Дисертації з теми "Grape seed extract (GSE)"

Dietary polyphenols have been positively correlated to the reduced risk of several chronic diseases, including Alzheimer' disease (AD). Grape (Vitis vinifera) seed contains 5-8 % polyphenols and annually, 2.5 million tonnes of grape seed is generated in the juice and wine industry. Grape seed extract (GSE) and its compounds gallic acid (GA), resveratrol (RSV) and epigallocatechin gallate (EGCG) have been shown to attenuate AD aetiological features including oxidative stress, protein aggregation, and mitochondrial dysfunction in cell and animal models. The main objectives of this work were to optimize the methods for grape seed polyphenol extraction and assess their antioxidant properties. Polyphenols from grape seeds were extracted using ethanol, methanol, and acetone as solvents. Total phenolic content (TPC), and total flavonoid content (TFC) of GSEs were determined by Folin-Ciocalteu’s and AlCl3 colorimetric methods. Free radical scavenging activities were measured using standard antioxidant assays (DPPH and ABTS) and levels of GA, EGCG, and RSV were determined by high performance liquid chromatography (HPLC). Findings from this study showed the highest TFC levels in acetone extraction that was consistent with previous studies. Ethanol showed improved extraction of phenolic compounds compared to acetone and better than methanol in overall polyphenol extraction. Ethanol extracted GSEs exhibited high free radical scavenging capacity measured using ABTS and DPPH demonstrating its potent antioxidant activity. Ethanol extracted GSEs displayed the highest ABTS scavenging ability as compared to acetone and methanol extractions. Ethanol extracted GSEs showed high free polyphenols content but low level of bound polyphenols. The TFC, TPC and radical scavenging properties of the free polyphenol fraction was significantly higher than those of the bound polyphenol fraction from GSE. Thus, potential therapeutic effects of GSE may be attributed to the free polyphenol fraction. GA and EGCG were detected in both unbound and bound phenolic extracts. However, RSV was only detected in bound polyphenol extract. Overall, the findings presented here have unravelled new insights into the polyphenol content of GSEs, solubility, efficiency of different extraction conditions and more importantly generated new knowledge that will be critical for developing industrial processes for developing GSE as a commercial food product for alleviating AD.

La sclérose en plaques (SEP) est une maladie auto-immune du système nerveux central générant de nombreux symptômes neurologiques, parmi lesquels la douleur chronique qui est très invalidante et fréquente. A ce jour, la SEP est une maladie incurable avec une étiologie complexe, multifactorielle et encore mal comprise. De nombreuses données suggèrent que les polyphénols végétaux pourraient avoir des bénéfices thérapeutiques en régulant le stress oxydant et la neuroprotection dans la SEP. Dans ce contexte, ce travail de thèse a pour objectif d’évaluer l'effet d’un traitement curatif chronique à l'extrait de pépins de raisin (GSE pour Grape Seed Extract) dans un modèle murin reproduisant certaines des caractéristiques cliniques et neuropathologiques de la SEP, l'encéphalomyélite auto-immune expérimentale (EAE). Dans un premier temps, la composition biochimique du GSE a été évaluée. Par la suite, le traitement des souris EAE par le GSE 10 jours après l’induction du modèle (J10) a montré une amélioration à la fois du score neurologique et des troubles sensitifs chez les souris. Des analyses biochimiques et moléculaires au niveau du cerveau et de la moelle épinière ont montré dès J20 une correction des anomalies du stress oxydant permettant une restauration des altérations de la myéline, de la prolifération astrogliale et microgliale et des niveaux d’expression des sirtuines. Enfin, une analyse protéomique a permis de confirmer ces résultats et d’envisager des mécanismes d’action bénéfiques supplémentaires du GSE, notamment la correction de la dégradation des lipides. L’ensemble des effets du GSE décrits au cours de cette thèse soutient fortement l'idée que le GSE pourrait être une approche thérapeutique efficace pour le traitement de la SEP
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system leading to many neurological symptoms, among which chronic pain is common and very disabling. To date, MS is an incurable disease with a complex, multifactorial and still poorly understood etiology. Numerous evidence suggest that plant polyphenols may have therapeutic benefits in regulating oxidative stress and providing neuroprotection in MS. In this context, this thesis work aimed to evaluate the effect of a chronic curative treatment with grape seed extract (GSE) in a mouse model reproducing some of clinical and neuropathological features of MS, the experimental autoimmune encephalomyelitis (EAE).First, the biochemical composition of GSE was evaluated. Subsequently, the treatment with GSE initiated from day 10 post-induction (D10) showed both an improvement in the neurological score and sensory disorders in mice. Biochemical and molecular analyzes in the brain and spinal cord showed from D20 a correction of oxidative stress abnormalities allowing restoration of myelin alterations, astroglial and microglial proliferation and levels of sirtuins expression. Finally, a proteomic analysis allowed to confirm these results and to identify new additional beneficial effect of GSE, such as the correction of lipid degradation. All the effects of GSE described during this thesis strongly supports the idea that GSE could be an effective therapeutic approach for the treatment of MS

Grape seed extract (GSE) and peanut skin extract (PSE) are waste products in the wine and peanut industries. Both extracts have high concentrations of polyphenols, known to possess antioxidant and antimicrobial properties. A subcategory of polyphenol is procyanidin, which can be divided into two types, type A and type B. Type A (PSE), contains two single bonds connecting the phenolic groups while type B (GSE), contains one single bond connecting the phenolic groups. The minimum inhibitory concentration (MIC) of the two extracts was evaluated for their antimicrobial effect on Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium using the pour plate method. GSE was found to have a significantly lower MIC (p ≤ 0.05) than PSE for L. monocytogenes (GSE=60.60ppm, PSE=not found), S. aureus (GSE=38.63ppm, PSE=51.36ppm), and S. Typhimurium (GSE=45.73ppm, PSE=60.60ppm). There was no significant difference in inhibition of E. coli O157:H7 (GSE=47.44ppm, PSE=51.13ppm). Since GSE, contributed to greater pathogen inhibition, its extract was fractionated into monomer and oligomers components. Growth curves of all four pathogens inoculated in the monomer and oligomer fractions were compared using the BioScreen method. Oligomers inhibited growth of L. monocytogenes, S. aureus, and E. coli O157:H7 while monomers inhibited growth of S. Typhimurium. These results indicate that an extract with type B procyanidins that are high in oligomers may be more effective as antimicrobials. Type B procyanidins have also been shown to prevent bacterial adhesion, as is the case with urinary tract infections, and may aid in the prevention of biofilms.
Master of Science in Life Sciences

