La technique des membranes induites par un conformateur en polymethylmethacrylate (PMMA) et greffe d’os spongieux autologue a été décrite par Masquelet pour la régénération des pertes de substance osseuse segmentaire des os longs. Cette technique en deux temps pourrait avantageusement être utilisée pour la reconstruction des pertes de substances osseuses segmentaires mandibulaires en cancérologie en cas d’échec ou de contre-indication des greffes osseuse revascularisées. Le premier objectif de ce travail était d’évaluer les propriétés histologiques et biologiques des membranes induites par un conformateur en PMMA avec radiothérapie, et de les comparer avec celles induites par un conformateur en silicone. Ce matériau plus souple que le PMMA a été choisi comme alternative au PMMA dans le but de faciliter l’ablation du conformateur. Les membranes induites par le PMMA ou le silicone en sous-cutané chez des rats Wistar avaient une structure histologique et des propriétés biologiques comparables mais les résultats étaient plus stables pour les membranes induites par le silicone. Le second objectif de ce travail était de proposer un procédé d’ingénierie tissulaire en alternative à la greffe d’os spongieux autologue. La néoformation osseuse au sein d’une membrane induite par un conformateur en silicone avec greffe de différentes combinaisons de céramique phosphocalcique biphasique macroporeuse (MBCP+™), de moelle osseuse totale, de simvastatine et de rhBMP-2 a été évaluée chez des rats Wistar en sous-cutané puis en site osseux fémoral. Les résultats n’ont pas permis de montrer de néoformation osseuse significative dans les groupes avec simvastatine. Par contre, une néoformation osseuse significative a été montrée dans les groupes avec rhBMP-2, avec ou sans radiothérapie. Un effet substantiel de l’adjonction de moelle osseuse totale a également été retrouvé. / Induced membrane technique with a polymethylmethacrylate (PMMA) spacer and autologous cancellous bone graft has been proposed by doctor Masquelet for segmental long bone reconstruction. This two stage technique could be proposed for segmental mandibular bone reconstruction in oncological situations in case of failure or contraindication of revascularized autologous bone graft. The first objective of this study was to evaluate the histological and biological properties of membranes induced by PMMA with radiotherapy, and to compare them to membranes induced by silicone. This material smoother than PMMA has been chosen to facilitate spacer removal. Membranes induced by both materials in subcutaneous models in rats had similar histological and biological properties, but membranes induced by silicone were less affected by radiotherapy. The second objective of this study was to propose a tissue engineering procedure as an alternative to autologous bone graft. The new bone formation inside silicone induced membranes has been analyzed after implantation of different combinations of macroporous biphasic phosphocalcic ceramic (MBCP+™), total bone marrow, simvastatine and rhBMP-2 in subcutaneous and femoral osseous models in rats. No significant new bone formation has been demonstrated in simvastatin groups. However, a significant new bone formation has been demonstrated in rhBMP-2 groups, with or without radiotherapy. An increased new bone formation has also been demonstrated with total bone marrow.

Background: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein known as the main initiator of blood coagulation. TF is over-expressed on many malignant cells and apart from increasing the risk of thrombosis, the presence of TF/FVIIa also promotes the progression of cancer and metastasis by intracellular signaling. TF expressing microparticles (MP) are, moreover, often found in the circulation of cancer patients. Aim: The aim of this thesis was to study different aspects of TF activity, e.g. the importance of procoagulant MP and TF-induced intracellular signaling pathways, with focus on cell migration (chemotaxis) and apoptosis. Results: The TF signaling complexes were shown to prevent apoptosis induced by serum starvation and TRAIL in cancer cells by reduced activation of caspase-8 in a PI3k/AKT-dependent manner. FVIIa also decreased transcription of pro-apoptotic genes in cancer cells treated with TRAIL. Simvastatin triggered apoptosis by transcriptional reduction of BCL-2 due to cytosolic retention of NFκB. Simvastatin also inactivated the PI3k/AKT pathway and reduced the production of the MP-like prostasomes which, respectively, impaired the anti-apoptotic signaling by TF and reduced the procoagulant activity in the vicinity of prostate cancer cells. Intracellular events conducted by the TF/FVIIa complex selectively enhanced PDGF-BB induced chemotaxis which was partly explained by the TF/FVIIa-induced transactivation of the PDGFβ-receptor. This was dependent on Src-family members and engagement of PAR2. Conclusions: The results presented in this thesis extend the current knowledge of TF-mediated signaling. We report the TF complexes to govern the extrinsic pathway of apoptosis, present data on FVIIa-dependent regulation of apoptosis-related genes, and exclude known surface proteins as transmitters of the anti-apoptotic signals. We moreover describe TF/FVIIa to transactivate the PDGFβ-receptor and play a decisive role in the potentiated chemotaxis toward PDGF-BB in a number of cell types. Finally, we explain the mechanism behind simvastatin-induced apoptosis in cancer cells and how statins interfere with TF-dependent signaling and coagulation.

