No description available.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 26, 2009). Includes bibliographical references.

La résistance à la chimiothérapie constitue un frein majeur à son efficacité. Notre équipe a récemment pu montrer que le masitinib, un nouvel inhibiteur de protéines tyrosine kinases, possède une activité de resensibilisation des cellules tumorales résistantes à la chimiothérapie lorsqu'il est combiné à certaines chimio-drogues. L'objectif des travaux de cette thèse est de déterminer les voies de signalisation, modulées par l'action du masitinib, qui sont impliquées dans la resensibilisation aux chimiothérapies et amélioration de l'activité anti-tumorale.Dans la première partie de la thèse, nous avons pu identifier la nucléoside kinase dCK (désoxycytidine kinase), protéine activatrice d'un grand nombre de chimiothérapies, comme nouvelle cible du masitinib. Cette première étude nous a permis de mettre en évidence un nouveau concept thérapeutique: le masitinib, un composé chimique de type inhibiteur de protéines tyrosine kinases, peut jouer en même temps le rôle d'activateur de nucléoside kinase.Nous avons pu mettre en évidence dans la deuxième partie de la thèse que le traitement combiné entre l'épi-drogue décitabine et le masitinib peut être plus efficace pour la réexpression de certains gènes non ou peu induits par la décitabine seule.En conclusion, ces travaux nous ont permis de mettre en évidence l'interaction entre un inhibiteur de protéine tyrosine kinases et une nucléoside kinase, dans un concept d'activation enzymatique qui pourra certainement servir de base pour l'élaboration de nouvelles petites molécules chimiques spécifiques de l'activation de dCK ou d'autres nucléosides kinases nécessaires à l'activation des drogues de chimiothérapie. / Resistance to chemotherapy is considered as one of the major blockers of its efficacy. Recently, our team demonstrated that masitinib, a new tyrosine kinases inhibitor, possesse a resensitization activity of cell lines resistant to chemotherapy when associated with chemodrugs.The aim of this work is to determine signaling pathways, modulated by masitinib action, that could explain the resensitization to chemotherapy and improvement of anti-tumoral activity.In the first part of this work, we identified the nucleoside kinase dCK (deoxycytidine kinase), a chemotherapy activating protein, as a new target of masitinib. In summary, this first part of the work allowed us to describe a new and never described concept: masitinib, a small molecule belonging to tyrosine kinases group, can also play a role as nucleoside kinase activator.We were able to demonstrate through the second part of the work that the combined treatment of the epidrug decitabine and masitinib can be more effective than decitabine treatment for the re-expression of some genes non or weakly induced by decitabine when used alone.In conclusion, These data allowed us to introduce an interaction between a tyrosine kinases inhibitor and a nucleoside kinase, as an enzymatic activation new concept. This could be used as a base for the design of new small chemical molecules specific for dCK or other nucleoside kinases essential for the activation of chemodrugs. This concept will obviously help to imagine and evaluate more potential therapeutic combinations of chemodrugs and small chemical molecules to overcome the resistance to chemotherapy dependent on nucleoside kinases.

Analyzing protein tertiary structure is an effective method to understanding protein function. In my thesis study, I aimed to understand how surface features of protein can affect the stability and specificity of enzymes. I focus on 2 proteins that are involved in human disease, Profilin (PFN1) and APOBEC3A (A3A). When these proteins are functioning correctly, PFN1 modulates actin dynamics and A3A inhibits retroviral replication. However, mutations in PFN1 are associated with amyotrophic lateral sclerosis (ALS) while the over expression of A3A are associated with the development of cancer. Currently, the pathological mechanism of PFN1 in this fatal disease is unknown and although it is known that the sequence context for mutating DNA vary among A3s, the mechanism for substrate sequence specificity is not well understood. To understand how the mutations in Profilin could lead to ALS, I solved the structure of WT and 2 ALS-related mutants of PFN1. Our collaborators demonstrated that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization was illuminated by my X-ray crystal structures of several PFN1 proteins. I found an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis. To characterize A3A’s substrate specificity, we solved the structure of apo and bound A3A. I then used a systematic approach to quantify affinity for substrate as a function of sequence context, pH and substrate secondary structure. I found that A3A preferred ssDNA binding motif is T/CTCA/G, and that A3A can bind RNA in a sequence specific manner. The affinity for substrate increased with a decrease in pH. Furthermore, A3A binds tighter to its substrate binding motif when in the loop region of folded nucleic acid compared to a linear sequence. This result suggests that the structure of DNA, and not just its chemical identity, modulates A3 affinity and specificity for substrate.

No description available.

