Hand proprioception is crucial for daily activities and may be compromised by diseases or injuries,impacting patients' independence. The lack of feasible, accurate, and affordable clinical tools forhand proprioception assessment poses a significant challenge, essential for identifying dysfunctionand evaluating treatment effects.The purpose of this study was to evaluate the concurrent validity of the LeapMotion controller(LMC) for assessing hand proprioception. We compared the LMC with a 3D camera system formotion analysis (Qualisys Motion Capture, QTM), known for its high measurement accuracy as thegold standard. Twenty participants (10 men, 10 women), 15 without, and 5 with hand injury or pain,took part in this cross-sectional study. Assessments included pointing acuity with open and closedeyes using the right and left hand. There were moderate to good correlations between LMC andQTM performed with closed eyes, with intraclass correlation coefficient (ICC) values of 0.6 and0.89. Contrary, tests with open eyes showed a poor overall correlation with ICC between 0.003 and0.3. Bland-Altman analysis showed median biases of≤ 1.5 mm between LMC and QTM with eyes open, and ≤ 5.1 mm with eyes closed. Limits ofagreement ranged from -0.4 to 3.5 mm with eyes open and -31.6 to 21.5 mm with eyes closed.The results indicate that the LMC could be a cost-effective and feasible tool for quantifying handproprioception with a clinically acceptable bias. Although the median biases were small formeasurements with eyes open, the ICCs were poor. This may be due to a high pointing acuity withinthe group combined with limited variability between the participants in the eyes open tests.

Submitted by admin tede (tede@pucgoias.edu.br) on 2017-02-06T16:52:19Z No. of bitstreams: 1 Caio Bruno Quinta de Souza Leal.pdf: 116273125 bytes, checksum: 5e247a988672d255dad21fa0aedcc5da (MD5) / Made available in DSpace on 2017-02-06T16:52:19Z (GMT). No. of bitstreams: 1 Caio Bruno Quinta de Souza Leal.pdf: 116273125 bytes, checksum: 5e247a988672d255dad21fa0aedcc5da (MD5) Previous issue date: 2014-01-31 / Monitoring of minimal residual disease (MRD) in patients with Chronic Myeloid Leukemia (CML) who receive treatment with Imatib mesylate (IM) is extremely important, because it enables the evaluation of the response to the treatment and the early diagnosis of possible recurrences. The objective of this study was to standardize molecular methods used in order to monitor MRD in patients with CML, on therapy with IM. Peripheral blood samples were collected from 11 patients diagnosed with CML in October 2012 to September 2013, in the Department of Hematology of Hospital Araújo Jorge of the Association to Combat Cancer in Goiás. Three months after starting treatment, patients underwent a new peripheral blood collection for evaluation of MRD. Detecting bcr-abl transcripts and endogenous controls (abl and β2m) employed reverse transcription methods associated with polymerase chain reaction (RT-PCR), while quantification of bcr-abl transcripts was achieved by using reverse transcription associated with real-time PCR (RQ-PCR) and Taqman probes. Specific oligonucleotides and probes recognizing e13a2 and e14a2 junctions of bcr-abl transcripts and to the abl endogenous control were used in this study. By the time of diagnosis, three patients (27.3%) expressed the b2a2 transcript, five patients (45.5%) expressed the b3a2 transcript, two patients (18.2%) expressed both transcripts and one patient (9%) did not express any of the transcripts. The endogenous controls analysis resulted in better amplification for the abl transcript, which was used in the RQ-PCR reactions. The assessment of DRM was possible in only eight patients, due to the loss of follow-up. Three months after starting treatment with IM, all patients presented complete hematologic response. However, only one patient (12.5%) presented the undetectable transcript, reaching the full molecular response, while the other seven patients (87.95%) presented MRD. One (12.5%) of the seven patients who presented MRD, reached complete molecular response, while six patients (75%) presented a reduction of two logs, achieving minor molecular response, and one patient (12.5%) presented only partial molecular