Dissertationen zum Thema „ApoE-/- mouse“

El Decabromodifenil èter (BDE-209) és un retardant de la flama àmpliament utilitzat i font de preocupació a causa de la toxicitat mostrada per altres Difenil Èters Polibromats (PBDEs). La presència de PBDEs en la llet materna fa preocupant la seva exposició durant el desenvolupament. Pensem que l’exposició primerenca a BDE-209 pot produir efectes a llarg termini i interactuar amb factors genètics, com el genotip de l’ApolipoproteinaE. Ratolins portadors de les diferents isoformeshumanes de l’ApoE foren tractats amb una dosi oral aguda de 0, 10 o 30 mg / kg de BDE-209 en el dia postnatal 10 i van ser avaluats per neurocomportament durant el desenvolupament, a l'edat adulta i la vellesa. L’exposició a BDE-209 indueix un retard en el desenvolupament físic i neuromotor i en la compactació de la mielina en els ratolins ApoE2, disminueix els nivells de tiroxina lliure en les femelles adultes i disminueix l'activitat en ratolins ApoE4. Els efectes més consistents durant tota la vida s'observen en ratolins ApoE3 i consisteixen en problemes d'aprenentatge als 4 mesos, i problemes d'aprenentatge i memòria i un augment de l'ansietat als 12 mesos.
Decabromodiphenyl Ether (BDE-209) is a flame retardant widely used and source of concern because ofthe toxicity showed by other Polibrominateddiphenyl ethers (PBDEs). The presenceof PBDEs in human’s breast milkmakes worrying its exposure during development. We hypothesised that an early exposure to BDE-209 can induce long-term impairments and interact with genetic factors, such as ApolipoproteinE genotype. Mice carrying the different Human ApoE isoforms treated with an acute oral dose of 0, 10 or 30 mg/kg of BDE-209 on postnatal day 10 were assessed for neurobehaviourduring development, in young adulthood and old age. BDE-209 exposure inducesadelay in physic and neuromotordevelopment and in myelin compaction in ApoE2 mice, decreases the levels of free thyroxin in adult femalesand decreases activity in ApoE4 mice. The most consistent effects across the lifespan are observed in ApoE3 mice and consist of impaired learning at 4 months, and impaired learning and memory and increased anxiety at 12 months.

Cyclodextrins (CDs) are naturally occurring cyclic oligosaccharides. Since it is believed that OxC blocks the removal of normal cholesterol from cells in the artery wall, it is possible that selective removal of OxC in the vessel wall in-vivo may prevent or reverse atherosclerosis.As a prelude to major studies, this research project was designed to answer two critical questions; 1/. What is the best route for delivery of CD. 2/. How do animals (apoE-/- mice) tolerate it. Pilot studies were established and results noted. These studies have provided valuable information in the apoE-/- mouse for subsequent studies to prevent or reverse atherosclerosis in this animal model.
Master of Science (Hons)

Abdominal Aortic Aneurysm (AAA) is a complex disease that leads to a localized dilation of the infrarenal aorta that develops over years. Longitudinal information in humans has been difficult to obtain for this disease, therefore mouse models have become increasingly used to study the development of AAAs. The objective of this study was to determine any changes that occur in the biomechanical response and fiber microstructure in the apolipoprotein E difficient (ApoE-/-) angiotensin II (AngII) infused mouse model of aneurysm during disease progression, as well as determine some of the underlying changes in biochemistry, and demonstrate a novel method of reducing any pathogenic protease activity. Using a Microbiaxial Opto-Mechanical Device (MOD), ex vivo studies included adult aortas of ApoE-/- AngII infused mice excised and tested for mechanical response simultaneously imaged using two-photon microscopy to assess the microstructure at multiple time points. In vitro and ex vivo studies have shown changes in protease concentrations with the use of FRET based proteolytic beacons able to provide a non-destructive method to quantify protease activity measured against mechanical and microstructural changes. In vitro studies have demonstrated protease activity can be reduced using a molecule providing a positive feedback mechanism for protease inhibition and possibly provide a reduction in aneurysm progression.

It is well established that atherosclerosis, an inflammatory response to chronic injury in the blood vessel wall, plays a leading role in the development and progression of cardiovascular disease. Mineralocorticoid receptor (MR) over-activation has been implicated in atherosclerosis. In mineralocorticoid-target tissues, 11β- Hydroxysteroid dehydrogenase type 2 (11β-HSD2) inactivates glucocorticoids, conferring aldosterone specificity upon the normally unselective MR. Recent evidence suggests that 11β-HSD2 may also afford protection of MR in the cells of the vasculature, providing possible mechanisms by which MR activation may directly promote atherosclerosis. Consistent with this, Apoe-/-/11β-HSD2-/- double knockout (DKO) mice show accelerated atheroma development. The present thesis tested the hypothesis that inactivation of 11β-HSD2, allowing inappropriate activation of MR in cells of the vasculature, accelerates atherogenesis through promotion of a pro-inflammatory environment with increased endothelial cell expression of adhesion molecules and subsequent macrophage infiltration into plaques. DKO mice received either the MR antagonist eplerenone (200mg/kg/day) or vehicle in normal chow diet from 2 months of age for 12 weeks. Eplerenone significantly decreased atherosclerotic burden in brachiocephalic arteries of DKO mice, an effect that was accompanied by alterations in the cellular composition of plaques such that a more stable collagen- and smooth muscle cell- rich plaque was formed. Eplerenone treatment was also associated with a reduction in vascular inflammation as demonstrated by a significant reduction in macrophage infiltration into DKO plaques. The accelerated atherogenesis in DKO mice was clearly evident by 3 months of age, a time point at which Apoe-/- mice were completely lesion free. By 6 months, some Apoe-/- mice had developed lesions whilst all DKO mice at this age showed much larger plaques. Compared to Apoe-/- mice, the cellular composition of DKO plaques was altered favouring vulnerability and inflammation, with increased macrophage and lipid content and decreased collagen content. To investigate the possible underlying mechanisms responsible for increased inflammatory cell content, the expression of vascular cell adhesion molecule 1 (VCAM-1) was compared in DKO and Apoe-/- brachiocephalic arteries. VCAM-1 immunostaining was significantly greater on the endothelial cells of DKO arteries at 3 months compared to age-matched Apoe-/- mice. At 6 months, DKO and Apoe-/- mice had similar expression of VCAM-1. Finally, mouse aortic endothelial cells (MAECs) were used to investigate the mechanism of adhesion molecule up-regulation in the absence of 11β-HSD2. Both aldosterone and TNF-α, included as a positive control, dramatically increased VCAM-1 expression in MAECs. Spironolactone pre-treatment blocked the effect of aldosterone, suggesting an MR-mediated mechanism. Corticosterone alone had no effect on VCAM-1 expression. However, inhibition of 11β-HSD2 by pre-treatment with glycyrrhetinic acid allowed corticosterone to induce a significant increase in the number of VCAM-1-stained MAECs, demonstrating functional expression of 11β- HSD2 in MAECs. Consistent with 11β-HSD2 involvement, VCAM-1 up-regulation by corticosterone in the presence of glycyrrhetinic acid was reversed by blockade of MR with spironolactone. In conclusion, loss of 11β-HSD2 activity leading to inappropriate activation of MR in atherosclerotic mice promotes plaque vulnerability and increases vascular infiltration of macrophages which accelerates plaque growth, possibly through enhanced MR- mediated endothelial cell expression of VCAM-1.