Les gènes suppresseurs de tumeurs (TSGs) contrôlent la prolifération cellulaire et leur inactivation joue un rôle important dans la leucémogénèse. Deux mécanismes épigénétiques majeurs sont impliqués dans la répression des TSGs: 1- la méthylation de l’ADN et 2- la déacétylation des histones des chromosomes. On les dit épigénétiques car ils n’affectent pas la séquence de l’ADN. Ces phénomènes sont réversibles, faisant donc d’eux des cibles thérapeutiques de choix. Dans le cadre de cette thèse, nous avons évalué le potentiel chimiothérapeutique de différents agents qui visent ces mécanismes épigénétiques et nous les avons administrés seuls et en combinaison dans le but d’améliorer leur efficacité. La 5-aza-2’-désoxycytidine (5-Aza-CdR) est un inhibiteur de la méthylation de l’ADN qui permet la ré-expression des TSGs. Cet agent s’est avéré efficace contre certaines maladies hématologiques et est d’ailleurs approuvé aux États-Unis dans le traitement du syndrome myélodysplasique depuis 2006. Cependant, le protocole d’administration optimal de cet agent, en termes de doses et de durée, n’est toujours pas établi. Nos recherches suggèrent que le celui-ci devrait être plus intensif que ce que rapporte la littérature. Les inhibiteurs des déacétylases des histones (HDACi) ont également montré une activité antinéoplasique intéressante. De récentes recherches ont montré que la combinaison d’agents ciblant à la fois la méthylation de l’ADN et la déacétylation des histones produit une réactivation synergique des TSGs, ce à quoi nous nous sommes intéressé. Nous avons observé que la co-administration d’un HDACi avec la 5-Aza-CdR potentialise son action anti-leucémique. Il est aussi possible d’augmenter l’activité de la 5-Aza-CdR en inhibant sa dégradation par l’enzyme cytidine (CR) désaminase. Nous avons observé que la co-administration du zebularine, un inhibiteur de la CR désaminase, avec la 5-Aza-CdR accroît son efficacité. Le zebularine est aussi un inhibiteur de la méthylation de l’ADN, ce qui pourrait contribuer à la potentialisation de la réponse anti-leucémique observée lors de la co-administration de ces deux agents. En résumé, il est possible d’augmenter l’efficacité anti-leucémique de la 5-Aza-CdR en : 1- intensifiant son protocole d’administration, en termes de doses et de durée, 2- la combinant avec un HDACi, et 3- diminuant sa dégradation par la CR désaminase. L’utilisation de ces résultats précliniques dans l’élaboration de protocoles cliniques pourrait être bénéfique à beaucoup de patients. / The silencing of tumor suppressor genes (TSG) that normally regulate cells proliferation plays an important role in leukemogenesis. Two major mechanisms are involved in TSG’s silencing: DNA methylation and histones deacetylation. Because those phenomenons are reversible, it makes them interesting therapeutic targets for chemotherapeutic agents. We evaluated the antineoplastic potential of different agents that target those events and we administered them alone or in combination with the goal of improving their efficiency. 5-aza-2’-deoxycytidine (5-Aza-CdR) is a DNA methylation inhibitor that can re-express TSGs that are silenced by methylations. This agent demonstrated its efficacy against hematological malignancies. Therefore, 5-Aza-CdR is used since 2006 in United States of America against myelodysplastic syndrome; but its optimal dose-schedule still needs to be established. Our researches suggest that the dose-schedule of 5-Aza-CdR should be more intensive than what is reported from the literature. Inhibitors of histones deacetylation (HDACi) also demonstrated some interesting antineoplastic activity. Recently, observations showed that combination of chemotherapeutic agent that targets both DNA methylation and histones deacetylation lead to a synergic reactivation of silenced TSG. This finding allowed us to observe that the co-administration of an HDACi with 5-Aza-CdR improve its antileukemic potential. Moreover, it is possible to increase the activity of 5-Aza-CdR by preventing its degradation by cytidine (CR) deaminase. We demonstrated that the co-administration of zebularine, an inhibitor of CR deaminase, with 5-Aza-CdR increases its activity. Zebularine is also an inhibitor of DNA methylation, which may contribute to the enhancement of the antileukemic action of this combination. In summary, our preclinical data indicate that the antileukemic activity of 5-Aza-CdR can be enhanced by: 1- increasing his dosage, 2- combining it with HDACi, and 3- preventing its inactivation by CR deaminase. The translation of those preclinical observations into clinical protocols may be effective in patients with advanced leukemia.

