Malgré plusieurs chimiothérapies suivies d’une transplantation et d’une immunothérapie, 40% des patients avec un neuroblastome (NB) à haut risque subissent une progression de la maladie ou une rechute. L’échec de ces traitements est attribué à la présence de cellules initiatrices de tumeur (TIC) qui expriment le marqueur CD133 et qui sont souvent résistantes aux agents chimiothérapeutiques. Les cellules Natural Killer (NK), qui possèdent un effet anti-tumoral, peuvent être utilisées dans le cadre du développement de nouvelles approches immuno-thérapeutiques. Nous posons l’hypothèse que les cellules NK activées éliminent efficacement les TIC et contribuent à la réduction des risques de rechute. De plus, il est possible d’augmenter l’effet anti-tumoral des cellules NK contre le NB. L’activité cytotoxique des cellules NK est augmentée par des cellules dendritiques plasmacytoïdes (pDC) activées. A la suite de la stimulation de leurs récepteurs Toll-like les pDC produisent de grandes quantités d'interféron-alpha (IFN-α). Nous avons étudié les propriétés lytiques des cellules NK contre des lignées cellulaires de NB à la suite de leur activation par l’IFN-α ou des pDC activées. Nos résultats révèlent une augmentation de l’activité cytolytique des cellules NK contre ces lignées en réponse à une stimulation par les pDC activées. De plus, les cellules de NB CD133+ ou celles résistantes à l’immunothérapie dirigée contre le GD2 sont sensibles à la lyse médiée par les cellules NK stimulées par les pDC. Nous avons examiné les mécanismes cellulaires impliqués dans la lyse des cellules de NB. Nous montrons que cette cytotoxicité est médiée en partie par TRAIL induisant l'apoptose et en partie par la libération des granules cytotoxiques. Ainsi, ces résultats permettent de proposer une nouvelle approche immuno-thérapeutique complémentaire au traitement par l’anticorps anti-GD2 pour les patients atteints de NB à haut risque. / Despite aggressive treatment by chemotherapy followed by transplantation and treatment with anti-tumor cell disialoganglioside (GD2) monoclonal antibody, IL-2, GM-CSF and retinoic acid, 40% of patients with high-risk neuroblastoma (NB) still undergo disease progression or relapse. Furthermore, tumor-initiating cells (TIC) expressing the CD133 marker are present in NB tumors and are more resistant to chemotherapy. To evaluate a new immunotherapeutic approach, we took advantage of the anti-tumor effect of Natural Killer (NK) cells. We hypothesized that activated NK cells would be a potent therapeutic strategy to eliminate TIC and reduce relapse of NB. We aimed to establish the best strategy to increase the NK cell mediated cytotoxicity against NB. NK cell cytotoxic activity is increased by cytokines, chemokines and activated plasmacytoid dendritic cells (pDC) which produce high amounts of interferon-alpha (IFN-α) upon Toll-like receptor stimulation. We investigated NK-cell lytic properties against NB cell lines following activation by IFN-α or activated pDC. Our results reveal an increased cytolytic activity of NK cells against NB cell lines after stimulation by activated pDC, CD133+ (TIC) as well as anti-GD2 resistant NB cells are sensitive to NK cell mediated cytotoxicity following stimulation by activated pDC. We also examined the cellular mechanisms involved in NK cell-mediated lysis of NB cell lines. The increased cytotoxicity is partially mediated by TRAIL induced apoptosis and as well as by the release of cytolytic granules. In conclusion, we propose a new immunotherapeutic approach that can be used in combination with the anti-GD2 therapy for the treatment of high-risk NB patients.