Cyclodextrins (CDs) are naturally occurring cyclic oligosaccharides. Since it is believed that OxC blocks the removal of normal cholesterol from cells in the artery wall, it is possible that selective removal of OxC in the vessel wall in-vivo may prevent or reverse atherosclerosis.As a prelude to major studies, this research project was designed to answer two critical questions; 1/. What is the best route for delivery of CD. 2/. How do animals (apoE-/- mice) tolerate it. Pilot studies were established and results noted. These studies have provided valuable information in the apoE-/- mouse for subsequent studies to prevent or reverse atherosclerosis in this animal model.

Alzheimer’s disease (AD) is the most common form of dementia and a growing disease burden that has seen pharmacological interventions primarily fail. Instead, it has been suggested that preventative measures such as a healthy diet may be the best way in preventing AD. Prebiotics are one such potential measure and are fermented into metabolites by the gut microbiota and acting as gut-brain axis components, beneficially impact the brain. However, the impact of prebiotics in AD prevention is unknown. Here we show that the prebiotic inulin increased multiple gut-brain axis components such as scyllo-inositol and short chain fatty acids in the gut, periphery, and in the case of scyllo-inositol, the brain. We found in E3FAD and E4FAD mice fed either a prebiotic or control diet for 4-months, that the consumption of the prebiotic inulin can beneficially alter the gut microbiota, modulate metabolic function, and dramatically increase scyllo-inositol in the brain. This suggests that the consumption of prebiotics can beneficially impact the brain by enhancing metabolism, helping to decrease AD risk factors.

Spectral karyotyping (SKY) is a widely used methodology to identify genetic aberrations. Multicolor fluorescence in situ hybridization using chromosome painting probes in individual colors for all metaphase chromosomes at once is combined with a unique spectral measurement and analysis system to automatically classify normal and aberrant chromosomes. Based on countless studies and investigations in many laboratories worldwide, numerous new chromosome translocations and other aberrations have been identified in clinical and tumor cytogenetics. Thus, gene identification studies have been facilitated resulting in the dissection of tumor development and progression. For example, different translocation partners of the TEL/ETV6 transcription factor that is specially required for hematopoiesis within the bone marrow were identified. Also, the correct classification of complex karyotypes of solid tumors supports the prognostication of cancer patients. Important accomplishments for patients with genetic diseases, leukemias and lymphomas, mesenchymal tumors and solid cancers are summarized and exemplified. Furthermore, studies of disease mechanisms such as centromeric DNA breakage, DNA double strand break repair, telomere shortening and radiation-induced neoplastic transformation have been accompanied by SKY analyses. Besides the hybridization of human chromosomes, mouse karyotyping has also contributed to the comprehensive characterization of mouse models of human disease and for gene therapy studies
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Spectral karyotyping (SKY) is a widely used methodology to identify genetic aberrations. Multicolor fluorescence in situ hybridization using chromosome painting probes in individual colors for all metaphase chromosomes at once is combined with a unique spectral measurement and analysis system to automatically classify normal and aberrant chromosomes. Based on countless studies and investigations in many laboratories worldwide, numerous new chromosome translocations and other aberrations have been identified in clinical and tumor cytogenetics. Thus, gene identification studies have been facilitated resulting in the dissection of tumor development and progression. For example, different translocation partners of the TEL/ETV6 transcription factor that is specially required for hematopoiesis within the bone marrow were identified. Also, the correct classification of complex karyotypes of solid tumors supports the prognostication of cancer patients. Important accomplishments for patients with genetic diseases, leukemias and lymphomas, mesenchymal tumors and solid cancers are summarized and exemplified. Furthermore, studies of disease mechanisms such as centromeric DNA breakage, DNA double strand break repair, telomere shortening and radiation-induced neoplastic transformation have been accompanied by SKY analyses. Besides the hybridization of human chromosomes, mouse karyotyping has also contributed to the comprehensive characterization of mouse models of human disease and for gene therapy studies.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015
As doenças que afectam o sistema cardiovascular e o osso estão entre as doenças que causam maior morbilidade e mortalidade na população, em especial a aterosclerose e a osteoporose. Vários estudos epidemiológicos têm demonstrado que estas duas doenças podem estar relacionadas de alguma forma, uma vez que ambas as patologias estão normalmente presentes no mesmo doente. Doentes com patologia cardiovascular apresentam um risco 1.2 a 6.7 vezes maior de sofrer de fraturas de fragilidade comparando com a população em geral, e esta associação também foi demonstrada em doentes com uma massa óssea reduzida que têm um risco aumentado de desenvolver doenças ao nível cardiovascular, em especial doentes com doenças autoimunes como lúpus eritematoso sistémico ou a artrite reumatoide. A aterosclerose é caracterizada pela acumulação de partículas lipídicas e células do sistema imunitário que levam ao estreitamento do lúmen das artérias, e numa fase mais avançada do processo pode ocorrer ruptura destas placas ateroscleróticas com possível formação de trombos. Estudos recentes têm demostrado o envolvimento do sistema imune na patogénese da aterosclerose. Este processo é iniciado pelos monócitos que posteriormente se diferenciam em macrófagos com capacidade de acumular lípidos no citoplasma formando as conhecidas foam cells, ou células espumosas, com capacidade de produzir citocinas pro-inflamatórias. Estas citocinas desempenham diversas funções no organismo, mas estão maioritariamente associadas a processos inflamatórios, processos estes que levam à progressão