Introdução: A artrite reumatoide é uma doença inflamatória autoimune associada à complicações sistêmicas como fadiga e perda muscular. Perda muscular pode estar relacionada com a ativação do sistema ubiquitina-proteossomo. O objetivo deste trabalho foi avaliar a perda muscular e o evolvimento do proteossomo no modelo de artrite induzida por colágeno (CIA), com ou sem o tratamento de metotrexato ou inibidor de TNF (etanercepte). Métodos: Camundongos DBA1/J machos foram divididos em 4 grupos (n=8 cada): CIA (salina); ETN (etanercepte, 5.5 /) e MTX (metotrexato, 35 /), tratados duas vezes por semana por 6 semanas, e um grupo controle saudável (CO). Tratamentos iniciaram uma semana após a injeção do booster. Escore clínico, edema da pata traseira e peso corporal foram analisados durante o período experimental. Músculo gastrocnêmio (GA) foi pesado após a morte e usado para quantificar a atividade, níveis proteicos e expressão de mRNA das diferentes subunidades do proteossomo através de ensaio fluorogênico, Western blot e rtPCR, respectivamente. Resultados: Tratamentos reduziram o desenvolvimento da doença, observado através do menor escore clínico e edema da pata traseira nos grupos ETN e MTX. ETN apresentou maior peso corporal do que MTX nas semanas 5 e 7. Músculo GA estava aumentado em ETN do que CIA e MTX, um resultado também observado no peso muscular normalizado. As propriedades catalíticas do proteossomo 26S muscular mostraram um aumento na atividade do tipo caspase nos grupos CIA e MTX. Tecidos musculares de animais MTX demonstraram maiores níveis proteicos das subunidades do proteossomo PSMB8 e PSMB9 e maior expressão gênica de Psmb5, Psmb8 e Psmb9. Por outro lado, a expressão de Psmb6 estava diminuída e de Psmb9 estava aumentada em CIA. Conclusões: Apesar de ambos os medicamentos melhorarem o escore da doença, ETN apresentou um afeito anti-artrítico mais forte e foi o único tratamento capaz de prevenir parcialmente a perda muscular. Ao contrário de ETN, CIA e o tratamento com MTX apresentaram perda muscular e atividade e expressão do proteossomo aumentadas. / Background: Rheumatoid arthritis is an autoimmune inflammatory disease associated with systemic complications like fatigue and muscle wasting. Muscle wasting could be related to the activation of the ubiquitin-proteasome system. The aim of this study was to evaluate muscle loss and involvement of the proteasome in collagen-induced arthritis (CIA), with or without treatment with methotrexate or a TNF inhibitor (etanercept). Methods: Male DBA1/J mice were divided into 4 groups (n=8 each): CIA (saline); ETN (etanercept, 5.5 /) and MTX (methotrexate, 35 /), treated twice a week for 6 weeks, and a healthy control group (CO). Treatments started one week after booster injection. Clinical score, hind paw oedema, and body weight were analysed during the experimental period. Gastrocnemius muscles (GA) were weighted after death and used to quantify proteasome activity, protein levels and mRNA expression of its subunits by Western blot and rtPCR, respectively. Results: Treatments slowed disease development, observed through smaller clinical score and hindpaw edema in ETN and MTX groups. ETN presented higher body weight compared to MTX group at weeks 5 and 7. GA weight was heavier in ETN than CIA and MTX, a result also observed in the normalized muscle weight. The catalytic properties of 26S proteasome showed an increase of caspase-like activity in CIA and MTX groups. Muscles tissues of MTX treated animals showed higher protein levels for proteasomal subunits PSMB8 and PSMB9 and higher gene expression for Psmb5, Psmb8 and Psmb9. In contrast, expression of Psmb6 was decreased and of Psmb9 was enhanced in CIA. Conclusions: Although both drugs improved the disease score, ETN presented a stronger anti-arthritic effect and was the only treatment able to partially prevent muscle wasting. In contrast to ETN, CIA and MTX treatment did not prevent muscles loss due to increased proteasome expression and activity.