Donats els problemes de salut associats al sobrepès, en aquesta tesi hem investigat el possible us d'un extracte de proantocianidines de pinyol de raïm (GSPE) com agent saciant, fent servir rates com a model d'experimentació. Hem observat que sota una pauta d'administració adequada, el GSPE disminueix la ingesta tant de manera aguda com de manera continuada, durant períodes de 8 dies consecutius. Aquestes propietats saciants, sumades a un efecte lipolític, resulten en un descens significatiu del pes corporal. A l'investigar les vies de senyalització implicades, hem observat que l'administració de GSPE modifica la producció i secreció de diverses hormones gastrointestinals que afecten l'apetit, entre les que destaquen el GLP-1, d'efectes saciants, i la ghrelina, inductora de l'apetit. En estudis amb antagonistes hem observat que de manera aguda l'administració de GSPE augmenta la concentració plasmàtica de GLP-1, i que l'efecte saciant del GSPE i d'un dels seus compostos, l'àcid gàlic, són directament mediats pel receptor de GLP-1. En estudis de 8 dies consecutius hem observat que els efectes saciants de l'àcid gàlic no es mantenen al llarg del temps, reforçant la importància d'altres compostos de l'extracte per a mantenir un efecte continuat. En aquests estudis subcrònics, l'administració de GSPE comporta un gran descens en la síntesi de ghrelina, un fet que hem observat estretament relacionat amb l'increment de senyalització de GLP-1 a l'hipotàlem, la inducció de la sacietat i l'efecte lipolític del GSPE. Esperem aquests estudis permetin iniciar estudis per a l'aplicació del GSPE en humans.
Dados los problemas de salud asociados al sobrepeso, en esta tesis hemos investigado el posible uso de un extracto de proantocianidinas de pepita de uva (GSPE) como agente saciante, utilizando ratas como modelo de experimentación. Hemos observado que bajo una pauta de administración adecuada, el GSPE disminuye la ingesta tanto de manera aguda como de forma continuada, durante períodos de 8 días consecutivos. Estas propiedades saciantes, sumadas a un efecto lipolítico, resultan en un descenso significativo del peso corporal. Al investigar las vías de señalización implicadas, hemos observado que la administración de GSPE modifica la producción y secreción de varias hormonas gastrointestinales que afectan el apetito, entre las que destacan el GLP-1, de efectos saciantes, y la ghrelina , inductora del apetito. En estudios con antagonistas hemos observado que de manera aguda la administración de GSPE aumenta la concentración plasmática de GLP-1, y que el efecto saciante del GSPE y de uno de sus compuestos, el ácido gálico, son directamente mediados por el receptor de GLP-1. En estudios de 8 días consecutivos hemos observado que los efectos saciantes del ácido gálico no se mantienen a lo largo del tiempo, reforzando la importancia de otros compuestos del extracto para mantener un efecto continuado. En estos estudios subcrónicos, la administración de GSPE conlleva un gran descenso en la síntesis de ghrelina, un hecho que hemos observado estrechamente relacionado con el incremento de señalización de GLP-1 en el hipotálamo, la inducción de la saciedad y el efecto lipolítico del GSPE. Esperamos estos estudios permitan iniciar estudios para la aplicación del GSPE en humanos.
Given the health problems associated with overweight, in this thesis we have investigated the possible use of a grape seed proanthocyanidin extract (GSPE) as a satiating agent, using rats as an experimental model. We have observed that under an adequate administration pattern, GSPE decreases intake both acutely and continuously along periods of 8 consecutive days. These satiating properties, added to a lipolytic effect, result in a significant decrease in body weight. In investigating the signaling pathways involved, we have observed that the administration of GSPE modifies the production and secretion of several gastrointestinal hormones that affect appetite, including GLP-1, with satiating effects, and the appetite-inducing hormone ghrelin. In studies with antagonists we have observed that the administration of GSPE increases the plasma concentration of GLP-1 and that the satiating effect of GSPE and one of its compounds, gallic acid, is directly mediated by the GLP-1 receptor. In studies of 8 consecutive days we have observed that the satiating effects of gallic acid are not maintained over time, reinforcing the importance of other compounds in the extract to maintain a continued effect. In these subchronic studies, GSPE administration leads to a large decrease in ghrelin synthesis, a fact that we have observed closely related to the increase in GLP-1 signaling in the hypothalamus, the satiety induction and the lipolytic effect of GSPE . We hope these studies will allow translational studies for the application of GSPE in humans.

Las procianidinas son compuestos bioactivos presentes en frutas y vegetales. Aunque se conocen los efectos beneficiosos de estos compuestos en la homeostasis de la glucosa, su acción en la funcionalidad de la célula β no es clara. La presente tesis doctoral se ha centrado en describir los efectos de las procianidinas en la síntesis y secreción de insulina. Nuestros resultados muestran la capacidad de las procianidinas de modificar la funcionalidad de la célula β aumentando la relación insulina plasmática/mRNA, aunque la efectividad del tratamiento depende de la situación fisiológica. En situaciones no patológicas, las procianidinas afectan la insulinemia modificando la síntesis, secreción y/o degradación de la insulina. En situaciones de resistencia a la insulina, el tratamiento crónico con procianidinas disminuye la síntesis y secreción de insulina gracias a su acción limitando el acúmulo de lípidos. En cambio, en un modelo más dañado (obesidad genética), las procianidinas ejercen efectos similares pero no son capaces de mejorar la hipersinulinemia. En conclusión, las procianidinas, en las dosis ensayadas, pueden utilizarse únicamente como compuestos bioactivos limitando la disfuncionalidad de la célula β en sus estados iniciales.
Les procianidines són compostos bioactius presents en fruites i vegetals. Tot i que es coneixen els efectes beneficiosos d’aquests compostos en l’homeòstasi de la glucosa, la seva acció en la funcionalitat de la cèl•lulaβ no és clara. La present tesi doctoral s’ha centrat en descriureels efectes de les procianidines en la síntesi i secreció d’insulina. Els nostres resultats mostren la capacitat de les procianidines de modificar la funcionalitat de la cèl•lula β augmentant la relació insulina plasmàtica/mRNA, tot i que l’efectivitat del tractamentdepèn de la situaciófisiològica. En situacions no patològiques, les procianidines afecten la insulinèmia modificant la síntesi, secreciói/o degradació d’insulina. En situacions de resistència a la insulina, el tractamentcrònicamb procianidines disminueix la síntesi i secreció d’insulina gràcies a la seva acció limitant l’acumulació de lípids. En canvi, en un model més danyat (obesitat genètica), les procianidines exerceixen efectes similars però no son capaces de millorar la hiperinsulinèmia. En conclusió, les procianidines, en les dosis assajades, podenutilitzar-seúnicament coma compostos bioactiuslimitant la disfuncionalitat de la cèl•lula β en els seus estats inicials.
Procyanidins are bioactive compounds found in fruits and vegetables widely consumed. It has been reported that procyanidins show some beneficial effects on glucose homeostasis, although their effects on β-cell functionality remain unresolved. This doctoral thesis is focus on describing the effects of procyanidins on insulin synthesis and secretion. Our results showed that procyanidins modify β-cell functionality through increasing the plasma insulin/mRNA ratio, although the effectiveness of the treatment depends on the physiological situation. Under non-pathological situation, procyanidins affected insulinaemia by modifying insulin synthesis, secretion and/or degradation activity. Under insulin-resistance situation, chronic procyanidins administration decreased insulin synthesis and secretion, thanks to its lipid-lowering effect. Otherwise in a more damaged model, Zucker fatty rat, procyanidins treatment is not able to reduce insulin plasma levels although they repress insulin expression. In conclusion, procyanidins could be used as bioactive compound to limit β-cell dysfunctions under high-palatable diets, but at the assayed doses, it is not enough to counteract a strong metabolic disruption.