da aterosclerose, mas também estão associados a outras patologias como a osteoporose. A osteoporose é uma doença resultante de um desequilíbrio no turnover ósseo, caracterizada pela perda de massa óssea, com alteração da microarquitectura do osso, diminuição da densidade mineral óssea e consequente aumento das fraturas de fragilidade. A associação entre estas duas patologias tem ganho robustez especialmente com o paradoxo da calcificação das placas com a baixa densidade óssea e também com a presença, no vaso, de várias proteínas associadas à remodelação óssea, como RANKL (Receptor Activator of Nuclear factor Kappa-B Ligand), OPG (Osteoprotegerin) e colagénio tipo 1. Estudos em modelos animais têm reforçado estes dados. Ratinhos knock-out para OPG, para além de apresentarem uma osteoporose generalizada, exibiram também um aumento da calcificação ao nível vascular. Este estudo centrou-se na hipótese da existência de mecanismos fisiopatológicos comuns aos dois sistemas. Pretendemos avaliar ao longo do tempo, utilizando um modelo animal (ApoE -/-) que é geneticamente propenso ao desenvolvimento de aterosclerose (por delecção do gene da apolipoproteína E), o efeito da inflamação sobre os ossos e os vasos. Os objectivos deste trabalho foram a avaliação dos níveis séricos de CTX-I (C-terminal cross-link telopeptide of type I collagen) e P1NP (N-propeptide of type I collagen), duas proteínas resultantes do processo de turnover ósseo (marcadores de reabsorção e de formação óssea, respectivamente), como também a microarquitectura óssea das vértebras lombares por histomorfometria, para compreender possíveis efeitos ao nível do osso trabecular. Pretendemos ainda avaliar a progressão das lesões ateroscleróticas e avaliar a relação entre a aorta e o osso pela determinação dos níveis de expressão de genes associados com a inflamação (interleucina (IL-) 1β; IL-6; IL-17A e TNF (Tumor Necrosis Factor)) e com a remodelação óssea (RANKL e OPG). Adicionalmente, como objectivo secundário deste estudo, pretendeu-se avaliar as variações no osso e no vaso, especialmente o desenvolvimento de lesões ateroscleróticas ao nível da aorta, num modelo animal inflamatório (K/BxAg7), que desenvolve uma artrite espontânea (com consequente desenvolvimento de uma osteoporose secundária) e que foi descrito como estando predisposto para o desenvolvimento de aterosclerose. Para cumprir com os objectivos foram formados 5 grupos correspondendo aos diferentes time-points (8, 16, 20, 24 e 28 semanas de idade) para as duas estirpes de ratinhos usados (ApoE -/-, como modelo de aterosclerose, e C57BL/6, como controlo saudável), com 10 animais em cada grupo, dos quais 5 foram destinados para os testes de expressão génica (osso e aorta) e histomorfometria, e os outros 5 para avaliação das lesões na aorta. Todos os ratinhos estiveram sob uma dieta gorda desde as 10 semanas. Para o objectivo secundário foram usados ratinhos K/BxAg7 divididos em dois grupos, num foi introduzida a dieta gorda às 10 semanas de idade (N=4) e noutro manteve-se com a dieta normal durante toda a experiência (N=5). Estes animais foram avaliados às 24 semanas de idade, com doseamento dos níveis séricos de CTX-I e P1NP, avaliação da expressão génica (osso e aorta) e da presença de lesões na aorta. A todos os animais, no momento da eutanásia foi recolhido sangue por punção cardíaca e posteriormente centrifugado para a obtenção do plasma. Após perfusão, as aortas foram recolhidas e conservadas em PBS (até serem congeladas com azoto líquido para posteriormente se proceder à extração de RNA) ou em paraformaldeído para a quantificação das lesões por marcação dos lípidos com Oil Red O. Foram ainda recolhidos e imediatamente congelados diversos ossos, incluindo um fémur (após remoção da medula) para extracção de RNA e as vértebras lombares para a histomorfometria. Os níveis séricos de CTX-I e P1NP foram determinados por ELISA (Enzyme-Linked Immunosorbent Assay) e as vértebras lombares (L3-L4) foram analisadas por histomorfometria para a determinação do volume de osso trabecular, espessura trabecular e também a separação entre trabéculas. Os fémures e aortas destinados à extração de RNA foram reduzidos a um pó fino, a extração foi efectuada com TRIzol®/clorofórmio e posteriormente foi realizada a síntese de cDNA para a análise da expressão génica utilizando SYBR®Green e primers desenhados especificamente para cada gene a avaliar. A quantificação foi feita por PCR (Polymerase Chain Reaction) quantitativo em tempo real e na análise final todos os genes foram normalizados para o rRNA 18S utilizado como housekeeping gene (gene de referência). Todos os animais utilizados neste estudo foram avaliados semanalmente e o seu peso registado. Adicionalmente, ao modelo de artrite, e como forma de avaliar a progressão da doença, foi aplicado um score clínico avaliado pela observação semanal dos animais. Pela avaliação dos dados histomorfométricos e dos níveis séricos dos marcadores de remodelação óssea verifica-se uma concordância entre os resultados obtidos, com uma diminuição significativa no volume ósseo, na espessura trabecular e níveis de P1NP ao longo do tempo, e um aumento na separação inter-trabecular e nos níveis séricos de CTX –I, sugerindo que ambos os grupos experimentais foram sofrendo de uma perda de massa óssea, com especial ênfase no grupo ApoE -/-, onde se verificaram algumas diferenças estatisticamente significativas em comparação com o grupo B6. Os resultados obtidos na expressão génica mostraram algumas diferenças estatisticamente significativas entre B6 e ApoE -/-, contudo, de uma maneira geral, foi o grupo dos ratinhos B6 que apresentou maiores níveis de expressão das citocinas, tanto na aorta como no osso. Não se encontrou uma associação com significância estatística na correlação da expressão das citocinas entre os dois tecidos. No entanto verificámos a existência de uma relação entre IL-1β e IL-6, e entre TNF e OPG no osso dos ApoE -/- às 16 semanas e no grupo dos B6 às 28 semanas apenas entre TNF e OPG, mas estas correlações foram pontuais, cingindo-se a apenas um ponto de avaliação, e não se verificou a continuidade destas correlações nos time-points seguintes. A falta de um forte suporte estatístico nestes resultados pode dever-se ao facto de a expressão dos genes avaliados, principalmente na aorta, apresentar valores muito dispersos, além de serem muito diferentes dos valores obtidos no osso. Esta discrepância poderá dever-se ao facto de a aorta ser