Ziel dieser Studie war es, die Häufigkeit der Expression von EGFR, HER-2 sowie COX-2 im Zervixkarzinom zu eruieren. Dabei galt es herauszufinden, ob Unterschiede hinsichtlich des Nachweises dieser drei, möglicherweise therapeutisch relevanten Moleküle zwischen den primären, nicht vortherapierten und operierten Karzinomen und den multimodal vorbehandelten Rezidiven gab. In der vorliegenden retrospektiven Arbeit wurden 45 TMMR-operierte Primärtumoren und 28 LEER-operierte Rezidivtumoren der Universitätsfrauenklinik Leipzig (Triersches Institut) einbezogen und zusätzlich hinsichtlich der prognostischen Überlebensanalyse durch das Tumorstadium, Lymphknotenmetastasen und Rezidivauftreten sowie histologischer Charakteristika untersucht. Dazu wurden Tissue - Microarrays angefertigt mit anschließender immunhistochemischer Untersuchung dieser. Die Ergebnisse zeigten, dass die TMMR-Operation die Überlebensprognose signifikant verbessert, denn lediglich bei den LEER-therapierten Rezidivtumoren erlitten die Patientinnen sowohl Fernmetastasen als auch erneute Rezidive. Weder die Expression der drei untersuchten Moleküle noch die histopathologischen Parameter haben eine prognostische Relevanz. Es gibt keine signifikanten Zusammenhänge zwischen der Häufigkeit der Expression von EGFR, HER-2 sowie COX-2 und Primär-, bzw. Rezidivtumoren, sodass diese Moleküle keine Targets für eine individualisierte, zielgerichtete Therapie beim Zervixkarzinom darstellen.

Dans le contexte du stockage profond des déchets radioactifs, il est important d'identifier l’hystérésis de saturation de la roche hôte, l'argilite du Callovo-Oxfordien, et sa capacité de scellement (en particulier, la porosité et la distribution de taille des pores). Tout d'abord, six cycles différents d'humidité relative sont destinés à évaluer l’hystérésis de saturation, qui n'est pas observée dans les cycles de faible amplitude. D'autre part, une nouvelle méthode est proposée pour la mesure de la porosité, qui utilise l'injection d'un gaz pour évaluer le volume des pores. Par rapport à la porosité par adsorption d'eau, l’injection de gaz fournit des porosités supérieures d’environ 5%. L'injection de gaz est également utilisée pour quantifier les isothermes de sorption-désorption, qui sont sensiblement différentes de celles obtenues par la méthode gravimétrique, avec un volume poreux accessible au gaz plus élevé pour une humidité relative <43%. Enfin, par Microscopie Electronique à Balayage couplée à un Faisceau Ionique Focalisé (FIB/MEB), on reconstruit le réseau poreux 3D de l'argilite à partir de séries d'images 2D espacées de 10nm : la porosité et la distribution de taille des pores sont quantifiés jusqu’à 20nm, ainsi que l’orientation et l'anisotropie. Avec une résolution plus élevée (jusqu’à moins de 1nm), la Microscopie Electronique à Transmission (MET) montre une grande quantité de pores de l’ordre de quelques nm, situés entre les agrégats d'argile. / In the context of deep underground storage of radioactive nuclear waste, it is important to identify the saturation hysteresis of the host rock, i.e. of Callovo-Oxfordian (COx) claystone, and its porosity and pore size distribution. Firstly, six different cycles of relative humidity are applied for saturation hysteresis, which is not observed in the cycles with low magnitude. Secondly, a new method is proposed for measuring porosity, which uses injection of gas to evaluate the pore volume. In contrast to porosity given by water adsorption, the gas injection method provides larger porosity values of around 5%. The gas injection method is also used to quantify the sorption-desorption isotherms of COx claystone, which are significantly different from those obtained by the gravimetric method, with a bigger pore volume accessible to gas in relative humidities < 43%. Finally, by Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM), we obtain 2D image stacks and 3D reconstructed pore volumes, by which porosity and pore size distribution are quantified down to 20nm, as well as pore orientation and anisotropy. At a higher resolution (below 1nm), Transmission Electron Microscopy (TEM) imaging reveals significant amounts of smaller pores (of a few nm) between clay aggregates.

