Le gène GALIG, interne à celui de la GALECTINE-3, permet la production de 2 protéines, les Galigines, qui interagissent avec des protéines de l’autophagie. Décrit comme un gène pro-apoptotique, il est inhibé par l’anti-apoptotique Mcl-1. Il est sous-exprimé dans la moelle osseuse (MO) de patients atteints de Leucémies Aiguës Myéloïdes (LAM) M2, pathologie caractérisée par un blocage de la différenciation. Au cours de cette thèse, j’ai pu montrer que le gène GALIG pourrait être impliqué dans la différenciation myéloïde. Son expression augmente de façon croissante chez les LAM en fonction de l’état de différenciation de la cellule leucémique. De plus, bien que faible au diagnostic, l’expression du gène augmente également dans la MO et le sang après traitement de chimiothérapie. Ces observations sont renforcées par des études in vitro qui indiquent que l’expression de GALIG augmente précocement lors de la différenciation en polynucléaires et en macrophages, avant l’apparition des caractéristiques de maturation terminale. La survie de ces cellules, suite à l’activité de GALIG pendant la différenciation, pourrait être assurée grâce à l’augmentation de l’expression du gène anti-apoptotique MCL1. Dans le sang de patients infectés par le Virus de l’Immunodéficience Humaine, traités et sans charge virale, à l’inverse des LAM, il est observé une surexpression de GALIG. Ceci suggère une perturbation du système immunitaire. En plus de l’activation de GALIG, il est noté une dérégulation de l’expression de plusieurs gènes impliqués dans l’autophagie. Ceci pourrait conduire à une inhibition du niveau basal de l’autophagie chez ces patients, qui pourrait être à l’origine du vieillissement prématuré des cellules sanguines et qui serait liée à l’inflammation chronique observée chez ces patients. En conclusion, ces résultats nous encouragent à poursuivre les études qui visent à comprendre les mécanismes de régulation de l’expression du gène GALIG et les mécanismes d’action des Galigines. / Embedded within the GALECTIN-3 gene, GALIG gene allows the production of 2 proteins, Galigins, which interact with autophagy proteins. Described as a pro-apoptotic gene, it is inhibited by Mcl-1, an anti-apoptotic protein. It is under-expressed in the bone marrow (BM) of patients with Acute Myeloid Leukemia (AML) M2, a pathology characterized by a differentiation blocking. During my thesis, I showed that the GALIG gene could be involved in myeloid differentiation. The expression of GALIG gene gradually increases in AML with the differentiation stage of the leukemic cell. In addition, although weak at diagnosis, GALIG gene expression also increases in BM and blood after chemotherapy treatment. These observations are reinforced by in vitro studies which indicate that GALIG expression increases early during polymorphonuclear and macrophages differentiations, before the onset of terminal maturation features. Cell survival during differentiation could be ensured by the increasing expression of the MCL1 which could counteract the apoptotic function of GALIG. In the blood of treated HIV-infected patients, without viral load, the transcription rate of GALIG is higher when compared to uninfected donors. This suggests a possible dysfunction of the immune system. In addition, several autophagy genes are dysregulated which could lead to the inhibition of the basal level of autophagy in these patients, which in turn could be a cause of the premature aging of blood cells and linked to the chronic inflammation reported for efficient treated HIV patients. In conclusion, these results encourage us to pursue studies deciphering the mechanisms of regulation of the GALIG gene expression and the mechanisms of action of the Galigins.