Disease- and drug-related bone disorders are rapidly increasing in the population. The drugs which are used for the treatment of neurodegenerative diseases and metabolic derangements, may have negative or positive effects on bone tissues. In the first study, the possible side-effects of Carbamazepine and epileptic seizures on bone structure and composition were investigated by FTIR and synchrotron-FTIR microspectroscopy, AFM and micro- and nano-hardness analysis. The effects on the blood parameters, bone turnover and vitamin D metabolism were also investigated by ELISA and western blot analysis. The current study provides the first report on differentiation of the effects of both epileptic seizures and AED therapy on bones. Besides Carbamazepine treatment, seizures also caused a decrease in the strength of bone. The biochemical data showed that both the epileptic and drug-treated groups decreased vitamin D levels by increasing the vitamin D catabolism enzyme / 25-hydroxyvitamin D-24-hydroxylase. In the second study, the possible pleiotropic (positive) effects of cholesterol lowering drug / Simvastatin on bones were investigated by ATR-FTIR spectroscopy. The current study provides the first report on dose-dependent effects of simvastatin on protein structure and lipid conformation of bones. ATR-FTIR studies showed that although both high and low dose simvastatin strengthen bones, low dose simvastatin treatment is much more effective in increasing bone strength. Neural network analysis revealed an increased antiparallel and aggregated beta sheet and random coil in the protein secondary structure of high dose group implying a protein denaturation. Moreover, high dose may induce lipid peroxidation which limit the pleiotropic effects of high dose treatment on bones. This study clearly demonstrated that using low dose simvastatin is safer and more effective for bone health than high dose simvastatin treatment.

The mevalonate pathway is an important biosynthetic pathway, found in all cells of virtually all known pro- as well as eukaryotic organisms. This thesis is an investigation into the use of two drugs, originally developed for different applications, but both affecting the mevalonate pathway, in to models of fracture repair. Using two different rodent models of fracture repair, a commonly used cholesterol lowering drug (statin) and two drugs used to treat osteoporosis (bisphosphonate) were applied both systemically as well as locally in order to enhance fracture repair. Papers I and II investigate the potential of simvastatin to improve the healing of femoral fractures in mice. Papers III and IV explore the use of two bisphosphonates to improve early fixation of stainless steel screws into rat bone. The statin simvastatin lead to an increased strength of the healing cellus. The application of bisphosphonates increased early screw fixation. It seems clear that both drugs have uses in orthopaedic applications. One interesting avenue of further research would be to combine the two classes of drugs and see if we can get the benefits while at the same time diminishing the drawbacks.