L’inflammation est un phénomène affectant des millions de personnes à travers le monde. Il existe une grande variété de médiateurs inflammatoires impliqués dans différentes fonctions biologiques, dont la cyclooxygénase-2. Bien que de nombreux inhibiteurs sélectifs de la COX-2 aient été développés et commercialisés, ceux-ci présentent des effets secondaires dont la gravité a entraîné, dans certains cas, l'arrêt de leur commercialisation.De nos jours, les méthodes in silico sont de plus en plus employées dans les stratégies de découverte de nouvelles molécules à visée thérapeutique. Au cours de ce projet, nous nous sommes appuyés sur les modèles pharmacophoriques et les méthodes de docking afin de guider et de prioriser la synthèse de molécules de structures diverses et originales, susceptibles de présenter les meilleures affinités pour la cible étudiée. Ainsi, des prédictions réalisées avec le logiciel TOMOCOMD-CARDD combinées à des tests biologiques, ont permis d’identifier le cyclocoumarol comme une molécule potentiellement anti-inflammatoire. Dans le cadre de ces travaux nous nous sommes intéressés à la synthèse et l’étude d’analogues du cyclocoumarol en tant qu’inhibiteurs sélectifs de la COX-2. La pharmacomodulation autour du cyclocoumarol et la mise en place de stratégies de synthèse judicieuses ont permis d’obtenir une série d’analogues. Divers outils bioinformatiques ont été utilisés : le logiciel LigandScout a permis de construire des pharmacophores sélectifs de la COX-2 et les études de docking ont permis de comprendre les modes de liaisons des différents composés. Enfin, le logiciel SeeSAR, a permis de prédire l’affinité des molécules les plus susceptibles d’inhiber sélectivement la COX-2. Les tests biologiques ont confirmé leur activité inhibitrice envers la COX-2 avec une inhibition non significative vis-à-vis de COX-1. Parmi les molécules synthétisées, le 4-OMe cyclocoumarol a démontré une activité et une sélectivité très intéressantes, comparables au NS-398, un inhibiteur sélectif connu de la COX-2. A partir des résultats biologiques obtenus, un travail de phamacomodulation autour de ces dérivés du cyclocoumarol a été réalisé en utilisant des outils in silico dans le but de prédire l’affinité de nouveaux composés et de découvrir de nouveaux inhibiteurs sélectifs de la COX-2.Mots clés : cyclocoumarol, benzalacétones, warfarines, pharmacophores, docking, criblage virtuel, COX-2, repositionnement / Inflammation is a phenomenon affecting millions of people throughout the world. There is a broad range of inflammatory mediators implied in different biological functions including the cyclooxygenase-2. Although many selective inhibitors selective of COX-2 have been developed and marketed, they have displayed diverse side effects leading, in some cases, to their with drawal from the market. Nowadays, in silico methods are more and more used in the drug discovery process. In this project, we have used pharmacophoric models and docking methods to guide and prioritize the synthesis of molecules, presenting different and original structures, with enhanced affinity for the biological target. Thus, predictions realized with the TOMOCOMD-CARDD software together with biological tests enable to identify the cyclocoumarol as a potential anti-inflammatory molecule. As part of these works, the synthesis of and the study of cyclocoumarol analogues as selective inhibitors of COX-2 have been realized. Pharmacomodulation of cyclocoumarol and development of synthesis strategies led to a serie of cyclocoumarol analogues. Several bioinformatics tools have been used: selective COX-2 pharmacophores were elucidated using LigandScout and docking studies (Surflex) were conducted to understand the binding mode of different compounds. Finally, SeeSAR enabled to predict the affinity of the molecules the most susceptible to inhibit selectively COX-2. Biological tests confirmed their inhibitory activity against COX-2 and showed no significant inhibition for COX-1. Among the synthesized molecules, the 4-OMe cyclocoumarol has demonstrated an activity and a selectivity very interesting, similar to NS-398, a known selective COX-2 inhibitor.Based on the biological results obtained, a pharmacomodulation study of cyclocoumarol derivatives has been realized using in silico tools in order to predict the affinity of new compounds and to discover new selective inhibitors of COX-2.Keywords : cyclocoumarol, benzalacetones, warfarines, pharmacophores, docking, virtual screening, COX-2, repositioning