3 ABSTRACT Title of project: Visitors of the Nature Park Ladronka and their interest in movement activities Aim of project: The goal of this Thesis is a socio-demographic analysis of the visitors of the Nature Park Ladronka and their motives for movement activities in this location in different season. Methods of project: All necessary data were collected by means of interviews directly in the Park Ladronka during this three weeks 25. - 31. 10. 2011 from 10:00-17:30, 23. - 29. 5. 2011 from 10:00-20:00 and 8. - 14. 8. 2011 from 10:00-20:00. The results were statistically processed using relative and absolute frequency. Results: The total number of respondents is 1,425. They are mostly - 32 % of visitors aged 20-29 - interested in the on-line skating track. The most common means of transport was for 40% of the visitors their car. The preferred sport for men (47 %) is running 42 % and gym 27 %, and for women (53 %) in-line skating 79 %. 14,5 % of visitors aged 20-29 are from housing estate. The park is visited mostly by students 32 % and people working on their computers 24 %. Key words: Natural fitness gym - sport for everyone - health grounds - outdoor activities - fitness trail - urban green - socio- demographic analysis

Sabe-se que TRAIL é capaz de matar células tumorais de forma seletiva e que TRAIL tem sua expressão reduzida em diversos tumores, porém pouco se sabe sobre os mecanismos responsáveis pela sua inibição. Tendo em vista que a expressão de TRAIL pode ser regulada pelo Ácido Retinóico; que PRAME é capaz de inibir a via do ácido retinóico através da proteína EZH2 e que nós observamos anteriormente que a expressão de TRAIL esta diminuída em pacientes com LMC, nós decidimos investigar a associação entre PRAME, EZH2 e TRAIL na LMC. Nós demonstramos que PRAME, mas não EZH2, tem sua expressão aumentada em células BCR-ABL+ e sua expressão está associada com a progressão da LMC. Alem disto, existe uma correlação positiva entre PRAME e BCR-ABL e negativa entre PRAME e TRAIL nestes pacientes. A inibição da expressão de PRAME ou EZH2 por RNAi induziu um aumento da expressão de TRAIL. Estes dados revelam um novo mecanismo de regulação responsável por diminuir a expressão de TRAIL, e geram novos possíveis alvos para a terapia da LMC e, possivelmente, também para outros tumores. / TRAIL was shown to selectively kill tumor cells. Not surprisingly, TRAIL is down-regulated in a variety of tumor cells, but the mechanism responsible for TRAIL inhibition remains elusive. Because TRAIL can be regulate by retinoic acid; PRAME was shown to inhibit transcription of retinoic acid receptor target genes through the polycomb protein EZH2; and we have found that TRAIL is inversely correlated with BCR-ABL in CML patients, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is up-regulated in BCR-ABL cells and is associated with the progression of disease in CML patients. In addition, PRAME expression is positively correlated with BCR-ABL and negatively with TRAIL in these patients. Importantly, knocking down of PRAME or EZH2 by RNA interference restores TRAIL expression. Our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A população de idosos cresce rapidamente no Brasil. A prevalência de hipertensão arterial sistêmica (HAS) e distúrbios cognitivos é elevada nesta população. Testamos a hipótese de que a HAS reduz o desempenho cognitivos em idosos. Foram selecionamos idosos hipertensos e normotensos com idade &#8805; 60 < 80. O desempenho cognitivo foi avaliado pelo Cambridge Cognitive Examination Revised (CAMCOG-R), por subtestes do Wechsler Adult Intelligence Scale v.3 (WAIS III), além do Rey Auditory Verbal Learning Test (RAVLT), e o dos Trail Making Tests A/B (TMT-A/B). O desempenho cognitivo avaliado pelo escore global do CAMCOG-R e do QI estimado do WAIS III está reduzido nos idosos hipertensos mesmo quando controlado pela escolaridade, depressão, estado geral de saúde e qualidade de vida. O desempenho cognitivo em diversos domínios específicos controlados para a escolaridade, depressão, estado geral de saúde e qualidade de vida, e avaliados pelo CAMCOG-R, WAIS III, TMT-A e RAVLT também está reduzido nos idosos hipertensos. O presente estudo sugere que a HAS está associada ao declínio do desempenho cognitivo global em idosos. Notadamente, o desempenho das funções executivas está reduzido nos idosos hipertensos. Especula-se que a HAS seja um fator de risco para o declínio progressivo do desempenho cognitivo e, portanto, para o desenvolvimento de demência. / The elderly population is growing rapidly in Brazil. The prevalence of hypertension and cognitive disorders is high in this population. We tested the hypothesis that hypertension is associated with decreased cognitive performance in the elderly. We selected normotensive and hypertensive subjects aged &#8805; 60 <80 years old. Cognition was assessed through Cambridge Cognitive Examination Revised (CAMCOG-R), subtests of the Wechsler Adult Intelligence Scale v.3 (WAIS III), the Rey Auditory Verbal Learning Test (RAVLT), and the Trail Making Tests A/B (TMT-A/B). The cognitive performance assessed by the CAMCOG-R global score and the estimated WAIS III. QI is reduced in elderly hypertensives even when controlled for education, depression, health status and quality of life. The cognitive performance in diverse cognitive domains assessed through CAMCOG-R, WAIS III, TMT-A e RAVLT is also reduced in elderly hypertensives when contolled for education, depression, health status and quality of life. These results suggest that hypertension is associated with reduced global cognitive performance in eldely hypertensives. Notably, executive functions are reduced in eldely hypertensives. We speculate that hypertension might be a risk factor for the progressive decline in cognitive function and, therefore, for the development of dementia.