response. By using molecular biology methods, our results have enabled the standardization and the establishment of a laboratory routine, according to the international guidelines, for monitoring MRD in patients with CML. / O monitoramento de doença residual mínima (DRM) em pacientes com leucemia meilóide crônica (LMC) que recebem tratamento com mesilato de imatibe (MI) é extremamente relevante, pois possibilita o acompanhamento da resposta e o diagnóstico precoce de eventuais recidivas da doença. O objetivo deste estudo foi padronizar métodos moleculares utilizados na avaliação de DRM em pacientes com LMC, em tratamento com MI. Amostras de sangue periférico foram coletadas de 11 pacientes diagnosticados com LMC, no período de outubro de 2012 a setembro de 2013, no Setor de Hematologia do Hospital Araújo Jorge da Associação de Combate ao Câncer em Goiás. Três meses após o início do tratamento, os pacientes foram submetidos a uma nova coleta de sangue periférico para avaliação de DRM. A detecção dos transcritos bcr-abl e controles endógenos (abl e β2m) empregaram os métodos de transcrição reversa associados à reação em cadeia da polimerase (RT-PCR), enquanto a quantificação dos transcritos bcr-abl foi feita por meio de transcrição reversa associada à PCR em tempo real (RQ-PCR), utilizando a metodologia de sondas de hidrólise (TaqMan). Oligonucleotídeos e sondas Taqman específicos para as junções e13a2 e e14a2 dos transcritos bcr-abl e para o controle endógeno (abl) foram usados neste estudo. Ao diagnóstico, três pacientes (27,3%) expressaram o transcrito b2a2, cinco pacientes (45,5%) o transcrito b3a2, dois pacientes (18,2%) expressaram ambos os transcritos e um paciente (9%) não expressou nenhum dos transcritos. A amplificação dos controles endógenos resultou em melhor amplificação para o transcrito abl, que foi usado nas reações de RQ-PCR. A avaliação de DRM foi possível em oito pacientes, devido à perda de seguimento dos demais. Três meses após o início do tratamento com MI, todos os pacientes apresentaram resposta hematológica completa. No entanto, apenas um paciente (12,5%) apresentou o transcrito bcr-abl indetectável, alcançando a reposta molecular completa, enquanto os outros sete pacientes (87,95%) apresentaram DRM. Dentre os sete pacientes que apresentaram DRM, seis (75%) apresentaram redução de um a dois logs, alcançando resposta molecular menor, enquanto um (12,5%) apresentou resposta molecular parcial. Nossos resultados possibilitaram a padronização e o estabelecimento de uma rotina segundo as diretrizes internacionais, para monitoramento da DRM em pacientes com LMC, utilizando métodos de biologia molecular.

Made available in DSpace on 2016-08-10T10:38:55Z (GMT). No. of bitstreams: 1 CAIO BRUNO QUINTA DE SOUZA LEAL - PARTE 1.pdf: 34304765 bytes, checksum: 476dda15021939ae8be633403ae9dabe (MD5) Previous issue date: 2014-01-31 / Monitoring of minimal residual disease (MRD) in patients with Chronic Myeloid Leukemia (CML) who receive treatment with Imatib mesylate (IM) is extremely important, because it enables the evaluation of the response to the treatment and the early diagnosis of possible recurrences. The objective of this study was to standardize molecular methods used in order to monitor MRD in patients with CML, on therapy with IM. Peripheral blood samples were collected from 11 patients diagnosed with CML in October 2012 to September 2013, in the Department of Hematology of Hospital Araújo Jorge of the Association to Combat Cancer in Goiás. Three months after starting treatment, patients underwent a new peripheral blood collection for evaluation of MRD. Detecting bcr-abl transcripts and endogenous controls (abl and β2m) employed reverse transcription methods associated with polymerase chain reaction (RT-PCR), while quantification of bcr-abl transcripts was achieved by using reverse transcription associated with real-time PCR (RQ-PCR) and Taqman probes. Specific oligonucleotides and probes recognizing e13a2 and e14a2 junctions of bcr-abl transcripts and to the abl endogenous control were used in this study. By the time of diagnosis, three patients (27.3%) expressed the b2a2 transcript, five patients (45.5%) expressed the b3a2 transcript, two patients (18.2%) expressed both transcripts and one