O objetivo deste trabalho foi comparar o desempenho do modelo de riscos proporcionais de Cox convencional, modelo de Cox modificado quando os riscos não são proporcionais e o modelo de análise de sobrevida baseado na teoria de processos de contagem. Para tanto utilizou-se uma coorte de 648 pacientes portadores de câncer de pênis, atendidos no Departamento de Cirurgia Pélvica do Hospital A. C. Camargo, no período de 1953 a 1985. Dessa coorte foram selecionadas três amostras com o objetivo de validar internamente os resultados da análise de sobrevida do banco de dados original. Os resultados do modelo de riscos proporcionais de Cox, no banco de dados original, foram confirmados por uma das amostras desse conjunto de dados. Apenas o estadiamento N foi confirmado como fator prognóstico também nas outras duas amostras. O modelo de riscos proporcionais de Cox e o modelo de análise de sobrevida baseado na teoria de processos de contagem apresentaram resultados semelhantes, na definição dos fatores prognósticos dessa coorte de pacientes com câncer de pênis. O modelo utilizando processos de contagem é mais sofisticado, do ponto de vista matemático. Porém o modelo de Cox está disponível em grande número de pacotes estatísticos e a interpretação de seus coeficientes se faz com maior facilidade. Por isso, talvez, continue a ser a técnica estatística mais utilizada quando o objetivo do estudo é definir fatores prognósticos e grupos de risco. Os fatores prognósticos para a sobrevida de pacientes com câncer de pênis foram os estadiamentos T e N e o grau de diferenciação do tumor. Esses resultados foram ajustados pelo ano de início de tratamento no Hospital A.C. Camargo. Os pacientes com prognóstico favorável foram os que apresentaram tumor pequeno, sem presença de linfonodos clinicamente positivos, e tumor bem diferenciado. / The aim of this study was to compare the performance of the Cox proportional hazards model, the Cox model with time-dependent covariates and the survival model using the counting process theory. These methods were applied in a cohort of 648 patients with penile cancer treated at the Department of Pelvic Surgery, Hospital A.C. Camargo (São Paulo-Brazil), between 1953 and 1985. Three samples were selected from the total database in order to check the internal validity. The prognostic factors selected using the Cox proportional hazards model were the same in one sample. The only prognostic factor selected in all samples was the N stage. The T and N stages, and the grade of differentiation were independent prognostic factors of survival using both the Cox proportional hazards model and the survival,model using the counting process theory. The statistical significance was the same and even the values of estimation of the coefficients were very close. The survival model using the counting process is more sophisticated from the mathematical point of view, but the Cox model is more available in statistical software, and, probably because of this, is more applied in survival analysis than the model using the counting processo Patients with small tumors, clinically negatives nodes and well differentiated tumors showed a favorable prognosis. These results were adjusted by year of the beginning in the study.

Esta Tese explora o Processo de Cox quando sua intensidade pertence a uma família de difusões afim. A forma da funçâo densidade de Probabilidade do Processo de Cox é obtida quando a intensidade é descrita por uma difusão fim d-dimensional arbitrária. Analisa-se também o acoplamento e convergência para o Processo de Cox com intensidade afim. Para ilustrar assume-se que a intensidade do Processo é governada por uma difusão de Feller e resultados mais detalhados são obtidos. Adicionalmente, os parâmetros da intensidade do Processo são estimados por meio do Filtro de Kalman conjugado com o estimador de Quase-Máxima Verossimilhança. / This Thesis deals with the Cox Process when its intensity belongs to a family of affine diffusions. The form of the probability density function of the Cox process is obtained when the density is described by an arbitrary d-dimensional affine diffusion. Coupling and convergence results are also addressed for a general Cox process with affine intensity. We adopted the Feller diffusion for driving the underlying intensity of the Cox Process to illustrate our results. Additionally the parameters of the underlying intensity processes are estimated by means of the Kalman Filter in conjunction with Quasi-Maximum Likelihood estimation.