um tecido menos robusto e que poderá estar mais susceptível à degradação do RNA durante o processamento da amostra. Procedeu-se posteriormente à comparação dos dados obtidos nas diversas análises efectuadas, medição de marcadores séricos, histomorfometria e expressão génica. Observou-se uma correlação negativa entre a espessura trabecular e os níveis de expressão da OPG no osso dos ratinhos ApoE -/- com 16 semanas, e também entre o volume ósseo e a separação trabecular no grupo B6 às 28 semanas, mas mais uma vez estas correlações foram pontuais, sem grande significado biológico. Estes resultados estão de acordo com o esperado face aos dados epidemiológicos e de estudos em modelo animal que reforçam a associação entre a progressão da aterosclerose e a perda de massa óssea com possível desenvolvimento de uma osteoporose. Contudo os dados de expressão génica não apoiam esta conclusão (chegando a ser discordantes), apesar de existirem alguns estudos com este modelo animal que reportam um aumento da formação óssea em ratinhos ApoE -/-, pelo que os dados com este modelo são ainda um pouco contraditórios. É importante ter em conta que o grupo controlo (B6) estava também sujeito a dieta gorda, uma vez que esta dieta potencia e acelera o desenvolvimento das lesões ateroscleróticas nos ratinhos ApoE -/-. A dieta gorda foi introduzida em ambos os grupos, a fim de uniformizar qualquer alteração introduzida pela dieta, pois um estudo prévio de Hirasawa et al. reportou que a dieta gorda induz uma redução da mineralização óssea. No entanto face aos resultados obtidos, este trabalho poderia beneficiar de uma análise dos parâmetros estudados, num grupo controlo (B6) sem dieta gorda de forma a verificar os níveis normais de todas as proteínas estudadas. Quanto ao objectivo secundário e ao modelo de inflamação (K/BxAg7), não foram encontradas lesões ateroscleróticas visíveis na aorta, contrariamente ao que foi reportado por Rose et al.. Este resultado poderá ter duas justificações: as condições de alojamento dos animais, visto que se encontravam num biotério de roedores SPF (specific pathogen-free), e o tempo de avaliação deste estudo. Relativamente às condições de alojamento, estas podem ter influência na microbiota o que, por sua vez, poderá ter um importante papel no desenvolvimento das lesões (foi já descrito como tendo uma elevada relevância no desenvolvimento de diversas doenças inflamatórias). Os dados preliminares obtidos não mostram nenhuma diferença estatisticamente significativa, contudo, de uma forma geral, ambos os grupos de K/BxAg7 mostraram níveis elevados da expressão dos genes analisados. No entanto, os níveis séricos apontam para uma remodelação óssea alterada no sentido da formação (aumento dos níveis de P1NP, e do ratio CTX-I/P1NP), contrariamente ao esperado, não só pela descrição do modelo, como pelo aumento do score clínico verificado nestas animais ao longo do tempo. Conclui-se com este trabalho que o modelo animal de aterosclerose apresenta algumas alterações ao nível do osso, ao longo do tempo, apesar dos resultados obtidos necessitarem ser confirmados para melhor compreensão do seu significado biológico. De forma a melhorar este trabalho seria interessante aumentar o número de animais em estudo (B6, ApoE -/- e K/BxAg7) para reforçar o poder estatístico dos resultados, como também para verificar se as tendências observadas se mantêm. Relativamente ao modelo animal de inflamação, em específico, seria importante fazer uma análise histomorfométrica para avaliar as alterações na microarquitectura do osso, num modelo caracterizado pelas conhecidas alterações ósseas, e uma vez que não se observaram lesões visíveis nos vasos destes ratinhos, e sabendo a importância do ambiente em que os animais estão alojados, seria importante testar os efeitos sobre os vasos deste modelo num ambiente menos limpo, colocando-os por exemplo, num biotério convencional. Será também interessante, se os ratinhos apresentarem lesões em ambos os tecidos (ossos e vasos), avaliar a progressão estudando os mesmos time-points definidos para os outros modelos experimentais.
Several epidemiological studies have revealed a link between atherosclerosis and osteoporosis, suggesting the existence of common underlying mechanisms. This relationship maybe due to the inflammatory processes that underlies both diseases. Thus, mechanisms regulating inflammation and also bone remodeling could represent potential links between these two diseases. Therefore this work focused on the evaluation of the alterations in bone and vessel of an animal model of atherosclerosis (ApoE -/-). Using different time-points, we measured the serum levels of CTX-I and P1NP to evaluate the systemic repercussions of bone remodeling, and performed histomorphometric analysis to evaluate bone behavior. We have also evaluated the relative gene expression, in bones and aortas, of genes associated with inflammation (IL-1β, IL-6, IL-17A and TNF) and also RANKL and OPG, associated with bone remodeling. The histomorphometric analysis and bone turnover proteins determination in serum shown concordant results, pointing towards bone loss, with the decrease levels of bone volume, trabecular thickness, and P1NP levels in serum coupled with an increase in trabecular separation, and CTX-I in serum. The expression of the genes associated with inflammation in ApoE -/- mice has, in general, lower levels, even in the later time-points, when compared to C57BL/6 mice. Additionally, a mouse model of inflammation (K/BxAg7) was used to evaluate the progression of both diseases in the same organism, these mice did not developed visible atherosclerotic lesions in the aorta and no significant differences were found comparing to a healthy group (C57BL/6 mice), despite the K/BxAg7 displayed a general increase in the expression of the inflammation related genes, in both aorta and bone. Our finding suggests that ApoE -/- mice suffer from bone alterations, and that could be associated with the atherosclerosis development although we did not find any correlation with inflammatory genes in the tissues affected. To complement this project it will be interesting to increase de number of animals in the study and also to include another group composed by B6 mice but without the fat diet. As for the model of inflammation, it will be interesting to house this animals in a conventional animal facility, with a more challenging environment, to see if any visible atherosclerotic lesion appear.