Procyanidins have been reported to modulate glucose homeostasis and β-cell functionality. However, their effects on pancreatic cell mass remain unknown. Therefore, this doctoral thesis focused on the study of the effects of a grape seed procyanidin extract (GSPE) on proliferation and apoptosis processes in pancreatic cells. Our results indicate that although the extract did not affect these processes under healthy conditions, it modulated β-cell proliferation and apoptosis under altered conditions. In vitro, GSPE enhanced the pro-apoptotic effects of high glucose and were antiproliferative under high glucose, insulin, and palmitate levels in the INS-1E cell line. In vivo, the effects of the extract depended on the dose, the duration of the treatment, and/or the gender. However, the extract tended to counteract the deleterious effects of the cafeteria diet or the Zucker Fatty genotype. Concerning the adenocarcinoma cell line MIA PaCa-2, GSPE exerted antiproliferative and pro-apoptotic effects, demonstrating anti-carcinogenic activity.
Les procianidines són compostos fenòlics abundants en plantes i vegetals. S’ha demostrat que aquests compostos bioactius tenen efectes beneficiosos per la salut, entre els quals destaquen les seves propietats antiinflamatòries i antioxidants. També s’ha vist que participen en l’homeòstasi dels lípids i la glucosa. En un estudi previ realitzat en el grup de recerca, es van avaluar els efectes d’un extracte de procianidines de pinyol de raïm (GSPE) en un model de resistència a la insulina induït per l’alimentació de rates femelles amb una dieta de cafeteria. Es va veure que el GSPE reduïa l’índex HOMA-IR i els nivells d’insulina plasmàtica, suggerint una millora de la resistència a la insulina en teixits perifèrics. A més a més, aquests resultats semblaven indicar que les procianidines podrien estar afectant el pàncrees, el principal òrgan responsable de l’homeòstasi dels nutrients, ja sigui millorant la funcionalitat o la massa de les cèl•lules β pancreàtiques. De fet, en una tesi doctoral duta a terme en paral•lel amb una altra, en la qual es va concloure que les procianidines actuen en el pàncrees modulant la síntesi, secreció i degradació de la insulina. Els individus amb diabetis del tipus 2 presenten hiperglucèmia i un metabolisme lipídic alterat, juntament amb resistència a la insulina, disfunció de les cèl•lules β i disminució de la massa β. Tot i que determinats factors genètics hi estan implicats, la diabetis del tipus 2 està estretament lligada a l’obesitat, i ambdós patologies estan assolint proporcions d’epidèmia a nivell mundial. En els primers estadis de la resistència a la insulina, la massa β s’incrementa per compensar la hiperglucèmia. Tot i així, quan les cèl•lules β ja no són capaces de compensar l’augment de la demanda d’insulina, la massa β es veu reduïda degut a un augment de l’apoptosi. A més a més, considerant el pàncrees, l’adenocarcinoma pancreàtic és un dels càncers més agressius, caracteritzat per una elevada resistència al tractament. L’acumulació d’alteracions genètiques resulta en un augment del creixement cel•lular i de la proliferació i en una inhibició de l’apoptosi. D’aquesta manera, l’obtenció d’informació sobre els compostos naturals amb efectes beneficiosos sobre la proliferació i l’apoptosi en les cèl•lules pancreàtiques, processos estretament lligats i alterats en les malalties mencionades anteriorment, és de gran interès. Els efectes de les procianidines sobre la proliferació i l’apoptosi han estat molt estudiats en diferents tipus cel•lulars. En línies cel•lulars de càncer les procianidines baixen els nivells de proliferació i incrementen l’apoptosi, actuant com a anticarcinogèniques. En altres tipus cel•lulars, les procianidines actuen com a eina terapèutica, protegint les cèl•lules del dany induït per factors ambientals o químics, disminuint l’apoptosi i estimulant el creixement cel•lular. Tot i així, existeix poca informació relativa als efectes de les procianidines en el pàncrees. Per tant, aquesta tesi doctoral es va centrar en l’estudi dels efectes de les procianidines sobre la proliferació i l’apoptosi de les cèl•lules pancreàtiques, avaluant la modulació d’aquests processos en situacions fisiològiques o patològiques. Per assolir els nostres objectius, vam utilitzar models in vivo de rates sanes, de rates amb obesitat induïda per la dieta i rates amb obesitat induïda genèticament; i models in vitro, usant la línia cel•lular d’insulinoma de rata INS-1E i d’adenocarcinoma de pàncrees MIA PaCa-2. La hiperglucèmia postprandial i la dislipèmia són factors comuns que tenen lloc prèviament al desenvolupament de la diabetis del tipus 2. L’exposició crònica a un ambient hiperglucèmic i a elevades concentracions d’àcids grassos causa la disfunció de les cèl•lules β pancreàtiques i la mort cel•lular, fenòmens anomenats glucotoxicitat i lipotoxicitat, respectivament. D’aquesta manera, quan vam exposar les cèl•lules INS-1E a elevades concentracions de glucosa i palmitat, ambdós nutrients van incrementar l’apoptosi. Quan, en aquestes condicions, les cèl•lules es van tractar amb GSPE, l’extracte va incrementar els efectes pro-apoptòtics de l’elevada glucosa, sense modificar la situació de lipotoxicitat. L’apoptosi induïda pel GSPE en situacions d’hiperglucèmia involucra la via intrínseca de l’apoptosi. In vivo, vam continuar l’estudi previ realitzat en rates femelles amb obesitat induïda per una dieta de cafeteria realitzat, i vam veure que GSPE modulava els marcadors d’apoptosi en el pàncrees d’aquestes rates, però els efectes eren dependents de la dosi i el període de tractament. Tot i així, el tractament semblava que tendia a contrarestar l’augment de l’apoptosi de les rates alimentades amb dieta de cafeteria. En canvi, quan els efectes de l’extracte es van analitzar en rates mascle, GSPE incrementava un marcador pro-apoptòtic, suggerint un increment de l’apoptosi en les rates tractades amb l’extracte. D’aquesta manera, es conclou que la modulació dels marcadors d’apoptosi per part del GSPE en rates alimentades amb dieta de cafeteria és dependent de la dosi, el període de tractament i/o el gènere. Pel que fa als efectes de GSPE sobre la proliferació, quan les cèl•lules β pancreàtiques es van exposar a elevats nivells de glucosa i insulina, els quals indueixen la proliferació, i nivells alts de palmitat, el qual inhibeix la proliferació, l’extracte va mostrar un clar efecte antiproliferatiu. Aquests efectes antiproliferatius són probablement a causa de les molècules d’alt pes molecular, les quals no es poden absorbir a l’intestí, de manera que cal tenir-ho en compte en el moment de comparar els efectes obtinguts in vitro amb els possibles efectes in vivo. De fet, en els experiments de rates alimentades amb una dieta de cafeteria, el GSPE no va modificar els marcadors de proliferació analitzats. Com a model d’obesitat induïda genèticament, es van utilitzar rates Zucker Fatty. Quan aquestes rates es van tractar crònicament amb GSPE, tot i que l’extracte contrarestava l’expressió de marcadors d’apoptosi i proliferació en comparació amb les rates obeses no tractades, els canvis moleculars induïts per les procianidines no van ser suficients per contrarestar l’efecte genètic de les rates Zucker Fatty a un nivell fisiològic, ja que tant l’apoptosi com els nivells plasmàtics de glucosa i insulina eren tan elevats com en les rates control. En aquest experiment, també es va analitzar el perfil proteic dels illots realitzant un estudi de proteòmica. Un dels processos biològics en els quals les proteïnes modificades per GSPE estaven involucrades era l’apoptosi i la mort cel•lular. Els nivells de la majoria de les proteïnes incloses en aquest grup contrarestaven els efectes del genotip Zucker Fatty, de la mateixa manera que es va observar en els marcadors d’expressió gènica. Per tant, tenint en compte les rates Zucker Fatty com a referència d’apoptosi, el GSPE tendia a millorar aquest procés, tot i que no va induir canvis als nivells finals d’apoptosi. Un cop analitzats els efectes de GSPE en les cèl•lules β en situacions patològiques, es van avaluar els seus efectes en situacions fisiològiques. El tractament de les cèl•lules INS-1E amb GSPE no va modificar ni l’apoptosi ni la proliferació d’aquestes cèl•lules. Aquests resultats in vitro coincideixen amb els observats in vivo, en els quals, el tractament crònic de rates amb GSPE no va modificar ni l’apoptosi ni la massa β. En aquest experiment, el perfil de microRNA també es va analitzar, ja que alguns microRNA s’ha vist que poden regular la funció pancreàtica, incloent la regulació de la síntesi i la secreció de la insulina i l’apoptosi. Tot i que vam trobar que els microRNAs dels illots pancreàtics eren diana de les procianidines, els modificats per l’extracte no estan involucrats en els processos de proliferació i apoptosi, fet que confirma el fet que el GSPE no altera aquests processos en condicions fisiològiques. Finalment, una altra situació patològica en la qual la proliferació i l’apoptosi estan alterats en cèl•lules pancreàtiques és en càncer, en el qual la proliferació està incrementada i l’apoptosi inhibida. D’aquesta menera, vam analitzar els efectes del GSPE en la línia cel•lular d’adenocarcinoma pancreàtic MIA PaCa-2 i vam veure que l’extracte inhibia la proliferació cel•lular i incrementava l’apoptosi, procés mediat per la modulació de proteïnes de la família de la Bcl-2 i per la despolarització de la membrana mitocondrial, implicant la via intrínseca de l’apoptosi. En aquest cas, els components de l’extracte amb més activitat antiproliferativa i pro-apoptòtica també van ser identificats. Tant l’epigal•locatequina gal•lat com l’àcid gàl•lic foren els components amb efectes antiproliferatius més elevats, però, considerant que la concentració d’àcid gàl•lic en l’extracte és més de 40 vegades més elevat que la d’epigal•logatequina gal•lat, es va considerar l’àcid fenòlic com un dels components de l’extracte responsables dels efectes observats. De la mateixa manera que el GSPE, l’àcid gàl•lic modulava l’expressió de proteïnes de la família de la Bcl-2 i promovia la despolarització de la membrana mitocondrial. En aquest estudi, es van utilitzar dues aproximacions per tal d’apropar-nos a una situació in vivo, ja que un cop ingerides, no tots els components de les procianidines són absorbides a l’intestí. Prèviament, són hidrolitzades en l’intestí prim i metabolitzades en l’intestí prim i el fetge. A més a més, les procianidines i els metabòlits que no són absorbits en l’intestí prim, poden ser absorbits en l’intestí gros posteriorment a l’acció de la microflora bacteriana. D’aquesta manera, per una banda, vam tractar les cèl•lules amb medis basolaterals que contenien els components de l’extracte absorbits i metabolitzats pels enteròcits humans Caco-2. Aquest sistema és novedós, tot i així, les cèl•lules Caco-2 s’han usat àmpliament per estudiar l’absorció i secreció intestinal de fàrmacs i compostos de la dieta. Tot i així, els compostos absorbits i metabolitzats per les cèl•lules Caco-2 no van modificar la taxa de proliferació de les cèl•lules MIA PaCa-2. De fet, l’anàlisi dels medis basolaterals va revelar que ni l’epigal•locatequina gal•lat ni l’àcid gàl•lic, les molècules més efectives en la inhibició de la proliferació, no eren absorbits per les cèl•lules Caco-2. Per altra banda, també vam tractar les cèl•lules MIA PaCa-2 amb sèrums de rata tractats amb GSPE. Aquesta aproximació, que segons el nostre coneixement no s’havia usat anteriorment, permet l’exposició de les cèl•lules als compostos fenòlics absorbits i metabolitzats en l’organisme i que aconsegueixen arribar al teixit diana in vivo. Tot i que lleument, el tractament amb els sèrum de les rates tractades amb GSPE van inhibir la proliferació de les cèl•lules d’adenocarcinoma de pàncrees. Per concloure, en aquesta tesi doctoral hem vist que el GSPE modula la proliferació i l’apoptosi de les cèl•lules pancreàtiques, tant in vitro com in vivo, però els seus efectes són dependents del model, la dosi i la durada del tractament. Hem utilitzat tècniques òmiques i diferents aproximacions in vitro per tal d’acostar-nos a situacions in vivo, a més d’identificar les molècules de l’extracte responsables dels efectes observats.