patient (9%) did not express any of the transcripts. The endogenous controls analysis resulted in better amplification for the abl transcript, which was used in the RQ-PCR reactions. The assessment of DRM was possible in only eight patients, due to the loss of follow-up. Three months after starting treatment with IM, all patients presented complete hematologic response. However, only one patient (12.5%) presented the undetectable transcript, reaching the full molecular response, while the other seven patients (87.95%) presented MRD. One (12.5%) of the seven patients who presented MRD, reached complete molecular response, while six patients (75%) presented a reduction of two logs, achieving minor molecular response, and one patient (12.5%) presented only partial molecular response. By using molecular biology methods, our results have enabled the standardization and the establishment of a laboratory routine, according to the international guidelines, for monitoring MRD in patients with CML. / O monitoramento de doença residual mínima (DRM) em pacientes com leucemia meilóide crônica (LMC) que recebem tratamento com mesilato de imatibe (MI) é extremamente relevante, pois possibilita o acompanhamento da resposta e o diagnóstico precoce de eventuais recidivas da doença. O objetivo deste estudo foi padronizar métodos moleculares utilizados na avaliação de DRM em pacientes com LMC, em tratamento com MI. Amostras de sangue periférico foram coletadas de 11 pacientes diagnosticados com LMC, no período de outubro de 2012 a setembro de 2013, no Setor de Hematologia do Hospital Araújo Jorge da Associação de Combate ao Câncer em Goiás. Três meses após o início do tratamento, os pacientes foram submetidos a uma nova coleta de sangue periférico para avaliação de DRM. A detecção dos transcritos bcr-abl e controles endógenos (abl e β2m) empregaram os métodos de transcrição reversa associados à reação em cadeia da polimerase (RT-PCR), enquanto a quantificação dos transcritos bcr-abl foi feita por meio de transcrição reversa associada à PCR em tempo real (RQ-PCR), utilizando a metodologia de sondas de hidrólise (TaqMan). Oligonucleotídeos e sondas Taqman específicos para as junções e13a2 e e14a2 dos transcritos bcr-abl e para o controle endógeno (abl) foram usados neste estudo. Ao diagnóstico, três pacientes (27,3%) expressaram o transcrito b2a2, cinco pacientes (45,5%) o transcrito b3a2, dois pacientes (18,2%) expressaram ambos os transcritos e um paciente (9%) não expressou nenhum dos transcritos. A amplificação dos controles endógenos resultou em melhor amplificação para o transcrito abl, que foi usado nas reações de RQ-PCR. A avaliação de DRM foi possível em oito pacientes, devido à perda de seguimento dos demais. Três meses após o início do tratamento com MI, todos os pacientes apresentaram resposta hematológica completa. No entanto, apenas um paciente (12,5%) apresentou o transcrito bcr-abl indetectável, alcançando a reposta molecular completa, enquanto os outros sete pacientes (87,95%) apresentaram DRM. Dentre os sete pacientes que apresentaram DRM, seis (75%) apresentaram redução de um a dois logs, alcançando resposta molecular menor, enquanto um (12,5%) apresentou resposta molecular parcial. Nossos resultados possibilitaram a padronização e o estabelecimento de uma rotina segundo as diretrizes internacionais, para monitoramento da DRM em pacientes com LMC, utilizando métodos de biologia molecular

Made available in DSpace on 2016-04-12T12:32:35Z (GMT). No. of bitstreams: 2 596.pdf: 1353914 bytes, checksum: bee2473adf5a37f943c4f715f4043519 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil / A Leucemia Mielóide Crônica (LMC) é uma neoplasia mieloproliferativa crônica, cuja incidência é de 1,5 casos por 100.000 habitantes ao ano, com predomínio do gênero masculino. A predisposição hereditária parece não existir e o único fator bem caracterizado de risco é a exposição à radiação ionizante. O geoprocessamento é uma ferramenta informacional de auxílio aos profissionais e estudiosos da área da saúde capaz de mapear as doenças e permitir a análise de riscos sócio-ambientais. O objetivo deste estudo foi caracterizar a distribuição dos casos de LMC e de exposição a tóxicos industriais dos pacientes residentes no estado