No presente trabalho analisa-se o comportamento do aço de reforço ante à corrosão, com o uso dos inibidores: nitrito de sódio, fosfato de sódio e etalonamina, na água de poros contaminada com cloreto, e no concreto com a finalidade de analisar seus resultados e seus mecanismos diferenciados. Para cumprir este objetivo o presente trabalho divide-se em duas etapas: uma primeira etapa baseada em simular sinteticamente a água de poro de um concreto, cuja solução é KOH 28g/l+NaOH 4g/l. Essa água de poro é simulada em ambiente marinho, cuja solução é KOH 28g/l + NaOH 4g/l+NaCl 35g/l, e a esta solução referência incorporamse os inibidores (20g/l da cada um). Realizaram-se ensaios de espectroscopia de impedância eletroquímica (EIE) (após 3 e 72 horas de imersão) e curvas de polarização (após 72 horas de imersão) com vistas a obter respostas da cinética da corrosão ante a cada solução. Obteve-se o melhor comportamento para a água de poros. No caso da água de poro contaminada por cloretos, o melhor comportamento se obteve para o inibidor nitrito de sódio. Na segunda etapa adotou-se apenas o inibidor nitrito de sódio, pois estatisticamente as eficiências dos três inibidores foram muito similares. Analisou-se o nitrito de sódio em amostras reais de concreto armado contaminado com cloreto de sódio. Para isso se elegeram dois tipos de cimentos (CP IV e CP V) e três relações água-cimento (a/c-0.4, a/c-0.5, a/c- 0.65). Para simular o ambiente marinho, realizaram-se ensaios acelerados de cloretos. Comparam-se métodos de análises simuladas sinteticamente e reais, concluindo-se em ambos meios, embora fossem um solido e outro líquido o inibidor Nitrito de Sódio aumento a sua eficiência com os ciclos de exposição. / In this paper it is analyzed the behavior of reinforcing steel against corrosion using inhibitors: sodium nitrate, sodium phosphate and ethanolamine in water contaminated with chlorides pore and concrete, in order to analyzing the results and different mechanisms. To meet the objective of this work, it was divided into two stages, a first stage based on synthetically simulate the pore water of a concrete, through the following solution KOH 28g/l+NaOH 4g/l, this same solution simulated pore water to a marine environment it would be KOH 28g/l + NaOH 4g/l+NaCl 35g/l, it is then incorporated into both reference solutions inhibitors in a proportion, (20g/l de cada um). Assays were performed electrochemical impedance spectroscopy (EIE) (last 3 hours and 72 hours of immersion) and polarization curves (last 72 hours of immersion) in order to obtain responses corrosion kinetics in each solution. the best performance was obtained in the pore water. In the case of water contaminated with chlorides pore, the best performance was obtained in the presence of sodium nitrite inhibitor. In the second step was performed only with the inhibitor sodium nitrate, as statistically efficiencies of the three inhibitors were similar. Sodium nitrate was analyzed in real samples of reinforced concrete contaminated with chlorides of sodium. So they were chosen two types of cement CP- IV and CP-V, cement water three relationships 0.4, a/c-0.5, a/c- 0.65. In this case to simulate the marine environment, accelerated tests were performed chloride. They were compared the methods of analysis, simulated synthetically and simulated in real concrete.

La surexpression de protéines d’efflux, et tout particulièrement la P-glycoprotéine, est impliquée dans la multidrug résistance. Dans cette thèse, nous démontrons que le vandetanib, inhibiteur de tyrosine kinase, est à la fois substrat et inhibiteur de la P-glycoprotéine et qu’il est capable de réverser in vitro la résistance à la doxorubicine liée à la surexpression de la P-glycoprotéine.Nous nous sommes également intéressés à l’étude du passage transplacentaire du vandetanib et nous montrons que ce médicament traverse la barrière placentaire. / Overexpression of ABC transporters, especially P-glycoprotein, is involved in multidrug resistance. In this study, we demonstrate that vandetanib, a tyrosine kinase inhibitor, is both substrate and inhibitor of P-glycoprotein and is able to reverse in vitro resistance to doxorubicin, linked to overexpression of P-glycoprotein.We also studied the placental transfer of vandetanib and we show that this drug crosses the placenta.

No description available.

Epóxido hidrolases (EHs) são enzimas que catalisam a hidrólise de epóxidos a seus correspondentes dióis, apresentam potencial aplicação biotecnológica (separação de enantiômeros na produção de fármacos), estão envolvidas no metabolismo de ácidos graxos poliinsaturados e inibidores de EHs estão sendo estudados para possível utilização no tratamento de doenças. Uma enzima epóxido hidrolase (TrEH) do fungo filamentoso Trichoderma reesei QM9414 foi clonada, expressa, purificada e caracterizada funcionalmente e estruturalmente. A atividade de TrEH foi determinada com o substrato óxido de estireno (racêmico), demonstrando maior atividade nas temperaturas de 23 a 50 °C, no pH 7,2 a 37 °C, e as constantes cataliticas Km= 4,6 mM e kcat= 336 s-1. A enzima recombinante mostrou ser enantiosseletiva, pois hidrolisa preferencialmente (S)-(-)-óxido de estireno, (R)-(-)- epicloridrina e (S)-(-)-1,2-epoxibutano. Moléculas inibidoras da atividade de TrEH foram identificadas e algumas delas inibem até 60% o crescimento de T. reesei. A estrutura terciária de TrEH (1,7 Å) foi determinada por cristalografia, apresenta dobramento α/β-hidrolase e não tem alta homologia com nenhuma outra estrutura de EH. TrEH é uma nova enzima epóxido hidrolase solúvel cujas propriedades mostram seu potencial de utilização em aplicações biotecnológicas. / Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to their corresponding diols, present a potential biotechnological application (separation of enantiomers for the production of drugs), they are involved in the metabolism of polyunsaturated fatty acids and EH inhibitors are being studied for possible use in the treatment of diseases. An epoxide hydrolase enzyme(TrEH) from the filamentous fungus Trichoderma reesei QM9414 was cloned, expressed, purified and functionally and structurally characterized. The activity of TrEH was determined with the substrate styrene oxide (racemic), showing higher activity at temperatures of 23 to 50 °C, at pH 7.2 at 37 °C, and the catalytic constants Km= 4.6 mM and kcat= 336 s-1. The recombinant enzyme has been shown to be enantioselective, because it preferably hydrolyzes (S)-(-)-styrene oxide, (R)-(-)-epichlorohydrin and (S)-(-)-1,2- epoxybutane. TrEH inhibitors have been identified and some of them inhibit up to 60% growth of T. reesei . The tertiary structure of TrEH (1.7 Å) was determined by crystallography, showing α/ β-hydrolase folding and low homology with any other EH structure. TrEH is a new soluble epoxide hydrolase enzyme whose properties show its potential for use in biotechnological applications.