Prolonged exposure to high ambient temperature without cooling causes heat stress (HS) resulting in altered growth, body composition and metabolic dysfunction in pigs. Grape seed extract (GSE) has been shown to reduce inflammation, and improve glucose transport and metabolism. Thus, GSE may be an effective supplement to combat the consequences of heat stress; however this possibility has not been evaluated in a large animal model. The objective of the current study was to examine the effect of GSE supplementation on pig performance and body composition during HS. Twenty-four female pigs (62.3± 8 kg BW) were randomly assigned to a 2X2 factorial experiment; thermal neutral (TN; 21-22°C) or heat stress conditions (HS; 33-34°C) for 7 days and fed either a control or a GSE supplemented diet (12mg/kg body weight). Body temperature (TB), respiration rate (RR) and feed intake (FI) were measured daily. Body composition was measured by dual-energy X-ray absorptiometry (DXA). Respiration rate and TB increased in the HS control group compared to the TN control group (p<0.05), however GSE did not alter these parameters compared to control for the duration of the 7 day period. HS decreased FI (P < 0.05). Fasting blood glucose concentrations were approximately 1.5-fold greater in the control diet compared to their GSE supplemented counterpart (p=0.067) on day 6 of the HS period, but did not differ between groups at the end of day 7 of HS. Body composition analysis indicated bone mineral density, bone mineral content, and percent change of fat remain unchanged between treatment groups. Percent change in weight was significantly reduced in HS. Lean tissue accretion was 45% greater in TN compared to HS groups (p<0.05). Endotoxin concentrations were approximately 2-fold lower in the HS-GSE group compared to the control (P=0.083). Grape seed extract supplementation does not appear to alter pig growth performance or body composition, but does appear to delay the onset of reduced feed intake by 1 day, reduce intestinal permeability, and improve insulin sensitivity during additional stress.
Master of Science in Life Sciences