Embora a transferência nuclear de células somáticas (TNCS) seja uma ferramenta promissora, seu amplo uso é impedido devido às altas taxas de mortalidade durante o desenvolvimento dos animais clonados. Acredita-se que a reprogramação epigenética anormal seja a principal causa desta baixa eficiência. Nós hipotetizamos que agentes modificadores de cromatina (AMCs) atingindo a acetilação das histonas e a metilação do DNA poderiam alterar a configuração da cromatina e torná-la mais facilmente reprogramável. Deste modo, fibroblastos bovinos foram tratados com 5-aza-2\'-deoxicitidina (AZA) mais tricostatina A (TSA) ou hidralazina (HH) mais ácido valpróico (VPA) enquanto, em outro experimento, zigotos bovinos clonados foram tratados com TSA. O tratamento dos fibroblastos com AZA+TSA ou HH+VPA aumentou a acetilação das histonas, mas não afetou o nível de metilação do DNA. Entretanto, o tratamento com HH+VPA diminuiu a viabilidade/proliferação celular. O uso destas células como doadoras de núcleo não mostrou efeitos positivos sobre o desenvolvimento pré- e pós-implantação. Em relação ao tratamento dos zigotos clonados com TSA, o tratamento destes mostrou um aumento nos padrões de acetilação das histonas, mas não reduziu o nível de metilação do DNA. Além disso, este tratamento não resultou em efeito positivo sobre o desenvolvimento pré- e pósimplantação. Este trabalho fornece evidências de que o tratamento de células doadoras de núcleo ou zigotos clonados com AMCs não tem efeito positive sobre o desenvolvimento pré- e pós-implantação de bovinos clonados. / Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatinmodifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2\'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and post-implantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and post-implantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and post-implantation development of cloned cattle.

Embora a transferência nuclear de células somáticas (TNCS) seja uma ferramenta promissora, seu amplo uso é impedido devido às altas taxas de mortalidade durante o desenvolvimento dos animais clonados. Acredita-se que a reprogramação epigenética anormal seja a principal causa desta baixa eficiência. Nós hipotetizamos que agentes modificadores de cromatina (AMCs) atingindo a acetilação das histonas e a metilação do DNA poderiam alterar a configuração da cromatina e torná-la mais facilmente reprogramável. Deste modo, fibroblastos bovinos foram tratados com 5-aza-2\'-deoxicitidina (AZA) mais tricostatina A (TSA) ou hidralazina (HH) mais ácido valpróico (VPA) enquanto, em outro experimento, zigotos bovinos clonados foram tratados com TSA. O tratamento dos fibroblastos com AZA+TSA ou HH+VPA aumentou a acetilação das histonas, mas não afetou o nível de metilação do DNA. Entretanto, o tratamento com HH+VPA diminuiu a viabilidade/proliferação celular. O uso destas células como doadoras de núcleo não mostrou efeitos positivos sobre o desenvolvimento pré- e pós-implantação. Em relação ao tratamento dos zigotos clonados com TSA, o tratamento destes mostrou um aumento nos padrões de acetilação das histonas, mas não reduziu o nível de metilação do DNA. Além disso, este tratamento não resultou em efeito positivo sobre o desenvolvimento pré- e pósimplantação. Este trabalho fornece evidências de que o tratamento de células doadoras de núcleo ou zigotos clonados com AMCs não tem efeito positive sobre o desenvolvimento pré- e pós-implantação de bovinos clonados. / Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatinmodifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2\'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and post-implantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and post-implantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and post-implantation development of cloned cattle.

