Thesis submitted in fulfilment of the requirements for the degree of Magister Technologiae: Agriculture in the Faculty of Applied Sciences at the Cape Peninsula University of Technology / Conjugated linoleic acid (CLA), a fatty acid (FA) present in the meat and milk of ruminant animals, is considered a functional nutrient for humans. The interest in CLA is attributed to its many health benefits, such as having anti-carcinogenic, anti-atherogenic, anti-diabetic and anti-adipogenic effects. Dairy products are a rich natural source of CLA, and preliminary research indicates that the levels in milk can vary based on cattle breed and feeding system. The Fleckvieh is a popular dual-purpose breed recently introduced to the Western Cape. It is used in crossbreeding programs with Holstein and Jersey herds to increase fertility and beef production. Holstein and Jersey cattle are the two most common South African dairy breeds, but little information is available on the effect of crossbreeding on FA content and CLA levels in the milk. The primary objective of this study was to compare milk FA composition and CLA content of these two dairy breeds and their respective Fleckvieh crosses. A second objective was to investigate the effect of lactation stage on CLA levels. Jersey (J) and Fleckvieh x Jersey (F×J) cows were kept in a pasture-based system (PBS) for the duration of the study. Holstein (H) and Fleckvieh x Holstein (F×H) cows were housed in a feedlot system and fed a total mixed ration (TMR) for the duration of the study. The FA and CLA content of feeds from these two feeding systems were also analysed and compared. All cows used in the study were housed at the Elsenburg Research Farm of the Western Cape Department of Agriculture. Milk samples were collected every 35 days from the cows in each feeding group, starting 10 days after calving. Samples were kept in a freezer at -20 °C until laboratory analyses. The total number of milk samples collected was 1044. The milk FA composition and CLA in particular were determined by gas chromatography. Differences between groups were statistically analysed by two-way repeated measures of analysis of variance (ANOVA). Results show that milk CLA levels are higher in Fleckvieh-crossbred cows when compared to purebred cows. The higher CLA content of milk from Fleckvieh crossbred cows demonstrates the feasibility of using Fleckvieh bulls in South African crossbreeding programs, and are also an indication that genetic selection for increased CLA content is possible. The analysis of lactation stage showed that milk CLA is higher during late lactation. A comparison of feed samples from the two feeding systems revealed that pasture feeding resulted in higher omega-3 FA in milk. Key words: Conjugated linoleic acid, milk fatty acids profile, diet, breed, health benefits, Fleckvieh, Holstein, Jersey, diet, pasture based system, total mixed ration system,

Três experimentos foram conduzidos com o objetivo de avaliar os efeitos do fornecimento de baixos teores de óleo de peixe em substituição parcial ao óleo de soja, sobre o consumo de matéria seca (CMS), produção e perfil de ácidos graxos do leite de ovelhas, ganho médio diário de peso corporal (GMD), características da carcaça e composição de ácidos graxos da carne de cordeiros, digestibilidade dos nutrientes, características de fermentação ruminal e metabolismo ruminal dos ácidos graxos. Os tratamentos consistiram de uma dieta controle (CONT), sem adição de óleo; e 4 dietas adicionadas com 4,0% de óleo, consistindo de 0,0% (0P); 0,25% (25P); 0,50% (50P) e 0,75% de óleo de peixe (75P), com o óleo de soja completando o teor de 4,0% de óleo adicionado (% MS). No Experimento I a dieta controle foi composta por 70% de concentrado e 30% de volumoso, nos Experimentos II e III a dieta controle foi composta por 90% de concentrado e 10% de volumoso. Experimento I: Foram utilizadas 50 ovelhas, distribuídas em delineamento experimental de blocos completos casualizados. Verificouse aumento linear na produção de leite das ovelhas e no GMD das crias com a inclusão de óleo de peixe nas dietas. As concentrações de ácido vacênico, CLA C18:2 trans-10, cis-12, ácido eicosapentaenóico (EPA) e ácido docosahexaenóico (DHA) também aumentaram linearmente com os teores crescentes de inclusão de óleo de peixe. Experimento II: Foram utilizados 50 cordeiros, distribuídos em delineamento experimental de blocos completos casualizados. O CMS expresso em % do peso corporal (PC) e em g/kg de PC0,75 aumentou linearmente com os teores crescentes de inclusão de óleo de peixe, o que resultou em aumento linear no GMD dos cordeiros. A concentração de ácido esteárico reduziu com os teores crescentes de substituição do óleo de soja pelo óleo de peixe. Verificou-se aumento linear na concentração de ácido vacênico à medida que o óleo de peixe foi adicionado à dieta. Em comparação ao tratamento controle, os animais alimentados com as dietas contendo óleo de peixe apresentaram maior concentração de CLA C18:2 cis-9, trans-11 na carne. Experimento III: Foram utilizados cinco borregos, canulados no rúmen e no duodeno, distribuídos em delineamento experimental quadrado latino 5 x 5. A suplementação com as fontes de óleo reduziu a digestibilidade da PB. A