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Contusion injuries cause significant muscle damage, activating a series of cellular events. Satellite cells (SC), the key role players in muscle regeneration, are activated to proliferate and develop into mature myoblasts, which could fuse to form new myotubes or to repair damaged fibres. Evidence suggests that anti-oxidants, such as those found in grape seed extract (GSE), enhance repair, but their effect on SCs is still unclear. This study aimed to harvest and culture primary rat myoblasts to investigate the effect of chronic in vivo GSE supplementation on SCs following a standardised crush injury. Using a modified pre-plate technique, myoblasts were harvested from rat muscle and then compared with the immortal C2C12 cell line for proliferation and differentiation competence. Several media options were compared: i) DMEM with or without L-glutamine, ii) Ham‘s F10 or iii) DMEM with L-glutamine and Ham‘s F10 combined. Primary myoblasts proliferated and differentiated at a much slower rate than C2C12 cells. The combined media was selected for further use. To investigate the effects of GSE on the recovery, rats were supplemented daily with GSE or placebo 14 days prior to a standardised mass-drop crush injury to the gastrocnemius. SCs were isolated and cultured from uninjured (NI, baseline) and from injured rats 4 hours (4h), 3 days (3d) or 14 days (14d) post-injury. Expression of myogenic proteins Pax7, M-cadherin, MyoD, CD56, desmin and CD34 was determined by flow cytometry. Myoblasts were sorted according to their CD56 and CD34 expression and three sub-sets were collected and re-cultured, namely CD56+/CD34-, CD56-/CD34+ and CD56+/CD34+. After 24 hours, sorted cells were stained for desmin expression. Pax7, M-cadherin and MyoD were present in 100% of isolated cells from all groups confirming their myogenic SC identity. For all groups, desmin was expressed only in ~80% of SCs. Lower adhesion competency in GSE supplemented groups resulted in lower yield obtained for culturing. Expression of CD56 increased significantly 3d post-injury in the placebo group. In contrast, with GSE, CD56 already increased 4h post-injury and decreased again 3d post-injury. Although CD34 expression did not differ dramatically, expression pattern resembled that of CD56. Immunocytochemistry revealed a range in morphology and desmin expression of sorted myoblasts. More myoblasts with high desmin expression were observed in the two CD56+ sub-sets (irrespective of CD34 expression), indicating that CD56 is still expressed in more mature myoblasts. Flow cytometry revealed a population of myoblasts expressing particularly high levels of desmin, primarily in the non-injured baseline GSE group. We hypothesise that this result is an indication of preparedness of myoblasts to respond earlier to injury, enabling quicker repair. This cell population with high desmin content is restored in skeletal muscle after repair (14d), only when supplemented with GSE. In conclusion, GSE attenuated adhesion competence of primary myoblasts in culture, but resulted in earlier maturation of SCs, possibly due to baseline preparedness of myoblasts in uninjured muscle for a quick response. Both reduced adhesion competence and early progression of myoblasts could enhance wound healing in skeletal muscle.
AFRIKAANSE OPSOMMING: Kneuswonde veroorsaak aansienlike skade aan skeletspier, wat ‘n reeks sellulêre prosesse in werking stel. Satellietselle, die hoofrolspelers tydens spierregenerasie, vermenigvuldig en ontwikkel tot volwasse mioblaste, wat saamsmelt om nuwe spiervesels te vorm. Antioksidante, soos die wat in druiwepit-ekstrak voorkom, bespoedig herstel, maar hul uitwerking op satellietselle is steeds onduidelik. Die doel van hierdie studie was om mioblaste uit rotspiere te isoleer en te kweek om die effek van langdurige in vivo aanvulling van druiwepit-ekstrak op satellietselle na ‘n kneusbesering te bepaal. 'n Aangepaste protokol is gebruik om primêre mioblaste te isoleer, wat daarna met C2C12 selle, ten opsigte van hul vermenigvuldigings- en differensiasievermoë vergelyk is. Verskeie groeimedia is gebruik: i) DMEM met of sonder L-glutamien, ii) Ham F10 en iii) ‘n kombinasie van DMEM, L-glutamien en Ham F10. Primêre mioblaste het stadiger vermenigvuldig en gedifferensieer as C2C12 selle. Die gekombineerde medium is vir verdere gebruik gekies. Om die uitwerking van druiwepit-ekstrak op spierherstel te ondersoek, is rotte vir 14 dae onderwerp aan daaglikse aanvullings van druiwepit-ekstrak of placebo voor ‘n gestandardiseerde kneusbesering aan die gastrocnemius. Satellietselle is geïsoleer vanuit onbeseerde spier (basiskontrole) en vanuit beseerde spier 4 ure (4h), 3 dae (3d) en 14 dae (14d) na die besering. Die uitdrukking van spierverwante proteïene Pax7, M-cadherin, MyoD, CD56, desmin en CD34 is vasgestel met 'n vloeisitometer. Mioblaste is daarna gesorteer op grond van hul CD56- en CD34-uitdrukking. Drie sub-groepe is versamel en verder gekweek, nl. CD56+/CD34-, CD56-/CD34+ en CD56+/CD34+. Na 24 uur is gesorteerde selle gekleur om desmin-uitdrukking te bepaal. Pax7, M-cadherin en MyoD is deur 100% satellietselle in alle groepe uitgedruk, wat hul spierverwante identiteit bevestig, alhoewel slegs 80% selle in alle groepe desmin uitgedruk. Druiwepit-ekstrak het die vermoë van selle om aan plate te heg onderdruk, wat gelei het tot ‘n laer opbrengs van mioblaste. Drie dae na die besering in die placebo groep het die CD56-uitdrukking beduidend toegeneem. In teenstelling hiermee het CD56-uitdrukking in die druiwepit-ekstrak groep 4 ure na die besering beduidend toegeneem en weer afgeneem na 3 dae. Hoewel daar nie sulke dramatiese verskille was tussen groepe ten opsigte van CD34-uitdrukking nie, was daar ‘n soortgelyke tendens as vir CD56-uitdrukking. Immunositochemie het ‘n verskeidenheid van morfologieë en variërende desminvlakke in gesorteerde mioblaste blootgestel. In die twee CD56+ groepe is meer mioblaste wat hoë desmin vlakke uitdruk gevind, wat aandui dat CD56 uitgedruk word deur meer volwasse mioblaste, ongeag van CD34-uitdrukking. Tydens vloeisitometrie is ‘n populasie selle wat hoë desminvlakke uitdruk, hoofsaaklik in die onbeseerde en 14d druiwepit-ekstrak groepe gevind. Dit is ‘n aanduiding dat sommige mioblaste voorbereid is om na 'n besering vinniger te reageer. Na die herstelproses word hierdie groep selle hernu in die teenwoordigheid van druiwepit-ekstrak-aanvulling. Die resultate het gevolglik daartoe gelei dat druiwepit-ekstrak die hegtingsvemoë van mioblaste verlaag, maar dat die aanvulling in vivo tot vroeër ontwikkeling van mioblaste lei, waarskynlik deur satellietselle voor te berei vir 'n vinnige respons na ‘n besering. Beide die onderdrukking van aanhegting aan kultuurplate en die vroeë ontwikkeling van mioblaste, kan die herstel van die skeletspier verbeter.
NRF and the Harry Crossley bursary for funding