A resistência natural do gambá Didelphis marsupialis ao veneno de serpentes se deve a fatores antibiotrópicos presentes em seu soro, do qual DM43 é um dos responsáveis pela inibição da atividade hemorrágica. Com o objetivo de entender melhor o mecanismo de ação desta proteína contra a metaloprotease contida no veneno pretendeu-se, neste trabalho, otimizar as condições de cristalização de DM43, na tentativa de determinar sua estrutura por difração de raios-X. Ensaios de cristalização revelaram que o sulfato de amônio é o precipitante mais eficiente na produção de cristais de DM43. A visualização e indexação dos padrões de difração destes cristais permitiram apenas a determinação dos parâmetros de sua cela unitária, devido a baixa qualidade dos mesmos. Deste modo, os seguintes parâmetros de cela unitária foram encontrados: a = b = 117.34 (0.03) Å, c = 193.39 (0.02)Å, ?=?=90° e ? = 120°, correspondentes ao sistema cristalino trigonal ou hexagonal. A recente determinação da sequência de aminoácidos de DM43, possibilitou modelar sua estrutura tridimensional por homologia utilizando o método de satisfação das restrições espaciais. Os três domínios tipo imunoglobulina de DM43 foram modelados em duas partes a partir da estrutura de referência KIR2DL1(1nkr) homóloga a dois domínios C-terminais de DM43, com apenas 27.72% de identidade. O domínio N-terminal denominado D0, foi modelado separadamente contra o domínio C-terminal de KIR2DL1 com um grau de identidade igual a 28.89%. A partir do programa GRASP e PATCHES, foram detectadas as prováveis regiões de ligação entre as duas partes da molécula e uma possível região responsável pela dimerização no domínio D2 de DM43. Interessantemente, resíduos polares em KIR2DLs que foram substituídos por hidrofóbicos em DM43, concentrando-se em uma face do domínio D2 ausente de sítios de glicosilação, o que coincide com as observações de dimerização do receptor do hormônio de crescimento. Um motivo estrutural característico de receptores hematopoiéticos identificados como WSXWS box, foi encontrado em todos os domínios de DM43, especialmente no domínio D2. Supõe-se que a sua existência esteja relacionada com a orientação relativa dos domínios por manter o primeiro triptofano do motivo posicionado exatamente na interface entre os domínios. Surge ainda uma fita extra (F´), no domínio D2, não pertencente ao enovelamento imunoglobulina devido a presença da Pro273, muito bem conservada, a três resíduos do motivo WSXWS. Esta fita parece desempenhar um papel importante na orientação do motivo pois além de formar ligações de hidrogênio com a fita G do domínio anterior, posiciona corretamente o primeiro triptofano do motivo na interface. Somando-se a isto, a presença de resíduos hidrofóbicos na interface dos domínios D1 e D2pode estar contribuindo para a orientação angular relativa menor que 90° entre os dois domínios. Uma interpretação análoga a de hormônios de crescimento sugere que, os loops entre as fitas AB, CC´ e EF do domínio 1, BC e FG do domínio 2 e o linker entre estes dois domínios, estejam envolvidos na interação da metaloprotease com DM43. / The natural resistance of the opossum, Didelphis marsupialis, towards snake venom is due to antibothropic factors present in the blood serum, of which DM43 is one. It is responsible for the inhibition of the hemorrhagic activity of the venom. With a view to better understanding the mechanism of action of this protein against venom metalloproteases, one of the aims of the present work was to optimize the crystallization conditions of DM43 in order to determine its three-dimensional structure by X-ray diffraction. Crystallization trials revealed that ammonium sulphate was the best precipitant. Visualization and indexing of the diffraction patterns from such crystals only allowed for the determination of the unit cell parameters due to their inherently low quality. The values obtained were a = b = 117.34 (0.03) \'ANGSTRON\', c = 193.39 (0.02) Å, ?=?= 90° e ? = 120°, corresponding to the trigonal or hexagonal crystal systems. The recent determination of the amino acid sequence of DM43 permitted the homology modelling of its three-dimensional structure by satisfaction of spatial restraints. The three immunoglobulin-like domains of DM43 were modelled in two separate parts from the reference structure KIR2DL1 (1nkr), homologous to the two C-terminal domains of DM43, with which its shares 27.72% sequence identity. The N-terminal domain, denominated D0, was separately modelled based on the C-terminal domain of KIR2DL1, with which it shares 28.89% identity. The programs PATCHES and GRASP were used to detect the probable interface region between the two separate parts of the molecule as well as a region probably responsible for dimerisation. Polar residues in KIR2DL1 which had been substituted by hydrophobic residues in DM43 are observed concentrated on one face of the molecule devoid of glycosilation sites (D2) coinciding with the dimerisation interface observed in the growth hormone receptor. A structural motif characteristic of the haematopoetic receptor family, identified as the WSXWS box, was observed to some extent in all three domains of DM43 and most evident in domain D2. It is speculated that the existence of this motif is related to the relative orientation of the domains, by maintaining the first tryptophan of the motif positioned at the domain interface. An additional ?