de Pernambuco com registro no Hospital Hemope nos últimos cinco anos. O estudo tipo série de casos incluiu todos os pacientes com diagnóstico de LMC, idade igual ou superior a 18 anos, de ambos os gêneros, atendidos no período de janeiro de 2004 a dezembro de 2009 e procedentes das diversas regiões de desenvolvimento do Estado. Para coleta e análise dos dados foram utilizados os registros dos prontuários de saúde e os aplicativos Excel 2003 e Terra View. No período do estudo foram diagnosticados 201 casos de LMC, com mediana de idade de 49 anos (extremos 18 a 93) e maior pico de incidência entre os 40 e 50 anos (24 por cento). Houve predomínio do sexo masculino (1,1:1) e de residentes de áreas urbanas (86 por cento). A incidência da LMC para as 12 regiões de desenvolvimento do Estado variou entre 0,5 e 3,8 casos por 100.000 habitantes/ano com média de 1,8 casos. As Regiões do Agreste Meridional e Sertão do Pajéu apresentaram maior incidência de casos de LMC, no entanto, não houve variação significante no número de casos ao longo dos anos e nem foram identificados determinantes sócio-ambientais relevantes associados. Apenas 10 por cento dos prontuários apresentaram registro sobre contato do pacientes com tóxicos industriais. A taxa de incidência da LMC encontrou-se dentro da frequência esperada, porém, com variações entre os municípios e as diferentes regiões de desenvolvimento do Estado. O estudo por geoprocessamento auxiliou na análise da distribuição espacial dos casos para uso dos dados como estudo de linha de base. A interface entre o profissional e o serviço de saúde pública é essencial para reconhecer e responder as preocupações com mudanças no perfil epidemiológico de doenças crônicas, frente aos novos cenários de desenvolvimento econômico

L'oncoprotéine BCR-ABL est responsable de la physiopathologie de la Leucémie Myéloïde Chronique (LMC). La fusion d'une partie de la protéine ABL et de BCR entraîne une dérégulation de l'activité kinase portée par ABL. Plusieurs domaines contenus dans ces deux protéines jouent un rôle important dans l'activation du pouvoir oncogène. L'un d'entre eux est une région de BCR, située en N-terminal, contenant un domaine de liaison au domaine SH2. Durant ce travail de thèse, j'ai caractérisé, pour la première fois, les propriétés structurales de cette région, à l'aide de plusieurs méthodes biophysiques: le domaine de BCR est intrinsèquement désordonné. En parallèle, j'ai étudié les interactions entre le domaine de liaison de BCR et les domaines SH d'ABL. J'ai identifié de nouveaux sites d'interactions avec ABL sur BCR. Enfin, j'ai évalué l'impact fonctionnel des nouveaux sites d’interactions au sein de l'oncoprotéines BCR-ABL, dans un modèle cellulaire. Les résultats préliminaires montrent que deux nouveaux sites auraient un rôle dans le pouvoir oncogénique de BCR-ABL. Ces résultats offrent la possibilité de développer de nouveaux médicaments complémentaires aux existants, qui cibleraient une nouvelle région de BCR-ABL, ainsi pourraient lutter contre les résistances apparues chez les patients vis-à-vis des traitements actuels. / The BCR-ABL oncoprotein is responsible for the pathogenesis of chronic myelogenous leukemia (CML). The fusion of a part of ABL and BCR leads to deregulation of kinase activity of ABL. Several domains in these two proteins play an important role in the activation of oncogenic properties. One of them is a BCR region, located at the N- terminal part, containing a SH2 domain binding. In this thesis , I have characterized for the first time , the structural properties of this region, using several biophysical methods : the domain of BCR is intrinsically disordered. In parallel, I have studied the interactions between the binding domain of BCR and theSH domains of ABL. I identified new sites of interaction with ABL into BCR. Finally, I evaluated the functional impact of new sites of interaction within the BCR- ABL oncoprotein in a cellular model. Preliminary results show that two new sites have a role in the oncogenic properties of BCR- ABL. These results offer the possibility to develop new drugs complementary to existing , that target a new region of BCR- ABL and could fight against the resistance occurred in patients vis-a-vis current treatments.

