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: 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.

Apesar da capacidade regenerativa da dentina já ser bem estabelecida, este processo pode ser insuficiente no caso de injúrias decorrentes de traumas e/ou lesões cariosas extensas, podendo levar à exposição pulpar e perda de sua vitalidade. Assim, pesquisas recentes no campo da engenharia tecidual têm identificado materiais e substâncias que poderiam ser utilizadas como biomodificadores da dentina e auxiliares na regeneração do complexo dentina-polpa. Entre eles estão os extratos ricos em proantocianidina, um composto fenólico bioativo presente no extrato de semente de uva (GSE). Assim, o objetivo deste trabalho foi avaliar o potencial estimulatório de quatro concentrações crescentes do GSE na atividade funcional de células odontoblastoides. Foram utilizadas células de camundongo da linhagem MDPC-23, cultivadas em garrafas de cultura até a subconfluência. Em seguida, as células foram cultivadas em placas de 24 poços em uma concentração de 104/poço e divididas em cinco grupos: células sem adição do GSE, células + 0.1 µg/mL de GSE; células + 1 µg/mL de GSE; células + 10 µg/mL de GSE; células + 20 µg/mL de GSE. Após 3, 7 e 10 dias, foram analisados os seguintes parâmetros: proliferação e viabilidade celular, detecção e atividade de fosfatase alcalina, quantidade de proteína total, detecção e quantificação de nódulos mineralizados, expressão quantitativa dos genes Alp, Col1a1 e Dmp1 por meio de PCR em tempo real e sua expressão proteica correspondente por meio de imunolocalização. Os dados obtidos foram analisados quanto à normalidade e submetidos aos testes estatísticos ANOVA e Kruskal-Wallis, com nível de significância estabelecido em 5%. Os resultados mostraram proliferação e viabilidade celular com as concentrações mais baixas de GSE (0,1 e 1 µg/mL), assim como a atividade de síntese de proteínas totais e da fosfatase alcalina. A deposição de nódulos mineralizados foi significativamente maior com a concentração de 1 µg/mL do extrato. Esta mesma concentração favoreceu a expressão quantitativa dos genes Alp, Col1a1 e Dmp1 e a secreção das proteínas correspondentes vistas por imunolocalização. Conclui-se que baixas concentrações do extrato de semente de uva podem influenciar e favorecer a atividade de células odontoblastoides, contribuindo para a regeneração dentinária, devido à suas características antioxidantes e biomineralizadoras / The process of dentinogenesis generally involves several steps of cell differentiation, culminating with an extracellular matrix very similar to bone tissue. Dentin regeneration may be necessary in cases of pulp exposure, root resorption and carious lesions. Studies on functional regeneration of lost tissues are being carried on recently, based on the presence of cells, scaffolds and/or substances that induce cell proliferation and differentiation. Among the several substances which can interact with cell metabolism are the extracts rich in proanthocyanidin, bioactive phenolic compounds present in fruits, vegetables and seeds. The purpose of this project is to evaluate the stimulatory potential of four different concentrations of grape seed extract (GSE) in the functional activity of odontoblast-like cells. MDPC-23 cell line was seeded in culture flasks until subconfluence followed by cell seeding in 24-well culture plates in the concentration of 104/well and divided in five groups: 1) cells without GSE, 2) cells + 0,1 µg/mL of GSE; 3) cells + 1 µg/mL of GSE; cells + 10 µg/mL of GSE and cells + 20 µg/mL of GSE. After 3, 7 and 10 days, there were evaluated the following parameters: cell proliferation and differentiation, ALP detection and activity, total protein content, mineralization detection and quantification, as well as the expression of genes Alp, Col1a1 and Dmp1 through Real Time PCR and their corresponding proteins by means of immunolocalization. Data were analyzed by ANOVA and Kruskal-Wallis statistical tests, with significance level set at 5%. The results demonstrated cell proliferation and viability with low GSE concentrations (0,1 and 1 µg/mL), as well as total protein content and ALP activity. Deposition of mineralized nodules was significantly increased with the GSE concentration of 1 µg/mL. The same concentration favoured the expression of genes Alp, Col1a1 and Dmp1 as well as the corresponding proteins. It is concluded that low concentrations of grape seed extract may influence functional activity of odontoblast- like cells, contributing to dentin regeneration by means of its antioxidant and biomineralizing properties

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 (0,5<n<0,89). / Aiming 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 <n<0.89).

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.

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 (0,5<n<0,89). / Aiming 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 <n<0.89).

The aim of this study was to develop a cost-effective process to produce a grape seed extract of high quality using only non-toxic extractants. When this study was started no grape seed extract was produced in South Africa. Large quantities were imported to supply the local demand in the human and animal herbal medicine industry. Grape seed extract is mainly used to boost the immune system of humans and animals based on its antioxidant activity. Initial work with different extractants established the polarity of the compounds with antioxidant activity. Antioxidant related activity was determined with five analysis techniques. Parameters such as the type, preparation and pre-treatment of grape seed, ratio of extractant to grape seed, composition of extractant, extraction time, extraction temperature, the interaction between temperature and time, drying temperature and subsequent treatment of extracts to remove compounds without antioxidant activity were evaluated. In all cases the cost implications of different methods used were kept in mind. Not only the quality but also the quantity extracted is important in establishing a viable extraction plant. According to the patent literature most techniques used to date produce yields of 0.5 to 2.5 %. The laboratory product went through five stages of development. The percentage extracted for our five laboratory products decreased from 12.0, 10.1, 6.0, 5.9 to 5.5 % whereas antioxidant activity for our product increased from 30, 55, 67 78 to 172 % compared to the best available commercial product. An important reason for the success of the procedure developed, is that we analyzed the different products developed with sophisticated procedures that gave information about the chemical composition of the extract. From this information procedures could be developed to increase the yield and activity. The procedure has been licensed to a private company that is in the process of establishing a factory for the large-scale production of grape seed extract. The detail regarding the procedure is confidential to protect the intellectual property and industrial exploitation of the process. / Dissertation (MSc)--University of Pretoria, 2004. / gm2014 / Paraclinical Sciences / unrestricted

Orientador: Giselle Maria Marchi Baron / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-26T22:21:22Z (GMT). No. of bitstreams: 1 Araujo_LarissaSgarbosaNapoleaode_D.pdf: 2549367 bytes, checksum: e7199299128873b8a98a1902b9a67070 (MD5) Previous issue date: 2015 / Resumo: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: The complete Abstract is available with the full electronic digital thesis or dissertation / Doutorado / Dentística / Doutora em Clínica Odontológica

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13055号 / 論農博第2839号 / 新制||農||1045(附属図書館) / 学位論文||H28||N5012(農学部図書室) / 33145 / (主査)教授 河田 照雄, 教授 保川 清, 教授 橋本 渉 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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