concentração de acetato, butirato e dos ácidos graxos de cadeia curta (AGCC) totais foi maior no conteúdo ruminal dos animais alimentados com a dieta controle em relação aos das dietas contendo óleo, como conseqüência, o pH ruminal destes animais foi inferior. O fluxo duodenal de C18:1 trans-11 e CLA C18:2 cis-9, trans-11 foi superior para os animais que receberam gordura suplementar. Observou-se aumento linear no fluxo duodenal de ácido C18:1 trans-11 em resposta a inclusão de óleo de peixe nas dietas. A inclusão de 0,75% de óleo de peixe na dieta misturado à 3,25% de óleo de soja mostrou-se como a melhor alternativa avaliada. / Three trials were conducted to evaluate the effects of small amounts of fish oil supply in partial replacement of soybean oil on dry matter intake (DMI), lactation performance and milk fatty acid composition of ewes, growth, carcass characteristics, and on meat fatty acid composition of feedlot lambs, some rumen constituents, and ruminal fatty acid metabolism. Treatments consisted of a control diet (CONT), and 4 diets with 4% added fat consisting of 0.0% (0FO), 0.25% (25FO), 0.50% (50FO) and 0.75% (75FO) fish oil with soybean oil providing the balance of 4% added fat. In trial I the control treatment consisted of 30:70 ratio of forage to concentrate (DM basis). In trials II and III the control treatment consisted of 10:90 ratio of forage to concentrate (DM basis). Trial I: Fifty Santa Inês ewes were penned individually and used in a randomized complete block design. Milk production and preweaning ADG of lambs increased linearly when fish oil replaced soybean oil. Vaccenic acid, CLA trans-10, cis-12, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased linearly with fish oil inclusion. Trial II: Fifty Santa Inês ram lambs were penned individually and used in a randomized complete block design. DMI (% of BW and g/kg of BW0,75) increased linearly when fish oil replaced soybean oil, as consequence ADG also increased. Stearic acid concentration decreased and vaccenic acid increased with fish oil inclusion. CLA C18:2 cis-9, trans-11 showed higher concentration in meat of animals fed diets containing fish oil compared to the control diet. Trial III: Five ram lambs cannulated in the rumen and proximal duodenum were assigned in a 5 x 5 Latin Square design. Soybean oil and fish oil supplementations decreased CP digestibility. Ruminal concentrations of acetate, butyrate and total SCFA were higher for animals fed the control diet. Ruminal pH was lower for animals fed the control diet compared to diets with oils. Duodenal flow of C18:1 trans-11 and CLA C18:2 cis-9, trans-11 was greater for diets containing supplemented oils. C18:1 trans-11 flow to the duodenum increased linearly with fish oil inclusion.The inclusion of 0.75% of fish oil in the diet mixed with 3.25% soybean oil was the best alternative evaluated.

O presente estudo teve como objetivo caracterizar o processo de cicatrização e investigar os efeitos da suplementação (gavagem) com OL e Li em estudos in vivo. Após anestesia, uma área de 10 mm2 de pele foi removida cirurgicamente da região dorsal dos animais. A suplementação com Li reduziu o tamanho das feridas no sétimo dia após a indução. Esse resultado foi acompanhado de aumento na produção de peróxido de hidrogênio (H2O2). O OL reduziu o conteúdo de H2O2 após 24 horas. Li acelerou a resposta migratória, aparecendo células inflamatórias uma hora após o ferimento. OL induziu ativação do fator de transcrição NF-<font face=\"Symbol\">kB e o Li ativou AP-1 após uma hora. Em 24 horas, ambos AGs inibiram o NF-<font face=\"Symbol\">kB e não alteraram o AP-1. Li elevou as concentrações de CINC-2<font face=\"Symbol\">a<font face=\"Symbol\">b e OL aumentou TNF-<font face=\"Symbol\">a, no homogenato, uma hora após a indução da ferida. Em 24 horas, ambos AGs reduziram a expressão e as concentrações de IL-1<font face=\"Symbol\">b, IL-6 e MIP-3<font face=\"Symbol\">a. A suplementação com OL e Li acelerou a fase inflamatória do processo de cicatrização. / This study aimed to characterize the healing process and to investigate the effects of supplementation (gavage) with OL and Li on the healing process through in vivo experiments. After anesthesia, an area of 10 mm2 of skin was surgically removed from the dorsum. Li reduced the size of the wounds on the seventh day after induction. This result was accompanied by increased production of hydrogen peroxide (H2O2). OL reduced the H2O2 content after 24 hours. Li accelerated the migratory response, since inflammatory cells were found one hour after the wound. OL activated the transcription factor NF-<font face=\"Symbol\">kB and Li increased the activation of AP-1 one hour after wound induction. Both fatty acids reduced the activation of NF-<font face=\"Symbol\">kB and did not alter AP-1 after 24 hours. Li raised the concentrations of CINC-2<font face=\"Symbol\">a<font face=\"Symbol\">b and OL increased TNF-<font face=\"Symbol\">a, in the homogenate, one hour after wound induction. In 24 hours, the FA reduced expression and concentrations of IL-1<font face=\"Symbol\">b, IL-6 and MIP-3<font face=\"Symbol\">a. Putting together, our results demonstrate that OL and Li accelerate the inflammatory phase of the wound healing.