Corynebacterium diphtheriae, Pseudomonas aeruginosa, Ricinus communis, Shigella dysentariae, and Vibrio cholerae produce AB toxins which share the same basic structural characteristics: a catalytic A subunit attached to a cell-binding B subunit. All AB toxins have cytosolic targets despite an initial extracellular location. AB toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against these toxins are therefore hard to develop because they use different surface receptors, entry mechanisms, enzyme activities, and cytosolic targets. We have found that grape seed extract provides resistance to five different AB toxins: diphtheria toxin (DT), P. aeruginosa exotoxin A (ETA), ricin, Shiga toxin, and cholera toxin (CT). To identify individual compounds in grape seed extract that are capable of inhibiting the activities of these AB toxins, we screened twenty common phenolic compounds of grape seed extract for anti-toxin properties. Three compounds inhibited DT, four inhibited ETA, one inhibited ricin, and twelve inhibited CT. Additional studies were performed to determine the mechanism of inhibition against CT. Two compounds inhibited CT binding to the cell surface and even stripped bound CT off the plasma membrane of a target cell. Two other compounds inhibited the enzymatic activity of CT. We have thus identified individual toxin inhibitors from grape seed extract and some of their mechanisms of inhibition against CT. This work will help to formulate a defined mixture of phenolic compounds that could potentially be used as a therapeutic against a broad range of AB toxins.
M.S.
Masters
Molecular Biology and Microbiology
Medicine
Biotechnology

Gliomas are rare intrinsic brain tumours which account for 2% of all cancers. Glioblastoma multiforme is the most malignant malignant glioma form and remains incurable. The biological features which preclude successful therapy include heterogeneity, diffuse invasive patterns and angiogenesis. Despite, advances in current conventional treatments the median survival time is only 14 months. Hence there is a need to investigate novel therapeutic approaches which can be included alongside conventional treatment. One such approach is the use of micronutrients in the management of glioblastoma multiforme. This study evaluated the affects of two micronutrient extracts, red clover extract (RCE) and red grape seed extract (RGSE), on human adult malignant brain tumours in vitro. Four primary (or short-term) cell cultures derived from human brain tumour biopsies, an established cell line and normal human brain cells from an epileptic pateint were used to measure the cell viability, anti-invasive, anti-angiogenic and pro-apoptotic potentials, following 48-hour treatment with the IC50s of either micronutrient extract. Both RCE and RGSE exhibited similar affects on the glioma cell cultures. They both appeared to reduce cell viability, invasive potential and angiogenesis potential though did not appear to have any significant affect on the apoptotic portential of the glioma cultures. For example, incubation with 0.007-1ug/ml RCE caused a significant (p < 0.05) reduction of in viability of glioma cells but did not affect viability of normal astrocytes. Similar results were obtained for RGSE. These doses also resulted in a significant decrease in invasion and angiogenesis (p<0.05). Effects varied between cell lines but in general decreased by 50-60%. This suggests that both RCE and RGSE do affect the development of glioma cell cultures in vitro and warrant further study into the pathways in which this may occur.