Atheroma, heart attacks and strokes continue to be a major cause of morbidity and mortality in our community. Atherosclerosis is a chronic inflammatory vascular disease, characterised by thickening of the vascular wall due to lipid accumulation, infiltration by circulating monocytes and T cells and proliferation of smooth muscle cells. Leukocyte adherence to the blood vessel wall is promoted by the up-regulation of cell adhesion molecules (CAM) by atherogenic substances such as tumour necrosis factor (TNF-α) and oxidised low density lipoprotein (oxidised-LDL). Recently our group has synthesised a novel polyunsaturated fatty acid, β-oxa 23:4n-6 which inhibits CAM up-regulation in blood vessel walls. It was therefore the objective of this thesis to determine whether this fatty acid protects against atherosclerosis. Advantage was taken of an experimental model of this disease, the apoE deficient mouse (apoE [superscript -/-]) which spontaneously develop atherosclerosis. To assist our studies on MP3, we established an appropriate classification of different stages of atherosclerotic lesions and defined the kinetics of development of the disease in this model. By examining of the sections at the level of aortic roots the atherosclerotic lesions were classified into six categories. This classification was based on the histological characteristics of the plaque component including the degree of macrophage infiltration and foam cells formation, the presence of cholesterol clefts and confluent lipid cores, calcification and ossification, the composition of the fibrous cap, the media involvement and the incipient/actual aneurysm formation and inflammation, including neutrophils. Kinetics of plaque development under the influence of a high fat and high cholesterol diet followed an exponential relationship of y= -e [ superscript -x ]. The asymptotic characteristic of this lesion development was however a function of compensatory aortic enlargement which accompanied the increase in lesion development and size. Thus it is concluded that the level of atherosclerosis needs to be gauged by the size of the lesion per se. This may be particularly important for the assessment of anti-atherogenic effects of drugs. Therefore attempts to develop a quantitative system to assess plaques revealed that expression of plaque size as % of occupation of blood vessel had limitations. Using this model we were able to demonstrate that injections of the novel polyunsaturated fatty acid, MP3 led to a significant reduction/inhibition (70%) of plaque area and a corresponding 60% inhibition of aortic size. As expected this inhibition was not as evident when results were expressed as % of aortic lumen size. The results also suggested that protection by MP3 was dependent on conditions which promoted increased uptake into tissues by, for example, preloading animals with MP3 prior to commencing the high fat high cholesterol diet. The protective effects of MP3 are consistent with a role for the activation of the transcriptional factor, NFkB and up-regulation of cell adhesion molecules in this disease, and the ability of MP3 to inhibit these targets. Thus the objective of this research has been achieved and the hypothesis proven.
Thesis (M.Med.Sc.)--Univesity of Adelaide, School of Paediatrics & Reproductive Health, 2005.

Atheroma, heart attacks and strokes continue to be a major cause of morbidity and mortality in our community. Atherosclerosis is a chronic inflammatory vascular disease, characterised by thickening of the vascular wall due to lipid accumulation, infiltration by circulating monocytes and T cells and proliferation of smooth muscle cells. Leukocyte adherence to the blood vessel wall is promoted by the up-regulation of cell adhesion molecules (CAM) by atherogenic substances such as tumour necrosis factor (TNF-α) and oxidised low density lipoprotein (oxidised-LDL). Recently our group has synthesised a novel polyunsaturated fatty acid, β-oxa 23:4n-6 which inhibits CAM up-regulation in blood vessel walls. It was therefore the objective of this thesis to determine whether this fatty acid protects against atherosclerosis. Advantage was taken of an experimental model of this disease, the apoE deficient mouse (apoE [superscript -/-]) which spontaneously develop atherosclerosis. To assist our studies on MP3, we established an appropriate classification of different stages of atherosclerotic lesions and defined the kinetics of development of the disease in this model. By examining of the sections at the level of aortic roots the atherosclerotic lesions were classified into six categories. This classification was based on the histological characteristics of the plaque component including the degree of macrophage infiltration and foam cells formation, the presence of cholesterol clefts and confluent lipid cores, calcification and ossification, the composition of the fibrous cap, the media involvement and the incipient/actual aneurysm formation and inflammation, including neutrophils. Kinetics of plaque development under the influence of a high fat and high cholesterol diet followed an exponential relationship of y= -e [ superscript -x ]. The asymptotic characteristic of this lesion development was however a function of compensatory aortic enlargement which accompanied the increase in lesion development and size. Thus it is concluded that the level of atherosclerosis needs to be gauged by the size of the lesion per se. This may be particularly important for the assessment of anti-atherogenic effects of drugs. Therefore attempts to develop a quantitative system to assess plaques revealed that expression of plaque size as % of occupation of blood vessel had limitations. Using this model we were able to demonstrate that injections of the novel polyunsaturated fatty acid, MP3 led to a significant reduction/inhibition (70%) of plaque area and a corresponding 60% inhibition of aortic size. As expected this inhibition was not as evident when results were expressed as % of aortic lumen size. The results also suggested that protection by MP3 was dependent on conditions which promoted increased uptake into tissues by, for example, preloading animals with MP3 prior to commencing the high fat high cholesterol diet. The protective effects of MP3 are consistent with a role for the activation of the transcriptional factor, NFkB and up-regulation of cell adhesion molecules in this disease, and the ability of MP3 to inhibit these targets. Thus the objective of this research has been achieved and the hypothesis proven.
Thesis (M.Med.Sc.)--University of Adelaide, School of Paediatrics & Reproductive Health, 2005.

