Les leucémies aigües myéloïdes (LAM) sont caractérisées par l'accumulation de blastes leucémiques pour lesquels les programmes de différenciation sont dérégulés. Les mutations des isocitrate déshydrogénases (IDH), présentes chez 15% des patients atteints de LAM à risque cytogénétique intermédiaire, participent à cette dérégulation en induisant la production de R-2-Hydroxyglutarate (R-2-HG), responsable d'une hyperméthylation globale de l'ADN. Nous avons identifié une signature génique spécifique de la mutation IDH1 R132H, caractérisée par une dérégulation des facteurs de transcription clés impliqués dans la différenciation granulocytaire, mais également par la dérégulation de gènes répondeurs à la différenciation induite par l'ATRA. Nous avons montré que la mutation IDH1 R132H sensibilise les lignées cellulaires et blastes d'échantillons primaires de patients à la différenciation induite par l'ATRA, à la fois in vitro et in vivo à des concentrations cliniquement relevantes. De plus, le traitement par une forme perméante du R-2-HG sensibilise la forme IDH1 WT à la différenciation induite par l'ATRA, alors que l'inhibition de la production du R-2-HG réduit de façon significative les effets de l'ATRA dans la lignée HL60 IDH1 R132H. L'ATRA permet de diminuer la viabilité cellulaire et augmenter l'apoptose spécifiquement en présence de la mutation IDH1 R132H, et conduit à une réduction drastique de la formation de colonies en milieu semi-solide. Il réduit également la prise tumorale de la lignée MOLM14 IDH1 R132H xénogreffées dans des souris NOD-Scid-IL2rynull et augmente de façon très significative la survie globale des souris, révélant l'effet potentiellement anti-leucémique de l'ATRA spécifiquement en présence de la mutation IDH1 R132H. Nous avons également mis en évidence un effet synergique entre l'ATRA et le dasatinib, spécifiquement en présence de la mutation IDH1 R132H, à la fois in vitro et in vivo. Pour finir, nous avons montré que la mutation IDH1 R132H réduit la survie de souris xénogreffées par rapport à la forme sauvage, ainsi que la sensibilité in vitro aux chimiothérapies classiquement utilisées pour le traitement des LAM (AraC et Idarubicine). Malgré tout, l'ajout d'ATRA permet d'augmenter l'efficacité des chimiothérapies, spécifiquement en présence de la mutation IDH1 R132H. Pour conclure, cette étude donne un rationnel pour tester ces combinaisons au cours de futurs essais cliniques dans ce sous-groupe de patients. / Acute myeloid leukemia (AML) is characterized by accumulation of malignant blasts with impaired differentiation programs due to recurrent mutations such as isocitrate dehydrogenase (IDH) mutations found in 15% of AML patients. These mutations result in the production of (R)-2-hydroxyglutarate (2-HG), leading to a hypermethylation phenotype that impairs hematopoietic differentiation. Here we identified mutant IDH1-specific gene signatures regulated by key transcription factors involved in myeloid differentiation and responsive to retinoids. Accordingly, we showed that the presence of the IDH1 mutation sensitized AML cell lines and primary patient samples to all-trans retinoic acid (ATRA)-induced differentiation both in vitro and in vivo using clinically achievable doses. Moreover, treatment with a cell-permeable form of 2-HG sensitized wild-type IDH1 AML cells to ATRA-induced myeloid differentiation, while inhibition of 2-HG production significantly diminished ATRA effects in mutant IDH1 cells. ATRA treatment specifically decreased cell viability and induced apoptosis of mutant IDH1 blasts in vitro. ATRA also reduced tumor burden of mutant IDH1 AML cells xenografted in NOD-Scid-IL2rynull mice and highly increased mice overall survival, revealing a potent anti-leukemic effect of ATRA in the presence of IDH1 mutation. Moreover, we showed a synergistic effect between ATRA and dasatinib specifically in the presence of IDH1 R132H mutation, both in vitro and in vivo. Finally, we demonstrated that IDH1 R132H reduces survival of mice xenografted with mutant cell lines compared to the WT, and reduces sensitivity to chemotherapies (AraC and Idarubicine) in vitro. Nevertheless, the association of ATRA to chemotherapies increases their activity specifically in the presence of IDH1 R132H mutation. These therapeutic strategies hold promise for this AML patient subgroup in future clinical studies.