Nucleosomes, the primary unit of chromatin structure, are positioned either statistically or specifically. The statistical positioning denotes the arbitrary positioning of nucleosomes on DNA agreeing with the nucleosome’s broad coverage of the genome—however, there is evidence that nucleosomes are also positioned specifically at controlled positions. DNA sequences determine the specific nucleosome positions, and the presence or depletion of nucleosomes affects the availability of the DNA region to other proteins. The DNA sequences of H2A and H2A.Z nucleosomes in Drosophila were analysed in search of nucleosome positioning patterns. Dinucleotide patterns with 10 bp periodicity were identified from the DNA sequences of H2A nucleosomes. Compared with the yeast patterns, the Drosophila patterns had the same periodicity but different dinucleotides near the dyad, which was related to the different H3 structure between them. The nucleosome positioning patterns from the H2A.Z nucleosomes implied the specific histone-DNA interaction as a result of the deviations of the patterns where the different amino acids of H2A and H2A.Z interact with the DNA. The Ly49 gene cluster was selected as a model system to study the interplay between nucleosomes and transcription factors. Ly49 proteins, the surface receptors on NK cells, display variegated expression patterns, and the bidirectional promoter Pro-1 is known as a key determinant of the stochastic expression of each Ly49 gene. The systematic analysis of nucleosome positions based on the genome sequences in the Ly49 gene cluster revealed that the repressing Pro-1 reverse promoters are open, while the activating forward Pro-1 promoters were covered by nucleosomes. Furthermore, specific nucleosome positions covered transcription factor binding sites. The covered factor binding sites were further examined by their periodic appearances on the nucleosome-covered sequences, which revealed the accessibility to the sites. The sequence analysis predicted that the regulation by the transcription factor AML-1 would be sensitive to the nucleosome coverage; the prediction was confirmed by cell line experiments. The 10 bp periodic nucleosome positioning patterns interact with histones specifically. The long nucleosome positioning patterns coexist with the short sequence motifs for transcription factor binding sites adding another layer of the control to the transcriptional regulation.

La leucémie aiguë myéloïde (LAM) est une hémopathie maligne à progression rapide, qui se caractérise par une expansion clonale de précurseurs myéloïdes présentant un contrôle défectueux de leur prolifération et de leur différenciation. Une rémission complète peut être obtenue chez environ 80% à 85% des patients en associant cytarabine et anthracycline qui sont respectivement un inhibiteur de la synthèse d’ADN et un agent intercalant. Néanmoins, les résultats globaux pour les patients atteints de LAM restent médiocres et le taux de survie à 5 ans des patients âgés de plus de 60 ans, est inférieur à 10%. Le paradigme bien accepté de la leucémogenèse est que la leucémie résulte de la transformation d'une cellule unique appelée cellule souche leucémique (SCL) ou cellule initiatrice de leucémie (LIC) qui se développe par autorenouvellement et engendre par division asymétrique les blastes leucémiques bloqués dans leur différentiation. Les LIC vont être responsables du maintien et de la rechute de la maladie après le traitement chimiothérapeutique car si les traitements actuels sont relativement efficaces contre les blastes leucémiques, ils échouent au niveau des LIC. Un autre facteur important impliqué dans la résistance aux traitements est le microenvironnement de la moelle osseuse qui forme la niche hématopoïétique. Des études ont montré que différents composants cellulaires de la niche peuvent transférer des mitochondries à des cellules normales soumises à un stress métabolique ou dans un contexte pathologique, vers des cellules cancéreuses. Durant ma thèse, j'ai pu montrer que les cellules murines de la lignée MS-5 et/ou des cellules stromales primaires humaines dérivées de la moelle osseuse, utilisées comme cellules nourricières dans des expériences de co-culture, sont capables de transférer des mitochondries fonctionnelles aux cellules leucémiques. En utilisant différentes approches moléculaires et d'imagerie, nous avons pu montrer que les cellules de LAM peuvent, par ce transfert, augmenter leur masse mitochondriale jusqu'à 14%. Dans la co-culture, les cellules LAM receveuses ont montré une augmentation de 1,5 fois de la production d'adénosine triphosphate (ATP) mitochondriale et se sont révélées moins sujettes à une dépolarisation mitochondriale après chimiothérapie, affichant une survie plus élevée. Ce transfert unidirectionnel, renforcé par certains agents chimiothérapeutiques, nécessite des contacts cellule-cellule et semble se dérouler par une voie endocytaire qui reste à déterminer. Enfin, nous démontrons que le transfert mitochondrial est observé in vivo dans un modèle de xénogreffe de souris immunodéficientes NSG et se produit également dans les cellules et les progéniteurs initiateurs de la leucémie humaine et leur conférant une capacité plus élevée à initier des cultures leucémiques à long terme. Nous avons ainsi apporté la preuve qu'un transfert horizontal de mitochondries provenant des cellules stromales de la niche hématopoïétique participe aux phénomènes de chimiorésistance des cellules