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
A aterosclerose é o principal substrato para a maioria das doenças cardiovasculares (DCVs), que são a maior causa de mortalidade a nível mundial. Os fatores de risco para o desenvolvimento de aterosclerose e de outras DCVs, além dos não modificáveis, relacionam-se com estilos de vida pouco saudáveis, incluindo dietas hipercalóricas e hiperlipídicas, bem como sedentarismo. Estes fatores estão associados a outras doenças, tais como obesidade e esteatose hepática. Abordagens não farmacológicas, nomeadamente intervenções nutracêuticas, poderão servir como terapias adjuvantes às terapêuticas farmacológicas atualmente disponíveis. A Dieta Mediterrânica (MDiet), baseada numa grande variedade de alimentos pouco refinados, tem sido considerada como um modelo dietético por várias organizações internacionais. O azeite, uma das principais fontes de gordura da MDiet, composto essencialmente por ácidos gordos monoinsaturados, apresenta uma pequena pequena fração de compostos fenólicos, que têm sido vistos como uma opção nutracêutica, exibindo efeitos marcantes na prevenção destas doenças. Um dos mais promissores é a oleaceína (OLEA), um polifenol do grupo dos secoroidoides que já tem mostrado in vitro efeitos antioxidantes, anti-inflamatórios, sensibilizadores de insulina e ainda potencial para propriedades hipolipemiantes e antiateroscleróticas.Neste sentido, formulámos a hipótese de que uma intervenção nutracêutica com OLEA poderia exercer efeitos hepato e ateroprotetores num modelo animal susceptível ao desenvolvimento de aterosclerose, o murganho knockout para a apolipoproteina E (APOE KO) alimentado com uma dieta aterogénica (ATD). Desta forma, definimos duas abordagens experimentais. A primeira usando murganhos C57BL/6J wild-type (WT) com o objetivo de comparar os efeitos metabólicos de três regimes alimentares – i) dieta controlo padronizada não refinada (STD, n=8), ii) dieta refinada com baixo teor de gordura (LF, n=8) e iii) dieta refinada aterogénica (ATD, n=8) - ao nível dos perfis glicémico, insulinémico e lipídico. A segunda avaliando o potencial efeito hepatoprotetor, antidislipidémico e anti-aterosclerótico da OLEA no murganho APOE KO, onde se definiram dois grupos - com e sem tratamento com 50 mg/kg/dia de OLEA (n=8 cada) – comparados com um grupo WT alimentado com ATD. Todos os grupos experimentais tiveram alimento e água fornecidos ad libitum.Os perfis glicémicos e insulinémicos foram avaliados nos tempos inicial e final e o perfil lipídico foi caracterizado em termos de valores séricos de colesterol total (c-Total), TGs, c-LDL e c-HDL, bem como de conteúdos hepáticos de TGs. Utilizou-se marcações histomorfológicas e observações de microscopia eletrónica de varrimento (SEM) para caracterizar as lesões hepáticas e vasculares e a deposição lipídica. Avaliou-se também os níveis séricos e hepáticos de SOD e quantificou-se a expressão hepática de SOD-1 e SOD-2 por RT-PCR.Os animais WT alimentados com ATD apresentaram um aumento significativo do peso corporal, sem alterações de tolerância à glicose ou de sensibilidade à insulina. Constatou-se existir no mesmo grupo um aumento da concentração sérica de c-Total, c-LDL e c-HDL, acompanhado de acumulação de TGs no fígado. Além disso, exibiram reduzida expressão hepática de ambas as isoformas de SOD, o que pode ser indicativo de um processo de stress oxidativo característico destas doenças.Os animais alimentados com a dieta LF apresentaram alterações metabólicas mais moderadas do que os submetidos a ATD, incluindo uma tendência para valores superiores de glucose circulante após 6 horas em jejum, bem como um aumento da concentração sérica de c-Total, c-LDL e c-HDL, e de TGs hepáticos, quando comparados com os animais alimentados com STD, sugerindo que a presença de ingredientes refinados nesta dieta, embora que com baixo teor de gordura, não impede um impacto negativo no metabolismo, comparativamente à dieta STD não refinada.Em relação à segunda abordagem experimental, constatou-se (por H&E e SEM) existirem lesões vasculares nos animais APOE KO compatíveis com a literatura. A OLEA apresentou um efeito marcante no perfil lipídico dos murganhos APOE KO, causando uma redução marcada dos valores séricos de c-Total, c-LDL, c-HDL e TGs, bem como uma forte atenuação da concentração hepática de TGs. Estes dados foram corroborados pelas marcações hepáticas com Oil Red O e H&E, verificando-se um efeito marcado anti-dislipidémico nos murganhos APOE KO. Não se observaram diferenças significativas nos perfis glicémicos e insulinémicos entre os 3 grupos.Globalmente, verifica-se que a suplementação com OLEA melhorou significativamente o perfil lipídico dos murganhos APOE KO, com forte proteção contra a deposição lipídica hepática e eventual efeito antioxidante. Mais estudos serão necessários para clarificar os mecanismos celulares e moleculares subjacentes à proteção contra o desenvolvimento de esteatose hepática neste modelo de hiperlipidema e aterosclerose.
Atherosclerosis is the main substrate for most cardiovascular diseases (CVDs) which are the main cause of mortality worldwide. The risk factors for the development of atherosclerosis and CVDs, in addiction to non-modifiable are related to unhealthy lifestyles, including hypercaloric and hyperlipidic diets, as well as sedentary lifestyle. These factors are associated with other diseases, such as obesity and hepatic steatosis. The non-pharmacological approaches, namely nutraceutical interventions, can serve as adjuvant approaches to the currently available pharmacologic therapies. The Mediterranean Diet (MDiet), based on a wide variety of unrefined foods, has been considered a dietary model by several international organizations. Olive oil, one of MDiet's main sources of fat, composed essentially of monounsaturated fatty acids, has a small fraction of phenolic compounds, which have been seen as a nutraceutical option, exhibiting marked effects in preventing these diseases. One of the most promising is oleacein (OLEA), a polyphenol from the secoiridoids group that has already shown in vitro antioxidant, anti-inflammatory, insulin sensitizing effects and even potential for lipid-lowering and anti-atherosclerotic properties.In this sense, we hypothesized that a nutraceutical intervention with OLEA could exert hepato- and atheroprotective effects in an animal model susceptible to the development of atherosclerosis, the apolipoprotein E (APOE KO) knockout mouse fed an atherogenic diet (ATD). In this way, we define two experimental approaches. The first using C57BL/6J wild-type (WT) mice to compare the metabolic effects of three diets – i) unrefined standardized control diet (STD, n=8), ii) refined low-fat diet (LF, n=8) and iii) atherogenic refined diet (ATD, n=8) - at the level of glycemic, insulinemic and lipid profiles. The second evaluating the potential hepatoprotective, anti-dyslipidemic and anti-atherosclerotic effect of OLEA in the APOE KO mouse, where two groups were defined - with and without treatment with 50 mg/kg/day of OLEA (n=8 each) - compared with a WT group fed with ATD. All experimental groups had food and water provided ad libitum.The glycemic and insulinemic profiles were evaluated in the initial and final times and the lipid profile was characterized in terms of serum values of total cholesterol (c-Total), TGs, c-LDL and c-HDL, as well as hepatic contents of TGs. Histomorphological markings and scanning electron microscopy (SEM) observations were used to characterize liver and vascular lesions and lipid deposition. Serum and hepatic SOD levels were also evaluated and the hepatic expression of SOD-1 and SOD-2 was quantified by RT-PCR.WT animals fed with ATD showed a significant increase in body weight, without changes in glucose tolerance or insulin sensitivity. It was found that in the same group there was an increase in the serum concentration of c-Total, c-LDL and c-HDL, accompanied by accumulation of TGs in the liver. In addition, they exhibited reduced hepatic expression of both SOD isoforms, which may be indicative of an oxidative stress process characteristic of these diseases.The animals fed the LF diet showed more moderate metabolic alterations than those submitted to ATD, including a trend towards higher values of circulating glucose after 6 hours of fasting, as well as an increase in the serum concentration of c-Total, c-LDL and c-HDL, and liver TGs, when compared to animals fed with STD, suggesting that the presence of refined ingredients in this diet, although low in fat, does not prevent a negative impact on metabolism, compared to the unrefined STD diet.Regarding the second experimental approach, it was found (by H&E and SEM) that there were vascular lesions in APOE KO animals compatible with the literature. OLEA had a marked effect on the lipid profile of APOE KO mice, causing a marked reduction in the serum values of c-Total, c-LDL, c-HDL and TGs, as well as a strong attenuation of the hepatic concentration of TGs. These data were corroborated by the liver markings with Oil Red O and H&E, showing a marked anti-dyslipidemic effect in the APOE KO mice. There were no significant differences in glycemic and insulinemic profiles between the 3 groups.Overall, it appears that supplementation with OLEA significantly improved the lipid profile of APOE KO mice, with strong protection against hepatic lipid deposition and an eventual antioxidant effect. Further studies will be needed to clarify the cellular and molecular mechanisms underlying protection against the development of hepatic steatosis in this model of hyperlipidema and atherosclerosis.