Herpes Zoster é uma doença causada pela reativação do vírus Varicela Zoster nos gânglios sensoriais, caracterizada pelo desenvolvimento de lesões na pele e dor. Não há modelos animais disponíveis para estudo da patofisiologia da doença. No entanto, um modelo murino que utiliza o HSV-1 tem sido usado para tal fim, visto que os animais desenvolvem lesões zosteriformes e desenvolvem hipersensibilidade na pata infectada. Não há dados na literatura acerca da resposta imune que se desenvolve nos gânglios da raiz dorsal destes animais. Logo, o objetivo deste trabalho foi o de avaliar células e mediadores inflamatórios presentes nos gânglios da raiz dorsal e sua relação com a hiperalgesia durante a infecção cutânea por HSV-1. Durante a fase aguda da infecção, os camundongos desenvolveram hiperalgesia nas patas ipsilaterais a partir do 3 dia pós-infecção, que perdurou até o 7 dia pós-infecção. A maior carga viral foi detectada nos gânglios L4, L5 e L6, os quais compõem o nervo ciático, que inerva a área infectada. O tratamento dos animais infectados com dexametasona ou fucoidina resultou na redução do comportamento de hiperalgesia, a partir do 5 dia pós-infecção, que corresponde ao período em que a migração de leucócitos passa a aumentar nos gânglios da raiz dorsal. Macrófagos, neutrófilos e linfócitos T CD4 foram detectados nos gânglios durante a infecção aguda. No entanto, linfócitos T CD8 estavam ausentes. A expressão do mRNA de TNF- e COX-2 estava aumentada nos gânglios, e o tratamento de animais infectados com drogas inibidoras de ambos resultou na redução da hiperalgesia. Os receptores do tipo Toll-like e da IL-1 não participam da geração da hipersensibilidade herpética. Após 50 dias da infecção, constatou-se que alguns animais apresentavam comportamento de hiperalgesia irreversível, semelhante à neuralgia pós-herpética humana (NPH). Não houve diferença significativa na incidência da NPH em animais de linhagens ou sexos diferentes. Ainda, o tratamento com drogas anticonvulsivantes e antidepressivas, mas não com morfina e anti-inflamatórios, resultou na redução transiente da hiperalgesia. Neste período, não há participação da inflamação na manutenção da hiperalgesia. A expressão de TNF- e COX-2 retorna aos níveis basais, e não são mais detectados neutrófilos e macrófagos. No entanto, a migração de linfócitos T CD4+ e CD8+ aos gânglios aumenta de maneira tempo-dependente. Durante a NPH, detectou-se uma intensa ativação das células satélites gliais, que contribuem para a manutenção da hiperalgesia pós-herpética. Nossos resultados demonstram que a manutenção hiperalgesia herpética é resultado da intensa resposta inflamatória que ocorre nos gânglios da raiz dorsal infectados, com aumento da produção de TNF- e COX-2, importantes mediadores para a hipersensibilidade. No entanto, durante a neuralgia pós-herpética, não há participação de células ou mediadores inflamatórios, mas de células da glia, as quais são importantes na manutenção da hiperalgesia. / Herpes Zoster is a disease caused by reactivation of varicella zoster virus in sensory ganglia, characterized by dermal rash and pain. There are no animal models available to study the pathophysiology of the disease. A murine model of HSV-1 infection on the hind paw skin has been used to study HZ, since mice develop HZ-like skin lesions and pain-related responses. There are no data available about the immune response in dorsal root ganglion (DRG) of these mice. Thus, the aim of this study was to evaluate cells and inflammatory mediators present in DRGs and its relationship with hiperalgesia during HSV-1 cutaneous infection. During the acute phase of infection, mice developed hyperalgesia in ipsilateral paws from 3 days post-infection, which persisted until 7 days post-infection. The highest viral load was detected in ganglia L4, L5 and L6. Treatment of infected mice with fucoidin or dexamethasone resulted in the reduction of hyperalgesic behavior, from the 5th post-infection