Os objetivos deste experimento foram os de realizar levantamento exploratório da produção, composição e perfil de ácidos graxos do leite de búfalas e dos alimentos utilizados em cinco fazendas da região de Sarapuí e Pilar do Sul, no Estado de São Paulo. Foram coletadas amostras mensais de leite de oito búfalas por fazenda e de alimentos durante os meses de abril a novembro de 2002. Das cinco fazendas, uma explorava sistema de confinamento total e quatro usavam sistemas de produção a pasto com suplementação volumosa no inverno (silagem de gramíneas e cana-de-açúcar) e concentrado (resíduo de cervejaria ou mistura comercial) o ano todo.Três propriedades realizavam duas ordenhas diárias, e as demais apenas uma. Os teores de gordura, proteína e lactose variaram de 5,4 a 8,6%, 3,7 a 4,9% e 4,5 a 5,6%, respectivamente, valores normais para bubalinos. Os teores de gordura e de proteína aumentaram ao longo da lactação, enquanto o teor de lactose acompanhou a curva de lactação. O teor de nitrogênio uréico no leite variou entre 5,6 e 27,3 mg/dL, com valor médio em todas as fazendas de 15,9 mg/dL, inferior ao observado na literatura para bubalinos. Do total de ácidos graxos na B. decumbens o ácido linoléico variou de 18,2% no inverno para 19,9% no verão e o ácido linolênico de 20,8% no inverno para 31,4% no verão. A B. ruziziensis apresentou teor de ácido linoléico 17,7% no inverno e 19,4% no verão, enquanto o ácido linolênico variou de 50,2% no inverno para 45,6% no verão. Observou-se grande variação individual no teor de CLA no leite de bubalinos (0,31-3,42%). Nos animais a pasto ou com suplementação volumosa de gramíneas frescas a variação foi maior (0,44 a 3,31%) que nos animais confinados (0,31 a 1,74%). Os teores obtidos de CLA no verão foram em média mais elevados (38,6%), que no inverno, nos rebanhos em pastejo. A Análise de Componentes Principais confirmou a correlação positiva entre os ácidos graxos de cadeia ímpar e de cadeia ramificada e alta correlação entre os ácidos capróico, caprílico e cáprico. Também indicou correlação negativa entre os ácidos graxos de cadeia longa com os de cadeia curta, média, ímpar e ramificada. O teor dos ácidos graxos hiper-colesterolêmicos (ácidos láurico, mirístico e palmítico) diminuiu à medida que houve elevação do teor de ácidos graxos de cadeia longa no leite. A atividade da enzima &#8710;9-dessaturase, medida indiretamente (relação produto x substrato), foi maior em animais que receberam resíduo de cervejaria em menor quantidade ao longo do ano. / The objective of this study was to determine milk production, composition and fatty acids profiles in five buffalo farms in the Sarapui and Pilar do Sul counties, in the Southeast region of the State of Sao Paulo. Monthly feed and milk (from eight animals per farm) samples were collected from April to November 2002. In one farm animals were housed in total confinement and were fed a TMR ration; wet brewers grains and a commercial grain mix and summer pasture and a mixture of grass silage plus chopped sugar cane during winter were offered in the remaining farms. Animals were milked twice daily in three farms. Milk fat, protein and lactose contents varied from 5.4 to8.6%, 3.7 to 4.9% and 4.5 to 5.6%, respectively. Milk fat and protein contents increased as the lactation progressed. Milk urea nitrogen showed a large variation among farms (5.6 to 27.3 mg/dL). The content of linoleic and linolenic acids in B. decumbens varied from18.2 and 20.8% (winter) to 19.9 and 31.4% (summer) and in B. ruziziensis varied from 17.7 and 50.2% (winter) to 19.4 and 45.6% (summer). "Milk CLA content showed a large variation among animals (0.31 to 3.42%). Pasture fed or green chopp supplemented animals showed a larger variation (0.44 to 3.31%) than TMR fed animals (0.31 to 1.74%). Milk CLA content was higher (39%) during summer than in winter, in pasture fed animals. Principle Component Analysis confirmed the positive correlation between odd chain and side chain fatty acids and a high correlation among caproic, caprilic and capric acids. It also showed a negative correlation between long chain fatty acids and short and medium chain and odd and side chain fatty acids. Milk hiper-colesterolemic fatty acids (lauric, miristic and palmitic acids) content decreased as the long chain fatty acids did increased. The indirectly measured Delta 9 dessaturase activity was higher in animals being offered small amounts of wet brewers grains throughout the lactation.