leucémiques receveuses. De ce fait, cibler ce transfert mitochondrial pourrait représenter une future cible thérapeutique originale pour un traitement adjuvant des LAM visant à interférer avec le soutien de leur microenvironnement. / Acute myelogenous leukemia (AML) is a heterogeneous group of hematopoietic malignancies arising from hematopoietic stem and/or progenitor cells that display defective control of their proliferation, differentiation, and maturation. Complete remission is achieved using anthracycline and cytarabine combination therapy in 80% to 85% of older patients. Nevertheless, the overall outcomes for AML patients remain poor, and the 5-year survival rate for patients over 60 is less than 10%. The well-accepted paradigm of leukemogenesis is that leukemia arises from the transformation of a single cell and is maintained by a small population of leukemic stem cells (LSC) or leukemia initiating cells (LICs). It is theorized that current treatments, although highly effective against the leukemic bulk, fail to eradicate the LICs that are therefore responsible for leukemia relapse. Another important factor involved in resistance to treatments is the microenvironment of the bone marrow, which is called the hematopoietic niche. Studies have shown that different niche cell components can transfer mitochondria to normal cells that undergo a metabolic stress and in a pathological context, to cancer cells. During my PhD we demonstrate that in an ex vivo niche-like coculture system, cells both primary and cultured AML cells take up functional mitochondria from murine or human bone marrow stromal cells. Using different molecular and imaging approaches, we show that AML cells can increase their mitochondrial mass by up to 14%. After coculture, recipient AML cells showed a 1.5-fold increase in mitochondrial ATP production and were less prone to mitochondrial depolarization after chemotherapy, displaying a higher survival. This unidirectional transfer enhanced by some chemotherapeutic agents required cell–cell contacts and proceed through an ill-defined endocytic pathway. Transfer was greater in AML blasts compared with normal cord blood CD34+ cells. Finally, we demonstrate that mitochondrial transfer was observed in vivo in an NSG immunodeficient mouse xenograft model and also occurred in human leukemia initiating cells and progenitors. As mitochondrial transfer provides a clear survival advantage following chemotherapy and a higher leukemic long-term culture initiating cell potential, targeting mitochondrial transfer could represent a future therapeutic target for AML treatment.

INTRODUCTION: Older patients with AML (> 60 years) often receive intensive EOL care including hospitalizations and chemotherapy close to death. Intensive EOL care has been shown to increase emotional and financial burdens for patients and families, while often not aligning with patients’ preferences. However, factors associated with the intensity of EOL care in this population are unknown. OBJECTIVES: There is a need to better understand the factors associated with intense EOL care, in hopes of providing more informed, high-quality EOL care in line with patient preferences and decreasing burdens associated with unnecessary healthcare. We aim to describe the associations between the intensity of EOL care, patient demographics, and baseline psychological distress in older patients with AML. METHODS: We conducted a secondary analysis of two supportive care studies including 168 deceased older patients with AML. We assessed patients’ demographics, quality of life (QOL) [Functional Assessment Cancer Therapy-Leukemia], and anxiety and depression symptoms [Hospital Anxiety and Depression Scale (HADS); Patient Health Questionnaire (PHQ-9)] at the time of diagnosis. We used multivariate logistic regression models to examine the association among demographic factors, patient-reported outcomes, and the following EOL care outcomes abstracted from the electronic health record: 1) hospitalizations in the last 7 days of life; 2) receipt of chemotherapy in the last 30 days of life; and 3) hospice utilization. RESULTS: The median age of the cohort was 69 (range 20-100), and the majority were males (63.7% 107/168). Overall, 66.7% (110/165) of patients were hospitalized in the last 7 days of life, 51.8% (71/137) received chemotherapy in the last 30 days of life, and 40.7% (70/168) utilized hospice services. In multivariate models, higher education (OR = 1.54, SE=0.24, P=0.006), and elevated depression symptoms [PHQ-9: OR=1.09, SE=0.04, P=0.028] at the time of diagnosis were associated with higher odds of being hospitalized in the last 7 days of life. In contrast, higher QOL at diagnosis [OR=0.98, SE=0.01, P=0.009] was associated with lower odds of being hospitalized in the last 7 days of life. Depression symptoms at the time of diagnosis as measured by the HADS was the only factor associated with the receipt of chemotherapy in the last 30 days of life [HADS-Depression: OR=1.10, SE=0.05, P=0.042]. Patients factors were not associated with hospice utilization. CONCLUSIONS: Older patients with AML who are more educated and report elevated depression symptoms and lower QOL at the time of diagnosis were more likely to receive intensive EOL care. These findings identify a population at the time of diagnosis of AML who are at higher risk for hospitalizations and chemotherapy use at the EOL and who may benefit from targeted supportive care interventions.