Les gènes suppresseurs de tumeurs (TSGs) contrôlent la prolifération cellulaire et leur inactivation joue un rôle important dans la leucémogénèse. Deux mécanismes épigénétiques majeurs sont impliqués dans la répression des TSGs: 1- la méthylation de l’ADN et 2- la déacétylation des histones des chromosomes. On les dit épigénétiques car ils n’affectent pas la séquence de l’ADN. Ces phénomènes sont réversibles, faisant donc d’eux des cibles thérapeutiques de choix. Dans le cadre de cette thèse, nous avons évalué le potentiel chimiothérapeutique de différents agents qui visent ces mécanismes épigénétiques et nous les avons administrés seuls et en combinaison dans le but d’améliorer leur efficacité. La 5-aza-2’-désoxycytidine (5-Aza-CdR) est un inhibiteur de la méthylation de l’ADN qui permet la ré-expression des TSGs. Cet agent s’est avéré efficace contre certaines maladies hématologiques et est d’ailleurs approuvé aux États-Unis dans le traitement du syndrome myélodysplasique depuis 2006. Cependant, le protocole d’administration optimal de cet agent, en termes de doses et de durée, n’est toujours pas établi. Nos recherches suggèrent que le celui-ci devrait être plus intensif que ce que rapporte la littérature. Les inhibiteurs des déacétylases des histones (HDACi) ont également montré une activité antinéoplasique intéressante. De récentes recherches ont montré que la combinaison d’agents ciblant à la fois la méthylation de l’ADN et la déacétylation des histones produit une réactivation synergique des TSGs, ce à quoi nous nous sommes intéressé. Nous avons observé que la co-administration d’un HDACi avec la 5-Aza-CdR potentialise son action anti-leucémique. Il est aussi possible d’augmenter l’activité de la 5-Aza-CdR en inhibant sa dégradation par l’enzyme cytidine (CR) désaminase. Nous avons observé que la co-administration du zebularine, un inhibiteur de la CR désaminase, avec la 5-Aza-CdR accroît son efficacité. Le zebularine est aussi un inhibiteur de la méthylation de l’ADN, ce qui pourrait contribuer à la potentialisation de la réponse anti-leucémique observée lors de la co-administration de ces deux agents. En résumé, il est possible d’augmenter l’efficacité anti-leucémique de la 5-Aza-CdR en : 1- intensifiant son protocole d’administration, en termes de doses et de durée, 2- la combinant avec un HDACi, et 3- diminuant sa dégradation par la CR désaminase. L’utilisation de ces résultats précliniques dans l’élaboration de protocoles cliniques pourrait être bénéfique à beaucoup de patients. / The silencing of tumor suppressor genes (TSG) that normally regulate cells proliferation plays an important role in leukemogenesis. Two major mechanisms are involved in TSG’s silencing: DNA methylation and histones deacetylation. Because those phenomenons are reversible, it makes them interesting therapeutic targets for chemotherapeutic agents. We evaluated the antineoplastic potential of different agents that target those events and we administered them alone or in combination with the goal of improving their efficiency. 5-aza-2’-deoxycytidine (5-Aza-CdR) is a DNA methylation inhibitor that can re-express TSGs that are silenced by methylations. This agent demonstrated its efficacy against hematological malignancies. Therefore, 5-Aza-CdR is used since 2006 in United States of America against myelodysplastic syndrome; but its optimal dose-schedule still needs to be established. Our researches suggest that the dose-schedule of 5-Aza-CdR should be more intensive than what is reported from the literature. Inhibitors of histones deacetylation (HDACi) also demonstrated some interesting antineoplastic activity. Recently, observations showed that combination of chemotherapeutic agent that targets both DNA methylation and histones deacetylation lead to a synergic reactivation of silenced TSG. This finding allowed us to observe that the co-administration of an HDACi with 5-Aza-CdR improve its antileukemic potential. Moreover, it is possible to increase the activity of 5-Aza-CdR by preventing its degradation by cytidine (CR) deaminase. We demonstrated that the co-administration of zebularine, an inhibitor of CR deaminase, with 5-Aza-CdR increases its activity. Zebularine is also an inhibitor of DNA methylation, which may contribute to the enhancement of the antileukemic action of this combination. In summary, our preclinical data indicate that the antileukemic activity of 5-Aza-CdR can be enhanced by: 1- increasing his dosage, 2- combining it with HDACi, and 3- preventing its inactivation by CR deaminase. The translation of those preclinical observations into clinical protocols may be effective in patients with advanced leukemia.

La plasticité cérébrale pathologique qui se met en place en réponse à l'administration répétée de drogue nécessite des modifications de l’expression des gènes, au moyen,entre autres, de mécanismes épigénétiques tels que la méthylation de l’ADN. Dans ces travaux, nous avons montré que l’inhibition des ADN méthyl transférases par la 5-aza-2’-désoxycytidine augmentait les propriétés renforçantes de la cocaïne dans un protocole d’auto-administration intraveineuse, et ce, sans affecter la motivation des rats pour la cocaïne, ni la réactivation du comportement de recherche après une période de sevrage.L’analyse du méthylome dans le cortex préfrontal médian nous a permis de caractériser près de 190000 régions génomiques différentiellement méthylées suite au traitement par la cocaïne, en association ou non avec la 5-aza-2’-désoxycytidine. Nous avons sélectionné une vingtaine de régions situées soit dans les promoteurs soit au sein de gènes participant à la plasticité neuronale. L’analyse de la transcription de ces gènes a permis, pour certains d’entre eux, de corréler les variations de méthylation avec celles d’expression, comme dans le cas du gène Hdac2. / Repeated drug administration lead to pathological brain plasticity that requires modifications of gene expression through, among others, epigenetic mechanisms such DNA methylation. Here, we showed that DNA methyltransferases inhibitors such 5-aza-2’-deoxycytidine increase reinforcing properties of cocaine in an intravenous self administration paradigm without affecting the motivation of rats for the drug, nor drug seeking after withdrawal. The analysis of the methylome in the medial prefrontal cortex allowed us to identify approximatively 190000 differentially methylated genomic regions in response to cocaine treatment, in association or not with 5-aza-2’-deoxycytidine. We selected around twenty regions within promoters or body of genes known to participate in neuronal plasticity. The study of the transcription of these genes permitted for some of them to correlate the modifications of the DNA methylation with the modifications of the expression, like, for example, in the case of the gene Hdac2.