Background and Aim: TRAIL has sparked a growing interest in oncology due to its ability to selectively trigger cancer cell death while sparing normal cells. The Fas/actin association through ezrin, a member of the ERM protein family, has been reported to regulate early steps of Fas-mediated apoptosis. In this project, we addressed the role of ezrin regarding TRAIL-induced cell death in B lymphoma cell lines, or adherent cancer cell lines (HeLa WT, HCT116, SW480). Methods: Molecular and biochemical approaches were employed to study the relevance of ezrin and its phosphorylation status in TRAIL signaling. Results: We found that ezrin displays a negative function towards TRAIL- and Fas-mediated apoptosis and that the ezrin-mediated TRAIL-induced cell death inhibition led to ezrin activation through phosphorylation/dephosphorylation events at serine 66 and tyrosine 353, but is mainly independent of TRAIL DISC (Death Inducing Signalling Complex) formation or activation. Mutations of these residues to alanine (S66A) or aspartic acid (Y353D) selectively enhanced TRAIL-induced cell death, whereas point mutations mimicking ezrin phosphorylation on S66 (S66D) or a nonphosphorylable variant on Y353 (Y353F) strongly protected cancer cells from apoptosis induced by TRAIL. Moreover, inhibition of the ezrin serine 66 PKA target site, using H89, increased cancer cell sensitivity to TRAIL, while treatment with 8bromocyclic AMP, a PKA activator, decreased TRAIL-induced cell death. In addition, combined TRAIL/cisplatin treatments abrogated ezrin-mediated inhibition of TRAIL-induced apoptosis.

A practicum report for the degree of Master of Environmental Science, Institute of Environmental Sciences, Miami University, 2007. / Title from first page of PDF document. Includes bibliographical references (p. 63-73).

La cytokine TRAIL est un candidat anticancéreux qui induit la mort spécifique de cellules tumorales. La liaison de TRAIL à ses récepteurs (DR) permet de former le complexe DISC qui induit la mort cellulaire. La raison de la mort sélective des cellules tumorales induite par TRAIL est inconnue. Nous avons découvert des partenaires de DR: chaînes lourdes de myosine IIa, IIb (NMHCIIa, NMHCIIb), chaîne légère régulatrice de myosine (MLC2) et ß-actine. Dans les cellules tumorales, la liaison de TRAIL abroge l'interaction NMHCII/DR, et DISC est activé. Au contraire, dans les cellules normales, l'interaction NMHCII/DR persiste et l'activation de DISC est incomplète. Affaiblir l'interaction NMHCII/DR par des inhibiteurs chimiques ou diminuer NMHCIIa permet d'augmenter l'apoptose liée à TRAIL. L'interaction réduite NMHCII/DR induit des niveaux altérés de phospho-MLC2 et de kinases régulant MLC2. Nous proposons que la résistance de cellules normales à TRAIL soit basée sur l'interaction DR/cytosquelette, déficiente dans des tumeurs. NMHCII étant aussi impliqué dans l'adhésion/migration cellulaire, il serait intéressant d'étudier les fonctions de NMHCII/DISC dans le détachement cellulaire, afin de mieux comprendre la résistance à TRAIL de certains cancers. / The cytokine TRAIL is a promising cancer therapeutic candidate as it induces apoptosis selectively in transformed cells. TRAIL-induced clustering of its receptors (DR) is essential for the DISC complex formation, which induces cell death. The mechanism for TRAIL’s tumour selective effect is largely unknown. We identified the cytoskeleton proteins non-muscle myosin heavy chain IIa, IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain (MLC2) and ß-actin as novel DR-interactors. An initially weak and TRAIL-induced abrogation of NMHCII/DR interaction correlated with efficient DISC formation in tumour cells. In contrast, a robust NMHCII/DR interaction that was sustained upon TRAIL stimulus was accompanied by incomplete DISC arrangement. Weakening the NMHCII/DR interaction in normal cells using chemical inhibitors enhanced TRAIL-induced apoptosis. Intriguingly, siRNA-mediated NMHCIIa- but not NMHCIIb depletion potently released TRAIL resistance in normal cells and influenced DISC composition. Reduced NMHCII/DR interaction in transformed cells was characterised by diminished MLC2 phosphorylation and altered protein expression of upstream regulatory kinases. Our results suggest that normal cell resistance to TRAIL-apoptosis is based on the interaction of cytoskeleton components with DR that is impaired upon transformation. Since NMHCII function in cell adhesion and migration, it will be interesting to study possible roles of the interaction in cell detachment and altered TRAIL sensitivity; moreover this link may provide clues as to the cause of TRAIL resistance in some cancers.