Most studies on the antihypertensive effects of bioflavonoids have reported short-term effects (within 7 weeks) at high concentrations (40–100 mg kg–1 day–1). The present study by contrast has investigated long-term effects of low concentrations of bioflavonoids on arterial blood pressure and left ventricular performance in spontaneously hypertensive rats (SHR). Spontaneously hypertensive rats were divided into a treated (n = 16) and a control (n = 16) group. The treated group received daily a grape seed proanthocyanidin extract (GSPE) at a concentration of 4mg kg–1day–1over six months. Arterial blood pressure (ABP) was measured once monthly on six randomly selected rats from both groups using an indirect tail-cuff method. After three months, the remaining rats underwent catheterizations to measure left ventricular performance and aortic pressure. The possible role of nitric oxide (NO) in the effects of GSPE was investigated by blocking NO synthase with N-nitro-Larginine methyl ester (L-NAME). Animals in the treated group had significantly lower arterial end-diastolic pressures (AEDP) after three months of treatment compared with control animals, and this trend continued until six months. In the treated group, left ventricular systolic pressures (LVSP) were reduced by 16.6% (P = 0.005), their dP/dtmax (left ventricular pressures) were reduced by 19.7% (P = 0.050), and cardiac work was reduced by 22.0% (P = 0.045) at the end of three months. Treatment with L-NAMEsuggested a contribution of NO to the effects of GSPE on blood pressure. A low concentration of GSPE administered over six months lowered AEDP significantly, and the L-NAME response suggested that NO is involved. The decreased AEDP had a lowering effect on left ventricular dynamics of hypertensive rats.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: This thesis aims to determine whether supplementation with a grape-seed derived antioxidant, oligomeric proanthocyanidin (PCO) or the glutathione precursor, N-acetylcysteine (NAC) may prove beneficial as treatment for exercise induced muscle damage (EIMD) in athletes. In this double-blind cohort study, 21 healthy, uninjured male rugby-players in mid-season training phase, aged between 18 and 25 years were randomly divided into three treatment groups. Participants received 210 mg PCO, NAC or placebo treatment for 9 consecutive days. The study comprised a 6-day wash-out period (protocol days: -12 to -7), followed by a 6-day supplement loading period (protocol days: -6 to -1) a plyometric exercise intervention (protocol day 0) and continued supplementation for 2 days (protocol days: 1 to 2). The exercise intervention comprised 15 sets of 10 near maximal, vertical plyometric squat jumps. Blood samples and delayed onset of muscle soreness (DOMS) scores were collected on protocol days: -6, 0, 1 and 2. Assessments included serum creatine kinase (CK) activity, oxygen radical absorbance capacity (ORAC), malondialdehyde (MDA) and soluble vascular cell adhesion molecule-1 (sVCAM-1) concentrations over time as well as a differential circulating leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils and basophils). Data analysis of CK activity revealed no significant differences between groups. However, PCO treatment prevented a significant peak in the CK response at 24 h (as seen in the placebo and NAC groups) when compared to baseline, pre and post readings (p<0.05). NAC supplementation significantly improved serum ORAC after the exercise intervention. By 48 h, serum ORAC had improved significantly from readings taken immediately post exercise (p<0.05) only in the NAC group. For all groups, absolute neutrophil counts peaked at 6 h post exercise from baseline or pre readings (p<0.05). In both NAC and placebo treated groups, neutrophil counts had decreased significantly in circulation by 24 h post exercise from the 6 h time-point (p<0.05). However, neutrophil counts only reached significantly lower levels by 48 h post exercise (p<0.05) in the group supplemented with PCO. The monocyte count also peaked significantly at 6 h post exercise when compared with other time-points before and after the exercise intervention (p<0.05) in all treatment groups. Neither antioxidant treatment significantly altered the responses of other leukocyte sub-populations, MDA or sVCAM-1 concentrations where main effects of plyometric exercise was evident. Although not statistically significant, a trend toward diminished sVCAM-1 expression with either antioxidant supplementation was apparent. These findings suggest that PCO supplementation (210mg/d) which includes a 7 day loading period may diminish plyometric EIMD by limiting (but not completely inhibiting) the neutrophil response. Secondary muscle damage may be prevented by partially blunting neutrophil infiltration, rather than only quenching free radicals released during the neutrophil oxidative burst. Furthermore, the finding that NAC supplementation improves serum ORAC only after exercise may provide added benefit when administered in combination with PCO.
AFRIKAANSE OPSOMMING: Hierde tesis is daarop gerig om vas te stel of aanvulling met ‘n druifsaadekstrak (DSE) gederiveerde antioksidant: pro-antosianiedoliese oligomeer (PSO), of die glutathione voorloopermolekule, N-asetielsistien (NAS) voordelig beskou kan word as behandeling vir atlete onderhewig aan spierskade veroorsaak deur oefening. Gedurende hierdie dubbelblinde kohort studie is 21 gesonde, manlike rugbyspelers sonder beserings tussen die ouderdom van 18 en 25 jaar in middel-seison fase ewekansig in drie behandelingsgroepe verdeel. Deelneemers het elk 210 mg PSO, NAS of placebo-aanvulling geneem vir nege agtereenvolgende dae. Die studie het bestaan uit ‘n 6-dag uitwasperiode (protokoldae: -12 tot -7), as ook ‘n 6-dag aanvullings periode (protokoldae: -6 tot -1), gevolg deur ‘n pliometriese oefeningsintervensie (protokol dag 0) en verdere aanvulling tot en met 2 dae na die oefening (protokol dae: 1 tot 2). Die oefeningsintervensie het 15 stelle van 10 naastenby maksimale, vertikale pliometriese hurkspronge behels. Bloedmonsters en vertraagde aanvang spierseerheid (VAS) tellings is op protokoldae: -6, 0, 1 en 2 geneem. Analiese het serum kreatien kinase (KK) aktiwiteit, suurstof radikaal absorpsie kapasiteit (SRAK), Malondialdahied (MDA) en oplospare vaskulêresel adhesie molekule-1 (oVAM-1) konsentrasie bepalings asook ‘n differentiële sirkulerende leukosiet seltelling ingesluit. KK aktiewiteit het geen merkwaardige verskil tussen groepe getoon nie. PSO aanvulling het wel gelei tot die voorkoming van ‘n merkwaardige piek in die KK response soos in die placebo en NAC behandelde groepe bevind is by die 24 h tydspunt in vergelyking met basislyn-, voor- en na-oefeningslesings (p<0.05). NAS het ‘n merkwaardige verbetering in serum SRAK getoon, maar eers teen 48 h na oefening. Slegs die NAS behandelde groep het op hierdie tydspunt ‘n betekenisvolle verbetering in SRAK getoon in vergelyking met lesings direk na oefening (p<0.05). Vir alle groepe is ‘n betekenisvolle toename in absolute neutrophiltellings waargeneem 6 h na oefening in vergelyking met basislyn- en vooroefeningslesings (p<0.05). Beide NAS en placebo-behandelde groepe het ‘n betekenisvolle afname in neutrophiltellings teen 24 h na oefening getoon in vergelyking met die 6 h tydspunt (p<0.05) maar met die PSO-behandelde groep word hierde afname eers teen 48 h waargeneem (p<0.05). Monosiettellings het in alle groepe 6 h na oefening ‘n betekinsvolle piek getoon (p<0.05). Waar slegs die hoofeffek van die pliometriese oefening betekenisvol was, het nie een van die twee antioksidant aanvullings ‘n merkwaardige verandering aan die respons van ander leukosiet sub-populasies, MDA of oVAM-1 konsentrasies getoon nie. Al kon statistiese beduidenheid nie bewys word nie, wil dit blyk dat ‘n verminderde oVAM-1 uitdrukking onstaan het in die geval van beide antioksidant-behandelde groepe. Tesame stel hierdie bevindinge voor dat PSO toediening (210mg/d) insluitende ‘n 7-dag aanvullingsperiode die vermoë verleen om die neutrophielrespons gedeeltelik te onderdruk (sonder om dit heeltemal te inhibeer) en sodoende spierskade verminder. Dus word verdere spierskade moontlik verlaag deur die voorkoming van neutrophil weefsel infiltrasie eerder as verwydering van reaktiewe spesies wat vrygestel word tydens oefening. Die bevinding dat NAS aanvulling serum SRAK eers na oefening merkwaardig verbeter, kan as voordelig beskou word, veral wanneer toegedien in samewerking met PSO om verdere spierskade te voorkom en herstelling vinniger te bewerkstellig.

Procyanidins (PCs) have been extensively investigated for their potential health protective activities, but the prospective bioactivities are limited by their poor bioavailability. The majority of the ingested dose remains unabsorbed and reaches the colon where extensive microbial metabolism occurs. The objectives of these studies are to better understand the roles and activities of PCs in the lower gastrointestinal tract. First, a new high-throughput Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry method was developed to efficiently analyze PCs and an extensive profile of their microbial metabolites. This method is sufficiently sensitive and effective in simultaneously extracting and measuring native PCs and their microbial metabolites in biological samples. Furthermore, administration of grape seed extract increased the expression of gut junction protein occludin and reduced levels of fecal calprotectin, which suggests an improvement of gut barrier integrity and a potential modulation of endotoxemia. Additionally, chronic supplementation of the diet with flavanols did not increase colonic tissue accumulation of PCs or their microbial metabolites over a 12 week feeding study. This was the first long-term study of its kind, and the results indicate that we still do not fully understand the outcome of ingested flavanols in the colon during chronic exposure rather than acute doses. Lastly, new understanding of the microbial metabolism of PCs in the colon has been reached by studying the colon as 4 segments, rather than as a complete unit as previous studies have done. Data show that a gradient is established along the length of the colon for both PCs and their metabolites, with PCs reaching highest concentrations within 3 h after ingestion, while metabolites reach maximum concentrations anywhere form 3-18 h after ingestion. Moreover, data indicate the progressive, step-wise degradation of PCs into small metabolites throughout the length of the colon. Overall, there is greater understanding of the colonic metabolism of dietary PCs derived from GSE and cocoa, the accumulation of these compounds, and their effect on gut permeability. Future work will build off of these novel studies, and will continue to advance the understanding of the health benefits of dietary PCs.
Ph. D.