Ácidos graxos conjugados (AGCs) é o termo geral para descrever os isômeros posicionais e geométricos dos ácidos graxos poliinsaturados com duplas ligações conjugadas. Inúmeros efeitos benéficos para a saúde como: anti-cancerígeno, anti-aterogênicos, anti-adipogênicos e anti-inflamatórios, tem sido atribuídos ao consumo dos AGCs. Entretanto, estudos sobre os efeitos dos AGCs no organismo ainda são inconclusivos e por isso o interesse em pesquisas visando a sua participação em processos fisiológicos. O objetivo deste estudo foi avaliar em ratos o efeito dos isômeros conjugados do ácido linoleico (9cis, 11trans e 10trans, 12cis) sobre o perfil lipídico tecidual e sua influência sobre parâmetros bioquímicos em processos oxidativos. Não foi possível detectar a atividade antioxidante in vitro dos CLAs. Os resultados obtidos demonstraram que o FFA-CLA foi capaz de reagir apenas com o radical DPPH. No experimento in vivo uma mistura comercial dos isômeros (9cis, 11trans e 10trans, 12 cis) foi utilizada como fonte de CLAs, e a influência da suplementação desses isômeros, foi avaliada e comparada com um grupo controle suplementado com água e com grupos experimentais suplementados com óleo de soja. O primeiro experimento foi realizado com animais Wistar saudáveis e teve duração de 40 dias, as amostras de ácidos graxos livres de CLAs, (FFACLAs) e óleo de soja foram fornecidas aos animais por meio de entubação orogástrica nas concentrações de 1%, 2% e 4% em relação ao consumo diário de dieta, o grupo controle recebeu 1% de água. A suplementação da dieta dos animais com os CLAs leva ao aumento nos níveis de triglicérides, mas não interfere nos parâmetros de estresse oxidativo, embora haja incorporação dose-dependente nos tecidos hepático, muscular e adiposo, não foram observadas alterações no volume das células adiposas, e na área e diâmetro do tecido muscular. No segundo experimento, com duração de 21 dias, ao avaliar o efeito da suplementação na dose de 2% de CLAs em ratos Wistar induzidos a peroxidação, pelo tratamento com tetracloreto de carbono. Pôde-se observar efeitos hepatoprotetor, dos CLAs atribuídos a sua prevenção na peroxidação lipídica e ao aumento da atividade das enzimas catalase (CAT), glutatina redutase (GR) e glutationa reduzida (GSH). / Conjugated fatty acids (CFAs) is the general term to describe positional and geometric isomers of polyunsaturated fatty acids with conjugated double bonds. Many beneficial health effects such as anti-cancer, anti-atherogenic, anti-adipogenic and anti-inflammatory, has been attributed to the consumption of the CFAs. However, studies on the effects of the CFAs in the body are still inconclusive and therefore the interest in research aimed at their participation in processes physiological. The objective of this study was to evaluate in rats the effect of conjugated linoleic acid isomers (9cis, 11trans and 10trans, 12cis) on lipid profile tissue and its effect on biochemical parameters in oxidative processes. It was not possible to detect the in vitro antioxidant activity of CLAs. The results showed that the free fatty acids of CLAs (FFA-CLAs) was able to react only with the DPPH radical. In in vivo experiment a commercial mixture of isomers (9cis, 11trans and 10trans, 12cis) was used as a source of CLAs and the influence of supplementation of these isomers was evaluated and compared with a control group supplemented with water and experimental groups supplemented with soybean oil. The first experiment was conducted with healthy Wistar and lasted 40 days, samples of FFA-CLA and soybean oil were delivered to animals via orogastric intubation at concentrations of 1%, 2 % and 4% in relation to daily diet, the control group received 1% water. Supplementing the diet with CLAs leads to increased levels of triglycerides, but does not interfere with oxidative stress parameters, although there is a dose-dependent incorporation into liver tissue, muscle and fat, no changes were observed in the volume of fat cells, and in the area and diameter of muscle tissue. In the second experiment, lasting 21 days, to evaluate the effect of supplementation at a dose of 2% of CLAs in Wistar, tha peroxidation induced by treatment with carbon tetrachloride. Could be observed hepatoprotective effects, attributed to the CLAs prevention lipid peroxidation and increased activity of the enzymes catalase (CAT), glutathione reductase (GR) and glutathione reduzida (GSH).

Os ácidos linoléicos conjugados (CLAs) consistem de um grupo de isômeros geométricos e posicionais do ácido linoléico aos quais tem sido atribuídos efeitos anticarcinogênico, antidiabético, antiaterogênico e modulador da composição corporal. As principais fontes naturais de CLA são o leite e a carne de ruminantes, uma vez que microorganismos ruminais são capazes de formar CLAs e seus precursores como intermediários da biohidrogenação. Para sua identificação e quantificação por cromatografia gasosa (CG) deve se tomar cuidado ao decidir um procedimento apropriado de metilação, a fim de evitar a isomerização dos CLAs. Dessa forma, o objetivo do presente trabalho foi de avaliar criticamente metodologias analíticas para caracterizar os isômeros conjugados c-9, t-11 e t-10, c-12 do ácido linoléico, identificando o melhor método para a esterificação e a melhor programação por cromatografia gasosa para a separação, identificação