Thesis advisor: X. Peter Zhang / Halogenated compounds are useful synthetic organic molecules. One valuable tool for synthesizing halogen containing molecules are atom transfer radical addition (ATRA) reactions which can difunctionalize olefins with a halogen moiety. Many transition complexes can catalyze these reactions but have drawbacks such as the need for harsh conditions and additives. Herein we describe the first ATRA reaction catalyzed by cobalt metalloradical catalysis (Co-MRC) which shows a broad substrate scope, moderate temperatures and uses no additives. This reaction showed excellent regioselectivity, when applicable, and low levels of enantioselectivity (up to 33% ee). / Thesis (MS) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

De par son rôle dans de nombreuses fonctions biologiques, la vitamine A est une molécule majeure et cruciale du développement embryonnaire à l’âge adulte. Celle-ci est aussi, à l’heure actuelle, déjà largement utilisée comme agent thérapeutique pour de nombreuses pathologies affectant principalement la peau et les yeux (cancer, acné, psoriasis,brûlures oculaires) et certains cancers. Son action pro-cicatrisante - bien que largement étudiée grâce à de nombreux modèles humains et animaux - reste encore aujourd’hui mal caractérisée quant aux mécanismes d’action moléculaire (régulation de gènes) et cellulaire(migration, prolifération…) qu’elle utilise.Dans le but d’améliorer et de mieux maîtriser l’utilisation de la vitamine A dans le traitement de lésions telles que les brûlures oculaires, ce travail visait d’une part, à étudier, plus en détail,l’effet de l’acide tout-trans rétinoïque ou ATRA - dérivé le plus actif - sur la cicatrisation de ’épithélium cornéen en utilisant un modèle in vivo de brûlures cornéennes par base chez la souris. Son but était également de déterminer par quels processus cellulaires, l’ATRA agit, en utilisant cette fois-ci un modèle in vitro (cellules épithéliales cornéennes humaines). Enfin,nous nous sommes intéressés à la régulation de gènes cibles par l’ATRA au niveau de la sphère oculaire, connus pour être impliqués dans la dynamique de la MEC.Ainsi, nous avons démontré que l’ATRA permettait la cicatrisation de l’épithélium cornéen en agissant principalement sur la migration cellulaire. Puis nous avons identifié un gène :LOXL4 - membre de la famille des lysyl oxydases - dont l’expression est induite par l’ATRA,et nous avons prouvé que son rôle était essentiel dans la cicatrisation cornéenne, décrivant ainsi pour la première fois un lien génique direct et protéique entre la vitamine A, substance active et son action clinique pro-cicatrisante. / Because of its role in many biological functions, Vitamin A is a major and crucialmolecule from development to adulthood. Currently, it is largely used as therapeutic agent inseveral eye or skin pathologies (acne, psoriasis, ocular burns) and cancers. Its pro-healingproperties have been largely studied in human and animal models but molecular (generegulations) and cellular (migration, proliferation…) mechanisms of the vitamin A actionhave to be more detailed.In order to better improve and control its use in the treatment of lesions such as ocular burns,this work aimed to study, more in details, the effects of atRA (all-trans-retinoic acid), activederivative form of vitamin A, on corneal epithelium healing using an in vivo model of alkaliocular burns in mouse and then, to determine by which cellular process atRA acts, by usingthis time, an in vitro model (human corneal epithelial cells). Finally, we were interested intargeting genes regulation by atRA in the ocular sphere, specially known to be implied in theECM dynamic.First, we demonstrated that atRA improves corneal epithelium wound healing, essentially byacting on migration. Then, we identified a gene, LOXL4, member of the lysyl oxidase family,which expression is induced by atRA and we proved that this one is essential in cornealwound healing, describing for the first time a direct gene and protein link between vitamin A,active substance, and its clinical pro-healing action.