Thesis deals with active magnetic bearing (AMB) levitation control design. Its prototype was done in FSI collaboration with FEKT VUT Brno. The research is focused on communication tools and mathematical model making as well as general AMB issues. Based on this the control design is done an experimentally verified.

La leucémie aiguë myéloïde (LAM) est une pathologie hématologique dont le pronostic reste très défavorable, malgré les progrès réalisés dans la compréhension des mécanismes physiopathologiques sous-tendant son développement. Identifier de nouvelles stratégies anti-leucémiques représente donc une étape clé dans la concrétisation des avancées thérapeutiques. Grâce à la combinaison de plusieurs approches de criblage génétiques et pharmacologiques, l’objectif de ma thèse a été de définir et valider de nouvelles cibles thérapeutiques dans les LAM. La première partie de ma thèse a eu pour but de transposer en clinique l’inhibition de la Glycogen Synthase Kinase 3 (GSK3). La stabilisation de la β-caténine secondaire à l'inhibition concomitante des deux paralogues de GSK3, représente un obstacle à l’utilisation clinique de cette classe thérapeutique. Mettant à profit la présence d'un «switch» Asp133 à Glu196 dans les domaines de liaison ATP de GSK3, nous avons identifié un inhibiteur sélectif du paralogue GSK3α et mené des études précliniques validant le BRD0705 comme nouveau traitement pro-différenciant dans les LAM. De plus, une combinaison de profilage métabolomique et d'approches de criblage haut débit à l’aide d’une banque de shRNA a permis d'identifier un nouveau lien entre EVI-1, la voie de la créatine kinase et la voie de signalisation GSK3. La deuxième partie de ma thèse a porté sur l'identification de nouvelles cibles thérapeutiques en utilisant une approche de criblage par banque de shRNA dans le modèle murin de LAM porteur de la translocation MLL-AF9. VCP, une AAA-ATPase, a ainsi été identifiée puis validée comme cible thérapeutique. Nous avons montré que VCP orchestre la génération d'une plateforme à ADN simple brin recouverte de RPA, ce qui entraîne l'activation de la kinase ATM et la HR. Dans leur ensemble, nos découvertes permettent une meilleure compréhension de la biologie des LAM et participeront ainsi à l’amélioration des traitements futurs de cette pathologie. / Despite the significant progress made in understanding Acute Myeloid Leukemia oncogenesis over the last decades, this disease remains devastating and the overall five-year survival does not exceed 17%. Developing new translational research strategies focused on the identification of druggable oncogenic targets is critical to continued progress in AML treatment. The goal of this work was to define and validate novel leukemia-specific dependencies using small-molecule inhibitors and RNA-interference-based high-throughput screening methods.The first part of my thesis work aimed at translating Glycogen synthase kinase 3 (GSK3) inhibition into the clinic. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent β-catenin stabilization, were a concern in the clinical translation of this target candidate. Specific knock-down of GSK3α or GSK3β alone does not increase β-catenin, thereby offering a conceptual resolution to GSK3 targeting. The design of selective ATP-competitive inhibitors posed a drug discovery challenge due to the high homology in the GSK3α and GSK3β ATP binding domains. Taking advantage of an Asp133 ® Glu196 “switch” in the GSK3 paralog hinge binding domains, we identified a first-in-class GSK3α selective inhibitor and conducted preclinical studies validating BRD0705 as a promising new differentiation therapy in AML. In addition, a combination of a metabolomic profiling and a pooled shRNA screening method identified a new interplay between the oncogene EVI-1, the creatine kinase pathway and GSK3 signaling. The second part of my studies focused on identification of new therapeutic targets using an in vivo pooled shRNA screening approach in the MLL-AF9-driven AML mouse model. VCP, an AAA-ATPase, was thus identified and validated as a top target. We demonstrated that VCP orchestrates RPA-coated-single-stranded-DNA platform generation, resulting in ATM kinase activation and subsequent HR. Taken together, our discoveries increased our understanding of AML biology and may therefore contribute to novel and more efficacious treatments for this highly aggressive and lethal disease.