e quantificação dos CLAs através de sua análise em suplementos, cortes bovinos e laticínios. Para tanto, foram empregadas as técnicas de esterificação ácida com trifluoreto de boro metanólico, esterificação alcalina com metóxido de sódio metanólico e uma esterificação mista usando a combinação dos dois catalisadores na metilação de uma amostra comercial de suplemento de ácido linoléico conjugado na forma de triacilglicerol, o Tonalin® CLA TG 80, da Cognis. Os ésteres metílicos foram identificados sob três condições cromatográficas CLA01, CLA02 e CLA03. Também foram realizadas quantificações dos isômeros c-9, t-11 e t-10, c-12 CLA no Tonalin® CLA TG 80 e FFA 80 através de ressonância magnética nuclear do carbono 13 (13C-RMN). Os resultados obtidos permitiram decidir pelo emprego da técnica de esterificação alcalina, seguido da análise cromatográfica pelo método CLA03 para análise de cinco cortes bovinos fracionados em músculo e capa de gordura, creme de leite, iogurte integral e três tipos de queijo. O método de esterificação alcalina, seguido de separação por CG pelo método CLA03 mostrou-se o mais adequado para uma quantificação segura do perfil de ácidos graxos de alimentos que contenham os isômeros conjugados do ácido linoléico, uma vez que os resultados obtidos concordam com a literatura atual. / Conjugated linoleic acids (CLAs) are a group of geometric and positional isomers of linoleic acid which have been attributed to present anticarcinogenic, antidiabetic, antiaterogenic and body composition modulation effects. Major natural sources of CLA are ruminants milk and meat, since rumen microorganisms are able to produce CLAs and their precursors as intermediates of biohydrogenation. Before identification and quantification by gas chromatography (GC), the methylation procedure should be carefully decided to avoid isomerization of CLAs. Thus, the objective of this study was to critically evaluate analytical methods for characterizing conjugated linoleic acid isomers c-9, t-11 and t-10, c-12, by identifying the best method for esterification and the best gas chromatography programming for separation, identification and quantification of CLAs through quantification of CLAs in supplements, beef and dairy products. In order to accomplish this objetive, the techniques of acid esterification with boron trifluoride methanol, alkaline esterification with methanolic sodium methoxide and mixed esterification using a combination of the two catalysts were employed in the methylation of a commercial sample of conjugated linoleic acid supplement in triacylglycerol form, the CLA Tonalin® TG 80, from Cognis. The methyl esters were identified in 3 chromatographic conditions CLA01, CLA02 and CLA03. CLA isomers c-9, t-11 and t-10, c-12 were also quantified in CLA Tonalin® TG 80 and FFA 80 by carbon 13 nuclear magnetic resonance (13C-NMR). The results indicated the alkaline esterification technique followed by chromatographic analysis by CLA03 method as the best method for analyzing five cuts of beef fractionated in muscle and fat layer, cream, full yoghurt and three types of cheese. The alkaline esterification method followed by GC separation method CLA03 proved to be the most suitable for safe quantification of fatty acid profile in foods that contain conjugated linoleic acid isomers, once the results agreed with the data available in the current literature.

Neste trabalho, foram desenvolvidas estratégias para modular a composição de elastômeros biodegradáveis da família dos polihidroxialcanoatos (PHA), produzidos por Pseudomonas, a partir de ácidos graxos ou óleos vegetais, para diversificar suas aplicações, sobretudo pela inserção de monômeros insaturados. A composição do PHA produzido variou com o tipo de ácido graxo fornecido e com sua proporção em misturas. Em genomas seqüenciados, detectaram-se dois genes fadH (codificador de 2,4-dienoilCoA hidratase) em P. aeruginosa e apenas um em outras Pseudomonas. Observou-se uma correlação entre o número de cópias de fadH no genoma e maior eficiência na oxidação de ácidos graxos insaturados. Mutantes afetados no metabolismo de ácidos graxos insaturados foram obtidos utilizando-se transposon, alguns destes mutantes apresentaram maior eficiência na incorporação de monômeros insaturados ao PHA. A clonagem e seqüenciamento de fragmentos de DNA interrompidos pelo transposon permitiram a identificação dos genes afetados nesses mutantes. / Different strategies were applied to modulate the composition of biodegradable elastomeric polyhydroxyalkanoates (PHA) accumulated by Pseudomonas from fatty acids and plant oils, in order to improve the applications of this material, mainly by unsaturated monomer insertion. PHA composition varied both with fatty acid type and fatty acids ratio in mixtures supplied. Analysis of genome sequences revealed two fadH (encoding 2,4-dienoyl-CoA hydratase) copies in Pseudomonas aeruginosa and only one in other Pseudomonas species. The number of fadH copies in the genome was related to the higher oxidation of unsaturated fatty acids. Transposon-induced mutants affected on unsaturated fatty acids metabolism were obtained, some of them showing higher efficiency to incorporate unsaturated monomers do the PHA. Cloning and sequencing of transposon-disrupted DNA regions allowed to identify the genes affected in those mutants.