A Doença Pulmonar Obstrutiva Crônica (DPOC) é caracterizada pela limitação persistente de trocas gasosas, usualmente progressiva e associada a uma resposta inflamatória crônica exacerbada das vias aéreas a partículas e gases nocivos. Apesar de prevenível e tratável, não se logrou até o presente uma terapêutica eficaz, que resulte na cura da doença. Neste cenário, a terapia celular apresenta-se como uma alternativa terapêutica potencialmente promissora em DPOC, bem como em outras doenças pulmonares degenerativas e de caráter inflamatório. Porém, vários aspectos da terapia celular carecem de um melhor entendimento. Um dos principais desafios ao sucesso da terapia celular são as baixas taxas de sobrevivência das células transplantadas. O Inibidor do Ativador de Plasminogênio 1 (Plasminogen Activator Inhibitor 1 - PAI-1) pode representar um potencial mediador da sobrevivência de células estromais mesenquimais (CTM) pós-transplante, pois tem sido proposto que anticorpos neutralizadores do PAI-1 auxiliam no aumento da sobrevivência de CTM no tecido-alvo da terapia celular. Desta forma, a diminuição dos níveis de PAI-1 possui um potencial terapêutico interessante, ao modular os principais processos envolvidos na criação de um ambiente pouco propício ao \"homing\" celular durante o processo de injúria. A diminuição dos níveis de PAI-1 é promovida, pela sinvastatina, fármaco da família das estatinas. Desta forma, objetivou-se com este trabalho analisar os efeitos da sinvastatina sobre a expressão do PAI-1, bem como sua influência na sobrevivência das células infundidas para terapia celular de enfisema pulmonar em modelo murino. Camundongos da linhagem FVB foram submetidos à instilação intranasal de elastase para indução de enfisema pulmonar e, posteriormente, tratados com CTM do tecido adiposo e sinvastatina. Os resultados mostraram que, quanto aos aspectos morfológicos e funcionais, considerando-se a análise conjunta de ambos os pulmões, não houve diferença estatisticamente significativa entre os grupos submetidos à instilação intranasal de elastase e submetidos à terapia celular com CTM tratados ou não com sinvastatina. Quando porém os pulmões foram analisados individualmente constatou-se que não houve diferença estatisticamente significativa entre os grupos controle e os resultados referentes ao lado direito do pulmão dos animais tratados com elastase e que receberam sinvastatina e infusão de CTM. Diferenças anatômicas entre os lados direito e esquerdo do pulmão, levaram a uma maior deposição de células no lado direito, como evidenciado pelos resultados obtidos nos ensaios de bioluminescência. Pode-se, portanto, inferir que a recuperação morfológica no lado direito do pulmão de animais com DPOC/enfisema poderia ser decorrente de um efeito regenerativo parácrino das CTM associadas à sinvastatina. / Chronic Obstructive Pulmonary Disease - COPD is characterized by the persistent limitation of gas exchange, is usually progressive, and associated to a chronic augmented inflammatory response of the airways to particles and noxious gases. Despite preventable e treatable, an effective, curative therapeutic approach is yet to be achieved. In this context, cell therapy presents itself as a promising therapeutic approach for COPD and other pulmonary inflammatory and degenerative diseases. However, many aspects of cell therapy with stem cells remain unclear. One of the major challenges to the success of cell therapy are the low survival rates of transplanted cells. The Plasminogen Activator Inhibitor 1 (PAI-1) is a potential mediator of the survival of mesenchymal stromal cells (MSC) after transplantation, since PAI-1 neutralizing antibodies have been shown to increase the survival rate of MSC in the target tissue of cell therapy. Thus, the decreased levels of PAI-1 has an interesting therapeutic potential to modulate key processes involved in creating an inhospitable environment, during the process of injury, to the homing of transplanted cells. Decreased levels of PAI-1 are promoted by simvastatin, a drug of the statins family. Thus, the goal of this work was to evaluate the effect of simvastatin in vivo on the expression of PAI-1, as well as its influence on the survival rate of infused cells in mice model of cell therapy for pulmonary COPD/emphysema. FVB mice were submitted pulmonary emphysema induction by means of intranasal instillation of elastase and than treated with adipose-derived mesenchymal stem cells and simvastatin. The results regarding morphological and functional aspects, when considering the analisys of both lungs, presented no statistically significant difference among the groups submitted to intranasal instillation of elastase and cell therapy with MSC, treated or not with simvastatin. However, when the lungs where analyzed individually, it was found that there was no statistically significant difference between the control group and the results regarding the right lung of animals treated with elastase and that received simvastatin and MSC infusion. Anatomical differences between the right and left sides of the lung lead to a higher deposition of cells in the right side, as observed in the bioluminescence assays. Thus, it is possible to infer that the morphological recovery in the right side of the lung of animals with DPOC/emphysema could be due to a regenerative paracrine effect of mesenchymal stem cells associated with simvastatin.