-strand (F) in D2, which is not characteristic of the immunoglobulin fold, is observed due to the presence of the conserved Pro273, three-residues prior to the WSXWS box. This strand appears to play an important role in correctly positioning the motif as it forms hydrogen bonds with strand G of the previous domain thus orientating the first tryptophan of the motif at the interface. The presence of hydrophobic residues at the interdomain interface, may also contribute to stabilizing the acute angular relationship between D1 and D2. An analogous interpretation to that given for the growth hormone receptor, suggests that the loops between ?-strands AB, CC\' and EF of domain D1 and between strands BC and FG do domain D2 plus the linker region, are involved in the binding of metalloproteinase by DM43.

Diversos estudos buscam identificar novas moléculas com ações regulatórias sobre a via de sinalização da insulina e consequentemente na homeostase da glicose. Assim, este trabalho visa avaliar o potencial de extratos de frutos no controle da homeostase glicêmica. Os frutos avaliados foram o morango (cv. Toianoca, Camarosa, Oso Grande e Camino Real), a amora-preta e a framboesa vermelha, em dois tempos de amostragem, sendo considerado como tempo A1 e tempo A2. As amostras foram caracterizadas quanto ao seu conteúdo de fenólicos totais, conteúdo de antocianinas monoméricas, capacidade antioxidante, avaliada pelos métodos DPPH e ORAC, ácido elágico total, capacidade de inibição da alfa-glicosidase e captação de glicose e lipólise em tecido adiposo de camundongos (ensaio explante). Dentre os frutos, no primeiro tempo de amostragem, a amora-preta e o morango, cv Oso Grande, foram os que apresentaram maior conteúdo de fenólicos totais (62,36 e 34,89 mg AG/g, respectivamente) no entanto não foram mantidos esses valores no segundo tempo de amostragem, com concentração 30% e 60% inferior, respectivamente; e maior concentração de antocianinas monoméricas (45,33 mg/g e 3,09 mg/g, respectivamente). Em relação a inibição da enzima alfa-glicosidase, avaliado em extrato metanólico, o fruto framboesa vermelha e o morango cv. Camino Real foram as que apresentaram alto potencial inibitório nos dois tempos de amostragem (IC50 0,47 mg FT e IC50 0,57 mg FT para framboesa vermelha e IC50 0,50 mg FT e IC50 0,46 mg FT para Camino Real). Quando avaliado os extratos enriquecidos em fenólicos, o valor de IC50 com maior potencial dentre os frutos avaliados foi da amora-preta, nos dois tempos A1 e A2 (0,0023 mg FT e 0,0021 mg FT, respectivamente). Para captação de glicose em tecido adiposo explate, ao utilizar a insulina para estimular a captação de glicose juntamente com o tratamento (extrato), esse estimulo foi efetivo no aumento da captação de glicose somente com as amostras cv. Camino Real e cv. Oso Grande. Isso pode ser explicado pela alta correlação encontrada de antocianinas identificadas no fruto, como pelargonidina-3-O-glicosídeo. Por outro lado, somente a amora-preta A1 aumentou a lipólise em condição basal, mas nenhuma fruta foi eficiente para reduzir a lipólise em condição estimulada pelo isoproterenol. Sendo assim, frutas vermelhas podem ser boas fontes de compostos bioativos, principalmente antocianinas, as quais podem ter corroborado positivamente com os resultados. / Several studies seek to identify new molecules with regulatory actions on the insulin signaling pathway and consequently on glucose homeostasis. Thus, this work aims to evaluate the potential of fruit extracts in the control of glycemic homeostasis. The fruits evaluated were strawberry (cv. Toianoca, Camarosa, Oso Grande and Camino Real), blackberry and red raspberry, in two sampling times, being considered as time A1 and time A2. Fruits were evaluated for total phenolic, monomeric anthocyanins and contents, antioxidant capacity, evaluated by DPPH and ORAC methods, alpha-glycosidase inhibition capacity and glucose uptake and lipolysis in adipose tissue of mice (explant assay). Among the fruits, in the first sampling period, blackberry and strawberry, cv. Oso Grande, showed the highest total phenolic content (62.36 and 34.89 mg AG / g, respectively), with a decrease of the 30% and 60%, respectively, in the second sampling time; and higher monomeric anthocyanins concentration (45.33 mg/g and 3.09 mg/g, respectively). The methanolic extracts of raspberry and the strawberry cv Camino Real (A1 and A2) presented the highest alpha-glucosidase inhibitory potential. Otherwise, the enriched-polyphenol extract of blackberry (A1 and A2) presented the highest potential among the evaluated fruits. Adipose tissue treated with strawberry cv. Camino Real and cv. Oso Grande was effective in increasing glucose uptake stimulated by insulin. This can be explained by the high correlation with the anthocyanins pelargonidin-3-O-glycoside identified in this fruit. In addition, blackberry A1 was the only sample to increase the lipolysis in basal condition, but all other fruits were not effective to decrease lipolysis in stimulated condition. Thus, berries could be a good sources of bioactive compounds to maintain the glucose homeostasis.