day, which corresponds to the period in which leukocyte migration increase in the dorsal root ganglia. Macrophages, neutrophils and CD4 + T lymphocytes were detected in the ganglia during acute infection. However, CD8 + T lymphocytes were absent. The mRNA expression of TNF- and COX-2 was increased in dorsal root ganglia, and the treatment of infected mice with drugs that inhibits both mediators resulted in reduced hyperalgesia. The Toll-like receptors and IL-1 does not participate in the generation of herpetic hypersensitivity. After 50 days of infection, it was found that some animals presented irreversible hyperalgesic behavior, like human post-herpetic neuralgia (PHN). There was no significant difference in the incidence of PHN in animals of different genders or strains. Furthermore, treatment with anticonvulsant and antidepressant drugs, but not morphine and anti-inflammatory, resulted in transient reduction of hyperalgesia. In this period, there is no participation of inflammation in the hyperalgesia maintenance of. The expression of TNF- and COX-2 returns to baseline levels, and neutrophils and macrophages are no longer detected. However, the migration of CD4 + and CD8 + to ganglia increases in a time-dependent manner. During NPH, an intense activation of glial cells satellites was detected, that contributes to the maintenance of post-herpetic hyperalgesia. Our results demonstrate that herpetic hyperalgesia maintenance is a result of an intense inflammatory response that occurs in the infected dorsal root ganglia, with increased production of TNF- and COX-2. However, during post-herpetic neuralgia, there is involvement of glial cells, which are important in hyperalgesia maintenance.

A transformação de Aspergillus nidulans, com RNA de macrófagos de ratos, resultou na linhagem RT2, produtora de uma glicoproteína antiinflamatória. Nosso objetivo foi avaliar esta linhagem genenomicamente e caracterizar esta glicoproteína quanto à natureza bioquímica e sua atividade. Para tal, foi realizado RAPD e análise fenotípica desta linhagem. A Nandina foi purificada e submetida à espectrometria de massa para sequenciamento e identificação dos carboidratos. Testes da atividade antiinflamatória in vivo foram realizados em peritonite e edema de pata e inibição dos receptores de glicocorticóides. Os testes in vitro, sobre a produção das COXs e de PGE2, foram realizados em cultura de macrófagos. Os resultados mostraram que a linhagem RT2 é resultante da UT448, mas contém diferenças em seu genoma. A proteína purificada possui 40KDa. A espectrometria de massa caracterizou dois fragmentos da proteína e sua glicosilação. Os testes in vivo mostraram que a proteína inibe o edema e o influxo leucocitário e que esta atividade não é dependente de glicocorticóides, mas sim da inibição in vitro de COX-2, mas não de COX-1 e nem de PGE2. / Aspergillus nidulans transformation with rat macrophage RNA results on RT2 strain, producer of an antiinflammatory glycoprotein. Our objective was to evaluate this strain genomically and characterize biochemically and activity of its glycoprotein. To this, RAPD and fenotipical analysis were performed. The Nandin was purified and mass spectrometry analyzed to sequencing and carbohydrates analysis. Antiinflammatory activity testes in vivo in peritonitis and edema, and glucocorticoid receptors inhibition were performed. The in vitro testes, over expression and activity of COXs and PGE2, were performed in macrophage culture. The results show that RT2 strain came from UT448, but have genomics differences. The purified glycoprotein has been 40KDa. The mass spectrometry sequenced two protein fragments and showed that glycosylation. The in vivo testes showed that the glycoprotein has antiinflammatory activity inhibiting the edema and leukocyte influx. The RU38486 experiments evidenced that activity is not glucocorticoid receptors dependent, but in vivo inhibition of COX-2, but not COX-1 neither its product PGE2.