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A população de idosos cresce rapidamente no Brasil. A prevalência de hipertensão arterial sistêmica (HAS) e distúrbios cognitivos é elevada nesta população. Testamos a hipótese de que a HAS reduz o desempenho cognitivos em idosos. Foram selecionamos idosos hipertensos e normotensos com idade &#8805; 60 < 80. O desempenho cognitivo foi avaliado pelo Cambridge Cognitive Examination Revised (CAMCOG-R), por subtestes do Wechsler Adult Intelligence Scale v.3 (WAIS III), além do Rey Auditory Verbal Learning Test (RAVLT), e o dos Trail Making Tests A/B (TMT-A/B). O desempenho cognitivo avaliado pelo escore global do CAMCOG-R e do QI estimado do WAIS III está reduzido nos idosos hipertensos mesmo quando controlado pela escolaridade, depressão, estado geral de saúde e qualidade de vida. O desempenho cognitivo em diversos domínios específicos controlados para a escolaridade, depressão, estado geral de saúde e qualidade de vida, e avaliados pelo CAMCOG-R, WAIS III, TMT-A e RAVLT também está reduzido nos idosos hipertensos. O presente estudo sugere que a HAS está associada ao declínio do desempenho cognitivo global em idosos. Notadamente, o desempenho das funções executivas está reduzido nos idosos hipertensos. Especula-se que a HAS seja um fator de risco para o declínio progressivo do desempenho cognitivo e, portanto, para o desenvolvimento de demência. / The elderly population is growing rapidly in Brazil. The prevalence of hypertension and cognitive disorders is high in this population. We tested the hypothesis that hypertension is associated with decreased cognitive performance in the elderly. We selected normotensive and hypertensive subjects aged &#8805; 60 <80 years old. Cognition was assessed through Cambridge Cognitive Examination Revised (CAMCOG-R), subtests of the Wechsler Adult Intelligence Scale v.3 (WAIS III), the Rey Auditory Verbal Learning Test (RAVLT), and the Trail Making Tests A/B (TMT-A/B). The cognitive performance assessed by the CAMCOG-R global score and the estimated WAIS III. QI is reduced in elderly hypertensives even when controlled for education, depression, health status and quality of life. The cognitive performance in diverse cognitive domains assessed through CAMCOG-R, WAIS III, TMT-A e RAVLT is also reduced in elderly hypertensives when contolled for education, depression, health status and quality of life. These results suggest that hypertension is associated with reduced global cognitive performance in eldely hypertensives. Notably, executive functions are reduced in eldely hypertensives. We speculate that hypertension might be a risk factor for the progressive decline in cognitive function and, therefore, for the development of dementia.

Sabe-se que TRAIL é capaz de matar células tumorais de forma seletiva e que TRAIL tem sua expressão reduzida em diversos tumores, porém pouco se sabe sobre os mecanismos responsáveis pela sua inibição. Tendo em vista que a expressão de TRAIL pode ser regulada pelo Ácido Retinóico; que PRAME é capaz de inibir a via do ácido retinóico através da proteína EZH2 e que nós observamos anteriormente que a expressão de TRAIL esta diminuída em pacientes com LMC, nós decidimos investigar a associação entre PRAME, EZH2 e TRAIL na LMC. Nós demonstramos que PRAME, mas não EZH2, tem sua expressão aumentada em células BCR-ABL+ e sua expressão está associada com a progressão da LMC. Alem disto, existe uma correlação positiva entre PRAME e BCR-ABL e negativa entre PRAME e TRAIL nestes pacientes. A inibição da expressão de PRAME ou EZH2 por RNAi induziu um aumento da expressão de TRAIL. Estes dados revelam um novo mecanismo de regulação responsável por diminuir a expressão de TRAIL, e geram novos possíveis alvos para a terapia da LMC e, possivelmente, também para outros tumores. / TRAIL was shown to selectively kill tumor cells. Not surprisingly, TRAIL is down-regulated in a variety of tumor cells, but the mechanism responsible for TRAIL inhibition remains elusive. Because TRAIL can be regulate by retinoic acid; PRAME was shown to inhibit transcription of retinoic acid receptor target genes through the polycomb protein EZH2; and we have found that TRAIL is inversely correlated with BCR-ABL in CML patients, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is up-regulated in BCR-ABL cells and is associated with the progression of disease in CML patients. In addition, PRAME expression is positively correlated with BCR-ABL and negatively with TRAIL in these patients. Importantly, knocking down of PRAME or EZH2 by RNA interference restores TRAIL expression. Our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML.