Les thérapies de différenciation sont une alternative prometteuse aux drogues génotoxiques utilisées en chimiothérapie pour le traitement de nombreux cancers. En particulier, l’acide tout-trans rétinoïque (ATRA) est utilisé avec succès pour traiter la leucémie aiguë promyélocytaire, un sous-type des leucémies aiguës myéloïdes (LAM). Malheureusement, son efficacité clinique est limitée dans les autres sous-types des LAM. Cela est en particulier du à une répression épigénétique des gènes de réponse à l’ATRA. Les SUMO constituent une famille de modificateurs post-traductionnels apparentés à l’ubiquitine dont la conjugaison sur de nombreuses protéines, appelée sumoylation, est impliquée dans la régulation de nombreux processus cellulaires, dont la transcription. Dans ce contexte, l’objective de ma thèse a été de comprendre le rôle de la sumoylation dans la réponse des LAM aux thérapies de différenciation. Nous avons pu montrer que la sumoylation réprime la différenciation induite par ATRA dans plusieurs lignées cellulaires, des cellules primaires de patients y compris celles résistantes à la chimiothérapie. L’inhibition de la sumoylation par les inhibiteurs pharmacologiques ou la surexpression des désumoylases augmente de façon remarquable la différenciation par ATRA et, à l’inverse l’augmentation de la sumoylation suite à une surexpression de SUMO ou son enzyme de conjugaison Ubc9 réduit fortement l’efficacité d’ATRA. L’ATRA synergise avec l’inhibition de la sumoylation pour limiter la prolifération des cellules de LAM in vitro et in vivo. D’un point de vue mécanistique, l’inhibition de la sumoylation favorise la différenciation des cellules de LAM en facilitant l’expression des gènes responsables de la différenciation myéloïde. Ainsi, cibler la sumoylation constitue une approche prometteuse pour sensibiliser la LAM aux thérapies de différenciation. / Differentiation therapies are a promising alternative to genotoxic-based chemotherapies in the treatment of many cancers. In particular, All-trans-retinoic acid (ATRA) is successfully used for Acute Promyelocytic Leukemias, a subtype of Acute Myeloid Leukemias. However, its clinical efficiency is very limited in the other AML subtypes, in particular because of epigenetic repression of ATRA-responsive genes. SUMOs are a family of post-translational modifiers related to ubiquitin and their conjugation, sumoylation, to their substrate proteins regulate many processes including gene transcription. The aim of my thesis was to understand the role of sumoylation in AML responses to treatments. I showed that sumoylation represses ATRA-induced differentiation in many AML cell lines and primary patient samples, including those resistant to chemotherapies. Inhibition of sumoylation with pharmacological inhibitors or overexpression of desumoylases markedly increased their differentiation by ATRA and increasing sumoylation by overexpression of SUMO or its conjugating enzyme Ubc9 strongly reduce ATRA efficiency. Inhibition of sumoylation synergize with ATRA to arrest AML cells proliferation both in vitro and in vivo. Mechanistically, inhibition of sumoylation primes AML cells for differentiation by facilitating the expression of master genes of the myeloid differentiation. Targeting the SUMO pathway thus constitute a promising approach to sensitize AML to differentiation therapies.

G-NNACI, a Naja naja atra chymotrypsin inhibitor consists of 57 amino acid residues cross-linked by three disulfide bridges and belongs to the Kunitz/BPTI superfamily, has been successfully cloned and expressed in our laboratory. Since snake venom non-neurotoxic Kunitz/BPTI inhibitors are most conserved in the core and in the N-terminal surface area, Ala-screening mutagenesis, deletion and Domain swapping on the N-terminus were carried out in this study to assess the role of N-terminus in G-NNACI. G-NNACI mutants with single amino acid substitution and deleted mutants were prepared. The secondary structure of all mutated proteins did not significantly alter as evidenced by CD spectra. Although all mutants are found to be functionally active as an inhibitor, their inhibitory potency against chymotrypsin differed. In contrast to G-NNACI and other mutants, R1A¡BP2A and ¡µN3 mutants had a propensity to alter their disulfide linkages under basic conditions. The results of thermal and urea denaturation suggested that amino acid substitution and deletion at the N-terminus lead to a change in the structural stability of G-NNACI. Consequently, the inhibitory potency of G-NNACI mutants along with time was affected. B chain of

Cancer has become the first among the 10 major death causing factors in Taiwan. Glioblastoma multiforme (GBM) is the most common malignant tumor in adult human brain tumors. Previously, heat shock or all trans retinoic acid (ATRA) treatment has been shown to be effective in inducing cell apoptosis and cell cycle arrest in several cancer cell lines. In this study, human brain tumor cell line GBM8401 was exposed to 43¢J for 30 min followed by incubation with ATRA. The treatment resulted in up to 50% inhibition of cell growth rate and 50% reduction of cell survival rate . Analysis of cell apoptosis related gene expression and protein expression with RT-PCR and Western blot has showen that p21, p27, pro-caspase 3, phospho-JNK and phospho-p38 were overexpressed after treatment of tumor cells with 43¢J for 30 min followed by addition of ATRA for 15 min to 8 hr. The immunocytochemistry assay revealed that overexpression of phospho-p53 in the nuclei after tumor cells were treated with 43¢J for 30 min followed by addition of ATRA for 8 hr. Results from this study show that treating tumor cells with heat shock before incubation with ATRA will enhance cell apoptosis and inhibit cell growth.