The aim of the research in this thesis was to study how environmentally friendly corrosion inhibitors for cooling water systems might be developed and used. Firstly, reduced toxicity inorganic corrosion inhibitors (i.e. nitrite/molybdate) were considered. Secondly, non-toxic inhibitors based on mono and di-basic salts of carboxylic acids were studied systematically as a function of carbon chain length. For nitrite inhibitor alone, a concentration of 7 mM NaNO2 was effective to inhibit carbon steel in chloride media of 10 mM NaCl, while 10 mM nitrite was needed in sulphate media of 3.66 mM Na2SO4. However, it was found possible to significantly reduce the concentration of nitrite by adding molybdate in synergy. This was attributed to the nitrite passivation combined with ferrous molybdate salt film pore plugging thus promoting a continuous and protective film on the material within these media. Thus, in pH 6-10 an inhibition efficiency of 97% was recorded with a mixture of 3 mM nitrite/2 mM molybdate in both chloride and sulphate media and at 25°C and 60°C. However as the solution pH decreased below pH 4 the inhibition efficiency decreased to about 47%.In the second part of the study, the use of sodium salts of carboxylic acids with different chain lengths has been investigated. In this part a summary of the performances and limitations of both mono- and di-sodium carboxylate inhibitors are presented. For mono-carboxylates, the inhibition efficiency reached a maximum value of 95% in stagnant aerated solutions at a chain length of C=4 with a critical inhibition concentration of 6 mM in 10 mM NaCl solution. However the inhibition efficiency gradually decreased as the number of carbon atoms in the chain length increased to more than 8, or less than 4, and this was in agreement with surface hydrophobicity and contact angle results. For lower chain lengths, the carboxylate anion becomes more acidic and complexing of the metal ion while for longer chain lengths, the carboxylate anion becomes less soluble and tends to micellise wherby the active groups are no longer available for surface adsorption. For di-carboxylates the inhibition efficiency improved in 10 mM NaCl at a given chain length compared with mono-carboxylates, and continued to increase to C=8 (sebacate), which achieved excellent inhibition efficiency. However, sebacate is costly so a blend with ethyl hexanoate was found to be economically favoured.

SAS-6 is the structural core of the forming centriole - a cylindrical protein complex, which is an essential component of the centrosome. Oligomerisation of SAS-6 is crucial for successful centriole duplication and is achieved through two dimerisation domains in the SAS-6 protein; a long C-terminal coiled-coil domain and a globular N-terminal dimerisation domain. As core components of the centrosome, centrioles help facilitate various cellular functions. They are involved in the anchoring of flagella and cilia to the membrane and in coordinating the spindle apparatus during chromosome segregation. A deeper insight into the molecular mechanisms at play in the centriole duplication process would have implications on our understanding of fundamental cell division processes and a number of related diseases. Here the involvement of an unstudied loop region in the C. elegans SAS-6 N-terminal domain dimerisation is described. Combining structural biology, biophysical and computational techniques, the molecular interactions of this loop were explored, contributing to the oligomerisation of SAS-6 at the N-terminal dimer interface. Furthermore, the screening and testing of small molecule inhibitors of the SAS-6 N-terminal domain dimerisation is described, targeting a hydrophobic pocket in the domain. Two candidate compounds are presented as a result of the screens and next steps towards structure based compound design are suggested, based on computational analysis. The search for inhibitory compounds includes a set-up of an in-house virtual screening pipeline, as well as in vitro screening efforts and a new crystallographic structure of the H. sapiens SAS-6 N-terminal domain. By investigating the making and breaking of the SAS-6 N-terminal domain dimerisation, light is shed on so far neglected details of this essential protein-protein interaction and advancements towards a SAS-6 oligomerisation inhibitor described, which could ultimately be used for new approaches in cell cycle research and might open up new avenues for medical research by binding a disease relevant target.