Diante da ausência de dados prévios, foi pretendido estabelecer uma correlação entre as possíveis modificações metabólicas e moleculares na resistência insulínica e inflamação em modelos animais de obesidade, induzida pela dieta, recebendo a suplementação de ômega 3/ALA. Além disto, pretendeu-se obter dados que permitam uma melhor elucidação dos mecanismos envolvidos na resistência insulínica neste modelo e um possível efeito preventivo da administração de ALA sobre este processo, podendo, desta forma, auxiliar no desenvolvimento de novas terapias. O objetivo do estudo foi demonstrar que a suplementação ALA reduz a resistência à insulina e a inflamação em modelo animal de obesidade. Foram divididos 40 camundongos machos (C57/BL6) em 4 grupos: controle (C), controle + ômega 3/ALA (CW), obesos (O) e obesas + ômega 3/ALA (OW). Por um período de oito semanas, os grupos O e OW receberam uma dieta hiperlipídica com 60% de lipídeos, enquanto o C e CW receberam ração padrão. Depois, os grupos CW e OW receberam suplementação de 10% de ômega 3/ALA liofilizado, extraído de semente de linhaça, diariamente, por mais 8 semanas. Observando os resultados, todos os grupos de animais tiveram o mesmo ganho de peso, assim como consumo alimentar, eficiência enérgica e eficiência alimentar. O uso do ALA diminuiu o peso da gordura subcutânea no grupo OW comparado ao O e manteve os valores semelhantes entre os outros grupos, no entanto, as comparações do peso do fígado, gordura epididimal, pâncreas e músculo gastrocnemio, foram semelhantes entre todos os grupos. Observou-se uma diminuição na resistência insulínica nos animais OW comparado ao O pelo teste de IPGTT, sendo a área sob a curva de glicose similar entre C e CW. Além disso, os níveis totais de gordura no fígado foram significativamente menores no CW e OW, em comparação com C e O, estes resultados são reforçados pela análise dos tecidos em avaliação histopatológica. Os níveis séricos de glicemia e insulina, ao final do estudo, mostraram uma redução importante em OW comparado a CW, porém, não foi observado diferenças entre os animais obesos suplementados ou não com ômega-3/ALA. Entretanto, quanto à avaliação de resistência insulínica pelo cálculo do HOMA IR, este mostrou-se menor comprando-se OW com O. Os níveis séricos de colesterol total e HDL foram maiores no grupo CW comparados ao C, sendo que os níveis de colesterol total foram menores, também, no grupo OW comparados ao O. No entanto, os valores de triglicérides séricos foram semelhantes entre todos os grupos, assim como valores de triglicérides hepáticos. Colesterol total hepático teve um aumento significativo entre OW e O. As dosagens séricas de IL1, IL6 e MCP1 mostraram uma redução importante em O comparadas com OW. Já quanto a IL17 e TNF, ambas foram equivalentes, nas comparações entre diferentes grupos. A avaliação da ativação do estresse do retículo endoplasmático mostrou que a proteína BIP teve um aumento importante tanto na comparação entre C e CW e também entre O e OW. A também chaperona HSP70 mostrou aumento significativo em ambas as comparações entre grupos, tanto em C e CW quanto em O e OW. GRP94 e IRE1 tiveram resultados semelhantes, sem diferenças entre os grupos, assim como a DAPK1. CHOP teve diminuição importante comparando-se C e CW, e O e OW. Em contrapartida, XBP1 teve diminuição importante na comparação entre os grupos C e CW. Por fim, a suplementação de ômega 3/ALA mostrou ser eficaz na prevenção de esteatose hepática, redução de resistência insulínica, diminuição do processo inflamatório, e redução da ativação do estresse do retículo endoplasmático em tecido hepático. / Given the absence of previous data, it is intended to establish a possible correlation between metabolic and molecular changes in insulin resistance and inflammation in animals receiving supplementation of -linoleic acid (ALA) found in flaxseeds oil. Furthermore, we intend to obtain data for an elucidation of the mechanisms involved in insulin resistance and preventive effect of administration of ALA on this process and may thus aid the development of new therapies. The aim of study was prove the ALA supplementation reduces insulin resistance and inflammation in an obesity animal model. 40 male mice (C57/BL6) were divided into 4 groups: control (C), control + omega 3/ALA (CW), obese (O) + omega 3/ALA and obese (OW). For a period of eight weeks, the groups O and OW received a high-fat diet with 60% fat, while the C and CW received regular chow. Then, the CW and OW groups were supplemented with 10% omega 3/ALA lyophilized, extracted from flaxseed daily, for another 8 weeks. Analyzing the results, all groups of animals had the same weight gain, and food consumption, food efficiency and energetic efficiency. The use of ALA decreased subcutaneous fat weight in group OW compared to O and remained similar values between the other groups, however, comparisons of the size of liver tissue, epididymal fat, pancreas and gastrocnemius muscle were similar among all groups. There was a decrease in insulin resistance in animals OW compared to O by GTT test, with an area under the curve of glucose similar between C and CW. Moreover, the total liver fat levels were significantly lower in CW and OW compared to C and O, these results are reinforced by histopathological tissues analysis. Serum glucose and insulin levels, at the end of the study, showed a significant reduction in OW compared to CW, however, no differences were observed between the obese animals supplemented or not with omega 3/ALA. Meanwhile, about the valuation of insulin resistance calculating by HOMA IR, this was lower in OW compared with O. Serum total cholesterol and HDL levels were higher in CW compared to the group C, and total cholesterol levels were also lower in the OW group compared to O. However, the serum levels of triglyceride were similar among all groups as well as hepatic triglycerides values. Hepatic total cholesterol increased significantly between OW and O. Serum levels of IL1, IL6 and MCP1 showed a significant reduction in O compared to OW. As for IL17 and TNF were similar for both comparisons between different groups. The evaluation of endoplasmic reticulum stress activation showed the BIP protein had a significant increase in the comparison between CW and C and also between OW and O. The also chaperone HSP70 showed a significant increase in both comparisons between groups, both in C and CW as for O and OW. GRP94 and IRE1 had similar results, with no differences between groups, as well as DAPK1. CHOP had significant decrease comparing CW and C, and O and OW. In contrast, XBP1 had significant reduction in the comparison between groups C and CW. Finally, omega 3/ALA supplementation showed to be effective in preventing hepatic esteatose, the reduction of insulin resistance, inflammation decrease, and reduction of activation of endoplasmic reticulum stress in liver tissue.

