receptor humano 5-HT3A. En resumen, en este trabajo de Tesis se profundizó en el conocimiento de distintas formas de modulación de los nAChRs y del receptor 5-HT3, ya sea por moléculas exógenas o por el entorno lipídico en el que se encuentran, brindando bases moleculares para el entendimiento de su función, su modulación y su afección en determinadas patologías como la EA. En este trabajo se lograron avances significativos, con resultados de gran relevancia, ya sea para el estudio o tratamiento de esta enfermedad. / The Cys-loop receptor family, which belongs to the Ligand-gated ion channel (LGIC) superfamily, includes cation channels, such as nicotinic acetylcholine receptors (nAChRs) and serotonin 5-HT3 receptors, and anion channels, such as those activated by GABA (GABAA) and glycine. These receptors are important in normal physiology and in various pathologies, among which neurodegenerative diseases, such as Alzheimer's disease (AD), are included. They are therefore important molecular targets for the development of new drugs, this being the reason why the knowledge of their function is indeed a very interesting aspect worth exploring. Cys-loop receptors are integral membrane proteins whose function is conditioned by their lipid environment as well as by different extrinsic factors. These receptors are formed by five subunits organized pseudo-symmetrically around a central pore. Three different domains can be distinguished: an extracellular domain (ECD), where the agonist and/or antagonist binding sites are localized, a transmembrane domain (TMD), which contains the ion pore and is in extensive contact with membrane lipids, and a small intracellular domain (ICD), which is important for the conductance of the channel and modulation. In this Doctoral Thesis, the function and modulation of two neurotransmitter receptors that belong to the Cys-loop receptor family, nAChRs and 5-HT3 receptors, were explored by using a large variety of fluorescence, confocal microscopy and electrophysiological approaches. In Chapter I, modulation of the muscle nAChR by its lipid environment was studied. Special attention was put on cholesterol (Chol), which displays different lateral and transmembrane asymmetries all throughout life as well as under health and disease conditions. This lipid is crucial for the proper functioning of the nAChR. Different model systems were used: large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), T. californica nAChR rich membranes and BOSC 23 cells heterologously expressing the muscle nAChR. The experimental systems were subjected to different treatments to modify their Chol conditions resulting in: i) an increment in Chol content, ii) an increment in sterols in the outer layer (after addition of cholesteryl hemisuccinate (CHEMS)), and iii) conversion of part of the Chol to cholestenone (through the enzyme Cholesterol Oxidase (ChOx)). Through fluorescence studies using DPH, TMA-DPH and Laurdan probes it was determined that the membrane order increased when CHEMS was incorporated. By using the fluorescent probe Crystal Violet (CrV) and patch-clamp recordings, conformational changes of the nAChR associated to changes in its activation properties were detected after incubation with either CHEMS or ChOx, but not with Chol. By treating LUVs with Triton, and subsequent centrifugation and separation by SDS-PAGE, it was determined that after incorporation of Chol or CHEMS to the system the nAChR was located mostly in ordered fractions, while after the conversion of Chol to cholestenone the receptor was found in disordered fractions. Confocal microscopy studies using GUVs showed that Chol alterations caused significant changes in the presence and distribution of ordered and disordered domains, and that the mere presence of a protein component in the system, specifically a peptide corresponding to the forth transmembrane segment of the nAChR (M4), led to a higher order of the entire system. According to our results, we conclude that modifications of membrane Chol lead to changes in membrane order and asymmetry, and, at the same time, in the location of the nAChR, a fact that would directly affect its functionality. The location of the nAChR in ordered domains causes a decrease in its activity whereas its location in more disordered domains enhances its function. In this way, the location of the nAChR in one or another domain fine-tunes its function and could explain the changes in the cholinergic signal reported in situations of aging and/or pathologies, such as AD. There is increasing evidence of functional interactions between β-Amyloid (Aβ) peptides, involved in Alzheimer's disease, and α7 nAChR. In Chapter II, modulation of α7 nAChR by Aβ1-40 and Aβ1-42 oligomers was studied. It has been postulated that the accumulation of these peptides in the brain causes the formation of senile plaques, which are directly related to the neurodegeneration and inflammation observed in AD. However, more recent evidence suggests that the most neurotoxic species are the oligomeric forms of Aβ. Fluorescence studies using the CrV probe demonstrated that Aβ oligomers cause conformational changes in the α7 receptor, which were dependent on the concentration. Accordingly, single channel recordings revealed that whereas at very low concentrations (pM to nM range) these oligomers are capable of activating the α7 receptor, at higher concentrations, which are compatible with those found in AD patients, they decrease the enhancement of α7 by positive allosteric modulators (PAMs). These results show a dual role of Aβ oligomers depending on their concentration, as agonists and as negative modulators of α7. The agonist effect, which enhances the cholinergic signal, could have an important role in normal physiology, while the inhibitory effect, given by the exacerbated production of Aβ, could contribute to the failure in memory and cognition associated with AD. Taking into account that Alzheimer’s is a multifactorial disease, the search for new molecules aims at discovering and developing “multitarget drugs”. Chapter III is focused on the discovery and optimization of new bifunctional molecules capable of activating the nAChR and inhibiting the AChE. Starting from a screening of extracts obtained from different medicinal plants and using chromatographic techniques and nuclear magnetic resonance, the caffeine molecule was identified as a highly interesting molecule for AD. In addition to its already known AChE inhibitory activity, using CrV fluorescence probe and patch-clamp techniques we postulated its agonist activity in the muscle and α7 nAChRs. After having characterized the activity of caffeine in the cholinergic system, five synthetic analogs of this molecule were obtained by rational design. It was determined that all the analogs have activity in both molecular targets, the AChE and the nAChRs, and that they have a greater potency than caffeine. Through docking studies, the sites of inhibition of caffeine analogs in the AChE and the sites of interaction with the nAChRs were proposed. These compounds are promising as new drugs for a therapeutic strategy for AD. The other receptor explored in this Thesis was the 5-HT3 receptor, one of the least known of the Cys-loop family, which has been recently proposed as a novel molecular target both in AD and in other neurodegenerative pathologies. In Chapter IV, by taking advantage of the high-conductance receptor 5-HT3AHC, differences between orthosteric and allosteric activation of the human 5-HT3A receptor were deciphered for the first time. By using the patch clamp technique, and combining macroscopic current recordings in the whole-cell configuration and single channel recordings in the cell-attached configuration, the activation of the human 5-HT3AHC receptor by the orthosteric agonists, 5-HT and tryptamine, and the allosteric activation, by carvacrol and thymol, were described. Both carvacrol and thymol were less effective agonists than 5-HT at the macroscopic level. However, at the single channel level, these two terpenoids activated 5-HT3A receptors more efficiently than 5-HT. Furthermore, it was demonstrated that in addition to acting as agonists, carvacrol and thymol behave as positive allosteric modulators of the human 5-HT3AHC receptor at low concentrations. In this way, these results made it possible to define the mechanistic bases underlying the differences between orthosteric and allosteric activation and potentiation of the human 5-HT3A receptor. Summing up, in this Doctoral Thesis we deepened into the knowledge of modulation of the nAChRs and the 5-HT3 receptor, either by extrinsic molecules or by the lipid environment in which they are located. Our findings provide molecular bases for the understanding of the function and modulation of these receptors and their implication in certain pathologies, such as AD. In this work, significant advances with highly relevant results were made for the study or treatment of this disease.