A Leucemia Mieloide Aguda (LMA) é uma neoplasia originada a partir da expansão clonal de blastos da linhagem mieloide em medula óssea, sangue periférico e outros tecidos. Entre as mutações mais frequentemente detectadas nas LMAs, se encontra a mutação do tipo duplicação interna em tandem (FLT3-ITD) que é detectada em aproximadamente 25% dos pacientes adultos. Esta mutação no receptor de tirosina quinase FLT3 é uma inserção de 3 a 400 pares de base na região juxtamembrana do receptor, a qual é responsável pelo controle da atividade enzimática dos domínios tirosina quinase. Quando esta mutação se encontra presente, a região juxtamembrana perde a capacidade de controlar a ativação dos domínios tirosina quinase e o receptor fica constitutivamente ativo conferindo uma vantagem proliferativa ao clone leucêmico. Esta mutação é considerada de mal prognóstico e já foram desenvolvidos inibidores de tirosina quinase específicos para o receptor FLT3 (FLT3 TKI). Porém, os resultados dos primeiros ensaios clínicos não apresentaram a efetividade esperada e continua a busca de novas combinações de drogas que contribuam a aumentar a efetividade destes inibidores. É por isso que este trabalho teve por objetivo testar a combinação de FLT3 TKIs com o ácido trans-retinoico (ATRA) já aplicado no tratamento da Leucemia Promielocítica Aguda (LPA) com PML-RARA. A combinação de FLT3 TKIs com ATRA induziu a morte celular programada de forma precoce tanto na linhagem de LMA MV4-11 como na MOLM-13. Esta morte celular observada foi inibida com pré- tratamento com inibidor de caspases QVD. O tratamento combinado in vivo em camundongos Nod Scid Gamma (NSG) transplantados com células MOLM-13, aumentou a sobrevida dos animais e diminuiu a percentagem de células CD45 humanas em medula óssea, baço e sangue periférico. No seu conjunto, nossos dados sugerem que o ATRA aumenta o efeito citotóxico dos FLT3-TKIs. Este achado poderá ser relevante para o tratamento de pacientes com LMA portadores de mutaçãoes ITD no gene FLT3 / Acute Myeloid Leukemia (LMA) is hematological disease that arises from the clonal expansion of a myeloid blast in bone marrow, peripheral blood and other tissues. Among LMAs mutations most frequently detected, 25% of adult patients carry the FLT3-ITD mutation. This mutation is a pair base insertion of 3 to 400 in the juxtamembrane domain of the receptor and leads to the constitutive activation of the kinase domains. It gives the leukemic clone a proliferative advantage and it is associated with a bag prognosis. FLT3 tyrosine kinase inhibitors (FLT3 TKI) were already developed in order to improved patients\' treatment. However, results from the first clinical trials were not as promising as expected. Therefore, there is still room for testing new drug combinations that could improve FLT3 TKIs efficacy. The main objective of this work was to test FLT3 TKIs in combination with all-trans retinoic acid (ATRA) already used in Acute Promyelocytic Leukemia (APL) with PMLRARA treatment. The combination of FLT3-TKIs and ATRA induced early programmed cell death in both the MV4-11 and MOLM-13 LMA lines. This early cell death was inhibited with QVD caspase inhibitor pre-treatment. In vivo combined treatment in Nod Scid Gamma (NSG) mice transplanted with MOLM-13 cells, increased animals survival and decreased the percentage of human CD45 cells in bone marrow, spleen and peripheral blood. Taken together, our data suggest that ATRA increases the cytotoxic effect of FLT3- TKIs. This finding may be relevant for the treatment of patients with AML with ITD mutations in the FLT3 gene