O ácido linoléico conjugado (CLA) é um ácido graxo poliinsaturado, encontrado nos produtos da alimentação. Estudos indicam que o CLA possui ações contra câncer, aterogênese e DM 2 e obesidade. O presente trabalho avaliou os efeitos da suplementação crônica com CLA em ratos Wistar machos e teve como objetivo investigar o desenvolvimento corporal e o perfil metabólico dos animais e dos adipócitos isolados do tecido adiposo branco periepididimal. Após quatro semanas de suplementação os animais apresentaram redução no ritmo de ganho de peso, acompanhado de redução da ingestão alimentar, redução da massa adiposa e do volume celular dos adipócitos. A menor incorporação dos substratos acetato e glicose em lipídeos, o aumento lipólise e diminuição da expressão do PPAR?, também contribuíram para menor adiposidade encontrada. A redução de massa adiposa foi acompanhada por resistência à insulina, elevados níveis de citocinas inflamatórias e desenvolvimento de esteatose hepática. Esses fatores estão relacionados com o desenvolvimento de síndrome lipodistrófica em animais. Portanto, a efetividade do CLA em reduzir massa adiposa foi comprovada no presente estudo, mas os efeitos devem ser considerados. / Conjugated linoleic acid (CLA) is a natural polyunsatured fatty acid found in many dietary sources. Animal studies demonstrated that CLA has properties against cancer, atherogenesis, diabetes and obesity. This work evaluated the effects with chronic CLA supplementation in young adults Wistar male rats for four weeks, aiming to investigate the possible changes in corporal development and metabolic profile as well as the effects in isolated adipocytes of periepidydimal white adipose tissue of these supplemented animals. We observed a reduction of the rhythym of body weight gain, followed by diminished food intake, regression of adipose mass, and also a reduction of adipocyte volume. The findings of low incorporation of acetate and glucose substrates into lipids, elevation on the lipolytic response and reduction of PPAR-gamma gene expression, also contributed to the lower adiposity. This reduction in adipose mass was followed by insulin resistance, high levels of inflammatory citokines and the development of hepatic steatosis, features related to the development of lipodystrophic syndrome. Therefore, this study demonstrated the CLA effect on reduction of adipose mass, although adverse effects associated with CLA chronic supplementation must be considered.

Lui Oi Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves [216]-240). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vii / 撮要 --- p.x / PUBLICATIONS --- p.xiii / TABLE OF CONTENTS --- p.xiv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis and Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoietic Development --- p.1 / Chapter 1.1.2 --- Leukemia --- p.8 / Chapter 1.1.2.1 --- General Diagnostic Tests for Leukemia --- p.9 / Chapter 1.1.2.2 --- Classification and Epidemiology of Leukemia --- p.10 / Chapter 1.1.2.3 --- Conventional Approaches to Leukemia Therapy --- p.17 / Chapter 1.1.2.4 --- Novel Approaches to Leukemia Therapy --- p.20 / Chapter 1.2 --- Conjugated Linoleic Acid --- p.23 / Chapter 1.2.1 --- Introduction: Historical Development and Occurrence of Conjugated Linoleic Acid --- p.23 / Chapter 1.2.2 --- Phytochemistry and Metabolism of Conjugated Linoleic Acid --- p.24 / Chapter 1.2.2.1 --- Chemical Structures of Conjugated Linoleic Acid Isomers --- p.24 / Chapter 1.2.2.2 --- Biosynthesis of Conjugated Linoleic Acid --- p.26 / Chapter 1.2.2.3 --- Metabolism of Conjugated Linoleic Acid --- p.30 / Chapter 1.2.2.4 --- Mode of Entry and Tissue Incorporation of Conjugated Linoleic Acid --- p.33 / Chapter 1.2.2.5 --- Toxicology of Conjugated Linoleic Acid --- p.33 / Chapter 1.2.3 --- Physiological Activities of Conjugated Linoleic Acid: Reported Health Benefits --- p.35 / Chapter 1.2.3.1 --- Anti-adipogenesis / Chapter 1.2.3.2 --- Anti-diabetogenesis --- p.36 / Chapter 1.2.3.3 --- Anti-atherosclerosis --- p.38 / Chapter 1.2.3.4 --- Anti-carcinogenesis --- p.39 / Chapter 1.2.3.5 --- Anti-tumor Activity --- p.40 / Chapter 1.2.3.6 --- Effects of Conjugated Linoleic Acid on Lipid Metabolism --- p.44 / Chapter 1.2.3.6.1 --- Actions on Phospholipids by Conjugated Linoleic Acid --- p.45 / Chapter 1.2.3.6.2 --- Conjugated Linoleic Acid as a Ligand for the PPAR System --- p.47 / Chapter 1.2.3.7 --- Immunomodulation --- p.47 / Chapter 1.3 --- Aims and Scopes of This Investigation --- p.50 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Animals --- p.52 / Chapter 2.1.2 --- Cell Lines --- p.52 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.52 / Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.54 / Chapter 2.1.5 --- Reagents and Buffers for Flow Cytometry --- p.58 / Chapter 2.1.6 --- Reagents