La modélisation des effets centre dans le cadre des données de survie repose souvent sur l'utilisation de modèles de Cox à effets mixtes. Tester un effet centre revient alors à tester à zéro la variance de l'effet aléatoire correspondant. La distribution sous l'hypothèse nulle des statistiques des tests paramétriques usuels n'est alors pas toujours connue. Les procédures de permutation ont été proposées comme alternative, pour les modèles linéaires généralisés mixtes.L'objectif est de développer, pour l'analyse des effets centre dans un modèle de survie de Cox à effets mixtes, une procédure de test de permutation pour les effets aléatoires.La première partie du travail présente la procédure de permutation développée pour le test d'un unique effet centre sur le risque de base, avec une application à la recherche d'un effet centre dans un essai clinique chez des patients atteints de leucémie myéloïde aiguë. La seconde partie porte sur l'extension de la procédure au test d'effets aléatoires multiples afin d’étudier à la fois des effets centre sur le risque de base et sur l'effet de variables, avec des illustrations sur deux cohortes de patients atteints de leucémie aiguë. Dans une troisième partie, les méthodes proposées sont appliquées à une cohorte multicentrique de patients en réanimation atteints d'hémopathies malignes, pour étudier les facteurs déterminant les effets centre sur la mortalité hospitalière. Les procédures de permutation proposées constituent une approche robuste et d'implémentation relativement aisée pour le test, en routine, d'effets aléatoires, donc un outil adapté pour l'analyse d'effets centre en épidémiologie clinique, afin de comprendre leur origine. / Centre effects modelling within the framework of survival data often relies on the estimation of Cox mixed effects models. Testing for a centre effect consists in testing to zero the variance component of the corresponding random effect. In this framework, the identification of the null distribution of usual tests statistics is not always straightforward. Permutation procedures have been proposed as an alternative, for generalised linear mixed models.The objective was to develop a permutation test procedure for random effects in a Cox mixed effects model, for the test of centre effects.We first developed and evaluated permutation procedures for the test of a single centre effect on the baseline risk. The test was used to investigate a centre effect in a clinical trial of induction chemotherapy for patients with acute myeloid leukaemia.The second part consisted in extending the procedure for the test of multiple random effects, in survival models. The aim was to be able to examine both center effects on the baseline risk and centre effects on the effect of covariates. The procedure was illustrated on two cohorts of acute leukaemia patients. In a third part, the permutation approach was applied to a cohort of critically ill patients with hematologic malignancies, to investigate centre effects on the hospital mortality.The proposed permutation procedures appear to be robust approaches, easily implemented for the test of random centre effect in routine practice. They are an appropriate tool for the analysis of centre effects in clinical epidemiology, with the purpose of understanding their sources.

NSAIDs are among the most used drugs in the world. It is estimated that 30 million people take NSAIDs daily world-wide, without including drugs sold over the counter. They are effective in alleviating pain and inflammation. Even though they are very common there does not appear to be any clear-cut guidelines to when which NSAID should be used. It has therefore been the purpose of this thesis to analyze if there is a need to differentiate between different NSAIDs according to contemporary evidence. Since the withdrawal of rofecoxib in 2004 there has been a general idea that coxibs as a group are cardiotoxic, recent evidence suggests that this holds true for all NSAIDs however. As such this work included 5 drugs, three common over the counter non-selective NSAIDs; naproxen, ibuprofen and diclofenac as well as the two coxibs currently on the Swedish market; celecoxib and etoricoxib. Pubmed and google scholar were searched for relevant studies on the subject. The results showed that there is a need to differentiate between NSAIDs, however the clinical setting is complex and a one-size fits all solution is difficult to come by. Naproxen and moderate doses of celecoxib (100 mg b.i.d.) show the best cardiovascular profiles whilst etoricoxib, celecoxib and diclofenac show the best gastrointestinal profiles. Coxibs show similar upper GI-profiles as tNSAIDs if combined with PPI however PPI are not without adverse events and the lower GI is not affected by PPI. Longer half-life is in general the better option in situations with lasting pain since it has been shown that lower dosing intervals increase adherence. In terms of pain management there does not appear to be any differences in efficacy amongst different NSAIDs

No description available.