FCT
FCT
FCT
Outro - COMPETE-FEDER funds (POCI-01-0145-FEDER-007440 and POCI-01-0145-FEDER-031712)
Outro - European Regional Development Fund (FEDER), through Programa Operacional Factores de Competitividade COMPETE2020 (CENTRO-01-0145-FEDER-000012-HealthyAging2020)

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
A aterosclerose é o principal substrato para a maioria das doenças cardiovasculares (DCVs), que são a maior causa de mortalidade a nível mundial. Os fatores de risco para o desenvolvimento de aterosclerose e de outras DCVs, além dos não modificáveis, relacionam-se com estilos de vida pouco saudáveis, incluindo dietas hipercalóricas e hiperlipídicas, bem como sedentarismo. Estes fatores estão associados a outras doenças, tais como obesidade e esteatose hepática. Abordagens não farmacológicas, nomeadamente intervenções nutracêuticas, poderão servir como terapias adjuvantes às terapêuticas farmacológicas atualmente disponíveis. A Dieta Mediterrânica (MDiet), baseada numa grande variedade de alimentos pouco refinados, tem sido considerada como um modelo dietético por várias organizações internacionais. O azeite, uma das principais fontes de gordura da MDiet, composto essencialmente por ácidos gordos monoinsaturados, apresenta uma pequena pequena fração de compostos fenólicos, que têm sido vistos como uma opção nutracêutica, exibindo efeitos marcantes na prevenção destas doenças. Um dos mais promissores é a oleaceína (OLEA), um polifenol do grupo dos secoroidoides que já tem mostrado in vitro efeitos antioxidantes, anti-inflamatórios, sensibilizadores de insulina e ainda potencial para propriedades hipolipemiantes e antiateroscleróticas.Neste sentido, formulámos a hipótese de que uma intervenção nutracêutica com OLEA poderia exercer efeitos hepato e ateroprotetores num modelo animal susceptível ao desenvolvimento de aterosclerose, o murganho knockout para a apolipoproteina E (APOE KO) alimentado com uma dieta aterogénica (ATD). Desta forma, definimos duas abordagens experimentais. A primeira usando murganhos C57BL/6J wild-type (WT) com o objetivo de comparar os efeitos metabólicos de três regimes alimentares – i) dieta controlo padronizada não refinada (STD, n=8), ii) dieta refinada com baixo teor de gordura (LF, n=8) e iii) dieta refinada aterogénica (ATD, n=8) - ao nível dos perfis glicémico, insulinémico e lipídico. A segunda avaliando o potencial efeito hepatoprotetor, antidislipidémico e anti-aterosclerótico da OLEA no murganho APOE KO, onde se definiram dois grupos - com e sem tratamento com 50 mg/kg/dia de OLEA (n=8 cada) – comparados com um grupo WT alimentado com ATD. Todos os grupos experimentais tiveram alimento e água fornecidos ad libitum.Os perfis glicémicos e insulinémicos foram avaliados nos tempos inicial e final e o perfil lipídico foi caracterizado em termos de valores séricos de colesterol total (c-Total), TGs, c-LDL e c-HDL, bem como de conteúdos hepáticos de TGs. Utilizou-se marcações histomorfológicas e observações de microscopia eletrónica de varrimento (SEM) para caracterizar as lesões hepáticas e vasculares e a deposição lipídica. Avaliou-se também os níveis séricos e hepáticos de SOD e quantificou-se a expressão hepática de SOD-1 e SOD-2 por RT-PCR.Os animais WT alimentados com ATD apresentaram um aumento significativo do peso corporal, sem alterações de tolerância à glicose ou de sensibilidade à insulina. Constatou-se existir no mesmo grupo um aumento da concentração sérica de c-Total, c-LDL e c-HDL, acompanhado de acumulação de TGs no fígado. Além disso, exibiram reduzida expressão hepática de ambas as isoformas de SOD, o que pode ser indicativo de um processo de stress oxidativo característico destas doenças.Os animais alimentados com a dieta LF apresentaram alterações metabólicas mais moderadas do que os submetidos a ATD, incluindo uma tendência para valores superiores de glucose circulante após 6 horas em jejum, bem como um aumento da concentração sérica de c-Total, c-LDL e c-HDL, e de TGs hepáticos, quando comparados com os animais alimentados com STD, sugerindo que a presença de ingredientes refinados nesta dieta, embora que com baixo teor de gordura, não impede um impacto negativo no metabolismo, comparativamente à dieta STD não refinada.Em relação à segunda abordagem experimental, constatou-se (por H&E e SEM) existirem lesões vasculares nos animais APOE KO compatíveis com a literatura. A OLEA apresentou um efeito marcante no perfil lipídico dos murganhos APOE KO, causando uma redução marcada dos valores séricos de c-Total, c-LDL, c-HDL e TGs, bem como uma forte atenuação da concentração hepática de TGs. Estes dados foram corroborados pelas marcações hepáticas com Oil Red O e H&E, verificando-se um efeito marcado anti-dislipidémico nos murganhos APOE KO. Não se observaram diferenças significativas nos perfis glicémicos e insulinémicos entre os 3 grupos.Globalmente, verifica-se que a suplementação com OLEA melhorou significativamente o perfil lipídico dos murganhos APOE KO, com forte proteção contra a deposição lipídica hepática e eventual efeito antioxidante. Mais estudos serão necessários para clarificar os mecanismos celulares e moleculares subjacentes à proteção contra o desenvolvimento de esteatose hepática neste modelo de hiperlipidema e aterosclerose.