for DNA Extraction --- p.59 / Chapter 2.1.7 --- Cell Death Detection ELISAPLUS Kit --- p.63 / Chapter 2.1.8 --- Reagents for Measuring Caspase Activity --- p.65 / Chapter 2.1.9 --- Reagents for Total RNA Isolation --- p.66 / Chapter 2.1.10 --- Reagents and Buffers for RT-PCR --- p.69 / Chapter 2.1.11 --- Reagents and Buffers for Gel Electrophoresis of Nucleic Acids --- p.74 / Chapter 2.1.12 --- "Reagents, Buffers and Materials for Western Blot Analysis" --- p.75 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.80 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Mouse Peritoneal Macrophages" --- p.81 / Chapter 2.2.3 --- Determination of Cell Viability --- p.82 / Chapter 2.2.4 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.83 / Chapter 2.2.5 --- In Vivo Tumorigenicity Study --- p.83 / Chapter 2.2.6 --- Analysis of Cell Cycle Profile / DNA Content by Flow Cytometry --- p.83 / Chapter 2.2.7 --- Measurement of Apoptosis --- p.84 / Chapter 2.2.8 --- Determination of the Mitochondrial Membrane Potential --- p.86 / Chapter 2.2.9 --- Measurement of Caspase Activity --- p.87 / Chapter 2.2.10 --- Study of Intracellular Accumulation of Reactive Oxygen Species (ROS) --- p.88 / Chapter 2.2.11 --- Study of the Scavenging Activity of Antioxidants --- p.88 / Chapter 2.2.12 --- Gene Expression Study --- p.89 / Chapter 2.2.13 --- Protein Expression Study --- p.92 / Chapter 2.2.14 --- Measurement of Cell Differentiation --- p.95 / Chapter 2.2.15 --- Statistical Analysis --- p.98 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI-TUMOR ACTICITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction / Chapter 3.2 --- Results --- p.99 / Chapter 3.2.1 --- Anti-proliferative Activity of CLA-mix and CLA Isomers on Various Myeloid Leukemia Cell Lines In Vitro --- p.101 / Chapter 3.2.2 --- Cytotoxic Effect of CLA-mix on the WEHI-3B JCS Cells In Vitro --- p.109 / Chapter 3.2.3 --- Cytotoxic Effect of CLA-mix on Primary Murine Myeloid Cells In Vitro --- p.111 / Chapter 3.2.4 --- Kinetic and Reversibility Studies of the Anti-proliferative Activity of CLA-mix on the WEHI-3B JCS Cells --- p.113 / Chapter 3.2.5 --- Effect of CLA-mix and its isomers on the Cell Cycle Profiles of the WEHI-3B JCS Cells In Vitro --- p.116 / Chapter 3.2.6 --- Effect of CLA-mix and its isomer on the Expression of Cell Cycle-regulatory Genes in the WEHI-3B JCS Cells --- p.123 / Chapter 3.2.7 --- Effect of CLA-mix and its isomer on the In V Tumorigenicity of the WEHI-3B JCS Cells ivo --- p.128 / Chapter 3.3 --- Discussion --- p.131 / Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING ACTIVITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 4.1 --- Introduction --- p.141 / Chapter 4.2 --- Results --- p.141 / Chapter 4.2.1 --- Induction of Apoptosis in Both Murine and Human Myeloid Leukemia Cells by CLA --- p.144 / Chapter 4.2.2 --- Effect of CLA and its Isomer on the Mitochondrial Membrane Potential of the WEHI-3B JCS Cells --- p.151 / Chapter 4.2.3 --- Effect of CLA-mix and its Isomer on the Expression of Apoptosis-regulatory Genes of the Bcl-2 Family in the WEHI-3B JCS Cells --- p.154 / Chapter 4.2.4 --- Effect of CLA-mix and its Isomer on the Expression of Apoptosis-regulatory Proteins in the WEHI-3B JCS Cells --- p.158 / Chapter 4.2.5 --- Effect of CLA-mix and its Isomer on the Induction of Caspase Activity in the WEHI-3B JCS Cells --- p.161 / Chapter 4.2.6 --- Effect of CLA-mix and its Isomer on the Induction of ROS in the WEHI-3B JCS Cells --- p.170 / Chapter 4.2.7 --- Effect of Antioxidants on the Induction of ROS by CLA-mix and its Isomer in the WEHI-3B JCS Cells --- p.173 / Chapter 4.2.8 --- Effect of Antioxidants on the Induction of Apoptosis by CLA-mix and its Isomer in the WEHI-3B JCS Cells --- p.176 / Chapter 4.2 --- Discussion / Chapter CHAPTER 5: --- STUDIES ON THE DIFFERENTIATION-INDUCING ACTIVITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 5.1 --- Introduction --- p.187 / Chapter 5.2 --- Results --- p.190 / Chapter 5.2.1 --- Morphological Alterations in CLA-mix- and CLA isomer-treated WEHI-3B JCS Cells --- p.190 / Chapter 5.2.2 --- Effects of CLA-mix on the Cell Size and Granularity of WEHI-3B JCS Cells --- p.196 / Chapter 5.2.3 --- Studies of the Surface Phenotypic Changes in the CLA-mix-treated WEHI-3B JCS cells --- p.198 / Chapter 5.2.4 --- Studies on the Induction of Monocytic Serine Esterase (MSE) Activity in the CLA-mix-treated WEHI-3B JCS Cells --- p.200 / Chapter 5.2.5 --- Studies on the Induction of Endocytic Activity in the CLA-mix-treated WEHI-3B JCS Cells --- p.201 / Chapter 5.2.6 --- Studies on the Expression of the Differentiation-regulatory Cytokine Genes in the CLA-mix-treated WEHI-3B JCS Cells --- p.202 / Chapter 5.3 --- Discussion --- p.204 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES REFERENCES --- p.208 / REFERENCES --- p.217