Treatment for lymphoma with alkylating therapy is known to increase the risk of secondary malignancies such as Acute Myeloid Leukemia (AML), although the risk is not fully understood. This study investigates the characteristics of AML that arise after lymphoma treatment in contrastto AML cases without a prior lymphoma. The study population consists of 115 individuals identified from the Swedish lymphoma register (SLR) with a diagnosis in the quality register for AML between 2000-2019, matched 1:1 to lymphoma-free comparators. A hierarchical clusteranalysis with Gower’s similarity measure and the k-prototypes clustering algorithm are employed to separately identify subgroups of those with a lymphoma history and the matched comparators. The survival of lymphoma patients is compared between subgroups in a Cox regression model. The findings suggests a two-cluster partition achieved by the hierarchical method for patients with a lymphoma history as well as for lymphoma-free patients (average Silhouette 0.853 and0.842, respectively). Both partitions completely separates patients with genetic information from those without. For AML patients with a preceding lymphoma, a subgroup defined by the hierarchical two-cluster partition is associated with an increased mortality rate (HR 2.40). A three-cluster partition achieved by the k-prototypes algorithm could be more clinically relevant, however only one subgroup is associated with increased mortality (HR 2.73).

Min ambition är att med aktuellt material samt med en handfull adekvata metoder pröva mintes om att det krävs en central europeisk myndighet, med såväl polis, åklagare som domstol,som är underställd justitiedepartementen med fullt mandat för att komma tillrätta med en utavvår tids största globala organiserade brottsligheter, Penningtvätt. Detta hotar samhälletsförtroende för kapitalmarknaderna, kan leda till finansiell systemkris och på det stora helasåledes vårt moderna sätt att leva. Samhällskontraktet mellan staten och dess medborgareutmanas. Människans innersta väsen, med en epigenetisk tillika predisponerad girighet står idirekt polemik med vårt sätt att på nations och unions vägar mitigera detta humana drag. Utanett skifte från förvaltningsrätten till straffrätten, med en verkställighet med tydligt preventivaoch direkt effektiva sanktioner som är större än ett ändrat arbetssätt och lite penningböter(som till slut diskonteras i Bankernas P/L kalkyler), så står inte dessa väl cementeradeinstitutioner till svars som en del utav en lösning. Inte heller är regleringen i form av lagar,regler och förordningar optimal, den borde kräva med [emfas] (direkt kausalitet) en vässadverkställighet och inte ett samordnande av artiklar, direktiv, regler och förslag. Bankerna,vågar jag påstå (stryk: att vi idag med all säkerhet kan stipulera), är en avgörande del avproblemet, vilket jag varken tror de vill eller skall så vara: de är en av nycklarna till lösningenpå problemet med penningtvätt. Alltså annorlunda uttryckt: hur får sheriffen kulor i revolvernoch hur får vi sheriffen att ta till mod för att stämma i bäcken? Sheriffen är härtillsynsmyndigheten. Hur skiftar vi paradigmet hos nyckelaktören: banken? Vad skulle kunnaskrämma banker och bankanställda till compliance/efterlevnad? Kan vi jämföra med och hurfår vi i så fall fram ett ställföreträdaransvar likt det sjukvården besitter?