冠狀動脈旁路移植術是治療合併左主幹及多隻冠狀動脈狹窄性病變患者的理想方法。然而靜脈橋失效極大地限制了冠脈搭橋手術的遠期療效。基於對靜脈橋失效潛在機制的研究，近年來開發出了多種針對性的防治手段。但是除了積極的降脂治療，目前尚未有其它療法獲得臨床證實可以有效改善靜脈橋遠期通暢率。所以，本研究旨在探索與防治靜脈橋再狹窄相關的新型生物標靶和防治策略。 / 我們應用豬大隱靜脈植入頸內動脈模型，觀察骨橋蛋白是否參與靜脈橋動脈化進程以及其與基質金屬蛋白酶功能活動的關係。我們發現骨橋蛋白表達在靜脈橋動脈化過程中顯著增加，並且與基質金屬蛋白酶2/9和增殖細胞數量的變化同步。此外，骨橋蛋白富集區域在靜脈橋內的再分佈與血管壁重構進程相關。這些結果表明, 骨橋蛋白積極參與了靜脈橋壁重構，而抑制骨橋蛋白表達作為防治靜脈橋失效的治療策略值得深入研究。 / 我們運用體外培養的方法研究了在高糖環境中骨成形蛋白4與靜脈內皮細胞舒張功能障礙的關係。我們發現，骨成形蛋白4在糖尿病患者的大隱靜脈與高糖培養的人臍靜脈內皮細胞中顯著增加；而骨成形蛋白4的高表達與靜脈血管內皮細胞依賴性舒張功能受損有關。本研究結果為解釋糖尿病患者有著較高的冠脈搭橋術後靜脈橋失效率提供了新證據，同時也為改善此類患者靜脈橋通暢率提出了潛在的治療靶點。 / 通過轉染金屬蛋白酶-3抑制物 (TIMP-3)基因來針對性地抑制血管中層平滑肌細胞的遷移和增殖，可以有效地減少靜脈橋新生內膜增生。基於前期研究，我們觀察了在豬模型中運用重組腺病毒轉載TIMP-3(RAdTIMP-3) 防治靜脈橋狹窄的遠期效果(3個月)。結果發現，即使在腺病毒載體已被清除的情況下，RAdTIMP-3對靜脈橋的良性保護作用仍持續存在。此外，我們通過比較術後7天與3個月獲取的橋血管中炎性標記物表達的差異，發現腺病毒轉染並未對靜脈橋造成長期的炎性損害。因此，我們認為RAd-TIMP3基因能夠安全有效地防治靜脈橋遠期狹窄。本研究結果為TIMP-3基因治療轉化至臨床實踐提供了可靠的前期證據。 / Coronary artery bypass grafting (CABG) remains the “gold standard“ for treating high-risk patients with unprotected left-main or multi-vessel coronary lesions. However, the long-term success of CABG is largely limited by an inadequate patency of saphenous vein grafts. To date, various therapeutic strategies targeting at the underlying mechanisms involved in the pathogenesis of vein graft failure (VGF) have been proposed and tested. However, apart from lipid-lowering therapy, no other intervention appears to have sustained benefits on improving vein graft patency in the clinical setting. Therefore, the aim of this study is to explore novel sets of molecular targets and effective therapeutic strategies to prevent VGF. / Novel molecules involved in the pathogenesis of vein graft failure / Using a porcine model, we assessed the involvement of osteopontin (OPN) in the venous arterialization and its relationship with the matrix metalloproteinases (MMPs). We found that the expression of OPN was significantly increased over the 3-month study period. Moreover, the expression of OPN at different time points well correlated with the fluctuating activities of MMP-2/9 and the number of proliferative cells. We also observed a time-dependent redistribution of OPN protein accumulating in different layers of the venous wall. These findings suggest a contributory role of OPN protein involved in the process of vein graft wall remodeling. / We used pig and human saphenous veins (SVs), as well as human umbilical endothelial cells (HUVECs), to investigate the changes of bone morphogenic protein-4 (BMP4) expression and its effects on endothelium-dependent relaxations (EDRs) under hyperglycemic conditions. Our results demonstrated a marked increase of BMP4 expression in SVs from diabetic patients and in HUVECs cultured with hyperglycemic medium. Moreover, such an increase of BMP4 contributes significantly to the impaired EDRs in venous conduits. Our findings add novel evidence that helps explain the high prevalence of VGF in diabetic patients undergoing CABG, and also suggest BMP4 as a potential therapeutic target to improve vein graft patency in this population. / Novel Therapeutic Strategy -- Gene Therapy / Aiming at blocking the development of neointima formation caused by vascular smooth muscle cells migration and proliferation, genetic transfection of tissue inhibitor of metalloproteinases-3 (TIMP-3) to vein grafts has shown promising results. Based on our previous study, we used recombinant adenoviruses that carry TIMP-3 (RAdTIMP-3) as a therapeutic gene to evaluate its long-term (3 months) effects on the pathological vein graft wall thickening in vivo. We found that the RAdTIMP-3-treated vein grafts had significantly reduced intimal and medial thickness compared with grafts from the control groups at 3 months, even after adenoviruses had already been cleared from transduced tissue. Furthermore, by assessing the amount of macrophages and the level of three inflammatory biomarkers within grafts harvested at 7 days and 3 months after implantation, we did not observe any detrimental effects of adenoviral transfection on the inflammatory status within the vein grafts. We therefore concluded that overexpression of TIMP-3 could effectively inhibit vein graft wall over-thickening in the longer-term. Our findings suggested the ex vivo RAdTIMP-3 gene therapy an attractive candidate for future clinical translation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Hu, Jia. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 109-143). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Declaration --- p.vii / Acknowledgement --- p.viii / Table of Contents --- p.x / List of Abbreviations --- p.xvi / List of Figures/Tables --- p.xviii / Chapter Chapter I --- INTRODUCTION --- p.1 / Chapter 1.1 --- SAPHENOUS VEIN GRAFTS IN CORONARY REVASCULARIZATION --- p.3 / Chapter 1.1.1 --- The use of venous conduits in CABG --- p.3 / Chapter 1.1.2 --- The long-term patency of saphenous vein grafts --- p.4 / Chapter 1.1.3 --- PCI for vein graft diseases --- p.6 / Chapter 1.1.4 --- Vein graft failure and adverse clinical outcomes --- p.7 / Chapter 1.2 --- MORPHOLOGY AND PHYSIOLOGY OF A NORMAL SAPHENOUS VEIN --- p.8 / Chapter 1.3 --- THE PATHOPHYSIOLOGY OF VEIN GRAFT FAILURE --- p.10 / Chapter 1.3.1 --- The quality of vein grafts prior to grafting --- p.10 / Chapter 1.3.1.1 --- Pre-existing endothelial dysfunction --- p.10 / Chapter 1.3.1.2 --- Surgical injuries --- p.11 / Chapter 1.3.2 --- Mechanisms of the pathological vein graft wall thickening --- p.12 / Chapter 1.3.2.1 --- Platelet activation and coagulant cascade --- p.13 / Chapter 1.3.2.2 --- Leukocytes recruitment and inflammation --- p.13 / Chapter 1.3.2.3 --- Hemodynamic forces --- p.14 / Chapter 1.3.2.4 --- Growth factors and VSMCs activation --- p.15 / Chapter 1.3.2.5 --- Contribution of adventitial and graft-extrinsic cells --- p.16 / Chapter 1.3.2.6 --- Oxidative stress --- p.17 / Chapter 1.3.2.7 --- Concomitant risk factors and vein graft atherosclerosis --- p.17 / Chapter 1.4 --- STRATEGIES FOR THE PREVENTION OF VEIN GRAFT FAILUR --- p.18 / Chapter 1.4.1 --- Minimizing surgical injuries --- p.18 / Chapter 1.4.2 --- Pharmacologic interventions --- p.19 / Chapter 1.4.3 --- External supports --- p.21 / Chapter 1.4.4 --- Genetic engineering of the vein graft --- p.23 / Chapter 1.4.4.1 --- Delivery systems --- p.23 / Chapter 1.4.4.2 --- Therapeutic strategies of the genetic modulation --- p.25 / Chapter 1.4.4.2.1 --- Antithrombotic and anticoagulant strategies --- p.25 / Chapter 1.4.4.2.2 --- Therapies for endothelial protection and regeneration --- p.27 / Chapter 1.4.4.2.3 --- Reducing inflammation and atherosclerosis --- p.28 / Chapter 1.4.4.2.4 --- Antioxidative therapy --- p.29 / Chapter 1.4.4.2.5 --- Therapies targeting at the cellular proliferation --- p.29 / Chapter 1.4.4.2.6 --- Inhibiting extracellular matrix reorganization --- p.31 / Chapter 1.5 --- CONCLUSIONS --- p.32 / Chapter Chapter II --- MATERIALS AND METHODS --- p.34 / Chapter 2.1 --- MATERIALS --- p.35 / Chapter 2.1.1 --- Reagents and equipment --- p.35 / Chapter 2.1.1.1 --- General materials and equipment for animal model --- p.35 / Chapter 2.1.1.2 --- General reagents and equipment for western blot --- p.35 / Chapter 2.1.1.3 --- General reagents and equipment for immunohistochemistry --- p.36 / Chapter 2.1.1.4 --- General reagents and equipment for venous ECs functional studies --- p.37 / Chapter 2.1.2 --- Buffers --- p.37 / Chapter 2.1.2.1 --- Buffers for human and animal samples --- p.37 / Chapter 2.1.2.2 --- Buffers for western blot --- p.38 / Chapter 2.1.2.3 --- Immunohistochemistry buffers --- p.39 / Chapter 2.1.2 --- Antibodies and adenoviral vectors --- p.41 / Chapter 