A ribonucleoproteína heterogênea nuclear K (hnRNP K) e o inibidor endógeno da fosfatase 2A (SET) são superexpressos e propostos como marcadores prognósticos em leucemia mieloide aguda e crônica. O objetivo do estudo foi caracterizar a participação das proteínas hnRNP K e SET na leucemogênese da leucemia promielocítica aguda (LPA), assim como na diferenciação celular induzida pelo ácido all-trans retinóico (ATRA). Os resultados iniciais de qRT-PCR demonstram que os níveis de RNAm de HNRNPK e SET estão aumentados em pacientes ao diagnóstico de LPA em comparação com amostras de indivíduos saudáveis e diminuem após indução e durante a manutenção do tratamento. Os resultados foram validados por Western blot, sugerindo hnRNP K e SET como marcadores diagnóstico e de resposta ao tratamento. O knockdown de hnRNP K e SET foi realizado em células de LPA sensível, NB4, e resistente ao ATRA, NB4-R2, utilizando RNA de interferência. Ambas as proteínas também foram testadas como alvo terapêutico com a utilização de inibidores de hnRNP K (U0126) e SET (OP449 e FTY720). A diminuição de hnRNP K nas células levou ao aumento da diferenciação celular granulocítica em ambas as células, principalmente na presença de ATRA, e portanto, foi capaz de reverter o fenótipo de resistência ao ATRA das células NB4-R2. O knockdown de hnRNP K, assim como o tratamento com U0126, levou a perda de viabilidade dessas células por indução de apoptose acompanhada da clivagem da proteína SET. O knockdown de SET em células LPA confirmou que a indução de apoptose em células com knockdown de hnRNP K ocorreu por clivagem e não pela diminuição da proteína SET nas células. Além disso, demonstrou também que SET prejudica a atuação do ATRA no processo de diferenciação celular. O modelo in vivo utilizando transplante de NB4-R2 em camundongos nude confirmou que o trióxido de arsênico (ATO) combinado a U0126 tem um maior potencial contra a progressão tumoral quando comparado ao tratamento isolado com ATO. FTY720 e OP449 tiveram efeito anti-leucêmico significativo com redução da viabilidade celular. Quando em associação, FTY720 e OP449, apresentaram efeito sinérgico significativo em NB4-R2 (CID<0,7). Concluímos que a superexpressão de hnRNP K e SET contribui para o bloqueio da diferenciação celular em promielócitos e prejudicam a atuação do ATRA no tratamento da LPA e, portanto, hnRNPK em associação com a proteína SET representam alvo terapêutico em potencial para terapia anti-leucêmica da LPA, principalmente para pacientes resistentes ao ATRA / Heterogeneous nuclear ribonucleoprotein K (hnRNP K) and endogenous inhibitor of phosphatase 2A (SET) are overexpressed and proposed as prognostic markers in acute and chronic myeloid leukemia. The study aim was to characterize the hnRNP K and SET proteins involvement in acute promyelocytic leukemia leukemia (APL) leukemogenesis as well as all-trans retinoic acid (ATRA) induced cell differentiation. Initial qRT-PCR results demonstrate that HNRNPK and SET mRNA levels are increased in patients diagnosed with APL compare to samples from healthy donors and decrease after induction and during maintenance of treatment. The results were validated by Western blot, suggesting hnRNP K and SET as diagnostic and response to treatment markers. The knockdown of hnRNP K and SET was performed on sensitive, NB4, and ATRA-resistant, NB4-R2, LPA cells using interfering RNA. Both proteins were also tested as a therapeutic target with a use of hnRNP K (U0126) and SET inhibitors (OP449 and FTY720). The decrease of hnRNP K in cells led to increased granulocyte cell differentiation in both cells, especially in the presence of ATRA, and thus was able to reverse the NB4-R2 cells resistance to ATRA phenotype. The hnRNP K knockdown, as well as the treatment with U0126, had a loss of cell viability by induction of apoptosis accompanied by cleavage of the SET protein. The SET knockdown in APL cells confirmed that an induction of apoptosis in cells with hnRNP K knockdown occurred by cleavage and not by the SET protein decrease in the cells. Furthermore, it has also shown that SET impairs the ATRA\'s performance in the cellular differentiation process. The in vivo model using NB4-R2 transplant in nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has a greater potential against tumor progression compared to the treatment isolated with ATO. FTY720 and OP449 have significant anti-leukemic effect reducing cell viability. When in combination, FTY720 and OP449, they had a significant synergistic effect on NB4-R2 (CDI <0.7). We conclude that overexpression of hnRNP K and SET contributes to block cell differentiation in promyelocytes and impair the performance of ATRA in the treatment of APL and therefore hnRNPK in association with a SET protein represent a potential therapeutic target for anti-leukemic therapy of APL, mainly for patients resistant to ATRA