Alzheimer's disease (AD) is the most common cause of dementia. No strong disease-modifying treatments are currently available. Amyloid-beta peptide (Abeta) appears to play a pivotal role in the pathogenesis of AD. We focused our interest on revealing the pathogenesis of the disease and developing novel therapeutic modalities. The thesis consists of three projects: 1. Prevention of AD by intramuscular delivery of an anti-Abeta single chain antibody (scFv) gene: Immunotherapy is effective in removing brain Abetaƒzbut was associated with detrimental effects. In the present study, the gene of an anti-Abeta scFv was delivered in the hind leg muscles of APPSwe/PS1dE9 mice with adeno-associated virus at three months of age. Six months later, we found that brain Abeta accumulation, AD-type pathologies and cognitive impairment were significantly attenuated in scFv-treated mice relative to enhanced green fluorescence protein (EGFP)-treated mice. Intramuscular delivery of scFv gene was well tolerated by the animals. These findings suggest that peripheral application of scFv is effective and safe in preventing the development of AD, and would be a promising non-inflammatory immunological modality for prevention and treatment of AD. 2. Prevention of AD with grape seed derived polyphenols: Polyphenols extracted from grape seeds are able to inhibit Abetaƒnaggregation, reduce Abeta production and protect against Abeta neurotoxicity in vitro. We investigated the therapeutic effects of a polyphenol-rich grape seed extract (GSE) in vivo. APPSwe/PS1dE9 transgenic mice were fed with normal AIN-93G diet (control diet), AIN-93G diet with 0.07% curcumin, or diet with 2% GSE beginning at 3 months of age for 9 months. Total phenolic content of GSE was 592.5 mg/g dry weight, including gallic acid, catechin, epicatechin and proanthocyanidins. Long-term feeding of GSE diet was well tolerated. The Abetaƒnlevels in the brain and serum of the mice fed with GSE were reduced by 33% and 44% respectively compared with the mice fed with the control diet. Amyloid plaques and microgliosis in the brain of mice fed with GSE were also reduced by 49% and 70% respectively. In conclusion, polyphenol-rich GSE is promising to be a safe and effective drug to prevent the development of AD. 3. Roles of p75NTR in the development of AD: P75NTR has been suggested to mediate Abeta induced neurotoxicity. However, its role in the development of AD is undetermined. APPSwe/PS1dE9 transgenic mice were crossed with p75NTR knockout mice to generate APPSwe/PS1dE9 mice with p75NTR gene deleted. P75NTR mainly expressed in the basal forebrain neurons and degenerative neurites in neocortex and hippocampus. Genetic deletion of p75NTR gene in APPSwe/PS1dE9 mice reduced soluble Abeta levels, but increased the insoluble Abeta accumulation and Abeta plaque formation in the brain. P75NTR deletion decreased Abeta production of cortical neurons in vitro. Recombinant extracellular domain of p75NTR attenuated the oligomerization and fibrillation of synthetic Abeta42 peptide in vitro, and reduced local Abeta plaques after hippocampus injection in vivo. Our data suggest that p75NTR plays an important role in AD development and may be a valid therapeutic target for the treatment of AD.

Orientador: Giselle Maria Marchi Baron
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Doutorado
Dentística
Doutora em Clínica Odontológica

Visando à obtenção de biomateriais que atuem como suporte que direcione e auxilie o processo de regeneração tecidual, materiais poliméricos naturais como a quitosana e o colágeno têm sido estudados. A utilização da quitosana e do colágeno se baseia em propriedades como: biocompatibilidade, ação antimicrobiana, capacidade de ativar macrófagos, estimular a proliferação celular e baixa antigenicidade. Também se tem buscado a utilização de fitoterápicos como, por exemplo, o extrato de semente de uva que no processo de cicatrização tecidual, atua estimulando o fator de crescimento endotelial vascular (angiogênese) e proliferação de fibroblastos. Este trabalho teve como objetivo o preparo e caracterização de esponjas de quitosana/colágeno e quitosana/colágeno/glicerol (1:1) e (1:2) contendo extrato de semente de uva. A caracterização foi feita por calorimetria exploratória diferencial (DSC), espectroscopia de absorção no infravermelho (FTIR), microscopia eletrônica de varredura (MEV), absorção de tampão fosfato salino (PBS) e liberação in vitro do extrato de semente de uva. Estudos de DSC mostraram que ocorre um aumento na temperatura de desnaturação do colágeno com o aumento da concentração do extrato, indicando um efeito de reticulação que é mais pronunciado nas esponjas (1:2) e na presença de glicerol. Os espectros FTIR mostraram que ocorre um deslocamento das bandas de amida I e II devido à interferência do anel aromático presente no extrato. O aumento da proporção de colágeno, de extrato e a adição do glicerol contribuíram para o aumento no número e diâmetro dos poros das esponjas, observados por MEV. A presença do extrato aumenta a capacidade de absorção de PBS das esponjas, o aumento da concentração de extrato aumenta a velocidade de absorção, mas diminui sua capacidade de absorção. Ensaios de liberação in vitro mostraram que as quantidades de extrato liberado em meio PBS aumentaram até as primeiras 24 h. A maior porcentagem de liberação ocorreu para a esponja Q1C2E2 (44%). A presença do glicerol influiu na liberação do extrato, diminuindo-a. Os valores de n mostraram que a liberação do extrato ocorreu por difusão, no qual os valores estão próximos 0,5 caracterizando um mecanismo de transporte Fickiano, exceto para as esponjas Q1C1E05 e Q1C2GE05, sendo por transporte anômalo (0,5Aiming to obtain biomaterials that act as a support to direct and assist the process of tissue regeneration, natural polymeric materials such as collagen and chitosan have been used. The use of chitosan and collagen is based on properties such as biocompatibility, antimicrobial activity, low antigenicity, ability to activate macrophages and stimulate cell proliferation. The use of herbal medicines, for example, grape seed extract in the process of tissue healing, acts by stimulating vascular endothelial growth factor (angiogenesis) and fibroblast proliferation. This work aimed to the preparation and characterization of chitosan/collagen and chitosan/collagen/glycerol sponges (1:1) and (1:2) containing grape seed extract. The characterization was made by differential scanning calorimetry (DSC), infrared absorption spectroscopy (FTIR), scanning electron microscopy (SEM), absorption of phosphate buffered saline (PBS) and in vitro release of grape seed extract. DSC studies have shown that there is an increase in denaturation temperature of collagen with increasing extract concentration, indicating a crosslinking effect that is more pronounced in the sponges (1:2) in the presence of glycerol. The FTIR spectra show that there is a displacement of bands I and amide II due to interference of the aromatic ring present in the extract. Increasing the proportion of collagen, extract and addition of glycerol contributed to the increase in the number and diameter of the pores of the sponges, observed by SEM. The presence of the extract increases the absorption capacity of PBS in the sponges, the concentration of the extract increases the absorption rate, but decreases its absorbent capacity. In vitro release tests showed that the released amounts of extract increased up to 24 h. The highest percentage of release was observed for the sponge Q1C2E2 (44%). The presence of glycerol influenced the release of the extract, decreasing it. The values of n showed that the release occurred by diffusion of the extract, in which the values are close to 0.5 featuring a Fickian transport mechanism, except for the sponges Q1C1E05 and Q1C2GE05, that occurred by anomalous transport (0.5