2.2 --- METHODS --- p.41 / Chapter 2.2.1 --- Animal model --- p.41 / Chapter 2.2.2 --- Functional studies --- p.44 / Chapter 2.2.3 --- Human endothelial cells culture --- p.44 / Chapter 2.2.4 --- Western blot analysis --- p.45 / Chapter 2.2.5 --- Immunochemistry and immunofluorescence --- p.46 / Chapter Chapter III --- ROLE OF BMP4 IN VENOUS ENDOTHELIAL DYSFUNCTION --- p.47 / Chapter 3.1 --- INTRODUCTION --- p.48 / Chapter 3.2 --- MATERIALS AND METHODS --- p.49 / Chapter 3.2.1 --- Patient characteristics --- p.49 / Chapter 3.2.2 --- Preparation of human vein segments --- p.51 / Chapter 3.2.3 --- Porcine saphenous veins culture --- p.51 / Chapter 3.2.4 --- Functional studies of vein segments --- p.52 / Chapter 3.2.5 --- Cell culture --- p.53 / Chapter 3.3.6 --- Western blot analysis of BMP4 --- p.53 / Chapter 3.3.7 --- ROS measurement by dihydroethidium fluorescence imaging --- p.54 / Chapter 3.2.8 --- Statistical analysis --- p.54 / Chapter 3.3 --- RESULTS --- p.54 / Chapter 3.3.1 --- ACh-induced EDRs are impaired in diabetic veins --- p.54 / Chapter 3.3.2 --- The expression of BMP4 is upregulated under hyperglycemic condition --- p.55 / Chapter 3.3.3 --- BMP4 induces venous endothelial dysfunction in diabetes --- p.56 / Chapter 3.3.4 --- BMP4 impairs EDRs in cultured porcine saphenous veins --- p.58 / Chapter 3.4 --- DISCUSSION --- p.59 / Chapter 3.5 --- CONCLUSIONS --- p.62 / Chapter Chapter IV --- ROLE OF OSTEOPONTIN IN VEIN GRAFT REMODELING --- p.63 / Chapter 4.1 --- INTRODUCTION --- p.64 / Chapter 4.2 --- MATERIALS AND METHODS --- p.66 / Chapter 4.2.1 --- Surgical procedures --- p.66 / Chapter 4.2.2 --- Immunohistochemistry --- p.67 / Chapter 4.2.3 --- Western blot --- p.68 / Chapter 4.2.4 --- Gelatin zymography --- p.69 / Chapter 4.2.5 --- Cell proliferation --- p.69 / Chapter 4.2.6 --- Statistical analysis --- p.69 / Chapter 4.3 --- RESULTS --- p.70 / Chapter 4.3.1 --- Expression and redistribution of OPN protein within the venous wall --- p.70 / Chapter 4.3.2 --- The fluctuating expression of the matrix metalloproteinases --- p.72 / Chapter 4.3.3 --- Vascular smooth muscle cells proliferation --- p.74 / Chapter 4.4 --- DISCUSSION --- p.75 / Chapter 4.5 --- CONCLUIONS --- p.79 / Chapter Chapter V --- TIMP-3 GENE THERAPY FOR NEOINTIMA FORMATION --- p.81 / Chapter 5.1 --- INTRODUCTION --- p.82 / Chapter 5.2 --- MATERIALS AND METHODS --- p.84 / Chapter 5.2.1 --- Materials --- p.84 / Chapter 5.2.2 --- Grafting of pig saphenous veins and adenoviral transfection --- p.84 / Chapter 5.2.3 --- Histologic and morphometric analysis of the vein graft --- p.87 / Chapter 5.2.4 --- Immunocytochemistry --- p.87 / Chapter 5.2.5 --- Data analysis and statistics --- p.88 / Chapter 5.3 --- RESULTS --- p.89 / Chapter 5.3.1 --- Histologic and morphometric analysis of the vein graft --- p.89 / Chapter 5.3.2 --- Overexpression of TIMP-3 in porcine interposition grafts --- p.91 / Chapter 5.3.3 --- Endothelial cell coverage and VSMCs content --- p.92 / Chapter 5.3.4 --- Inflammation in vein grafts --- p.92 / Chapter 5.4 --- DISCUSSION --- p.97 / Chapter Chapter VI --- SUMMARY AND DISCUSSION OF MAJOR FINDINGS --- p.103 / Chapter 6.1 --- SUMMARY AND DISCUSSION --- p.104 / Chapter 6.1.1 --- The role of BMP4 in the pathogenesis of venous endothelial dysfunction --- p.104 / Chapter 6.1.2 --- The involvement of osteopontin in the process of vein graft remodeling --- p.105 / Chapter 6.1.3 --- Sustained benefits of adenoviruses-mediated TIMP-3 gene transfer in reducing vein graft neointima formation --- p.106 / Chapter 6.1.4 --- The inflammatory responses induced by adenoviral transfection --- p.106 / Chapter 6.1.5 --- Perspectives: novel therapeutic targets and clinical translation --- p.107 / Chapter 6.2 --- CONCLUSIONS --- p.108 / REFERENCES --- p.109 / PUBLICATION LIST --- p.144