In Sweden you will find the coot chiefly in the southern parts. The species winters in the southern part of the Baltic Sea and along the coasts of northern Europe. The number of coots is heavily decreased by harsh winters. The population of the coots in Sweden is estimated to 20 000 to 30 000 pairs. The region of Dalarna is the random zone for the breeding of coots in Sweden . During the summer of 2006 the coot was reported to reproduce in at least 31 lakes and rivers preferably in the southern part of the Dalarna region. From Lake Brunnsjön at least 50 pairs of coots were reported. Lake Brunnsjön is by far the most nutrient rich lake in Dalarna.From the three lakes that are included in the study, namely Lake Glistjärn, Lake Limsjön and Lake Kyrkbytjärn, the water analysis shows high pH values, well-buffered waters and meso- trophic waters.The number of successful breedings was eleven. Lake Glistjärn zero breeding, Lake Limsjön four and Lake Kyrkbytjärn seven breedings. The northernmost lake in Dalarna where breeding occurred is Lake Limsjön.

Telomeres are important structural and functional components of chromosomes, serving to provide stability and enabling full replication of the chromosomes. However, a shortening of the telomeres occurs with each cell division that can be fixed by a polymerase activity provided by telomerase, preventing this loss which would otherwise eventually lead to chromosome end-to-end fusions, senescence and cell death. The telomerase activity is present in stem cells and germ line cells, but absent or barely noticeable in adult somatic cells. However, in approximately 80-90% of transformed somatic cells the telomerase activity is recovered, resulting in a "telomerase positive phenotype". This phenotype has been a prime target in cancer research, and recently a novel mechanism for regulating telomerase levels has been uncovered. Makorin 1 RING finger protein (MKRN1) was found to be an E3 ubiquitin ligase for hTERT, the rate-limiting catalytic component of telomerase, leading to the ubiqutin-mediated 26s proteasomal degradation of hTERT and reduced telomerase activity. So, MKRN1 plays a role in telomere homeostasis. In this study we looked at the expression of MKRN1 in numerous tumor cell lines (Hela, HCT116, HL60) and the normal diploid fibroblasts (WI-38). In the latter cell line, basal levels of MKRN1 were found to increase 6-fold when the cells were serum starved and arrested in G1/G0. In contrast, the cancer cell lines expressed MKRN1 at low levels or undetectable. This would indicate that MKRN1 is up-regulated in resting or G1 arrested cells.In one cell line the promyelocytic leukemia, HL-60, showed no protein levels of MKRN1. This cell line is able to be terminally differentiated upon ATRA treatment, when cells are arrested at G1. In this model system of cellular differentiation hTERT mRNA levels and telomerase activity decrease drastically and quickly. We hypothesized that the differentiation of HL-60 induced by ATRA would be accompanied by an increase in MKRN1 levels. MKRN1 mRNA and protein levels were strongly up-regulated during the ATRA-mediated differentiation of HL-60 cells. Although, a decrease in hTERT mRNA is a contributor to telomerase inhibition during cellular differentiation; our data indicate that the up-regulation of MKRN1 ensures the effective removal of residual telomerase activity by the ubiquitin-mediated degradation pathway at the proteasome.