The Magellanic Clouds (MCs) consist of a pair of galaxies, the Large Magellanic Cloud (LMC) and the Small Magellanic Cloud (SMC), which are located at a distance of 50 kpc and 60 kpc, with stellar masses of 1010 M and 109 M , respectively. Morphologically they are categorized as irregular type galaxies. The MCs are gas rich and metal poor (Z=0.008 for LMC, and 0.004 for SMC) as compared to the Milky Way (MW), and have active star-forming regions. Their proximity and location at high galactic latitude enable us to resolve their individual populations as well as detect faint stellar populations. It is well known that the MCs are interacting with each other, as well as with the MW. The interaction is supported by the presence of the Magellanic Bridge and the Magellanic Stream. The evolved stellar populations in the MCs help us to understand their evolution and interaction process. The MCs host both Population I as well as Population II stars. This extended range of star formation is a valuable source of information to understand the formation and evolution of galaxies in general, and the MCs in particular. Evolved stellar popu-lation means the stars that have evolved o the main sequence and the giants, such as red giants (RGs), red clump stars, and asymptotic giant branch stars. There is a dominant population of evolved stars present in the MCs, in star clusters as well as in the eld. The aim of the thesis is to study the evolved stellar populations for one of the component of the MCs, the LMC. The study is primarily divided into two parts. (1) Study of sparse star clusters in the LMC: To increase our understanding of sparse star clusters in the LMC, with well estimated parameters, using deep Washington photometric data for 45 LMC clusters. (2) To estimate a metallicity map of LMC: In order to understand the metallicity variation across the galaxy. This is done by creating a high spatial resolution metallicity map of the LMC, using red giant branch (RGB) stars, with the help of photometric data and calibrated using spectroscopic studies of RGs in eld and star clusters. The introduction to the thesis study along with the aim are described in Chapter 1 of the thesis. The three sets of photometric data used for this study are described in Chapter 2. The data sets are: CT1 Washington photometric data for 45 star clusters within the LMC, the VI photometric data from the Optical Gravitational Lensing Experiment Phase-III survey (OGLE III), and the Magellanic Cloud Photometric Survey (MCPS). Study of sparse star clusters in the LMC: A systematic study is per-formed to analyse the 45 cluster candidates, to estimate their parameters (radius, reddening, and age) using the main-sequence turn-o (MSTO), as well as the evolved portion of the colour{magnitude diagram (CMD). The basic parameters were estimated for 33 genuine clusters, whereas the other 12 cluster candidates have been classi ed as possible clusters/asterisms. The study of 33 star clusters are presented in Chapter 3. These clus-ters are categorized as genuine star clusters based on their strong density enhancement and cluster features with respect to their surrounding eld regions. Out of the 33 clusters, 23 are identi ed as single clusters and 10 are found to be members of double clusters. Detailed discussions of all the individual clusters are presented. The estimated parameters for the single and double clusters are listed in two di erent tables. About 50% of the clusters are in the age range 100{300 Myr, the rest of them being older or younger. Comparison with previous age estimates shows some agreement as well as some deviation. The remaining 12 clusters which could not be categorized as genuine star clusters are studied in Chapter 4. These clusters have poor (/suspi-cious) density enhancement and cluster features when compared to their surrounding elds. It is important to study such cluster candidates, as these objects probe the lower limit of the cluster mass function. Detailed discussion on these individual objects are presented and their estimated parameters are tabulated in this chapter. A detailed discussion based on the study of all the 45 inconspicuous clusters is presented in this chapter, including the estimated sizes (radii 2{10 pc), reddening with respect to eld, and location in the LMC. The mass limit estimated for genuine clusters is found to be 1000 M , whereas for possible clusters/asterisms it is few 100 M , using synthetic CMDs. The study of sparse clusters enlarged the number of objects con rmed as genuine star clusters (33) and estimated their fundamental parameters. The study emphasizes that the sizes and masses of the studied sample are found to be similar to that of open clusters in the MW. Thus, this study adds to the lower end of cluster mass distribution in the LMC, suggesting that the LMC, apart from hosting rich clusters, also has formed small, less massive open clusters in the 100{300 Myr age range. The 12 cases of possible clusters/asterisms are worthy of attention, in the sense that they can throw light on the survival time of such objects in the LMC. Photometric metallicity map of the LMC using RGB stars: A