Atherosclerosis is the main substrate for most cardiovascular diseases (CVDs) which are the main cause of mortality worldwide. The risk factors for the development of atherosclerosis and CVDs, in addiction to non-modifiable are related to unhealthy lifestyles, including hypercaloric and hyperlipidic diets, as well as sedentary lifestyle. These factors are associated with other diseases, such as obesity and hepatic steatosis. The non-pharmacological approaches, namely nutraceutical interventions, can serve as adjuvant approaches to the currently available pharmacologic therapies. The Mediterranean Diet (MDiet), based on a wide variety of unrefined foods, has been considered a dietary model by several international organizations. Olive oil, one of MDiet's main sources of fat, composed essentially of monounsaturated fatty acids, has a small fraction of phenolic compounds, which have been seen as a nutraceutical option, exhibiting marked effects in preventing these diseases. One of the most promising is oleacein (OLEA), a polyphenol from the secoiridoids group that has already shown in vitro antioxidant, anti-inflammatory, insulin sensitizing effects and even potential for lipid-lowering and anti-atherosclerotic properties.In this sense, we hypothesized that a nutraceutical intervention with OLEA could exert hepato- and atheroprotective effects in an animal model susceptible to the development of atherosclerosis, the apolipoprotein E (APOE KO) knockout mouse fed an atherogenic diet (ATD). In this way, we define two experimental approaches. The first using C57BL/6J wild-type (WT) mice to compare the metabolic effects of three diets – i) unrefined standardized control diet (STD, n=8), ii) refined low-fat diet (LF, n=8) and iii) atherogenic refined diet (ATD, n=8) - at the level of glycemic, insulinemic and lipid profiles. The second evaluating the potential hepatoprotective, anti-dyslipidemic and anti-atherosclerotic effect of OLEA in the APOE KO mouse, where two groups were defined - with and without treatment with 50 mg/kg/day of OLEA (n=8 each) - compared with a WT group fed with ATD. All experimental groups had food and water provided ad libitum.The glycemic and insulinemic profiles were evaluated in the initial and final times and the lipid profile was characterized in terms of serum values of total cholesterol (c-Total), TGs, c-LDL and c-HDL, as well as hepatic contents of TGs. Histomorphological markings and scanning electron microscopy (SEM) observations were used to characterize liver and vascular lesions and lipid deposition. Serum and hepatic SOD levels were also evaluated and the hepatic expression of SOD-1 and SOD-2 was quantified by RT-PCR.WT animals fed with ATD showed a significant increase in body weight, without changes in glucose tolerance or insulin sensitivity. It was found that in the same group there was an increase in the serum concentration of c-Total, c-LDL and c-HDL, accompanied by accumulation of TGs in the liver. In addition, they exhibited reduced hepatic expression of both SOD isoforms, which may be indicative of an oxidative stress process characteristic of these diseases.The animals fed the LF diet showed more moderate metabolic alterations than those submitted to ATD, including a trend towards higher values of circulating glucose after 6 hours of fasting, as well as an increase in the serum concentration of c-Total, c-LDL and c-HDL, and liver TGs, when compared to animals fed with STD, suggesting that the presence of refined ingredients in this diet, although low in fat, does not prevent a negative impact on metabolism, compared to the unrefined STD diet.Regarding the second experimental approach, it was found (by H&E and SEM) that there were vascular lesions in APOE KO animals compatible with the literature. OLEA had a marked effect on the lipid profile of APOE KO mice, causing a marked reduction in the serum values of c-Total, c-LDL, c-HDL and TGs, as well as a strong attenuation of the hepatic concentration of TGs. These data were corroborated by the liver markings with Oil Red O and H&E, showing a marked anti-dyslipidemic effect in the APOE KO mice. There were no significant differences in glycemic and insulinemic profiles between the 3 groups.Overall, it appears that supplementation with OLEA significantly improved the lipid profile of APOE KO mice, with strong protection against hepatic lipid deposition and an eventual antioxidant effect. Further studies will be needed to clarify the cellular and molecular mechanisms underlying protection against the development of hepatic steatosis in this model of hyperlipidema and atherosclerosis.
FCT
FCT
FCT
Outro - COMPETE-FEDER funds (POCI-01-0145-FEDER-007440 and POCI-01-0145-FEDER-031712)
Outro - European Regional Development Fund (FEDER), through Programa Operacional Factores de Competitividade COMPETE2020 (CENTRO-01-0145-FEDER-000012-HealthyAging2020)

碩士
淡江大學
數學學系碩士班
101
In this thesis, we want to use Morse Theorem to prove Reeb''s Theorem. Before showing the proof of these theorems, we need to review some basic properties of a differentiable manifold M with a differentiable structure. In general, if we define some functions from M (or its subspace) to real value, the difference between manifold and coordinate space should be considered. Every point we choose must send to a coordinate subspace first. So defining a coordinate system is helpful to deal with any functions on manifold M. The main result we review is to prove Reeb''s Theorem using Morse Lemma and Morse Theorem. Here we use a surgery lemma to prove disjoint union of two spaces, matched along their common boundary. We also show how to construct a homotopy equivalence between manifold M and a n-sphere, for all dimension n is larger or equal to 1.

碩士
朝陽科技大學
應用外語研究所
99
With the high development of internet technology, it appears more and more consumer product discussions in the internet. Consumers tend to read consumer’s review online before they buying an unfamiliar product for helping decision making. Word-of-mouse has the characteristics of lasting longer time, no geographic restriction, and anonymity. However, anonymity of Internet occurs the doubt of the credibility of word-of-mouse. The purpose of this study is to explore the relationships among source credibility of word-of-mouse, consumers’ trust, involvement and purchase intention. This research distributed questionnaire at online forums. Totally, 400 of participants were included. Descriptive statistics analysis, reliability, validity analysis, factor analysis, and regression analysis were employed to testify the relationships among the variables. The results implies that source credibility of word-of-mouse has a significantly positive effect on purchase intention. Moreover, consumers’ trust has a significantly positive effect on the relationship between trustworthiness and purchase intention. Also, consumers’ involvement has a significantly positive effect on the relationship between trustworthiness and purchase intention.