O objetivo deste estudo foi avaliar o efeito da sinvastatina, isolada e associada ao α-tocoferol e à L-arginina, sobre os inibidores endógenos da óxido nítrico sintase, os metabólitos do óxido nítrico e tióis, em pacientes hipercolesterolêmicos. Analisou-se um grupo de 16 pacientes hipercolesterolêmicos que seguiram o seguinte protocolo: período de washout (sem medicação), 1 mês; sinvastatina (20mg/dia), 2 meses; sinvastatina (20mg/dia) + α-tocoferol (400U/dia), 2 meses; sinvastatina (20mg/dia, washout), 1 mês; sinvastatina (20mg/dia) + L-arginina (7g/dia), 2 meses. A sinvastatina reduziu significativamente as concentrações do colesterol total e LDL-colesterol e a razão LDL-colesterol/HDL-colesterol. O tratamento com sinvastatina, isolada e associada ao α-tocoferol, promoveu diminuição nas concentrações de S-nitrosotióis. A L-arginina associada à sinvastatina, aumentou os níveis de colesterol total quando comparada com a sinvastatina isoladamente. As concentrações plasmáticas de α-tocoferol e L-arginina não aumentaram em decorrência da suplementação, devido à grande dispersão dos dados obtidos, embora as medianas das concentrações plasmáticas de Larginina e α-tocoferol tenham sido mais elevadas após as suplementações. O tratamento com sinvastatina, isolada ou associada à L-arginina e ao α-tocoferol, não alterou as concentrações dos inibidores endógenos da óxido nítrico sintase (ADMA e SDMA), dos metabólitos do óxido nítrico, da nitrotirosina total e dos tióis analisados. / The aim of this study was to evaluate the effect of sinvastatin, isolated and associated to α-tocopherol and to L-arginine, on the endogenous inhibitors of nitric oxide synthase, on nitric oxide metabolytes and thiols, in hypercholesterolemic patients. A group of 16 hypercholesterolemic patients were analysed, acconting to the protocol: a washout period (without medication) of 1 month, sinvastatin (20 mg/day) for 2 months; sinvastatin (20 mg/day) + α-tocopherol (400U/day) for 2 months; sinvastatin (20 mg/day) for 1 months (washout period), sinvastatin (20 mg/day) + L-arginine (7g/day) for 2 months. Sinvastatin significantly reduced the concentrations of total cholesterol and LDL-cholesterol, as well as the LDL-cholesterol/HDLcholesterol ratio. The treatment with sinvastatina, alone and associate to α-tocoferol, resulted in a reduction of RSNO concentration. The L-arginine associated with sinvastatin, increase the level of total cholesterol as compared with simvastatin alone. The plasma concentrations of a-tocopherol and Larginine did not increase following supplementation due to the large dispersion of the data obtained, even though the median plasma concentrations of L-arginine and a-tocopherol were elevated after supplementation. Treatment with simvastatin, alone or associated to L-arginine and a-tocopherol did not alter the concentrations of the endogenous inhibitors of nitric oxide synthase (ADMA and SDMA), or that of nitric oxide metabolytes, total nitrotyrosine or the thiols analysed.

Breast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models. / text

No description available.

Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Drug-drug interactions (DDIs) refer to situations where one drug affects the pharmacokinetics or pharmacodynamics of another. DDIs represent a major cause of morbidity and mortality. A common adverse drug reaction (ADR) that can result from, or be exacerbated by DDIs is drug-induced myopathy. Identifying DDIs and understanding their underlying mechanisms is key to the prevention of undesirable effects of DDIs and to efforts to optimize therapeutic outcomes. This dissertation is dedicated to identification of clinically important myopathic DDIs and to elucidation of their underlying mechanisms. Using data mined from the published cytochrome P450 (CYP) drug interaction literature, 13,197 drug pairs were predicted to potentially interact by pairing a substrate and an inhibitor of a major CYP isoform in humans. Prescribing data for these drug pairs and their associations with myopathy were then examined in a large electronic medical record database. The analyses identified fifteen drug pairs as DDIs significantly associated with an increased risk of myopathy. These significant myopathic DDIs involved clinically important drugs including alprazolam, chloroquine, duloxetine, hydroxychloroquine, loratadine, omeprazole, promethazine, quetiapine, risperidone, ropinirole, trazodone and simvastatin. Data from in vitro experiments indicated that the interaction between quetiapine and chloroquine (risk ratio, RR, 2.17, p-value 5.29E-05) may result from the inhibitory effects of quetiapine on chloroquine metabolism by cytochrome P450s (CYPs). The in vitro data also suggested that the interaction between simvastatin and loratadine (RR 1.6, p-value 4.75E-07) may result from synergistic toxicity of simvastatin and desloratadine, the major metabolite of loratadine, to muscle cells, and from the inhibitory effect of simvastatin acid, the active metabolite of simvastatin, on the hepatic uptake of desloratadine via OATP1B1/1B3. Our data not only identified unknown myopathic DDIs of clinical consequence, but also shed light on their underlying pharmacokinetic and pharmacodynamic mechanisms. More importantly, our approach exemplified a new strategy for identification and investigation of DDIs, one that combined literature mining using bioinformatic algorithms, ADR detection using a pharmacoepidemiologic design, and mechanistic studies employing in vitro experimental models.