metallic-ity map of the LMC is estimated using OGLE III and MCPS photometric data. This is a rst of its kind map of metallicity up to a radius of 4{5 de-grees, derived using photometric data and calibrated using spectroscopic data of RGB stars. The RGB is identi ed in the V, (V I) CMDs of small areal subregions of varying sizes in both data sets. The slope of the RGB is used as an indicator of the average metallicity of a subregion, and this RGB slope is calibrated to metallicity using spectroscopic data for eld and cluster RGs in selected subregions. The metallicity map estimated using OGLE III photometric data is presented in Chapter 5. A method to identify the RGB of small subre-gions within the LMC and estimate its slope by using a consistent and automated method was developed. The technique is robust and indepen-dent of reddening and extinction. The details of calibrating the RGB slopes to metallicities, using previous spectroscopic results of RGs in eld and star clusters are presented. The OGLE III metallicity maps are pre sented, based on four cut-o criteria to separate regions with good ts. The OGLE III map has substantial coverage of the bar, the eastern and western LMC, but does not cover the northern and southern regions. The OGLE III metallicity map shows the bar region to be metal rich whereas the eastern and western regions to be relatively metal poor. The mean metallicity is estimated for three di erent regions within the LMC. For the complete LMC the mean [Fe/H] is = 0.39 dex ( [Fe/H] = 0.10); for the bar region it is = 0.35 dex ( [Fe/H] = 0.9); and for the outer LMC it is = 0.46 dex ( [Fe/H] = 0.11). The metallicity histogram for these di erent regions are also estimated. A radial metallicity gradient is estimated in the de-projected plane of the LMC. The metallicity gradient is seen to remain almost constant in the bar region (till a radius of 2.5 kpc) and has a shallow gradient of 0.066 0.006 dex kpc 1 beyond that till 4 kpc. In Chapter 6 the metallicity map based on MCPS photometric data is estimated. The MCPS data covers more of the northern and south-ern LMC (less of eastern and western regions) and is important to be analysed in order to reveal the metallicity trend of the overall disk. The systematic di erences between the lter systems of MCPS and OGLE III are corrected, and the MCPS slopes are then calibrated using the OGLE III slope{metallicity relation. The MCPS metallicity maps are presented, based on four cut-o criteria to separate regions with good ts. The bar region is found to be metal rich as was found using OGLE III data, whereas the northern and southern regions are marginally metal poor. The mean metallicity estimated for the complete LMC is = 0.37 dex ( [Fe/H] = 0.12); and for the outer LMC it is = 0.41 dex ( [Fe/H] = 0.11). The metallicity histogram for these di erent regions are estimated and compared with the OGLE III distribution. The metallicity range of the complete LMC is found to be almost similar for both data sets. The metallicity distribution within the bar has a narrow range as found using both data sets. The slight di erence between mean metallicity of outer LMC for the two data sets is attributed to their coverage. We suggest that the northern and southern regions of the LMC could be marginally more metal rich than the eastern and western regions. The metallicity gradient of the LMC disk, estimated from MCPS data is found to be shallow 0.049 0.002 dex kpc 1 till about 4 kpc. We also constructed a metallicity map of outliers using both OGLE III and MCPS data, and identi ed subregions where the mean metallic-ity di ers from the surrounding areas. We suggest further spectroscopic studies in order to assess their physical significance. The detailed conclusion of the thesis and future work are presented in Chapter 7. From the study of sparse star clusters in the LMC, it is concluded that LMC has open cluster like star cluster systems. It is important to include them to understand the cluster formation history (CFH) and their survival time scale. Presently, our understanding of the CFH is dominated by rich clusters. The bar of the LMC is found to be the most metal rich region, and the LMC metallicity gradient though shallow, resembles the gradient seen in spiral galaxies. The gradient is also similar to that found in our Galaxy. The higher metallicity in the bar region might indicate an active bar in the past.