Bien que constitué anatomiquement et physiologiquement pour la course d’endurance, l’Homme est considérablement exposés à diverses blessures musculo-squelettiques liées à la répétition de contraintes mécaniques. Le coureur de trail running par exemple est soumis à de nombreux impacts ainsi qu’à une fatigue et des dommages musculaires sévères. Ces chocs répétitifs et dommages musculaires réduiraient la tolérance du coureur face aux contraintes mécaniques le poussant ainsi à altérer sa cinématique de course. Par conséquent, minimiser les dommages musculo-squelettiques serait déterminant pour la performance en trail running. Des évidences montrent que la pose de pied altère la localisation et l’intensité des contraintes appliquées au système musculo-squelettique. L’objectif de ce travail de thèse a été d’étudier l’influence du pattern de pose de pied sur l’impact et la fatigue neuromusculaire en trail running. Les phases de descente ont été tout particulièrement étudiées du fait qu’elles sont les plus traumatisantes. En effet, ce travail de thèse a mis en évidence qu’en situation de trail running, l’intensité de l’impact augmente lorsque la pente diminue, et que la fatigue neuromusculaire périphérique est aussi sévère à la suite d’une descente isolée qu’après un ultra-trail de plusieurs heures. En étudiant l’influence de la pose de pied adoptée au cours d’une descente en situation de trail running, il a été observé qu’attaquer le sol par l’avant du pied augmentait la fatigue neuromusculaire aux extenseurs du genou. Cependant, une importante variabilité dans les patterns de pose de pied adoptés au cours de la descente a été associée à une baisse de la fatigue neuromusculaire aux extenseurs du genou et fléchisseurs plantaires. L’influence de la pose de pied sur l’intensité du choc et le contenu vibratoire le long des axes axial et transversal a également été démontrée : adopter une attaque talon diminue la sévérité du choc axial mais réduit l’intensité du choc transversal. La principale conclusion est qu’aucun pattern de course ne saurait être universellement recommandé du fait que « changer de pose de pied » est synonyme de « changer la localisation et la magnitude des contraintes appliquées au système musculo-squelettique ». En ce sens, alterner entre différents patterns de course serait une stratégie efficiente en trail running / Although Humans are “born” anatomically and physiologically adapted to long distances run, they are substantially exposed to various musculoskeletal overuse injuries. Trail runners sustain a high number of foot-to-ground contacts and develop severe muscle fatigue and damages. Repetitive shocks and muscle damages would reduce the runners’ tolerance to mechanical strains leading to changes in running kinematics. Minimizing musculoskeletal damages is therefore considered paramount for performance in trail running. Numerous studies highlighted that the foot strike pattern alters the localization and magnitude of the mechanical strains applied on the musculoskeletal system. The main purpose of this thesis was to study the influence of the foot strike pattern on impact and neuromuscular fatigue in trail running. Downhill sections were mainly investigated since they are the most mechanically stressful. Indeed, it was observed from this thesis’ work that, in real trail running practice, the impact intensity increases as the slope decreases, and that the neuromuscular fatigue induced by a single downhill run is as severe as the one induced by an ultratrail race that lasts several hours. Investigating the effect of the foot strike pattern adopted during a downhill trail run on fatigue, it was observed that forefoot striking increases the neuromuscular fatigue at knee extensors. However, a high variability in foot strike patterns adopted was associated to a lower neuromuscular fatigue at both knee extensors and plantar flexors. The effect of the foot strike pattern on axial and transversal shock and vibration content was also demonstrated: heel striking was correlated to a lower impact severity along the axial axis of the skeleton but a greater one along its transversal axis. The main conclusion of this thesis is that no single foot strike pattern should be universally advised due to “changing of foot strike” means “changing the localization and magnitude of the mechanical stress applied on the musculoskeletal system”. Switching between different running patterns might be an efficient strategy in trail running