As the financial system has grown ever more computerized and complex, so has the problem of money laundering, and thus also the need for anti-money laundering (AML). However, as the financial system is hard for states to access, they have, encouraged by international organizations such as Financial Action Taskforce (FATF), delegated a large portion of the responsibility for anti-money laundering efforts to private financial institutions through the use of regulation. This study therefore investigates how people who work with AML in financial institutions see their role as executers of public policy, what they see as their responsibilities, and how they experience the system of delegated responsibility. The analysis is based on ten interviews with AML-professionals from three Swedish banks.  The study shows that AML-professionals in Swedish banks are positive to their role as public policy executers and see their role as not just rule-following, but also as a part of the crime preventing infrastructure. They therefore exhibit commitment towards three responsibilities: to bear the banks profit margins in mind, to ensure that AML-laws are followed, and to secure that the AML-efforts counteracts money laundering in an impactful way, as they see money laundering as a pressing problem. These three responsibilities have the possibility to conflict and hence require prioritization. The results of this study showcase how regulation with the intent of societal improvement can lead to the regulated not just following rules, but also internalizing the purpose of the regulation.

Indiana University-Purdue University Indianapolis (IUPUI) / FMS-like tyrosine receptor kinase-internal tandem duplications (FLT3-ITDs) are the most frequent deleterious mutations found in acute myeloid leukemia (AML) and portend a poor prognosis. Currently, AML patients typically achieve disease remission, yet undergo high rates of disease relapse, implying a residual post-treatment reservoir of resistant malignancy-initiating cells. This begs for new therapeutic approaches to be discovered, and suggests that targeting multiple cellular compartments is needed for improved therapeutic approaches. We have shown that the protein tyrosine phosphatase, Shp2, associates physically FLT3-ITD at tyrosine 599 (Y599) and positively regulates aberrant STAT5 activation and leukemogenesis. We also demonstrated that genetic disruption of Ptpn11, the gene encoding Shp2, increased malignancy specific survival of animals transplanted with FLT3-ITD-transduced cells, suggesting that Shp2 may regulate the function of the malignancy-initiating cell. Taken together, I hypothesized that inhibiting Shp2 can target both FLT3-ITD+ AML tumor cells as well as FLT3-ITD-expressing hematopoietic stem cells. To study this hypothesis, I employed two validation models including genetic inhibition of Shp2 interaction with FLT3-ITD in 32D cells or genetic disruption of Shp2 in FLT3-ITD-expressing HSCs. Using FLT3-ITD-expressing 32D cells as an AML tumor model, I found that mutating the Shp2 binding site on FLT3-ITD (Y599) reduced proliferation in vitro and increased latency to leukemia onset in vivo. Further, pharmacologic inhibition of Shp2 preferentially reduced proliferation of FLT3-ITD+ primary AML samples compared to FLT3-ITD- samples, and cooperated with inhibition of the lipid kinase, phospho-inositol-3-kinase (PI3K), and of the tyrosine kinase, Syk, to reduce proliferation of both FLT3-ITD+ and FLT3-ITD- AML samples. To evaluate the stem cell compartment, I crossed a murine locus-specific knock-in of FLT3-ITD with Shp2flox/flox; Mx1-Cre mice to generate FLT3-ITD; Shp2+/- mice and found that Shp2 heterozygosity dramatically inhibits hematopoietic stem cell engraftment in competitive transplant assays. Further, I found that lineage negative cells from FLT3-ITD; Shp2+/- mice demonstrated increased senescence compared to control mice, suggesting that Shp2 may regulate senescence in FLT3-ITD-expressing hematopoietic stem cells. Together, these findings indicate a cooperative relationship between the tyrosine phosphatase, Shp2, and the kinases PI3K and Syk in AML tumor cells, and indicate that Shp2 plays a positive role in the stem cell compartment to promote stem cell function of the malignancy-initiating cell in AML. Therefore, targeting Shp2 may hold therapeutic benefit for patients with FLT3-ITD+ AML.