La leucémie myéloïde chronique (LMC) est une hémopathie maligne causée par l‘apparition du chromosome Philadelphie dans la cellule souche hématopoïétique (CSH), conduisant à l‘expression de la protéine de fusion BCR-ABL1. L‘activité tyrosine kinase dérégulée de cette oncoprotéine provoque l‘activation de plusieurs voies de signalisation critiques dans la leucémogenèse. Si les inhibiteurs de tyrosine kinase (ITK) ciblant BCR-ABL1 représentent des traitements dans l'ensemble très efficaces, plusieurs études montrent que les cellules leucémiques les plus immatures de la moelle osseuse y sont insensibles. Cette thèse propose de compléter les connaissances relatives aux effets de BCR-ABL1 dans la cellule, et plus généralement aux propriétés des CSH de LMC. Notre intérêt s‘est focalisé sur le rôle potentiel des microARN. Dans un premier travail, nous nous sommes intéressés à l‘effet de l‘activité BCR-ABL1 sur le protéome et sur l‘expression des microARN dans la lignée cellulaire K562. Les résultats montrent que BCR-ABL1 régule l'expression d'un microARN fréquemment surexprimé dans les cancers, miR-21. Cet effet dépend du facteur de transcription STAT5, cible bien connue de l'activité kinase de BCR-ABL1. Dans une seconde partie, nous avons montré que dans la moelle osseuse des patients LMC, la fraction cellulaire enrichie en cellules souches (les cellules CD34+CD38low) exprime quatre microARN particuliers: mir-10a, mir-150, miR-155 et miR-146a. Deux de ces microARN (miR-150 et miR-155) sont trouvés spécifiquement dans les cellules des patients, et pas dans celles des individus sains. / In chronic myeloid leukemia (CML), the activity of the constitutively active tyrosine kinase BCR-ABL1 drives the activation of the PI3K/AKT, JAK/STAT, and RAS/RAF/MEK/ERK pathways. Among other consequences, activated or inhibited transcription factors induce important modifications of the CML cells gene expression pattern that could impact cell cycle control, apoptosis and genetic instability, leading to the expansion of the oncogene-transformed cells and to the acquisition of potentially harmful de novo mutations. However, indirect BCR-ABL1-dependant regulations might also occur, for instance through the action of microRNAs (miRNAs). Among the ~2000 miRNAs reported in humans, numerous species are up- or down-regulated in various cancer models. In the context of CML however, there is no clear consensus regarding the role of specific miRNAs, despite several studies. The first aim of this thesis was to study the effects of a clinically relevant concentration of imatinib, a tyrosine-kinase inhibitor (TKI) that blocks BCR-ABL1, on the CML cell line K562: both the microRNA expression profile and the cells proteome were analyzed. Using microarray hybridization, RT-qPCR experiments and a functional assay, we identified miR-21 as one of the most significantly down-regulated microRNA in cells that were treated with imatinib. In parallel, a semi-quantitative proteomic approach identified the tumor suppressor programmed cell death protein 4 (PDCD4) as the most over-expressed protein in imatinib-treated cells. We showed that miR-21 can bind to PDCD4 3'UTR and decrease its expression. The STAT5 - miR-21 - PDCD4 pathway was conserved in CML primary CD34+ cells, and to some extent in acute myeloid leukemia (AML) models as well; the known functions of miR-21 and PDCD4 suggest that their regulation by BCR-ABL1 could participate in the antileukemic response triggered by tyrosine kinase inhibitors. In the second part of this manuscript, we was interested in the immature stem cells population that cannot be eliminated by TKI. The underlying mechanisms of this resistance are not fully understood. The TKI-resistant CML stem cells reside in the CD34+/CD38low subpopulation, that can be sorted from the mononuclear cells fraction using FACS. In this project, we propose to describe the microRNA repertoire of the CML CD34+/CD38low cells to highlight the potential role of microRNA in the resistance mechanisms by identifying some of their targets, using bioinformatic and experimental approaches. This combination of miRNome and functional analysis would allow to increase the knowledge of the biology of the TKI-resistant CML stem cells. Our results have shown that the cellular fraction enriched in stem cells (CD34+CD38low) expressed specifically four microRNA: miR-10a, miR-146, miR-150 and miR-155. It is also interested to notice that only two of them, miR-150 and miR-155, are highly expressed in CML-patient CD34+CD38low cells compared to normal cells.

La leucémie myéloïde chronique (LMC) a été le premier cancer humain associé à une anomalie chromosomique : le chromosome de Philadelphie. Le gène de fusion BCR-ABL1 résultant code pour une tyrosine kinase ayant une activité dérégulée. Les inhibiteurs de tyrosine kinase (ITKs), qui inactivent la protéine BCR-ABL1, représentent la thérapie ciblée la plus efficace pour la LMC en phase chronique. Cependant, la LMC en phase avancée ne répond pas bien au traitement par les ITKs. Les mécanismes sous-jacents à la progression de la LMC ne sont pas bien compris. Par conséquent, la découverte de gènes qui coopèrent avec l’oncogène BCR-ABL1, et qui pourraient expliquer la progression de la LMC vers des phases avancées, est nécessaire pour l’identification de nouvelles cibles thérapeutiques de la LMC. Nous proposons d'établir un modèle cellulaire humain permettant l'identification de gènes capables de coopérer avec l'oncogène BCR-ABL1 pour une transformation tumorale complète. Ce système repose sur l'expression de BCR-ABL1 et l'inactivation d'autres gènes en particulier à l'aide de la technologie CRISPR-Cas9. L'identification des gènes coopérant avec BCR-ABL1 permettra la création de modèles cellulaires pour faciliter la sélection de médicaments capables de traiter la LMC dans les phases finales. Dans un second temps, une étude approfondie du gène TP53 a été menée. Ce gène étant muté dans plus de 50% des cancers, il est important de déterminer les conséquences de son inactivation dans des fibroblastes humains non tumoraux. La technologie CRISPR-Cas9 a été utilisée afin d’inactiver ce gène en particulier. Puis les cellules exprimant la forme sauvage ou la forme inactivée de p53 ont subi un traitement à la nutline-3a. Cette molécule empêche l’interaction du facteur de transcription p53 avec son inhibiteur MDM2. Des analyses transcriptomiques ont alors permis d’identifier d’une part les cibles aspécifiques de la nutline-3a et d’autre part les gènes cibles de p53 dans cette lignée de fibroblaste. / Chronic myeloid leukemia (CML) was the first human cancer to be consistently associated with a chromosomal abnormality: the Philadelphia chromosome. The resulting BCR-ABL1 fusion gene encodes a tyrosine kinase with deregulated activity. Tyrosine kinase inhibitors (TKIs) inactivating the BCR-ABL protein represent the most successful targeted therapy for CML in chronic phase. However, advanced CML does not respond well to TKIs treatment. The mechanisms underlying the progression of CML are not well understood. Therefore, the discovery of genes that cooperate with the BCR-ABL1 oncogene, which could explain the progression of CML to advanced phases, is required for the identification of novel therapeutic targets of CML. We propose to establish a human cellular model system that allows the identification of genes that are able to cooperate with the oncogene BCR-ABL1 for full tumoral transformation. This system relies on the expression of BCR-ABL1 and the generation of gene knock-out by using the CRISPR-Cas9 technology. Identification of genes cooperating with BCR-ABL1 will permit the creation of cellular model systems for identifying drugs that are able to treat CML in final phases. Secondly, we performed a detailed study of TP53 function. This gene is mutated in more than 50% of all cancer types. It is therefore crucial to understand the impact of its inactivation in human fibroblast cells. The CRISPR/Cas9 system was used to inactivate this gene. Wild-type and TP53 knock-out cells subsequently underwent nutlin-3a treatment. This molecule blocks the interaction between p53 and its regulator: MDM2. Transcriptomic analyses were performed to further study p53 regulated genes, and also to discover other potential nutlin-3a targets.

La lutte contre le cancer est certainement l’un des défis majeurs de ce 21ème siècle. Les résistances qui émergent contre les agents de thérapie ciblée présentent un aspect particulièrement épineux de cette problématique. La thèse présentée ici s’inscrit dans ce cadre. Elle vise à développer des molécules bioactives pouvant circonvenir les résistances apparues contre les traitements de certaines hémopathies malignes : la leucémie myéloïde chronique (LMC) et le syndrome myélodysplasique (SMD). Après avoir mis au point une méthodologie de synthèse monotope permettant de transformer un azoture en un 5-alcynyl-1,2,3-triazole, nous avons synthétisé deux séries de produits : nucléosidique et non nucléosidique. Pour chacune de ces séries, des relations structure-activité ont été établies. Après plusieurs cycles d’optimisation, trois composés lead très efficaces contre des lignées cellulaires résistantes de LMC et SMD, ont été sélectionnés. De surcroît, leur mode d’action s’est révélé très intéressant : il repose (partiellement ou entièrement, suivant le composé) sur un processus cellulaire qui connaît un véritable regain d’intérêt, à savoir l’autophagie. Une évaluation in vivo a été réalisée et a permis de valider l’activité prometteuse de notre composé lead nucléosidique. Par ailleurs, des études visant à déterminer la localisation intracellulaire et les cibles moléculaires de nos produits sont actuellement en cours / The fight against cancer is certainly one of the biggest challenges of the 21st century. Resistance that comes up against targeted therapy agents presents a particularly important aspect of this issue. The thesis presented here takes part within that framework. It aims at developing bioactive molecules able to circumvent resistance that have emerged against the treatment of certain hematological malignancies: chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS). Having developed a one-pot synthesis methodology that converts azides into 5-alkynyl-1,2,3-triazole, we synthesized two series of products: nucleosidic and non-nucleosidic. For each of these series, structure-activity relationships have been established. After running several cycles of optimization, three lead compounds particularly active on resistant cell lines of CML and MDS were selected. Further, their mode of action proved to be very interesting. It is based (partially or fully, depending on the compound) on a cellular process, which is experiencing a real renewed interest, the autophagy. An in vivo evaluation confirmed the promising activity of our nucleosidic lead compound. Moreover, studies aiming at determining the intracellular localization and molecular targets of our products are currently in progress

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